Pharmacokinetics of small doses of methotrexate in patients with psoriasis.

The pharmacokinetics of methotrexate was studied in 23 patients with chronic plaque psoriasis. Ten patients received a single oral does and 13 patients a single intramuscular injection of 25 mg methotrexate. Serum methotrexate was measured for 24 hours following administration of the drug using a magnetizable solid-phase radio-immunoassay. The disappearance of methotrexate from the serum fitted a two-compartment model with a distribution phase half-life of 1.18 +/- 0.12 h and an elimination phase half-life of 5.35 +/- 0.62 h following the oral dose and 1.45 +/- 0.22 h and 4.71 +/- 0.32 h, respectively following the intramuscular dose. Peak serum methotrexate concentrations varied greatly between patients but the mean area below the curve for the two routes of administration did not differ significantly. In a further 18 psoriatic patients receiving long-term maintenance methotrexate therapy there was no consistent relationship between salivary and serum methotrexate levels.

Magnetizable solid-phase fluoroimmunoassay of phenytoin in disposable test tubes.

We developed a fluoroimmunoassay for phenytoin in serum or plasma, based on the magnetic separation technique. The method involves sheep anti-phenytoin serum coupled to magnetizable cellulose/iron oxide particles, with a fluorescein-labeled phenytoin analog as tracer. After magnetic sedimentation of the solid phase from assay mixtures, the free fraction of the tracer is aspirated, removing endogenous fluorophores and other interfering components of the sample. The antibody-bound tracer is then eluted from the solid phase into a methanolic buffer medium and quantitated fluorometrically. The entire procedure, including fluorometry, is performed within disposable polystyrene test tubes. The assay involves only simple reagents and equipment, and correlates closely with established radioimmunoassay (r = 0.97) and gas-liquid chromatographic (r = 0.98) techniques.

Novel 125I-labeled nortriptyline derivatives and their use in liquid-phase or magnetizable solid-phase second-antibody radioimmunoassays.

Nortriptyline derivatives prepared by reaction with fluorescein isothiocyanate or conjugation to N-acetyl-L-histidine were radioiodinated and the products purified with Sephadex LH-20 columns to obtain two novel nortriptyline radioligands. Antisera were raised in rabbits by immunization with nortriptyline conjugated to succinylated ovine albumin. By use of the iodinated fluorescein derivative we developed a liquid-phase second-antibody radioimmunoassay that gives results correlating closely (r = 0.98) with those by an established radioimmunoassay of similar specificity in the assay of apparent total amitriptyline and its metabolite nortriptyline in serum or plasma from patients being treated with these drugs. With the iodinated N-acetyl-L-histidine derivative we developed a magnetizable solid-phase second-antibody radioimmunoassay. The cross reactivities of amitriptyline and nortriptyline could be made equal by performing the assay at pH 9.0, which makes it possible to measure true total active drug concentrations in patients receiving amitriptyline.

Preparation of 125iodine-labelled methotrexate and its use in a magnetisable particle solid-phase radioimmunoassay.

Two techniques for the iodination of methotrexate are describes, involving covalent linkage to the drug of 125I-labelled N-succinimidyl-3-(4-hydroxyphenyl)propionate (Bolton and Hunter reagent), or to 125I-labelled histamine. A rapid highly specific radioimmunoassay for methotrexate was developed, employing a specific antiserum covalently linked to magnetisable particles, and 125I-labelled methotrexate as tracer. Incubation times for the assays were 60 and 10 min for the Bolton and Hunter reagent-linked methotrexate and 125I-labelled histamine-linked methotrexate respectively. Separation of bound from free antigen was achieved by a rapid magnetic separation system. Results obtained for serum samples correlated closely with those using an enzymatic (dihydrofolate reductase) competitive protein binding assay for methotrexate. A major advantage of the assay is its potential for processing large numbers of samples rapidly, making it highly suitable for routine clinical use.

Separation fluoroimmunoassay methods for phenytoin in serum.

A separation fluoroimmunoassay system for phenytoin was established based on the use of a specific rabbit antiserum, a fluorescein-labelled ligand, and precipitation of the antibody-bound fraction of the labelled ligand with sodium sulphate. Simple measures were taken to obviate non-specific binding and matrix effects. Either the free fraction (in the supernatant) or thebound fraction of the labelled ligand was quantitated fluorimetrically. Assays of patient serum samples by either method correlated well with established gas-liquid chromatographic and radioimmunoassay techniques. Advantages of a separation based procedure as compared with previously described non-separation hapten fluoroimmunoassay techniques are that only simple instrumentation and assay reagents are required, and that the separation step may enable the removal of any interfering intrinsic fluorescence of serum samples.

Anti-drug sera raised by immunisation with a readily available drug-enzyme conjugate.

A phenytoin-enzyme conjugate, readily available as an enzyme-immunoassay reagent, is shown to be a convenient immunogen for production of anti-phenytoin sera for immunoassay purposes.