Ocular toxicity of AUY922 in pigmented and albino rats

Toxicology and Applied Pharmacology 309 (2016) 55–62

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Ocular toxicity of AUY922 in pigmented and albino rats Danielle Roman a,⁎, James VerHoeve b,c, Heiko Schadt a, Axel Vicart a, Ursula Junker Walker a, Oliver Turner d, Terrilyn A. Richardson e,1, Suzanne T. Wolford f, Paul E. Miller g, Wei Zhou h, Hong Lu i,1, Mikhail Akimov j, William Kluwe d a

Preclinical Safety, Novartis Pharma AG, Basel, Switzerland Ocular Services on Demand, Madison, WI, USA University of Wisconsin School of Medicine and Public Health, Madison, WI, USA d Preclinical Safety, Novartis Pharmaceuticals Corporation, East Hanover, NJ, USA e Pharmaceutical Sciences, College of Pharmacy, Northeast Ohio Medical University, Rootstown, OH, USA f Covance Laboratories Inc., Madison, WI, USA g Comparative Ophthalmic Research Laboratory (CORL), University of Wisconsin, Veterinary Medical Teaching Hospital, Madison, WI, USA h Drug Metabolism and Pharmacokinetics, Novartis Pharmaceuticals Corporation, East Hanover, NJ, USA i Biologics Clinical Pharmacology, Janssen BioTherapeutics at Johnson & Johnson, Spring House, PA, USA j Oncology Global Development, Novartis Pharma AG, Basel, Switzerland b c

a r t i c l e

i n f o

Article history: Received 30 March 2016 Revised 24 August 2016 Accepted 26 August 2016 Available online 28 August 2016 Keywords: HSP90 inhibitor AUY922 Ocular toxicity Rodents Histopathology Electroretinography

a b s t r a c t AUY922, a heat shock protein 90 inhibitor is associated with ocular adverse events (AEs). To provide a better understanding of ocular AEs in patients, 4 investigative studies were performed in a step-wise approach to assess retinal structure and function in pigmented (Brown Norway) and albino (Wistar) rats. In rats administered 30 mg/kg of AUY922, the AUC0–24 h and Cmax are comparable to that in patients at 70 mg/m2. AUY922 at ≥30 mg/kg was poorly tolerated by rats with morbidity or mortality generally after the third weekly treatment. Electroretinography (ERG) changes were observed at doses ≥30 mg/kg. The ERG changes were dose dependent, consistent with an effect on the photoreceptors, and fully reversible. The ERG effects could not be minimized by decreasing the Cmax while maintaining AUC. Histopathological changes were seen mainly when rats were administered AUY922 at 100 mg/kg. The 2-hour infusion of AUY922 at 100 mg/kg caused disorganization of the outer segment photoreceptor morphology in male Brown Norway rats; the severity of the disorganization increased with the number of administrations, but was reversible during a 4-week posttreatment period. There was no major difference in ocular response between Brown Norway and Wistar rats. No changes in serum iron levels, and no changes in rhodopsin, PDE6α, β-transducin concentrations, or retinal pigment epithelium-specific protein RPE65 expression were observed after single and multiple infusions of AUY922 at 100 mg/kg compared to vehicle-treated controls. AUY922 retinal toxicity in rats recapitulates and further characterizes that reported in patients and is shown to be reversible, while a precise molecular mechanism for the effect was not determined. © 2016 Published by Elsevier Inc.

1. Introduction Heat shock protein 90 (HSP90) is a molecular chaperone that plays an important role in the posttranslational stability and activation of several oncogenic client proteins (e.g., HER2, EGFR, ER/PR, and EML4-ALK) that are critical for cell growth, differentiation and survival (Taipale et al., 2010). Inhibition of HSP90 results in misfolding and degradation of client proteins via the ubiquitin proteasome pathway (Pearl et al.,

⁎ Corresponding author at: Preclinical Safety, Novartis Pharma AG, Werk Klybeck, Postfach, CH-4002 Basel, Switzerland. E-mail address: [email protected] (D. Roman). 1 Terrilyn A. Richardson and Hong Lu were employees of Novartis Pharmaceuticals Corporation at the time of conduct of these studies.

http://dx.doi.org/10.1016/j.taap.2016.08.025 0041-008X/© 2016 Published by Elsevier Inc.

2008). Due to these properties, HSP90 has emerged as an attractive target for anticancer treatment (Banerji, 2009; Kim et al., 2009). First-generation HSP90 inhibitors belonged to the benzoquinone ansamycin class and include geldanamycin and its derivatives 17DMAG (17-dimethylaminoethylamino-17-demethoxygeldanamycin), 17-AAG (17-allylamino-17-demethoxygeldanamycin), and IPI-504 (retaspimycin) (Taldone et al., 2008). However, these derivatives are not only difficult to formulate but also are dependent on NADPH:quinone oxidoreductase activity. Furthermore, first-generation HSP90 inhibitors are also associated with hepatotoxicity (Goetz et al., 2005; Grem et al., 2005; Banerji et al., 2005). Second-generation HSP90 inhibitors are based on a variety of chemical scaffolds (including resorcinol, purine, and benzamide structures) and include AUY922, ganetespib, BIIB021, DS-2248, SNX-5422, and AT13387 (Taldone et al., 2008; Biamonte et al., 2010). AUY922 is an

