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.     

Protection against cisplatin nephrotoxicity by prochlorperazine

Summary In pentobarbital anesthetized rats that received 4 mg/kg i.v. methotrexate (MTX) or 7-hydroxymethotrexate (7-OH-MTX), the pharmacokinetics of the two drugs were similar. Plasma concentrations of both drugs declined biexponentially, with terminal half-lives of 90.6 min for MTX and 97.2 min for 7-OH-MTX. The total clearance values were 9.2 and 9.6 mlxkg^-1xmin^-1, respectively. With MTX, 48.2% of the dose was excreted in the urine within 200 min and another 31.6% was recovered from the bile; 5.8% was metabolized to 7-OH-MTX and appeared in the bile. Plasma concentrations of the metabolite 7-OH-MTX after MTX administration were below the detection limit. Injected 7-OH-MTX was predominantly excreted into the bile (72.8% of the dose); only 11.2% could be recovered from the urine. Differences between the physicochemical properties of MTX and 7-OH-MTX or different affinities for active transport systems may account for the unequal importance of these two excretion pathways for the two compounds.     

Inhibition of 7-hydroxymethotrexate formation by amsacrine

Summary The inhibition of methotrexate (MTX) biotransformation to 7-hydroxymethotrexate (7-OH-MTX) by 4-(9-acridinylamino)-methanesulfon-m-anisidide (mAMSA) was studied in bile-drained rats in vivo and in incubates of isolated rat hepatocytes and rat-liver homogenate in vitro. In vivo, i.v. administration of 10 mg/kg mAMSA prior to [³H]-MTX infusion (50 mg/kg) led to a significant alteration in 7-OH-MTX kinetics. 7-OH-MTX peak concentrations and AUC in bile and serum were reduced by 75% and the recovery of MTX as 7-OH-MTX in bile and urine decreased by 70%, whereas MTX pharmacokinetics remained unaltered. In suspensions of isolated hepatocytes. 10 m mAMSA led to a 54% decrease in 7-OH-MTX formation. However, the hepatocellular influx and efflux of MTX was not perturbed by mAMSA. Preincubation of rat-liver homogenates with 1.25–10 m mAMSA reduced the formation of 7-OH-MTX by up to 73%. mAMSA appeared to inhibit MTX hydroxylation competitively, exhibiting aK _iof 3 m. Due to its inhibition of the MTX-oxidizing system, mAMSA may be beneficial in combination chemotherapy with MTX by reducing 7-OH-MTX-associated toxicity and, possibly, enhancing the cytotoxic effects of MTX.This study was financially supported by the Norwegian Cancer Society and the Erna and Olav Aakre Foundation for Cancer Research     

Pharmacokinetics of methotrexate and 7-hydroxy-methotrexate in rabbits

Summary In rabbits the IV kinetics of MTX (1.33, 4 and 12 mg/kg) could be described by a linear three-compartment model with a terminal half-life between 2.4 and 3.6 h. During 8 h 50% of the dose was excreted into urine in unchanged form and 15% as the metabolite 7-OH-MTX. These fractions remained constant with increasing dose. In continuous infusion experiments (9–900 g/kg x min MTX IV) a decrease of the renal MTX clearance with increasing plasma concentration was observed. This effect was nearly compensated by an increase of the extrarenal MTX clearance. After short-term infusion of 7-OH-MTX (4 mg/kg) a biexponential decline of 7-OH-MTX plasma concentrations was observed with a terminal half-life of 0.45 h. About 80% of the dose was regained from urine during 5 h. From the combined pharmacokinetic data a linear model was constructed for the calculation of 7-OH-MTX plasma concentrations after short-term MTX infusion. For the first 4 h after MTX application the predicted values were in good accordance with the 7-OH-MTX concentrations actually measured.     

