Neurobiomarkers: Basic Aspects and Their Relevance in Nonclinical Studies

Abstract

The second session of the 2024 European Society of Toxicologic Pathology (ESTP) Congress highlighted the significance of neural biomarkers and functional endpoints in nonclinical studies for detecting acute or delayed peripheral (PNS) and central nervous system (CNS) alterations and /or injury caused by drugs during development. The session emphasized the potential clinical translation of these biomarkers and endpoints and critical role of pathologists in correlating these biomarkers with the microscopic findings. Key neural biomarkers discussed included fluid-based biomarkers such as Neurofilament Light Chain (NF-L), Nonspecific Enolase (NSE), Tubulin Associated Unit (TAU), and Glial Fibrillar Associated Protein (GFAP) in blood and/or cerebrospinal fluid (CSF). These were evaluated in 15 in-vivo studies conducted with CNS and PNS toxicants. Safety pharmacology evaluation, such as the Irwin screen/the functional observation battery (FOB), were presented for detecting drug effects on behavior, motor and sensory functions in both rodents and nonrodent species, with or without histopathological correlate. Follow-up tests like nerve conduction velocity assessment were also highlighted. The session underscored the usefulness of noninvasive imaging modalities, including magnetic resonance imaging (MRI), nuclear imaging techniques, X-ray computed tomography, and ultrasound in preclinical studies. Overall, integrating neural biomarkers, safety pharmacology endpoints, advanced imaging modalities, and detailed histopathological analysis aids in better predicting neurotoxicity.

Keywords

Neurobiomarkers fluid-based biomarkers circulating biomarkers CSF safety pharmacology Irwin test functional observation battery nerve conduction velocity pathology imaging MRI X-rays ultrasound tomography

Introduction

Development, validation, and implementation of noninvasive neural biomarkers are needed to detect and/or predict nervous system changes in nonclinical and clinical studies. These biomarkers can be used to evaluate the efficacy of new drugs, monitor neurological diseases, and better characterize neuropathology findings. They should be sensitive and specific to a neurologic alteration, and detectable in body fluids (blood, cerebrospinal fluid [CSF], urine) or by clinical observations and/or imaging modalities. Finding specific biomarkers has been challenging in the neurology field. The best example is the release of neurofilament light chain (NF-L), an intermediate filament protein present mostly in axons, in blood and CSF after axonal and/or neuronal injury. Neurofilament light chain is a nonspecific but sensitive neural biomarker for multiple neurologic indications^14,19,20 that has shown good correlations with histopathology findings in nonclinical studies.^10,13,18,36 During this session, an overview of existing biomarkers—including fluid-based markers, safety pharmacology assessment, and imaging techniques—was discussed, with a focus on their use in rats as the nonclinical species, with an emphasis on their clinical translation and correlation with microscopic findings.

Performance of Biomarkers NF-L, NSE, TAU, and GFAP in Blood and CSF in Rats for the Detection of Nervous System Injury

Dr. Warren Glaab delivered the first lecture of this session and reviewed the results of a large study performed by a group of scientists of the European Union’s Innovative Medicines Initiative (IMI). The IMI has two projects, one dedicated to Translational Safety Biomarker Pipeline (TransBioLine) and the second directed toward Neurotoxicity De-Risking in Preclinical Drug Discovery (NeuroDeRisk), designed to support the NeuroDeRisk IMI initiative.³⁸ Neurotoxicity is a common cause of test item attrition during nonclinical and clinical development. Therefore, inclusion of safety biomarkers in nonclinical studies provides an opportunity for early detection and monitoring of potential neurotoxic liabilities that might have translational value for clinical trials. In this large IMI study, four fluid-based biomarker candidates for nervous system injury—NF-L, glial fibrillary acidic protein (GFAP), neuron-specific enolase (NSE), and total Tau—were evaluated in plasma and CSF for 15 in vivo rat toxicity studies performed that assessed model nervous system toxicants. Parallel assessment of neural function and structure were conducted to evaluate the sensitivity and specificity of conventional safety endpoints (neurobehavioral testing for function, macroscopic and microscopic pathology evaluation for structure) to detect and characterize nervous system (NS) damage. A statistical approach utilizing receiver operator characteristic (ROC) curves was used to compare the relative utility of these candidate biomarkers. These curves plot the fold change values versus a binary “yes/no” histopathology endpoint and allow the generation of sensitivity and specificity values for a given biomarker. In addition, the area under the curve (AUC) values represent the degree of positive (or negative) correlation for various biomarkers with respect to their abilities in demonstrating NS injury. Plasma NF-L was the single best predictor of damage to both the peripheral (PNS) and central nervous system (CNS; AUC of 0.97-0.99). For CSF, Tau was the most effective marker CNS damage (AUC 0.97) but did not perform well for PNS injury; CSF levels of NSE and GFAP were also suitable for monitoring CNS injury but had reduced sensitivity compared to NF-L. For rat nonclinical studies NF-L has proven to be a sensitive and specific biomarker for detecting test item-related CNS and PNS toxicity. Importantly, measurements of any single biomarker do not indicate the site of neural injury, but parallel analysis of multiple biomarkers in both blood and CSF can provide additional context about the location of the NS injury. Taken together, these results highlight the growing utility of fluid-based (“noninvasive”) safety biomarkers as tools for detecting drug-induced neural injury in nonclinical species that can be translated to monitor potential patient responses in the clinical setting.