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isoxazole-based compound (Fig. 1) that competitively inhibits HSP90 ATPase activity and has significant antitumor activity in a range of primary and animal tumor models (Brough et al., 2008; Eccles et al., 2008; Jensen et al., 2008). Administration of AUY922 as a single agent or in combination with targeted therapy has shown clinical activity in different cancer types including specific molecular subtypes of non–small cell lung cancer, breast cancer, and hematological malignancies (Biamonte et al., 2010; Sessa et al., 2013; Kong et al., 2012; Garon et al., 2012; Felip et al., 2012; Schroder et al., 2011). Preclinical and clinical experiences have shown that the HSP90 inhibitors 17-DMAG and AUY922 are associated with ocular adverse events (AEs) including blurred vision, dry eye syndrome, visual disturbances, and delayed dark adaptation (Zhou et al., 2011; Samuel et al., 2010). HSP90 ocular toxicity is more difficult to demonstrate in common toxicological species, but can be detected in rats at high doses that none-the-less allow assessment of ocular function. We assessed ocular toxicity associated with AUY922 in pigmented and albino rats to provide a better understanding of the ocular AEs associated with AUY922 in clinical studies and their prognosis. 2. Materials and methods A series of 4 investigative studies were performed (studies 1, 2, and 4 at Covance Laboratories Inc., and study 3 at Novartis Laboratory) in a step-wise approach to assess retinal structure and function in pigmented (Brown Norway) and albino (Wistar) rats. All studies were conducted in accordance with Covance standard operating procedures and generally recognized good laboratory practices. All procedures used were in compliance with the United States Department of Agriculture Animal Welfare Regulations, the Guide for the Care and Use of Laboratory Animals, and the Public Health Service Policy on Humane Care and Use of Laboratory animals; and were approved by the Institutional Animal Care and Use Committee. 2.1. Animals Brown Norway and Wistar rats were obtained from Charles River Laboratories, Raleigh, North Carolina. At initiation of dosing, age of the animals was ≥10 weeks but not N16 weeks and weight of the animals ranged from 150 to 500 g. Animals were individually housed in stainless steel cages. Animals were fed with Certified Rodent Diet #20s16C (Harlan Laboratories, Inc.) ad libitum unless otherwise specified. Water samples were routinely analyzed for specified microorganisms and environmental contaminants. Environmental controls for the animal

room were set to maintain 18 °C to 26 °C, a relative humidity of 30% to 70%, a minimum of 10 air changes/h, and a 12-h light/12-h dark cycle; a light level of 25 to 40 fc was used. The animals were weighed on the day of arrival. They were infused with sterile isotonic (0.9%) saline on the day prior to initiation of dosing (acclimation). The maintenance rate was 0.20 ml/h for male animals, and 0.15 ml/h for female animals. 2.2. Study drug preparation AUY922 (drug content: 94.1%; salt: 1.284, conversion factor, base to salt: 1.365 [the impurity profile is not anticipated to contribute to the toxicity profile]) was administered as a 2.5-mg/ml solution in 5% dextrose injection, USP. AUY922 was stored in a freezer, set to maintain −10 °C to −30 °C, protected from light in sealed packaging. 2.3. Preclinical studies Details of these studies are given in Table 1. Initially, study 1 and study 2 were performed. Study 3 was conducted in order to follow-up on the histopathological changes seen in the initial 4-week study, and study 4 was conducted to assess if the electroretinography (ERG) effects could be minimized by blunting Cmax. 2.3.1. Study 1. The purpose of this study was to determine the maximum tolerable dose level of AUY922 when administered once weekly via intravenous (iv) infusion to rats for at least 4 weeks (4 doses) and to assess whether ERG changes were observed at the highest dose level achieved. 2.3.2. Study 2. Since ERG changes were observed in study 1, the main goal of study 2 was to assess if these ERG changes were reversible after a recovery phase of at least 4 weeks. 2.3.3. Study 3. According to the histopathological atrophic changes of the photoreceptors noted after repeated dosing at the limit dose of 100 mg/kg in study 1, study 3 was initiated in order to further investigate the mechanism of the ocular toxicity. 2.3.4. Study 4. The purpose of study 4 was to evaluate the effect of infusion time and the shape of the exposure vs. time curve (AUC) on the ocular toxicity and toxicokinetics of AUY922. 2.4. Assessments 2.4.1. Expression of phototransduction and visual cycle proteins (study 3 only). 2.4.1.1. Retina isolation. The retina/soft tissue of the right eye or left eye was used for the determination of proteins of the phototransduction/visual cycle and pharmacodynamics using Western blot analysis. The weight of retina and soft tissue was determined. After removal, the whole retina was put in 6 ∗ 16 mm round bottom glass tubes (KbioScience, No. 520045), capped with snap-caps, and immediately frozen on dry ice (same procedure separately for the soft tissue). Visual inspection of samples and Western blot before further sample processing indicated that the quality of the retinas was heterogeneous; retinas were contaminated with sclera and vitreous body. Available samples were thawed and retinas were cleansed of contaminating adjacent tissues; in addition, a small sample was separated for possible mRNA examination. The samples were stored at − 70 °C or below until analysis.

Fig. 1. Structure of AUY922 (5-(2,4-dihydroxy-5-isopropylphenyl)-N-ethyl-4-(4(morpholinomethyl)phenyl)isoxazole-3-carboxamide).

2.4.1.2. Retina protein extraction. After addition of 50 μl ice cold RIPA buffer (Pierce 89900) supplemented with inhibitors of proteases and phosphatases (Sigma P8340, P2850, P5726 - 1/100 each), retinas were homogenized at 4 °C using a E210 (Covaris) ultrasonicator (duty cycle 10% intensity level: 5; time: 5 s; cycle burst: 500, power tracking).

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Table 1 Details of preclinical investigative studies. Details

Study 1

Study 2

Study 3

Study 4

Species

Male/female Brown Norway and Wistar rats 100, 60

Male Brown Norway and Wistar rats

Male Brown Norway rats

Male Wistar rats

60, 30

100

30

Doses (mg/kg) once a week Dose volume

40 ml/kg (20 ml/kg/h) for 100 mg/kg 24 ml/kg (12 ml/kg/h) or 12 ml/kg (6 40 ml/kg dose and 24 ml/kg (12 ml/kg/h) for ml/kg/h) to provide a dose of 60 60 mg/kg dose mg/kg or 30 mg/kg, respectively

AUY922 administered as 2.5-mg/ml AUY922 administered as 2.5-mg/ml Dose administration solution in 5% dextrose injection, USP solution in D5W. Doses administered a (D5W) on days 1, 8, 15, and 22. Animals of AUY922 administered with 30 mg/kg/dose were dosed on days 29, 36, 43, and 50 Infusion time (h) Study duration (weeks) Assessment of posttreatment recovery Main investigations

Toxicokinetic evaluation N

12 ml/kg (2 ml/kg/h administered over approximately 6 h), and 24 ml/kg/h administered over approximately 0.5 h AUY922 was administered as a 2.5-mg/ml solution in D5W

