Insmed Incorporated, 10 Finderne Avenue, Building 10, Bridgewater, NJ 08807, USA.
Insmed Incorporated, 10 Finderne Avenue, Building 10, Bridgewater, NJ 08807, USA.
Pulm Pharmacol Ther. 2018 Apr;49:95-103. doi: 10.1016/j.pupt.2018.01.012. Epub 2018 Feb 2.
Idiopathic pulmonary fibrosis is a progressive and lethal disease and while there are now two approved drugs (Esbriet and Ofev) additional effective treatments are still needed. Recently, prostacyclin analogs such as iloprost and treprostinil (TRE) have been shown to exert some protection against bleomycin-induced pulmonary fibrosis in mice when administered in a prophylactic regimen. In this study, we evaluated the effect of the inhaled treprostinil prodrug hexadecyl-treprostinil (C16TR) formulated in a lipid nanoparticle (INS1009) administered therapeutically in a fibrotic rat model. Male Fischer 344 rats challenged with intra-tracheal saline instillation were then treated with daily inhaled phosphate buffered saline (PBS) while rats challenged with bleomycin sulfate (3.5-4.0 mg/kg) instillation were treated with either daily inhaled PBS, daily inhaled INS1009 (10, 30, or 100 μg/kg), or twice-daily orally with the anti-fibrotic compound pirfenidone (100 mg/kg). Dosing started on day 10 post-bleomycin challenge and continued until day 27 after bleomycin. Lungs were harvested 24 h after the last dose of treatment for evaluation of lung hydroxyproline content and pulmonary histology. Lung hydroxyproline content increased from 421 μg/lung lobe in saline challenged and PBS treated animals to 673 μg/lung lobe in bleomycin challenged and PBS treated rats. Treatment of bleomycin challenged rats with 10, 30, or 100 μg/kg INS1009 dose-dependently reduced lung hydroxyproline content to 563, 501, and 451 μg/lung lobe, respectively, and pirfenidone decreased hydroxyproline content to 522 μg/lung lobe. Histologically, both INS1009 (100 μg/kg) and pirfenidone (100 mg/kg) reduced the severity of subepithelial fibrosis. Single dose pharmacokinetic (PK) studies of inhaled INS1009 in bleomycin challenged rats showed dose-dependent increases in lung C16TR concentration and plasma TRE on day 10 post-bleomycin challenge. Multiple dose PK studies of inhaled INS1009 showed dose-dependent increases only in lung C16TR concentration on day 27 post-bleomycin challenge. We also investigated the effects of TRE on the cytokine transforming growth factor-β (TGF-β)-stimulated collagen gene and protein expressions in cultured human lung fibroblasts, assessed by real-time PCR and Sirius Red staining, respectively. In human fibroblasts, TRE (0.001-10 μM) inhibited TGF-β (20 ng/mL)-induced expression of collagen mRNA and protein in a concentration-dependent manner. These results demonstrated that inhaled INS1009, administered in a therapeutic dosing paradigm, dose-dependently (10-100 μg/kg) inhibited bleomycin-induced pulmonary fibrosis in rats. This effect may involve direct actions of TRE in suppressing collagen expression in lung fibroblasts.
特发性肺纤维化是一种进行性和致命性疾病，虽然现在有两种批准的药物（Esbriet 和 Ofev），但仍需要额外的有效治疗方法。最近，前列腺素类似物如伊洛前列素和曲前列尼尔（TRE）在预防性方案中给药时，已被证明对博莱霉素诱导的小鼠肺纤维化具有一定的保护作用。在这项研究中，我们评估了吸入性曲前列尼尔前药十六烷基曲前列尼尔（C16TR）在脂质纳米颗粒（INS1009）中的疗效，该药物在纤维化大鼠模型中以治疗性方式给药。然后，用每日吸入磷酸盐缓冲盐水（PBS）治疗用气管内盐水滴注挑战的雄性 Fischer 344 大鼠，而用博莱霉素硫酸盐（3.5-4.0mg/kg）滴注挑战的大鼠用每日吸入 PBS、每日吸入 INS1009（10、30 或 100μg/kg）或每天两次口服抗纤维化化合物吡非尼酮（100mg/kg）治疗。从博莱霉素挑战后第 10 天开始给药，直到博莱霉素后第 27 天结束。在最后一次治疗后 24 小时收获肺脏，用于评估肺羟脯氨酸含量和肺组织病理学。在盐水挑战和 PBS 处理的动物中，肺羟脯氨酸含量从 421μg/肺叶增加到博莱霉素挑战和 PBS 处理的大鼠的 673μg/肺叶。用 10、30 或 100μg/kg INS1009 剂量依赖性地治疗博莱霉素挑战的大鼠，肺羟脯氨酸含量分别降低至 563、501 和 451μg/肺叶，吡非尼酮降低羟脯氨酸含量至 522μg/肺叶。组织学检查显示，INS1009（100μg/kg）和吡非尼酮（100mg/kg）均降低了亚上皮纤维化的严重程度。博莱霉素挑战大鼠单次吸入 INS1009 的药代动力学（PK）研究表明，博莱霉素后第 10 天肺 C16TR 浓度和血浆 TRE 呈剂量依赖性增加。博莱霉素后第 27 天的多次剂量 PK 研究表明，仅在博莱霉素后第 27 天，肺 C16TR 浓度呈剂量依赖性增加。我们还研究了 TRE 对体外培养的人肺成纤维细胞中转化生长因子-β（TGF-β）刺激的胶原基因和蛋白表达的影响，分别通过实时 PCR 和 Sirius Red 染色进行评估。在人成纤维细胞中，TRE（0.001-10μM）以浓度依赖性方式抑制 TGF-β（20ng/mL）诱导的胶原 mRNA 和蛋白表达。这些结果表明，以治疗性剂量方案给予吸入性 INS1009 可剂量依赖性地（10-100μg/kg）抑制博莱霉素诱导的大鼠肺纤维化。这种作用可能涉及 TRE 直接作用于抑制肺成纤维细胞中胶原的表达。
