Quantitation of 6-thioguanine residues in peripheral blood leukocyte DNA obtained from patients receiving 6-mercaptopurine-based maintenance therapy.

The antimetabolite 6-mercaptopurine is widely utilized in maintenance therapy for childhood acute lymphoblastic leukemia. Following p.o. administration, this prodrug undergoes extensive biotransformation, resulting in the generation of a plethora of metabolites including 2'-deoxy-6-thioguanosine triphosphate. Incorporation of 6-thioguanine (6-TG) bases into DNA is generally considered to be central to thiopurine-mediated cytotoxicity. We have developed a novel precolumn derivatization HPLC technique for quantifying 6-TG base accumulation into leukocyte DNA obtained from acute lymphoblastic leukemia patients receiving 6-mercaptopurine maintenance therapy. The method is based on enzymatic degradation of DNA to 2'-deoxyribonucleosides and the derivatization of released 2'-deoxy-6-thioguanosine with a thiol-reactive reagent containing a 7-amino-4-methylcoumarin-3-acetic acid fluorophore. The 2'-deoxy-6-thioguanosine-7-amino-4-methylcoumarin-3-acetic acid adduct is resolved by reversed-phase HPLC and quantified fluorometrically. Assay response is linear from 15 pmol to 60 fmol 6-TG bases/microgram DNA with a limit of quantitation corresponding to the incorporation of 1 6-TG residue per 50,000 bases. In a small cohort of acute lymphoblastic leukemia patients receiving p.o. 6-mercaptopurine-based maintenance therapy, significant interindividual variation in the accumulation of 6-TG bases into leukocyte DNA was revealed. The determined levels of drug base incorporation ranged from 95 to 710 fmol 6-TG bases/microgram DNA (6-TG base:nucleotide ratio 1:32,000 to 1:4,000). The assay may provide a novel methodology for pharmacological monitoring of thiopurine therapy either in the routine clinical setting or during studies of alternative routes of drug delivery.

Protective effects of tumor necrosis factor on murine hematopoiesis during cycle-specific cytotoxic chemotherapy.

Tumor necrosis factor (TNF) is a pleiotropic cytokine which exerts a wide range of effects when administered in vivo. Using a murine model, we have investigated the effect of pretreatment with 1 microgram (2.6 x 10(4) units) per mouse of recombinant murine TNF-alpha on hematopoietic recovery following administration of cyclophosphamide, 5-fluorouracil, methotrexate, or vinblastine. TNF pretreatment results in enhanced regeneration of circulating neutrophils and hematopoietic progenitors, as measured by in vivo and in vitro assays, in animals given cycle-specific chemotherapeutic agents. The results may suggest that TNF affects cycle kinetics in hematopoietic progenitor cell populations, thus making these cells less prone to the cytocidal effects of the chemotherapeutic agents. As myeloablation is a frequent and often critical side effect following cancer treatment, these findings may have clinical implications.

Dose-dependent pharmacokinetics of methotrexate and 7-hydroxymethotrexate in the rat in vivo.

The pharmacokinetics of methotrexate (MTX) and 7-hydroxymethotrexate (7-OH-MTX) in bile, urine, and serum was studied in rats in vivo after short-time infusions of 10, 50, 250, and 1000 mg/kg MTX. All animals were anesthetized and drained of bile during experiments. The biliary secretion rate of MTX approached saturation when serum MTX levels surpassed 700-800 microM, causing a significant reduction in biliary recovery as the parent compound (49 to 32%) at MTX doses exceeding 50 mg/kg. The hepatic metabolism of MTX to the 7-hydroxy metabolite was not saturated at the doses used. Serum MTX pharmacokinetics demonstrated dose dependency, inasmuch as doses exceeding 10 mg/kg were accompanied by a reduced total body clearance (Clr) and biliary clearance (ClB). A significant finding in relation to acute hepatotoxicity reported after high-dose MTX in humans was the occurrence of cholestasis 30-90 min after drug infusion and the observation of macroscopic precipitations in the bile duct in five of six animals treated with 1000 mg/kg MTX. In these five animals, cessation of bile secretion occurred at similar bile 7-OH-MTX levels [9800 +/- 1100 (SD) microM], while the single rat that secreted bile throughout the experiment had a 5-fold lower peak 7-OH-MTX concentration in bile. Analysis of biliary precipitates formed in vivo and in vitro found 7-OH-MTX to constitute 97% and MTX 3% of the drug content of the precipitated material.

