Nose-to-Brain Delivery in NHPs: 125I SPECT Case Study

The blood-brain barrier (BBB) is one of the main constraints in central nervous system (CNS) drug development. It limits the entry of many small molecules and most biologics into the brain parenchyma. Intranasal, or nose-to-brain delivery has therefore attracted interest because the nasal cavity is anatomically connected to the CNS through the olfactory and trigeminal pathways. These pathways may provide access to the brain that is not explained by systemic circulation alone.

The scientific challenge is not simply to select an intranasal route. It is to demonstrate where the administered material goes, how much signal is associated with the brain, how that signal changes over time, and whether the observation reflects the intact test article rather than a detached label or metabolite. Plasma pharmacokinetics (PK) cannot answer these questions. Cerebrospinal fluid (CSF) sampling provides information from a limited compartment and a limited number of time points. Terminal brain analysis provides direct tissue measurements, but each animal generally contributes one terminal observation.

Radiolabeled molecular imaging provides a complementary way to address these questions in non-human primates (NHPs). By combining a radiolabeled test article with serial SPECT/CT, investigators can follow tracer-associated signal in the brain and throughout the body in the same animal over time. This article discusses an iodine-125-labeled peptide case presented during the Prisys Biotech molecular imaging webinar and explains what the study design can, and cannot, demonstrate.

Why Plasma PK Is Not Sufficient to Evaluate CNS Delivery

Systemic exposure is not equivalent to brain exposure. A compound may produce measurable or even substantial plasma concentrations while reaching only limited concentrations in the brain. The relationship is particularly difficult to interpret after intranasal administration, because the administered material may be retained in the nasal cavity, cleared into the respiratory tract, absorbed systemically, or transported toward the CNS through local anatomical pathways.

CSF sampling adds useful information, but CSF is not a direct surrogate for every brain compartment. Sampling is invasive, provides sparse time points, and may not represent regional parenchymal exposure. Terminal brain tissue analysis is more direct, yet a longitudinal retention curve requires separate animals for each terminal time point. This increases the number of animals required and makes the time course more dependent on between-subject variability.

Neither plasma PK nor a single terminal measurement provides a complete spatial picture. For an intranasal CNS program, investigators may also need to know whether the test article remains in the nasal cavity, reaches the lower airways, distributes to other organs, or shows a regional pattern within the brain. These are distribution questions as much as they are concentration questions.

How Radiolabeled SPECT Supports Nose-to-Brain Assessment

A molecular imaging study begins with selection of a radionuclide and a labeling strategy that fit the test article and the expected biological time course. In the webinar case, the peptide was labeled with iodine-125 (125I), a radionuclide with a physical half-life of approximately 59.4 days. Its relatively long physical half-life is compatible with serial observation over hours to days, which is relevant when brain retention may extend beyond the short imaging window of commonly used short-lived PET radionuclides.

The labeled material is administered at tracer level. This type of microdose design is intended to provide a detectable imaging signal while minimizing pharmacological perturbation by the labeled material. It does not, by itself, establish that the labeled preparation behaves identically to the unlabeled therapeutic. That question depends on the labeling site, specific activity, radiochemical purity, formulation, and preservation of the parent molecule's biological properties.

SPECT/CT combines radionuclide-derived molecular information with anatomical co-registration. Serial scans can be used to evaluate:

• whether brain-associated signal is detectable after intranasal administration;
• the anatomical distribution of signal within the imaged CNS regions;
• the change in brain-associated signal over time; and
• concurrent disposition in the nasal cavity, airways, lungs, and other organs within the field of view.

The resulting measurements are best interpreted as tracer-associated signal or radiolabeled-material distribution. They should not automatically be described as intact parent-drug concentration unless label stability and ex vivo bioanalysis support that interpretation.

Case Study: Serial 125I SPECT After Intranasal Peptide Administration in NHPs

The case was conducted by Prisys Biotech's NHP translational research capabilitiesa and radiochemistry and molecular imaging capabilities. The study used an iodine-125-labeled peptide administered intranasally to NHPs.

