Determination of L-6-Keto-piperidine-2-carbonyl-L-leucyl-L-proline Amide (RGH-2202), a New Analog of Thyrotropin-Releasing Hormone, in Plasma by Radioimmunoassay

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A sensitive and specific radioimmunoassay has been developed for the determination of L-6-keto-piperidine-2-carbonyl-L-leucyl-L-proline amide (RGH-2202), a new analog of thyrotropinreleasing hormone (TRH), in plasma. An antiserum was produced in guinea pigs immunized with RGH-2202 which was conjugated to bovine serum albumin by an N-succinimidyl ester method. Iodine-125 labeled L-6-keto-piperidine-2-carbonyl-L-leucyl-L-prolyltyramine ([^<125>I] RGH-2202) was used as a tracer. Polyethylene glycol was used to separate bound and free [^<125>I] RGH-2202 in the reaction mixture. The assay of RGH-2202 in plasma was possible over a concentration range from 0.1 to 6.4 ng/ml, using 0.1 ml of plasma without the need for an extraction procedure. The antiserum used for the assay was highly specific for RGH-2202,and did not cross-react with TRH and the hydrolyzed derivatives of RGH-2202 such as L-6-keto-piperidine-2-carbonyl-L-leucyl-L-proline, L-6-keto-piperidine-2-carbonyl-L-leucine and L-leucyl-L-proline amide, which were assumed to be present in plasma as metabolites. The coefficients of variation were 4.6-6.7% for within-assay and 6.0-8.8% for between-assay. Plasma levels of RGH-2202 in rats were determined after an intravenous administration of RGH-2202 at a pharmacologically effective dose (0.625 mg/kg).
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