Development and application of a radioimmunoassay for physostigmine

Antiphysostigmine antibodies were produced in rabbits using a physostigmine analog, 1,3-dimethyl-3-[2- [N-methyl-N-(7-carboxyheptanoyl)] aminoethyl]-5-(N-methyl-carbamoyloxy)-2,3-dihydroindole hydrochloride, conjugated to keyhole limpet hemocyanin. These antibodies were used to develop a radioimmunoassay ranging from 0.2 to 15.0 ng/ml of physostigmine in a 0.1-ml plasma sample. A typical standard curve gave an r2 value of 0.992. This assay measures physostigmine in plasma with better sensitivity and much greater through-put than do current state-of-the-art, high-performance liquid chromatography techniques. In addition, only small volumes (100 microliters) of the plasma samples are required. Precision represented by within and among day coefficients of variance was less than 20% for 1.0 to 50.0 ng/ml and less than 22% for 0.2 ng/ml. Accuracy for the 1.0 to 50.0 ng/ml range varied less than 15% and was 22% for 0.2 ng/ml. Plasma levels of physostigmine were determined in the rat after i.m. administration of physostigmine salicylate to give a free base equivalency of 27 micrograms/kg. Estimates of the various pharmacokinetic parameters were calculated using the computer program PCNONLIN. The results were as follows: apparent volume of distribution = 5.9 liters/kg, absorption rate half-life = 2.7 min. elimination rate half-life = 17.4 min, area under the curve = 118 ng x min/ml, maximal plasma concentration = 3.5 ng/ml and time to maximal plasma concentration = 7.7 min.