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http://tpx.sagepub.com/content/early/2013/11/27/0192623313505781The online version of this article can be found at:

 DOI: 10.1177/0192623313505781

published online 28 November 2013Toxicol PatholDoorten, James E. Ridings, Marshall S. Scicchitano, Jérémy Silvano and Jennie Woodfine

Gales, Richard Haworth, Shaun R. Maguire, Rosanna C. Mirabile, David Mullins, Bernard Palate, Yolanda Ponstein-Simarro Kendall S. Frazier, Cécile Sobry, Victoria Derr, Mike J. Adams, Cathaline Den Besten, Sjef De Kimpe, Ian Francis, Tracy L.

OligonucleotideLesions in Mice and Monkeys Following Chronic Administration of a Second-generation Antisense

Species-specific Inflammatory Responses as a Primary Component for the Development of Glomerular  

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Species-specific Inflammatory Responses as a PrimaryComponent for the Development of Glomerular Lesions inMice and Monkeys Following Chronic Administration of a

Second-generation Antisense Oligonucleotide

KENDALL S. FRAZIER1, CECILE SOBRY

2, VICTORIA DERR3, MIKE J. ADAMS

4, CATHALINE DEN BESTEN5, SJEF DE KIMPE

5,

IAN FRANCIS4, TRACY L. GALES

1, RICHARD HAWORTH4, SHAUN R. MAGUIRE

4, ROSANNA C. MIRABILE1, DAVID MULLINS

1,

BERNARD PALATE2, YOLANDA PONSTEIN-SIMARRO DOORTEN

5, JAMES E. RIDINGS4, MARSHALL S. SCICCHITANO

1,

JEREMY SILVANO2, AND JENNIE WOODFINE

4

1Department of Safety Assessment, GlaxoSmithKline, King of Prussia, Pennsylvania, USA2CiToxLab, Evreux, France

3Pathology, Microbiology and Immunology, University of California–Davis, California, USA4Departments of Safety Assessment and Scinovo, GlaxoSmithKline, Ware, United Kingdom

5Prosensa Therapeutics, Leiden, The Netherlands

ABSTRACT

Chronic administration of drisapersen, a 20-OMe phosphorothioate antisense oligonucleotide (AON) to mice and monkeys resulted in renal

tubular accumulation, with secondary tubular degeneration. Glomerulopathy occurred in both species with species-specific characteristics. Glomer-

ular lesions in mice were characterized by progressive hyaline matrix accumulation, accompanied by the presence of renal amyloid and with sub-

sequent papillary necrosis. Early changes involved glomerular endothelial hypertrophy and degeneration, but the chronic glomerular amyloid and

hyaline alterations in mice appeared to be species specific. An immune-mediated mechanism for the glomerular lesions in mice was supported

by early inflammatory changes including increased expression of inflammatory cytokines and other immunomodulatory genes within the renal cor-

tex, increased stimulation of CD68 protein, and systemic elevation of monocyte chemotactic protein 1. In contrast, kidneys from monkeys given

drisapersen chronically showed less severe glomerular changes characterized by increased mesangial and inflammatory cells, endothelial cell hyper-

trophy, and subepithelial and membranous electron-dense deposits, with ultrastructural and immunohistochemical characteristics of complement and

complement-related fragments. Lesions in monkeys resembled typical features of C3 glomerulopathy, a condition described in man and experimental

animals to be linked to dysregulation of the alternative complement pathway. Thus, inflammatory/immune mechanisms appear critical to glomerular

injury with species-specific sensitivities for mouse and monkey. The lower observed proinflammatory activity in humans as compared to mice and

monkeys may reflect a lower risk of glomerular injury in patients receiving AON therapy.

Keywords: monkey pathology; mouse pathology; renal; safety assessment; glomerulonephritis; amyloid; hyaline glomerulopathy.

INTRODUCTION

Antisense oligonucleotides (AONs) are single-stranded,

synthetic deoxy-, or ribonucleotide sequences designed to

hybridize to specific and complimentary messenger RNA

(mRNA) sequences and inhibit their expression. Currently,

numerous AONs are being evaluated in clinical trials for treat-

ing cancer, inflammation (allergic, autoimmune, and other

inflammatory diseases), metabolic diseases (diabetes and high

cholesterol), neuromuscular disorders, or viral diseases. Newer

generation AONs have benefitted from structural chemical

modifications to improve their stability, potency, and bioavail-

ability. The toxicologic target organ profiles of these AONs are

relatively similar, although there may be significant quantita-

tive differences in the expected toxicities between specific

structural groups (Henry et al. 2008). The second-generation

AONs, including a compound (drisapersen) in development

by GlaxoSmithKline (GSK) and Prosensa as a potential treat-

ment for Duchenne’s muscular dystrophy, have stereotypical

The author(s) declared the following potential conflicts of interest with

respect to the research, authorship, and/or publication of this article: Several

of the authors are employees of GlaxoSmithKline or Prosensa, and these two

companies are jointly developing GSK2402968 as a potential therapeutic

treatment.

The author(s) received no financial support for the research, authorship,

and/or publication of this article.

Address correspondence to: Kendall S. Frazier, Department of Safety

Assessment, 709 Swedeland Road, Mail Stop UE0376, King of Prussia, PA

19406, USA; e-mail: kendall.s.frazier@gsk.com.

Abbreviations: AONs, antisense oligonucleotides; CRP, C-reactive pro-

tein; DAPI, 40,6-diamidino-2-phenylindole; EDD, electron-dense deposit;

FFPE, formalin-fixed paraffin embedded; FITC, fluorescein isothiocyanate;

FSGS, focal segmental glomerulosclerosis; GMS, Gomori methenamine silver;

H&E, hematoxylin and eosin; IF, immunofluoresence; IHC, immunohisto-

chemistry; IL-6, interleukin 6; LOQ, limit of quantitation; MCP-1, monocyte

chemotactic protein 1; MPGN, membranoproliferative glomerulonephritis;

mRNA, messenger RNA; OCT, optimal cutting temperature; PAS, periodic

acid Schiff; TLRs, toll-like receptors; vWF, von Willebrand’s factor.

