Toxicological Characterization of Exon Skipping Phosphorodiamidate Morpholino Oligomers (PMOs) in Non-human Primates

Abstract

Background: Phosphorodiamidate morpholino oligomers (PMOs) are a class of exon skipping drugs including eteplirsen, which has shown considerable promise for treatment of the degenerative neuromuscular disease, Duchenne musculardystrophy (DMD).

Objective: Toxicity studies in non-human primates (NHPs) of 12 weeks duration with two new PMOs for DMD, SRP-4045 and SRP-4053, along with results from a chronic study in NHPs of 39 weeks duration for eteplirsen, are described here.

Methods: PMOs were administered once-weekly by bolus intravenous (IV) injections to male NHPs. Endpoints evaluated included plasma exposures, clinical observations, body weight/food consumption, eye exams, electrocardiograms, male reproductive hormones/endpoints, complement alternative pathway, clinical pathology, urinalysis, and macroscopic/light microscopic pathology.

Results: Findings in these studies were limited to the kidneys, with a common presentation of tubular basophilia, vacuolation, and/or minimal degeneration that was considered non-adverse. No necrosis, glomerular lesions, or effects on renal function tests such as serum creatinine or urea nitrogen were observed, suggesting that PMO-related kidney findings are not likely to develop into frank nephrotoxicity. There were no adverse effects on other potential target organs after repeated IV injections at the highest dose levels tested, 320 mg/kg.

Conclusions: Nonclinical results in NHPs for these three PMOs, together with the excellent clinical safety established for eteplirsen to date, suggest that once-weekly IV administration of PMOs for lifetime durations at therapeutic doses will be well tolerated by patients with DMD.

Keywords

Duchenne muscular dystrophy eteplirsen AVI-4658 phosphorodiamidate morpholino oligomers exon skipping splice switching oligonucleotides toxicity studies non-human primates kidney toxicity

INTRODUCTION

PMOs are a class of exon skipping drugs considered to be promising, disease-modifying, therapies to treat DMD, an X-linked, degenerative, neuromuscular disorder with a worldwide incidence of approximately 1 in 3,500 newborn boys [1 –7]. DMD is caused by mutations in the dystrophin gene that disrupt the reading frame of the encoded dystrophin messenger ribonucleic acid (mRNA), preventing the translation of functional dystrophin protein, which is critical to the structural stability of myofibers in skeletal, smooth, and cardiac muscle [5, 7]. Approximately 80% of DMD patients have mutations that may be addressed by the mechanism of exon skipping during mRNA processing [1 , 8]. Eteplirsen (AVI-4658), SRP–4045, and SRP-4053 are PMOs with nucleobase sequences designed to skip exons 51, 45, and 53, respectively. Skipping of these three exons is expected to restore the dystrophin mRNA reading frame, resulting in production of shortened but functional dystrophin proteins in patients with certain dystrophin gene mutations, and provide a therapeutic benefit to approximately 30% of the DMD patient population [8].

The molecular structure and the nucleobase sequence of the most extensively characterized PMO to date, eteplirsen, has been previously published [9]. Eteplirsen was shown to have the desired pharmacodynamic effect (exon 51 skipping) in vitro and in vivo [10]. In proof-of-concept clinical trials in which boys with DMD who had dystrophin mutations amenable to exon 51 skipping were given single doses of eteplirsen, either by intramuscular (IM) injection or once weekly by intravenous (IV) infusion for 12 weeks, eteplirsen demonstrated the expected pharmacodynamic effect of exon 51 skipping and restored dystrophin expression, resulting in improvements in the various clinical outcomes assessed [11 –14].

In order to support first-in-human trials of two related PMO drug candidates, as well as to support the continuing clinical development of eteplirsen, 12-week toxicity studies in NHPs with SRP-4045 and SRP-4053 and a 39-week chronic study with eteplirsen in NHPs were recently completed and are reported here. SRP-4045 and SRP-4053 are structurally similar to eteplirsen, with the same morphilinyl groups and uncharged phosphorodiamidate linkages in their backbones. The main differences are in the number and combination of nucleobases that comprise their unique sequences, which are designed to hybridize with specific sequences in dystrophin pre-mRNA to address the subsets of dystrophin gene mutations targeted by each PMO. SRP-4045 is a 22-mer (approx. MW of 7,600 daltons), SRP-4053 is a 25-mer (∼8,600 daltons), and eteplirsen is a 30-mer (∼10,000 daltons).

Results from the 12-week toxicity studies reported here for SRP-4045 and SRP-4053, as well as for the 39-week eteplirsen study, are generally consistent with previously published observations from a 12-week study of eteplirsen [15] in NHPs. Where they occurred, findings for PMOs in repeat-dose studies of 12 weeks duration in NHPs have been almost entirely restricted to the kidneys. Renal findings of tubular basophilia, which occasionally presented as basophilic granules with or without vacuolation and/or tubular degeneration and dilatation, were typically minimal to mild even at the highest dose levels tested (320 mg/kg). In addition, the morphological changes observed were partially or completely reversibile 4 to 8 weeks after the last dose, as evidenced by lower incidences and/or lower severities of these findings in the recovery animals.

