(T1130-03-20) Enhancing Drug Penetration Across the Blood Ocular Barrier Using Peptide Transporters: Expression and Pharmacokinetic-Based Analysis

Purpose: The structure of the eye poses a great challenge to the delivery of xenobiotics, mainly because of various anatomical and physiological ocular barriers present in the eye. These barriers restrict the entry of xenobiotics to such an extent that they do not reach effective therapeutic concentration at site of action. Drug transporters have been proposed to play an important role in the absorption, distribution and disposition of therapeutic agents. Peptide transporters are important plasma membrane proteins that facilitate the cellular translocation of dipeptides and tripeptides, in addition to a variety of peptidomimetic molecules. Increasing number of peptide-based drug therapy are having potential for the development of a newer class of drugs for the treatment of various ocular diseases. Therefore, studying peptide transporters in ocular tissues is important to understand their role in the ocular pharmacokinetics of xenobiotics.

Methods: The expression of peptide transporters viz. Peptide Transporter 1 (PEPT1), Peptide Transporter 2 (PEPT2), Peptide/Histidine Transporter 1 (PHT1) and Peptide/Histidine Transporter 2 (PHT2) was studied in ocular tissues i.e. cornea, sclera, conjunctiva, iris-ciliary body (ICB), and retina-choroid of rabbits. Tissue samples were collected and expression was characterized by PCR. To determine the substrate specificity, transcorneal penetration of glycylsarcosine (glysar) was evaluated at different concentrations and pH. Further, rabbits (n=4) were divided into two groups: 1) Glysar (substrate), 2) Glysar with losartan (blocker) pretreatment. The treatment was given via topical, intravenous (IV) and intravitreal (IVT) route. After topical instillation and IV administration of drugs, tear and aqueous humor (AH) were collected. Various ocular fluids and tissues were collected following IVT at 15, 30, 60 and 120 mins. The drug levels were analyzed using Liquid Chromatography–Tandem Mass Spectrometry (LC-MS/MS) and pharmacokinetic evaluation was performed using ModVizPoP.

Results: The study revealed the expression of peptide transporters in ocular tissues. Highest expression of PEPT1 was observed in retina-choroid, conjunctiva, and sclera. Whereas PEPT2 was present in all ocular tissues. Interestingly, ocular tissues such as ICB and retina-choroid were found to have the expression of PHT2 and ocular tissues such as ICB, conjunctiva, cornea and sclera were found to have the expression of PHT1. Topically applied glysar (68.42 µmol/mL) showed a maximum AH concentration of 3 nmol/mL at 30 min. Upon blockage of transcorneal peptide transporters, the conc. of glysar was reduced to 0.3 nmol/mL (p < 0.001). Tear kinetics of glysar showed a rapid decline at 30 min upto 1 hr followed by a gradual precorneal elimination. After blocker pre-treatment, the precorneal residence of glysar (0.09 nmol/mL) decreased significantly (p < 0.001). However, after IV administration of glysar, its conc. in AH of blocker pre-treated group was reduced to 0.38 nmol/mL (p < 0.01) and in tear increased to 2.79 nmol/mL (p < 0.05). After IVT administration of glysar, its conc. in cornea, lens, ICB, conjunctiva and plasma of blocker pre-treated group was reduced to 9.6 nmol/mL (p < 0.01), 1.07 nmol/mL (p < 0.01), 10 nmol/mL (p < 0.01), 16.38 nmol/mL (p < 0.05) and 0.25 nmol/mL (p < 0.001) respectively. Whereas, conc. was significantly increased in vitreous humor, retina and sclera to 2.04 µmol/mL (p < 0.01), 331 nmol/mL (p < 0.01) and 34 nmol/mL (p < 0.05) respectively.

Conclusion: This study, for the first time gives insight into the presence and functional importance of peptide transporters involved in ocular uptake of peptide substrates. Regional variation of the expression of peptide transporters in eye indicates the possibility of alteration in the ocular pharmacokinetics. The involvement of peptide transporters in the ocular disposition of glysar was demonstrated. Inhibition of peptide transporters by losartan at a concentration of 2.36 µmol/mL was confirmed through transcorneal transport experiments using glysar as a model substrate. This study would be critical for the development of peptide transporter targeted drug delivery to the various ocular tissues.

Acknowledgements: We would like to thank Indian Council of Medical Research (ICMR) for providing senior research fellowship to Mr. Tapas Kumar Roy (F.No. 45/10/2022-PHA/BMS).