(T1130-06-37) Establishment of an LC-MS/MS Method for Quantification of Lifitegrast in Rabbit Ocular Tissues and Its Application to Pharmacokinetic Study

Purpose: Lifitegrast (Xiidra^®, ophthalmic solution 5%), lymphocyte function-associated antigen 1 (LFA-1) antagonist, was approved by the FDA for the treatment of dry eye disease. The active sites are the cornea and conjunctiva, and the drug concentration in these tissues seems to have a direct relationship with its efficacy. Nevertheless, analytical methods for the determination of lifitegrast in these tissues have been limitedly reported so far. In the present study, we aimed to develop a sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for the determination of lifitegrast in rabbit plasma and ocular tissues (cornea, conjunctiva, and sclera). In addition, the developed assay was applied to characterize pharmacokinetics of lifitegrast in rabbits.

Methods: For chromatographic separations, the Atlantis dC18 column (150 × 2.1 mm, 5.0 µm) was used. The mobile phase consisted of a mixture of distilled water and acetonitrile with 0.1 % formic acid (50:50 v/v%). To detect the compounds using tandem mass spectrometry, a turbo ion spray interface with positive ion multi reaction monitoring (MRM) mode was used. The mass transitions were monitored at 615.2→145.0 for lifitegrast, and 621.2→145.0 for IS (lifitegrast-d₆). The biological samples were prepared by protein precipitation using acetonitrile. The accuracy, precision, and stability of developed assay were validated according to the FDA guidance. Following i.v. and ophthalmic administration of lifitegrast in rabbits, blood and ocular tissues were collected at predetermined time points. Drug concentrations in these samples were determined by established LC-MS/MS assay.

Results: Overall, lifitegrast and IS in plasma and ocular tissues (cornea, conjunctiva, and sclera) were selectively separated from endogenous interferences. The linearity of the calibration curves was accomplished for plasma and ocular tissues over the concentration range from 2 to 500 ng/mL and 5 to 500 ng/mL, respectively. The intra- and inter-day accuracy and precision of the developed assay were 95.03-111.75% and 0.75-12.40%, respectively. The stability of lifitegrast in biological samples including plasma, cornea, conjunctiva, and sclera was estimated as 89.68-107.00% for long-term stability, 89.48-102.50% for short-term stability, 96.08-105.83% for freeze-thaw stability, and 94.13-109.00% for auto-sampler stability. The pharmacokinetic parameters of lifitegrast were calculated by non-compartmental analysis of plasma concentration-time data. Following ophthalmic administration, the bioavailability of lifitegrast in plasma was estimated to be about 3%. The tissue concentrations were found to be highest in the conjunctiva, followed by cornea, and sclera.

Conclusion: In the present study, a simple and sensitive LC-MS/MS method was developed for the determination of lifitegrast in rabbit plasma and ocular tissues. The assay showed appropriate accuracy, precision, sensitivity, linearity, and stability. Also, the method was successfully applied to the pharmacokinetic study of lifitegrast in rabbits. The developed assay is expected to be used in further pharmacokinetic studies and formulation development.

Acknowledgements: This research was supported by the Industrial Strategic Technology Development Program (20008799, Development of drug delivery application technology using contact lenses to increase the absorption rate of dry eye treatment drug (lifitegrast)), funded by the Ministry of Trade, Industry, and Energy (MOTIE, Korea).