Freeze-Drying Process Design for Radiopharmaceutical Development of Peptide-Based FAPI and PSMA Ready-to-Use Kits for Labeling with Technetium-99m Including Stability Studies on Batch Formulations

echnetium-99m radiopharmaceuticals are indispensable in modern medical imaging, offering high-quality images with relatively low risk, aiding in diagnosing, staging, and following various diseases, particularly in oncology, cardiology, and neurology. Their unique properties and the continuous development of new Tc-99m labelled compounds expand their potential in medical diagnostics.
Tc-99m is the most frequently used SPECT radionuclide. The optimal energy of emitted photons for imaging, its wide availability, and the low costs of 99Mo/99mTc generators have facilitated its frequent use in diagnostic imaging.
Radioactive peptides that recognize tumor cells are already being used for diagnosis and treatment of malignant diseases, causing image or tumor cell death by radiation toxicity through specific binding to the cancer cells.

In our previous work, we indicated the possibility of introducing available technology for the "ready to use" preparation of a cold kit freeze-dried formulation of conjugated antibodies (rituximab, trastuzumab) and peptide-based radiopharmaceuticals for labeling with different radioisotopes. At the same time, we established and standardized the methods for identification and characterization of the obtained products, suggesting quality control and demonstrating the stability of the final product.
For this project proposal, we aim to design and evaluate novel Technetium-99m peptide based radiopharmaceuticals for selective targeting, focusing on the following pharmacophores:
- Fibroblast activation protein inhibitor (FAPI), a peptide based on the molecular targeting of the FAP, which is known as a serine proteinase highly expressed on the surface in the major cell population in tumor stroma on the subpopulation of activated fibroblasts, termed cancer-associated fibroblasts.
- Prostate cancer-specific membrane antigen (PSMA), type II transmembrane glycoprotein (100 kDa) expressed in prostate epithelial cells and demonstrates glutamic carboxypeptidase (GCP-II) and folic acid hydrolase activity. PSMA is highly overexpressed on the surface of 90% of PCa cells, 1000 times more than in normal tissue. The expression of PSMA is further increased in high-risk PCa, metastatic PCa and castration-resistant prostate cancer (CRPC).

This project proposal will be focused on enhancing cancer imaging by designing selective and targeted radiopharmaceuticals based on the FAPI and the PSMA protein, labeled with Technetioum-99m, for clinical use in malignant diseases. Thorough evaluation of the proposed formulations will facilitate further diagnostic development, promising targeted and effective cancer and metastasis discovering. An important aspect of this project is to make advanced cancer treatments accessible in developing countries, thereby improving global access to cutting-edge medical interventions.
To achieve a stable formulation of a potential radiopharmaceutical using Technetium-99m as a radionuclide, it is crucial to introduce a suitable molecule and chelators specific for Technetium-99m labelling. These steps are executed before the radionuclide is added in the final stage, therefore saving many half-lives of radioactivity and obtain interpretable image.
In this project proposal we suggest using several molecules of FAPI and PSMA, published in the literature and commercially available. This will result in identifying the best composition for the final product and employ procedure for the labeling with Tc-99m. Tc-99m (t1/2 = 6.0 hours) is a pure γ–ray emitting radionuclide rendering this isotope suitable for SPECT imaging.
Because peptides, as biopharmaceutical formulations are often unstable liquids having a poor shelf-life, freeze-drying is one way to convert these sensitive molecules into stable formats. Acoording to that, we propose to employ previously established and standarized protocols for preparation of home-made freeze-dried radiopharmaceuticals in a stable, shippable after designing the final formulation. The final reeze-dryed product, as potential radiopharmaceuticals will be evaluated using standardized techniques for quality control of nonradioactive (“cold”) formulated kit and radioactive form after the labeling with Tc-99m. All kits will be prepared according GMP/GMRP.

The very important segment in our proposed project concerns defining the final formulation that should be ready for use in clinical studies in human medicine, but preferably also in veterinary medicine, considering the similar mechanism of expression of pathological changes. In accordance with this, an important part is the realization of stability studies in the same way as they are carried out for all biological preparations in the pharmaceutical industry. We expect these studies to provide information on the length of use of potential whales for use and at the same time be a guarantee of quality.

The last part of our project proposal will consist of determining the pharmacokinetic parameters of potential radiopharmaceuticals using in silico modeling. These predictions are helpful in optimizing the design and dosing strategies of radiopharmaceuticals, improving their therapeutic efficacy, minimizing potential adverse effects, and may provide a cost-effective and time-efficient approach to obtain critical information on the behavior of radiopharmaceuticals. This ultimately contributes to more informed decision-making in their clinical development and application.