TOXICOLOGICAL SCIENCES, 145(2), 2015, 383–395 doi: 10.1093/toxsci/kfv059 Advance Access Publication Date: March 13, 2015 Research Article

Contribution of Membrane Trafficking Perturbation to Retinal Toxicity Su Khoh-Reiter*, Sharon A. Sokolowski†, Bart Jessen*, Mark Evans*, Deepak Dalvie‡, and Shuyan Lu*,1 *Drug Safety Research and Development, Pfizer Inc., San Diego, California, †Drug Safety Research and Development, Pfizer Inc., Groton, Connecticut, and ‡Department of Pharmacokinetics, Dynamics and Metabolism, Pfizer Inc., San Diego, California 1

To whom correspondence should be addressed. Fax: 1-858-678-8290. E-mail: [email protected].

ABSTRACT The retina is a highly structured tissue that is formed by layers containing 7 different cell types. The photoreceptor cell is a specialized type of neuron in the retina that is capable of absorbing and converting light into electrophysiological signals. There is a constant renewal process for photoreceptors consisting of intermittent shedding of the distal tips of the photosensitive outer segment and subsequent phagocytosis (uptake, degradation and recycling) by retinal pigmented epithelial (RPE) cells. This rebuilding process is essential for vision and the survival of photoreceptors and RPE cells. Drugs with a basic moiety have the potential to accumulate in the lysosome and impair its functions including the phagocytosis process, which could hinder clearance of outer segments and ultimately induce retinopathy. To determine the prevalence of this cellular mechanism in retinal toxicity, a collection of proprietary compounds associated with retinal toxicity were subjected to a battery of in vitro tests using the human adult retinal pigmented epithelium cell line, ARPE-19. The tests included a phagocytosis assay, and lysosomal and autophagosomal staining. The compounds that induced retinopathy clustered in the basic and lipophilic region, which drives lysosomal sequestration. This accumulation coincided with phagocytosis inhibition and an increase in autophagosome staining, suggesting a blockage of the membrane trafficking process. A correlation between the physicochemical properties and in vitro lysosomal pathway effects was established. These data reveal the importance of physicochemical properties of compounds and lysosome accumulation as a potential mechanism for drug-induced retinopathy and demonstrate the usefulness of in vitro screening in predicting this liability. Key words: retinal toxicity; lysosomotropic; phagocytosis

The retina is a thin layer of light-sensitive neural tissue that lines the back of the eye. It is highly structured with multiple intricate layers responsible for conversion of visible light into the electrochemical signal interpreted by the brain as vision. The retinal pigmented epithelial (RPE) is a specialized monolayer epithelium that forms the outermost layer of the retina and is positioned between the neuroretina and choroid. RPE performs multiple functions including transport of nutrients, light absorption, and phagocytosis of shed photoreceptor membranes, which are essential for the homeostasis of the neural retina (Bok, 1993). RPE cells are equipped with numerous long microvilli along the apical side facing the photoreceptor outer segment (POS) allowing one RPE cell to support 30–50

photoreceptors, which shed approximately 5% of their outer segment mass daily (Bonilha, 2008). The shed membranes are phagocytosed and digested in phago-lysosomes within the RPE (Strauss, 2005). The RPE constitutes the outer blood-retina barrier while the inner blood-retina barrier is mainly comprised of endothelial cells. Both the vascular endothelium and pigment epithelium possess well-developed tight junctions, which prevent plasma components from freely entering into the retina and control the exchange of metabolites and waste products. The blood-retinal barrier may also prevent many systemically administrated drugs from entering the eye. However, many systemically administered drugs for nonocular conditions have been reported

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to gain access to the eye and trigger various ocular toxicological manifestations (Mecklenburg and Schraermeyer, 2007). Toxic compounds can either affect the photoreceptor or ganglion cells directly or affect the neural retina through effects on the RPE. However, the molecular mechanisms of retinal toxicity are still poorly understood. Lysosomes are membrane-enclosed cytoplasmic organelles filled with acid hydrolytic enzymes (eg, cathepsins) that constitute the primary degradative compartments of cells. For the optimal activity of the acid hydrolases the lysosomal luminal pH needs to be maintained near 5.0. Lysosomes fuse with vesicles from multiple pathways including endocytosis, phagocytosis and autophagy, and digest macromolecules or organelles generated externally and internally. RPE cells, which are among the most phagocytically active animal cells, are responsible for clearing engulfed outer segments of photoreceptors (Young, 1971) and are thus especially susceptible to lysosomal dysfunction. A strong link between lysosomal abnormalities and pathological change in RPE has been demonstrated in lysosomal storage disease (LSD). For instance, in the mouse model of Npc1 and Npc2 (Niemann–Pick disease, type C) mutations, retinal degeneration and modulation of autophagy and lipofuscin accumulation within RPE were observed (Claudepierre et al., 2010). Lysosomal overload and dysfunction in RPE have also been suggested as an early cause of age-related macular degeneration (AMD) (Ramkumar et al., 2010). Lysosomal dysfunction has also been shown to play a role in drug induced retinopathy. Quinoline derivatives, such as chloroquine and hydroxychloroquine, are used to treat malarial and autoimmune diseases and have been demonstrated to affect the retina in humans as well as in various animal models (Abraham and Hendy, 1970; Gregory et al., 1970; Kazi et al., 2013; Rosenthal et al., 1978). Chloroquine is considered a lysosomotropic agent due to its lysosomal accumulation by way of pH partitioning which is driven by its basic and lipophilic characteristics (Reijngoud and Tager, 1976). The increase in lysosomal pH and perturbation of membrane trafficking associated with lysosomal accumulation of chloroquine are likely related to chloroquine induced vacuole formation, lipid accumulation, and decreased dextran uptake (indicating lysosomal dysfunction) in ARPE-19 cells(Chen et al., 2011). In order to understand whether lysosomal dysfunction could play a role in retinopathy induced by other experimental drugs, we identified 12 proprietary compounds that have been associated with retinal lesions in vivo (defined as positive retinal toxicants) and 8 proprietary compounds that were not toxic to the retina as negative controls. We also included chloroquine, a classic lysosomotropic compounds associated with retinopathy (Mahon et al., 2004) in the in vitro studies as our positive control. Strikingly, all of positive compounds including chloroquine are basic lipophilic and the majority of them increase lysosomal mass, and perturb the autophagy and phagocytosis processes, whereas most of negative compounds had no findings for those endpoints. We thus propose a novel concept of lysosomotropism as a potential general mechanism for drug induced retinopathy by basic lipophilic compounds.

