Distribution of Small Molecular Weight Drugs into the Porcine Lens: Studies on Imaging Mass Spectrometry, Partition Coefficients, and Implications in Ocular Pharmacokinetics

Lens is the avascular tissue in the eye between the aqueous humor and vitreous. Drug binding to the lens might affect ocular pharmacokinetics, and the binding may also have a pharmacological role in drug-induced cataract and cataract treatment. Drug distribution in the lens has been studied in vitro with many compounds; however, the experimental methods vary, no detailed information on distribution between the lens sublayers exist, and the partition coefficients are reported rarely. Therefore, our objectives were to clarify drug localization in the lens layers and establish partition coefficients for a wide range of molecules. Furthermore, we aimed to illustrate the effect of lenticular drug binding on overall ocular drug pharmacokinetics. We studied the distribution of 16 drugs and three fluorescent dyes in whole porcine lenses in vitro with imaging mass spectrometry and fluorescence microscopy techniques. Furthermore, we determined lens/buffer partition coefficients with the same experimental setup for 28 drugs with mass spectrometry. Finally, the effect of lenticular binding of drugs on aqueous humor drug exposure was explored with pharmacokinetic simulations. After 4 h, the drugs and the dyes distributed only to the outermost lens layers (capsule and cortex). The lens/buffer partition coefficients for the drugs were low, ranging from 0.05 to 0.8. On the basis of the pharmacokinetic simulations, a high lens-aqueous humor partition coefficient increases drug exposure in the lens but does not significantly alter the pharmacokinetics in the aqueous humor. To conclude, the lens seems to act mainly as a physical barrier for drug distribution in the eye, and drug binding to the lens affects mainly the drug pharmacokinetics in the lens.


Introduction to lens structure
Lens epithelium and fibers. In the anterior lens, the epithelial cell monolayer regulates the transport and permeability of water, electrolytes and other compounds between aqueous humor and the lens 1 . Lens epithelial cells possess tight and adherens junctions and active transport for electrolytes and amino acids. During lens growth and aging, anterior to lens equator, the epithelial cells undergo mitosis and migrate to the lens equator, and then differentiate and elongate into lens fiber cells, which do not have nuclei or other organelles. The lens fiber cells lay over the older cells, forming a core-like structure. The lens fibers can be further divided into lens cortex and nucleus: the cortex comprises of the youngest lens fibers and is loose and soft in consistency, whereas the lens nucleus contains the oldest lens fibers in a compact arrangement, resulting in a dense consistency.
The lens capsule. Epithelial and fiber cells are completely enveloped by the lens capsule, the thickest basement membrane in the body, which has various roles in lens mechanics and cell survival 2 . Moreover, the lens is suspended by the capsule and the collagenous zonule fibers from the ciliary muscles. The lens capsule, produced by the lens epithelial cells and the fiber cells, is a lamellar structure consisting of laminin, type IV collagen, entactin/nidogen and proteoglycans 2 . The capsule is the thickest just anterior and posterior to lens the equator, in the regions where zonules attach to it, and thins towards the anterior and posterior poles 1 . In the pole areas, the capsule thickness is 5-10 fold greater in the anterior than posterior side. As an example, in adult human the capsule thickness ranges from 4 μm (posterior pole) to 23 μm (near equator).

Lens composition.
The lens composes mostly of water (65% of wet weight) and protein (34% of wet weight), most of which are various crystallins, and 1% of other compounds, such as lipids, inorganic ions, glucose, ascorbic acid and amino acids. The water, protein and lipid content in the lens varies between the cortex and nucleus. The cortex, consisting of the younger, softer fiber cells, has higher water content and lower protein and lipid contents than the nucleus. In contrast, the lens nucleus with the tightly-packed fiber cells contains more protein and protein-bound lipids and less water than the cortex. In addition to the anatomical volume of the lens, the K p values were also calculated using the estimated true distribution volume in the surface layer of the lens. Based on the MALDI-IMS images of drug distribution in lens section for atropine, pindolol, propranolol, pilocarpine and tizanidine, it seems that despite the five drugs display different logD 7.4 values, they had roughly the same spatial distribution: most of the lens section does not contain drugs, and the drugs distribute only to a small volume in the outer lens. This assumption was extended also to the other compounds. In the calculations, we assumed that the diameter of the lens area clear from the drugs is roughly 90% of the total lens diameter. Thus, the true distribution volume can be calculated by subtracting the unoccupied volume from the total volume. Supplementary equation 1 was reduced to a numerical value of the ratio between the true distribution volume and anatomical lens volume.  (1) where A is the equatorial diameter and B is the polar diameter.

Pharmacokinetic simulations
The pharmacokinetic simulation model was built on a model of topical timolol instillation 4 , where a lens compartment, separate from the reservoir compartment, was added. Experimental concentration-time data, extracted with Webplotdigitizer (v4.2, https://automeris.io/WebPlotDigitizer), in the aqueous humor and lens after topical dosing of timolol 5 was used as a basis for the lens compartment.
The timolol distribution clearance between aqueous humor and lens (QLENS), lens-buffer partition coefficient (K p ) and clearance from tear fluid to cornea (CLTF,CO) were obtained by comparing the simulated curves with experimental in vivo data on timolol distribution in the rabbit lens 5 . The parameter values were adjusted manually until a good fit between the simulated and the observed concentration in the lens was achieved (Supplementary figure 6). Based on the final K p and QLENS values, the distribution clearance between aqueous humor and reservoir (QRESERVOIR) and volume of the reservoir (VRESERVOIR) were changed to match the corresponding parameters in the original model 4 (Supplementary table 1