青蒿酯和青蒿素的放射免疫测定法

青蒿素是一种新型抗疟药,青蒿酯是其活性衍生物之一。药理研究和临床试验均证明二者对一般恶性疟、脑型疟和间日疟有较好疗效,且对耐氯喹株疟原虫感染也有效。为了深入进行青蒿酯的代谢和临床药理研究,我们建立了放射免疫测定法,并用此法研究了青蒿酯在狗体内的血药时程。

RADIOIMMUNOASSAY OF QINGHAOSU AND ARTESU NATE

Qinghaosu (artemisinine), the active principle of the Chinese herb Qinghao (Artemisia annua L.) is a sesquiterpene peroxide. Artesunate is the hemisuccinate of dihydroqinghaosu (reduced qinghaosu). Laboratory and clinical studies indicated that both qinghaosu and artesunate have a high level of activity against falciparum and vivax malarial infections even in chloroquine resistant cases. For pharmacokinetic studies on these antimalarials a rapid, sensitive and specific method of assay would be most advantageous. Methods hither-to available require solvent extraction and evaporation in addition to ample amount of sample of biological specimen but lack sensitivity. This paper reports the development of a radioimmunoassay method.Since we have found that the artesunate-BSA adduct failed to produce antibodies to artesunate and qinghaosu, dihydroqinghaosu-12-O-acetic acid (3) was conjugated to bovine serum albumin (BSA). Antiqinghaosu serum was obtained by immunizing a sheep with the conjugate (4) plus complete Freund's adjuvant. The titer of the antiserum was measured to be about 1800 using ~3H-dihydroqinghaosu as the labeled antigen. This radioimmunoassay method can measure as little as 2-3 ng of qinghaosu. The antiserum bound qinghaosu, artesunate, dihydroqinghaosu and artemether, another active derivative of qinghaosu, equally well. These four compounds differ only by the substitucnts on the O-12 position. Since BSA was conjugated to the site very close to the O-12 position, it is not surprising that the antibody fails to differentiate between these four compounds. The peroxide linkage within the molecule is critical in determining the antibody specificity since deoxyqinghaosu, which lacks the peroxide linkage and is devoid of antimalarial activity, was bound to a much lesser degree.The radioimmunoassay technique reported here is rapid and sensitive and should be useful for the determination of qinghaosu, artesunate and artemether in clinical pharmacokinetic studies. As a preliminary test of the method, artesunate, 4 mg/kg and 14 mg/kg, was given to two dogs i. v.. The plasma drug concentration-time curves were found to fit a one compartment model with half-lives of 33 rain and 20 min. Since dihydroqinghaosu, known to be an active metabolic intermediate of artesunate and qinghaosu, may also be detected by the antiserum, the result of the assay would be the total of unchanged drug and active metabolites.