Title of the project
Imaging of PSMA-targeted radiopharmaceuticals for cancer
NTS Identifier
NTS-NO-188773 v.1, 23-02-2024
NTS National Identifier
Field will not be published.
EU Submission
Field will not be published.
Yes
Project duration expressed in months.
48
Keywords
Diagnostikk
PET/SPECT imaging
prostate cancer
Targeted Radiopharmaceuticals
Purpose(s) of the project
Translational and applied research: Human Cancer
Describe the objectives of the project (for example, addressing certain scientific unknowns, or scientific or clinical needs).
There is a significant unmet need for treatment options for cancer patients. Highly expressed surface molecules on cancer cells (and low expression on normal cells) are ideal targets for radioligand therapy (TRT). TRT has gained a lot of interest the past few years as it enables specific delivery of toxic radiation to cancer cells while minimising the exposure to normal cells. In 2022, 177Lu-PSMA-617 was approved for the treatment of metastatic prostate cancer. We work on developing other radioligands for the treatment of cancer. However, in addition to testing the therapeutic effect of the radioligand, being able to track them in different tissues and follow the expression of the targeted molecule during uptake of the radioligand can be useful to better understand the distribution of the radioligand. Multimodality imaging of tumor microenvironment is available to optimize personalized treatment. The aim of this study is to evaluate the pharmacokinetic profile, biodistribution and diagnostic capabilities of our novel tumor targeting peptide by PET/SPECT-CT in mouse models of cancer. Peptides have been synthesized, radiolabelled and screened in vitro. PET/SPECT-CT imaging will be performed to evaluate the radiopeptides' potential as imaging agents in relevant tumor models of cancer.
We have previously shown therapeutic efficacy of our radioligand products through animal experiments at KPM-Rad. In these studies, we have also found that multiple injections improves therapeutic efficacy, and we are interested in seeing wheter the uptake of the radioligand or the expression of the target is part of the reason for this. PET/SPECT-CT allow non-invasive in vivo examination in real time of the tumor targeting radiopeptides providing quantitative data of their biodistribution, pharmacology as well as specificity for relevant oncological targets.
Therefore, the aim of this research is to investigate the uptake of radioligands in mice with subcutaneous or metastatic xenografts using preclinical SPECT-CT and autoradiography, whereas the expression of the targeted molecule on the cancer cells will be investigated by preclinical PET.
What are the potential benefits likely to derive from this project? Explain how science could be advanced, or humans, animals or environment may ultimately benefit from the project. Where applicable, differentiate between short-term benefits (within the duration of the project) and long-term benefits (which may accrue after the project is finished).
Metastatic cancer is responsible for 90% of cancer-related deaths worldwide. The radioligand AB001 is targeting the prostate specific membrane antigen and is a highly promising treatment for metastatic prostate cancer. This compound will be tested in a Phase 1 clinical study in 2024, and towards that, optimisation of treatment regimens and gaining knowledge about the characteristics of the radioligand in vivo will give us valuable results.
The proposed research addresses important issues in developing better treatment strategies for patients with metastatic prostate cancer failing available therapies. Preclinical imaging tools offer non-invasive insights into tumor characteristics, aiding in the development of more effective and personalized cancer treatments, while minimising side-effects. By achieving these objectives, suitable PET/SPECT imaging targets with the potential to enhance diagnosis, staging and treatment will be selected for further applications. The ultimate goal is to translate findings into innovative and targeted cancer treatments for clinical use.
In what procedures will the animals typically be used (for example, injections, surgical procedures)? Indicate the number and duration of these procedures.
The procedures reported in this section are described in detail in Attachment "Summary of experimental groups”.
Ear marking: Animals will be ear marked with an ear punch before the experiment. Frequency: 1 time. Duration: 1-2 min.
Establishment of human prostate tumor xenografts, and metastatic prostate cancer in mice will be performed subcutaneous or by intracardiac injection of prostate cancer cells, respectively (day 0). The C4-3 and PC-3 cells that will be injected into animals has been tested for murine viruses and mycoplasma before injection (attachments 35 and 36).