Maximum tolerated doses of methotrexate and 7-hydroxy-methotrexate in a model of acute toxicity in rats

Purpose: After more than 50 years of methotrexate (MTX) treatment of acute lymphoblastic leukaemia (ALL), it is currently believed that as long as dose escalations are followed by adequate leucovorin rescue guided by monitoring MTX serum concentrations, hydration and urinary alkalinization, high-dose MTX (HD-MTX) can be tolerated without life-threatening toxicity. However, our recent experimental animal studies of the major metabolite of MTX, 7-OH-MTX, indicate that this concept may have some limitations. Animals with levels of 7-OH-MTX of 1 mM, which is below the levels routinely found in patients on HD-MTX, demonstrate intolerable toxicity and some animals die within 8 h. Electron microscopy indicates that endothelial cell and platelet functions are perturbed. Since animal data are lacking, and interspecies differences not known, we wanted to investigate the maximum tolerated doses of MTX and 7-OH-MTX in a rat model of short-term effects. The maximum tolerated dose was chosen instead of LD₅₀ for reasons of animal welfare. Methods: We infused MTX and 7-OH-MTX into anaesthetized male Wistar rats and monitored the animals for 8 h. The drugs were given as a bolus plus continuous infusion. The dose-finding ranges were 1.8–11.3 g/kg MTX and 0.1–1.2 g/kg 7-OH-MTX. Results: The maximum tolerated dose was between 3 and 5 g/kg for MTX and lower than 0.1 g/kg for 7-OH-MTX. The mean serum concentrations of MTX and 7-OH-MTX in animals that did not survive the 8-h period were 21.9 and 1.6 mM, respectively. The animals that received the highest MTX or 7-OH-MTX doses and concentrations died after sudden reductions in heart rate and blood pressure. Conclusions: We demonstrated a lower maximum tolerated dose of 7-OH-MTX than of MTX in rats after 8 h. The 7-OH-MTX concentrations were in the therapeutic range after HD-MTX. If the rat/human interspecies differences are not large, our data may indicate that HD-MTX regimens should not be further dose intensified, due not so much to the effects of MTX as to those of 7-OH-MTX. Received: 28 July 1999 / Accepted: 25 January 2000     

Cephalosporins increase the renal clearance of methotrexate and 7-hydroxymethotrexate in rabbits

Summary Anaesthetized rabbits were infused with methotrexate (MTX; 30 μg × kg⁻¹ × min⁻¹ for 4h. Constant plasma concentrations of MTX and its main metabolite 7-hydroxymethotrexate (7-OH-MTX) were achieved 40–60 min after the start of the infusion. In all, 50% of the infused MTX was eliminated by the kidney; another 15%–30% was hydroxylated and excreted as 7-OH-MTX in the urine. A concomitant infusion of penicillin G (3.96 mg × kg⁻¹ × min⁻¹) decreased the renal clearance of MTX and 7-OH-MTX, probably by competitive antagonism at the common tubular secretion site. In contrast, the four cephalosporins ceftriaxone, ceftazidime, ceftizoxime and cefoperazone all increased the renal clearance of MTX and 7-OH-MTX. At similar plasma concentrations, ceftriaxone and ceftazidime were almost equipotent, ceftizoxime was less effective and cefoperazone seemed to have a biphasic effect, depressing the clearance of MTX and 7-OH-MTX at higher drug concentrations. The effects are best explained by an inhibition of the tubular reabsorption of the cytostatic and its metabolite. The results suggest that cephalosporins are a better choice than penicillin for antibiotic treatment during MTX therapy.     