MATERIALS AND METHODS

12-week studies of SRP-4045 and SRP-4053in NHPs

The 12-week toxicity studies of SRP-4045 and SRP-4053 were conducted according to Good Laboratory Practice (GLP) regulations at contract research organizations that fully complied with the U.S. National Research Council’s Guide for the Care and Use of Laboratory Animals or its European equivalent [European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes (Strasbourg, Germany)]. These animal studies were conducted under a study protocol approved by each facility’s Institutional Animal Care and Use Committee (IACUC). Naïve, sexually mature, male cynomolgus monkeys (Macaca fascicularis) of Chinese origin were received from Covance Research Products Inc. (Alice, TX) for use in these studies. At initiation of dosing, the animals were 4 to 8 years old, with body weights ranging from 3.8 Kg to 9.6 Kg. Animals were housed in stainless steel cages and, following establishment of compatible commingling partners/groups, animals were commingled (two animals/cage) or group-housed continuously (socially housed), unless separation was needed for study-related procedures. Water was provided ad libitum. Animals were offered Certified Primate Diet #2055C (Harlan Laboratories, Inc.) two times daily unless fasted for study procedures. Environmental controls were set to maintain the following animal room conditions: temperature range of 20 to 26°C, relative humidity range of 30 to 70%, 10 or greater air changes/hour, and a 12-hour light/12-hour dark cycle. The light/dark cycle was interrupted for study-related activities. Animals were given various cage-enrichment devices and fruit, vegetable, or dietary enrichment.

SRP-4045 and SRP-4053 were administered in separate studies by IV injection at 0 (vehicle control), 5, 40, or 320 mg/kg (9 males/group) once weekly for 12 weeks. Both PMOs were formulated as a solution for injection in 1X Dulbecco’s phosphate buffered saline (PBS) vehicle and the dose volume was 3.2 mL/kg. Animals were dosed via slow bolus injection (over a period of at least 1 minute) into the saphenous vein followed by a sterile saline flush of approximately 3 mL. An attempt was made to alternate the leg for dosing each week. Animals were dosed once weekly for 12 weeks during the dosing phase (a total of 12 doses) and the last injection site was marked and maintained for collection at necropsy. Three animals/group were maintained without further dosing for a 4-week recovery period. Plasma toxicokinetics (TK) were assessed on Day 1 and after the last dose at Week 12. Blood samples (at least 1.5 mL each) were collected from the femoral vein on Days 1 and 78 of the dosing phase. Samples were collected pre-dose (within 15 minutes prior to dosing) and approximately 0.25, 0.5, 1, 2, 4, 12, 36, and 48 hours post-dose based on the end time of the dose for each animal. Animals were not fasted for TK sample collections unless fasted for other procedures (e.g., clinical pathology). Blood for TK analyses was processed to plasma, which was analyzed for SRP-4045 or SRP-4053 content using liquid chromatography/tandem mass spectrometry methods. Calculated TK parameters included peak plasma concentration (C_max) and area under the plasma concentration-time curve (AUC).

Toxicity assessments in both studies at the end of the terminal and recovery phases included mortality; clinical observations; body weight (BW) and food consumption measurements; ophthalmoscopic evaluations; electrocardiogram (ECG) evaluations; serum reproductive hormones: testosterone, luteinizing hormone (LH), and follicle-stimulating hormone (FSH); serum complement alternative pathway (CAP) activation parameters: Bb, C3a des Arg, C5a des Arg, and C5b-C9 (if warranted, see below); male reproductive endpoints: sperm counts, motility, and morphology; clinical pathology: clinical chemistries, hematology, and coagulation parameters; urinalysis; and macroscopic and light microscopic pathology.

Ophthalmic examinations were conducted for each animal once during the pre-dose phase and during Week 11 of the dosing phase by a board-certified veterinary ophthalmologist using an indirect ophthalmoscope and a slit-lamp biomicroscope. Animals were anesthetized with ketamine, and the eyes were dilated with a mydriatic agent prior to examination. Ophthalmic examinations during the recovery phase were to be conducted only if abnormalities were observed in the eyes during the dosing phase. ECG’s were collected once during the pre-dose phase, on Days 70 (approximately 24 hours prior to the last dose) and 71 (approximately 1 hour after the last dose) of the dosing phase, and on Day 26 of the recovery phase. Recordings were performed on animals anesthetized with ketamine. At each time point, eight-lead ECGs [16] were recorded for at least 30 continuous seconds. At least five consecutive ECG waveforms and the associated RR intervals deemed most representative were selected for quantitative analysis at each time point. The ECG interval measurements were made on a single lead. The measured waveforms at each time point were averaged and reported. A heart rate correction for the QT interval (QTc) was calculated using the Bazett (QT/[RR]_1/2) method [17, 18]. Quantitative measurements (PR interval, QRS duration, QT interval, QTc interval, RR interval, and heart rate) were evaluated and the ECG recordings were qualitatively reviewed for rhythm abnormalities and disturbances including normal sinus rhythm variations, abnormal sinus rhythms, conductance or repolarization abnormalities, bradycardia, and tachycardia [19].