Frequency: 1 time. Duration: 5 min for subcutaneous, 5-10 min for intracardiac.
Intravenous injection of therapeutic radiopharmaceutical: 7 days after subcutaneous or intracardiac inoculation of cells (day 0), the mice will be injected intravenously with radiopharmaceuticals to the tail vein (0.05–0.15 ml). Mice will be injected up to 6 times (day 0, 3, 6, 9, 12, 15 for the 3-day interval gr; day 0, 7, 14, 21, 28, 35 for the 7-day interval gr; day 0, 14, 28 for the 14-day interval gr). Frequency: 1-6 times. Duration of restrain and injection: 5 min.
Intraperitoneal injection of D-luciferin and optical imaging using MILab imaging system: Weekly, mice with metastatic tumors will receive 150 μg/g body weight luciferin (see attachment "Luciferin preparation" and "Intraperitoneal injection") 10-15 minutes prior to optical imaging (see attachment “Optical imaging of mice”) to track tumor progression. Mice will be injected once per week up to experimental endpoint after 3 months. Frequency: 1-8 times. Duration of restrain and injection: 1-3 min. Duration of imaging: up to 5 min
Intravenous injection of PET tracer and PET imaging using MILabs optical imaging system: 1 day before TRT administration, the mice will be injected intravenously with PET tracer to the tail vein (0.05-0.15 ml). Frequency: max 1 time per mouse. Duration of restrain and injection: 5 min. Duration of PET imaging: up to 1h.
SPECT/CT and optical imaging using MILabs optical imaging system: SPET/CT imaging will be performed at 2 and 24 h after TRT administration. Frequency: max 1 time per mouse. Duration of imaging: up to 1h.
Euthanasia day: Mice will be euthanized by cervical dislocation. Several organs including tumour, kidneys, femur, and salivary glands will be collected for autoradiography or histopathology analysis.
What are the expected impacts/adverse effects on the animals, for example pain, weight loss, inactivity/reduced mobility, stress, abnormal behaviour, and the duration of those effects?
Subcutaneous injection: Sore or hematoma formation at the injection site. Wiping the injection site with a soaked paper in ethanol and correct technique will help preventing this plausible outcome. Duration of a complication can be between 1 day and several days (long-lasting mild pain). If the mouse does not recover, it will be sacrificed.
Intracardiac injection: Injury to the coronary artery and hemorrhage within the pericardium. Duration will be 5-10 minutes immediately after the injection (short term moderate to severe pain). A too deep injection can puncture the lungs. After waking up from the anasthesia, these possible failures will be seen immediately (short term moderate pain). If so, the animal will be euthanized. Vetergesic will always be used as an analgesic. If normal behavior is not observed after the mice wakes up, they will be euthanized.
Intravenous injection: Restraining device, which can be stressful. The maximum duration is 5 minutes per mouse (short term moderate distress) and the animals are expected to recover fast without any impairment on the wellbeing.
The compounds administered in the intravenous injection will be radioactive, but no lethal or toxic dose will be used. Therefore, this procedure is only expected to cause mild clinical abnormality within the time-scale of the study according to the relevant radionuclide.
Intraperitoneal injection: Bleeding at injection site (solution: apply pressure until the bleeding stops and clean with gauze and water), peritonitis – inflammation or infection of the peritoneal cavity (if gut is punctured or a non‐sterile substance injected), laceration of abdominal organs and internal bleeding or infection, injection into the gastrointestinal tract or bladder. It is not expected to observe any urgent harm from these possible effects, but long-lasting mild pain (for instance from not recovering at the injection site for days) must be followed up if it occurs.
Imaging: Animals will be anesthetized using gas anasthesia. No harmful effects are expected on the animals.
What species and numbers of animals are expected to be used? What are the expected severities and the numbers of animals in each severity category (per species)?
Species
Total number
Estimated numbers per severity
Non recovery
Mild
Moderate
Severe
What will happen to the animals kept alive at the end of the procedure?
Species
Estimated numbers of animals to be reused, to be returned to habitat/husbandry system or to be rehomed
Reused
Returned
Rehomed
Please provide reasons for the planned fate of the animals after the procedure.