MATERIALS AND METHODS Test Compounds All proprietary compounds were synthesized internally and were obtained from Pfizer’s central raw materials group. Twelve proprietary compounds that have been associated with retinal

lesions in animals studies (positive retinal toxicants) and 8 proprietary compounds that did not cause retina toxicity in animal studies (negative controls) were identified for the study. Dimethylsulfoxide (DMSO), chloroquine and bafilomycin A (BFA) were purchased from Sigma Aldrich (St. Louis, Missouri). In vivo Studies Male Sprague-Dawley rats from Charles River Laboratories International Inc. (Wilmington, Massachusetts) were used for the in vivo retinal studies. Rats were housed in an environmentally controlled room on a 12-h light/12-h dark cycle and provided ad libitum Rodent Diet 5001 and water that was purified by reverse osmosis. The studies were conducted in accordance with the current guidelines for animal welfare (as amended in 1970, 1976, and 1985, and 1990, and the Animal Welfare Act implementing regulations in title 9, CFR chapter 1, subchapter A, parts 1–3). The procedures used in this study were reviewed and approved by the Institutional Animal Care and Use Committee. Rats (5 rats per group) were orally gavaged with vehicle control and proprietary compounds (100–500 mg/kg) for 7–14 consecutive days. Upon necropsy the eyes were preserved by immersion in 10% neutral-buffered formalin. After trimming, the eye samples were embedded in paraffin, sectioned, and stained with hematoxylin and eosin. These eye sections were microscopically analyzed by a Diplomate of the American College of Veterinary Pathologists. Cell Culture ARPE-19 cells (CRL-2302) were purchased from American Type Culture Collection (Manassas, Virginia). These cells were maintained in DMEM: F12 (Life Technologies, Carlsbad, California) media supplemented with 10% (v/v) heat-inactivated FBS, 100 U/ml of Penicillin/Streptomycin, and 2 mM L-Glutamine (Thermo Fisher Scientific, Waltham, Massachusetts) at 37 C in a humidified incubator with 5% CO2. LysoTracker Red Measurement ARPE-19 cells were seeded in 96-well plates at 4000 cells/well and allowed to attach overnight. Cells were treated with the compounds of interest at concentrations of 100.0, 33.3, 11.1, 3.7, 1.2, 3.1, and 1.5 mM in triplicate wells for 24 h. The final DMSO concentration was 0.5% in vehicle control wells and compound treated wells. Cells were then stained with LysoTracker Red (LTR) probes (Life Technologies, Carlsbad, California) containing 60 nM LTR and 5 mg/ml Hoechst 33342 (Life Technologies, Carlsbad, California). The resultant images were captured and quantified using Thermo Fisher Scientific ArrayScan VTI (Thermo Fisher Scientific, Waltham, Massachusetts). The result is calculated as fold increase over vehicle control. Phagocytosis Evaluation Seeding and treatment of the ARPE-19 cells were similar to that with LTR. For the phagocytosis assay, pHrodo Escherichia Coli BioParticles Conjugate from Life Technologies (Carlsbad, California) was used. ARPE-19 cells were treated with compounds for 24 h. Uptake buffer (HBSS (Hank’s Balanced Salt Solution) with calcium, magnesium and 20 mM Hepes) (Life Technologies, Carlsbad, California) was added to a vial of pHrodo Red E. Coli Bioparticle conjugates and sonicated for 5 min. The prepared suspension was added to each well and incubated in the dark for 3 h in a humidified incubator for 37 C in the absence of CO2. In the last 20 min of incubation, the cells were counterstained with Hoechst dye at a final concentration

