Birnbaum D T, Kilcomons M A, DeFelippis M R, Beals J M
Division of Biopharmaceutical Development, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46285, USA.
Pharm Res. 1997 Jan;14(1):25-36. doi: 10.1023/a:1012095115151.
Investigations into the kinetic assembly and dissociation of hexameric LysB28ProB29-human insulin (LysPro), a rapid-acting insulin analog produced by the sequence inversion of amino acids at positions B28 and B29, were designed to explain the impact that the sequence inversion has on the formulation and pharmacokinetics of the insulin analog.
The kinetics of phenolic ligand binding to human insulin and LysPro were studied by stopped-flow spectroscopy. The kinetics of R6 hexamer disruption were studied by extraction of Co(II) with EDTA.
Phenolic ligand binding to human insulin yielded rate constants for a fast and slow phase that increased with increasing ligand concentration and are attributed to the T6 --> T3R3 and T3R3 --> R6 transitions, respectively. However, the kinetics of phenolic ligand binding with LysPro was dominated by rates of hexamer assembly. The kinetic differences between the insulin species are attributed to alterations at the monomer-monomer interface in the dimer subunit of the LysPro analog. The extraction of Co(II) from both hexameric complexes by EDTA chelation is slow at pH 8.0 and highly dependent on ligand concentration. Cobalt extraction from LysPro was pH dependent. Of the various phenolic ligands tested, the relative affinities for binding to the human and LysPro hexamer are resorcinol > phenol > m-cresol.
The extraction data support the formation of an R6-type LysPro hexamer under formulation conditions, i.e., in the presence of divalent metal and phenolic ligand, that is similar in nature to that observed in insulin. However, the formation kinetics of LysPro identify a radically different monomeric assembly process that may help explain the more rapid pharmacokinetics observed with the hexameric formulation of LysPro insulin relative to human insulin.
对六聚体LysB28ProB29 - 人胰岛素（LysPro）（一种通过B28和B29位氨基酸序列倒置产生的速效胰岛素类似物）的动力学组装和解离进行研究，旨在解释序列倒置对胰岛素类似物的制剂和药代动力学的影响。
通过停流光谱法研究酚类配体与人胰岛素和LysPro的结合动力学。通过用EDTA萃取Co(II)研究R6六聚体解离的动力学。
酚类配体与人胰岛素结合产生了快速和慢速相的速率常数，它们随配体浓度增加而增加，分别归因于T6→T3R3和T3R3→R6转变。然而，酚类配体与LysPro结合的动力学主要由六聚体组装速率主导。胰岛素种类之间的动力学差异归因于LysPro类似物二聚体亚基中单体 - 单体界面的改变。在pH 8.0时，通过EDTA螯合从两种六聚体复合物中萃取Co(II)很慢，并且高度依赖于配体浓度。从LysPro中萃取钴依赖于pH。在测试的各种酚类配体中，与人和LysPro六聚体结合的相对亲和力为间苯二酚>苯酚>间甲酚。
萃取数据支持在制剂条件下（即在二价金属和酚类配体存在下）形成R6型LysPro六聚体，其性质与在胰岛素中观察到的相似。然而，LysPro的形成动力学确定了一个截然不同的单体组装过程，这可能有助于解释相对于人胰岛素，LysPro胰岛素六聚体制剂观察到的更快药代动力学。