The effects of antibiotics and uricosuric drugs on the renal elimination of methotrexate and 7-hydroxymethotrexate in rabbits

Summary In anesthetized rabbits, continuous infusion of methotrexate (MTX; 30 g kg^-1 min^-1) established steadystate plasma concentrations for MTX and the metabolite 7-hydroxymethotrexate (7-OH-MTX) within 40 min. Fifty percent of the infused dose was eliminated unchanged by the kidneys and the renal MTX clearance was slightly higher than the inulin clearance. Another 15%–30% was metabolized and excreted as 7-OH-MTX in the urine. Infusions of 7-OH-MTX, furosemide or benzarone had no influence on the clearance of MTX or 7-OH-MTX. Infusions of probenecid or piperacillin decreased the renal clearance of MTX and 7-OH-MTX mainly by reducing the tubular secretion of both compounds. In contrast, infusions of the antibiotics ceftriaxone and sulfamethoxazole increased the renal elimination of MTX and 7-OH-MTX. An increase was also observed during the infusion of the uricosuric drugs sulfinpyrazone and benzbromarone. These results are consistent with competition for tubular reabsorption between MTX, ceftriaxone and sulfamethoxazole. The pharmacokinetic drug interactions observed occurred with therapeutic drug concentrations and thus may be clinically relevant.     

High-dose 7-hydroxymethotrexate: Acute toxicity and lethality in a rat model

To elucidate mechanisms for methotrexate (MTX)-induced renal and hepatic toxicity, we investigated the acute effects of bolus plus continuous infusion of up to 0.4 g/kg 7-hydroxymethotrexate (7-OH-MTX) in the rat. We demonstrate for the first time in any species the occurrence of acute lethal toxicity within a few hours after 7-OH-MTX administration. Serum concentrations of 7-OH-MTX measured at the time of death were 1.4 mM (mean), about one-half of those achieved in some patients after infusion of high-dose MTX (HD-MTX) in the clinic. The data suggest an approximate LD50 (the dose lethal to 50% of the study population) of 0.3 g/kg and a steep dose/ lethality curve for 7-OH-MTX. Moreover, acute renal and hepatic toxicity occurred as evidenced by severe morphological findings and increased serum levels of creatinine and liver transaminases. In all rats subjected to continuous infusion of 7-OH-MTX, yellow microscopic precipitations were apparent in the kidney tubules. Crystallization was also seen in bile ducts of the liver in some of the rats. These results further support that the formation of 7-OH-MTX is disadvantageous and that reported attempts to prevent its formation during MTX treatment are warranted.     

Interactions between 7-hydroxymethotrexate and methotrexate at the cellular level in the Ehrlich ascites tumor in vitro

Studies were undertaken to characterize the cellular pharmacology of 7-hydroxymethotrexate (7-OH-MTX) in Ehrlich ascites tumor cells, compare it to that of methotrexate (MTX), and define the interactions between the parent compound and its catabolite. Transport of 7-OH-MTX is mediated by the MTX-tetrahydrofolate cofactor carrier, with a Km of 9 microM in comparison to the MTX Km of 5 microM. Both compounds mutually inhibit their influx and steady-state levels of free drug accumulated. While influx of 7-OH-MTX is slower than influx of MTX, 7-OH-MTX efflux is likewise slower, so that the steady-state level of 7-OH-MTX achieved is comparable to that of MTX. Influx of 7-OH-MTX is inhibited by extracellular 5-formyltetrahydrofolate and trans-stimulated in cells preloaded with this tetrahydrofolate cofactor. The energetics of 7-OH-MTX transport is similar to that of MTX in the influx and net transport are stimulated by sodium azide, while net transport is reduced by glucose. As observed for MTX, 7-OH-MTX transport is sensitive to the anionic composition of the extracellular compartment and was shown to be inhibited by organic and inorganic phosphates. 7-OH-MTX does not, alone, inhibit [3H]deoxyuridine incorporation into DNA at concentrations of up to 50 microM. However, the catabolite reduces MTX inhibition of deoxyuridine metabolism, presumably due to the reduction in the free level of intracellular MTX achieved. These findings support the possibility that when 7-OH-MTX accumulates to high levels relative to MTX in clinical regimens, it may modulate the pharmacological effects of MTX.     

