Morath Volker, Maurer Stefanie, Feuchtinger Annette, Walser Rebecca, Schlapschy Martin, Bolze Florian, Metzler Thomas, Bruder Johanna, Steiger Katja, Walch Axel, Klingenspor Martin, Skerra Arne
Chair of Biological Chemistry, School of Life Sciences, Technical University of Munich, Freising 85354, Germany.
Department of Nuclear Medicine, School of Medicine and Health, Technical University of Munich, Munich 81675, Germany.
Mol Pharm. 2025 Jun 2;22(6):3017-3032. doi: 10.1021/acs.molpharmaceut.4c01503. Epub 2025 May 7.
Despite the multifaceted role of leptin for energy homeostasis and its broad therapeutic potential, the FDA/EMA-approved metreleptin constitutes the only leptin drug to date. To translate the promising results from previous studies on murine PASylated leptin with improved solubility and extended plasma half-life using PASylation technology─a biological alternative to PEGylation─we have developed a second-generation human leptin drug candidate and tested it rigorously and . To this end, the exposed hydrophobic Trp residue at position 100 in human leptin was replaced by Gln, which, together with the genetic fusion with a 600-residue PAS polypeptide, yielded a protein with high solubility, folding stability and receptor-stimulatory activity. In a pharmacokinetic (PK) study with wild-type mice, this modified human leptin showed an extended plasma half-life of 18.8 ± 3.6 h after subcutaneous (s.c.) injection. Furthermore, leptin-deficient mice were dosed s.c. with the modified human leptin carrying two different PAS fusion tags, PAS#1 or P/A#1, each comprising 600 residues. After only four doses, the disease phenotype, including morbid adiposity, hyperphagia, and hepatic steatosis, was completely reversed by both PASylated leptin versions, but not by the non-PASylated leptin if administered at the same dose. To assess its tissue distribution, P/A(200)-huLeptin was doubly labeled with two fluorescent dyes, which were specifically attached to the leptin and the PAS moiety, respectively. Analysis of relevant mouse organs by light sheet fluorescence microscopy after clearance revealed colocalized signals in the kidney and liver, thus indicating general stability of the PAS-leptin fusion protein . However, discrete signals were observed in the hypothalamic region, only with leptin detectable in the choroid plexus, which implies cleavage of the PAS tag during transcytosis across the physiological barriers. This study should pave the way toward a second-generation leptin drug enabling prolonged dosing intervals.
尽管瘦素在能量平衡中具有多方面作用且具有广泛的治疗潜力，但美国食品药品监督管理局（FDA）/欧洲药品管理局（EMA）批准的美曲普明是迄今为止唯一的瘦素药物。为了将先前关于使用聚唾液酸化（PASylation）技术（一种聚乙二醇化的生物替代方法）制备的具有改善的溶解度和延长的血浆半衰期的小鼠PAS化瘦素的研究成果转化应用，我们开发了一种第二代人瘦素候选药物并对其进行了严格测试。为此，将人瘦素第100位暴露的疏水性色氨酸残基替换为谷氨酰胺，这与与一个600个残基的PAS多肽的基因融合一起，产生了一种具有高溶解度、折叠稳定性和受体刺激活性的蛋白质。在对野生型小鼠进行的药代动力学（PK）研究中，这种修饰后的人瘦素皮下注射后血浆半衰期延长至18.8±3.6小时。此外，给瘦素缺乏的小鼠皮下注射携带两种不同PAS融合标签（PAS#1或P/A#1，每种包含600个残基）的修饰后人类瘦素。仅注射四剂后，两种PAS化瘦素版本均完全逆转了包括病态肥胖、食欲亢进和肝脂肪变性在内的疾病表型，但相同剂量的非PAS化瘦素则没有这种效果。为了评估其组织分布，P/A(200)-huLeptin用两种荧光染料进行了双重标记，这两种染料分别特异性地连接到瘦素和PAS部分。清除后通过光片荧光显微镜对相关小鼠器官进行分析，在肾脏和肝脏中发现了共定位信号，从而表明PAS-瘦素融合蛋白具有总体稳定性。然而，仅在下丘脑区域观察到离散信号，仅在脉络丛中可检测到瘦素，这意味着在跨生理屏障的转胞吞过程中PAS标签被裂解。这项研究应为第二代瘦素药物的研发铺平道路，使其能够延长给药间隔。
