Barcode lipids for absolute quantitation of liposomes in ocular tissues.

Lipid-based drug formulations are promising systems for improving delivery of drugs to ocular tissues, such as retina. To develop lipid-based systems further, an improved understanding of their pharmacokinetics is required, but high-quality in vivo experiments require a large number of animals, raising ethical and economic questions. In order to expedite in vivo kinetic testing of lipid-based systems, we propose a barcode approach that is based on barcoding liposomes with non-endogenous lipids. We developed and evaluated a liquid-chromatography-mass spectrometry method to quantify many liposomes simultaneously in aqueous humor, vitreous, and neural retina at higher than ±20% precision and accuracy. Furthermore, we showed in vivo suitability of the method in pharmacokinetic evaluation of six different liposomes after their simultaneous injection into the rat vitreal cavity. We calculated pharmacokinetic parameters in vitreous and aqueous humor, quantified liposome concentrations in the retina, and quantitated retinal distribution of the liposomes in the rats. Compared to individual injections of the liposome formulations, the barcode-based study design enabled reduction of animal numbers from 72 to 12. We believe that the proposed approach is reliable and will reduce and refine ocular pharmacokinetic experiments with liposomes and other lipid-based systems.

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.

Altering distribution profile of palbociclib by its prodrugs.

Palbociclib, a cyclin-dependent kinase (CDK) 4/6 inhibitor, is currently used clinically for treating hormone receptor-positive and human epidermal growth factor receptor 2 negative breast cancer. Additionally, it has the potential to be utilized in the treatment of various tumors, including malignant glioblastoma. Previous research has indicated that palbociclib is a substrate for two efflux transporters, P-glycoprotein (P-gp; MDR1) and breast cancer-resistant protein (BCRP), which restrict the brain exposure of palbociclib. In the present study, our objective was to alter the brain distribution pattern of palbociclib by creating and assessing two novel prodrugs through in vitro, in situ, and in vivo evaluations. To this end, we synthesized two prodrugs of palbociclib by attaching it to the tyrosine promoiety at the para- (PD1) and meta-(PD2) position via a carbamate bond. We hypothesized that the prodrugs could bypass efflux transporter-mediated drug resistance by leveraging the l-type amino acid transporter (LAT1) to facilitate their transport across the blood-brain barrier (BBB) and into cancer cells, such as glioma cells that express LAT1. The compounds PD1 and PD2 did not show selective binding and had limited inhibitory effects on LAT1 in three cell lines (MCF-7, U87-MG, HEK-hLAT1). However, PD1 and PD2 demonstrated the ability to evade efflux mechanisms, and their in vitro uptake profiles were comparable to that of palbociclib, indicating their potential for effective cellular transport. In in situ and in vivo studies, brain uptake was not significantly improved compared to palbociclib, but the pharmacokinetic profiles showed encouraging enhancements. PD1 exhibited a higher AUCbrain/plasma ratio, suggesting safer dosing, while PD2 showed favorable long-acting pharmacokinetics. Although our prodrug design did not significantly improve palbociclib brain delivery due to the potential size limitation of the prodrugs, the study provides valuable insights for future prodrug development and drug delivery strategies targeting specific transporters.

Aldehyde oxidase 1 activity and protein expression in human, rabbit, and pig ocular tissues.

Aldehyde oxidase (AOX) is a cytosolic drug-metabolizing enzyme which has attracted increasing attention in drug development due to its high hepatic expression, broad substrate profile and species differences. In contrast, there is limited information on the presence and activity of AOX in extrahepatic tissues including ocular tissues. Because several ocular drugs are potential substrates for AOX, we performed a comprehensive analysis of the AOX1 expression and activity profile in seven ocular tissues from humans, rabbits, and pigs. AOX activities were determined using optimized assays for the established human AOX1 probe substrates 4-dimethylamino-cinnamaldehyde (DMAC) and phthalazine. Inhibition studies were undertaken in conjunctival and retinal homogenates using well-established human AOX1 inhibitors menadione and chlorpromazine. AOX1 protein contents were quantitated with targeted proteomics and confirmed by immunoblotting. Overall, DMAC oxidation rates varied over 10-fold between species (human ˃˃ rabbit ˃ pig) and showed 2- to 6-fold differences between tissues from the same species. Menadione seemed a more potent inhibitor of DMAC oxidation across species than chlorpromazine. Human AOX1 protein levels were highest in the conjunctiva, followed by most posterior tissues, whereas anterior tissues showed low levels. The rabbit AOX1 expression was high in the conjunctiva, retinal pigment epithelial (RPE), and choroid while lower in the anterior tissues. Quantification of pig AOX1 was not successful but immunoblotting confirmed the presence of AOX1 in all species. DMAC oxidation rates and AOX1 contents correlated quite well in humans and rabbits. This study provides, for the first time, insights into the ocular expression and activity of AOX1 among multiple species.