Blood samples (approximately 1.5 mL each) for reproductive hormone analysis were collected from each animal twice during the pre-dose phase, once during Week 6 of the dosing phase, on the day of scheduled terminal sacrifice, and within 7 days of the recovery sacrifice. Animals were fasted for sample collections and these samples were processed to serum samples for analysis of testosterone, LH, and FSH. The LH and FSH assays used standards and antibodies specific to each analyte in radioimmunoassays (RIA) to determine the amount of labeled LH and FSH in the bound fraction by interpolation from standard curves. The lower limit of quantitation for LH was 0.8 ng/mL and the lower limit of quantitation for FSH was 1.6 ng/mL. Testosterone was analyzed by solid-phase chemiluminescent immunoassay, using the Siemens Immulite analyzer (Los Angeles, California), with a lower limit of quantitation of 20 ng/dL.

Blood samples (approximately 1.5 mL each) for analysis of CAP components were collected from each animal during the pre-dose phase; on Days 1, 8, and 78 of the dosing phase and once prior to recovery sacrifice. Blood samples collected during the pre-dose phase were collected approximately the same time of day as each time point (four total time points) during Day 1 of the dosing phase. Blood samples collected on Days 1, 8, and 78 were collected pre-dose and at approximately 10 or 15 minutes, 1 hour, and 4 or 6 hours post-dosing. The recovery phase collection occurred once, at approximately the same time of day as the time of the last dose administered during the dosing phase. Animals were not fasted for sample collections unless fasted for other procedures (e.g., clinical pathology). Blood was processed to plasma, which was analyzed for each CAP component using methods appropriate for use with NHPs. Bb split product was measured by enzyme-linked immunosorbent assay (ELISA) using microtiter plates precoated with a specific monoclonal antibody against Bb (Quidel, San Diego, CA). C3a des Arg was measured by ELISA using microtiter plates precoated with specific monoclonal antibodies against C3a (BD Pharmingen optEIA^TM, Singapore). C5a des Arg was determined using a competitive RIA (Institute of Isotopes, Budapest, Hungary). C5b-C9 analyses were only performed if C5a des Arg results indicated complement activation occurred.

Blood samples for hematology, coagulation, and clinical chemistry were collected from animals fasted overnight prior to scheduled collections. Blood samples were collected twice during the pre-dose phase, on Day 29 of the dosing phase, and on the days of scheduled sacrifices. In addition, blood for serum creatinine only (approximately 0.5 mL) was collected on Days 2, 8, 23, and 79 of the dosing phase. On the days of urine collection (described below), blood was collected to coincide with the end of the 18-hour urine collection periods. The anticoagulants were sodium citrate for coagulation tests and tri-potassium (K₃) EDTA for hematology tests. Samples for clinical chemistry and serum creatinine were collected without anticoagulant. Hematology parameters assessed included hematocrit; erythrocyte, leukocyte, reticulocyte, and platelet counts; total and mean corpuscular hemoglobin; blood cell morphology; and coagulation (prothrombin time, activated partial prothrombin time). Clinical chemistry parameters were determined using an automated system (Modular P Chemistry Analyzer, Roche Diagnostics; Indianapolis, IN) and included standard tests for renal function (creatinine and urea nitrogen), liver function (alanine aminotransferase, aspartate aminotransferase, gamma glutamyl transferase and total bilirubin), and other tissue damage (creatine kinase, aspartate aminotransferase and alkaline phosphatase), as well as other circulating indicators of general metabolic and physiological state (e.g., total protein, glucose, cholesterol, triglycerides, calcium, albumin and globulin).

Urine samples from overnight cage pan collections on wet ice, collected once during the pre-dose phase; on Days 2, 8, 23, 29, and 79 of the dosing phase; and on the day of recovery sacrifice, were used for urinalysis. Animals were fasted overnight for scheduled collections and urinalysis tests included volume, specific gravity, pH, color/appearance, protein, glucose, bilirubin, ketones, blood, urobilinogen, sediment microscopy, creatinine, and creatinine clearance.

At both the terminal and recovery necropsies, a complete battery of organs and tissues was examined macroscopically for gross lesions, organ weights were assessed, and at least 40 different tissues, representing all major organs/systems, were collected from each animal. Tissues were preserved in 10% neutral-buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E) for light microscopic evaluation.