Formation and elimination of 7-hydroxymethotrexate in the rat in vivo after methotrexate administration.

Bile, urine, and serum concentrations of methotrexate (MTX) and 7-hydroxy-methotrexate (7-OH-MTX) were monitored in rats in vivo following a short-time infusion of 10 mg/kg [3H]MTX. The experiments were performed in one group of anesthetized, bile-drained rats and in two control groups, one anesthetized and one unanesthetized, that were not bile-drained. Peak biliary levels of MTX (3.8 x 10(-3) M) and 7-OH-MTX (1.8 x 10(-4) M) appeared within 15 min after cessation of infusions. For two log ranges of serum MTX concentrations, biliary levels remained 180-fold higher. High bile 7-OH-MTX levels appeared few min after start of MTX administration, and were 720 times higher than the peak serum concentrations, indicating that the liver is a major site of 7-OH-MTX formation in the rat. 7-OH-MTX concentrations in bile declined monophasically with a half-life of 29.4 min, while MTX showed a biphasic elimination with initial and second phase half-lives of 23.1 and 86.4 min, respectively. Bile was the major excretory route for MTX and 7-OH-MTX, with 50% of the dose recovered as the parent compound and 3.6% as the metabolite. There was no difference in urinary recovery of MTX in bile-drained and control animals, indicative of insignificant enterohepatic circulation of MTX. This was further corroborated by the finding of just 2.1% urinary recovery of MTX in rats who received previously collected MTX-containing bile through a duodenal catheter. Serum concentration curves were analyzed according to a three-compartment open model with an initial elimination half-life of 1.7-3.3 min, a second phase half-life of 15.4-21.0 min, and a terminal phase half-life of 119-240 min. Our finding of 7-OH-MTX formation and high biliary levels of the metabolite in the rat, can be used as basis for studies of interactions with in vivo MTX conversion to the 7-hydroxy metabolite.

Induction of HL-60 cell differentiation by 3-deaza-(+/-)-aristeromycin, an inhibitor of S-adenosylhomocysteine hydrolase.

The metabolically stable inhibitor of S-adenosylhomocysteine hydrolase (AdoHcyase), 3-deaza-(+/-)-aristeromycin (dzAri) has recently been shown to induce differentiation in HL-60 cells. The present study was undertaken to characterize the cytostatic, cytotoxic, and differentiation inducing properties of dzAri in HL-60 cells and to investigate biochemical consequences of AdoHcyase inhibition. A dye exclusion test and a clonogenic assay were used to test cytotoxic and cytostatic properties. dzAri had reversible cytostatic effects on HL-60 cells at concentrations lower than 10 microM and partially reversible cytotoxic effects above 10 microM. The induction of differentiation was dependent upon concentration and time of exposure, with maximal effect after 6 days incubation with 5-10 microM dzAri. Washout experiments demonstrated that the cells were not committed to differentiation after 48 h of incubation with dzAri. The AdoHcyase of HL-60 cells was inhibited with a Ki of 20 nM. The concentration of S-adenosylhomocysteine increased dose dependently 48 h after incubation with 0.1-100 microM dzAri. The incorporation of [3H]methyl from [methyl-3H]methionine into 5-methylcytosine of DNA was reduced by 26% at 5 microM dzAri. The findings indicate that continuous presence of dzAri is necessary to induce differentiation and inhibit proliferation in HL-60 cells. The inhibition of AdoHcyase perturbs levels of transmethylation metabolites and the incorporation of [3H]methyl into 5-methyl-cytosine of DNA.

Induction of a reversible block in murine CFU-C differentiation by exposure to nitrous oxide.