1

Toxicologic Pathology, XX: 1-13, 201X

Copyright # 2013 by The Author(s)

ISSN: 0192-6233 print / 1533-1601 online

DOI: 10.1177/0192623313505781

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class-related toxicity responses, including proinflammatory

activity and renal toxicity that are relatively independent of

sequence and may be highly correlated with the agent’s phar-

macokinetic properties. Accumulation of cytoplasmic granules

in epithelial cells from a range of organs and tissues is a class

effect of AONs observed in all species (Henry et al. 2008).

Ultrastructural studies and immunohistochemical staining have

demonstrated that the granular material found in epithelial cells

represents the test compound or associated material contained

within endosomal or lysosomal vesicles or vacuoles (Monteith

et al. 1999). Renal effects in short-term toxicity studies with

second-generation AONs have been observed in association

with this renal accumulation of test material in the form of

basophilic granules, most often identified in the proximal

tubules and only rarely in the glomeruli (Henry et al. 2008).

GSK has recently completed chronic administration of drisa-

persen to mice and monkeys over periods of 27 and 39 weeks,

respectively. This article describes additional renal pathology

involving the glomeruli, which is observed in mice and monkey

following chronic administration of drisapersen, including

dedicated mechanistic investigations to better understand the

pathogenesis and its clinical translation.

MATERIALS AND METHODS

Study Descriptions

Drisapersen was administered as a subcutaneous injection

for 27 weeks in male CD-1 mice followed by a 20-week

(high-dose group) or 35-week (control and other dose groups)

off-dose period. The doses were 0 (control), 6, 18, or 72 mg/kg/

injection, and the test compound was administered subcuta-

neously twice a week for the first 2 weeks, then once weekly

at the end of the treatment period. The vehicle was 20 mM

phosphate buffer in 0.8% saline for injection. Mice from each

group were killed and examined at necropsy at the end of the

treatment period and after the off-dose period. Plasma samples

taken in weeks 5, 13, and 26 of treatment were examined for

monocyte chemotactic protein 1 (MCP-1) and interleukin 6

(IL-6) cytokine levels as well as samples for clinical pathology

to assess kidney function.

Drisapersen was administered to male CD-1 mice as 10

repeated subcutaneous injections at weekly intervals at doses

of 0 (control), 30, 100, and 300 mg/kg mg/kg/injection. The

vehicle was 20 mM phosphate buffer in 0.8% saline for injec-

tion. Mice from each group were killed and examined at

necropsy at the end of the treatment period.

Drisapersen was administered as a subcutaneous injection

to male cynomolgus monkeys for 39 weeks followed by a

39-week off-dose period at doses of 0 (control), 2, 6, and

12 mg/kg/injection twice weekly for the first 2 weeks and then

weekly. The vehicle was 20 mM phosphate buffer in 0.8% sal-

ine for injection. Monkeys from each group were killed and

examined at necropsy at the end of the treatment period and

after the off-dose period. Samples of right kidney from 6 con-

trols, four 6 mg/kg/injection, and one 12 mg/kg/injection mon-

keys were used for immunohistochemistry (IHC) and electron

microscopy. The animals in drisapersen-treated groups were

selected on the basis of the presence of glomerulopathology

observed by H&E in the original study. Plasma samples were

taken on weeks 1, 5, 7, 9, 11, 15, 18, 22, 26, 31, 35, and 39 and

examined for a range of inflammatory biomarkers as well as

samples for clinical pathology to assess kidney function. In the

off-dose period, blood sampling was performed monthly. Uri-

nalysis was performed monthly using both standard parameters

and urinary albumin levels.

All animal studies were ethically reviewed and carried out

in accordance with European Directive 86/609/EEC and the

GSK Policy on the Care, Welfare and Treatment of animals

or after review by Institutional Animal Care and Use Commit-

tee in accordance with the GSK Policy on the Care, Welfare

and Treatment of Laboratory Animals and were in accordance

with the Guide for the Care and Use of Laboratory Animals.

Kidney Sampling and Preparation

Kidneys from two mouse studies and one monkey study

were routinely processed in buffered formalin and embedded

in paraffin, with a section of right kidney cortex bisected and

half embedded in optimal cutting temperature (OCT) com-

pound and the other half processed for electron microscopy

as described subsequently. The cortex in the monkey was

collected separately for electron microscopy. Formalin-fixed

kidneys were embedded in paraffin wax, sectioned, and stained

with hematoxylin and eosin (H&E). For the periodic acid Schiff

(PAS) procedure, formalin-fixed paraffin-embedded (FFPE)

sections were placed on the Sakura DRS, deparaffinized,

rehydrated, and oxidized with 0.5% periodic acid and

incubated in cold PAS reagent (PolyScientific R&D Corp,

Bayshore, NY) for 15 min. Sections stained with Masson’s

trichrome or Congo red (Sigma Aldrich, St Louis, MO) were

placed on the Artisan Staining System (Dakocytomation,

Carpenteria, CA), deparaffinized, rehydrated, and either

oxidized (Gomori methenamine silver [GMS]) or treated with

mordant (Masson’s trichrome) overnight at room tempera-

ture. Separate sections were also stained with toluidine blue

to better define cytoplasmic granules. Age-matched normal

multitissue controls were included in each staining run and

examined to validate the procedure.

Immunohistochemistry

FFPE or OCT frozen-embedded mouse kidney sections

were placed on the Ventana Discovery XT System1 (Ventana

Medical Systems, Inc, Tucson, AZ), deparaffinized, and incu-

bated with primary antibody (rat anti-mouse CD68; Abcam

Ltd, Cambridge, MA, 0.1 mg/mL; rabbit anti–von Willebrand’s

factor [Vwf]; Dakocytomation, 1:1,000; rabbit antisynapto-

podin; Sigma, 3 mg/mL or guinea pig antinephrin; Progen

Biotechnik GmbH, Heidelberg, Germany, 10 mg/mL; and

anti-mouse immunoglobulin (Ig) G and IgM; Vector Labora-

tories, Inc, Burlingame, CA) for 1 hr. Slides stained for nephrin

were subjected to an avidin/biotin block prior to application of

primary antibody. Slides stained for synaptopodin, nephrin,

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and vWF were subjected to either an enzyme (protease 1) or

heat-induced epitope retrieval using an EDTA-based buffer

(CC1). Sections were incubated with appropriate secondary

horseradish peroxidase–conjugated antibodies and reacted with

DAB CMTM (Ventana Medical Systems, Inc) chromagen

before counterstaining with hemotoxylin. Age-matched normal

mouse multitissue controls were included in each staining run

and examined to validate the procedure.