Serum monitoring of methotrexate (MTX) and 7-hydroxymethotrexate concentrations in patients treated with MTX using high-pressure liquid chromatography (HPLC) and comparison of serum MTX levels between HPLC method and fluorescence polarization immunoassay (FPIA)

In order to measure simultaneously the serum levels of methotrexate (MTX) and its major metabolite, 7-hydroxymethotrexate (7-OH-MTX), in samples obtained from patients treated with MTX, we have investigated the reversed-phase high-pressure liquid chromatographic assay using ion-pairing reagents. The mobile phase consisted of 77.5% solution of 0.005M tetrabutylammonium and 22.5% acetonitrile. SEP-PAK C18 Cartridges were used for the precolumn. The detectable range of MTX and 7-OH-MTX were 0.02-0.03 and 1.0 mumol/l respectively. A significant positive correlation was observed (r = 0.983) between FPIA and HPLC methods. Serum MTX levels with FPIA were significantly (p less than 0.05) higher than those of HPLC method. The serum 7-OH-MTX levels at 24 hr and 48 hr were 4.857 +/- 1.383 (n = 10) and 1.835 +/- 0.286 (n = 6) mumol/l respectively with the dosage of 400 mg/m2. The serum 7-OH-MTX levels at 48 hr were 6.254 +/- 3.053 mumol/l (n = 5) with the dosage of 3,000 mg. The serum half lives of MTX and 7-OH-MTX were 8.05 +/- 1.03 (n = 4) and 14.8 +/- 1.35 (n = 6) hours respectively between 24 hr and 48 hr after administration. The T1/2 7-OH-MTX/MTX ratio was 1.8. Percent cross-reactivity of 7-OH-MTX with concentrations ranging from 1-10 mumol/l were 0.6-2.0% by FPIA. However, patients' serum levels of 7-OH-MTX were 15-85 times (n = 21) higher than those of MTX. MTX levels of containing both MTX and 7-OH-MTX (7-OH-MTX/MTX ratio was 50/1) were significantly higher than those of containing MTX alone by FPIA.     

Influence of piperacillin on the pharmacokinetics of methotrexate and 7-hydroxymethotrexate

The influence of concomitant administration of piperacillin (PIP) on the pharmacokinetic parameters of methotrexate (MTX) and 7-hydroxymethotrexate (7-OH-MTX) was studied in rabbits. Six rabbits received an initial i.v. bolus (0.21 mg kg⁻¹) followed by a constant-rate i.v. infusion of the drug (5 μg min⁻¹ kg⁻¹) for 240 min. The PIP dose (30 mg kg⁻¹) was repeated every 30 min until the end of the infusion period. The control group consisted of four rabbits treated the same way except for the addition of PIP. There were significant increases in the mean residence times found for MTX (MRT_inf) and 7-OH-MTX (MRT_m,inf) following PIP administration. Concomitant administration of PIP with MTX also produced significant 1.5- and 2.8-fold increases in the area under the curve of MTX and 7-OH-MTX, respectively. The total body clearance of MTX and the operative total body clearance of 7-OH-MTX significantly decreased, but in a less than proportional manner. The study demonstrates that the interaction between MTX and PIP is mainly due to the reduced clearance of both MTX and 7-OH-MTX combined with a slight increase in the formation clearance of the metabolite. Received: 9 March 1998 / Accepted: 14 May 1998     

Formation of 7-hydroxymethotrexate polyglutamyl derivatives and their cytotoxicity in human chronic myelogenous leukemia cells, in vitro

