Toxicologic Pathology Forum: Opinion on Interpretive Challenges for Procedure-Related Effects Associated With Direct Central Nervous System Delivery of Oligonucleotides to Rodents, Dogs, and Nonhuman Primates.

Direct delivery of therapeutics to the central nervous system (CNS) greatly expands opportunities to treat neurological diseases but is technically challenging. This opinion outlines principal technical aspects of direct CNS delivery via intracerebroventricular (ICV) or intrathecal (IT) injection to common nonclinical test species (rodents, dogs, and nonhuman primates) and describes procedure-related clinical and histopathological effects that confound interpretation of test article-related effects. Direct dosing is by ICV injection in mice due to their small body size, while other species are dosed IT in the lumbar cistern. The most frequent procedure-related functional effects are transient absence of lower spinal reflexes after IT injection or death soon after ICV dosing. Common procedure-related microscopic findings in all species include leukocyte infiltrates in CNS meninges or perivascular (Virchow-Robin) spaces; nerve fiber degeneration in the spinal cord white matter (especially dorsal and lateral tracts compressed by dosing needles or indwelling catheters), spinal nerve roots, and sciatic nerve; meningeal fibrosis at or near IT injection sites; hemorrhage; and gliosis. Findings typically are minimal to occasionally mild. Findings tend to be more severe and/or have a higher incidence in the spinal cord segments and spinal nerve roots at or close to the site of administration.

Range of Neurological Signs in Cynomolgus Monkeys After Intrathecal Bolus Administration of Antisense Oligonucleotides.

Intrathecal (IT) dosing (ie, injection into the subarachnoidal space at the lumbar region) is a common route of administration in cynomolgus monkey preclinical safety studies conducted for antisense oligonucleotides (ASO) that target central nervous system diseases. Herein we report on neurological signs that have been observed in 28 IT studies conducted in 1,016 cynomolgus monkeys. Neurological signs were classified into 5 groups: (1) A nonadverse transient absence of lower spinal reflexes. This observation occurred at low incidence in nontreated animals and in those that were injected artificial cerebrospinal fluid. The incidence increased in animals that were injected an ASO. Reflexes were present again at 24 hours or 48 hours after dosing. The incidence appeared to increase with dose. (2) Test-article-related adverse muscle tremor or muscle spasticity occurring during the injection procedure or immediately thereafter. In one-third of animals this finding responded to treatment with diazepam, in two-third it required euthanasia. (3) Neurological findings occurring between 30 minutes and 4 hours after dosing were characterized by any combination of ataxia, paresis, nystagmus, urinary incontinence, or muscle tremor. Those conditions either spontaneously resolved or they slowly worsened, eventually resulting in a poor general condition. (4) Neurological findings due to spinal cord injury were characterized by rapidly progressing paralysis of hind limbs. Magnetic resonance imaging revealed a focal hyperintense lesion, indicative of spinal cord necrosis. (5) Test-article-related adverse hind limb paresis or paralysis that occurred between 2 and 18 days after dosing. Those findings were rare and resulted in a poor general condition requiring euthanasia.

Save Your Maximum Tolerated Dose: How to Diagnose Procedure-Related Spinal Cord Lesions After Lumbar Intrathecal Bolus Administration of Oligonucleotides in Cynomolgus Monkeys.

Many potential drugs for treatment of neurodegenerative diseases, particularly antisense oligonucleotides (ASOs), are administered via lumbar intrathecal injection, because these drugs do not cross the blood-brain barrier. Intrathecal injection is a well-established method in cynomolgus monkeys, a species that is used in preclinical safety assessment when other nonrodent species cannot be used. The authors completed intrathecal ASO administration in over 30 preclinical safety studies (>1000 animals and >4500 dose administrations) during which we observed 3 cases of procedure-related spinal cord necrosis (incidence <0.1%). We describe clinical symptoms, diagnostic approaches, morphological features, and prognosis of this rare injury, and compare these findings with typical drug-related findings of ASOs dosed by intrathecal injection. The low incidence of procedure-related and dose-limiting lesions confines this analysis to a small sample set. The pattern of effects is similar across all monkeys despite differences in age, body weight, and intrathecal injection site. All 3 cases presented a combination of the following findings: blood in cerebrospinal fluid at time of injection, clinical signs that increase in severity within a day of dosing, lameness of both hind limbs, reduced muscle tone, and loss of patellar, foot grip, and/or anal reflexes. In all cases, magnetic resonance imaging (MRI) showed a linear hyperintense lesion in the lumbar spinal cord. In 2 cases, this hyperintensity was associated with evidence of spinal cord edema. We conclude that a pattern of in-life and pathology findings, including noninvasive MRI assessment, is indicative of procedure-related effects.