Patients subjected to prolonged exposure to nitrous oxide (N2O) often develop megaloblastic bone marrow changes. This toxicity is due to the N2O-mediated inactivation of cobalamin-dependent enzymes with resultant perturbations in cell metabolism. The effect of N2O on the behavior of murine colony-forming units-cytokine (CFU-C) in vitro was studied by incubating granulocyte/macrophage colony-stimulating factor (GM-CSF)-stimulated bone marrow cultures for 7 days in an atmosphere of either 5% CO2 in air or 50% N2O/5% CO2 in air. Exposure of bone marrow cells in agarose to N2O resulted in an approximately 50% reduction in colony formation when compared with cultures incubated in air. In contrast, when residual CFU-C numbers were determined in bone marrow liquid cultures after 7 days of incubation in the presence of GM-CSF, exposure to N2O was found to dramatically enhance CFU-C recovery. Since these liquid cultures contain a strong differentiation inducer, and are unable to support CFU-C generation, the enhancement of CFU-C recovery in N2O-exposed cultures appears to be related to its ability to induce a reversible block in CFU-C differentiation. The reversible block in CFU-C maturation seen in vitro parallels clinical observations where a rapid hematologic recovery is seen in N2O-exposed patients treated with hydroxycobalamin. These observations would suggest that N2O is not markedly cytotoxic to CFU-C and that its action is, at least in part, cytostatic in nature.

Effect of methotrexate on murine bone marrow cells in vitro: evidence of a reversible antiproliferative action.

Methotrexate (MTX) acts by inducing cellular depletion of reduced folates, which ultimately leads to an inhibition of DNA synthesis. Like many anticancer drugs, this antimetabolite has little selectivity for tumor cells, and its effectiveness is limited by toxicity to normal tissues, particularly gastrointestinal epithelium and bone marrow. Previous studies have shown that MTX inhibits colony formation of the hematopoietic progenitor cells (CFU-C) in vitro. Whether this effect is due to a cytotoxic or a cytostatic mechanism has not been resolved. The present study was undertaken to eludicate the mechanism by which MTX inhibits CFU-C formation. Bone marrow cells in agarose cultures supplemented with recombinant murine granulocyte-macrophage colony-stimulating factor (rmGM-CSF) were incubated for 7 days in the presence or absence of MTX. Exposure to 33 nM to 1 microM MTX reduced colony formation by more than 80% when compared to control cultures. When bone marrow suspension cultures supplemented with rmGM-CSF were incubated for 5 days in the presence or absence of MTX, exposure to 10 nM to 1 microM MTX resulted in a 60 to 80% reduction in cell numbers when compared to untreated cultures. Residual CFU-C numbers were determined in the same cultures by replating into agarose. Exposure to 10 nM MTX was found to enhance CFU-C recovery three-fold as compared to controls and cultures exposed to higher MTX concentrations. Addition of 10 microM of the reduced folate leucovorin (LV; 5-formyl-tetrahydrofolate) prevented CFU-C accumulation in the presence of 10 nM MTX. The kinetics of LV rescue of CFU-C, pre-exposed to 100 nM MTX, were investigated in clonogenic assays. The addition of 1 microM LV to semisolid bone marrow cultures preincubated with 100 nM MTX for up to 8 days completely abolished the inhibition of colony formation seen with 100 nM MTX alone. When the dose range of MTX was expanded from 33 nM to 3.3 microM, we found that administration of 10 microM LV on day 5 rescued the hematopoietic progenitors from MTX inhibition in all groups. These observations suggest that MTX is not cytotoxic to hematopoietic progenitors over its entire dose range but that it can induce a reversible block in the proliferation and differentiation of cells in the progenitor compartment.

Interactions with the protein binding of 7-hydroxy-methotrexate in human serum in vitro.