Behavioral Tests in Drug Safety Assessment—Are They Relevant for Pathologists?

The second lecture was delivered by Dr. Andrea Greiter-Wilke and covered the behavioral tests that are performed in drug research and development and its relevance for pathologists.

Prior to testing new drug entities in human clinical trials, the International Council for Harmonization (ICH) S7A guideline recommends that a core battery of safety pharmacology tests be conducted to evaluate potential acute drug effects on behavior, coordination, sensory/motor reflex responses, motor activity, and body temperature in nonclinical studies.¹⁶

Dr. Greiter-Wilke described various tests to assess the behavioral effects of drugs, the most common ones being the Irwin screen or the functional observation battery (FOB). The Irwin test was originally developed to detect and distinguish psychoactive compounds in mice,¹⁷ whereas observations in the FOB, originally developed for neurotoxicity testing in rats in the chemical/agrochemical industries, focused more on responses to reflex testing. However, no clear distinctions between the two test batteries exist, and each laboratory employs its own “toolbox” to assess behavior.³⁰ Most of these assessments are done in mice or rats, however successful implementation of parts of the test battery has also been described in dogs,¹¹ cynomolgus monkeys,¹² and the minipig.⁴⁰ Adaptations to reflect the anatomy, physiology, and ease of manual handling of the relevant species need to be considered. General principles are followed for all species, such as performing a sequence of tests from the least (observation) to the most stressful events (manipulation, body temperature measurements) and repeating assessments most commonly over 24 hours to evaluate reversibility or even rebound effects. Since these are subjective tests, the experienced observer should be blinded.

In rodents, additional testing for balance and coordination can be achieved by evaluating their performance on the rotarod that measures the time animals are able to remain on a rotating rod at different speeds over 2 minutes and after a defined number of attempts. The “beam walking” test reflects a more natural rodent behavior of traversing a defined length of a beam of various shapes and diameters, with the time needed and the number of foot slips recorded as parameters.

Another frequent test for behavioral integrity in rodents is the locomotor assessment with automated measurement of voluntary locomotion and rearing in a novel environment, commonly done over 30 to 60 minutes at expected maximal drug exposures. Digital capabilities to assess locomotion in the animal’s home cage have emerged in recent years, providing the opportunity to not only evaluate changes in behavior during the day but also at nighttime, which is the most active period in rodents. This enables long-term and repeated assessment of effects in an automated and hence very objective way without disturbing the animals’ nocturnal activities.^29,37

Dr. Greiter-Wilke also explained that this traditional safety pharmacology evaluation according to the requirements outlined in the ICH guideline S7A (2001) is performed after a single treatment at several dose levels to ascertain human safety in phase 1 studies. Even if adverse effects are noted (e.g. changes in body temperature, arousal, sedation, loss of coordination, reduced response to reflex testing, convulsions, etc.), these hardly ever have a histopathological correlation. Pathology evaluation is therefore not routinely performed after such single-dose studies. The situation is different in repeat dose toxicity studies, where the inclusion of an Irwin screen or the FOB might reveal tolerance, worsening, or even late onset of behavioral changes after longer-term treatment²⁸ that can be associated with degenerative changes in the CNS and/or PNS, or the musculature and would also be reflected in the histopathological evaluation.