Differential responses to JNJ-37822681, a specific and fast dissociating dopamine D2 receptor antagonist, in cynomolgus monkey and Sprague-Dawley rat general toxicology studies: clinical observations, prolactin levels, mammary histopathology findings and toxicokinetics.

JNJ-37822681 is a potent, specific and fast dissociating dopamine D2 receptor antagonist intended for the treatment of schizophrenia. Its nonclinical toxicological profile was investigated in a series of general repeat dose toxicity studies in cynomolgus monkeys and Sprague-Dawley rats. The maximum duration of treatment was 9 and 6 months, respectively. Interspecies differences were noted in the response to JNJ-37822681 in terms of extrapyramidal (EPS)-like clinical signs and prolactin-mediated tissue changes in the mammary gland. Monkeys showed severe EPS-like clinical signs such as abnormal posture, abnormal eye movements and hallucination-like behavior at relatively low exposures compared to those associated with EPS in patients with schizophrenia. The high sensitivity of the monkey to JNJ-37822681-induced EPS-like signs was unexpected based on the fast dissociating properties of the compound. Rats, however, were not prone to EPS. Elevated serum prolactin levels were found in rats and monkeys. While rats showed slight to moderate prolactin-related tissue changes upon histopathological examination in all studies, which among others affected the mammary gland, only minor mammary gland tissue changes were noted in monkeys. Prolactin levels were only slightly increased in patients with schizophrenia receiving relatively high dose levels of JNJ-37822681. The monkey toxicology studies did not provide an exposure-based safety margin, while in rats adverse effects were only noted at exposures considerably higher than those achieved at efficacious plasma concentrations in the clinic. Overall, the available data suggest that the cynomolgus monkey showed better predictivity towards the nature of JNJ-37822681-associated adverse events in humans than the Sprague-Dawley rat.

Safe and Effective Cynomolgus Monkey GLP-Tox Study with Repetitive Intrathecal Application of a TGFBR2 Targeting LNA-Gapmer Antisense Oligonucleotide as Treatment Candidate for Neurodegenerative Disorders.

The capability of the adult central nervous system to self-repair/regenerate was demonstrated repeatedly throughout the last decades but remains in debate. Reduced neurogenic niche activity paralleled by a profound neuronal loss represents fundamental hallmarks in the disease course of neurodegenerative disorders. We and others have demonstrated the endogenous TGFß system to represent a potential pathogenic participant in disease progression, of amyotrophic lateral sclerosis (ALS) in particular, by generating and promoting a disequilibrium of neurodegenerative and neuroregenerative processes. The novel human/primate specific LNA Gapmer Antisense Oligonucleotide "NVP-13", targeting TGFBR2, effectively reduced its expression and lowered TGFß signal transduction in vitro and in vivo, paralleled by boosting neurogenic niche activity in human neuronal progenitor cells and nonhuman primate central nervous system. Here, we investigated NVP-13 in vivo pharmacology, safety, and tolerability following repeated intrathecal injections in nonhuman primate cynomolgus monkeys for 13 weeks in a GLP-toxicology study approach. NVP-13 was administered intrathecally with 1, 2, or 4 mg NVP-13/animal within 3 months on days 1, 15, 29, 43, 57, 71, and 85 in the initial 13 weeks. We were able to demonstrate an excellent local and systemic tolerability, and no adverse events in physiological, hematological, clinical chemistry, and microscopic findings in female and male Cynomolgus Monkeys. Under the conditions of this study, the no observed adverse effect level (NOAEL) is at least 4 mg/animal NVP-13.

Reconditioning the Neurogenic Niche of Adult Non-human Primates by Antisense Oligonucleotide-Mediated Attenuation of TGFß Signaling.