Chronic study of eteplirsen in NHPs

The chronic, 39-week toxicity study of eteplirsen was conducted according to GLP regulations at an Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) accredited CRO that fully complied with the U.S. National Research Council’s Guide for the Care and Use of Laboratory Animals, under an IACUC-approved study protocol. Naïve, sexually mature, male cynomolgus monkeys (Macaca fascicularis) of Chinese origin were obtained from Covance Research Products Inc. (Alice, TX). Animals were housed individually in stainless steel cages and were 5 to 6 years old at initiation of dosing, with body weights ranging from 5.3 Kg to 7.2 Kg. Water was provided ad libitum, Certified Primate Diet #2055C (Harlan Laboratories, Inc.) was offered one to two times daily, unless fasted for study procedures, and various cage-enrichment devices and fruit, vegetable, or dietary enrichment (that did not require analyses) were provided. Environmental conditions were similar to those used in the 12-week SRP-4045 and SRP-4053 studies and the light/dark cycle was occasionally interrupted for study-related activities.

Eteplirsen was administered at 0 (Dulbecco’s PBS 1x), 5, 40, or 320 mg/kg (8/group) by slow bolus IV injection, once weekly for 39 weeks. The dose volume in all groups was 3.2 mL/kg. Individual doses were administered over a period of at least 1 minute via the saphenous vein once weekly for 39 weeks (dosing phase, a total of 39 doses) at a dose volume of 3.2 mL/kg. The first dose for each animal was considered the animal’s respective Day 1 of the dosing phase. Dosing began on dosing phase Day 1 and continued through 2 days prior to the terminal sacrifice (Day 1 of the recovery phase). Doses were based on the most recently recorded scheduled body weight and the last injection site was marked and maintained for collection at necropsy. At the end of the 39-week dosing phase, necropsies were conducted on 5 males/group, and the remaining 3 males/group were maintained without further dosing for an 8-week recovery period.

Plasma TK was assessed on Day 1 and Week 39. Blood samples (at least 1 mL) for TK analyses were collected in tubes containing K₃ EDTA via a femoral vein or saphenous vein on Day 1 and on the last day of dosing pre-dose and approximately 0.25, 0.5, 1, 2, 4, 12, and 36 hours post-dose. Blood collection time was based on the end of the dose administration for each animal. Animals were not fasted for blood collections. Blood for TK analyses was processed to plasma, which was analyzed for eteplirsen content using anion exchange, high-pressure liquid chromatography with fluorescence probe hybridization detection methods. Calculated TK parameters included C_max and AUC.

Assessments of toxicity were based on mortality, clinical observations, BW, food consumption, ophthalmoscopic evaluations, ECG evaluations, urinalysis, and/or clinical pathology. Effects of eteplirsen on male reproductive parameters, reproductive hormones (testosterone, LH, and FSH), and CAP functional activity (quantification of Bb, C3a, and C5a fragments) were also evaluated in this study. Ophthalmic exams were conducted as described above for the 12-week studies, once during the pre-dose phase and during Week 38 of the dosing phase using the same procedures described above for the SRP-4045 and SRP-4053 12-week studies. Ophthalmic examinations during the recovery phase were conducted only if abnormalities were noted during the dosing phase. Eight-lead ECGs were recorded under ketamine anesthesia for at least 30 continuous seconds, once during the pre-dose phase, during Week 38 of the dosing phase pre-dose and approximately 1 to 2 hours post-dosing, and once within the last 8 days of the recovery phase. Post-dose collections were based on last animal dosed/group. The ECG recordings were collected and analyzed using the same procedures described above for the 12-weekstudies.

Semen and femoral vein blood samples for reproductive hormone analyses were obtained twice during the pre-dose phase; during Weeks 13, 26, and 38 of the dosing phase; and within 7 days of the recoverysacrifice. Volume, sperm number (total sperm number/collected sample), concentration, percent motility, and sperm morphology were determined, calculated, and analyzed statistically. Blood samples were processed to serum, which was analyzed for male reproductive hormones using the same assays described above for the 12-week studies.

Blood samples (approximately 1 mL) for analysis of CAP components in plasma and serum were collected on Days 1 and 8 of the dosing phase, during Weeks 13 and 39 of the dosing phase, and once prior to the recovery sacrifice from animals that were not fasted. Dosing phase collections were approximately 10 minutes, 1 hour, and 6 hours post-dose. The recovery phase samples were collected once at approximately the same time as the 1-hour post-dose time point from the last dose administered during the dosing phase. Dosing phase plasma samples on Day 1 were analyzed for CAP components Bb, C3a-des-Arg, and C5a-des-Arg using MicroVue^® enzyme immunoassay kits (Intertek ALTA Analytical Laboratory; San Diego, CA). Additional blood samples were collected in serum separator tubes. Dosing phase serum samples on Day 8, Week 13, Week 39, and after the recovery phase were analyzed for the terminal pathway component C5b-C9, using the Wieslab™ enzyme immunoassay kit (Euro-Diagnostica AB, Medeon, Sweden), adapted for qualitative determination of C5b-C9 assay in complement-preserved NHP serum.