Mass spectrometry in ocular drug research.

Mass spectrometry (MS) has been proven as an excellent tool in ocular drug research allowing analyzes from small samples and low concentrations. This review begins with a short introduction to eye physiology and ocular pharmacokinetics and the relevance of advancing ophthalmic treatments. The second part of the review consists of an introduction to ocular proteomics, with special emphasis on targeted absolute quantitation of membrane transporters and metabolizing enzymes. The third part of the review deals with liquid chromatography-MS (LC-MS) and MS imaging (MSI) methods used in the analysis of drugs and metabolites in ocular samples. The sensitivity and speed of LC-MS make simultaneous quantitation of various drugs and metabolites possible in minute tissue samples, even though ocular sample preparation requires careful handling. The MSI methodology is on the verge of becoming as important as LC-MS in ocular pharmacokinetic studies, since the spatial resolution has reached the level, where cell layers can be separated, and quantitation with isotope-labeled standards has come more reliable. MS will remain in the foreseeable future as the main analytical method that will progress our understanding of ocular pharmacokinetics.

In vitro identification of decreased function phenotype ABCG2 variants.

Reduced activity of efflux transporter ABCG2, caused e.g., by inhibition or decreased function genetic variants, can increase drug absorption and plasma levels. ABCG2 has one clinically significant single nucleotide variant Q141K (c.421C>A), which leads to decreased protein levels and transport activity. In addition to Q141K, ABCG2 has over 500 rare (<1% minor allele frequency) nonsynonymous variants, but their functionality remains unknown. We studied the transport activity and abundance of 30 rare ABCG2 variants. The variants were transiently expressed in HEK293 cells. Transport activity and protein abundance were measured from inside-out crude membrane vesicles. Results were normalised to the reference ABCG2, while Q141K was used to categorise variants into decreased and normal function phenotypes based on their apparent transport activity. Fourteen variants (G80E, D128V, T434M, Q437R, C438R, C438W, C438Y, L479S, P480L, S486N, T512N, S519P, G553D and K647E) had similar or lower apparent transport activity than Q141K and thus were categorised as having a decreased function phenotype. Protein abundance could not explain all of the observed changes in transport activity: Only six variants (D128V, Q437R, C438R, S519P, G553D, and K647E) had similar or lower abundance compared to Q141K. The decreased function variants may increase systemic drug exposure and therefore cause interindividual variability in pharmacokinetics. In the future, in vitro phenotype classification may help to design personalised drug treatments.

Comparison of barrier properties of outer blood-retinal barrier models - Human stem cell-based models as a novel tool for ocular drug discovery.

The retinal pigment epithelial (RPE) cell monolayer forms the outer blood-retinal barrier and has a crucial role in ocular pharmacokinetics. Although several RPE cell models are available, there have been no systematic comparisons of their barrier properties with respect to drug permeability. We compared the barrier properties of RPE secondary cell lines (ARPE19, and ARPE19mel) and both primary (hfRPE) and stem-cell derived RPE (hESC-RPE) cells by investigating the permeability of nine drugs (aztreonam, ciprofloxacin, dexamethasone, fluconazole, ganciclovir, ketorolac, methotrexate, voriconazole, and quinidine) across cell monolayers. ARPE19, ARPE19mel, and hfRPE cells displayed a narrow Papp value range, with relatively high permeation rates (5.2-26 × 10-6 cm/s). In contrast, hESC-RPE cells efficiently restricted the drug flux, and displayed even lower Papp values than those reported for bovine RPE-choroid, with the range of 0.4-32 cm-6/s. Therefore, ARPE19, ARPE19mel, and hfRPE cells failed to form a tight barrier, whereas hESC-RPE cells restricted the drug flux to a similar extent as bovine RPE-choroid. Therefore, hESC-RPE cells are valuable tools in ocular drug discovery.