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of 5 lg/ml. Cells were rinsed in HBSS several times before microscopic observation. LC3 Assessment Seeding and treatment of the ARPE-19 cells were similar to that with LTR. The assessment was conducted using the LC3B (an autophagosome marker for monitoring autophagy) Detection Kit from Thermo Fisher Scientific (Waltham, Massachusetts) following manufacturer’s protocol. Briefly, cells were fixed with 4% paraformaldehyde for 15 min. Primary antibody was added for 1 h at room temperature after initial permeabilization and blocking step. Cells were washed twice in 150 ml of dPBS (Life Technologies, Carlsbad, California) prior to the addition of secondary antibody, Dylight 488 goat anti-rabbit and nuclear counterstain, Hoechst dye. The cells were visualized after several washes in 150 ml of dPBS. Image capture and quantification. Culture plates from LTR, LC3 and phagocytosis assessment were read in an Array Scan VTI automated fluorescence imager (Thermo Fisher Scientific, Waltham, Massachusetts). Cells were photographed using a 20  objective in the Hoechst and Tritc (XF-93) channels for LTR and phagocytosis evaluation. LC3 signal were captured in the FITC (XF-93) channel. Quantification of LTR fluorescence intensity was conducted using Target Activation algorithm and LC3 with Spot Detector algorithm. Up to 250 cells from 6 fields was used for the analysis. For phagocytosis multiple images was visually inspected to observe the difference between control and treated wells. SQSTM1/p62 Evaluation SQSTM1/p62, as a substrate for autophagy degradation, is commonly used as a marker for autophagic flux. ARPE-19 cells were seeded at 7  104 cells in 6 well, tissue culture treated, flat bottom plates (Corning, Corning, New York) and incubated overnight prior to dosing. Selected compounds (30 mM for proprietary compounds and 25 nM for BFA) were added into wells. Twenty 4 h after compound addition, cells were lysed using 500 ml freshly prepared complete lysis buffer comprised of the following; RIPA Cell Lysis Buffer with 1  Halt protease and phosphatase inhibitors (Thermo Fisher Scientific, Waltham, Massachusetts). Protein concentrations were determined using bicinchoninic acid protein assay (Thermo Fisher Scientific, Waltham, Massachusetts). Approximately 20 mg of protein was resolved in a NuPAGE Bis-Tris Gel (Thermo Fisher Scientific, Waltham, Massachusetts) by electrophoresis. Proteins were transferred to a 0.45 mm nitrocellulose membrane and blocked with blocking buffer for fluorescent western blotting (Rockland, Limerick, Pennsylvania), then incubated overnight at 4 C with SQSTM1/p62 antibody (Cell Signaling, Danvers, Massachusetts). The human b-actin antibody (Santa Cruz Biotechnology, Santa Cruz, California) was used as loading control. After 3 washes with 1  TBS Tween-20 buffer (KPL, Gaithersburg, Maryland), the membranes were probed with secondary infrared antibodies in 1  TBS Tween-20 buffer for 1 h, and the signal was visualized using Odyssey infrared scanner (Li-Cor Biosciences, Lincoln, Nebraska). in vitro Metabolism of CP-346086 CP-346086 (10 mM), was incubated with human liver S-9 fractions (protein concentration 2.5 mg/mL) (BioreclamationIVT, Westbury, New York), MgCl2 (3.3 mM), in the presence of NADPH (3.0 mM) in a total volume of 1.0 ml potassium phosphate (0.1 M, pH 7.4). Incubations were started by addition of NADPH and

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shaken in a water bath set at 37 C. Control experiments were carried out in a similar manner except that the NADPH solution was substituted with phosphate buffer. After 1 h, the incubations were quenched with acetonitrile (5 ml), centrifuged and the supernatant was evaporated to dryness in a Turbo-Vap under nitrogen. The residue was reconstituted in 200 ml of acetonitrile and water mixture (1:3) and 25 ml aliquot was injected onto the column for the identification of the oxidation products using LC-MS/MS. Statistical Analysis of Data The dose-response relationship in LTR and LC3 staining was analyzed using GraphPad Prism software (GraphPad, La Jolla, California). Experimental data were subjected to 1-way analysis of variance analysis (ANOVA) with Dunnett’s post hoc test.

RESULTS Compound Selection The purpose of the study was to determine whether lysosomal dysfunction could contribute to chemical-induced retinopathy. Twelve proprietary compounds that have been associated with retinal lesions in animals studies (positive retinal toxicants) and 8 proprietary compounds that did not cause retina toxicity in animal studies (negative controls) were identified for the study. Animals treated with negative control compounds showed regular retinal layers with normal appearing RPE, whereas retinal lesions by the positive compounds were characterized by disrupted RPE with multiple, round-to-oval vacuoles of variable size in RPE cytoplasm (Fig. 1). The twelve positive compounds listed in Table 1 causes a very similar histopathology finding as that demonstrated in Figure 1 after 7–14 days of oral administration of 100–500 mg/kg and are hereafter referred to as retinal positive. The remaining 8 negative compounds were identified from different discontinued drug development programs that did not demonstrate any retinal toxicity findings following similar in vivo toxicity assessment, and are hereafter referred to as retinal negative. The physicochemical properties including clogP (the calculated partition coefficient of the neutral species of the compound between octanol and water) and basic pKa (the logarithm of the dissociation constant of the most basic center of the compound) were analyzed for all compounds. Remarkably, all positive compounds carry a basic moiety with basic pKas ranging from 6.02 to 10.01 and are lipophilic with clogP spanning from 3.64 to 7.0, whereas most of the negative compounds do not meet both criteria (basic and lipophilic). The physicochemical property difference is visibly demonstrated by the scatter plot in Figure 2, where positive compounds all clustered in upper right quadrant (basic and lipophilic). Lysosomal Staining A fluorescent dye, LTR DND-99, which has been reported to accumulate in lysosomes (Lemieux et al., 2004) was used to study the lysosomal change with the compound treatment. The LTR analysis was automated using a high-content screening instrumentation programmed to detect and quantify the LTR signal. After 24 h exposure ranging from 100 to 3.6 mM, an increase of LTR staining was observed by visual inspection and representative images from CP-237228, CP-237916, and positive control chloroquine are shown in Figure 3D–F. However, retinal negative compounds did not show any obvious LTR difference compared with control well, exemplified by CP-651802 and CP470711 (Fig. 3B and C). Quantitative LTR fluorescence intensity

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FIG. 1. H and E staining of representative retinal tissue sections from control rats (A) and from rats exposed to CP-237228 for 14 days (B). A, Control tissue shows normal retinal layers. B, After exposure to CP-237228 for 14 days, retinal injury included disrupted RPE by multiple, round-to-oval vacuoles of variable size in RPE cytoplasm (arrow). Magnification: 40.

of treated wells was employed to calculate the fold difference in the LTR intensity from control samples. All of the positive compounds exhibited concentration-dependent activity ranging from 3- to 10-fold greater LTR staining at their peak concentrations (Fig. 3G). In addition, these compounds induced 2-fold LTR intensity increase at several concentrations (LTR positive), with the exception of CP-346086 with a 2-fold increase of LTR staining at only a single concentration (LTR weak positive). In contrast, 5 out of 8 negative compounds did not trigger an increase in LTR staining at the tested concentrations or the increase was 2-fold increase in LTR staining at 100 mM (LTR weak positive). However, the negative compound CP655066 is the exception, with increased LTR staining >2-fold at the highest 3 concentrations (LTR positive). In summary, all retinal positive compounds were indeed either positive or weak positive for LTR and 6 out of 8 retinal negative compounds have negative response for LTR (Table 2).