2

2

AUY922 was dissolved in 5% glucose injection solution and administered to 12 groups of 10 or 5 BN male rats at weekly intervals either once (day 1), or twice (days 1 and 8), or 4 times (days 1, 8, 15, and 22) 2 0.5 and 6

4

4 (60 mg/kg); 8 (30 mg/kg)

1–2 and 4

4

No

Yes (recovery period: 4 weeks)

Yes (recovery period: 4 weeks)

No

Slit-lamp biomicroscopy, indirect ophthalmoscopy, IOP, ERG and histopathology

Slit-lamp biomicroscopy, indirect ophthalmoscopy, ERG, and histopathology

Slit-lamp biomicroscopy, indirect ophthalmoscopy, ERG, and histopathology

Yes

No

Histopathology, biochemistry, protein expression, PD, protein expression (retina), biochemistry (blood, and retina) Yes

60 toxicity rats (5 males and 5 females at each dose [0, 60, and 100

80 male rats (10 rats at 0 and 30

mg/kg] of Wistar and Brown Norway strains) and 108 toxicokinetic rats (9 males and 9 females at each dose [0, 60, and 100 mg/kg] of both Wistar and Brown Norway strains)

mg/kg dose and 20 rats at 60 mg/kg of both Wistar and Brown Norway strains)

100 Brown Norway rats (10 rats each treated at 0 and 100 mg/kg, single dose, terminated on study day 8; 10 rats each treated at 0 and 100 mg/kg, single dose, terminated on study day 15; 10 rats each treated at 0 and 100 mg/kg, multiple dose, terminated on study day 29; 10 rats each treated at 0 and 100 mg/kg, multiple dose, terminated on study day 58; 5 rats each treated at 0 and 100 mg/kg, single dose, terminated on study day 1 (about 2 h post dose); and 5 rats each treated at 0 and 100 mg/kg, dosing on days 1 and 8, terminated on study day 9)

Yes 60 male Wistar rats (10 toxicity and 10 toxicokinetic rats treated at 0 mg/kg dose for 6 h; 10 toxicity and 10 toxicokinetic rats treated at 30 mg/kg dose for 30 min; 10 toxicity and 10 toxicokinetic rats treated at 30 mg/kg dose for 6 h).

Abbreviations: ERG, electroretinography; IOP, intraocular pressure; PD, pharmacodynamics. a Control animals (0 mg/kg group) received D5W at an equivalent dose volume of 24 ml/kg (12 ml/kg/h).

Homogenates were transferred to precooled Eppendorf™ tubes (Eppendorf) and incubated on a thermomixer (Eppendorf) at 4 °C for 30 min. Lysates were then centrifuged for 10 min at 20 000 g at 4 °C, and the supernatant was collected and stored at −80 °C. Protein concentration was measured with the MicroBCA Assay Kit (Pierce, #23235) in a 384 well format (Hamilton Robotics Star). Absorbance was measured at 562 nm on an Envision® plate reader (Perkin Elmer). 2.4.1.3. Western blot for phototransduction and visual cycle proteins. 5 μg of retina protein extracts were loaded on Nu-PAGE 4–12% Bis-Tris mini gels and proteins blotted on nitrocellulose membranes (I-blot, Invitrogen® Life Technologies, Corp.). Proteins of interest were detected using antibodies targeting HSP90 (R&D MAB3286), HSP70 (R&D AF1663), Rhodopsin (Santa-Cruz sc-53991), PDE6α (Proteintech 21200-1-AP), β-transducin (Abcam ab3504), RPE65 (Santa-Cruz sc53489), β-Actin (Sigma A5060) and fluorescent secondary antibodies (LI-COR). Quantification was performed after blot scanning (Odyssey Infrared Imaging System, LI-COR). 2.4.2. Analytical biochemistry (study 3). About 0.25 ml blood was collected sublingually (prior to first dosing and approximately 24 h after second dose) into K3-EDTA tubes and plasma was obtained for vitamin A (retinol) determination by high performance liquid chromatography

(HPLC). Plasma proteins were precipitated with ethanol and retinol was extracted with 800 μl of n-hexane containing butylhydroxytoluol. After centrifugation, the supernatant was evaporated and reconstituted in mobile phase prior to injection (100 μl) to a C18 reversed-phase HPLC-fluorescence detection system for analysis. Chromatographic separation of retinol was achieved by isocratic chromatography with a mobile phase consisting of acetonitrile/tetrahydrofuran/methanol/ ammonia acetate solution 1% (684/220/68/28) according to a published method (Aebischer et al., 1999). Retina samples were collected into cryogenic tubes and immediately snap frozen in liquid nitrogen for determination of all-trans retinal, retinol, retinoic acid and the bisretinoid dimer A2E by HPLC coupled to mass spectrometry (LC-MS/MS, Agilent 1200 HPLC coupled to an Agilent® 6410 triple-quadrupole tandem mass spectrometer (Agilent, Corp.) operated in positive electrospray ionization mode). Samples were homogenized and extracted with acetonitrile (ACN). The extract was centrifuged and the supernatant evaporated. After reconstitution in 60% ACN + 0.1% formic acid (FA), 20 μl were injected into the C18-reversed-phase LC-MS/MS system for analysis. Chromatographic separation was achieved applying an isocratic chromatography with 65% ACN + 0.1% FA. Multiple reaction monitoring (MRM) transitions were 269 → 119 (retinol), 285.5 → 161 (retinal), 301 → 159 (retinoic acid), 592.5 → 188 (A2E).

