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.

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.

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.

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.

Ocular Intracameral Pharmacokinetics for a Cocktail of Timolol, Betaxolol, and Atenolol in Rabbits.

The mechanisms of drug clearance from the aqueous humor are poorly defined. In this study, a cocktail approach was used to simultaneously determine the pharmacokinetics of three ß-blocker agents after intracameral (ic) injection into the rabbit eyes. Aqueous humor samples were collected and analyzed using LC-MS/MS to determine drug concentrations. Pharmacokinetic parameters were obtained using a compartmental fitting approach, and the estimated clearance, volume of distribution, and half-life values were the following: atenolol (6.44 µL/min, 687 µL, and 73.87 min), timolol (19.30 µL/min, 937 µL, and 33.64 min), and betaxolol (32.20 µL/min, 1421 µL, and 30.58 min). Increased compound lipophilicity (atenolol < timolol < betaxolol) resulted in higher clearance and volume of distributions in the aqueous humor. Clearance of timolol and betaxolol is about 10 times higher than the aqueous humor outflow, demonstrating the importance of other elimination routes (e.g., uptake to iris and ciliary body and subsequent elimination via blood flow).

Intravitreal Pharmacokinetics in Mice: SPECT/CT Imaging and Scaling to Rabbits and Humans.

Preclinical in vivo tests of retinal drug responses are carried out in mice and rats, often after intravitreal injections. However, quantitative pharmacokinetics in the mouse eye is poorly understood. Ocular pharmacokinetics studies are usually done in rabbits. We investigated elimination of three compounds ([99mTc]Tc-pentetate, [111In]In-pentetreotide, [99mTc]Tc-human serum albumin with molecular weights of 510.2 Da, 1506.4 Da, and 66.5 kDa, respectively) from mouse vitreous using imaging with single photon emission computed tomography/computed tomography (SPECT/CT). Increasing molecular weight decreased elimination of the compounds from the mouse eyes. Half-lives of [99mTc]Tc-pentetate, [111In]In-pentetreotide, and [99mTc]Tc-human serum albumin in the mouse eyes were 1.8 ± 0.5 h, 4.3 ± 1.7 h, and 30.0 ± 9.0 h, respectively. These values are 3-12-fold shorter than half-lives of similar compounds in the rabbit vitreous. Dose scaling factors were calculated for mouse-to-rabbit and mouse-to-man translation. They were 27-90 and 38-126, respectively, for intravitreal injections in rabbit and man. We show ocular pharmacokinetic parameters for mice and interspecies scaling factors that may augment ocular drug discovery and development.

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.

Alzheimer's Disease Phenotype or Inflammatory Insult Does Not Alter Function of L-Type Amino Acid Transporter 1 in Mouse Blood-Brain Barrier and Primary Astrocytes.

PURPOSE: The study aim was to evaluate the effect of Alzheimer's disease (AD) and inflammatory insult on the function of L-type amino acid transporter 1 (Lat1) at the mouse blood-brain barrier (BBB) as well as Lat1 function and expression in mouse primary astrocytes. METHODS: The Lat1 function and expression was determined in wildtype astrocytes with and without lipopolysaccharide (LPS)-induced inflammation and in LPS treated AD APP/PS1 transgenic astrocytes. The function of Lat1 at the BBB was evaluated in wildtype mice with and without LPS-induced neuroinflammation and APP/PS1 transgenic mice by in situ brain perfusion. RESULTS: There were 2.1 and 1.6 -fold decreases in Lat1 mRNA and protein expression in LPS-treated wildtype astrocytes compared to vehicle-treated astrocytes. In contrast, Lat1 mRNA and protein expression were increased by 1.7 and 1.2 -fold (not statistically significant) in the transgenic cells. A similar trend was observed in the cell uptake of [14C]-L-leucine. There were no statistically significant differences in [14C]-L-leucine BBB permeation between the groups. CONCLUSIONS: The results showed that neither LPS-induced inflammation or the presence of APP/PS1 mutations alters Lat1 function at the mouse BBB as well as Lat1 protein expression and function in mouse primary astrocytes.

Disease-Induced Alterations in Brain Drug Transporters in Animal Models of Alzheimer's Disease : Theme: Drug Discovery, Development and Delivery in Alzheimer's Disease Guest Editor: Davide Brambilla.