Phagocytosis Assessment The phagocytic function of RPE is critical to the maintenance of retinal health. Ready-made pHrodo E. coli BioParticles conjugates were employed to study the effect of the test compounds on phagocytosis. Cells from vehicle control treated conditions demonstrate bright punctate staining in the cytoplasm (Fig. 4A). Two retinal negative compounds CP-650812 and CP-470711 (Fig. 4B and C), had very similar staining pattern as the vehicle control, which is defined as phagocytosis negative. In contrast, retina positive compounds have a strikingly different staining pattern, characterized by diffuse cytoplasmic staining as shown for CP-237228, CP-237916, and chloroquine (Fig. 4D–F). Because the fluorescence of the novel pHrodo dye dramatically increases as pH decreases from neutral to acidic, the diffuse weak staining pattern potentially indicates a higher pH of the phagosome, which is presumably due to either disturbed fusion of phagosome and lysosome, or defective acidification of the phagosome. The compounds with diffuse weak staining are classified

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TABLE 1. Physicochemical Properties, SMILES String and in vivo Retinal Finding for the Tested Compounds Compound

SMILES (Simplified Molecular-Input Line-Entry System)

CP-138973

CCN(CC)C1CCN(CC1)C(¼O)C[C@@H](Cc2ccccc2)C(¼O)N[C@@H] (CSC)C(¼O)N[C@@H](CC3CCC(CC3)(F)F)[C@H]([C@H](CC(C)C)O)O CC(C)OC(¼O)[C@@H]([C@H](CC1CCCCC1)NC(¼O)[C@H](CSC)NC(¼O) [C@H](Cc2ccccc2)CC(¼O)N3CCC(CC3)NC)O c1ccc(c(c1)c2ccc(cc2)C(F)(F)F)C(¼O)Nc3ccc4c(c3)CCNC4 CCN(C)C1CCN(CC1)C(¼O)O[C@@H](Cc2ccccc2)C(¼O)N[C@@H](CSC)C(¼O)N [C@@H](CC3CCC(CC3)(F)F)[C@H]([C@H](CC(C)C)O)O CCC[C@@H](C(¼O)N[C@@H](CC1CCCCC1)[C@H]([C@H](CCC(F)(F)F)O)O)NC (¼O)[C@H](Cc2ccccc2)CC(¼O)N3CCC(CC3)N(C)C COC(¼O)NCCN1CCc2cc(ccc2C1)NC(¼O)c3ccccc3c4ccc(cc4)C(F)(F)F CN(C)[C@H]1[C@H]2[C@@H]1CN(C2)C(¼O)C[C@@H](Cc3ccccc3)C(¼O)N[C@@H] (CSC)C(¼O)N[C@@H](CC4CCCCC4)[C@H]([C@H](CCC(F)(F)F)O)O CCC[C@@H](C(¼O)N[C@H](CC1CCCCC1)C([C@@H](CCC(F)(F)F)O)O)NC(¼O)[C@H] (Cc2ccccc2)CC(¼O)N3C[C@@H]4[C@H](C3)[C@H]4N(C)C CC(C)C[C@@H]([C@@H]([C@H](CC1CCC(CC1)(F)F)NC(¼O)[C@H](CSC)NC(¼O)[C@H] (Cc2ccccc2)CC(¼O)N3CCC(CC3)N4CCOCC4)O)O c1ccc(c(c1)c2ccc(cc2)C(F)(F)F)C(¼O)Nc3ccc4c(c3)CCN(C4)Cc5[nH]ccn5 CN1CCN(CC1)S(¼O)(¼O)C[C@@H](Cc2ccccc2)C(¼O)N[C@@H](CSC)C(¼O)N[C@@H] (CC3CCCCC3)[C@H]([C@H](CCC(F)(F)F)O)O c1ccc(c(c1)c2ccc(cc2)C(F)(F)F)C(¼O)Nc3ccc4c(c3)CCN(C4)Cc5nc[nH]n5 c1cc(c(cc1Oc2ccc(cc2C3CCCN3)F)Cl)Cl CC[C@@H](CO)Nc1c2c(cn(c2nc(n1)C)c3c(cc(cc3C)Br)C)C COC(¼O)Cc1ccc(cc1)OCCNC[C@@H](c2ccc(nc2)N)O CC(C)(C)CN1c2ccc(cc2[C@H](S[C@@H](C1¼O)CC(¼O)N3CCC(CC3)C(¼O)O)c4cccc5c4cccc5)Cl CC(C)c1cc(cc(c1NC(¼O)NS(¼O)(¼O)c2cccc(c2)C(C)(C)O)C(C)C)Cl c1cc(sc1C(¼O)C2c3cc(c(cc3N(C2¼O)C(¼O)N)Cl)F)F CC1CN(CC(N1c2ccnc(n2)[C@@H](C)O)C)c3ccnc(n3)[C@@H](C)O c1ccc(cc1)[C@@H](c2ccccn2)NC(¼O)c3cc4ccc(cc4nc3)NC(¼O)c5ccccc5c6ccc(cc6)C(F)(F)F

CP-108671 CP-237228 CP-144470 CP-237916 CP-467688 CP-230813 CP-239971 CP-133333 CP-319340 CP-162765 CP-346086 CP-655066 CP-247106 CP-331683 CP-340868 CP-424174 CP-449122 CP-470711 CP-651802

Basic pKa

cLogP

In vivo Retinal Finding

10.0

5.6

POS

10.0

3.6

POS

9.4 9.3

4.5 5.9

POS POS

9.1

4.8

POS

8.1 8.1

5.0 6.5

POS POS

8.1

7.0

POS

7.4

4.6

POS

7.0 6.2

4.7 5.5

POS POS

6.0 10.1 6.3 7.8 0.0 0.0 0.0 7.5 3.7

4.5 5.5 6.2 0.5 5.8 6.0 1.9 20.3 6.8

POS NEG NEG NEG NEG NEG NEG NEG NEG

Note: POS, positive; NEG, negative.