Adult neurogenesis is a target for brain rejuvenation as well as regeneration in aging and disease. Numerous approaches showed efficacy to elevate neurogenesis in rodents, yet translation into therapies has not been achieved. Here, we introduce a novel human TGFß-RII (Transforming Growth Factor-Receptor Type II) specific LNA-antisense oligonucleotide ("locked nucleotide acid"-"NVP-13"), which reduces TGFß-RII expression and downstream receptor signaling in human neuronal precursor cells (ReNcell CX® cells) in vitro. After we injected cynomolgus non-human primates repeatedly i.th. with NVP-13 in a preclinical regulatory 13-week GLP-toxicity program, we could specifically downregulate TGFß-RII mRNA and protein in vivo. Subsequently, we observed a dose-dependent upregulation of the neurogenic niche activity within the hippocampus and subventricular zone: human neural progenitor cells showed significantly (up to threefold over control) enhanced differentiation and cell numbers. NVP-13 treatment modulated canonical and non-canonical TGFß pathways, such as MAPK and PI3K, as well as key transcription factors and epigenetic factors involved in stem cell maintenance, such as MEF2A and pFoxO3. The latter are also dysregulated in clinical neurodegeneration, such as amyotrophic lateral sclerosis. Here, we provide for the first time in vitro and in vivo evidence for a novel translatable approach to treat neurodegenerative disorders by modulating neurogenesis.

Feasibility of intravitreal injections and ophthalmic safety assessment in marmoset (Callithrix jacchus) monkeys.

To safeguard patients, regulatory authorities require that new drugs that are to be given by the intravitreal (IVT) route are assessed for their safety in a laboratory species using the same route of administration. Due to the high similarity of ocular morphology and physiology between humans and nonhuman primates (NHPs) and due to the species specificity of many biotherapeutics, the monkey is often the only appropriate model. To this end, intravitreal administration and assessment of ocular toxicity are well established in cynomolgus monkeys (Macaca fascicularis). In contrast, the common marmoset monkey (Callithrix jacchus) is not a standard model for ocular toxicity studies due to its general sensitivity to laboratory investigations and small eye size. It was the purpose of the present work to study whether the marmoset is a useful alternative to the cynomolgus monkey for use in intravitreal toxicological studies. Six marmoset monkeys received repeated (every 2 weeks for a total of four doses) intravitreal injections of 10 or 20  µ L of a placebo. The animals were assessed for measurements of intraocular pressure (IOP), standard ophthalmological investigations and electroretinography (ERG). At the end of the dosing period, the animals were sacrificed and the eyes were evaluated histologically. ERG revealed similar results when comparing predose to end-of-study data, and there was no difference between the two dose volumes. A transient increase in the IOP was seen immediately after dosing, which was more pronounced after dosing of 20  µ L compared to 10  µ L. Ophthalmologic and microscopic observations did not show any significant changes. Therefore, it can be concluded that 10  µ L as well as 20  µ L intravitreal injections of a placebo are well tolerated in the marmoset. These results demonstrate that the common marmoset is an alternative to the cynomolgus monkey for intravitreal toxicity testing.

Safety Evaluation of CNS Administered Biologics-Study Design, Data Interpretation, and Translation to the Clinic.

Many central nervous system (CNS) diseases are inadequately treated by systemically administered therapies due to the blood brain barrier (BBB), which prevents achieving adequate drug concentrations at sites of action. Due to the increasing prevalence of neurodegenerative diseases and the inability of most systemically administered therapies to cross the BBB, direct CNS delivery will likely play an increasing role in treatment. Administration of large molecules, cells, viral vectors, oligonucleotides, and other novel therapies directly to the CNS via the subarachnoid space, ventricular system, or parenchyma overcomes this obstacle. Clinical experience with direct CNS administration of small molecule therapies suggests that this approach may be efficacious for the treatment of neurodegenerative disorders using biological therapies. Risks of administration into the brain tissue or cerebrospinal fluid include local damage from implantation of the delivery system and/or administration of the therapeutic and reactions affecting the CNS. Preclinical safety studies on CNS administered compounds must differentiate between the effects of the test article, the delivery device, and/or the vehicle, and assess exacerbations of reactions due to combinations of effects. Animal models characterized for safety assessment of CNS administered therapeutics have enabled human trials, but interpretation can be challenging. This manuscript outlines the challenges of preclinical intrathecal/intracerebroventricular/intraparenchymal studies, evaluation of results, considerations for special endpoints, and translation of preclinical findings to enable first-in-human trials. Recommendations will be made based on the authors' collective experience with conducting these studies to enable clinical development of CNS-administered biologics.