Frozen OCT-embedded monkey kidney sections were cut

at 7 mm and incubated with the following primary antibodies:

C4d (LifeSpan Biosciences, Inc, Seattle, WA), C3c (Dako North

America, Inc, Carpinteria, CA), IgG (Dako), and IgM (AbD

Serotec, Raleigh, NC). A polymer-based chromogenic method

was used for C4d, and immunofluoresence (IF) was used for the

other antibodies using either direct (C3c fluorescein isothiocya-

nate [FITC]) or indirect methods (Alexa Fluor 488 conjugated

secondary antibodies). For IF staining, 40,6-diamidino-2-

phenylindole (DAPI) was used as a nuclear counterstain.

Transmission Electron Microscopy

Formalin-fixed tissues in paraffin-embedded blocks were

examined from selected control and high-dose mice from the

27-week mouse study. Blocks were melted, and the tissues

were removed for deparaffinizing in xylene. After rehydration

in serial alcohols and phosphate buffer, the samples were fixed

in 2.5% glutaraldehyde in phosphate buffer, postfixed in 1%osmium tetroxide, stained en bloc with uranyl acetate, dehy-

drated, and embedded in epoxy resin. In the investigative 8-

week mouse study and 39-week study in monkey, fresh kidney

was harvested at necropsy. A portion of 1 kidney from selected

animals, including controls, in all 3 studies was trimmed to

approximately 1 mm3 pieces and fixed with 2.5% glutaralde-

hyde/2% formaldehyde in 0.1 M cacodylate buffer for 24 hr,

postfixed with osmium tetroxide, dehydrated, and embedded

in epoxy resin. Sections were cut, stained with uranyl acetate

and lead citrate, and examined with either an FEI Tecnai

20 transmission electron microscope operating at 120 kV or a

Hitachi 7500 transmission electron microscope operating at

80 kV. Representative digital images were captured using

either a Gatan UltraScanTM 1000 (Gatan, Inc, Pleasanton,

CA) or an AMT XR41TM camera (for transmission electron

microscopy), Woburn, MA.

Gene Expression Analysis (TaqManTM)

Mouse kidneys were separated into 3 groups: controls, those

given 300 mg/kg/injection of drug with histologic evidence of

glomerular injury, and those given 300 mg/kg drug without evi-

dence of glomerular injury by routine microscopic examina-

tion. A portion of fresh kidney from mice treated for 8 weeks

was placed in OCT media. Eight 10-mm OCT sections from 6

control animals, 6 histologically affected treated animals, and

3 histologically unaffected treated were cut onto slides using

an RNase/DNase-free microtome. Excess OCT was removed

from the slides using a sterile razor before the tissue was

scraped into a labeled, sterile 1.5-mL microcentrifuge tube. All

samples were homogenized for *1 min in 300 ml of working

lysis buffer. RNA was isolated and concentrated using the

Absolutely RNA Microprep Kit and the RNA Clean & Concen-

tratorTM (Products Division, La Jolla, CA) with quality assess-

ment using Agilent RNA 6000 Bioanalyzer (Agilent

Technologies, Inc, Santa Clara, CA) and quantity assessment

using the Quant-iTTM RiboGreen1 Kit. The RNA was quanti-

fied using the Quant-iT RiboGreen Kit (Invitrogen, Carlsbad,

CA) and qualitated using the Agilent RNA 6000 Nano

Reagents and Analyzer (Agilent Technologies). Complemen-

tary DNA (cDNA) was created from the RNA using the

High-Capacity RNA-to-cDNA Kit and then was loaded onto

the Taqman1 Gene Signature Immune Array for gene analysis.

The Taqman raw data were evaluated qualitatively using RQ

Manager Software and quantitatively assessed using the Data

AssistTM Software (Life Technologies, Grand Island, NY)

using the comparative CT (DDCT) method for calculating

relative quantitation of gene expression. The Ct values for all

samples were normalized to 18S, and b-actin and the fold

changes were created by comparing the control to treated. Taq-

Man Universal PCR Master Mix (2�; Applied Biosystems,

Foster City, CA) and 7900HT Real-Time Fast PCR System

(Applied Biosystems) were used to perform TaqMan Analysis

according to the manufacturer instructions. TaqMan was

performed for each gene using 50 ng of amplified single-

stranded cDNA as previously described (Dalmas et al. 2005,

2008). Negative controls included a no template control for

each gene of interest. Data were evaluated using the DDCT

method and as described in Applied Biosystems User Bulletin

2: ABI Prism 7700 Sequence Detection System and reported as

fold change relative to respective controls.

RESULTS

In general, the results of these nonclinical studies were

consistent with the results from other AON compounds and

have been well described in the literature (Henry et al. 2008;

Monteith and Levin 1999) and for brevity therefore will not

be presented in this article except where relevant to the kidney

findings.

Macroscopic Pathology and Light Microscopy

Drisapersen administration induced renal findings typical of

the class of (modified) phosphorothioate AONs. These findings

were evident against a class-related background of widespread

treatment-related inflammatory activity typified by activated

granular macrophages in multiple tissues.

Mouse

Mice given 300 mg/kg/injection drisapersen for 8 weeks had

notable renal pathology. Minimal to mild basophilic granules

were seen within the proximal convoluted tubules, and in a few

animals, there was also minimal secondary degeneration of the

tubular epithelium. These tubular changes consisted of baso-

philia, rare cell sloughing, and occasional dilation of tubules.

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Glomerular changes were only noted in 6 of the 22 animals by

routine H&E staining and were characterized by slightly

increased mesangial matrix, increased glomerular cellularity,

occasional inflammatory cells or nuclear debris, and rare intra-

glomerular basophilic granules (Figure 1A–F). Basophilic

granules were better visualized using toluidine blue staining

as compared to routine H&E. In addition to granules within

most proximal tubules corresponding to those noted with

H&E, toluidine blue staining also revealed occasional dark

blue-staining granules within endothelial cells, podocytes, or

mesangial cells of glomeruli. The glomeruli were negative for

amyloid by Congo red stains and negative for fibrosis/collagen

deposition by Masson’s trichrome stains. GMS and PAS stains

both demonstrated slightly thickened basement membranes in the

glomeruli of a few mice. No renal effects were seen at 100 mg/kg.

