Compounding of Tc-labeled antimicrobial peptide for molecular imaging of bacterial infections

In clinical practice it is often very challenging to discriminate bacterial infection in bone, soft tissue and orthopedic devices from sterile inflammation, due to the common unspecific symptoms. The most useful diagnostic procedures for detecting infection in such conditions are histopathology and imaging studies. Nuclear Medicine imaging techniques have been widely used to detect functional, metabolic, as well as biochemical changes at molecular level. In vitro radiolabeled autologous leukocytes have been considered “gold standard” for visualization of suspecting infections (Palestro et al., 2009), especially in orthopedic surgery. Besides 99m Tc and 111 In – labeled leukocytes, used as single photon emitting (SPECT) radiopharmaceuticals (RF), 18 FFDG and recently 64 Cu as positron emitting tomography (PET) labels are introduced to improve the quality of leukocyte imaging studies (Rini and Palestro, 2006). Nevertheless, they have all been proved to be non-specific in discrimination between infection and sterile inflammation (Bunyaviroch et al., 2006). Radiolabeled antimicrobial peptides (AMPs) are new generation of radiopharmaceuticals, developed to overcome the lack of specificity issue. One of these peptides is derivate (29-41 fraction) of ubiquicidine (UBI), a natural AMP found in all mammal tissues, which specifically binds to bacterial membrane, enters the cell and remains within the cytoplasm. Our aim was to present the compounding of 99m Tc-UBI (29-41) and quality control of the radiopharmaceutical, as primary step of product introduction in Nuclear Medicine Department settings.


Introduction
In clinical practice it is often very challenging to discriminate bacterial infection in bone, soft tissue and orthopedic devices from sterile inflammation, due to the common unspecific symptoms. The most useful diagnostic procedures for detecting infection in such conditions are histopathology and imaging studies.
Nuclear Medicine imaging techniques have been widely used to detect functional, metabolic, as well as biochemical changes at molecular level. In vitro radiolabeled autologous leukocytes have been considered "gold standard" for visualization of suspecting infections (Palestro et al., 2009), especially in orthopedic surgery. Besides 99m Tc and 111 Inlabeled leukocytes, used as single photon emitting (SPECT) radiopharmaceuticals (RF), 18 F-FDG and recently 64 Cu as positron emitting tomography (PET) labels are introduced to improve the quality of leukocyte imaging studies (Rini and Palestro, 2006). Nevertheless, they have all been proved to be non-specific in discrimination between infection and sterile inflammation (Bunyaviroch et al., 2006).
Radiolabeled antimicrobial peptides (AMPs) are new generation of radiopharmaceuticals, developed to overcome the lack of specificity issue. One of these peptides is derivate (29-41 fraction) of ubiquicidine (UBI), a natural AMP found in all mammal tissues, which specifically binds to bacterial membrane, enters the cell and remains within the cytoplasm. Our aim was to present the compounding of 99m Tc-UBI (29-41) and quality control of the radiopharmaceutical, as primary step of product introduction in Nuclear Medicine Department settings.

Results and discussion
Successful 99m Tc-UBI compounding process and the stability of the RF are dependent of alkaline pH. During the 99m Tc UBI freeze-dried kit compounding, the formation of radioactive complex occurred at pH 9. After adjustment with saline, final pH was 7.5. In all three "in-situ" compounded formulations the pH value, measured after incubation, was between 9 and 10. After obtaining the final volume, the pH remained within the same range.
Quality control results obtained for the 99m Tc UBI "in-situ" formulations reveal high RCP in all 3 RFs and had not been affected by the increased quantity of the ligand and alkalizing agent. RCP by ITLC and HPLC was 97.9-98.6% and 99.5-99.8% respectively.

Conclusion 99m
Tc UBI compounding was successfully performed with freeze-dried kit and "in-situ" formulations, both with high PCP. In Nuclear Medicine Department settings without GMP premises for in-house kit production, compounding of the RF could be performed in Shielded Biosafety LAF cabinet using "in-situ" unit dose formulations.