FIG. 2. Correlation of physicochemical properties with retinal toxicity. A scatter plot showing the distribution of compounds associated with retinal toxicity (red circle) and compounds without retinal toxicity (green circle) within the clogP-basic pKa physicochemical property space. Compounds with retinal toxicity are clustered within the basic lipophilic area with basic pKa ranging from 6.0 to 10.0 and clogP spanning from 3.6 to 7.0. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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FIG. 3. Lysosomal assessment by LTR staining. ARPE-19 cells were treated with compounds for 24 h. LTR staining was conducted to monitor the lysosomal change. Representative LTR images from A, control; B, CP-651802 100 mM; C, CP-470711 100 mM; D, CP-237228 33 mM; E, CP-237916 33 mM; and F, Chloroquine 33 mM are shown. Concentration response of LTR change after quantitative image analysis was plotted for retinal positive compounds (G) and retinal negative compounds (H). The fold change of LTR intensity compared with control was plotted for each compound and data are expressed as mean 6 SD from a representative experiment (the assay was repeated twice). For each compound the concentration range tested is from 100 mM (left) to 3.6 mM (right). Dotted line depicts 2-fold change of LTR intensity. One-way ANOVA with Dunnett’s post test was conducted for statistical analysis comparing to DMSO control (*P < .05; **P < .01; ***P < .001)

as phagocytosis positive. The phagocytosis result for each tested compound was visually evaluated and summarized in Table 2. Eleven out of 12 retinal positive compounds were positive in the phagocytosis assay while the 12th compound, CP346086, was phagocytosis negative. Six out of 8 retinal negative compounds were negative with phagocytosis assay; the

exceptions being CP-655066 and CP-247106, which were phagocytosis positive. Autophagy Evaluation Autophagy is an evolutionarily conserved self-catabolizing process by which cytoplasmic components including

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TABLE 2. Correlation of in vivo and in vitro Findings Compound

in vivo Retinal Finding

Phagocytosis

LTR

LC3

Chloroquine CP-138973 CP-108671 CP-237228 CP-144470 CP-237916 CP-467688 CP-230813 CP-239971 CP-133333 CP-319340 CP-162765 CP-346086 CP-655066 CP-247106 CP-331683 CP-340868 CP-424174 CP-449122 CP-470711 CP-651802

POS(Mahon, Anderson, Gardiner, et al., 2004) POS POS POS POS POS POS POS POS POS POS POS POS NEG NEG NEG NEG NEG NEG NEG NEG

POS POS POS POS POS POS POS POS POS POS POS POS NEG POS POS NEG NEG NEG NEG NEG NEG

POS POS POS POS POS POS POS POS POS POS POS POS wPOS POS wPOS NEG NEG NEG NEG NEG NEG

POS POS POS POS POS POS POS POS POS POS POS POS POS POS wPOS NEG POS NEG wPOS NEG NEG

Note. POS, positive; NEG, negative; wPOS, weak positive. Pearson correlation between in vivo and in vitro result was analyzed. There is a statistically significant relationship between in vivo retinal finding with phagocytosis (r ¼ 0.69 and P < .01), LTR (r ¼ 0.81 and P < .01) and LC3 (r ¼ 0.62 and P < .01).

FIG. 4. Phagocytosis modulation by the compounds. ARPE-19 cells were treated with compounds for 24 h. pHrodo E. Coli BioParticles were used to evaluate phagocytosis alterations. Representative phagocytosis images from A, control; B, CP-651802 33 mM, C, CP-470711 100 mM; D, CP-237228 11 mM; E, CP-237916 11 mM; and F, Chloroquine 33 mM are shown. The assay was repeated twice.

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FIG. 5. Autophagy modulated by tested compounds. ARPE-19 cells were treated with compounds for 24 h. LC3 immunofluorescence staining was conducted to monitor autophagy change. Representative LC3 images from A, control; B, CP-650812 100 mM; C, CP-470711 100 mM; D, CP-237228 33 mM; E, CP-237916 33 mM; and F, Chloroquine 33 mM are shown. Concentration response of LC3 change after quantitative image analysis was plotted for retinal positive compounds (G) and retinal negative compounds (H). The fold change of LC3 intensity compared with control was plotted for each compound and data are expressed as mean 6 SD from a representative experiment (the assay was repeated twice). For each compound the concentration range tested is from 100 mM (left) to 3.6 mM (right). Dotted line depicts 2-fold change of LC3 intensity. One-way ANOVA with Dunnett’s post test was conducted for statistical analysis comparing to DMSO control (*P < .05; **P < .01; ***P < .001).

macromolecules (eg, long-lived proteins) and organelles (eg, mitochondria), are delivered to lysosomes and degraded (Doria et al., 2013). As a hallmark morphological feature of this dynamic process, double-membrane-bound autophagosomes go through a maturation process to sequester various substrates and fuse with lysosomes to form autolysosomes. When the autophagosome is formed, the cytosolic Atg 8 protein, also known as LC3, is recruited to the membrane of nascent autophagosomes and controls autophagosome expansion. LC3 is the most widely monitored autophagy-related protein (Klionsky

et al., 2012). RPE cells have a relatively high rate of autophagy, the dysregulation of which can lead to the development of AMD (Kaarniranta et al., 2013). To assess whether the selected compounds have any effect on autophagy, localized levels of LC3 were evaluated in ARPE-19 using immunofluorescent staining. Visual inspection of images revealed a lack of LC3 puncta in the negative control well and cells treated with retinal negative compounds CP-651802 and CP-470711 (Fig. 5A–C), while an apparent accumulation of LC3 puncta was observed in the cells treated with positive compounds such as CP-237228, CP-237916,