All mice in this study will be injected with cancer cells. The radioligand injected to observe uptake in different organs is not necessarily toxic, and the animal can therefore live further and be re-used if an additional imaging procedure would be relevant. Mice that for some reason will be treated or end up having no tumor take will be euthanized once the studies are finished as they can’t be re-used due to the uncertainty of their tumour status.
1. Replacement
State which non-animal alternatives are available in this field and why they cannot be used for the purposes of the project.
Optimisation of imaging protocols with the nuclides desired to use herein have been performed throughout the last year using mouse phantoms as a replacement for animals. This reduces the number of scans per animal, as the setting and tools already are optimized.
2. Reduction
Explain how the numbers of animals for this project were determined. Describe steps that have been taken to reduce the number of animals to be used, and principles used to design studies. Where applicable, describe practices that will be used throughout the project to minimise the number of animals used consistent with scientific objectives. Those practices may include e.g. pilot studies, computer modelling, sharing of tissue and reuse.
Advances in preclinical imaging technologies have been achieved the last year, such as higher resolution imaging or more sensitive detectors. This contributes to increased data quality. This improved sensitivity may allow researchers to obtain meaningful results with smaller sample sizes, reducing the overall number of animals needed for a study. Moreover, optimisation of imaging protocols have been performed throughout the last year using mouse phantoms. This reduces the number of scans per animal, as the setting and tools already are optimized.
Instead of using separate groups of animals for each time point or experimental condition, researchers can opt for sequential imaging studies on the same group of animals. This allows longitudinal data collection over time, reducing the total number of animals needed for the study.
Utilizing robust statistical design and conducting power analyses will help researchers determine the optimal sample size needed to achieve statistically significant results. By ensuring that experiments are adequately powered, researchers can avoid using an excessive number of animals while still obtaining reliable and meaningful data.
When appropriate, we will use a shared control group across multiple experimental conditions rather than having a separate control group for each condition. This approach allows for a reduction in the overall number of animals used while maintaining experimental rigor.
Since we already have preformed multiple therapy studies on this radioligand in mice before (FOTS numbers 22197 and 30148), establishments of the number of studies needed for imaging can mentioned as a reduction strategy. We have built upon existing datasets, reducing the need for additional experiments to answer related research questions.
3. Refinement
Give examples of the specific measures (e.g., increased monitoring, post-operative care, pain management, training of animals) to be taken, in relation to the procedures, to minimise welfare costs (harms) to the animals. Describe the mechanisms to take up emerging refinement techniques during the lifetime of the project.
Using appropriate anesthetic agents (herein; sevofluran), monitoring vital signs, and ensuring proper recovery procedures will be done to contribute to the overall well-being of the animals.
Optimisation of imaging protocols have been performed throughout the last year using mouse phantoms. This reduces the number of scans and the time animals spend under anesthesia, as the setting and tools already are optimized.
All animals will be provided with a stimulating and enriched environment when not undergoing imaging procedures. Enrichment activities, such as the provision of toys, tunnels, or social interactions, contribute to the overall well-being of the animals and may mitigate stress associated with research activities.
Refinement extends beyond the laboratory to data sharing and collaboration. Researchers with long experience in the imaging techniques will provide protocols, and insights, reducing the need for redundant experiments. Collaboration allows the scientific community to collectively benefit from the knowledge gained without unnecessarily repeating studies.
Regularly assessing and reassessing protocols, incorporating feedback from animal care specialists, and staying informed about advancements in imaging technology will be done, which contribute to ongoing refinement efforts.
Explain the choice of species and the related life stages.
Athymic nude mice will be used for all studies. Athymic nude mice lack T-cells and NK cells, but contain functional B-cells and is better for short term toxicity, therapy and biodistribution studies. We have long experience with this breed and are familiar with the expected responses using these mice with our cancer cells and radioactive compounds.
Male mice are chosen because we are only studying prostate cancer. Mice will be delivered with an age between 3 and 5 weeks, and we wait minimum 1-3 weeks (until they are >25 g), before we start the experiments.