Mice given 72 mg/kg/injection drisapersen for 27 weeks had

tubular basophilic granules and some secondary degenerative

changes associated with drug accumulation as well as findings

in the glomeruli and papilla. Increased glomerular mesangial

matrix was characterized by diffuse, segmental to global accu-

mulation of a homogenous, eosinophilic material in the glo-

merular tufts with normal or decreased glomerular cellularity.

Weakly positive Congo red stains and minimally to mildly

increased Masson’s trichrome and PAS staining at the

27-week time point suggested this matrix was a mixture of

amyloid, fibrosis, and other hyaline material. Minimal to

marked papillary necrosis was also noted in a few mice in asso-

ciation with amyloid deposits within the interstitium. Two mice

that died or were euthanized prior to the end of 27 weeks of

treatment had marked bilateral papillary necrosis, with loss

of collecting ducts, thin loops of Henle, and vasa recta through-

out the distal portion of the medullary papilla. These marked

lesions were associated with casts, marked tubular basophi-

lia/atrophy in the cortex, minimal single-cell necrosis of tubu-

lar cells and slight tubular dilation (ascending necrosis from

functional nephron loss), and the presence of tubules lined by

low cuboidal epithelium with cytoplasmic basophilia, nuclear

crowding, and occasional mitoses (regeneration and cellular

repair processes). An increased incidence of tubular cysts was

considered a consequence of the chronic degenerative changes.

Basophilic granules were also noted in the proximal tubules of

most mice given 18 mg/kg/injection, but there was no evidence

of degenerative tubular changes or glomerular lesions in these

kidneys. Activated granular macrophages were found within

multiple organs of mice given �18 mg/kg/injection, including

the kidney. No drug-related renal changes were noted (includ-

ing no basophilic granules) in mice given 6 mg/kg/injection.

At the end of the 20-week off-dose period, the glomerular

changes in mice given 72 mg/kg/injection for 27 weeks were

more severe than seen in those killed at the end of the treatment

period, with mild to marked matrix deposition and loss of cel-

lularity. These changes were associated with moderate to

marked papillary edema and necrosis, interstitial amyloidosis,

and tubular degeneration. Tubular basophilic granules were

still noted at the end of the off-dose period and a few contained

activated granular macrophages. Tubular basophilic granules

were rare at the end of 35 weeks’ off-treatment period in mice

given 18 mg/kg/injection but treatment-related changes in the

glomeruli (increased matrix) were noted with a higher inci-

dence (6 of 10 vs. 3 of 10) and severity as compared to controls

and similar staining characteristics to animals given 72 mg/kg/

injection. In contrast, mice given 6 mg/kg/injection and sacri-

ficed after 35 weeks’ off-treatment period had glomerular

changes of similar incidence (3 of 10), severity, and character

(minimally thickened membranes in tufts only) as controls and

were therefore considered most likely spontaneous, age-related

glomerular membrane changes. H&E and toluidine blue staining

of the slides from the most severely affected mice demonstrated

marked global diffuse glomerular changes including enlarged

acellular glomeruli filled with homogenous material that stained

eosinophilic with H&E or PAS stains and light blue with Tolui-

dine blue (Figure 1A and B). Some small areas of light pink con-

gophilic material were noted in glomeruli and in the interstitium

with Congo red stains, but green birefringence was largely neg-

ative under polarized light, suggesting amyloid is not the princi-

pal accumulating component. Seven mice given 72 mg/kg/

injection died during the off-dose period, with most having mod-

erate to severe kidney lesions and/or amyloid-related papillary

necrosis, which were considered the cause of death. As noted

previously, minimal amyloid was also noted in the kidneys of

a few control mice at the end of the off-dose period with special

stains in conjunction with age-related glomerular changes.

Monkey

In addition to the presence of basophilic granules in tubular

epithelial cells (without secondary degenerative tubular chan-

ges), there was minimal to slight glomerulopathy noted in 2

of the 6 monkeys given 12 mg/kg/injection and 4 of the 8 mon-

keys given 6 mg/kg/injection for 39 weeks. This change was

multifocal or diffuse and segmental to global. The glomeruli

were enlarged and characterized by variably increased cellular-

ity of the tufts and increased mesangium. Some basophilic

granules and small numbers of neutrophils were occasionally

present in the affected glomeruli. At the end of the 39-week

off-treatment period, minimal thickening of the mesangium

was noted in only a single monkey given 6 mg/kg/injection,

with no other accompanying renal changes, suggesting at least

partial reversibility of the glomerular lesion. In addition, there

was a marked decrease in the incidence and severity of the

basophilic granules in tubular epithelial cells, as they were

noted largely confined to macrophages of the lymph nodes and

rarely in macrophages in other organs.

There were no significant alterations in routine clinical chem-

istry parameters referable to the kidney at any time during the

study, but there were significant changes in systemic proinflam-

matory markers as noted subsequently. Neither quantitiative nor

qualitative urinalysis mean parameters were significantly altered

in treated versus control monkeys for the duration of the study.

At week 39, only a single animal with minimal glomerulopathy

(observed at the end of the off-dose period) had increases in urin-

ary albumin levels.

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FIGURE 1.—Mouse kidney. (A) H&E control mouse kidney with normal glomeruli. (B) H&E. Note marked accumulation of eosinophilic material

effacing glomeruli in mouse given 72 mg/kg/injection for 27 weeks with 20-week off-treatment period. (C) PAS of control mouse kidney, with

negative staining of glomeruli. (D) Note PAS-positive material in the glomeruli of a mouse given 72 mg/kg/injection for 27 weeks with 20-week

off-treatment period. (E) Immunohistochemical stain for IgG/IgM in mouse control kidney, demonstrating generally negative or very light back-

ground staining in glomeruli. (F) IgG/IgM immunostaining in mouse given 72 mg/kg/injection drisapersen for 27 weeks with 8-week off-

treatment period. Note strong dark staining in glomeruli, consistent with murine hyaline glomerulopathy as well as staining in interstitium.