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and chloroquine (Fig. 5D–F). The LC3 signal was quantified and comparisons were made between compound and vehicle control-treated conditions. All retinal positive compounds exhibited concentration-dependent peak activity ranging from 16- to 91-fold greater punctate LC3 fluorescence relative to the vehicle control (Fig. 5G). In addition, all positive compounds showed a >2-fold increase of LC3 staining at multiple concentrations (LC3 positive). As shown on Figure 5H, although 4 out of the 8 retinal negative compounds did not increase LC3 more than 2-fold (LC3 negative), CP-655066 and CP-340868 induced >2-fold increase of LC3 relative to the vehicle control (LC3 positive). In addition, CP247106 and CP-449122 had only slightly more than 2-fold increase of LC3 at the highest concentration tested (LC3 weak positive). The LC3 data is summarized in Table 2. SQSTM1/p62 Assessment A steady-state increase in the number of endogenous LC3 puncta does not necessarily reflect a stimulation of autophagic activity as treatment with downstream blockers of the autophagy process can produce very similar results. To determine whether the increase of LC3 puncta is indeed due to downstream suppression of autophagy, we evaluated the change of abundance of SQSTM1/p62 with compound treatment. The SQSTM1/p62 (hereafter p62) is a multifunctional protein that links LC3 to polyubiquitinated substrates during autophagosome formation and is primarily degraded in autolysosomes (Ichimura et al., 2008). Therefore, the abundance of p62 protein could be used as a representative marker for the autophagic flux; induction of autophagy leads to decreased p62 abundance, whereas inhibition of autophagy correlates with increased levels of p62. In addition to the vehicle control and positive control (BFA), a selected set of the compounds were tested for their effects on p62 protein levels using western-blotting. In the vehicle control cells, there was relatively low expression of p62 while BFA, which is a known autophagy inhibitor, increased p62 level by approximately 10-fold (Fig. 6). Retinal positive compounds CP237228, CP-237916, CP-138973, CP-108671, and CP-239971 showed at least a 5-fold increase in p62 expression compared with the vehicle control, indicating LC3 increase by these compounds are attributed to the autophagy inhibition. One retinal positive outlier, CP-346086, did not cause substantial increase of p62, while the retinal negative compound, CP-449122, that was weakly positive for LC3, did not trigger a substantial change in p62 expression as well. Correlation of Physicochemical Properties, in vitro Data and Histopathology The physicochemical properties, in vivo retinal findings, and summary of LTR, phagocytosis and autophagy data for the test compounds are summarized in Tables 1 and 2. All of the compounds that have basic pKa >6 are LTR positive except CP-331683 and CP-470711, which have a low clogP value (0.52 and 0.26, respectively). Similarly, all of the compounds that were positive for LTR were also LC3 positive and phagocytosis positive with the exception of CP-346086. The Table 2 listings also reveal the association between the in vitro and in vivo findings. Eleven out of twelve retinal positive compounds were positive for phagocytosis, LTR and LC3 endpoints with the exception of CP-346086, which is LC3 positive, but weak LTR positive and phagocytosis negative. Four out of 8 retinal negative compounds are negative for all in vitro endpoints tested. CP-340868 and CP-449122 only have 1 endpoint (LC3) as positive or weak positive respectively. Two retinal negative compounds,

FIG. 6. Effects on SQSTM1/p62 with compound treatment. ARPE19 cells were incubated with compounds for 24 h (30 mM for proprietary compounds and 25 nM for BFA). Total lysates were prepared, and western blots were probed with a specific anti-SQSTM1/p62 antibody. (A) b-actin probing served as a loading control. The bar graph (B) shows p62 quantification normalized against the b-actin level for each sample.

CP-655066 and CP-247106, are positive for all 3 endpoints, although CP-247106 has weak positive finding for LTR and LC3. Pearson correlation coefficient analysis (Table 2) established the statistically significant correlation between in vivo retinal finding and all 3 individual in vitro endpoints. Identification of Metabolites With less optimal physicochemical properties for lysosomal accumulation, CP-346086 (basic pKa 6.0) was shown to induce retinal lesion in vivo (Table 1). Additionally in the in vitro screening assays CP-346086 was only positive for LC3 (Table 2). To assess if metabolism could play a role in the in vivo retinal finding, assessment of metabolites in the rats was performed by incubating CP-346086 with rat liver S9. Mass spectral analysis of the incubation mixture revealed 4 products (Fig. 7). These were tentatively identified as the N-oxide of CP-346086 (M1), N-dealkylated metabolite (M2), the dihydroquinolinium (M3), and the quinolinium metabolite (M4). The formation of M3 and M4 was consistent with previous reports on tetrahydroisoquinoline (or its congener) containing compounds (Dalvie and O’Connell, 2004; Obach and Dalvie, 2006). M2, which is CP-237228, has a higher basic pKa of 9.4 compared with CP-346086. As shown previously CP-237228 had positive findings in vivo and in all in vitro endpoints (Table 2), therefore, the in vivo retina finding by CP346086 is likely driven by its metabolite CP-237228.

DISCUSSION The aim of this study was to determine if lysosome dysfunction plays a role in chemical-induced retinopathy. Strikingly, the analysis of the physicochemical properties of the test compounds demonstrated a strong relationship between basicity and lipophilicity, and the formation of retinal lesions (Fig. 2) This is consistent with previous observations that other basic

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FIG. 7. An overview of the main metabolic routes of CP-346086. Multiple metabolites including CP-237228 were produced from CP-346086.