7-Hydroxy-methotrexate (7-OH-MTX), the major extracellular methotrexate (MTX) metabolite, is 90-95% bound in human serum, with albumin (HSA) as the major binding protein. Reports of an interaction with concomitantly administered non-steroidal antiinflammatory drugs (NSAIDs) during MTX therapy led us to investigate whether these compounds could reduce the binding of 7-OH-MTX in vitro. Equilibrium dialysis experiments demonstrated that naproxen and indomethacin concentration dependently reduced the binding of 1 microM 7-OH-MTX. After ingestion of 1000 mg naproxen, per cent unbound 7-OH-MTX in sera from volunteers increased 2-3-fold in vitro, positively correlated to naproxen concentrations (P less than 0.00015). In addition, etacrynic acid, bilirubin, sulphamethizole and acetylsalicylic acid displaced 7-OH-MTX from its binding protein(s) in a competitive manner. The data suggest that 7-OH-MTX interacts with several exogenous and endogenous substances associated with HSA in human serum. Displacement of 7-OH-MTX from HSA may contribute to the interaction between NSAIDs and MTX.

The effect of vindesine on methotrexate hydroxylation in the rat.

The effect of vindesine (VDS) on methotrexate (MTX) disposition was studied in bile-drained rats administered VDS prior to [3H]MTX, and in isolated rat hepatocytes and rat liver homogenate concomitantly incubated with MTX and VDS at 37 degrees. In vivo, 7-hydroxylation was reduced by 0.65 mg/kg VDS. In VDS-treated animals, biliary recovery of the MTX dose (50 mg/kg) as 7-hydroxymethotrexate (7-OH-MTX) (1.75 +/- 0.2%, mean +/- SEM) was significantly reduced compared to controls (2.83 +/- 0.57%). In vitro, hydroxylation of MTX (10-200 microM) in hepatocytes was reduced by 14.3 and 66.4% (means) at 12.5 and 100 microM VDS, respectively. With increasing VDS concentrations up to 100 microM, a reduction in intracellular MTX accumulation could account for the decreased MTX hydroxylation. Experiments in a cell free system gave no evidence of inhibition of 7-OH-MTX formation by VDS. In vitro MTX transport studies demonstrated that VDS inhibited the hepatocellular influx of MTX, as (1) the accumulation of MTX corresponded inversely to increasing VDS concentrations and (2) the MTX efflux was not increased by VDS. The apparent Ki for VDS inhibition of MTX influx was 57 microM. We suggest that VDS, by reducing the 7-OH-MTX formation in liver cells, may have implications for combination chemotherapy regimens which include MTX.

On the prognostic value of systemic methotrexate clearance in childhood acute lymphocytic leukemia.

The prognostic value of systemic methotrexate clearance (ClMTX) during high-dose therapy was evaluated in a cohort of 42 children with acute lymphocytic leukemia (ALL). As part of an extensive chemotherapy protocol, they had received a total of 293 methotrexate (MTX) infusions in the 6-8 g/m2 dose range. At the termination of the study, when they had all been followed up for 3.5 years or more, 26 of these patients were still in continuous complete remission, whereas 16 had suffered relapse. The intrapatient variability in ClMTX during the eight courses was up to six-fold. In 67% of the patients, the maximum level of ClMTX reached at least twice the minimum value. The coefficients of variation for the intra- and interindividual variability in ClMTX were 9-57% and 26-41%, respectively. The cumulative probability of relapse, estimated by the Kaplan-Meier procedure, was increased for patients with a high ClMTX during the initial treatment course, but the difference was not significant on a 5% level. There was no significant relationship between high individual median ClMTX and subsequent relapse of ALL. However, ClMTX during the initial infusion, the time-dependent mean for ClMTX, and the individual patient's median ClMTX, were significant predictors for event-free survival in a Cox proportional hazards regression analysis. The present study demonstrates gross pharmacokinetic variability and unpredictable values of ClMTX in subsequent courses after standardized administration of MTX to paediatric patients with ALL. In spite of the association between ClMTX and prognosis shown by some of the analyses, estimates of ClMTX rates may not necessarily be related to disease outcome in a way that can be exploited to the benefit of the individual patient.

Variability in methotrexate serum and cerebrospinal fluid pharmacokinetics in children with acute lymphocytic leukemia: relation to assay methodology and physiological variables.

