Abstract

Transthyretin (TTR) dissociation is the rate limiting step for both aggregation and subunit exchange. Kinetic stabilisers, small molecules that bind to the native tetrameric structure of TTR, slow TTR dissociation and inhibit aggregation. One such stabiliser is the non-steroidal anti-inflammatory drug (NSAID), diflunisal, which has been repurposed to treat TTR polyneuropathy. Previously, we compared the efficacy of diflunisal, tafamidis, tolcapone, and AG10 as kinetic stabilisers for transthyretin. However, we could not meaningfully compare diflunisal because we were unsure of its plasma concentration after long-term oral dosing. Herein, we report the diflunisal plasma concentrations measured by extraction, reversed phase HPLC separation, and fluorescence detection after long-term 250 mg BID oral dosing in two groups: a placebo-controlled diflunisal clinical trial group and an open-label Japanese polyneuropathy treatment cohort. The measured mean diflunisal plasma concentration from both groups was 282.2 $\mu$M $\pm$143.7 $\mu$M (mean $\pm$ standard deviation). Thus, quantification of TTR kinetic stabilisation using subunit exchange was carried out at 100, 200, 300, and 400 $\text{μM diflunisal}$ concentrations, all observed in patients after 250 mg BID oral dosing. A 250 $\mu$M diflunisal plasma concentration reduced the wild-type TTR dissociation rate in plasma by 95%, which is sufficient to stop transthyretin aggregation, consistent with the clinical efficacy of diflunisal for ameliorating transthyretin polyneuropathy.

摘要

甲状腺素运载蛋白 (TTR) 解离是聚集和亚基交换的速率限制步骤。动力学稳定剂是与 TTR 天然四聚体结构结合的小分子，可减缓 TTR 解离并抑制聚集。其中一种稳定剂是非甾体抗炎药 (NSAID) 二氟尼柳，它已被重新用于治疗 TTR 多发性神经病。之前，我们比较了二氟尼柳、他法米迪、托卡朋和 AG10 作为转甲状腺素蛋白动力学稳定剂的功效。然而，我们无法有意义地比较二氟尼柳，因为我们不确定长期口服给药后其血浆浓度。在此，我们报告了两组长期 250 mg BID 口服给药后通过提取、反相 HPLC 分离和荧光检测测定的二氟尼柳血浆浓度：安慰剂对照二氟尼临床试验组和开放标签的日本多发性神经病治疗队列。测得的两组二氟尼柳平均血浆浓度均为 282.2$\mu$中号$\pm$143.7$\mu$M（平均$\pm$标准差）。因此，在 100、200、300 和 400 下进行了使用亚基交换的 TTR 动力学稳定性定量 $\text{μM 二氟尼柳}$所有浓度均在 250 mg BID 口服给药后的患者中观察到。一个250$\mu$M二氟尼柳血浆浓度使血浆中野生型TTR解离率降低95%，足以阻止转甲状腺素蛋白聚集，与二氟尼柳改善转甲状腺素蛋白多发性神经病的临床疗效一致。