Determination of unbound fraction of dorzagliatin in human plasma by equilibrium dialysis and LC-MS/MS and its application to a clinical pharmacokinetic study

• A novel method was established for determining the unbound fraction of dorzagliatin in human plasma. • A solution was provided to address the issue of lack of the “true unbound fraction” value. • Method reproducibility was demonstrated in incurred sample reanalysis. Dorzagliatin, a novel glucokinase (GK) activator targeting both pancreatic and hepatic GK, is currently in late-stage clinical development for treatment of type 2 diabetes (T2D). For the optimization of dosing regimens to ensure adequate safety and efficacy, it is critical to have a deep understanding of pharmacokinetic (PK) and pharmacodynamic (PD) profiles of the drug in various targeting patient populations, considering the fact that T2D adversely affects a vast patient population who often times also suffer from a wide range of comorbidities including severe liver and/or kidney damage. Since drug efficacy seems to be closely related to unbound drug concentrations at the site of action, therefore, the determination of plasma unbound concentrations/fractions of dorzagliatin is of crucial importance, especially when performing the PK/PD assessment in those special populations. In the current study, a method was developed and validated for determining the unbound fraction (f u ) of dorzagliatin in human plasma by using equilibrium dialysis for the separation of the bound and unbound drug, and LC–MS/MS for subsequent quantification. We have successfully addressed two widely recognized challenges for determination of the f u , i.e., the lack of knowledge on the “true f u ” and the difficulty in assessing the accuracy and reproducibility of the measurement. Using this method, a 0.2 mL aliquot of human plasma samples were first dialyzed against 0.35 mL of phosphate buffered saline buffer at 37 °C for 5 h in the equilibrium dialysis device to separate the unbound dorzagliatin. Afterwards, post-dialysis samples were extracted by protein precipitation using acetonitrile. Separation of dorzagliatin and potential interferences were achieved using a Gemini C18 column coupled with gradient elution. Subsequent detection was carried out on tandem mass spectrometer operated by multiple reaction monitoring in positive mode using electrospray ionization. The standard curve over the concentration range of 0.125−250 ng/mL exhibits good linearity. The method was fully validated meeting the requirements in current bioanalytical guidance and was successfully applied in a clinical PK study of dorzagliatin in healthy volunteers and patients with renal function impairment. Method reproducibility was demonstrated in incurred sample reanalysis. With demonstrated accuracy, stability and reproducibility, reliable analytical results were obtained from clinical samples for PK/PD interpretation, providing valuable insight for the development of dorzagliatin.