PURPOSE: Alzheimer's disease (AD) may disturb functions of the blood-brain barrier and change the disposition of drugs to the brain. This study assessed the disease-induced changes in drug transporters in the brain capillaries of transgenic AD mice. METHODS: Eighteen drug transporters and four tight junction-associated proteins were analyzed by RT-qPCR in cortex, hippocampus and cerebellum tissue samples of 12-16-month-old APdE9, Tg2576 and APP/PS1 transgenic mice and their healthy age-matched controls. In addition, microvessel fractions enriched from 1-3-month-old APdE9 mice were analyzed using RT-qPCR and Western blotting. Brain transport of methotrexate in APdE9 mice was assessed by in vivo microdialysis. RESULTS: The expression profiles of studied genes were similar in brain tissues of AD and control mice. Instead, in the microvessel fraction in APdE9 mice, >2-fold alterations were detected in the expressions of 11 genes but none at the protein level. In control mice strains, >5-fold changes between different brain regions were identified for Slc15a2, Slc22a3 and occludin. Methotrexate distribution into hippocampus of APdE9 mice was faster than in controls. CONCLUSIONS: The expression profile of mice carrying presenilin and amyloid precursor protein mutations is comparable to controls, but clear regional differences exist in the expression of drug transporters in brain.

Prediction of Ocular Drug Distribution from Systemic Blood Circulation.

Systemically circulating drugs may distribute to ocular tissues across the blood-ocular barriers. Ocular distribution is utilized in the treatment of ocular diseases with systemic medications, but ocular delivery of systemic drugs and xenobiotics may also lead to adverse ocular effects. Ocular distribution after systemic drug administration has not been predicted or modeled. In this study, distribution clearance between vitreous and plasma was obtained from a previous QSPR model for clearance of intravitreal drugs. These values were used in a pharmacokinetic simulation model to describe entry of unbound drug from plasma to vitreous. The simulation models predicted ocular distribution of 10 systemic drugs in rabbit eyes within 1.96 mean fold error and the distribution of cefepime from plasma to vitreous in humans. This is the first attempt to predict ocular distribution of systemic drugs. Reliable predictions were obtained using systemic concentrations of unbound drug, computational value of ocular distribution clearance, and a simple pharmacokinetic model. This approach can be used in drug discovery to estimate ocular drug exposure at an early stage.

Intracellular PK/PD Relationships of Free and Liposomal Doxorubicin: Quantitative Analyses and PK/PD Modeling.

Nanomedicines are widely studied for intracellular delivery of cancer drugs. However, the relationship between intracellular drug concentrations and drug responses are poorly understood. In this study, cellular and nuclear concentrations of doxorubicin were quantified with LC/MS after cell exposure with free and liposomal doxorubicin (pH-sensitive and pegylated liposomes). Cellular uptake of pegylated liposomes was low (∼3-fold extracellular concentrations) compared with doxorubicin in free form and pH-sensitive liposomes (up to 280-fold extracellular concentrations) in rat glioma (BT4C) and renal clear cell carcinoma (Caki-2) cells. However, after the cell exposure with pegylated liposomes, intracellular doxorubicin was distributed into the nuclear compartment in both cell types. Despite high drug concentrations in the nuclei, Caki-2 cells showed strong resistance toward doxorubicin. A model was successfully built to describe PK/PD relationship between drug concentrations in nucleus and cytotoxic responses in BT4C cells. This model is the first step to link target site concentration of doxorubicin into its effect and can be a useful part of more comprehensive future in vivo PK/PD models.

Challenges of using in vitro data for modeling P-glycoprotein efflux in the blood-brain barrier.

The efficacy of central nervous system (CNS) drugs may be limited by their poor ability to cross the bloodbrain barrier (BBB). Transporters, such as p-glycoprotein, may affect the distribution of many drugs into the CNS in conjunction with the restricted paracellular pathway of the BBB. It is therefore important to gain information on unbound drug concentrations in the brain in drug development to ensure sufficient drug exposure from plasma at the target site in the CNS. In vitro methods are routinely used in drug development to study passive permeability and p-glycoprotein efflux of new drugs. This review discusses the challenges in the use of in vitro data as input parameters in physiologically based pharmacokinetic (PBPK) models of CNS drug disposition of p-glycoprotein substrates. Experience with quinidine demonstrates the variability in in vitro parameters of passive permeability and active pglycoprotein efflux. Further work is needed to generate parameter values that are independent of the model and assay. This is a prerequisite for reliable predictions of drug concentrations in the brain in vivo.

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.

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.

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.

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.

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.

Prediction of the vitreal half-life of small molecular drug-like compounds.

PURPOSE: To build a fast, user-friendly computational model to predict the intravitreal half-lives of drug-like compounds. METHODS: We used multivariate analysis to build intravitreal half-life models using two data sets, one with experimental data derived from both pigmented and albino rabbits and another including only data from experiments with albino rabbits. RESULTS: The final models had a Q(2) value of 0.65 and 0.75 for the mixed and albino rabbit models, respectively. The models performed well in predicting the intravitreal half-life of an external test set. In addition, the models are physiologically interpretable, containing mainly hydrogen bonding and lipophilicity descriptors. CONCLUSION: The developed models enable reliable predictions of intravitreal half-lives for use in the early drug development stages, without the need for prior experimental data.