(T1130-05-29) Integrating Targeted Proteomics and Ocular Pharmacokinetics to Support PBPK Modeling of Carboxylesterase-Mediated Drug Metabolism in the Eye: A Case Study with Latanoprost Ophthalmic Solution

Purpose: The increasing global prevalence of diabetes and aging population have significantly increased the burden of eye diseases such as glaucoma, diabetic retinopathy, and age-related macular degeneration, which led to a rapid growth in the ophthalmic drug market. Physiologically based pharmacokinetic (PBPK) modeling has become a valuable tool in drug development, guiding clinical trial design and supporting regulatory evaluation. However, current ocular PBPK models mainly consider the formulation properties of the drug and the physiological characteristics of the eye, which has limited utility for drugs that undergo local metabolism and transport within the eye.¹ Unlike the liver, where cytochrome P450 (CYP) enzymes dominate, the eye shows minimal expression of CYP enzymes.² Instead, hydrolases, especially carboxylesterases (CESs), are abundantly expressed across rabbit ocular subtissues,³ and they are known to play important roles in activating many ophthalmic prodrugs such as latanoprost.⁴ This study aims to quantify CES protein expression levels in rabbit ocular subtissues and integrate them with pharmacokinetics (PK) of latanoprost ophthalmic solution in a rabbit model. These data will provide key inputs for building advanced ocular PBPK models that incorporate CES-mediated local metabolism, supporting better translational predictions for ophthalmic drug disposition and efficacy in the eye.

Methods: Ocular subtissues including cornea, aqueous humor (AH), iris-ciliary body (ICB), lens, vitreous humor (VH), and retina-choroid complex (RC) were collected from New Zealand white rabbit (8-12 weeks old). Liver samples were included as a reference. Whole tissue homogenates were prepared using beads homogenization in lysis buffer (50 mM Tris-HCl, 150 mM KCl, 2 mM EDTA), and protein concentration was determined using the BCA assay. Tryptic digestion was performed as described previously.⁵ Two tryptic signature peptides were selected for each rabbit CES isoform: one previously reported in literature (named pep1),⁶ and another selected in silico (pep2). Synthetic unlabeled signature peptides were used as calibration standards and isotope-labeled signature peptides were used as internal standards. In vitro latanoprost depletion assays were conducted in rabbit liver and ocular tissue to provide input value for scaling intrinsic clearance. For the ocular PK study, microdialysis probes were surgically implanted into the AH and VH of the rabbit’s right eye. The probes were perfused with phosphate buffer containing calibrator at a flow rate of 1 𝜇l/min. After a 2-hour stabilization, 50 𝜇l of 0.005% latanoprost ophthalmic solution was applied topically. Dialysate samples were collected every 30 minutes over 7 hours. Tear and plasma samples were also collected to explore the local metabolism and systemic exposure. Latanoprost and latanoprost acid were quantified in those samples by HPLC-MS/MS.

Results: Quantification of CES1 using the CES1_pep1 resulted in extremely low abundance ( < 1 pmol/mg in all tissues). In contrast, CES1_pep2 produced robust and reproducible data, with an average expression of 502 ± 150 pmol/mg in rabbit liver, which is comparable to reported values in human liver.⁷ CES2 quantification was consistent across both peptides, yielding 35 ± 10 pmol/mg in rabbit liver. CES3 expression was low in rabbit liver (~0.5 pmol/mg) and undetectable in ocular tissues. In ocular tissues, CES1 expression levels (Figure 1) were highest in the ICB (28.5 ± 5.8 pmol/mg), followed by the cornea (14.3 ± 4.1 pmol/mg), VH (5.6 ± 1.4 pmol/mg), lens (0.9 ± 0.5 pmol/mg), and AH, 0.5 ± 0.1 pmol/mg. CES2 expression was ~3 pmol/mg in the cornea, AH, and ICB, while VH and lens exhibited lower levels, averaging around 1 pmol/mg. Preliminarily date on substrate depletion studies revealed rapid latanoprost hydrolysis in liver S9 (t₁/₂ = 23 min at 0.01 mg/mL protein), while metabolism was substantially slower in ocular S9 (t₁/₂ = 231 min at 0.2 mg/mL protein), in vitro substrate depletion study on ocular subtissues are ongoing. PK analysis in the tear film showed that latanoprost was rapidly eliminated following first-order kinetics, with a half-life of 5.1 ± 0.9 minutes. In contrast, latanoprost acid exhibited much slower elimination from the tear film, with a half-life of 3.2 ± 0.3 hours. The prodrug latanoprost was not detected in either AH or VH, whereas latanoprost acid was only detected in the AH, reaching a maximum concentration (Cmax) of 142 ± 25 nM approximately 3 hours post-dose.

Conclusion: This study demonstrates that appropriate selection of signature peptides is critical for accurate CES quantification by targeted proteomics. In rabbit eye, CES1 was highly expressed in the ICB and cornea, but showed minimal expression levels in AH, consistent with previous report in human eye,⁸ highlighting the translational relevance between rabbit and human. The latanoprost PK profile in rabbit AH closely mirrored published human data,⁹ indicating the suitability of using in-situ microdialysis for ocular PK study. In vitro metabolism data will further inform CES-mediated clearance and will support mechanistic scaling in model development. Together, these data, combined with published ocular anatomical and physiological parameters, provide a solid foundation for establishing an advanced Ocular-PBPK model that accounts for CES-mediated local metabolism.

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Acknowledgements: This study was supported by a COBRE pilot project award (PI: Michael Zhuo Wang) from the Center for Molecular Analysis of Disease Pathway (CMADP) at the University of Kansas funded by the National Institute of General Medical Sciences (P30GM145499; PI: Susan Lunte). Animal study protocol (AUS# 210-04) was approved by the Institutional Animal Care & Use Committee at the University of Kansas.

Figure 1. The abundance of rabbit CES1 and CES2 in rabbit ocular subtissues. AH, aqueous humor; ICB, iris-ciliary body; VH, vitreous humor. Data are presented as mean ± SD.

Figure 2. Concentration-time profile of latanoprost and latanoprost acid in rabbit tear film. Topical application of 2.5 μg latanoprost in rabbit eye.

Figure 3. Concentration-time profile of latanoprost acid in rabbit aqueous humor. Topical application of 2.5 μg latanoprost in rabbit. Time point represents for the dialysate collection time.