Methotrexate (MTX) steady state concentrations were evaluated in 42 children who had received high-dose infusions (6-8 g/m2) for acute lymphocytic leukemia. Concentrations in serum and cerebrospinal fluid (CSF) measured by immunoassay were found to be highly variable. Reanalysis by a reference high-pressure liquid chromatography method ruled out analytical factors as a source of this variability. The correlation coefficient between the analytical methods was 0.77 for the serum data and 0.88 for the CSF data. The variability of serum and CSF concentrations was higher in younger patients (serum; P = 0.05 and CSF; P = 0.18), and the CSF concentration decreased with decreasing age and in later courses. Body surface area, body mass index, weight, and gender were not significantly related to MTX variability. We conclude that the pronounced pharmacokinetic variability seen during MTX infusions remains largely unexplained.

Reversal of sexual impotence in male patients with chronic obstructive pulmonary disease and hypoxemia with long-term oxygen therapy.

Erectile impotence is commonly encountered in male patients with respiratory failure and hypoxia. In this study, 42% of the patients experienced reversal of sexual impotence during long-term oxygen therapy (LTOT). We examine the association between sexual impotence, gonadal axis hormones, hypoxia, and oxygen therapy. Nineteen sexually impotent male patients eligible for LTOT (pO2 < 7.3 kPa during stable disease) and with sexual impotence received oxygen therapy for 1 month (n = 12) or 24 h (n = 7). pO2, LH, FSH, testosterone, and SHBG (sex hormone binding globulin) were monitored. Five of 12 patients receiving oxygen for 1 month regained sexual potency. The responders showed a significant increase in arterial pO2 and serum testosterone, and a decline in SHBG compared to non-responders. None of the patients receiving oxygen for 24 h experienced reversal of sexual impotence, despite a significant increase in pO2. In these patients, serum testosterone did not increase significantly. Reversal of sexual impotence may be achieved in some patients with respiratory failure. The oxygen therapy must, however be administered for an adequate length of time.

Gonadal endocrine dysfunction in patients with lung cancer: relation to responsiveness to chemotherapy, respiratory function and performance status.

Male lung cancer patients with poor performance status [Eastern Cooperative Oncology Group (ECOG) index 3-4] have an endocrinological dysfunction as assessed by serum testosterone and sex hormone-binding globulin (SHBG) levels. Patients who respond to therapy regain normal free testosterone levels within 12 weeks post chemotherapy, whereas non-responders continue to exhibit subnormal levels. The perturbations of endocrinological variables in patients with lung cancer is not due to development of hypoxia, as patients with respiratory failure maintain a significantly lower testosterone level compared to cancer patients. The development of a deficiency in total testosterone concentrations in lung cancer patients is correlated to their performance status, and not to the presence of metastatic disease. The mechanisms responsible for the endocrinological dysfunction in patients with lung cancer remain unknown.

Pharmacokinetics and metabolism of doxorubicin after short-term infusions in lymphoma patients.

PURPOSE/METHODS: Twenty-four patients (17 males and 7 females with a mean age of 54 years) with malignant lymphoma participated in a study of doxorubicin pharmacokinetics after 50 mg/m(2) as 10-min infusions. In addition to plasma samples, serial leukocyte samples and - in one subject - serial biopsy specimens from lymphoma infiltrates were obtained. The samples were analysed by reversed-phase high-performance liquid chromatography. RESULTS: In contrast to several previous studies, the data suggested that 7-deoxydoxorubicinolone, and not doxorubicinone, is a metabolite of doxorubicin in humans. Doxorubicin, but no metabolites, was present in significant and fairly constant concentrations in circulating leukocytes. These levels may reflect the drug levels in lymphoma infiltrates. The data further suggest that metabolism to 7-deoxydoxorubicinone is subject to large interindividual variation, possibly due to a genetic polymorphism, and that significant levels of a metabolic product which may be a doxorubicin glucuronide can be recovered from plasma of patients treated with doxorubicin.

Quantitation of cell-associated doxorubicin by high-performance liquid chromatography after enzymatic desequestration.

