Institute for Laboratory Medicine, Stadtspital Triemli, Zurich CH-8063, Switzerland.
J Pharm Biomed Anal. 2013 Mar 5;75:192-8. doi: 10.1016/j.jpba.2012.11.040. Epub 2012 Dec 5.
The tridecapeptide afamelanotide (Scenesse®) is a congener of α-melanocyte stimulating hormone (α-MSH). Upon binding to the melanocortin 1 receptor (MC1R) on the surface of pigment cells of the skin, the melanocytes, α-MSH or afamelanotide trigger the synthesis of cAMP, which stimulates the synthesis of melanin and therefore induces skin tanning. In a recent trial, afamelanotide administered as controlled release implants protected erythropoietic protoporphyria (EPP) patients from sunlight induced phototoxic skin reactions. Administration of biological therapeutic peptides may elicit unwanted immunogenic responses in recipients of these products. Although in a previous study using ELISA technique we excluded any newly developed immunogenicity during prolonged exposure to afamelanotide, we confirmed the previously published existence of low titers of antibodies against α-MSH in drug-naïve individuals that cross-reacted with afamelanotide. In order to investigate whether such antibodies are neutralizing, i.e. could block the biological effect of afamelanotide, we developed a cell culture-based bioassay. The basis of our assay was the measurement of afamelanotide-induced cAMP formation in a strain of the B16 mouse melanoma cell line, G4F-7, expressing the transfected human MC1R. Average half-effective concentrations of the natural hormone α-MSH and its congener afamelanotide were 38.8 ± 10.6 and 10.9 ± 7.17 nM (n=5), respectively. Neutralizing antibodies would reduce the cAMP formation. Two neutralizing anti-α-MSH antibodies served as positive controls. cAMP formation in the G4F-7 cells after addition of sera of drug-naïve (n=6) and of drug-exposed EPP patients (n=17) was significantly lower than after that from healthy volunteers (n=13). There was no difference between drug-naïve and drug-exposed patients. Using forskolin as a hormone-independent stimulator of cAMP formation, we excluded an unspecific interference of EPP sera with cAMP formation. We conclude that afamelanotide even after prolonged application to EPP patients did not elicit neutralizing antibodies. Further, the low titer immunoreactivity observed in sera of some drug-naïve individuals had no effect on the biological activity of afamelanotide.
十三肽阿法美拉汀（Scenesse®）是α-促黑素细胞激素（α-MSH）的同系物。当与皮肤色素细胞（黑素细胞）表面的黑素皮质素 1 受体（MC1R）结合时，α-MSH 或阿法美拉汀会触发环磷酸腺苷（cAMP）的合成，从而刺激黑色素的合成，从而诱导皮肤晒黑。在最近的一项试验中，作为控释植入物给予的阿法美拉汀可保护红细胞生成性原卟啉症（EPP）患者免受阳光引起的光毒性皮肤反应。给予生物治疗性肽可能会在这些产品的接受者中引起不需要的免疫原性反应。尽管在以前使用 ELISA 技术的研究中，我们排除了在长时间接触阿法美拉汀期间新产生的免疫原性，但我们证实了在药物初治个体中存在针对 α-MSH 的低滴度抗体的先前发表的存在，这些抗体与阿法美拉汀发生交叉反应。为了研究这些抗体是否具有中和作用，即可以阻断阿法美拉汀的生物学效应，我们开发了一种基于细胞培养的生物测定法。我们的测定法的基础是测量在表达转染的人 MC1R 的 B16 小鼠黑素瘤细胞系 G4F-7 中阿法美拉汀诱导的 cAMP 形成。天然激素 α-MSH 和其同系物阿法美拉汀的平均半效浓度分别为 38.8 ± 10.6 和 10.9 ± 7.17 nM（n=5）。中和抗体将减少 cAMP 的形成。两种中和抗-α-MSH 抗体用作阳性对照。加入药物初治（n=6）和药物暴露的 EPP 患者（n=17）的血清后，G4F-7 细胞中的 cAMP 形成明显低于健康志愿者（n=13）的血清。药物初治患者和药物暴露患者之间没有差异。使用 forskolin 作为 cAMP 形成的激素非依赖性刺激物，我们排除了 EPP 血清对 cAMP 形成的非特异性干扰。我们得出的结论是，即使在长时间给予 EPP 患者后，阿法美拉汀也没有引起中和抗体。此外，在一些药物初治个体的血清中观察到的低滴度免疫反应对阿法美拉汀的生物学活性没有影响。
