Ocular Pharmacokinetics of Intravitreally Injected Protein Therapeutics: Comparison among Standard-of-Care Formats.

The current standard of care for antivascular endothelial growth factor (VEGF) treatment requires frequent intravitreal (IVT) injections of protein therapeutics, as a result of limited retention within the eye. A thorough understanding of the determinants of ocular pharmacokinetics (PK) and its translation across species is an essential prerequisite for developing more durable treatments. In this work, we studied the ocular PK in macaques of the protein formats that comprise today's anti-VEGF standard of care. Cynomolgus monkeys received a single IVT injection of a single-chain variable fragment (scFv, brolucizumab), antigen-binding fragment (Fab, ranibizumab), fragment crystallizable-fusion protein (Fc-fusion, aflibercept), or immunoglobulin G monoclonal antibody (IgG, VA2 CrossMAb). Drug concentrations were determined in aqueous humor samples collected up to 42 days postinjection using immunoassay methods. The ocular half-life (t1/2) was 2.28, 2.62, 3.13, and 3.26 days for scFv, Fab, Fc-fusion, and IgG, respectively. A correlation with human t1/2 values from the literature confirmed the translational significance of the cynomolgus monkey as an animal model for ocular research. The relation between ocular t1/2 and molecular size was also investigated. Size was inferred from the molecular weight (MW) or determined experimentally by dynamic light scattering. The MW and hydrodynamic radius were found to be good predictors for the ocular t1/2 of globular proteins. The analysis showed that molecular size is a determinant of ocular disposition and may be used in lieu of dedicated PK studies in animals.

Influence of Melanin Characteristics on Drug Binding Properties.

Melanins are biopolymers encompassing a high degree of chemical heterogeneity. Binding of small-molecule drugs to ocular melanin significantly affects the ocular pharmacokinetics, and could serve as a strategy for prolonged drug retention in the eye. The influence of the structural and physical characteristics of melanins originating from different sources on their drug binding properties has not yet been methodically investigated. We performed physical characterization of Sepia officinalis, synthetic and porcine melanin. The particle size distribution was analyzed by laser diffractometry. A dynamic vapor sorption method, requiring small amounts of the material, was developed to analyze the differences in the specific surface area of the melanins. The extent of melanin binding at equilibrium was determined for a set of 34 small-molecule drugs and compared across different melanin types. Despite systematic shifts in the extent of binding within a twofold range, binding data were highly correlated across the melanins. These moderate differences in binding could not be directly explained by the substantial differences in particle size and were more in line with the relatively similar specific surface area of these different melanin materials. Overall, these results suggest that the specific surface area reflects the actual accessibility of a small molecule in the melanin structure and could serve as a surrogate to explain the binding differences observed for the respective melanin materials.

Establishment of an In Vitro-In Vivo Correlation for Melanin Binding and the Extension of the Ocular Half-Life of Small-Molecule Drugs.

A large variety of drugs bind effectively to melanin, and this binding influences their ocular pharmacokinetic and distribution profiles. We aimed to establish a correlation between in vitro melanin binding and in vivo ocular pharmacokinetics (PK). The extent of melanin binding in vitro was determined for a set of model drugs; binding kinetics and binding isotherms were generated and fitted to a mechanistic model to derive the drug-melanin binding parameters (Bmax, KD, kon, and koff). In addition, in vitro ADME properties such as cellular permeability, P-glycoprotein-mediated efflux, plasma protein binding, and octanol partition coefficients were determined. Moreover, cellular uptake was measured in the nonpigmented ARPE-19 cells and in lightly pigmented human epidermal melanocytes. Finally, in vivo ocular PK studies were performed in albino and pigmented rats using intravenous injections. Substantial drug enrichment accompanied by a very long residence time was observed in pigmented ocular tissues, which could be linked to the melanin binding determined in vitro and to the intracellular drug uptake into the pigmented cells. The resulting ocular PK profile is shown to be a consequence of the interplay of melanin binding with concurrent processes such as systemic clearance, plasma protein binding, cellular permeation, P-glycoprotein efflux, pH partitioning, and tissue binding. Understanding this interplay at a mechanistic level could help in the rational design and development of new small-molecule drug candidates with the desired PK/pharmacodynamic profile to target the back of the eye.