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2.4.3. Clinical biochemistry (study 3). About 0.25 ml blood was collected (pretest, about 24 h postdose [second dose]) using no anticoagulant for iron determination. 2.4.4. Histopathology (all studies). The eyes were fixed in Davidson's solution for 1.5 to 3 h, trimmed and further fixed in Davidson's for 24 h (or in ethanol 70% during the weekend) before embedding in paraffin wax. Eyes were then sectioned at nominally 2.5 μm, and 2 sections per eye were stained with hematoxylin and eosin (H&E). All sections of Davidson-fixed eyes from animals after terminal or interim necropsies were examined microscopically by a board-certified veterinary pathologist. Autofluorescence of the subretinal bodies in Davidson-fixed and H&E stained retinal sections from selected treated animals was investigated using a Zeiss Axiophot® Fluorescence microscope with objective lenses at 40× and a FITC filter. Some eyes were also fixed in formaldehyde (about 24 h or 48 h) and were stained with Prussian blue (Perl's technique) for detection of possible iron deposits in the retina. 2.4.5. Ophthalmic examination (studies 1, 2, and 4). Ophthalmic examinations were performed following pharmacologic mydriasis by a Diplomate of the American College of Veterinary Ophthalmologist once during the predose phase and on day 24 (study 1); days 9 and 28 (study 2); and days 3, 10, 17, and 24 (study 4) of the dosing phase using a Kowa hand-held slit lamp biomicroscope and an indirect ophthalmoscope. Examination findings at each timepoint were recorded on a separate examination sheet for each animal. For intraocular pressure (IOP) measurements (study 1 only), a TonoLab rebound tonometer was used once during the predose phase and on days 23, 25, and 28 of the dosing phase (same days as, but prior to, conducting ERGs). The ERG testing was done once during the predose phase and on various days of the dosing phase. Scotopic tests were recorded after dark adaptation (DA) of at least 2 h using stimuli as follows: dim rodisolating flash strength of 0.01 cds/m2, (DA 0.01); intermediate luminance flash (DA 0.06); a bright flash, mixed rod-cone stimulus (DA 2.5); and oscillatory potentials (OPs), high frequency components digitally band-pass filtered between 70 and 180 Hz from the DA 2.5 condition). 2.4.6. Toxicokinetics (studies 1, 3, and 4). 2.4.6.1. Study 1. Blood was taken (3 animals/sex/timepoint) at predose (day 22 only), 1 h after the start of infusion, within 2 min after the end of infusion and 0.5 h, 2 h, 6 h, and 24 h after the end of infusion on days 1 and 22. 2.4.6.2. Study 3. Blood was taken (1 animal/timepoint) at the following timepoints: end of the 2-hour infusion, as well as 5 min, 15 min, 0.5 h, and 2 h after the end of infusion. 2.4.6.3. Study 4. For the 0.5-hour infusion, blood was taken during infusion and at the end of infusion, as well as 0.5 h and 1.5 h after the end of infusion. For the 6-hour infusion, blood was taken during infusion and at the end of infusion, as well as 2 h after the end of infusion. At each timepoint, approximately 0.5 ml of blood was withdrawn into a tube containing sodium-EDTA anticoagulant. The blood specimens were frozen immediately in a freezer and maintained at approximately −70 °C or below. Analysis was done by LC-MS/MS and Cmax as well as AUC was determined. 2.5. Statistical Methods The ERG waveforms were first quantified by placement of cursors on the A and B waves of the ERG waveform in the 3 flash intensity conditions using LKCLab Version 2.0 software. Oscillatory potentials were measured as the sum of the amplitudes of the elicited wavelets using automated software routines (LKCLab Version 2). Means, covariate-

adjusted means, and standard deviations of the ERG A- and B-wave amplitudes and peak latencies and the summed amplitude and latency-tofirst peak of the oscillatory potentials were presented in tables prepared by Covance. The analyses were based on average of ERG waveform measures for both eyes. Separate analyses of covariance (ANCOVA) were performed for males and females for each ERG condition using the baseline ERG measure as a covariate; number of surviving Brown Norway females were insufficient; hence Brown Norway females were not included for this analysis. 3. Results Multiple doses of AUY922 at ≥30 mg/kg was generally not well tolerated by rats as seen by the high morbidity or mortality incidence (data not presented). However, careful comparison indicated no strict association between general morbidity/mortality and the presence of ocular effects, indicating the two were parallel, independent toxic phenomena. Animals that reached a humane endpoint prior to scheduled necropsy were humanely euthanized in accordance with applicable Animal Welfare Regulations, Guides, and Policies. Other investigations done are reported here for individual studies. 3.1. Study 1 3.1.1. Ophthalmic examinations. No discernable ocular abnormalities were noted on slit lamp biomicroscopy or indirect ophthalmoscopy. Corneal dystrophy was a common spontaneous background finding in the Wistar rats, both prior to dosing and during the dosing phase of the study. The relatively mild degree of corneal opacity in animals was unlikely to have had a detectable effect on ERG testing. On day 24 of the dosing phase, multiple, linear, white streaks (also known as focal chorio-retinal atrophy or focal linear retinopathy) were present in the right retina of 1 rat (AUY922 at 60 mg/kg, male). This was considered spontaneous background finding in this species as it has been previously reported to be a background finding in rats (Hubert et al., 1994) and was not observed in any other eye in this study. 3.1.2. Clinical observations - eyes. Treatment-related clear eye discharge was observed in Brown Norway rats given 60 or 100 mg/kg dose. The finding of squinted eyes was seen in only 1 Wistar rat given 60 mg/kg and is of uncertain relationship to treatment, although this animal later died. 3.1.3. Intraocular pressure measurements. There was no evidence that iv administration of either the vehicle or AUY922 at 60 or 100 mg/kg resulted in an abnormal elevation of IOP (N 31 mm Hg) in any individual animal, or that an increase (or decrease) in IOP would be of sufficient magnitude to alter the ERG findings. 3.1.4. Toxicokinetics. Toxicokinetic parameters of AUY922 in rat whole blood are shown in Supplementary Table 1. In rats treated with AUY922 60 mg/kg, the AUC0–24 h was comparable to that achieved in patients at 70 mg/m2 (recommended phase 2 dose of AUY922) (Sessa et al., 2013) whereas the Cmax was 3 to 7 times higher. At 100 mg/kg in rats, the AUC0–24 h was 1.5 times the exposure attained in patients at 70 mg/ m2 and Cmax was 6 to 10 times higher. 3.1.5. ERG changes. AUY922 at 100 mg/kg resulted in a significant reduction of the ERG in albino and pigmented rats on day 23, 25, or 28 (Table 2). A downward trend in ERG amplitude was also observed at 60 mg/kg. Electroretinography was depressed in both nonpigmented and pigmented rats, although the amplitude of the nonpigmented Wistar strain in general, including predose value, was generally much lower than the pigmented Brown Norway strain. Although group analysis could not be performed on animals administered the 60 mg/kg/ dose, both nonpigmented and pigmented rats showed that the postdose