Blood samples (femoral vein) were collected for hematology, coagulation, and clinical chemistry. Urine samples from overnight cage pan collections on wet ice were used for urinalysis and urinary biomarker assessments. Blood was collected twice during the pre-dose phase; during Weeks 13, 26, and 39 of the dosing phase approximately 24 hours post-dose; and on the day of the recovery sacrifice. The 24-hour urine samples were collected once during the pre-dose phase and 0 to 24 hours after dosing on Days 1, 85, 176, and 260. In addition, urine was collected in cage pans on wet ice from animals fasted during the 24 hours prior to dosing on Days 8, 92, 183, and 267 and prior to recovery sacrifice (collection started on Days 7, 91, 182, and 266 of the dosing phase and Day 57 or 58 of the recovery sacrifice). Hematology, coagulation, clinical chemistry, and standard urinalysis tests were similar to those described above for the SRP-4045 and SRP-4053 12-week studies.

Additional urine chemistry tests consisted of urinary biomarkers for renal function assessment (creatinine, cystatin C/creatinine ratio, and KIM-1/creatinine ratio), with urine samples analyzed using validated, multi-analyte profile methods for quantifying cystatin C and KIM-1 (kidney injury molecule 1) in urine samples from NHPs. Streptavidin-Phycoerythrin was purchased from Molecular Probes^® (Thermo Fisher Scientific Life Technologies, Grand Island, NY). All buffers, reagents, capture microsphere multiplexes, multiplexed cocktails of biotinylated reporter antibodies, and multiplexed standards and controls were prepared by Myriad Rules Based Medicine (Austin, TX). Urine samples were centrifuged for clarification and an aliquot of each sample was introduced into one of the capture microsphere multiplexes. These mixtures of sample and capture microspheres were incubated at room temperature for 1 hour, multiplexed cocktails of biotinylated reporter antibodies were then added, followed by an additional 1 hour incubation at room temperature. Multiplexes were developed using an excess of streptavidin-phycoerythrin solution, followed by vacuum filtration and dilution into matrix buffer for analysis in a Luminex 100/200 instrument. Assays were run in high density multiplexed panels and quantitated using an eight-point standard curve with appropriate quality control standards for each analyte.

At both the terminal and recovery necropsies, a complete battery of organs and tissues was examined macroscopically for gross lesions, organ weights were assessed, and at least 50 different tissues, representing all major organs/systems, were collected from each animal. Eyes, optic nerves, and testes were collected in modified Davidson’s fixative, and all other tissue samples were preserved in 10% neutral-buffered formalin, embedded in paraffin, sectioned, and stained with H&E for light microscopic evaluation.

RESULTS

12-week study of SRP-4045 in NHPs

All animals survived to the scheduled necropsy. There were no clinical observations or effects on BW, food consumption, ophthalmic examinations, hematology, coagulation, urinalysis, organ weights, or macroscopic evaluations related to SRP-4045 administration. Slight, but statistically significant, changes observed in several clinical chemistry parameters (glucose, urea nitrogen, creatinine, and phosphorus concentrations; aspartate aminotransferase) were not considered toxicologically relevant, since the values were still within the range of historical control values for this species and there was no dose-response for these changes. All ECG parameters, including heart rate, RR interval, PR interval, QRS duration, QT interval, and QTc interval, were considered quantitatively normal for cynomolgus monkeys. The occurrence of infrequent ventricular premature complexes (VPCs) was observed in 4 animals, but a clear relationship to SRP-4045 administration was not established. Intermittent periods of atrial premature complexes (APCs) were also observed in 3/9 animals at 320 mg/kg. One of these three animals given 320 mg/kg/dose continued to have infrequent atrial premature complexes during the recovery phase.

Dose-dependent increases in Bb and C3a des Arg complement fractions, the highest of which occurred on Day 8 at the 320 mg/kg dose level (3.4-fold and 9.3-fold, respectively, at 10 minutes post-dose, and 3.4-fold and 3.7-fold, respectively, at 1 hour post-dose), suggested that CAP activation may have occurred. However, these changes were transient, returning to pre-dose levels by 6 hours post-dose, and the expected concommitant increases in C5a des Arg were not observed.

All animals were confirmed to be sexually mature at study initiation by the presence of sperm at one or more of the pre-dose phase semen collections. No SRP-4045-related effects were observed on testicular volume measurements, sperm density, total sperm count, percent motility, or sperm morphology during the dosing or recovery phase evaluations and no effects on circulating male reproductive hormones LH, FSH, or testosterone were observed.

