Cholinesterase studies with (R) (+)- and (S)(-)-5-(1,3,3-trimethylindolinyl)-N-(1-phenylethyl)carbamate.

A limited number of carbamates have been found useful for treatment of cholinergic symptoms with pyridostigmine and physostigmine being the main focus. In recent years 5-(1,3,3-trimethylindolinyl)N,N-dimethylcarbamate (I) has received considerable attention in the Chinese literature for a similar role. We report on the first synthesis of stereoisomers of an analog of (I). The isomers prepared were (R)(+)-5-(1,3,3-trimethylindolinyl)-N-(1-phenylethyl)carbamate (II) and (S)(-)-5-(1,3,3-trimethylindolinyl)-N-(1-phenylethyl)carbamate (III). The pKa value for each isomer was 6.8. Eel acetylcholinesterase inhibition studies were carried out at 25.0 degrees C over the pH range of 6.0 to 9.0. They reflect the first pH profiles using enantiomorphs of a cholinesterase inhibitor. The inhibition potencies for (II) and (III) over the range examined were similar. At pH 7.60 the ki for II = 7.38 x 10(3) M-1 min-1 (SD = 398) and for (III) the ki = 6.67 x 10(3) M-1 min-1 (SD = 355). In accord with the findings of Wilson and Bergmann20 on physostigmine our results indicate that the protonated form of (II) and (III) is the more potent inhibitor.

Anticholinesterase activity of potential therapeutic 5-(1,3,3-trimethylindolinyl) carbamates.

Six N-alkyl and N-aryl 5-(1,3,3-trimethylindolinyl) carbamates were synthesized and studied for their structure-activity relationships in inhibiting eel acetylcholinesterase (AChE). The carbamates were 5-(1,3,3-trimethylindolinyl)N,N-dimethylcarbamate (Cui Xing Ning) (I), 5-(1,3,3-trimethylindolinyl)N,N-diethylcarbamate (IV), 5-(1,3,3-trimethylindolinyl)N-ethylcarbamate (III), 5-(1,3,3-trimethylindolinyl)N,N-diethylcarbamate (IV), 5-(1,3,3-trimethylindolinyl)N-heptylcarbamate (V), and 5-(1,3,3-trimethylindolinyl)N-(3-chlorophenyl)carbamate (VI). The inhibition studies were carried out at 25.0 degrees C at pH 7.60. The rank order of the ki values for eel AChE inhibition is II > V > I > III > VI > IV. Compound II has a greater affinity for the enzyme than any irreversible inhibitor cited in the literature (Kd = 7.14 x 10(-8) M). Our findings should aid in the application of these carbamates (1) for counteracting the cholinergic problems associated with various diseases, and (2) for developing potential pretreatment compounds for organophosphate poisoning.

Intramuscular administration of atropine in the rat: jet spray versus conventional needle injection.

The characteristics of atropine plasma levels after jet spray injection were compared to those after conventional needle injection (i.m.) in 12 male rats, six per group. Blood samples were sequentially collected from the tip of the tail over a 7h period. Injection of atropine sulfate (8.0 mg kg-1) using the jet spray resulted in mean peak plasma levels of 650 ng ml-1 (95 per cent C.I. = 90) compared to 488 ng mg-1 (95 per cent C.I. = 64) using a conventional needle. Times to reach maximum concentration were 30 min (95 per cent C.I. = 12) and 58 min (95 per cent C.I. = 6) for the jet spray and needle, respectively. Histopathologic examination (5 days post-injection) of target muscle showed that minimal fiber damage resulted from using the low pressure setting on the jet spray. The results suggest that the jet spray may offer a means of increasing the antidotal benefit over that achieved with conventional techniques using presently available therapeutic formulations for acetylcholinesterase poisoning.

pH effects in the spontaneous reactivation of phosphinylated acetylcholinesterase.

Previous studies on the spontaneous reactivation of phosphorylated and phosphonylated cholinesterases report bell-shaped curves with reaction rate maxima between pH values of 7 and 9. By way of contrast, we found reactivation rate minima in the same pH region for a phosphinylated bovine erythrocyte acetylcholinesterase and three phosphinylated eel acetylcholinesterases. To further elucidate these observations, eel acetylcholinesterase was inhibited with racemic 4-nitrophenyl ethyl(phenyl)phosphinate. The spontaneous reactivation of the inhibited enzyme over the pH range 6.00 to 9.00 was monitored following 1. both inhibition and spontaneous reactivation at the same pH, and 2. inhibition at pH 7.60 followed by spontaneous reactivation at the selected pH. The combined plots of both studies gave overlapping pH curves with minima around pH 7.60. The results indicate that the minima in the rates of the spontaneous reactivation of phosphinylated acetylcholinesterases are not the consequence of a pH-controlled change in the relative inhibition rates of the P(+)- and P(-)-enantiomers participating in the inhibition reaction. Our results suggest that the orientation of the phosphinyl group in the active site of phosphinylated acetylcholinesterase is quite different from that of the inhibitor groups in phosphonylated or phosphorylated enzyme.

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.

A radioimmunoassay for pyridostigmine.

Antipyridostigmine antibodies were produced in rabbits using a pyridostigmine analog conjugated to keyhole limpet hemocyanin. These antibodies were used for development of a radioimmunoassay that has a linear standard curve (r2 = 0.986) ranging from 0.5 to 10.0 ng/ml of pyridostigmine bromide in a 0.1-ml plasma sample. This assay measures pyridostigmine 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 microliter) of the plasma samples are required. Plasma levels of pyridostigmine were determined in the rat after intramuscular administration (0.056 mg/kg) of pyridostigmine bromide. Estimates of the various pharmacokinetic parameters were calculated using the computer program NONLIN84. The results were as follows: apparent volume of distribution = 1.97 l/kg, absorption rate constant = 0.277 min-1, elimination rate constant = 0.0273 min-1, area under the curve = 1010 ng x min/ml, absorption rate half-life = 2.41 min, elimination rate half-life = 24.8 min, maximal plasma concentration (Cmax) = 21.3 ng/ml and time to Cmax = 9.02 min.