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Table 2 Analysis of covariance effects in Wistar and Brown Norway rats. Sex Wistar rats Females

Males

Brown Norway rats Males

Test

A-wave amplitude

A-wave latency

B-wave amplitude

B-wave latency

DA 2.5 DA 0.06 DA 0.01 OP DA 2.5 DA 0.06 DA 0.01 OP

T T × T (25)

T T

T T × T (23, 28)

T × T (25, 28)

T T T T T × T (23, 25, 28) T × T (25, 28) T × T (28) T × T (23, 25, 28)

NA T × T (23, 25) T × T (25) T T×T T × T (23, 25) T

DA 2.5 DA 0.06 DA 0.01 OP

T × T (23, 25, 28)

T × T (23, 25, 28) T T × T (23, 25, 28) T

T T × T (23, 25, 28) T T

T × T (23, 25, 28) T × T (25, 28) T × T (23, 25)

Abbreviations: OP, oscillatory potential; T, treatment effect only; T × T, treatment × time interaction (days significant are in parentheses).

ERG A-wave and B-wave amplitudes trended downward when compared to their predose values.

One Wistar rat given 30 mg/kg/dose had squinted eyes on the day of scheduled necropsy.

3.1.6. Histopathologic changes in retina. Minimal focal or diffuse retinal atrophy was observed at 100 mg/kg in male Wistar rats (Table 3). The outer nuclear and the photoreceptor layers were affected. In female rats, minimal to slight focal or diffuse retinal atrophy affecting outer nuclear and photoreceptor layers was also seen in a number of control animals. However, the incidence was increased in animals administered with AUY922 at 100 mg/kg and an individual female administered with 100 mg/kg had an increase in severity of these alterations. No retinal atrophy was seen at 60 mg/kg. In Brown Norway rats, minimal focal or diffuse retinal atrophy was only seen in animals administered with AUY922 at 100 mg/kg, but not in control animals or those administered with 60 mg/kg. Similar to Wistar rats, outer nuclear and photoreceptor layers were affected. Immunohistochemical staining for rhodopsin and retinal S confirmed the retinal atrophy as seen with hematoxylin and eosin staining. Staining for neurofilament did not reveal any differences between control and treated animals.

3.2.3. ERG changes. Individual ERG waveforms from each eye showed the expected increase in B-wave amplitude with increasing flash intensity in all dose groups at baseline. The 2 low-intensity flashes (DA 0.01, DA 0.06) elicited the expected positive voltage B-wave with a minimal preceding negative voltage A-wave. The bright flash (DA 2.50) elicited a large negative voltage A-wave preceding the positive voltage Bwave. Both strains of rats evidenced significant decrements in ERG function during the dosing phase of the study. At each of the 3 flash intensities (DA 0.01, DA 0.05, DA 2.5), the peak latency of the A- and B-waves increased and A- and B-wave amplitudes decreased. The significant differences between control and dosed groups evident during the dosing phase were no longer present following the recovery phase.

3.2. Study 2 3.2.1. Ophthalmic examination findings. No ocular abnormalities were attributable to iv administration of the vehicle (D5W) or AUY922 at 30 mg/kg or 60 mg/kg. Corneal dystrophy was a common spontaneous background finding in Wistar rats prior to dosing and during the dosing phase. The relatively mild degree of corneal opacity in animals was unlikely to have had a detectable effect on ERG testing.

3.2.2. Clinical observations - eyes. Pale eyes and clear discharge from eyes was seen only in animals that died prior to scheduled sacrifice.

Table 3 Summary of microscopic findings. Microscopic finding

Dose (mg/kg) 0

Wistar rats No. of animals examined Retinal atrophy, focal Retinal atrophy, diffuse Brown Norway rats No. of animals examined Retinal atrophy, focal Retinal atrophy, diffuse

60

100

Male

Female

Male

Female

Male

Female

12

14 4 2

5

5

14 1 1

13 9

14

13

5

5

14 1 7

10 2 5

3.2.4. Histopathologic changes in retina. There were no conclusive retinal changes that could be clearly attributed to AUY922 administration. Noteworthy changes were only observed in 2 Wistar rats. In 1 animal (60 mg/kg, sacrificed after 4 weeks), there was minimal focal thinning (retinal atrophy) of the photoreceptor and outer nuclear layers; its association with AUY922 administration was inconclusive. In the other animal (30 mg/kg, 4 weeks of dosing plus 2 additional doses), there was a focal retinal dysplasia of the outer plexiform layer, characterized by disorganized morphology and variably sized abnormal cells that was considered to be an incidental finding and not associated with AUY922 administration.

3.3. Study 3 3.3.1. Histopathologic changes in retina. The morphological changes were observed in the photoreceptor outer segments (POS) after ≥2 intravenous administrations of AUY922. No iron deposits were detected in the formalin-fixed retinas of animals. On days 8 and 15 following a single administration of AUY922, no morphological difference was evident between the photoreceptors of the treated and control retinas. A very small number of retinal eosinophilic bodies were noted in 2 of 5 animals. Following the second administration of AUY922 (day 9), the palisade-like arrangement of the photoreceptors was maintained in 3 of 5 animals, nevertheless the POS appeared thicker than in the control eyes, suggesting swelling. This subtle change was focal/multifocal in 2 animals and was nearly diffuse in 1 animal. More advanced alterations were noted in the remaining 2 animals, which showed swelling alternating with multifocal disorganization of the POS and a very small