lipophilic compounds, including chloroquine, tamoxifen, amiodarone, clomipramine, and imipramine (Lullmann-Rauch, 1976), cause retinal lipidosis. However, the functional implications of this retinal epithelial alteration remain to be elucidated. In this study, the 20 test compounds and positive control chloroquine were thoroughly evaluated for their effects on the lysosome and membrane trafficking processes. All compounds that increase LTR carry a basic moiety and are also lipophilic (Table 1). This association is consistent with previous findings which showed that basic lipophilic compounds increase LTR following 24–48 h of treatment in primary hepatocytes (Logan et al., 2014; Lu et al., 2012). The increase in lysosomal staining is likely related to the tendency of these compounds to accumulate in lysosomes by pH partitioning, thus deriving the designation lysosomotropic. Interestingly, both compounds that are weakly positive for LTR (CP-346086 and CP-247106), have basic pKas around 6, in comparison to all other LTR positive compounds with basic pKa >7. In addition, 2 basic compounds with clogP 0.5 or lower (CP-331683 and CP-470711) are negative for LTR endpoint. It has been shown that both properties contribute to the lysosomal accumulation (de Duve et al., 1974). Generally, clogP is associated with compound’s permeability and determines the kinetics of compound trapping whereas the basic pKa influences the extent of accumulation. It was shown that lysosomal concentration increased when basic pKa increased from 4 to 9 (Kaufmann and Krise, 2007), and basic pKa of 8 has been proposed to be optimal for compound accumulation (de Duve et al., 1974). Possibly, the negative LTR result by CP-331683 and CP-470711 is attributed to low clogP whereas CP-346086 and CP247106 may be less potent due to their non-optimal basic pKa.

Although the LTR signal is not an exact measure of the collective size of the lysosomes, it is often used as a surrogate marker for lysosomal mass (Funk and Krise, 2012; Logan et al., 2014). Interestingly an increase of LTR is often associated with cells with lysosome dysfunction. LSDs are a group of approximately 50 genetic diseases and lysosomal dysfunction associated with LSD is caused by mutations in genes encoding lysosomal digestion enzymes and lysosomal membrane proteins involved in trafficking of cellular macromolecules. An increase of LTR staining has been noted in the fibroblasts derived from patients from multiple LSDs such as neuronal ceroid lipofuscinoses, mucolipidosis type VI, and NPC (Cao et al., 2011; Curcio-Morelli et al., 2010; Xu et al., 2012). The increase of LTR observed in LSDs and with compounds in our study is likely a compensatory attempt to overcome lysosomal dysfunction. Notably, decrease of LTR has also been used as a phenotypic screen to identify compounds that restore lysosomal function (Xu et al., 2014). A decrease in the efficiency of lysosomal egress (trafficking of cargo out of lysosomes) has also been observed with the treatment of basic lipophilic compounds (Logan et al., 2013), and could contribute to the increase of LTR staining. In addition, recent data demonstrated that various lysosome stressors including chloroquine, has been shown to induce a striking nuclear accumulation of TFEB (Settembre et al., 2012), which is a master transcription regulator for lysosome biogenesis. Possibly, basic lipophilic compounds in this study can also activate TFEB and increase lysosome size and number (influx enhancement). This notion of lysosomal dysfunction by the LTR positive compounds was further supported by the membrane trafficking assessment including autophagy and phagocytosis. Although not

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completely concordant, the majority of compounds that increased LTR also decreased phagocytosis and increased LC3 staining. The increase of p62 further indicates that the accumulation of LC3 puncta is due to downstream inhibition rather than upstream enhancement since an increase of p62 is typically associated with autophagy inhibition. Phagocytosis inhibition has been seen for other lysosomotropic compounds, including lidocaine and diphenylamine using polymorphonuclear leucocytes as a model (Vandenbroucke-Grauls et al., 1984). Autophagy inhibition by the compounds tested in the current study agrees with autophagy modulation reports for other basic lipophilic compound treatment (Ashoor et al., 2013). The lack of effects on phagocytosis with the weakly LTR positive compound CP-346086 could be related to the less optimal pKa for accumulation. Multiple mechanisms potentially play a part in the lysosome dysfunction by these basic lipophilic compounds. Many lysosomotropic compounds, including chloroquine, have been shown to increase lysosomal pH dramatically (Chen et al., 2011; Nadanaciva et al., 2011). This pH increase could decrease lysosomal degradation capability, since acidic pH is optimal for all acid hydrolases in the lysosomal lumen (Kawai et al., 2007). However, the increase of LTR at 24 h requires an acidic environment for optimal staining and therefore does not indicate an increase of lysosomal luminal pH, suggesting pH might not be a critical factor in decreasing lysosomal function. Recently, it was demonstrated that multiple lysosomotropic compounds can redistribute the mannose 6-phosphate receptor from the transGolgi network to endosomes and concomitantly increase the secretion of lysosomal enzymes, resulting in a decline of intracellular lysosomal enzyme levels (Ikeda et al., 2008), which could contribute to the decrease of lysosomal function. In addition, the activity of lysosomal enzymes, such as acid sphingomyelinase, has been shown to be directly inhibited by some lysosomotropic drugs (Kornhuber et al., 2008, 2011), further decreasing the degradation capability of lysosomes. Additionally, lysosomotropic agents such as sphingosine and hydroxychloroquine have been shown to compromise lysosomal membrane integrity (Boya and Kroemer, 2008; Villamil Giraldo et al., 2014), which has been demonstrated to promote the cytosolic aggregate formation (Micsenyi et al., 2013) indicating inhibition of lysosomal function. Further research effort is warranted to understand the connection between accumulation of lysosomotropic compounds, lysosomal membrane permeabilization, and subsequent lysosomal dysfunction. The lysosomal dysfunction and phagocytosis inhibition observed in this study could play significant roles in the in vivo retinal findings. The RPE is a polarized post-mitotic pigmented cell that forms part of the blood/retina barrier. The apical surfaces have microvilli that surround the outer segment of photoreceptor. Light-sensitive POSs go through a constant destruction due to photo-oxidative damage. To maintain vision, the POS are constantly renewed by shedding the destroyed tips of the POS which are cleared by the phagocytic activity of the RPE (Kevany and Palczewski, 2010). As one of the more phagocytically active cell types, RPE are particularly vulnerable to lysosomal dysfunction and phagocytosis inhibition. Severely abnormal retinal findings, including numerous large and small vacuoles present in RPE and loss of photoreceptors were observed in cathepsin D (a lysosomal aspartyl protease) knockout mice (Koike et al., 2003), indicating the critical function of lysosomes in retinal maintenance. Abnormal retinal manifestation is also often associated with LSD. For instance, glycosaminoglycans deposition within retinal pigment epithelial cells and retinal degeneration were observed in mucopolysaccharidosis patients