SRP-4045-related microscopic findings occurred in the kidney at ≥40 mg/kg and were limited to non-adverse, tubular basophilia/microvacuolation and tubules containing basophilic luminal material. Four weeks after the last dose, these microscopic observations were noted only in the 320 mg/kg group and at a lower severity, suggesting reversibility. The no observed adverse effect level (NOAEL) for repeated IV administration of SRP-4045 in this study was considered to be the highest dose level tested (320 mg/kg).

12-week study of SRP-4053 in NHPs

All animals survived to the scheduled necropsy and there were no effects on clinical signs, BW, ECG parameters (including QT intervals), ophthalmology exams, or clinical pathology parameters related to SRP-4053 administration. Transient increases in Bb (up to 3.2-fold in 5/9 animals at 320 mg/kg) and C3a (up to 12-fold in 6/9 animals at 320 mg/kg) CAP components occurred at 15 minutes post-dosing on Day 8, along with sporadic increases in C5a (up to 2.8-fold in 2/9 animals at 320 mg/kg) 1 hour post-dose on Day 78 only, but no consistent pattern of CAP activation or activation of the terminal complement pathway was observed.

A statistically significant increase in testicular weight (testicle weight:BW ratio approximately 50% greater than that of controls) was seen at 320 mg/kg. Slightly lower LH concentrations at ≥5 mg/kg and FSH concentrations at ≥40 mg/kg were seen relative to controls, while testosterone was not affected. These changes in testicular weights and hormones levels were not observed 4 weeks after the last dose. No associated effects on other reproductive endpoints (sperm counts, sperm motility, and morphology) were noted and there were no histopathology correlates for the organ weight change, so these reversible findings were considered non-adverse. The only SRP–4053 related histopathology finding noted was minimal diffuse follicular cell hypertrophy in the thyroid gland of 1/6 animals at 320 mg/kg, which was also considered non-adverse. The NOAEL for repeated IV administration of SRP-4053 in this study was considered to be the highest dose level tested(320 mg/kg).

Chronic study of eteplirsen in NHPs

All animals in the 39-week eteplirsen study survived to the scheduled necropsy. No adverse effects on clinical observations, BW changes, ophthalmoscopic or ECG evaluations, or male reproductive parameters were associated with exposure to eteplirsen. An eteplirsen-related, mild decrease in total leukocyte, neutrophil, and monocyte counts on dosing Day 268 that resolved after the 8-week recovery was noted at 40 and 320 mg/kg. No changes attributed to eteplirsen occurred in serum chemistry, coagulation, urinalysis, or urine chemistry, including urinary total protein:creatinine or urinary KIM-1:creatinine ratios.

Quantitation of the Bb complement fragment suggested minimal to moderate CAP activation at 10 minutes (34%) and 1 hour (65%) post-dose on Day 1 at 320 mg/kg. In contrast, C3a des Arg concentrations, which would be expected to increase following activation of the classical, alternative, or lectin complement pathways, was variable and decreased slightly (up to 29%) at these intervals. The C5a des Arg fragment, which remained below detectable levels at all intervals on Day 1, and serum C5b-C9 analyses on Day 8, Week 13, and Week 39 demonstrated that the complement terminal pathway was not activated in NHPs by eteplirsen.

The animals were sexually mature as evidenced by semen analysis (prior to and during the study) and testicular histology analysis at the end of the 39-week dosing phase. There were no effects of eteplirsen on male reproductive parameters based on assessments of animal age, BW, reproductive organ weights (epididymis, prostate, seminal vesicles and testes), testicular histology (conducted in a stage-aware manner), testicular volume, or semen parameters (sperm density, motility, and morphology). Testosterone concentrations were detectable in all animals throughout the entire study period and showed large variability, but no effects of eteplirsen on testosterone were observed. LH and FSH concentrations were typically low or below the lower limit of quantitation and were also not affected by eteplirsen administration.

A dose-related increased incidence and severity of minimal to moderate, multifocal, basophilic cytoplasm in the renal tubules was observed microscopically in all dose groups at the end of the dosing phase (Fig. 1). These kidney findings were still present 8 weeks after the last dose in recovery animals from the 320 mg/kg group, although the severity was reduced, and were not present at lower doses, suggesting reversibility. In addition, 2 of 3 animals in the recovery group at 320 mg/kg in this study also had minimal or slight multifocal dilatation of the renal tubules, which were lined by degenerated and/or attenuated epithelium, contained basophilic granular material in the lumen, and were occasionally associated with mononuclear inflammation in the surrounding areas (Fig. 1). As can be seen below in Table 1, minimal to mild tubular basophilia is a common finding in NHPs dosed repeatedly with PMOs. Since degenerative kidney changes at the recovery phase were of minimal or slight severity, and no correlative clinical pathology findings suggesting renal dysfunction were noted, all kidney findings were considered non-adverse. Finally, chronic vascular/perivascular inflammation was observed microscopically at the injection site at the end of the dosing phase in 1/5 vehicle control animals, 2/5 at 5 mg/kg, 0/5 at 40 mg/kg, and 3/5 at 320 mg/kg, which recovered 8 weeks after the last dose and was considered non-adverse. The NOAEL for chronic IV administration (39 weeks) of eteplirsen in this study was considered to be the highest dose level tested (320 mg/kg).