Functional Characterization of Six SLCO1B1 (OATP1B1) Variants Observed in Finnish Individuals with a Psychotic Disorder.

Variants in the SLCO1B1 (solute carrier organic anion transporter family member 1B1) gene encoding the OATP1B1 (organic anion transporting polypeptide 1B1) protein are associated with altered transporter function that can predispose patients to adverse drug effects with statin treatment. We explored the effect of six rare SLCO1B1 single nucleotide variants (SNVs) occurring in Finnish individuals with a psychotic disorder on expression and functionality of the OATP1B1 protein. The SUPER-Finland study has performed exome sequencing on 9381 individuals with at least one psychotic episode during their lifetime. SLCO1B1 SNVs were annotated with PHRED-scaled combined annotation-dependent (CADD) scores and the Ensembl variant effect predictor. In vitro functionality studies were conducted for the SNVs with a PHRED-scaled CADD score of >10 and predicted to be missense. To estimate possible changes in transport activity caused by the variants, transport of 2',7'-dichlorofluorescein (DCF) in OATP1B1-expressing HEK293 cells was measured. According to the findings, additional tests with rosuvastatin and estrone sulfate were conducted. The amount of OATP1B1 in crude membrane fractions was quantified using a liquid chromatography tandem mass spectrometry-based quantitative targeted absolute proteomics analysis. Six rare missense variants of SLCO1B1 were identified in the study population, located in transmembrane helix 3: c.317T>C (p.106I>T), intracellular loop 2: c.629G>T (p.210G>V), c.633A>G (p.211I>M), c.639T>A (p.213N>L), transmembrane helix 6: 820A>G (p.274I>V), and the C-terminal end: 2005A>C (p.669N>H). Of these variants, SLCO1B1 c.629G>T (p.210G>V) resulted in the loss of in vitro function, abolishing the uptake of DCF, estrone sulfate, and rosuvastatin and reducing the membrane protein expression to 31% of reference OATP1B1. Of the six rare missense variants, SLCO1B1 c.629G>T (p.210G>V) causes a loss of function of OATP1B1 transport in vitro and severely decreases membrane protein abundance. Carriers of SLCO1B1 c.629G>T might be susceptible to altered pharmacokinetics of OATP1B1 substrate drugs and might have increased likelihood of adverse drug effects such as statin-associated musculoskeletal symptoms.

Functional in vitro characterization of SLCO1B1 variants and simulation of the clinical pharmacokinetic impact of impaired OATP1B1 function.

Organic Anion Transporting Polypeptide 1B1 is important to the hepatic elimination and distribution of many drugs. If OATP1B1 function is decreased, it can increase plasma exposure of e.g. several statins leading to increased risk of muscle toxicity. First, we examined the impact of three naturally occurring rare variants and the frequent SLCO1B1 c.388A>G variant on in vitro transport activity with cellular uptake assay using two substrates: 2', 7'-dichlorofluorescein (DCF) and rosuvastatin. Secondly, LC-MS/MS based quantitative targeted absolute proteomics measured the OATP1B1 protein abundance in crude membrane fractions of HEK293 cells over-expressing these single nucleotide variants. Additionally, we simulated the effect of impaired OATP1B1 function on rosuvastatin pharmacokinetics to estimate the need for genotype-guided dosing. R57Q impaired DCF and rosuvastatin transport significantly yet did not change protein expression considerably, while N130D and N151S did not alter activity but increased protein expression. R253Q did not change protein expression but reduced DCF uptake and increased rosuvastatin Km. Based on pharmacokinetic simulations, doses of 30 mg (with 50% OATP1B1 function) and 20 mg (with 0% OATP1B1 function) result in plasma exposure similar to 40 mg dose (with 100% OATP1B1 function). Therefore dose reductions might be considered to avoid increased plasma exposure caused by function-impairing OATP1B1 genetic variants, such as R57Q.

Retraction: Hellinen et al. Drug Flux across RPE Cell Models: The Hunt for an Appropriate Outer Blood-Retinal Barrier Model for Use in Early Drug Discovery. Pharmaceutics 2020, 12, 176.

The published article [...].

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.

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.

The Effect of Single Nucleotide Variations in the Transmembrane Domain of OATP1B1 on in vitro Functionality.