Serial SPECT imaging was performed at 1, 24, 48, 72, and 96 hours after administration. The imaging sequence was designed to examine the presence and distribution of brain-associated radiolabeled signal over a multi-day period rather than relying on a single terminal measurement.

The case provided three related types of information. First, brain-associated signal was observed after intranasal dosing, supporting further investigation of CNS delivery. Second, the distribution of the signal could be assessed across anatomical brain regions rather than treated as a single whole-brain value. Third, the serial scans showed how the brain-associated signal changed during the 96-hour observation period, including retention of detectable signal for at least 72 hours as presented in the webinar.

The same acquisitions also provided information outside the CNS. Signal was evaluated in the nasal region, cheek, bronchial tract, and lung. This matters because local deposition and respiratory clearance can influence the fraction of an intranasal dose that remains available for potential nose-to-brain transport. An imaging design that examines both CNS-associated and respiratory disposition can therefore help investigators interpret the delivery route as a whole rather than focusing only on the final brain image.

What the Study Design Can Answer

1. Is brain-associated signal detectable after intranasal dosing?

Serial SPECT provides in vivo evidence that radiolabeled material is present in the imaged brain region after administration. This is stronger than inferring CNS delivery from plasma exposure alone. The result should still be described precisely: imaging demonstrates brain-associated tracer signal, but it does not by itself prove the exact transport route or establish that the intact peptide entered a particular brain cell type.

2. Where does the signal accumulate?

SPECT/CT provides spatial information at the level permitted by the modality, registration quality, reconstruction method, and study-specific resolution. Regional analysis can show whether signal is concentrated in particular anatomical areas. This is important for CNS programs in which a whole-brain average could conceal regional differences in exposure or retention.

3. How does brain-associated signal change over time?

The 1- to 96-hour sequence provides a within-subject time course. Investigators can examine the time to the highest observed signal, persistence over the observation window, and apparent washout. These imaging-derived observations can be considered alongside plasma PK, CSF data, pharmacodynamic endpoints, and ex vivo tissue measurements.

4. Where does the remainder of the dose go?

Whole-body or extended-field imaging can reveal concurrent distribution outside the brain. For intranasal dosing, the nasal cavity and respiratory tract are especially relevant. Peripheral organ distribution can also help identify exposure patterns that may be relevant to safety, clearance, or interpretation of the apparent CNS delivery fraction.

5. Can the study reduce dependence on terminal sampling?

Serial imaging allows repeated observation in the same subject, which may reduce the need to assign separate terminal cohorts to every time point. The extent of any reduction depends on the study objectives, the validation plan, the number of animals, the imaging schedule, and the need for direct ex vivo confirmation. Molecular imaging should therefore be viewed as a way to improve the information obtained from an animal study, not as an automatic substitute for terminal analysis.

What Imaging Does Not Establish on Its Own

The first limitation is spatial and cellular resolution. SPECT/CT can localize radiolabeled signal to anatomical regions, but it cannot determine whether the material is inside neurons, glial cells, vascular compartments, or the extracellular space. Cellular and subcellular localization requires methods such as autoradiography, histology, immunohistochemistry, or other terminal assays.

The second limitation concerns molecular identity. The camera detects radioactive decay from the isotope. It does not distinguish intact peptide from a radiolabeled metabolite or free iodine if the label is released in vivo. Label stability should therefore be assessed before and during the study where appropriate. Selected terminal time points can be paired with ex vivo bioanalysis to determine how closely the imaging signal represents the parent test article.

The third limitation is route attribution. Brain-associated signal after intranasal dosing is compatible with CNS delivery, but SPECT alone cannot definitively distinguish direct nose-to-brain transport from systemic absorption followed by subsequent BBB passage, or from a combination of routes. Route-specific conclusions require integration with study design, formulation data, plasma and tissue PK, CSF measurements where appropriate, and relevant controls.

These limitations do not reduce the value of imaging. They define how the data should be used. Serial imaging can identify the regions and time points that warrant detailed terminal investigation, allowing ex vivo work to be focused on specific questions rather than distributed across an arbitrary time course.

