Comprehensive Pharmacokinetic Evaluation of High Melanin Binder Levofloxacin in Rabbits Shows Potential of Topical Eye Drops for Posterior Segment Treatment.

Purpose: The purpose of this work was to understand the impact of melanin binding on ocular pharmacokinetics after administration of a high-binder model drug via different administration routes. Methods: We applied levofloxacin to pigmented and albino rabbits as eye drops (single and multiple), as well as by intravitreal and intravenous injections. Ocular tissues and plasma were analyzed for levofloxacin concentrations with liquid chromatography-mass spectrometry (LC-MS/MS), and pharmacokinetic parameters were calculated. Results: The data show enrichment of levofloxacin and weeks-long retention in pigmented tissues. Upon intravitreal injection, the area under the curve (AUC) values in pigmented tissues were about 9 to 15 times higher than the respective values in the albino rabbits, but this difference expanded to 255- to 951-fold following topical eye drop administration. Multiple dosing of eye drops led to substantial accumulation of levofloxacin in the pigmented tissues: AUC values were 3 to 12 times higher than after intravitreal injection. The AUCs were much lower after single topical or intravenous drug administrations. High drug levels (0.1-35 µM) were always observed in the neural retinas of pigmented eyes; the highest exposure was seen after intravitreal administration followed by multiple doses of topical drops. Single topical instillation and intravenous injections to the albino rabbits resulted in vitreal bioavailability values of 0.009% and 0.003%, respectively. Conclusions: Melanin binding can be used to achieve targeted drug delivery and extended retention in pigmented ocular tissues. The results from topical multiple dosing experiments suggest that eye drop treatment may yield drug exposures and responses comparable to intravitreal delivery, even in the retinal pigment epithelium and choroid.

Mechanisms of cellular retention of melanin bound drugs: Experiments and computational modeling.

Melanin binding of drugs is known to increase drug concentrations and retention in pigmented eye tissues. Even though the correlation between melanin binding in vitro and exposure to pigmented eye in vivo has been shown, there is a discrepancy between rapid drug release from melanin particles in vitro and the long in vivo retention in the pigmented tissues. We investigated mechanisms and kinetics of pigment-related drug retention experimentally using isolated melanin particles from porcine retinal pigment epithelium and choroid, isolated porcine eye melanosomes, and re-pigmented ARPE-19 cells in a dynamic flow system. The experimental studies were supplemented with kinetic simulations. Affinity and capacity of levofloxacin, terazosin, papaverine, and timolol binding to melanin revealed Kd values of ≈ 50-150 µM and Bmax ≈ 40-112 nmol.mg-1. The drugs were released from melanin in <1 h (timolol) or in 6-12 h (other drugs). The drugs were released slower from the melanosomes than from melanin; the experimental differences ranged from 1.2-fold (papaverine) to 7.4-fold (timolol). Kinetic simulations supported the role of the melanosomal membrane in slowing down the release of melanin binders. In release studies from the pigmented ARPE-19 cells, drugs were released from the cellular melanin to the extracellular space in ≈ 1 day (timolol) and ≈ 11 days (levofloxacin), i.e., much slower than the release from melanin or melanosomes. Simulations of drug release from pigmented cells in the flow system matched the experimental data and enabled further sensitivity analyses. The simulations demonstrated a significant prolongation of drug retention in the cells as a function of decreasing drug permeability in the melanosomal membranes and increasing melanin content in the cells. Overall, we report the impact of cellular factors in prolonging drug retention and release from melanin-containing cells. These data and simulations will facilitate the design of melanin binding drugs with prolonged ocular actions.

Liposomal sunitinib for ocular drug delivery: A potential treatment for choroidal neovascularization.

Choroidal neovascularization (CNV) is a prevalent vision-threatening vascular disorder in aging population. CNV is associated with several diseases in the posterior segment of the eye such as age-related macular degeneration (AMD). In this study we developed sunitinib-loaded liposomes to block the neovascularization signalling pathway through inhibition of tyrosine kinase of vascular endothelial growth factor receptors (VEGFRs). Liposomal sunitinib formulations were prepared by thin film hydration method and studied for their encapsulation efficiency (EE), loading capacity (LC) and drug release profile in buffer andvitreous. Our finding showed that the liposomes (mean size 104 nm) could effectively entrap sunitinib (EE ≈ 95%) at relatively high loading capacity (LC ≈ 5%) and release sunitinib over at least 3 days. Intravitreal sunitinib-loaded liposomes revealed inhibitory effect on established neovascularization in laser-induced CNV mouse model while the intravitreal injection of sunitinib solubilized with cyclodextrin was inefficient in management of neovascularization. Accordingly, liposomal sunitinib is a promising drug delivery system that should be further studied to inhibit the CNV related to AMD.

Microscale Thermophoresis as a Screening Tool to Predict Melanin Binding of Drugs.

Interactions between drugs and melanin pigment may have major impacts on pharmacokinetics. Therefore, melanin binding can modify the efficacy and toxicity of medications in ophthalmic and other disease of pigmented tissues, such as melanoma. As melanin is present in many pigmented tissues in the human body, investigation of pigment binding is relevant in drug discovery and development. Conventionally, melanin binding assays have been performed using an equilibrium binding study followed by chemical analytics, such as LC/MS. This approach is laborious, relatively slow, and limited to facilities with high performance quantitation instrumentation. We present here a screening of melanin binding with label-free microscale thermophoresis (MST) that utilizes the natural autofluorescence of melanin. We determined equilibrium dissociation constants (Kd) of 11 model compounds with melanin nanoparticles. MST categorized the compounds into extreme (chloroquine, penicillin G), high (papaverine, levofloxacin, terazosin), intermediate (timolol, nadolol, quinidine, propranolol), and low melanin binders (atropine, methotrexate, diclofenac) and displayed good correlation with binding parameter values obtained with the conventional binding study and LC/MS analytics. Further, correlation was seen between predicted melanin binding in human retinal pigment epithelium and choroid (RPE-choroid) and Kd values obtained with MST. This method represents a useful and fast approach for classification of compounds regarding melanin binding. Thus, the method can be utilized in various fields, including drug discovery, pharmacokinetics, and toxicology.

Release of functional dexamethasone by intracellular enzymes: A modular peptide-based strategy for ocular drug delivery.

Tissue barriers limit drug delivery in the eye. Therefore, retinal diseases are treated with intravitreal injections. Delivery systems with reduced dosing frequency and/or cellular drug delivery properties are needed. We present here a modular peptide-based delivery system for cell targeted release of dexamethasone in the retinal pigment epithelial cells. The peptide-dexamethasone conjugates consist of cell penetrating peptide, enzyme cleavable linker and dexamethasone that is conjugated with hydrazone bond. The conjugates are chemically stable in the vitreous, internalize into the retinal pigment epithelial cells and release dexamethasone intracellularly by enzymatic action of cathepsin D. In vitro binding assay and molecular docking confirm binding of the released dexamethasone fragment to the human glucocorticoid receptor. In vivo rabbit studies show increased vitreal retention of dexamethasone with a peptide conjugate. Modular peptide conjugates are a promising approach for drug delivery into the retinal cells.