Manipulating Aromaticity: characterization of an ultra-rapid insulin analog

描述(申请人提供)：我们寻求开发一种治疗糖尿病的超快胰岛素类似物配方。超快药代动力学/动力学(PK/PD)有望使泵疗法(连续皮下胰岛素输注；CSII)具有更高的安全性和与连续血糖监测仪(CGM)更强大的集成，从而实现卓越的性能。我们的产品承诺提高算法控制的闭环系统(“人工胰腺”)的安全性和有效性；超快的PK/PD也将促进传统多次注射方案的餐后血糖控制。基于胰岛素分子的非标准突变，提出了一种创新的结构方法。本申请已根据研究科的建议进行了修订，最突出的是加强了对稳定性的关注，并提供了支持动物的数据。目前阻碍胰岛素锌六聚体快速皮下吸收的障碍是它的分解速度。这一速率由六角体二聚体界面上反平行层的稳定性控制。皮下注射后加速分解的部分进展是在20世纪90年代通过在这张纸的外围进行传统的定点突变而实现的。门冬氨酸胰岛素(Novolog(R)；诺和诺德的活性成分)和Lispro(Humalog(R)的活性成分；礼来公司的活性成分)含有这样的部分不稳定突变(Novolog中的ProB28-&GT；Asp；Humalog中的ProB28-&gt；Lys和LysB29-&gt；Pro(“KP”))。然而，由于它们的设计受到传统突变的限制，这两种产品都不能充分利用结构策略来促进胰岛素六聚体中核心-折叠的分解。胰岛素六聚体的核心是8个保守的芳香环(TyrB16、PheB24、PheB25、TyrB26和它们的二聚体相关伴侣)的汇合。该界面的热力学和动力学稳定性受一系列复杂的芳香族相互作用的调节。我们发现，PheB24被其非芳香族类似物环己基丙氨酸(CHA)取代后，与天然结构和功能相容，但显著提高了KP-胰岛素的分解速度。自提交原始申请以来，我们还发明了这种模拟的超稳定版本，其中包含通过A8-&gt；Glu额外的A链取代。因此，我们建议合成和表征ChaB24-KP-胰岛素和ChaB24-GluA8-胰岛素作为候选超快速制剂。B·弗兰克博士(首席研究员)是Humalog(R)的共同发明人，他之前在礼来公司工作过。Thermarin糖尿病有限责任公司拥有ChaB24相关IP的独家许可证，该IP由CWRU拥有。我们感谢研究科的指导。 与公共健康相关：通过操纵胰岛素分子中关键侧链的芳香性，我们已经加速了胰岛素六聚体的分解。在猪的模型中，本发明似乎提供了胰岛素制剂在血液中的超快速吸收，因此有望使糖尿病患者实现增强的血糖控制，同时降低与膳食相关的高血糖或低血糖的风险。在将这一想法付诸实践后，我们开发了Cyclolog-1和Cyclolog-1E(延长保质期)；与我目前使用的或(据我们所知)正在开发的应用于胰岛素泵和闭环系统的安全性和有效性的其他快速作用类似物相比，这两种药物有望提供显著的临床益处。该项目将完成Cyclolog-1和Cyclolog-1E的可行性试验。

DESCRIPTION (provided by applicant): We seek to develop an ultra-fast insulin analog formulation for the treatment of diabetes mellitus. Ultra-fast pharmacokinetic/dynamic (PK/PD) promises to enable superior performance of pump therapy (continuous subcutaneous insulin infusion; CSII) with enhanced safety and more robust integration with continuous glucose monitors (CGM). Our product promises to improve the safety and efficacy of algorithm-controlled closed-loop systems (the "artificial pancreas"); ultra-fast PK/PD would also facilitate post-prandial glycemic control in conventional multi-injection regimens. An innovative structural approach is proposed based on non-standard mutagenesis of the insulin molecule. This application has been revised based on the suggestions of the Study Section, most saliently to enhance its focus on stability and to provide supporting animal data. The current barrier to more rapid subcutaneous absorption is the rate of disassembly of the zinc insulin hexamer. This rate is controlled by the stability of an anti-parallel ¿-sheet at the dimer interface of the hexamr. Partial progress toward accelerated disassembly following subcutaneous injection was achieved in the 1990s by conventional site-directed mutagenesis at the periphery of this ¿-sheet. Insulin aspart (the active component of Novolog(R); Novo-Nordisk) and insulin lispro (the active component of Humalog(R); Eli Lilly) contain such partially destabilizing mutations (ProB28->Asp in Novolog; ProB28->Lys and LysB29->Pro in Humalog ("KP")). Because their designs were limited by conventional mutagenesis, however, neither of these products could fully exploit structural strategies to promote disassembly of the core ¿-sheet in the insulin hexamer. The core ¿ -sheet of the insulin hexamer is remarkable for the confluence of 8 conserved aromatic rings (TyrB16, PheB24, PheB25, TyrB26, and their dimer-related mates). The thermodynamic and kinetic stability of this interface is regulated by an intricate series of aromatic-aromatic interactions. We have discovered that the substitution of PheB24 by its non-aromatic analog cyclohexanylalanine (Cha) is compatible with native-like structure and function, but markedly enhances the rate of disassembly of KP-insulin. Since submission of the original application, we have also invented an ultra-stable version of this analog containing additional A-chain substitution ThrA8->Glu. We therefore propose to synthesize and characterize ChaB24-KP-insulin and ChaB24- GluA8-insulin as candidate ultra-rapid formulations. Dr. B. Frank (principal investigator) was co-inventor of Humalog(R) during his prior career at Eli Lilly. Thermalin Diabetes, LLC has an exclusive license to ChaB24- related IP, which is owned by CWRU. We thank the Study Section for its guidance. PUBLIC HEALTH RELEVANCE: By manipulating the aromaticity of a key side chain within the insulin molecule, we have conferred accelerated disassembly of the insulin hexamer. In a pig model this invention appears to provide ultra-rapid absorption of the insulin formulation into the blood stream and so promises to enable patients with diabetes mellitus to achieve enhanced glycemic control with reduced risk of meal-related hyper- or hypoglycemia. Having reduced this idea to practice, we have developed Cyclolog-1 and Cyclolog-1E (extended shelf life); these promise to provide significantly improved clinical benefits relative to other fast-acting analogs i present use or (to our knowledge) in development with application to the safety and efficacy of insulin pumps and closed-loop systems. This project will complete feasibility testing on Cyclolog-1 and Cyclolog-1E.