H&E ¼ hematoxylin and eosin; Ig ¼ immunoglobulin; PAS ¼ periodic acid Schiff.

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Electron Microscopy

Mouse

Ultrastructural examination of the glomeruli from mice

given 300 mg/kg/injection drisapersen for 8 weeks revealed

occasionally thickened and irregular endothelial cell linings,

although fenestrations were maintained. Endothelial cells

were hypertrophied with expanded cytoplasm and/or pyknotic

nuclei. Frequently, endothelial cells, mesangial cells, and

podocytes had numerous membrane-bound lysosomal vesicles

containing electron-dense material. Basement membranes were

mildly thickened and contained cellular debris and occasionally

demonstrated longitudinal ‘‘layering’’ or ‘‘splitting’’ and/or

reduplication. Rarely, the basement membrane and intercellular

junctions between the endothelium and the podocytes contained

electron-dense material, and electron-dense deposit (EDD) and

electron-lucent deposit were also seen adjacent to these areas

of altered glomerular basement membrane. No ultrastructural

abnormalities were detected in the kidneys of the control mice

after 8 weeks of dosing.

Ultrastructurally, the glomeruli of some mice treated with

72 mg/kg/injection drisapersen for 27 weeks were effaced by

curvilinear fibrils of approximately 12 to 15 nm in diameter

and approximately 200 to 800 nm in length, sometimes in

layers, whorls, or fingerprint patterns, and consistent with the

murine syndrome of hyaline glomerulopathy (Figure 2). These

fibrils were noted along basement membranes and filled with

the mesangium, separating remnant cell populations and effa-

cing glomerular tufts. Glomeruli generally lacked cellularity,

and some less affected glomeruli contained slightly thickened

basement membranes and rare EDDs. In rare areas of a few glo-

meruli, there were masses of randomly arranged straight fibrils

7 to 10 nm wide of various lengths embedded in a granular

matrix and consistent with amyloid (Figure 2C). Collagen

fibers were noted rarely in glomeruli, distributed in a few col-

lagen fibrils between mesangial cells and their associated base-

ment membrane. Tubules contained intralysosomal inclusions

within proximal epithelial cells. No findings were noted in the

glomeruli of controls examined ultrastructurally.

Monkey

Ultrastructural examination of the kidneys from monkeys

treated with drisapersen for 39 weeks demonstrated a spectrum

of changes that varied slightly between animals (Figure 3A–F).

There was an increased cellularity within the glomeruli charac-

terized by an increased number of mesangial cells, with occa-

sional infiltration of inflammatory cells including mononuclear

cells (lymphocytes, macrophages, and plasma cells) and rare

neutrophils, and this was accompanied by hypertrophy of the

endothelial cells, and less commonly, hypertrophy of podo-

cytes. The plump endothelial cells often contained tubuloreti-

cular bodies and/or enlarged lysosomes. The mesangium was

thickened, and there were intramembranous and subepithelial

amorphous, finely granular, EDDs noted along the basement

membrane, which resulted in some areas with effacement of

podocytes and foot processes. Dense lysosomal inclusion bod-

ies were occasionally noted within the cytoplasm of multiple

cell types of the glomeruli. Osmiophilic lysosomal inclusion

bodies were also noted in proximal tubule epithelium, typical

of those associated with the basophilic granules of drug sub-

stance. Vacuolation of podocytes was rarely observed. There

were no significant ultrastructural abnormalities noted in kid-

neys from control monkeys.

Immunohistochemistry

Mouse

No differences between controls and mice given 300 mg/kg

drisapersen for 8 weeks were noted for immunohistochemical

stains for nephrin or synaptopodin, suggesting that podocytes

were not significantly affected by the compound at this time

point. Immunohistochemical stains for vWF, which stains glo-

merular and interstitial endothelial cells, demonstrated increased

staining within glomeruli in several mice given 300 mg/kg as

compared to the controls. These areas of increased staining

likely corresponded to areas of endothelial hypertrophy, degen-

eration, or pooling of vWF protein within cytoplasmic spaces.

CD68 immunohistochemical staining was markedly positive in

treated mouse kidneys, including those with no histologic evi-

dence of glomerular changes, while controls were largely neg-

ative for the same marker. Staining was most prominent in the

peritubular interstitium, with only minimal staining within

glomeruli. Positive CD68 immunostaining correlated with in-

creased CD68 mRNA in the genomic data.

In mice given drisapersen for 27 weeks, IHC was strongly

positive for IgG/IgM throughout the affected glomeruli of

treated mice as well as within the cortical interstitium and ves-

sels (Figure 1E and F). Control mice shared some of the inter-

stitial and vascular staining but generally lacked staining of

glomerular elements other than endothelium.

Monkey

In monkey, anti-C3c IHC revealed an increase in linear,

granular-type staining within the glomeruli of test article-

treated animals (Figure 4A–D). This was particularly prominent

in 3 monkeys given 6 mg/kg/injection. IHC staining with anti-

C4d, IgG, and IgM demonstrated no difference in glomerular

staining between control and GSK2402968-treated animals

Gene Expression

In mice given 300 mg/kg/injection drisapersen for 8 weeks,

an increase in immune response was seen in the following

genes: IL-10, Ccl3, Ccl2, IL-6, IL-1a, Gzmb, Cxcl10, Cd68,

IL-17, Tnf, IL-1b, Ptprc, Cd4, Nos2, Stat1, Ifng, Ccl5, Csf2,

Socs1, Ctla4, Csf3, Vcam1, Csf1, C3, B2m, IL-12a, Sele, Cd8a,

and Ptgs2 based on at least a 2-fold change in message when

normalized to 18S and b-actin (Figure 5). The histologically

unaffected (no evidence of glomerular injury) treated group

followed the same trend as the histologically affected treated

but with slightly less magnitude.

