Previously we determined that levamisole (LMS), when stored for a period of time, breaks down to three degradation products at neutral and alkaline pH. At low concentrations (10-6 M), Product 1 inhibits the lymphocyte response to concanavalin A (Con A), Product 2 enhances the response and Product 3 has no effect. At higher concentrations (10-5 M) all three products inhibit the response. To determine if these products are formed in culture media under culture conditions (e.g. in RPMI-1640 bicarbonate buffered medium, 37°C, pH 7.0-7.5, during a 72 h culture period), we added freshly prepared LMS solutions to culture media with and without lymphocytes present and maintained the pH at 7.0, 7.25 or 7.5 by varying the amount of CO2 present. Periodically over a 72 h period, aliquots of the media were removed and analyzed for the presence of LMS and the three degradation products. Within 4 h, two of the degradation product began to form in culture media with or without lymphocytes present. Product No. 1, 3-(2-mercaptoethyl)-5- phenylimidazolidine-2-one or dl-2-oxy-3-(2-mercaptoethyl)-5-phenylimidazolidine (OMPI), which inhibits the lymphocyte response to concanavalin A (Con A) at concentrations above 0.4 μg/ml, was formed at pH 7.0, 7.25 and 7.5, but the compound did not reach inhibitory concentrations in the lymphocyte cultures during the 72 h culture period. Product No. 2, 6-phenyl-2,3-dihydroimidazo (2,1-b) thiazole, which enhances the Con A response between concentrations of 0.5 and 10 μg/ml, was detected at concentrations between 2.5 and 3.5 μg/ml at pH 7.25 and 7.5. Product 2 was not detected in cultures at pH 7.0 and subsequently when we cultured lymphocytes with freshly prepared LMS and maintained the pH at 7.0, no significant enhancement of the Con A response was observed. Product No. 3, bis[3-(2-oxo-5-phenylimidazolidin-1-yl)ethyl] disulfide was not detected in the culture media during the 72 h period. A solution of LMS which had been stored for 2 weeks in RPMI-1640 medium at 4°C (in which Product 2 was detected) significantly enhanced the Con A response of cells cultured at pH 7.0, while a similar LMS solution which had been stored for 2 weeks at a higher temperature, 37°C (which contained both Products 1 and 2), inhibited the response. The results indicate that the enhancement of the Con A response by LMS is primarily due to an oxidation product of LMS which is formed in small, but stimulatory amounts during the 72 h culture period and suggest that the varied results reported with LMS in cell culture (enhancement, no effect or inhibition) may be due to the formation of these stimulatory and inhibitory products, the relative concentration of which may vary depending upon the culture conditions used and/or the method of preparing and/or storing LMS solutions.
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