Ronald G. Stoller

Use of Plasma Pharmacokinetics to Predict and Prevent Methotrexate Toxicity

To correlate the pharmacokinetics and toxicity of methotrexate, we measured the drugs clearance from plasma after 395 high-dose, six-hour infusions given to 78 patients. After 375 infusions, 48 hour methotrexate levels fell within 2 standard deviations of the mean for nontoxic infusions, and myelosuppression did not occur. Methotrexate concentrations exceeded the range for nontoxic patients (mean +/- 2 standard deviations) after 20 infusions. Serious myelosuppression occurred after six of these 20 infusions, including five of 12 infusions associated with 48-hour drug concentrations above 9 X 10(-7) M. In seven patients with 48-hour concentrations above 9 X 10(-7) M, the absence of toxicity could be attributed to subsequent rapid clearance of the drug; four of these patients also received large doses of supplemental leucovorin (50 to 100 mg per square meter every six hours). Determination of methotrexate concentration in plasma thus identified patients at high risk of toxicity, a group that may benefit from supplemental leucovorin rescue.

7-Hydroxymethotrexate as a urinary metabolite in human subjects and rhesus monkeys receiving high dose methotrexate.

Human subjects and rhesus monkeys receiving the antitumor agent methotrexate at the high dose levels recently introduced into clinical use (greater than 50 mg/kg) excrete significant amounts of the metabolite 7-hydroxymethotrexate. The metabolite is not detected in these species after methotrexate therapy at conventional dose levels. The evidence indicates that in primates, the in vivo conversion of methotrexate to 7-hydroxymethotrexate is a dose-dependent phenomenon, with the enzyme system(s) catalyzing the reaction having a low affinity for the drug.

Analysis of cytidine deaminase and tetrahydrouridine interaction by use of ligand techniques.

Ligand techniques, as employed in radio-immunossay and radioreceptor assay, offer a sensitive and precise method for characterizing the interaction of enzymes and tight-binding inhibitors. Tetrahydrouridine (H4U) inhibition of human liver cytidine deaminase (EC 3.5.4.5) has been examined by direct measurement of enzyme-inhibitor (EI) binding and release. With partially purified enzyme from human liver, the EI complex was found to have a dissociation constant (KD) of 4.5 × 10−8M at 37°, in close agreement with estimates based on inhibition of enzyme activity by H4U. The presence of steady state conditions during competitive binding analysis was confirmed by direct measurement of the rate constants for EI binding at 25° and 37° (Kon 1.7 × 104 and 5.6 x ×04M−1sec−1 respectively). The rate constant for EI release at 37° was also determined experimentally (Koff = 4.0 × 10−3sec−1), and was in close agreement with the Koff value calculated from the experimentally determined KD and Kon values (KD = Koff/Kon). Scatchard analysis of H4U-enzyme binding, both in the presence and in the absence of 10−3M cytidine, showed no variation in total enzyme concentration (ET) but a decrease in apparent inhibitor affinity for enzyme, suggesting that cytidine and H4U compete for the same binding sites on cytidine deaminase, and confirming the competitive inhibition suggested by Lineweaver-Burk analysis. The turnover number for cytidine deaminase based on per H4U binding sites was 3.9 × 103 min−1. Thermodynamic constants for cytidine deaminase-tetrahydrouridine binding were derived from data on the temperature dependence of binding and included an enthalpy change (ΔH) = −12.7 kcal/mole, entropy change (ΔS) = −8.68 cal/deg/mole and Gibbs free energy change (ΔG) = −9.96 kcal/mole at 37.1°. This study indicates that ligand techniques can be applied to the difficult problem of characterizing the interaction of enzymes and tight-binding inhibitors.

Methotrexate Disposition in Humans: Case Studies in Ovarian Cancer and Following High-Dose Infusion

(1978). Methotrexate Disposition in Humans: Case Studies in Ovarian Cancer and Following High-Dose Infusion. Drug Metabolism Reviews: Vol. 8, No. 1, pp. 107-117.