Comprehensive ocular and systemic pharmacokinetics of dexamethasone after subconjunctival and intravenous injections in rabbits.

Even though subconjunctival injections are used in clinics, their quantitative pharmacokinetics has not been studied systematically. For this purpose, we evaluated the ocular and plasma pharmacokinetics of subconjunctival dexamethasone in rabbits. Intravenous injection was also given to enable a better understanding of the systemic pharmacokinetics. Dexamethasone concentrations in plasma (after subconjunctival and intravenous injections) and four ocular tissues (iris-ciliary body, aqueous humour, neural retina and vitreous) were analysed using LC-MS/MS. Population pharmacokinetic modelling for plasma data from both injection routes were used, and for first time the constant rate of absorption of dexamethasone from the subconjunctival space into plasma was estimated (ka,plasma = 0.043 min-1, i.e. absorption half-life of 17.3 min). Non-compartmental analysis was used for the ocular data analysis and resulting in ocular drug exposure of iris-ciliary body (AUC0-∞= 41984 min·ng/g) > neural retina (AUC0-∞= 25511 min·ng/g) > vitreous (AUC0-∞= 7319 min·ng/mL) > aqueous humour (AUC0-∞= 6146 min·ng/mL). The absolute bioavailability values after subconjunctival injection, reported for the first time, were 0.74 % in aqueous humour (comparable to topical dexamethasone suspensions), and 0.30 % in vitreous humour (estimated to be higher than in topical administration). These novel and comprehensive pharmacokinetic data provide valuable information on the potential for exploiting this route in ocular drug development for treating both, anterior and posterior segment ocular diseases. Moreover, the new generated dexamethasone-parameters are a step-forward in building predictive pharmacokinetic models to support the design of new subconjunctival dexamethasone formulations, which may sustain drug effect for longer period of time.

5-(Sulfamoyl)thien-2-yl 1,3-oxazole inhibitors of carbonic anhydrase II with hydrophilic periphery.

Hydrophilic derivatives of an earlier described series of carbonic anhydrase inhibitors have been designed, prepared and profiled against a panel of carbonic anhydrase isoforms, including the glaucoma-related hCA II. For all hydrophilic derivatives, computational prediction of intraocular permeability routes showed the predominance of conjunctival rather than corneal absorption. The potentially reactive primary or secondary amine periphery of these compounds makes them suitable candidates for bioconjugation to polymeric drug carriers. As was shown previously, the most active hCA II inhibitor is efficacious in alleviating intraocular pressure in normotensive rabbits with efficacy matching that of dorzolamide.

Understanding dexamethasone kinetics in the rabbit tear fluid: Drug release and clearance from solution, suspension and hydrogel formulations.

Rapid precorneal loss of topically applied eye drops limits ocular drug absorption. Controlling release and precorneal residence properties of topical formulations may improve ocular drug bioavailability and duration of action. In this study, we evaluated in vivo ocular pharmacokinetics of dexamethasone in rabbits after application of a drug solution (0.01%), suspension (Maxidex® 0.1%), and hydrogels of 2-hydroxyethyl methacrylate (HEMA) and acrylic acid (AAc) copolymers. The rabbits received a single eyedrop (solution or suspension) or dexamethasone-loaded hydrogel topically. Dexamethasone in tear fluid was sampled with glass capillaries and quantitated by LC-MS/MS. Higher dexamethasone exposure (AUC) in the tear fluid was observed with the suspension (≈3.6-fold) and hydrogel (12.8-fold) as compared to the solution. During initial 15 min post-application, the highest AUC of dissolved dexamethasone was seen after hydrogel application (368 min*µg/mL) followed by suspension (109.9 min*µg/mL) and solution (28.7 min*µg/mL. Based on kinetic simulations, dexamethasone release from hydrogels in vivo and in vitro is comparable. Our data indicate that prolonged exposure of absorbable dexamethasone in tear fluid is reached with hydrogels and suspensions. Pharmacokinetic understanding of formulation behavior in the lacrimal fluid helps in the design of dexamethasone delivery systems with improved ocular absorption and prolonged duration of action.

Ocular metabolism and distribution of drugs in the rabbit eye: Quantitative assessment after intracameral and intravitreal administrations.

Quantitation of ocular drug metabolism is important, but only sparse data is currently available. Herein, the pharmacokinetics of four drugs, substrates of metabolizing enzymes, was investigated in albino rabbit eyes after intracameral and intravitreal administrations. Acetaminophen, brimonidine, cefuroxime axetil, and sunitinib and their corresponding metabolites were quantitated in the cornea, iris-ciliary body, aqueous humor, lens, vitreous humor, and neural retina with LC-MS/MS analytics. Non-compartmental analysis was employed to estimate the pharmacokinetic parameters of the parent drugs and metabolites. The area under the curve (AUC) values of metabolites were 12-70 times lower than the AUC values of the parent drugs in the tissues with the highest enzymatic activity. The ester prodrug cefuroxime axetil was an exception because it was efficiently and quantitatively converted to cefuroxime in the ocular tissues. In contrast to the liver, sulfotransferases, aldehyde oxidase, and cytochrome P450 3A activities were low in the eye and they had negligible impact on ocular drug clearance. With the exception of esterase substrates, metabolism seems to be a minor player in ocular pharmacokinetics. However, metabolites might contribute to ocular toxicity, and drug metabolism in various eye tissues should be investigated and understood thoroughly.