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number of eosinophilic bodies, sandwiched between the POS and the retinal pigment epithelium (RPE). One week after the fourth administration of AUY922, 1 animal still showed both multifocal swelling and disorganization of the POS (Fig. 2), whereas disorganization of the POS was diffuse in the remaining 4 animals of the group. A very small number of retinal eosinophilic bodies were observed in all 5 animals. These small rounded to oval bodies (size range, 3–5 μm) were typically located on the apical surface of the RPE, sandwiched between the POS, and the RPE. Isolated eosinophilic bodies were also observed occasionally in the subretinal space of control animals, suggesting a physiological deposition of POS debris resulting from the continuous breakdown and renewal of the outer segment discs. Similar findings were recorded in an animal sacrificed moribund on day 20 shortly before the third administration of AUY922. This animal also showed an eosinophilic body in the photoreceptor inner segment and outer nuclear layer. Only 3 of 5 animals survived till the end of the 4-week recovery period following 4 weekly administrations of AUY922, and the left eye of 1 of these animals was inadvertently perforated during necropsy and could not be evaluated microscopically. Examination of the retinas of the remaining 2 animals revealed neither morphological alteration of the POS nor the presence of retinal eosinophilic bodies, suggesting a full reversibility of the changes observed at the end of treatment. 3.3.2. Iron and retinoid levels, phototransduction, and visual cycle proteins expression. Ocular toxicity might results from hypoxic conditions in the retina driven by an iron chelating effect. No changes in iron levels were observed after single and multiple infusions of AUY922 at 100 mg/kg. No statistical significant changes in HSP70 and HSP90 protein expression in the retina were present (Supplementary Fig. 1). However,

it has to be noted that HSP70 assessment was done in the animals from samples that were collected at least 7 days after last dose. HSP90 inhibition could induce misfolding of key retina proteins with subsequent up/down-regulation of these proteins, leading to a disturbance in the visual phototransduction and visual cycle processes. No statistically significant changes in rhodopsin, phosphodiesterase PDE6α, βtransducin, or retinal pigment epithelium-specific 65 kDa protein (RPE65) expression were observed following AUY922 infusions at 100 mg/kg (Supplementary Fig. 2). There were no treatment-related plasma vitamin A changes after 2 infusions (days 1 and 8) at 100 mg/ kg AUY922 on day 9. In addition, there were no statistically significant changes in the levels of retinal, retinol, and the pyridinium bisretinoid A2E in the retina, and retinoic acid was not detected. 3.3.3. Toxicokinetics. The PK data (AUC, Cmax) in study 3 were similar to study 1 (Supplementary Table 2). 3.4. Study 4 3.4.1. Clinical observations - eyes. seen in 3 euthanized animals.

Treatment-related pale eyes were

3.4.2. Ophthalmic examination findings. Corneal dystrophy was noted during the predose and dosing phases in Wistar rats. The relatively mild degree of corneal opacity in animals was unlikely to have had a detectable effect on ERG testing. Similarly, a small white vitreous floater (likely representing a hyaloid artery remnant) in the left eye was present during the predose and dosing phases in a single animal. This background finding also had no detectable effect on ERG testing. On day 24, three animals given 30 mg/kg for 6 h had pale irides and choroidal and retinal vascular attenuation. These changes were most consistent with profound systemic hypotension or anemia and not a primary chorioretinal disorder as iridal vessels were also involved.

A. Normal POS in a control animal

B. Disorganized POS and eosinophilic bodies after 4 infusions at 100 mg/kg AUY922

Fig. 2. Photoreceptor outer segment. A. Normal POS in a control animal. B. Disorganized POS and eosinophilic bodies after 4 infusions at 100 mg/kg AUY922.

3.4.3. ERG changes. Although ERG amplitudes and latencies in animals administered with 30 mg/kg for a 0.5-hour or 6-hour infusion were consistently depressed relative to controls, no difference in ERG effects was noted between animals administered with a relatively rapid infusion over 0.5 h and those a relatively slow infusion over 6 h that minimized initial plasma drug concentrations. On days 9 and 16 of the dosing phase, the DA 2.5 A-wave amplitude was significantly reduced in animals administered with the 0.5-hour or 6-hour infusions relative to controls. The peak latency of the DA 2.5 A-wave was prolonged on days 2, 9, 16, and 23 in animals administered with the 0.5-hour or 6-hour infusion. On day 23 of the dosing phase, animals administered with 30 mg/kg over 0.5 h or 6 h had similar differences in A-wave latency. The DA 2.5 Bwave amplitude was reduced on days 9 and 16. On day 23 of the dosing phase, B-wave amplitude reached statistical significance for animals administered with the 6-hour infusion. The DA 2.5 B-wave latency increase was significant for animals administered with the 0.5-hour infusion, with no specific dosing days reaching statistical significance. OP amplitude was reduced and peak time prolonged in animals administered with 0.5-hour or 6-hour infusion on days 2, 9, 16, and 23. For A- and B-wave amplitudes of ERG to the DA 0.06 flash strength, B-wave amplitudes were significantly reduced in animals administered with 0.5-hour or 6-hour infusion on days 9 and 16. The DA 0.06 A-wave peak latency was prolonged in animals administered with the 0.5hour or 6-hour infusion, without reaching statistical significance on specific dosing days. The peak latency of the B-wave was prolonged on days 2, 9, 16, and 23 in animals administered with either the 0.5-hour or the 6-hour infusion. For the dim DA 0.01 flash strength, the negative voltage A-wave was small and variable. The A-wave to the DA 0.01 flash strength was reduced across timepoints in animals administered with the 6-hour infusion. The B-wave was reduced in amplitude in animals administered the 0.5-hour or 6-hour infusion. The peak latency for the