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FIGURE 2.—Transmission electron micrographs of glomeruli of a control mouse (A) and a mouse given 72 mg/kg/injection for 27 weeks followed

by an 8-week off-treatment period (B). Note the intact basement membrane (BM) surrounding acellular areas within tufts effaced by bundles and

whorls of curvilinear fibrils (*) have replaced normal architecture. Note the characteristic randomly arranged straight fibrils of amyloid measuring

7 to 10 nM in diameter within granular background observed in a treated mouse (C). Higher magnification (D) of curvilinear fibrils (*) in treated

mice revealed *15 nM diameter lamellae characteristic of mouse hyaline glomerulopathy. Both hyaline glomerulopathy (arrows) and minimal

collagen accumulation characterized by larger diameter banded fibers (arrowhead) were observed (E). (F) Electron-dense deposits and redundant

or remnant membranes on the subendothelial side of the basement membrane were seen in the treated mice (arrows). A ¼ amyloid fibrils; BM ¼basement membrane; BS ¼ Bowman’s space; MC ¼ mesangial cell; Pa ¼ parietal epithelial cell; PC ¼ podocyte.

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FIGURE 3.—Electron microscopy of monkey kidneys in the 39-week toxicity study. (A and B) Normal glomerulus from vehicle control. (C and D)

Glomerulus from monkey treated with drisapersen for 39 weeks. Note subepithelial-dense deposits (arrowheads) within and along the thickened

basement membrane characteristic of immune deposition. (E and F) Glomerulus from monkey treated with drisapersen for 39 weeks. Note

endothelial tubuloreticular bodies (arrows), dilated lysosomes, and an osmiophilic, membrane bound, electron-dense deposit (EDD), potentially

representing drug accumulation, adjacent to the nucleus. BM ¼ basement membrane; EC ¼ endothelial cell; FP ¼ foot processes (podocyte ped-

icels); P ¼ podocyte; RBC ¼ red blood cell.

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Systemic Inflammatory Markers

Mice

In mice treated for 27 weeks, there was evidence of systemic

inflammatory activity in the form of a dose-related increase in

MCP-1 at doses of 6 mg/kg and more (Table 1). IL-6 was unaf-

fected (data not shown). Significant increases in MCP-1 levels

were noted in all samples from mice given 72 mg/kg/injection

in weeks 5 to 26 at all time points. Increased MCP-1 was also

noted in weeks 5 and 13 in mice given 18 mg/kg/injection, and

in week 26 in mice given 6 mg/kg/injection. Levels of MCP-1 and

number of animals affected increased with prolonged duration of

treatment at all dose levels. By the end of the off-dose period,

MCP-1 levels were below the limit of quantitation (LOQ) in all

animals. Values for control animals always fell below the LOQ.

Monkey

In the 39-week study in monkeys, evidence of an inflamma-

tory response was initially noted at 12 mg/kg/injection with the

earliest significant changes occurring in MCP-1 at week 4

(Table 2). Mild to marked increases in MCP-1 were noted at

multiple time points in all monkeys given 12 mg/kg/injection,

but MCP-1 serum levels in control monkeys generally hovered

near the baseline (Table 2 and Figure 6). By week 26, most

monkeys given �6 mg/kg/injection demonstrated moderate

to marked increases in multiple systemic serum inflammatory

markers as compared to pretreatment values. In monkeys

given 12 mg/kg/injection, maximal increases were noted in

C-reactive protein (CRP; 36-fold), MCP-1 (16-fold), haptoglo-

bin (5.5-fold), and fibrinogen (2.4-fold) and maximal decreases

were noted in the albumin/globulin ratio (0.46-fold). Animals

given 6 mg/kg/injection had correspondingly less pronounced

increases (CRP increase of 1.93-fold and haptoglobin increase

of 1.5-fold). There was a reproducible trend for lower mean

complement activation (CH50) and higher complement frac-

tion (C3a) during the course of the study in monkeys given

6 or 12 mg/kg/injection. Reversibility was seen after the

off-dose period.

FIGURE 4.—Kidney sections of monkey stained with C3C (A) or FITC-conjugated isotype control antibody (B) compared with kidney sections of

monkey treated with 6 mg/kg drisapersen for 39 weeks stained with C3C (C) or FITC-conjugated isotype control antibody (D). FITC staining

green, DAPI (nuclear) staining blue. Note moderate positive C3C staining only in (C). FITC ¼ fluorescein isothiocyanate; DAPI ¼ 40,6-diami-

dino-2-phenylindole.

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DISCUSSION

Due to the overwhelming past experience of AON-related

kidney toxicity being derived from subacute and subchronic

studies, the descriptions and proposed pathogenesis have

largely focused on tubular effects (Henry et al. 1999, 2008;

Marquis and Grindel 2000; Monteith et al. 1999). AONs are

primarily excreted by the kidneys and accumulate within

the proximal tubule cell lysosomes, resulting in susbstantial

renal tissue levels. In the toxicity studies with GSK2402968

described in this publication, lysosomal drug accumulation in

the form of basophilic granules was abundant within proximal

tubular epithelium of kidneys in both mice and monkeys, with

slight tubular degenerative changes seen only in mice. In con-

trast, basophilic granules within glomeruli were not commonly

identified at light microscopy in either mice or monkeys given

drisapersen.

Minimal glomerular changes noted in several mice given

300 mg/kg/injection as early as 8 weeks after starting AON

treatment were not associated with any evidence of matrix

accumulation. Only at or after treatment of mice for 27 weeks

did glomeruli demonstrate significant matrix accumulation and

loss of cellularity, and these lesions progressed further during

the off-dose period, indicating that matrix effects in mice are

chronic, irreversible sequelae to initial glomerular injury. Glo-

merular lesions in monkeys given drisapersen for 39 weeks

were much less severe than in the mouse, lacking the pro-

nounced matrix accumulation and demonstrating (partial)

reversibility following an off-dose period (see Figure 3 for

comparison of species). Drisapersen has an elimination half-

life of approximately 27 days based on the mean plasma data,

and therefore it should be noted that exposure continued during

the off-dose period.

Glomerular matrix accumulation in chronically treated mice

was predominantly composed of fibrils of *15 nM diameter

that are characteristic of murine hyaline glomerulopathy. The

hyaline glomerulopathy was confirmed by negative Congo red

stains, positive PAS, Masson’s trichrome, and strong positive

IgG/IgM immunostaining. Similar fibrils and fingerprint-like

patterns have been noted as a spontaneous age-related lesion

in mice that are considered to be formed from Ig fragments

(Frazier et al. 2012; Wojcinski, Albassam, and Smith 1991).