A Novel Ferroptosis Inhibitor UAMC-3203, a Potential Treatment for Corneal Epithelial Wound.

Corneal wound, associated with pain, impaired vision, and even blindness, is the most common ocular injury. In this study, we investigated the effect of a novel ferroptosis inhibitor, UAMC-3203 (10 nM-50 µM), in corneal epithelial wound healing in vitro in human corneal epithelial (HCE) cells and ex vivo using alkali-induced corneal wounded mice eye model. We evaluated in vivo acute tolerability of the compound by visual inspection, optical coherence tomography (OCT), and stereomicroscope imaging in rats after its application (100 µM drug solution in phosphate buffer pH 7.4) twice a day for 5 days. In addition, we studied the partitioning of UAMC-3203 in corneal epithelium and corneal stroma using excised porcine cornea. Our study demonstrated that UAMC-3203 had a positive corneal epithelial wound healing effect at the optimal concentration of 10 nM (IC50 value for ferroptosis) in vitro and at 10 µM in the ex vivo study. UAMC-3203 solution (100 µM) was well tolerated after topical administration with no signs of toxicity and inflammation in rats. Ex-vivo distribution study revealed significantly higher concentration (~12-38-fold) and partition coefficient (Kp) (~52 times) in corneal epithelium than corneal stroma. The UAMC-3203 solution (100 µM) was stable for up to 30 days at 4 °C, 37 °C, and room temperature. Overall, UAMC-3203 provides a new prospect for safe and effective therapy for corneal wounds.

Mucoadhesive properties of nanogels based on stimuli-sensitive glycosaminoglycan-graft-pNIPAAm copolymers.

Mucoadhesive formulations capable of situ gelation are promising for improving ocular drug delivery. Here we investigated two types of nanogels based on anionic glycosaminoglycans with grafted thermo-responsive poly(N-isopropylacrylamide) chains. One type of nanogels were formed by thermo-induced gelling of heparin-graft-poly(N-isopropylacrylamide) and chondroitin sulfate-graft-poly(N-isopropylacrylamide) copolymers. Another type of nanogels was based on the same copolymers, but terminal groups of thermosensitive macromolecular chains were modified to form covalent disulfide cross-links. All types of nanogels were studied towards their ability to encapsulate and release model drug - dexamethasone. Mucoadhesivity of both thermo-gelled and covalently cross-linked polymeric systems, as well as their ability to interact with dexamethasone, was assessed by microscale thermophoresis (MST). Mucoadhesion properties were also evaluated by isothermal titration calorimetry (ITC), which were in good correlation with MST data. The presence of disulfide linkages and thiol groups were shown to favor improved binding of cross-linked nanogels to mucin. Moreover, in vivo intraocular pressure studies showed that presence of polymers in solution can alter the ocular absorption of carbonic anhydrase inhibitor from eyedrops. The pharmacological effect was in line with mucoadhesive properties of these copolymers.

Partitioning and Spatial Distribution of Drugs in Ocular Surface Tissues.

Ocular drug absorption after eye drop instillation has been widely studied, but partitioning phenomena and spatial drug distribution are poorly understood. We investigated partitioning of seven beta-blocking drugs in corneal epithelium, corneal stroma, including endothelium and conjunctiva, using isolated porcine tissues and cultured human corneal epithelial cells. The chosen beta-blocking drugs had a wide range (-1.76-0.79) of n-octanol/buffer solution distribution coefficients at pH 7.4 (Log D7.4). In addition, the ocular surface distribution of three beta-blocking drugs was determined by matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) after their simultaneous application in an eye drop to the rabbits in vivo. Studies with isolated porcine corneas revealed that the distribution coefficient (Kp) between the corneal epithelium and donor solution showed a positive relationship and good correlation with Log D7.4 and about a 50-fold range of Kp values (0.1-5). On the contrary, Kp between corneal stroma and epithelium showed an inverse (negative) relationship and correlation with Log D7.4 based on a seven-fold range of Kp values. In vitro corneal cell uptake showed a high correlation with the ex vivo corneal epithelium/donor Kp values. Partitioning of the drugs into the porcine conjunctiva also showed a positive relationship with lipophilicity, but the range of Kp values was less than with the corneal epithelium. MALDI-IMS allowed simultaneous detection of three compounds in the cornea, showed data in line with other experiments, and revealed uneven spatial drug distribution in the cornea. Our data indicate the importance of lipophilicity in defining the corneal pharmacokinetics and the Kp values are a useful building block in the kinetic simulation models for topical ocular drug administration.