The rapid synthesis of poly-gamma-glutamyl derivatives of 7-hydroxymethotrexate (7-OH-MTX) and their selective intracellular retention are reported in human chronic myelogenous leukemia cells, K-562. After a 30-min exposure to 5 microM [3H]7-OH-MTX, three different polyglutamyl derivatives were detected by high-performance liquid chromatography. When extracellular 7-OH-MTX was removed, the 7-OH-MTX diglutamate level declined slowly in comparison to the monoglutamate, but the higher polyglutamyl derivative levels increased. Within 10 min after exposure of cells to 7-OH-MTX, the level of these polyglutamyl derivatives far exceeds the dihydrofolate reductase binding capacity. Gel filtration or charcoal binding analysis followed by high-performance liquid chromatography analysis of the bound component showed intracellular binding of virtually all 7-OH-MTX tetraglutamate at a level 4-fold higher than that of the dihydrofolate reductase binding capacity. No bound 7-OH-MTX diglutamate or triglutamate could be detected. Treatment of the 7-OH-MTX tetraglutamate: protein complex with 100 microM unlabeled methotrexate (MTX) for 15 min resulted in only a partial dissociation of this complex to an extent compatible with the dihydrofolate reductase level. The residual 7-OH-MTX tetraglutamate remained bound to a site with a molecular weight of approximately 25,000 to 35,000 as assessed by Bio-Gel P-60 analysis and could not be displaced by folic acid, 5-formyltetrahydrofolate, 7-OH-MTX, or the tetraglutamate of MTX. 7-OH-MTX and MTX cytotoxicities were compared by clonogenic assay in agar and by their effects on cell growth. After a 2-hr exposure, the 50% inhibitory concentrations for 7-OH-MTX and MTX in cells growing in agar were 10(-5) and 10(-6) M, respectively. A 10-fold difference in cytotoxicity was also observed in cells growing in suspension. Continuous exposure to glycine: adenosine: thymidine completely protects cells from a sustained exposure to 7-OH-MTX over the entire period of clonal growth. However, even a brief exposure to 7-OH-MTX also requires continuous exposure to glycine: adenosine: thymidine for protection. This suggests that, as observed for MTX, the 7-OH-MTX polyglutamyl derivatives that are retained within the cells have a sustained cytotoxic effect after the monoglutamate is removed.     

Interactions between 7-hydroxymethotrexate and folinic acid in RAJI cells, in vitro

HPLC analysis of plasma samples obtained from patients included in a high-dose methotrexate-folinic acid Rescue (HD-MTX-CF) protocol, allowed the simultaneous determination of MTX, CF and their respective plasma metabolites, 7-hydroxymethotrexate (7-OH-MTX) and 5-methyltetrahydrofolate (5-CH3-FH4). These 4 compounds interact at the cellular level to ensure the selective effectiveness of the HD-MTX-CF rescue protocol. An in vitro study has been investigated in RAJI cells to better describe the interference of CF on uptake, accumulation and metabolism of [3H]7-OH-MTX. Results about uptake and accumulation of CF were also obtained using [3H]CF, in the absence or the presence of unlabeled 7-OH-MTX. The rate of [3H]7-OH-MTX influx in RAJI cells (Km = 25.30 +/- 7.75 microM, n = 3) was competitively inhibited by the presence of 10 microM CF with a Ki value of 6.00 +/- 1.94 microM (n = 2). Intracellular 7-OH-MTX accumulation was decreased by approximately 30% when extracellular CF concentration was twice as high as that of 7-OH-MTX, and 70% when CF extracellular concentration was 5 times higher. The metabolism of 7-OH-MTX to its intracellular polyglutamyl derivatives was depressed by 90% when 10 microM CF were incubated for 2 h with equimolar [3H]7-OH-MTX, and it was completely abolished in the presence of 100 microM CF.     

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 M, and concentrations of 7-OH-MTX declined triphasically, showing at1/2 value of 2.45 min, at1/2 value of 30.5 min, and a terminal half-life (t1/2) of 240 min. The total clearance value was 14.5 ml min^–1 kg, and the postdistributional volume of distribution (V) was 5070 ml/kg. Our results may indicate a direct toxic effect of 7-OH-MTX on kidney and liver cells.This study was supported financially by the Norwegian Cancer Society     