Why NHPs Are Relevant for Nose-to-Brain Development

Rodents remain useful for formulation screening, mechanism studies, and early proof-of-concept work. However, the nasal anatomy and respiratory physiology of rodents differ from those of humans. Rodents are obligate nose breathers, and the olfactory epithelium occupies a proportionally larger part of the nasal cavity. These features may influence deposition and may lead to a higher apparent contribution from olfactory transport than would be expected in humans.

NHPs provide a closer anatomical and physiological context for selected translational questions, including nasal cavity geometry, airway organization, breathing pattern, CNS anatomy, and compatibility with clinical-equivalent imaging. NHP data do not eliminate uncertainty in human translation, and they should not be interpreted as a direct prediction of clinical exposure. Their value is that they allow route, regional distribution, retention, and systemic disposition to be evaluated in a model with greater relevance to human anatomy than standard rodent studies for some delivery questions.

The choice of species should remain driven by the development question. An NHP study is most informative when it is designed to resolve a decision that cannot be adequately addressed in vitro or in rodents, such as formulation selection, regional CNS exposure, delivery-device assessment, or the relationship between local deposition and systemic exposure.

Integrating Imaging With PK, Bioanalysis, and Pharmacology

Molecular imaging is most useful when it is planned as one component of an integrated translational dataset. A nose-to-brain study may combine serial SPECT/CT with plasma PK, CSF sampling, nasal and respiratory assessments, tissue bioanalysis, biomarker measurements, and pharmacodynamic endpoints.

This integration allows several layers of evidence to be compared:

• Systemic exposure: plasma concentration, exposure, and clearance;
• Spatial exposure: regional brain and peripheral-organ distribution of the radiolabeled material;
• Temporal exposure: change in signal from the early post-dose period through later retention or washout;
• Molecular identity: the proportion of signal attributable to intact parent material or relevant metabolites; and
• Biological effect: target engagement, biomarker response, or functional pharmacology.

No single layer is sufficient for every program. The translational interpretation becomes stronger when the imaging signal is linked to the measured test article, the expected mechanism, and a biological response that is relevant to the therapeutic hypothesis.

Prisys Biotech's Role in NHP Nose-to-Brain Imaging Studies

Prisys Biotech conducts NHP translational studies that can incorporate intranasal dosing, serial molecular imaging, PK/PD, biomarker analysis, and complementary tissue assessments. The workflow can include radionuclide selection, radiolabeling, post-labeling preparation and purification, quality control, large-animal SPECT/CT or PET/CT, and quantitative image analysis.

The webinar materials describe labeling and imaging workflows applicable to small molecules, peptides, proteins, macromolecules, nanomaterials, and antibodies. The appropriate radionuclide and modality depend on the test article, labeling chemistry, expected kinetics, required observation window, sensitivity, and study objectives. These parameters should be established during study design rather than selected solely on the basis of instrument availability.

For nose-to-brain programs, the practical objective is to connect the delivery procedure with a defensible evidence package: where the material is detected, how its distribution changes over time, what proportion of the signal can be attributed to the parent molecule, and whether the exposure is associated with a pharmacological effect.

Conclusion

Serial 125I SPECT provides a way to evaluate brain-associated and respiratory-system distribution after intranasal peptide administration in NHPs. In the webinar case, scans at 1, 24, 48, 72, and 96 hours showed how a single tracer-level administration could generate longitudinal and spatial information, including detectable brain-associated signal for at least 72 hours as presented by the study team.

The method does not independently prove the exact nose-to-brain transport mechanism, cellular localization, or intact-parent concentration. Those questions require complementary controls, label-stability assessment, bioanalysis, and, where necessary, terminal tissue methods. Its value lies in adding a repeated in vivo view of distribution and retention to the conventional PK and tissue-analysis framework.

For CNS drug development, the most informative design is usually not imaging instead of bioanalysis. It is imaging together with bioanalysis, pharmacology, and appropriate translational endpoints. This combination can support more precise decisions about formulation, dosing, regional CNS exposure, retention, and the next stage of nonclinical development.