A method for measuring cellular concentrations of the anthracycline doxorubicin was developed. The assay involves cell lysis and protein degradation by detergent and proteinase K treatment followed by DNA hydrolysis using DNase I. Prior to high-performance liquid chromatography, samples are deproteinized by the addition of ZnSO4 and methanol. The assay is linear with respect to both the cellular drug content and the number of cells assayed over the ranges tested, and drug recovery is close to 100%. The method has a limit of detection of 50 fmol injected doxorubicin. Within run and between-day coefficients of variation have consistently been found to be in the 5% and 10% range, respectively, in different cell lines exposed to doxorubicin in vitro. The method has been evaluated in analyses of doxorubicin levels in mononuclear blood cells of patients. The assay offers several advantages over commonly used organic extraction techniques and may improve cellular drug monitoring during anthracycline therapy in patients.

Evaluation of methotrexate tissue exposure by in situ microdialysis in a rat model.

The feasibility of using a microdialysis technique to obtain pharmacokinetic data on tissue exposure to methotrexate (MTX) was investigated. Microdialysis probes were implanted in the jugular vein, femoral muscle, and liver of anesthetized male Wistar rats. MTX (100 mg/kg) was given as a bolus injection through an indwelling venous catheter, and blood samples were obtained through a second venous access and by microdialysis for a total of 6 h. Heparinized plasma, ultrafiltered plasma, and microdialysis effluent from tissue and venous probes were analyzed by high-performance liquid chromatography. Centrifugal ultrafiltration of rat plasma spiked in vitro with MTX (1-100 microM) revealed a mean binding to plasma proteins of 21%. In vitro microdialysis of this spiked plasma resulted in 23% relative recovery of the unbound fraction. In rats receiving MTX, plasma protein binding was 23% and the relative drug recovery as assessed with venous microdialysis probes was 18%. Plotting of unbound (i.e., ultrafiltrate) MTX concentrations in the blood against venous microdialysis perfusate values in the blood gave a good linear correlation with a coefficient of correlation (r2) of 0.98. There was also a linear correlation between the total MTX concentrations in venous blood and the drug levels in microdialysis samples from muscle and liver (r2 = 0.93 and 0.74, respectively). Area under the curve estimations were consistent with an MTX exposure of 30% and 46% for the muscle and liver as compared with the circulation. The present study demonstrates that the microdialysis technique can provide reproducible data on tissue exposure to MTX in an animal model and indicates that the methodology is adaptable to clinical settings.

Pharmacokinetics of different doses of methotrexate at steady state by in situ microdialysis in a rat model.

We used a microdialysis technique to monitor extracellular methotrexate (MTX) levels during the steady state in a rodent model. Microdialysis probes were implanted in the muscle, liver, and kidney of anesthetized male Wistar rats. MTX (18.75-500 mg/kg) was given as a continuous infusion through a venous catheter, and blood samples were obtained through a second venous catheter. Heparinized plasma, ultrafiltered plasma, microdialysis effluent from tissues, and tissue samples (obtained at the end of experiments) were analyzed for MTX content by high-performance liquid chromatography (HPLC). Steady state was demonstrated in the blood and tissues from 2 h until the end of the experiments (6 h). Extracellular drug levels in muscle and liver displayed a linear correlation with doses, whereas kidney levels reached a plateau at an MTX dose of 150 mg/kg per 6 h. Microdialysis-fluid endpoint levels for muscle, liver, and kidney were positively correlated to the endpoint total tissue levels (r2 = 0.80, 0.85, and 0.68, respectively). In the kidneys, the maximal relative tissue MTX accumulation was measured at a total dose of 75 mg/kg per 6 h. At higher doses, the relative drug sequestration declined to less than half of the values observed at this dose. This study demonstrates that the microdialysis technique can provide reproducible data on MTX tissue exposure in an animal model and that it offers a means of serial and reproducible monitoring of extracellular-tissue MTX levels at steady state and over a wide dose range. Pending additional studies, microdialysis may be a helpful technique for elucidating the kinetics of drug delivery to both targeted and toxicity-prone tissues during chemotherapy.

Continuous intratumoral microdialysis during high-dose methotrexate therapy in a patient with malignant fibrous histiocytoma of the femur: a case report.