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B-wave in the DA 0.01 flash condition was prolonged for animals administered with 0.5-hour or 6-hour infusion on days 9, 16, and 23. 3.4.4. Toxicokinetics. No difference in total exposure (AUC) to AUY922 was observed on day 1 or day 22 in rats receiving a 0.5-hour or 6-hour infusion (Supplementary Table 3). As expected, the Cmax of AUY922 was higher (approximately 9-fold or 7-fold) in animals receiving the 0.5-hour infusion compared to the 6-hour infusion. 4. Discussion We performed 4 investigative studies in a step-wise approach to assess the retinal structure and function in pigmented and albino rats to provide a better understanding of ocular AEs associated with AUY922 in clinical studies. Administration of AUY922 to cancer patients over the dose range of 40–70 mg/m2, iv (once each 28 day cycle) was associated with reversible visual disturbance characterized by night blindness, photopsia, blurred vision and visual impairment (Sessa et al., 2013; Seggewiss-Bernhardt et al., 2015); associated Cmax values and AUC over this dose range were 714–1278 ng/ml, and 9262– 13,457 ng·h/ml, respectively (Sessa et al., 2013). While the specific mechanism by which HSP90 inhibitors impair visual function is not clear, the similarities in measured effects in AUY922-treated patients and rats suggest the rat as an appropriate model for further study of the phenomenon with relevance to humans. Recent work in a P23H transgenic rat model suggests that HSP90 inhibitors enhance visual function and delay photoreceptor degeneration, and this is associated with induction of HSP expression and reduced rhodopsin aggregation (Aguilà et al., 2014). Thus, visual symptoms reported with HSP90 inhibition such as blurred vision and slowed dark adaptation may be due to a pharmacodynamic effect on the visual cycle. In the preclinical study by Zhou et al. (2013), the HSP90 inhibitors, 17-DMAG and AUY922, induced marked photoreceptor cell death; however, these effects were not produced by either 17-AAG or ganetespib treatment. In our mechanistic studies, corneal dystrophy was noted during the predose and dosing phases in Wistar rats. However, corneal dystrophy has previously been reported to be an extremely common background finding in this strain of albino rats (Bellhorn et al., 1988; Hashimoto et al., 2013; Wojcinski et al., 1999). It primarily affects the nasal and axial regions of the cornea and relatively spared the perilimbal and temporal regions. Animals with such changes are not considered to represent compromised biologic test systems for non-corneal effects (Bruner et al., 1992). Histopathological changes were seen mainly when rats were administered AUY922 at 100 mg/kg (atrophic changes of the retina in study 1 and disorganization of the outer segment photoreceptor morphology in study 3). The severity of the alterations in study 3 increased with the number of administrations, and recovered after a 4-week recovery period. The dose level of 100 mg/kg was used in study 1 and we went as high as possible in order to assess if ERG changes would occur at all. Besides the ERG changes at 100 mg/kg, we also observed histopathological changes. In study 3, we only used that dose level in order to further assess the mechanistic endpoints (PDE6, rhodopsin, iron, retinoid etc.). Our studies also assessed changes in ocular function. At doses ≥30 mg/ kg, ERG changes were observed; these changes were dose dependent, consistent with an effect on the photoreceptors, fully reversible, and could not be minimized by decreasing the Cmax. Drug binding to melanin is not predictive of retinal toxicity (Leblanc et al., 1998). In our studies, no major difference in ocular response between pigmented and nonpigmented rats was demonstrated. Published preclinical studies have evaluated retinal morphology, HSP70 expression, apoptotic induction, and pharmacokinetic drug exposure analysis of 17-DMAG, 17-AAG, AUY922, SNX-5422, and ganetespib (Zhou et al., 2013; Zhou et al., 2012; Kanamaru et al., 2014). Both 17DMAG and AUY922 elicited strong retinal HSP70 upregulation and promoted marked photoreceptor cell death 24 h after the final dose, whereas

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neither 17-AAG nor ganetespib produced any detectable apoptotic photoreceptor injury. Both 17-DMAG as well as AUY922 showed substantial retinal accumulation, with high retina/plasma (R/P) ratios and slow elimination rates; 51% of 17-DMAG and 65% of AUY922 present at 30 min post injection were retained in the retina 6 h post dose. The retinal elimination was rapid for 17-AAG and ganetespib (90% of 17-AAG and 70% of ganetespib were eliminated from the retina at 6 h) and correlated with lower R/P ratios. Recent studies suggest that rhodopsin kinase (GRK1) and PDE6 are HSP90 client photo-transduction proteins that may be affected by HSP90 inhibitor treatment. The HSP90 co-chaperone, aryl hydrocarbon receptor interacting protein-like 1 (AIPL1) is expressed by cone and rod photoreceptor cells and plays a critical role in cellular viability, and is also a potential source of “on-target” effects (Zhou et al., 2013). HSP70 is known to be upregulated upon HSP90 inhibition (Dakappagari et al., 2010). As all the samples that could be assessed were taken 7 days after the AUY922 administration, it could be the possibility that the HSP70 levels were elevated on day 1 after treatment but were back to normal after 7 days, as seen in preclinical study by Zhou et al. (2013). In that study, for the HSP90 inhibitor 17-DMAG, HSP70 induction was maximal after 1 day of treatment and almost back to normal after 5 days. Further, even if AUY922 seems to stay longer in retina than 17-DMAG, 35% of AUY922 is eliminated from retina after 6 h and therefore it is unlikely that there is much left after 7 days. In the present study, in rats at 30 mg/kg AUC0-24h and Cmax were comparable to that reported for patients at 70 mg/m2 (Sessa et al., 2013). At 60 mg/kg, the AUC0-24h was comparable to that attained in patients at 70 mg/m2 whereas the Cmax was 3 to 7 times higher. At 100 mg/kg, the AUC0–24 h in rats was 1.5 times the AUC exposure attained in patients at 70 mg/m2 and Cmax was 6 to 10 times higher. AUY922 at ≥30 mg/kg was generally not well tolerated by rats, despite the use if these comparable doses in cancer patients. In summary, these investigative studies indicate that at an exposure equivalent to the human exposure at a therapeutic dose of 70 mg/m2, fully reversible ERG changes occurred in AUY922-treated rats. Retinal histopathological changes were observed at higher AUY922 doses associated with substantial general toxicity and mortality, and consisted of either atrophy (reversibility not assessed) or disorganization in the outer segment photoreceptor morphology (also reversible upon discontinuation of the treatment). In clinical studies, most of the ocular AEs in patients treated with AUY922 at recommended dose (70 mg/m2) clinical studies were grade 1 or 2, and were reversible upon study drug interruption or discontinuation. No grade 4 or irreversible ocular toxicities were reported (Kong et al., 2012; Sessa et al., 2013; Seggewiss-Bernhardt et al., 2015). Thus, ocular toxicity of AUY922 at dose N30 mg/kg in pigmented and albino rats correlates with the clinical findings and the ERG changes were observed at the clinical relevant dose and were fully reversible. These data provide a better understanding of the human risk associated with therapeutic use of HSP90 inhibitors and the relevance of ocular function testing to clinical safety monitoring. Although there are no preclinical studies of AUY922 ongoing, future studies are warranted to investigate deeply the underlying functional mechanism of the ocular toxicity of HSP90 inhibitors. Future studies are also warranted in immune compromised animals as HSP90 inhibitors are given to cancer patients where the immune system is already emaciated. Alternative approaches e.g. global gene expression analysis, quantitative metabolite (including neurotransmitter and lipids) and proteomic profiling of the retina, or more sophisticated techniques like high spatial resolution quantitative imaging analysis based on mass spectrometry might provide additional mechanistic insights (Anderson et al., 2014; Magharious et al., 2011; Tan et al., 2016).