In the spontaneous form, immunological processes in normal

aging mice produce circulating antigen: Ig complexes that

localize in kidneys along glomerular capillary walls and are

0

1

10

100

Il10

Ccl3

Ccl2 Il6 Il1a

Gzm

bCx

cl10

Cd68 Il1

7Tn

fIl1

bPt

prc

Cd4

Nos

2St

at1

Ifng

Ccl5

Csf2

Socs

1Ct

la4

Csf3

Vcam

1Cs

f1 C3B2

mIl1

2aSe

leCd

8aPt

gs2

log

Fold

Cha

nge

Affected Treated Unaffected Treated

FIGURE 5.—Relative mRNA levels after extraction from mouse renal cortex. Immune response observed in the affected treated and unaffected

treated when normalized to 18S and b-actin when compared to controls. mRNA ¼ messenger RNA.

TABLE 1.—Mean MCP-1 serum levels in mice given drisapersen for

27 weeks.

Dose level (mg/kg/injection) 0 6 18 72

Week 5

4 hr — 0 0 834 (4/6)

24 hr 0 0 189 (2/7) 832 (7/7)

96 hr 0 0 0 639 (6/7)

Week 13

Predose — 0 341 (6/11) 658 (8/11)

24 hr 0 0 386 (5/11) 979 (11/11)

Week 26

Predose — 355 (5/11) 808 (9/11) 1161 (10/11)

24 hr 0 339 (5/11) 690 (9/11) 1250 (9/9)

Note: Incidence of values above limit of quantitation (LOQ) in parentheses.

10 FRAZIER ET AL. TOXICOLOGIC PATHOLOGY

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only cleared slowly (if at all) through phagocytosis by mesan-

gial cells (Linder, Pasternack, and Edgington 1972). There is a

distinct human syndrome of immunotactoid glomerulopathy,

which has also been referred to as hyaline glomerulopathy, but

it is characterized by larger sized (20–30 nM) microtubular

structures within the mesangium and therefore is unrelated to

the syndrome in mice (Schwartz 2007b). PAS-positive hyaline

fragments have also been noted in some cases of focal segmen-

tal glomerulosclerosis (FSGS) in people, but FSGS lacks other

morphologic features of the mouse syndrome. Although anti-

body fragments are noted in human diseases such as light chain

amyloidosis and Bence Jones protein glomerulopathy with

myeloma, the ultrastructural morphology and staining charac-

teristics are also quite different, and murine hyaline glomerulo-

pathy is therefore considered a rodent-specific syndrome

(Frazier et al. 2012; Wojcinski, Albassam, and Smith 1991).

This is further supported by the absence of glomerular matrix

accumulation in monkeys receiving chronic treatment with

drisapersen.

Amyloidosis is one of the major disorders of aging mice

(especially CD-1 strains), where the kidney is frequently a tar-

get organ. Although some amyloid accumulation may be

expected in mice of this age, it is more likely that, like hyaline

glomerulopathy, interstitial amyloidosis is a result of the

ongoing, treatment-related, immune stimulation in these mice.

Existing amyloid fibrils can act as a seed for further amyloid

progression, and many factors including age, strain, concurrent

inflammation, and amyloid/precursor protein levels in the

serum can all affect fibril formation and deposition within tis-

sues (Gise, Christ, and Bohle 1981). The combination of glo-

merular dysfunction and interstitial deposition of amyloidosis

likely resulted in the secondary papillary changes that occurred

with drisapersen and resulted in premature deaths. Interstitial

amyloidosis is a well-recognized cause of papillary necrosis

in mice (Frazier et al. 2012), possibly related to ischemia of the

distal medulla via progressive loss of the vascular supply

through occlusion of the medullary vasculature (Frazier and

Seely 2013). Lysosomes are intimately linked with amyloid

fibril formation and provide an environment conducive to the

transition from helical to b-pleated sheet structure. Hence, there

may be some connection between lysosomal accumulation of

AONs and the eventual concentration of amyloidosis in the

cortex and medulla. The amyloidosis and papillary necrosis

seen in mice are unlikely to occur following clinical dosing

as amyloid is not deposited in human kidneys in response to

inflammatory activity without concurrent genetic (familial)

predisposing abnormalities (Faccini, Abbot, and Paulus 1990).

TABLE 2.—Serum MCP-1 data in control monkeys and those given 12 mg/kg/injection drisapersen.

Animal number Wk 1 Wk 4 Wk 9 Wk 13 Wk 17 Wk 22 Wk 26 Wk 30 Wk 35 Wk 39

Grp1-1 241 127 185 227 185 215 258 187 202 144

Grp1-2 563 324 277 247 329 100 247 257 321 201

Grp1-3 294 134 207 153 192 292 259 219 237 213

Grp1-4 153 262 266 272 242 374 326 258 294 244

Grp1-5 234 225 195 192 195 185 142 136 161 280

Grp1-6 228 200 257 174 180 218 389 252 209 152

Grp4-1 228 1000 300 443 1300 614 520 1674 636 373

Grp4-2 306 448 235 144 916 285 215 328 364 314

Grp4-3 284 483 479 352 503 1441 1327 NA NA NA

Grp4-4 313 667 463 1195 204 101 177 419 184 179

Grp4-5 88 505 122 200 433 382 315 646 666 NA

Grp4-6 169 540 317 362 1878 372 398 1921 305 146

Grp4-7 396 1762 633 843 1295 960 885 1135 659 525

Grp4-8 648 1096 767 2451 2286 1649 1835 1908 876 1051

Grp4-9 298 586 617 552 1639 593 615 1405 627 366

Gp4-10 292 616 180 437 935 554 493 654 387 407

Gp4-11 586 1389 694 3903 823 567 188 423 443 350

Note: Grp1 ¼ control monkeys; Grp4 ¼ monkeys treated with 12 mg/kg/injection drisapersen; NA ¼ sample not available.

FIGURE 6.—Mean serum MCP-1 values of control (group 1) and

monkeys treated with 12 mg/kg/injection Drisapersen (group 4) for

39 weeks. MCP-1 ¼ monocyte chemotactic protein 1.