A case example of neurotoxicity caused by artemisinin where Irwin screen was performed was discussed by Dr. Greiter-Wilke. Very efficacious derivatives of artemisinin used as antimalarial treatment have been linked to various neurotoxic effects including ototoxicity, tremors, and autonomic, and motor impairments. A long-term study with beta-arteether (bAE) assessed sensorimotor function in 7-day-old rats and tested the same animals as adults in the FOB and for motor activity. Both functional (behavioral and motor) changes and structural (brainstem) findings developed in a dose- and time-related fashion. Repeated dosing of rats with 1 or 5 mg/kg bAE induced subtle motor deficits (e.g., slightly deficient righting reflex with uncoordinated landing), while intermittent cycles of 10 mg/kg bAE produced additional motor and behavioral changes (increased vocalization, resistance to handling, abnormal gait, and less rearing). Rats administered 1 mg/kg bAE developed no neuropathologic changes while some at 5 mg/kg bAE and all given 10 mg/kg bAE had brainstem lesions characterized by progressive motor neuron necrosis with gliosis and satellitosis in the trapezoid, vestibular, and olivary nuclei.⁹

Case examples from the literature as well as company experience also support the need for additional tests to assess CNS and PNS function such as nerve conduction velocity test (NCV). This test has been adapted from human to nonclinical species in drug development to measure the speed of sensory and motor neurons upon stimulation. Negative effects on NCV aided in assessing clinical signs, their progression, and recovery (or lack thereof) for neurological deficits in the case report of a 39-week toxicity study in cynomolgus monkeys, as described by Bopst et al.⁷ In this case, observing clinical signs and their apparent improvement in recovery would have been misleading in judging the reversibility of peripheral nerve damage, since animals learned to adapt foot placement despite their loss of peripheral sensation. The NCV assessment revealed the lack of functional neuronal recovery even 15 weeks after treatment cessation. This was confirmed by the histopathological assessment of axonal degeneration in the mid- and particularly the distal sections of several peripheral nerves and minimal to moderate degeneration in the gracile fasciculus in the cervical section of the spinal cord.

In conclusion, the classical one-day dosing Irwin test/FOB rarely has a histopathological correlation. Its inclusion in repeat dose toxicity studies might aid in picking up effects (or tolerance to them) after prolonged dosing and could support potential histopathological findings. Even though it is most frequently applied in rodents, it is also possible to perform the FOB in the commonly employed nonrodent species in drug development (dogs, cynomolgus monkeys, and minipigs), when adapted to species physiology and anatomy. In certain cases, follow-up additional tests, such as the NCV assessment, might be of advantage to judge the progress or recovery of CNS or PNS lesions. Even though detection of the most common clinical signs in phase 1 studies (headache, nausea, dizziness, fatigue/somnolence, and pain) is limited by the typical nonclinical FOB/Irwin,²² the truly serious and life threatening events (e.g. convulsions or ataxia) can commonly be picked up. The FOB/Irwin has been validated against known CNS-modulating agents, and the assays are sensitive for those less common but potentially more serious adverse events.^2,17,23,31 For the detection of agents inducing peripheral neuropathies, the nonclinical NCV test has proven to be well clinically translatable, as functional properties of peripheral nerves are conserved across species due to the shared structural features of the brain, spinal cord, and peripheral nerves.^1,8,33,35

Neuropathology Through the Lens of Translational Imaging: Opportunities for Preclinical Therapy Assessment

Dr. Nicolau Beckmann gave the last presentation about integration of imaging modalities like magnetic resonance imaging (MRI), nuclear tomographic imaging techniques, X-ray computed tomography and ultrasound in nonclinical and clinical studies. In the past, these imaging techniques were primarily used in clinical settings for diagnostic purposes.