Kinetics of 7-hydroxy-methotrexate after high-dose methotrexate therapy

Summary Kinetics of 7-hydroxy-methotrexate (7-OH-MTX) excretion after high-dose methotrexate (MTX) (12 g/m²) therapy were monitored in 93 consecutive drug cycles of 19 adolescent patients with osteosarcoma. A reversed-phase HPLC method was used. Serum elimination was found to be monophasic with a mean half-life of 5.5 h. Shortly after the 4-h MTX infusion period 7-OH-MTX levels exceeded those of the parent compound. By 12 h after MTX infusion 7-OH-MTX levels were 16.5 times higher than those of MTX itself.Autostimulation of MTX metabolism leading to enhanced 7-OH-MTX production after repeated drug cycles was not observed. The production of 7-OH-MTX decreased significantly from the first to the last high-dose MTX cycle of the adjuvant chemotherapy protocol.     

Plasma pharmacokinetics of high-dose oral melphalan in patients treated with trialkylator chemotherapy and autologous bone marrow reinfusion

The pharmacokinetics of melphalan following high-dose p.o. administration were determined in 17 patients with various malignancies for the purpose of assessing interpatient and intrapatient pharmacokinetic variability. All patients underwent bone marrow harvest on day -8 (relative to bone marrow reinfusion). On days -7, -6, and -5, melphalan was given p.o. and the dose was escalated on each cohort consisting of at least 3 patients (beginning at 0.75 mg/kg). On days -6, -4, and -2, cyclophosphamide at 2.5 g/m2 and thiotepa at 225 mg/m2 were given i.v. On day -7 the peak melphalan concentration was 1.64 +/- 0.89 (SD) microM with a terminal half-life of 1.56 +/- 0.86 h. The area under the plasma concentration time curve (AUC) and oral clearance were 217.9 +/- 115.1 microM/min and 30.2 +/- 14.2 ml/min/kg. There was only a moderate correlation between the melphalan dose and both the peak concentration (r = 0.50, P less than 0.05) and AUC (r = 0.64, P less than 0.01) over the dosage range of 0.75-2.5 mg/kg. There was a trend towards greater interpatient variability in peak concentration, AUC, and oral clearance observed at the higher doses of melphalan. Analysis of intrapatient pharmacokinetic variability in 8 patients showed a significant difference between the doses given on days -7 and -5 in the peak concentration (2.09 versus 1.07 microM, P = 0.02), AUC (264.9 versus 134.8 microM/min, P = 0.01), and oral clearance (25.1 versus 53.1 ml/min/kg, P = 0.05) but no significant difference in the time to peak and terminal half-life. We conclude that there is marked interpatient and intrapatient variability in melphalan pharmacokinetics following high-dose p.o. administration. The data are consistent with saturable absorptive pathways for melphalan, which might be especially sensitive to concurrent high-dose chemotherapy.     

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

Summary Concentrations of methotrexate (MTX) and 7-hydroxymethotrexate (7-OH-MTX) were determinded 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 porportional 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.     

Reduction of methotrexate toxicity with improved nutritional status in tumor-bearing animals

The administration of chemotherapy in clinical situations is limited frequently because of the associated toxicity to normal bone marrow cells, gastrointestinal epithelium, and other host tissues. Although nutritional support has been advocated to reduce chemotherapy-related toxicity in cancer patients, few studies substantiate this clinical impression. The current study was performed to determine the role of nutritional status and enteral nutrient intake as determinants of methotrexate (MTX) toxicity in a well-controlled, tumor-bearing animal model. After subcutaneous mammary tumor (AC-33) inoculation, 56 female Lewis/Wistar rats were assigned randomly to one of the following two nutritional regimens for 14 days: (1) protein-depleted chow (PC) (0.03% protein; 4.27 kcal/g) or (2) standard chow (RC) (22.0% protein; 3.50 kcal/g). After 7 days of dietary control, all animals received one of three weight-adjusted doses of MTX (5, 10, or 20 mg/kg intramuscularly [IM] ) or placebo. All animals received leucovorin rescue (0.6 mg IM) at 6 and 24 hours after MTX injection. Improved nutritional status was associated with a significant reduction in objective measures of MTX-related morbidity and mortality. At low doses of MTX (5 and 10 mg/kg), the mean duration of clinical signs of toxicity (i.e., hair loss, lethargy, and diarrhea) and severity of leukopenia were greater in protein-depleted (PD) animals. With high-dose MTX (20 mg/kg), mortality was increased significantly in PD animals (100%) compared with well-nourished animals (0%). Equivalent tumor response was observed in PD and well-nourished animals. Thus, improved nutritional status by enteral nutrition reduced the morbidity and mortality associated with MTX significantly in this tumor-bearing animal model.     