We used a microdialysis technique to assay intratumoral methotrexate (MTX) levels during high-dose (12 g/m2 given as a 4-h infusion) therapy in a 43-year-old man with a malignant fibrous histiocytoma in the medial femoral condyle. Additional microdialysis probes were implanted in muscle tissue contralateral to the tumor and in an antecubital vein. Microdialysis was attempted during the initial two high-dose courses, but the two latter probes were removed at the start of the second treatment cycle due to leakage. No attempt to correct for microdialysis recovery was made. The intratumorally localized probe gave reproducible data on tumor MTX exposure of 9.3-14% of unbound systemic MTX. There was a close correlation between tumor and systemic levels for both MTX and its major extracellular metabolite 7-hydroxymethotrexate. Although limited to the study of MTX pharmacokinetics in a single subject, the experiment demonstrates that intratumoral microdialysis may provide data on tumor drug exposure, although of an indirect nature and dependent on the probe characteristics, the flow rate, and, possibly, the time after probe implantation. We propose that the application of microdialysis may prove useful for elucidation of the relationship between local drug exposure and the therapeutic response in normally inaccessible compartments during cancer pharmacotherapy.

Determination of extracellular methotrexate tissue levels by microdialysis in a rat model.

We used a microdialysis technique to determine tissue methotrexate (MTX) levels during steady state in a rodent model. Two different approaches were employed to measure the actual extracellular MTX concentrations in muscle, liver, and kidney tissues of anesthetized Wistar rats. With the reduced-perfusion-rate technique, the flow in the microdialysis perfusate was gradually decreased toward zero to permit calculation of zero-flow intercepts. Using the net change technique, microdialysis probes were perfused with different MTX concentrations to allow an assessment of equilibrium drug levels. For these two methods to be used, drug concentrations in the matrix to be analyzed must remain unchanged during the experimental procedure. In the animal model, steady state was attained after 1.5 h and maintained throughout the rest of the experiments by the administration of MTX as continuous infusions through a venous catheter. In vitro and in vivo, both the reduced-perfusion-rate and net change techniques gave reproducible data that permitted the estimation of extracellular drug concentrations in the dialyzed tissue compartments.The data suggest that the level of unbound MTX in the circulation is fairly similar to the extracellular concentrations in the muscle and liver. In the kidney, MTX levels were measured to be 3-8 times higher than those of unbound, circulating MTX, and a considerable discrepancy between the two methods used for estimations was apparent. These results demonstrate that both the net change and reduced-flow microdialysis techniques can produce reproducible and precise data. The results may constitute a basis for determining recoveries and, thus, true extracellular drug levels during in vivo microdialysis of MTX. This may be of importance in delineation of the relationship between tissue MTX levels and outcome in a variety of normally inaccessible compartments during cancer pharmacotherapy.

7-Hydroxymethotrexate concentrations in serum and cerebrospinal fluid of children with acute lymphoblastic leukemia.

Concentrations of methotrexate (MTX) and 7-hydroxymethotrexate (7-OH-MTX) were determined by HPLC in the serum and cerebrospinal fluid (CSF) of 29 children with acute lymphoblastic leukemia. CSF and serum samples were obtained at the end of 104 infusions of MTX given in a dose range of 0.5-8.0 g/m2. Concentrations, distribution ratios in serum and CSF for MTX and 7-OH-MTX, and the metabolic index were analyzed with regard to the MTX dose, age and clinical state of the patients. A wide inter-patient (2- to 12-fold) but narrower (1.1- to 3.5-fold) intra-patient variability of the concentrations was observed. A dose-proportional increase in the metabolite concentration was found in serum. On the other hand, the elevation of the level of metabolite in CSF was less than proportional to the dose. The CSF/serum distribution data suggest the existence of a saturable carrier system for MTX and 7-OH-MTX between serum and CSF that has lower affinity for 7-OH-MTX. No correlation was found between concentrations of MTX and 7-OH-MTX in the serum of patients receiving the same dose of MTX. No significant difference was observed in the values for metabolic index between relapsed patients and those who were in continuous complete remission. A significant correlation was found between age and metabolic index: the younger the patient, the higher the metabolite concentration measured in serum.