(M1230-02-09) Comparative Transcriptomic Profiling of Drug Metabolizing Enzymes and Drug Transporters in the Rabbit Ocular Sub-tissues

Purpose: The increasing prevalence of diabetes and aging population has led to a growing market demand for ophthalmic pharmaceutical drugs. However, the development of ophthalmic drugs is hampered by the poor understanding of drug metabolizing enzymes (DMEs) and drug transporters (DTs) in the ocular sub-tissues. DMEs play important roles in the bioactivation of prodrugs and the detoxication of drugs, while DTs can facilitate or limit drug exposure to the site of action¹. Rabbit is the most commonly used animal model to study ocular diseases and develop ophthalmic drugs. However, rabbit ocular DMEs and DTs have not been systematically characterized for their expression in ocular sub-tissues or compared to other major organs of drug disposition, e.g., liver and duodenum. This knowledge gap hinders precise extrapolation of ocular pharmacokinetic and pharmacodynamic data from rabbits to humans. As such, the goal of this study was to determine the localization and relative gene expression levels of DMEs and DTs in various rabbit ocular sub-tissues, including cornea, iris-ciliary body (ICB), vitreous humor (VH), and retina/choroid complex (RC), and to compare them to the rabbit liver and duodenum using RNA-seq.

Methods: Ocular sub-tissues, liver, and duodenum were collected from healthy New Zealand White rabbits (n=6, comprising 3 females and 3 males) weighing 2.0-3.0 kg and aged 8-10 weeks. The isolated tissues were homogenized with TRIzol Reagent (1 mL/100 mg tissue) using a Mini Bead Mill Homogenizer and the total RNA was extracted according to the TRIzol Reagent instructions. The total RNA was quantified using the Qubit assay and the RNA integrity number (RIN) was determined using the Agilent RNA ScreenTape assay. The 36-plex mRNA library pool was prepared using NEBNext Directional RNA Library Prep Kit with PolyA Selection Module starting with 1000 ng of total RNA for each sample. Illumina NextSeq2000 P3 SR-100bp sequencing was employed to sequence the 36-plex pool. The resulting RNAseq reads were aligned with STAR and quantified by using RSEM to generate raw counts and TPM (transcripts per million) for downstream analysis². A panel of candidate genes (n=755) for rabbit DMEs and DTs were assembled through manual screening of the Ensembl database for pertinent genes. Differential expression analysis was performed with DESeq2^3,4 using raw counts identify transcriptome-wide differences in relative expression of DMEs and DTs among the rabbit cornea, ICB, VH, RC, liver, and duodenum. Heatmaps with the log₂ (TPM + 1) values were plotted to highlight the top highly expressed DMEs and DTs within each tissue type.

Results: A total of 380 DME and 346 DT genes were identified in this study, and the variability of their expression patterns among all samples was assessed by multidimensional scaling, as depicted in Figure 1. For within-tissue type comparison, the top-highly expressed DMEs and DTs within each tissue type are listed in Table 1. Among the ocular sub-tissues, the top-highly expressed DMEs include the aldehyde dehydrogenases, glutathione S-transferases, glutathione peroxidases, and epoxide hydrolases, while the top-highly expressed DTs are from the solute carrier 6 (SLC6), SLC3, SLC22, and SLC7 families. For cross-tissue type comparison, tissue-specific DMEs (cutoff: FDR-adjusted P-value < 0.01 and log₂ fold-change > 1, compared to all other tissue types) were determined in the cornea (n=22), ICB (n=5), VH (n=6), RC (n=2), liver (n=91), and duodenum (n=26) (data not shown). Similarly, tissue-specific DTs were determined in the cornea (n=15), ICB (n=11), VH (n=3), RC (n=7), liver (n=46), and duodenum (n=34) (data not shown). In addition, the relative mRNA expression levels of DMEs and DTs across all tissue types was visualized using heatmaps. For instance, the relative mRNA expression heatmap of cytochrome P450s (CYPs) across all tissue type is shown in Figure 2. Notably, our study recapitulated previous findings regarding the expression of CYP3A6, CYP1A, CYP2C, and CYP2D in the rabbit liver and duodenum.^5,6 Additionally, our study confirmed the mRNA expression of CYP1B1, CYP4B1, and CYP4V2 in the rabbit eye, which are known to play crucial roles in the metabolism of endogenous compounds in the human eye.¹

Conclusion: A systematic transcriptomic expression analysis has been carried out to compare the expression profiles of DMEs and DTs among rabbit ocular sub-tissues, liver, and duodenum. Overall, the expression patterns of DMEs and DTs across ocular sub-tissues are more similar to each other, but markedly different from those observed in the liver and duodenum. Furthermore, tissue-specific DMEs and DTs have been identified for ocular sub-tissues, liver, and duodenum, which could be useful for elucidating tissue-specific drug responses. Additionally, the relative gene expression levels of DMEs and DTs across ocular sub-tissues, liver, and duodenum were assessed, which is expected to aid the development of ocular physiological based pharmacokinetics (O-PBPK) models and improve interspecies extrapolation between rabbits and humans.

References: (1) Nakano, M.; Lockhart, C. M.; Kelly, E. J.; Rettie, A. E. Ocular Cytochrome P450s and Transporters: Roles in Disease and Endobiotic and Xenobiotic Disposition. Drug Metabolism Reviews 2014, 46 (3), 247–260.
(2) Ewels, P. A.; Peltzer, A.; Fillinger, S.; Patel, H.; Alneberg, J.; Wilm, A.; Garcia, M. U.; Di Tommaso, P.; Nahnsen, S. The Nf-Core Framework for Community-Curated Bioinformatics Pipelines. Nature Biotechnology 2020, 38 (3), 276–278.
(3) Love, M. I.; Huber, W.; Anders, S. Moderated Estimation of Fold Change and Dispersion for RNA-Seq Data with DESeq2. Genome Biol 2014, 15 (12), 550.
(4) Zhao, S.; Ye, Z.; Stanton, R. Misuse of RPKM or TPM Normalization When Comparing across Samples and Sequencing Protocols. RNA 2020, 26 (8), 903–909.
(5) Pineau’, T.; Maurel, P. Developmental Expression of Rabbit Cytochrome P450 CYPIAI, CYP1A2 and CYP3A6 Genes. Eur J Biochem. 1991 Apr 10;197(1):145-53.
(6) Nakamura, T.; Okada, K.; Nagata, K.; Yamazoe, Y. Intestinal Cytochrome P450 and Response to Rifampicin in Rabbits. Japanese Journal of Pharmacology 2000, 82 (3), 232–239.

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). Transcriptome analysis was supported by the IDeA Program of the National Institute of General Medical Sciences (P20 GM103418), as well as the University of Kansas Center for Genomics. This study was approved by the Institutional Animal Care & Use Committee (IACUC) at the University of Kansas.


Figure1. Multidimensional scaling (MDS) plots accessing the variability and similarity of gene expression pattern among all samples for (A) DMEs and (B) DTs. The six biological replicates from each tissue type cluster together and are well separated from other tissue groups of biological replicates.


Table 1. List of top-highly expressed DMEs and DTs within rabbit ocular sub-tissues, liver, and duodenum.


Figure2. Heatmap of the mRNA expression level of cytochrome P450 genes across rabbit ocular sub-tissues, liver, and duodenum. The color code represents the log2-transformed DESeq2 normalized counts.