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Further support for an immune-based mechanism in mice is

provided by marked upregulation of many immune system-

related cytokines (noted in the murine renal microarray data

and increased CD68 immunostaining) and the increased sys-

temic cytokine levels in animals treated for 8 weeks. The proin-

flammatory potential of AONs is well recognized (Henry et al.

1999), with known quantitative differences based on the back-

bone structure and/or base sequence (Krieg 1998). Immunomo-

dulatory effects involve stimulation of multiple receptors of the

innate immune system, such as Toll-like receptors (TLRs),

leading to stimulation of the innate immune system with resul-

tant release of cytokines and chemokines and complement acti-

vation via the alternative pathway. Cellular patterns of TLR

expression vary widely between different species, such that

results of TLR stimulation preclinically may not be predictive

of what will occur in humans or even in another preclinical spe-

cies (Richardt-Pargmann and Vollmer 2009), and this may be

an explanation for the somewhat different histomorphologic

and systemic expression of inflammatory stimuli between mice

and monkeys in our studies.

The initial cellular site of injury in the mouse glomerulus

after 8 weeks of treatment appears to be the glomerular

endothelial cell, rather than the podocyte or mesangial cell. The

endocapillary endothelial cell represents a potential target for

circulating cytokine-mediated injury, whereas podocytes

(which appeared much less affected in mice at this early stage)

are affected only secondarily via basement membrane effects.

Alterations in the basal lamina include the presence of EDDs

along the basement membrane. This type of change is indica-

tive of an immune-mediated pathogenesis of glomerular injury

(Jones et al. 1984; Sachs, Zhous, and Sheerin 1996) rather than

reflecting direct nephrocytoxicity. The marked increase in

CD68 immunostaining correlated with increased CD68 gene

data in mice, demonstrating concordance of genomic and pro-

teomic upregulation. Importantly, many of the upregulated

genes including CD68 have been associated with both TLR

activity and amyloid formation, suggesting that immune stimu-

lation has both an important role in the early pathogenesis of

glomerular injury in the mouse and a possible role in the ten-

dency for the mouse to later develop progressive hyaline glo-

merulopathy and renal amyloidosis.

The glomerular changes in the monkey are less pronounced

than in the mouse and resemble features of human syndromes of

both membranous (MGN) and membranoproliferative glomeru-

lonephritis (MPGN). MGN is characterized by intramembranous

and subepithelial EDDs of complement and complement frag-

ments and has been associated with a variety of drug-induced

glomerulopathies, including those due to penicillamine, cap-

topril, and lithium (Schwartz 2007a, 2007b). Thickened tuft

walls, increased mesangial matrix, and hypercellularity are all

features shared between drisapersen-treated monkeys and the

human diseases. In particular, tubuloreticular structures within

endothelium noted in monkey have been identified previously

in human cases of idiopathic and hepatitis B-related MGN

(Schwartz 2007a). Inflammatory cell infiltrates, as seen in the

monkey glomeruli, are however generally absent in human

MGN but are a recognized feature of human syndromes of

MPGN.

The glomerular lesions in the monkey stain positively for

complement C3 fragments but not for Igs or complement C4

fragments. This differential staining pattern is a common fea-

ture of C3 glomerulopathies described in man (Barbour, Pick-

ering, and Cook 2013; Pickering and Cook 2008) and animal

models of factor H deficiency. The pathogenesis is linked to

dysfunction of the alternative complement pathway via inter-

ference with a key regulator protein, factor H (e.g., through

genetic defects and/or autoantibodies). It is noteworthy that the

proinflammatory effects of AONs in the monkey are character-

ized by selective activation of the alternative complement path-

way, through transient inhibition of factor H (Henry et al. 1997,

2008), leading to increased circulating complement split fac-

tors and resulting in a progressive decline in complement C3

activity upon chronic treatment. The commonality in dysfunc-

tion of factor H and specific morphologic characteristics of the

kidney pathology (i.e., dense deposits, selective C3 staining

with no Ig staining) between the monkey and C3 glomerulopa-

thies recognized in man and animal models of factor H defi-

ciency makes it tempting to classify the glomerular lesions in

the monkey as a C3 glomerulopathy. Monkeys are particularly

sensitive to AON-induced complement activation (Kwoh

2007) and a similar direct AON-induced complement activa-

tion has not been observed in humans or other species.

Proteinuria has been occasionally noted in animals as well

as in humans in clinical trials with AONs (Rao et al. 2004).

Lysosomal accumulation of drug within tubules is an expected

consequence of AON therapies and at high doses can induce

mild, reversible tubular injury. Tubular effects and proteinuria

may therefore potentially occur in patients, but it should not be

assumed that glomerular injury will also accompany tubular

changes when proteinuria is observed. Given the distinct

mechanisms of injury between tubular degeneration and glo-

merular lesions associated with AON administration, the two

types of renal lesions probably occur independently. It is

important clinically to attempt to distinguish between tubular

and glomerular injury when proteinuria is identified in a patient

receiving AON treatment. Large increases (e.g., >1.5 g/L) or

the presence of large molecular weight proteins in urine may

aid in identifying glomerular origin and signal a risk of clinical

glomerulonephritis. A kidney biopsy would then normally be

considered. The characteristic expression of glomerulopathy

may well depend on the inflammatory response specific for

each species, and this differs both in character and in magni-

tude between humans and laboratory animals with AON treat-

ment (Henry et al. 1997, 2008; Monteith et al. 1999). A lower

risk of glomerular injury in patients with this type of therapy

may therefore parallel lower proinflammatory activity in

humans as compared to either mice or monkeys. However, clin-

ical risk assessment necessitates monitoring, as in other cases

where preclinical renal risks are identified. Since glomerular

damage in preclinical species appears to be so intimately related

to immune pathogenesis, monitoring systemic inflammatory

activity/markers in addition to routine renal and glomerular

12 FRAZIER ET AL. TOXICOLOGIC PATHOLOGY

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functional assays is essential in AON clinical trials to assess the

potential for drug-related glomerular injury.

ACKNOWLEDGMENTS

The authors wish to thank Jan Kane, Roberta Thomas, Deon

Hildebrand, and Anna Hughes for technical assistance and

especially the staff at CiToxLab and HLS for their help in run-

ning the 9-month monkey toxicity study, 6-month mouse toxi-

city study, and male fertility study in mice.

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