Effects of enteral and parenteral nutrition on tumor response to chemotherapy in experimental animals

The effects of oral and intravenous nutrition on host and tumor responses to graded doses of methotrexate (MTX) were evaluated in 150 adult Sprague-Dawley rats. All animals were inoculated with Walker-256 carcinosarcoma and were fed a regular diet for ten days before assigning them to three dietary groups. Group I (n = 64) received a constant intravenous infusion of 30% dextrose-5% amino acids (IVH), group II (n = 64) received an identical solution orally ad libitum, and group III (n = 22) received a regular diet ad libitum. Animals in groups I and II were then divided into three subgroups each that received either 20 mg/kg, 40 mg/kg, or 60 mg/kg of MTX intramuscularly. Ten days later, all surviving animals were killed. Animals fed the 30% dextrose-5% amino acid diet orally and given 20 mg/kg and 40 mg/kg of MTX lost slightly less body weight when compared with their IVH counterparts. In the 60 mg/kg treatment group, orally fed animals lost 52 gm of body weight compared with 23 gm in IVH animals. IVH rats given 20 mg/kg and 40 mg/kg of MTX demonstrated significant inhibition of tumor growth and decreased tumor weight/body weight ratios when compared with orally fed rats. No improvement in tumor response to 60 mg/kg of MTX was observed, however, when IVH animals were compared with orally fed rats. In a second study, nutrient intake was maintained at a constant level by intravenous infusion in one group and intrajejunal infusion in another group of tumor-bearing rats. Host and tumor responses to 20 mg/kg of MTX were similar in both groups of animals.     

Effect of the nonimmunosuppressive cyclosporin analog SDZ PSC-833 on colchicine and doxorubicin biliary secretion by the rat in vivo

Colchicine and doxorubicin are secreted into bile as a major pathway of their elimination. Colchicine and doxorubicin are also substrates for P-glycoprotein, and P-glycoprotein has been demonstrated to be present at the liver canalicular membrane. Cyclosporin (CsA) inhibits colchicine biliary secretion in vivo. In the present study, the effects of SDZ PSC-833, a nonimmunosuppressive cyclosporin D analog, on the biliary secretion of colchicine and doxorubicin were investigated. SDZ PSC-833 given at a bolus dose of 2 mg/kg promptly decreased colchicine biliary clearance from 9.05±0.2 to 2.41±0.43 ml min^–1 kg^–1 (P<0.001) and the colchicine bile/plasma ratio from 146±8 to 35±5 (P<0.001). SDZ PSC-833 also inhibited doxorubicin biliary clearance (basal: 10.5±3 vs post-SDZ PSC-833: 2.48±0.94 ml min^–1 kg^–1;P=0.06) and the doxorubicin bile/plasma ratio (basal: 228±64 vs post-SDZ PSC-833: 48±22;P<0.01). Colchicine renal secretion was completely inhibited by SDZ PSC-833. Thus, SDZ PSC-833 inhibits the constitutive transport of the multidrug-resistance substrates colchicine and doxorubicin and is more potent than cyclosporin in this regard. The possibility of increased toxicity to normal tissues because of impaired elimination of cytotoxic agents will need to be considered if SDZ PSC-833 is used to chemosensitize cancer cells.This work was supported in part by the Research Service, Department of Veterans Affairs