PURPOSE: Organic Anion Transporting Polypeptide 1B1 (OATP1B1) mediates hepatic influx and clearance of many drugs, including statins. The SLCO1B1 gene is highly polymorphic and its function-impairing variants can predispose patients to adverse effects. The effects of rare genetic variants of SLCO1B1 are mainly unexplored. We examined the impact of eight naturally occurring rare variants and the well-known SLCO1B1 c.521C > T (V174A) variant on in vitro transport activity, cellular localization and abundance. METHODS: Transport of rosuvastatin and 2,7-dichlorofluorescein (DCF) in OATP1B1 expressing HEK293 cells was measured to assess changes in activity of the variants. Immunofluorescence and confocal microscopy determined the cellular localization of OATP1B1 and LC-MS/MS based quantitative targeted absolute proteomics analysis quantified the amount of OATP1B1 in crude membrane fractions. RESULTS: All studied variants, with the exception of P336R, reduced protein abundance to varying degree. V174A reduced protein abundance the most, over 90% compared to wild type. Transport function was lost in G76E, V174A, L193R and R580Q variants. R181C decreased activity significantly, while T345M and L543W retained most of wild type OATP1B1 activity. P336R showed increased activity and H575L decreased the transport of DCF significantly, but not of rosuvastatin. Decreased activity was interrelated with lower absolute protein abundance in the studied variants. CONCLUSIONS: Transmembrane helices 2, 4 and 11 appear to be crucial for proper membrane localization and function of OATP1B1. Four of the studied variants were identified as loss-of-function variants and as such could make the individual harboring these variants susceptible to altered pharmacokinetics and adverse effects of substrate drugs.

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.

Biopharmaceutics of Topical Ophthalmic Suspensions: Importance of Viscosity and Particle Size in Ocular Absorption of Indomethacin.

Eye drops of poorly soluble drugs are frequently formulated as suspensions. Bioavailability of suspended drug depends on the retention and dissolution of drug particles in the tear fluid, but these factors are still poorly understood. We investigated seven ocular indomethacin suspensions (experimental suspensions with two particle sizes and three viscosities, one commercial suspension) in physical and biological tests. The median particle size (d50) categories of the experimental suspensions were 0.37-1.33 and 3.12-3.50 µm and their viscosity levels were 1.3, 7.0, and 15 mPa·s. Smaller particle size facilitated ocular absorption of indomethacin to the aqueous humor of albino rabbits. In aqueous humor the AUC values of indomethacin suspensions with different particle sizes, but equal viscosity, differed over a 1.5 to 2.3-fold range. Higher viscosity increased ocular absorption 3.4-4.3-fold for the suspensions with similar particle sizes. Overall, the bioavailability range for the suspensions was about 8-fold. Instillation of larger particles resulted in higher tear fluid AUC values of total indomethacin (suspended and dissolved) as compared to application of smaller particles. Despite these tear fluid AUC values of total indomethacin, instillation of the larger particles resulted in smaller AUC levels of indomethacin in the aqueous humor. This suggests that the small particles yielded higher concentrations of dissolved indomethacin in the tear fluid, thereby leading to improved ocular bioavailability. This new conclusion was supported by ocular pharmacokinetic modeling. Both particle size and viscosity have a significant impact on drug concentrations in the tear fluid and ocular drug bioavailability from topical suspensions. Viscosity and particle size are the key players in the complex interplay of drug retention and dissolution in the tear fluid, thereby defining ocular drug absorption and bioequivalence of ocular suspensions.

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.

The effect of prolyl oligopeptidase inhibitors on alpha-synuclein aggregation and autophagy cannot be predicted by their inhibitory efficacy.

Previous studies have shown that prolyl oligopeptidase (PREP) negatively regulates autophagy and increases the aggregation of alpha-synuclein (αSyn), linking it to the pathophysiology of Parkinson's disease. Our earlier results have revealed that the potent small molecular PREP inhibitor KYP-2047 is able to increase autophagy and decrease dimerization of αSyn but other PREP inhibitors have not been systematically studied for these two protein-protein interaction mediated biological functions of PREP. In this study, we characterized these effects for 12 known PREP inhibitors with IC50-values ranging from 0.2 nM to 1010 nM. We used protein-fragment complementation assay (PCA) to assess αSyn dimerization and Western Blot of microtubule-associated protein light chain 3B II (LC3B-II) and a GFP-LC3-RFP expressing cell line to study autophagy. In addition, we tested selected compounds in a cell-free αSyn aggregation assay, native gel electrophoresis, and determined the compound concentration inside the cell by LC-MS. We found that inhibition of the proteolytic activity of PREP did not predict decreased αSyn dimerization or increased autophagy, and we also confirmed that this result did not simply reflect concentration differences of the compounds inside the cell. Thus, PREP ligands regulate the effect of PREP on autophagy and αSyn aggregation through a conformational stabilization of the enzyme that is not equivalent to inhibiting its proteolytic activity.