Development of a Unified Dissolution and Precipitation Model and Its Use for the Prediction of Oral Drug Absorption.

Drug absorption is a complex process involving dissolution and precipitation, along with other kinetic processes. The purpose of this work was to (1) establish an in vitro methodology to study dissolution and precipitation in early stages of drug development where low compound consumption and high throughput are necessary, (2) develop a mathematical model for a mechanistic explanation of generated in vitro dissolution and precipitation data, and (3) extrapolate in vitro data to in vivo situations using physiologically based models to predict oral drug absorption. Small-scale pH-shift studies were performed in biorelevant media to monitor the precipitation of a set of poorly soluble weak bases. After developing a dissolution-precipitation model from this data, it was integrated into a simplified, physiologically based absorption model to predict clinical pharmacokinetic profiles. The model helped explain the consequences of supersaturation behavior of compounds. The predicted human pharmacokinetic profiles closely aligned with the observed clinical data. In summary, we describe a novel approach combining experimental dissolution/precipitation methodology with a mechanistic model for the prediction of human drug absorption kinetics. The approach unifies the dissolution and precipitation theories and enables accurate predictions of in vivo oral absorption by means of physiologically based modeling.

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.

Understanding the Half-Life Extension of Intravitreally Administered Antibodies Binding to Ocular Albumin.

The burden associated with frequent injections of current intravitreal (IVT) therapeutics may be reduced by long-acting delivery strategies. Binding to serum albumin has been shown to extend the ocular half-life in rabbits, however, the underlying molecular mechanisms and translational relevance remain unclear. The aim of this work was to characterize the in vitro and in vivo formation of complexes between human serum albumin (HSA) and an antigen-binding fragment of a rabbit antibody linked to an anti-HSA nanobody (FabA). The ocular and systemic pharmacokinetics of 3H-labeled FabA (0.05 mg/eye IVT) co-formulated with HSA (1 and 15 nmol/eye) were assessed in Dutch belted rabbits. Next, FabA was incubated in vitreous samples from cynomolgus monkeys and human donors (healthy and diseased) supplemented with species-specific serum albumin. Finally, the FabA-albumin complexes formed in vitro and in vivo were analyzed by radio-size exclusion chromatography. A 3-fold increase in FabA vitreal exposure and half-life was observed in rabbits co-administered with 15 nmol HSA compared to 1 nmol and a control arm. The different pharmacokinetic behavior was explained with the formation of higher molecular weight FabA-albumin complexes. The analysis of vitreous samples revealed the existence of predominantly 1:1 complexes at endogenous or low concentrations of supplemented albumin. A shift towards 1:2 complexes was observed with increasing albumin concentrations. Overall, these results suggest that endogenous vitreal albumin concentrations are insufficient for half-life extension and warrant supplementation in the dosing formulation.

Understanding Molecular Drivers of Melanin Binding To Support Rational Design of Small Molecule Ophthalmic Drugs.

Binding of drugs to ocular melanin is a prominent biological phenomenon that affects the local pharmacokinetics and pharmacodynamics in the eye. In this work, we report on the development of in vitro and in silico tools for an early assessment and prediction of melanin binding properties of small molecules. A robust high-throughput assay has been established to study the binding of large sets of compounds to melanin. The extremely randomized trees approach was used to develop an in silico model able to predict the extent of melanin binding from the molecular properties of the compounds. After the last iteration of the model, strong melanin binders could prospectively be identified with 91% accuracy. On the basis of in vitro data generated for approximately 3400 chemically diverse drug-like small molecules, pronounced correlations were observed between the extent of melanin binding and the basicity, lipophilicity, and aromaticity of the compounds.

Extension of the dissolution-precipitation model for kinetic elucidation of solvent-mediated polymorphic transformations.

Thorough understanding and control of the different crystal forms of a drug product is key for fine chemistry and materials science; it ultimately determines the product's physicochemical properties and performance. In this work, we extend the application of a mechanistic dissolution-precipitation model to solvent-mediated solid form transformations. To address the relevance of the model, various kinetic solvent-mediated polymorphic transition studies were retrieved from the literature. Our model succeeds in accurately describing the experimental data, shedding light on the molecular steps driving the polymorphic conversion. Given its simplicity and mechanistic character, the model can be viewed as a useful tool to monitor, predict and optimize the solvent-mediated transformations of solid forms.