Overall PMO comparison

In 12-week NHP studies conducted to date, which include a previously published eteplirsen study [15], there were no PMO-related adverse effects on in-life parameters such as survival, BW, food consumption, clinical observations, ophthalmoscopic exams, male reproductive assessments, cardiovascular (ECGs), and complement activation assessments. There were also no PMO-related clinical chemistry, hematology, urinalysis, or organ weight changes indicative of a target-organ toxicity. The NOAEL for 12 weeks of repeated IV administration of all three PMOs (eteplirsen, SRP-4045, and SRP-4053) to NHPs was the highest dose level tested (320 mg/kg).

The consistency of plasma exposures for PMOs is illustrated below (Fig. 2), which shows mean plasma concentrations vs. time on Day 1 and during Week 12 for all three PMOs at the doses administered in the 12-week NHP studies (5, 40, and 320 mg/kg). Plasma concentrations at each time point were remarkably consistent on both days of dosing, regardless of the PMO administered and a clear dose-dependent increase in exposures can be seen for all three PMOs. Dose-normalized exposures from these studies were compared among tested PMOs, overall, and by study day. As can be seen in Table 2, TK parameters for these PMOs in NHPs were comparable, with none showing major quantitative differences among the PMOs. Overall variability in C_max and AUC_0 - t parameters was low, with average coefficients of variation less than 25%, and no differences in systemic exposures were observed for eteplirsen, SRP-4045, or SRP-4053 as a function of day of exposure (i.e., no evidence of significant plasma accumulation after repeated dosing).

Findings in the 39-week eteplirsen study reported here were similar to findings in the 12-week studies and comparison of light microscopic observations in the kidney demonstrates remarkable consistency among PMOs in NHPs, as described below in Table 3. A common feature is the presence of renal tubular basophilia, and occasionally the formation of basophilic granules, along with associated cytoplasmic vacuolation/microvacuolation, which occurred with low severity scores (typically minimal to mild). Histopathologic evidence of major kidney injury, such as moderate or marked tubular dilatation, degeneration, and/or regeneration, or associated changes in clinical pathology parameters were not observed. In all studies, there was evidence of reversibility for the primary lesions (tubular basophilia and vacuolation), consisting of either reduced severity, reduced incidence, or both, in the recovery groups 4 weeks (12-week studies) or 8 weeks (39 week study) after the last dose.

DISCUSSION

Results from studies reported here are consistent with those from a previously published 12-week study of eteplirsen [15], demonstrating that PMOs have almost no effects on a wide range of potential target organs of toxicity after repeated IV administration of high doses (up to 320 mg/kg) in NHPs, including cardiovascular, respiratory, ocular, immune, male reproductive, neurobehavioral, hematopoietic, and hepatic systems. Although both VPCs and APCs were observed in the SRP-4045 study, these effects were not clearly related to SRP-4045 exposure, since these arrhythmias are normal variants in NHPs and can sometimes be an incidental observation in primate studies [20], and older animals are considered to be more prone to incidental arrhythmias. No incidences of VPC or APC were observed in the SRP-4053 study or in the 39-week eteplirsen study.

The kidneys were generally identified as a target organ of toxicity for PMOs, but the renal effects of PMOs in NHPs have been limited to relatively minor microscopic changes (i.e., renal tubular basophilia and vacuolation or microvacuolation) that were non-adverse, showed evidence of reversibility, and did not produce any corresponding alterations in renal function tests such as serum creatinine, urea nitrogen, or total bilirubin assessments. Extending the dosing duration from 12 weeks to chronic administration (39 weeks) with eteplirsen resulted in a slight progression of microscopic observations in the kidneys, but again these changes did not affect renal function and were considered non-adverse. Furthermore, target organs other than kidneys were not identified in the chronic eteplirsen study, despite continued high eteplirsen exposures for the additional 27 weeks of dosing.