Funding These studies were sponsored by Novartis Pharmaceuticals Corporation.

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Conflict of interest DR, HS, AV, UJW, MA are employees of Novartis Pharma AG, Basel, Switzerland. OT, WK, and WZ are employees of Novartis Pharmaceuticals Corporation, East Hanover, New Jersey, USA. JVH reports personal fees from OSOD, LLC, during the conduct of the study. STW reports and would like to disclose that her company, Covance Inc., was paid by the sponsor, Novartis Pharmaceuticals Corporation, for conduct of the indicated studies. PEM reports personal fees from OSOD, during the conduct of the study; personal fees and non-financial support from OSOD, outside the submitted work. All other authors have nothing to disclose. Transparency document The Transparency document associated with this article can be found, in online version. Acknowledgments We thank Pushkar Narvilkar and Shiva Krishna Rachamadugu, Novartis Healthcare Pvt. Ltd. for providing medical editorial assistance with this manuscript. The authors also thank Jean-Philippe Gasser, Gregory Guillemain, Marianne Schwald, and Audrey Fischer for running the analysis for phototransduction and visual cycle proteins, and Mireille Court for the histological evaluation of study 3. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.taap.2016.08.025. References Aebischer, C.P., Schierle, J., Schüep, W., 1999. Simultaneous determination of retinol, tocopherols, carotene, lycopene, and xanthophylls in plasma by means of reversedphase high-performance liquid chromatography. Methods Enzymol. 299, 348–362. Aguilà, M., Bevilacqua, D., McCulley, C., Schwarz, N., Athanasiou, D., Kanuga, N., Novoselov, S.S., Lange, C.A., Ali, R.R., Bainbridge, J.W., Gias, C., Coffey, P.J., Garriga, P., Cheetham, M.E., 2014. Hsp90 inhibition protects against inherited retinal degeneration. Hum. Mol. Genet. 23, 2164–2175. Anderson, D.M., Ablonczy, Z., Koutalos, Y., Spraggins, J., Crouch, R.K., Caprioli, R.M., Schey, K.L., 2014. High resolution MALDI imaging mass spectrometry of retinal tissue lipids. J. Am. Soc. Mass Spectrom. 25, 1394–1403. Banerji, U., 2009. Heat shock protein 90 as a drug target: some like it hot. Clin. Cancer Res. 15, 9–14. Banerji, U., O'Donnell, A., Scurr, M., Pacey, S., Stapleton, S., Asad, Y., Simmons, L., Maloney, A., Raynaud, F., Campbell, M., Walton, M., Lakhani, S., Kaye, S., Workman, P., Judson, I., 2005. Phase I pharmacokinetic and pharmacodynamic study of 17-allylamino, 17demethoxygeldanamycin in patients with advanced malignancies. J. Clin. Oncol. 23, 4152–4161. Bellhorn, R.W., Korte, G.E., Abrutyn, D., 1988. Spontaneous corneal degeneration in the rat. Lab. Anim. Sci. 38, 46–50. Biamonte, M.A., Van de Water, R., Arndt, J.W., Scannevin, R.H., Perret, D., Lee, W.C., 2010. Heat shock protein 90: inhibitors in clinical trials. J. Med. Chem. 53, 3–17. Brough, P.A., Aherne, W., Barril, X., Borgognoni, J., Boxall, K., Cansfield, J.E., Cheung, K.M., Collins, I., Davies, N.G., Drysdale, M.J., Dymock, B., Eccles, S.A., Finch, H., Fink, A., Hayes, A., Howes, R., Hubbard, R.E., James, K., Jordan, A.M., Lockie, A., Martins, V., Massey, A., Matthews, T.P., McDonald, E., Northfield, C.J., Pearl, L.H., Prodromou, C., Ray, S., Raynaud, F.I., Roughley, S.D., Sharp, S.Y., Surgenor, A., Walmsley, D.L., Webb, P., Wood, M., Workman, P., Wright, L., 2008. 4,5-Diarylisoxazole Hsp90 chaperone inhibitors: potential therapeutic agents for the treatment of cancer. J. Med. Chem. 51, 196–218. Bruner, R.H., Keller, W.F., Stitzel, K.A., Sauers, L.J., Reer, P.J., Long, P.H., Bruce, R.D., Alden, C.L., 1992. Spontaneous corneal dystrophy and generalized basement membrane changes in Fischer-344 rats. Toxicol. Pathol. 20, 357–366. Dakappagari, N., Neely, L., Tangri, S., Lundgren, K., Hipolito, L., Estrellado, A., Burrows, F., Zhang, H., 2010. An investigation into the potential use of serum Hsp70 as a novel tumour biomarker for Hsp90 inhibitors. Biomarkers 15, 31–38. Eccles, S.A., Massey, A., Raynaud, F.I., Sharp, S.Y., Box, G., Valenti, M., Patterson, L., de Haven Brandon, A., Gowan, S., Boxall, F., Aherne, W., Rowlands, M., Hayes, A., Martins, V., Urban, F., Boxall, K., Prodromou, C., Pearl, L., James, K., Matthews, T.P., Cheung, K.M., Kalusa, A., Jones, K., McDonald, E., Barril, X., Brough, P.A., Cansfield, J.E., Dymock, B., Drysdale, M.J., Finch, H., Howes, R., Hubbard, R.E., Surgenor, A.,

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