Pyrimethamine significantly lowers cerebrospinal fluid Cu/Zn superoxide dismutase in amyotrophic lateral sclerosis patients with SOD1 mutations.

OBJECTIVE: Cu/Zn superoxide dismutase (SOD1) reduction prolongs survival in SOD1-transgenic animal models. Pyrimethamine produces dose-dependent SOD1 reduction in cell culture systems. A previous phase 1 trial showed pyrimethamine lowers SOD1 levels in leukocytes in patients with SOD1 mutations. This study investigated whether pyrimethamine lowered SOD1 levels in the cerebrospinal fluid (CSF) in patients carrying SOD1 mutations linked to familial amyotrophic lateral sclerosis (fALS/SOD1). METHODS: A multicenter (5 sites), open-label, 9-month-duration, dose-ranging study was undertaken to determine the safety and efficacy of pyrimethamine to lower SOD1 levels in the CSF in fALS/SOD1. All participants underwent 3 lumbar punctures, blood draw, clinical assessment of strength, motor function, quality of life, and adverse effect assessments. SOD1 levels were measured in erythrocytes and CSF. Pyrimethamine was measured in plasma and CSF. Appel ALS score, ALS Functional Rating Scale-Revised, and McGill Quality of Life Single-Item Scale were measured at screening, visit 6, and visit 9. RESULTS: We enrolled 32 patients; 24 completed 6 visits (18 weeks), and 21 completed all study visits. A linear mixed effects model showed a significant reduction in CSF SOD1 at visit 6 (p < 0.001) with a mean reduction of 13.5% (95% confidence interval [CI] = 8.4-18.5) and at visit 9 (p < 0.001) with a mean reduction of 10.5% (95% CI = 5.2-15.8). INTERPRETATION: Pyrimethamine is safe and well tolerated in ALS. Pyrimethamine is capable of producing a significant reduction in total CSF SOD1 protein content in patients with ALS caused by different SOD1 mutations. Further long-term studies are warranted to assess clinical efficacy. Ann Neurol 2017;81:837-848.

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.

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.