Prodrug Approach for Posterior Eye Drug Delivery: Synthesis of Novel Ganciclovir Prodrugs and in Vitro Screening with Cassette Dosing.

Because of poor ocular drug bioavailability, intravitreal injections have become the gold standard for drug delivery to the posterior eye. The prodrug approach can be used for optimizing the biopharmaceutical properties of intravitreal drugs. The preclinical screening of prodrugs' properties, such as hydrolysis and bioconversion, should be conducted in a resource-efficient way for an extensive set of synthesized compounds with validated methods. Our objective was to explore cassette dosing in in vitro prodrug hydrolysis and bioconversion studies in buffer, vitreous, and retinal pigment epithelium (RPE) homogenate for rapid medium-throughput screening. Moreover, our aim was to correlate the prodrug structure with hydrolytic behavior. We synthesized 18 novel ganciclovir prodrugs and first studied their hydrolysis in aqueous buffer and porcine vitreous in vitro with cassette dosing for 35 h. A method for vitreous homogenate pH equilibration to a physiological level by using buffer and incubation under 5% carbon dioxide was validated. The hydrolysis of the prodrugs was evaluated in porcine RPE homogenate in vitro with cassette dosing, and five prodrugs were assayed individually to examine their bioconversion into ganciclovir in RPE after 2 h. Lastly, the prodrugs' binding to melanin was studied in vitro. The prodrugs showed a wide spectrum of hydrolysis rates, ranging from a few percentages to 100% in the vitreous and RPE; in general, hydrolysis in RPE was faster than in vitreous. Prodrugs with long carbon chains and disubstitution showed lability in the tissue homogenates, whereas prodrugs with branched carbon chains and aromatic groups were stable. All five prodrugs chosen for the bioconversion study in RPE were hydrolyzed into ganciclovir, and their hydrolytic behavior matched results from the cassette mix experiment, supporting the cassette mix approach for hydrolysis and bioconversion studies. None of the prodrugs bound highly to melanin (<50% bound). In conclusion, cassette dosing proved useful for the rapid screening of prodrug hydrolysis and bioconversion properties. Analyzing several compounds simultaneously can complicate the analytics, and thus, choosing the compounds of the cassette mix should be done carefully to avoid mutual interference of the compounds with the results. The methodology and results of the work are applicable in ocular drug research and prodrug design.

Drug Flux Across RPE Cell Models: The Hunt for An Appropriate Outer Blood-Retinal Barrier Model for Use in Early Drug Discovery.

The retinal pigment epithelial (RPE) cell monolayer forms the outer blood-retinal barrier and has a crucial role in ocular pharmacokinetics. Although several RPE cell models are available, there have been no systematic comparisons of their barrier properties with respect to drug permeability. We compared the barrier properties of several RPE secondary cell lines (ARPE19, ARPE19mel, and LEPI) and both primary (hfRPE) and stem-cell derived RPE (hESC-RPE) cells by investigating the permeability of nine drugs (aztreonam, ciprofloxacin, dexamethasone, fluconazole, ganciclovir, ketorolac, methotrexate, voriconazole, and quinidine) across cell monolayers. ARPE19, ARPE19mel, and hfRPE cells displayed a narrow Papp value range, with relatively high permeation rates (5.2-26 × 10-6 cm/s. In contrast, hESC-RPE and LEPI cells efficiently restricted the drug flux, and displayed even lower Papp values than those reported for bovine RPE-choroid, with the range of 0.4-32 cm-6/s (hESC-RPE cells) and 0.4-29 × 10-6 cm/s, (LEPI cells). Therefore, ARPE19, ARPE19mel, and hfRPE cells failed to form a tight barrier, whereas hESC-RPE and LEPI cells restricted the drug flux to a similar extent as bovine RPE-choroid. Therefore, LEPI and hESC-RPE cells are valuable tools in ocular drug discovery.