The mechanism for the renal responses of NHPs to PMOs described here and previously for eteplirsen [15], is unknown, but may involve uptake by the kidneys during renal excretion. Other classes of exon skipping oligomers (e.g., phosphorothioates) have been shown to be preferentially excreted through the urinary system, where they are absorbed across the brush border or transported via the basolateral membrane into the proximal tubules and are incorporated into lysosomes as basophilic granules. The granules are digested only slowly and tend to accumulate with repeated exposure leading to microscopically evident tubular basophilia in H&E stained kidney sections. Degeneration of some of the affected cells can occur, and detachment of granule-laden cells into the tubule lumen, with formation of basophilic casts containing basophilic granules could result in passage of these casts through the renal drainage system for elimination [21 –24]. Separate findings of tubular basophilia or basophilic granules, vacuolation or microvacuolation, and tubular inflammation, dilatation, or degeneration (chronic eteplirsen study only) were recorded at the highest doses of eteplirsen and SRP-4045 (320 mg/kg), suggesting that these findings may be the result of a similar renal elimination process as that described for phosphorothioates. It is unclear why these morphological changes did not occur with SRP-4053 in the 12-week study, since overall plasma exposures were similar to eteplirsen and SRP-4045 exposures, but it may be due to a difference in kinetics of renal uptake and elimination of SRP-4053. In the absence of more data linking PMO tissue levels, which were not assessed in these studies, to their plasma exposures, it is not possible to determine a definitive cause for the disparate kidney findings with SRP-4053 in NHPs.

Although renal tubular basophilia and vacuolation typically occur at much lower doses (as low as 6 mg/kg with the recently discontinued development candidate for treatment of DMD, drisapersen) with other classes of exon skipping oligomers, these findings are considered to be evidence of cellular uptake, not adverse events [25]. Slight, inflammation-related glomerulopathies are also sometimes seen with these other classes [26 –29], whereas no glomerular involvement or other evidence of a pro-inflammatory response (i.e., activated granular macrophage accumulation, lymphoid organ hyperplasias, and/or vasculitis in multiple tissues) has been observed with PMOs tested to date. In addition, several other target organ toxicities are generally known to occur for other classes of exon skipping oligomers in animal studies. These class-specific toxicities, such as delayed thrombocytopenia; coagulopathies; proinflammatory effects, including complement activation and vasculitis; and hepatic accumulation with or without Kuppfer cell basophilia and/or hyperplasia [25 –30], were not observed with PMOs in the studies reported here.

The potential of eteplirsen to activate complement was evaluated by immunoassay of critical components of the alternative pathway and, although transient PMO-related CAP activation was observed in these studies, these changes did not lead to significant activation of the terminal pathway. Complement activation is well documented for other classes of exon skipping oligomers and, along with inhibition of clotting (transient and reversible increases in aPTT), is considered a major, dose-limiting toxicity for phosphorothioates [25 , 28]. Intravenous doses of phosphorothioates as low as 20 mg/kg have caused death in NHPs and aPTT increases have been observed in humans after 2-hour IV infusions at doses as low as 1 mg/kg [26]. Moreover, these types of dose-limiting, immune-mediated toxicities are believed to be related to high peak plasma concentrations achieved after IV doses [26], which may explain why subcutaneous injection has become the preferred route of administration for other exon skipping oligomers, including drisapersen [31]. The mechanism of the effects of drisapersen on the immune system of mice and NHPs has been described in detail [29].

Many of the toxicities observed in animals for other classes of exon skipping oligomers have translated into related safety findings in clinical trials conducted with these agents [32, 33]. The relatively mild renal effects and absence of other class-specific toxicities (listed above) after once-weekly IV doses up to 320 mg/kg in the animal safety data described here for PMOs are consistent with the lack of safety concerns with long-term clinical experience at IV eteplirsen doses up to 50 mg/kg in boys with DMD [11 , 14]. In addition, our findings with three different PMO sequences, one of which (eteplirsen) has the same mechanism of action but a toxicity profile that is dramatically different than that of the 2′-O-methylphosphorothioate drisapersen, add to the body of knowledge suggesting that the toxicity of exon skipping oligomers is driven by their backbone chemistries and not their primary mechanism of action [34]. Thus, when taken together with the excellent clinical safety profile established for eteplirsen to date [11 , 14], the nonclinical results in NHPs reported here suggest that once-weekly IV administration of eteplirsen and other PMOs for lifetime durations at therapeutic dose levels will be well tolerated by patients with DMD.

CONFLICT OF INTEREST STATEMENT

Michael P. Carver, Peter Sazani, Jay S. Charleston, Courtney Shanks, and Jianbo Zhang were all employees of Sarepta Therapeutics, Inc. (Cambridge, MA) at the time this manuscript was prepared. Sarepta Therapeutics, Inc. provided all financial and material support for this research. Mark Mense, Alok K. Sharma, and Harjeet Kaur of Covance Laboratories, Inc. (Madison, WI) have no financial conflicts of interest to report.

Footnotes

ACKNOWLEDGMENTS

The authors thank Steve Van Adestine of Covance Laboratories, Inc. and Johannes Dworzak of Sarepta Therapeutics, Inc. for assisting with the figures.

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