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(Summers and Ashworth, 2011). Accumulation of lipofuscin in the retinal pigment epithelium layer and degeneration of POSs was also observed in the mouse model of Niemann-Pick type C disease (Claudepierre et al., 2010). Phagocytosis and lysosome dysfunction have also been suggested as a mechanism for AMD (Chen et al., 2009, 2011), further supporting the negative impact of phagocytosis inhibition in retinal disease. Several lysosomotropic drugs have been reported to be associated with retinal abnormality eg, characteristic bull’s eye maculopathy and severe phototoxic retinopathy. This includes aminoquinolines such as chloroquine and hydroxychloroquine, antipsychotic agents such as chlorpromazine and thioridazine, and tamoxifen (Li et al., 2008). Lysosomal dysfunction triggered by these compounds possibly plays a role in the retinal toxicity manifestation. Rats with chloroquine retinopathy indeed showed an increased number and size of lysosomes or lysosomes related organelles in RPE, bipolar and ganglion cells (Gregory et al., 1970; Ivanina et al., 1983) supporting lysosomal dysfunction as a mechanism of retinopathy. Another noteworthy observation is the inhibition of autophagy by the majority of the compounds associated with in vivo retinal findings in this study. Autophagy is a conserved cellular pathway that controls protein and organelle degradation, and has essential roles in cellular homeostasis. This process is especially vital for terminally differentiated cells, and has been shown to be highly active in the RPE (Kaarniranta et al., 2013). Failure of the RPE cells to employ the autophagy process can result in accumulation of aggregation-prone proteins and cellular degeneration (Ryhanen et al., 2009). In addition, a study using Drosophila as a model demonstrated that the degradation of the outer segment occurs via cross talk between autophagy and the phagocytosis/lysosomal pathway (Midorikawa et al., 2010), further suggesting that autophagy inhibition could exacerbate the decreased retinal function by these basic lipophilic compounds. Besides the trafficking perturbation by the lysosomotropic compounds, high tissue exposure due to the basic and lipophilic properties potentially contributes to the retinal toxicity as well. Basic lipophilic compounds have been shown to extensively accumulate in various tissues, leading to a high volume of distribution (Bickel et al., 1983) with tissue exposure generally higher than that in blood. This is especially the case with organs that are known to have abundant lysosomes, such as liver, lung, and kidney. Indeed, in animal studies, chloroquine was found at 700–1600 times the plasma concentration in the liver, spleen, kidney, and lung after 1 month of 40 mg/kg/day administration (McChesney et al., 1967). Presumably, retinal positive compounds may have high retinal tissue exposure due to their basic lipophilic nature, increasing the potential for toxicity. Further studies to understand the retinal exposure can certainly corroborate this notion. The concentrations (up to 100 mM) used in our in vitro study might seem extreme relative to systemic exposure. However, due to the potential for asymmetrical tissue accumulation and the longer duration of in vivo treatment relative to that of in vitro exposure could provide the rationale for the high concentrations used. Although there is a robust correlation between in vitro and in vivo for majority of the compounds (Table 2), discordance was observed from some compounds (eg, retinal negative compounds CP-655066 and CP-247106 are all positive for all 3 in vitro endpoints). The lack of translation from positive in vitro results to in vivo pathology may reflect the limited exposure attained in vivo relative to the high concentrations used in vitro. Another possibility is that these compounds require a longer duration of in vivo exposure for the morphological manifestations in the

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RPE to occur. Conversely, CP-346086, a retinal positive compound, was only positive for 2 in vitro endpoints (LTR weak positive and LC3 positive), which could be attributable to its less than optimal basic pKa (6.02) for lysosomal accumulation. The in vivo positive finding for CP-346086 is at least partly related to its active metabolite CP-237228, which has a more optimal pKa and was positive for all 3 in vitro endpoints. This finding reveals the significance of drug metabolism considerations which is often not determined. Understanding the biotransformation of experimental compounds could certainly add value for toxicity prediction. It should also be noted that other unknown properties of the compounds, such as off-target interaction with receptors or enzymes, could influence the translation between the in vitro and in vivo effects. Although the toxicological manifestations are mostly limited to the lesion in the RPE layer in the 14-day duration of dosing in this report, with an extended dosing period, it is likely that the photoreceptor layers would be affected. Indeed, photoreceptor cell degeneration has been demonstrated with chloroquine after 12 weeks of treatment (Duncker and Bredehorn, 1996). In atrophic AMD, dysfunction of RPE is the initial occurrence and can lead to the dysfunction and loss of photoreceptors during the progression of AMD (Bhutto and Lutty, 2012). Likely, these compounds can prompt photoreceptor changes with extended dosing and additional studies are warranted to support the notion. In conclusion, we established a correlation between physicochemical properties, LTR change, membrane trafficking perturbation (autophagy and phagocytosis), and retinal pathology. Our study not only identifies lysosomal dysfunction and membrane trafficking impairment as a mechanism for retinal toxicity, but also establishes a battery of screening assays for future compound testing. Assays of these types might best be used to identify safer back-up molecules when retinal lesions have been observed within a drug discovery program. In addition, high retinal exposure due to lysosomal trapping could also play a role in the toxicity manifestation. Toxicity associated with metabolites should also be taken into consideration in in vitro screening as well as in evaluating translation to the in vivo setting. Hypothetically, tissue accumulation and lysosomal disruption could occur in other organs and may manifest in tissuesspecific functional deficits that depend on the role of such pathways in the particular organs affected.

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