Ocular pharmacokinetics of atenolol, timolol and betaxolol cocktail: Tissue exposures in the rabbit eye.

Quantitative understanding of pharmacokinetics of topically applied ocular drugs requires more research to further understanding and to eventually allow predictive in silico models to be developed. To this end, a topical cocktail of betaxolol, timolol and atenolol was instilled on albino rabbit eyes. Tear fluid, corneal epithelium, corneal stroma with endothelium, bulbar conjunctiva, anterior sclera, iris-ciliary body, lens and vitreous samples were collected and analysed using LC-MS/MS. Iris-ciliary body was also analysed after intracameral cocktail injection. Non-compartmental analysis was utilized to estimate the pharmacokinetics parameters. The most lipophilic drug, betaxolol, presented the highest exposure in all tissues except for tear fluid after topical administration, followed by timolol and atenolol. For all drugs, iris-ciliary body concentrations were higher than that of the aqueous humor. After topical instillation the most hydrophilic drug, atenolol, had 3.7 times higher AUCiris-ciliary body than AUCaqueous humor, whereas the difference was 1.4 and 1.6 times for timolol and betaxolol, respectively. This suggests that the non-corneal route (conjunctival-scleral) was dominating the absorption of atenolol, while the corneal route was more important for timolol and betaxolol. The presented data increase understanding of ocular pharmacokinetics of a cocktail of drugs and provide data that can be used for quantitative modeling and simulation.

Pharmacokinetics of intravitreal macromolecules: Scaling between rats and rabbits.

Rats are widely used to study ocular drug responses, whereas rabbits are the most widely used preclinical model of ocular pharmacokinetics. Despite their wide use in evaluation of intravitreally injected drugs, translational information about pharmacokinetics and dose scaling between rats and rabbits is missing. In this study, we investigated intravitreal pharmacokinetics in rats and rabbits using non-invasive ocular fluorophotometry. Fluorescein and fluorescently labeled molecules (dextrans) with different molecular weights (376 Da, 10, 150 and 500 kDa), were injected into the vitreous of rabbits and rats. Intravitreal concentrations of the compounds were determined and pharmacokinetic parameters were calculated. Overall, the elimination half-lives of the macromolecules in rat vitreous were 5-6 times shorter than in rabbits, and the half-lives were prolonged at increasing molecular weights. The apparent volumes of distribution for tested compounds in rats and rabbits were in the range of the anatomical vitreal volumes. In both species, anterior route of elimination was predominant for the dextrans, whereas fluorescein was mainly eliminated via posterior route. Rabbit-to-rat ratios for intravitreal clearance were in the range of 2 to 5 for dextrans. Therefore, 2-5 times higher doses are needed for similar drug exposure in rabbits than in rats. Also, the shorter half-lives of macromolecules in the rat vitreous must be taken into account in translation to rabbit and human studies. The scaling factors presented herein will augment translational drug development for eye diseases.

Topical ocular pharmacokinetics and bioavailability for a cocktail of atenolol, timolol and betaxolol in rabbits.

Ocular bioavailability after eye drops administration is an important, but rarely determined, pharmacokinetic parameter. In this study, we measured the pharmacokinetics of a cocktail of three beta blockers after their topical administration into the albino rabbit eye. Samples from aqueous humour were analysed with LC-MS/MS. The pharmacokinetic parameters were estimated using compartmental and non-compartmental analyses. The ocular bioavailability was covering broad range of values: atenolol (0.07 %), timolol (1.22%, 1.51%) and betaxolol (3.82%, 4.31%). Absolute ocular bioavailability presented a positive trend with lipophilicity and the values showed approximately 60-fold range. The generated data enhances our understanding for ocular pharmacokinetics of drugs and may be utilized in pharmacokinetic model building in ophthalmic drug development.

Highly hydrophilic 1,3-oxazol-5-yl benzenesulfonamide inhibitors of carbonic anhydrase II for reduction of glaucoma-related intraocular pressure.

Four inhibitors of human carbonic anhydrase II (hCA II) were designed based on the previously reported subnanomolar 1,3-oxazole-based sulfonamide inhibitors of the enzyme to incorporate primary and secondary amine functionality in the carboxamide side chain. The new hydrophilic compounds were found to inhibit the target isoform in sub-nanomolar to low nanomolar range with a good degree of selectivity to several other hCA isoforms. The hydrophilic character of these compounds is advantageous for intraocular residence time but not for corneal permeability which generally requires that a drug be sufficiently lipophilic. Two of the four compounds investigated, however, were found to exert comparable efficacy as 1% eye drops in PBS to that of the clinically used 2% dorzolamide (Trusopt®) eye drops. This indicated that the absorption of the compounds may occur via alternative route across conjunctiva and sclera.