During this lecture, selected examples were used to illustrate the usefulness of noninvasive imaging in preclinical models of neurological disorders in small rodents. Imaging is an increasingly important tool for detecting and quantifying pathology and its progression. In the context of pharmacological investigations, it supports the investigation of drug targets, the assessment of compound effects, and the validation of novel efficacy and/or safety biomarkers.^5,6,27,32

Imaging to examine small rodent models provides many distinct advantages. First, functional, metabolic, molecular, or structural changes can be examined with minimal distress to the animals. Importantly, imaging may be employed to generate information that is not accessible to ex vivo or postmortem approaches. Second, disease progression and responses to therapy may be monitored longitudinally. Third, repeated measurements including one or more baseline (pretreatment) time points allow each animal to serve as its own control. This enhances the statistical power of the studies and can result in an estimated reduction in animal use of more than 80%. Fourth, imaging is significantly less time-consuming than dissection and histopathological examinations, greatly reducing the effort needed for tissue analysis thus accelerating evaluation of drug candidates. Fifth, imaging can supply key details on the optimal treatment regimen therefore improving the design of studies. Importantly, stratification based on imaging data prior to initiating treatment is achievable. Sixth, imaging offers the opportunity for early detection and quantification of potential toxic liabilities.^15,25 Seventh, images may be re-analyzed to address new biological questions without having to run additional animal experiments. Finally, imaging modalities are readily applicable to both the nonclinical and clinical settings, thereby facilitating translational research. Moreover, back-translation of insights from clinical imaging studies can contribute to refining animal models therefore improving the relevance of preclinical studies.

Analyzing potential liabilities of therapies in relevant small rodent models using the same imaging technique as in the clinics facilitates drug candidate selection. Figure 1 illustrates the power of translational imaging in the context of toxicological analyses. Several studies in animals and patients support targeting amyloid-β (Aβ) as a therapeutic strategy for Alzheimer’s disease (AD). Both active and passive anti-Aβ immunotherapies have been demonstrated in transgenic mice and patients to reduce the brain load of amyloid plaques.²¹ However, in some cases T₂*-weighted MRI detected the induction of microhemorrhages during the course of anti-Aβ immunotherapy (Figure 1A),³ thereby raising safety concerns for therapeutic approaches directed toward removing Aβ.³⁴ In a preclinical study, T₂*-weighted MRI has been used in the context of long-term administration of a beta-site amyloid precursor protein cleaving enzyme 1 (BACE) inhibitor to investigate microhemorrhages in an amyloid precursor protein (APP) transgenic murine model,⁴ where APP23 mice naturally develop extensive cerebral amyloid angiopathy and microhemorrhages during aging³⁹ (Figure 1B). Animals were treated for 3 months with vehicle (nonrelevant control antibody), a β1 mouse monoclonal IgG2a antibody known to induce microhemorrhages in APP23 mice,²⁶ or with a brain-penetrable BACE inhibitor. A strong increase in microhemorrhage volume was detected in the brains of APP23 animals receiving the β1 antibody, while treatment with vehicle or BACE led to a similar lesion development as detected during natural aging (Figure 1C). These data attested to the safety of the BACE inhibitor, which went into further clinical testing.²⁴

Figure 1.

Potential safety issues investigated using translational imaging. (A) Several punctate microhemorrhages in the right parietal region (circle) detected by T₂*-weighted MRI in an AD patient after 19 weeks of treatment with an IgG1 antibody. Reproduced with permission from Barakos et al.³ ©American Society of Neuroradiology. (B) Left: Representative T₂*-weighted image of a 15.5-month-old APP23 mouse, displaying round areas of signal void (arrowheads) corresponding to microhemorrhages. Right: natural increase of microhemorrhages during aging. (C) Effects of compound treatment on microhemorrhages quantified noninvasively by T₂*-weighted MRI. Modified from Beckmann et al.⁴ ©The authors. Published by Elsevier Inc.

In conclusion, this session highlighted the significance of neural biomarkers and functional endpoints in nonclinical studies for detecting acute or delayed peripheral (PNS) and CNS alterations and /or injury caused by drugs during development. Integration of these neural biomarkers in conjunction with detailed histopathological analysis aids in better predicting neurotoxicity in nonclinical studies.

Footnotes

Acknowledgements

The authors thank the European Society of Toxicologic Pathology’s (ESTP) Scientific Organizing Committee for their support of this program. They thank the staff of the ESTP for organizational support in producing the materials and arranging the meeting space. The authors thank Dr. Brad Bolon for providing editorial comments to this paper.

Author Contributions

The authors are solely responsible for the contents and drafting of this paper.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

ORCID iDs

Andrea Greiter-Wilke

Nicolau Beckmann

Diethilde J. Theil

Ingrid D Pardo

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