Nucl Med Biol. 2014 May-Jun;41(5):390-400.

Peptide synthesis, characterization and ⁶⁸Ga-radiolabeling of NOTA-conjugated ubiquicidin fragments for prospective infection imaging with PET/CT.

Ebenhan T1, Chadwick N2, Sathekge MM3, Govender P2, Govender T4, Kruger HG4, Marjanovic-Painter B5, Zeevaart JR6.
  • 1School of Chemistry, University of KwaZulu-Natal, Durban, South Africa; Radiochemistry, The South African Nuclear Energy Corporation, Pelindaba, South Africa.
  • 2School of Life Sciences, University of KwaZulu-Natal, Durban, South Africa.
  • 3Department of Nuclear Medicine, University of Pretoria and Steve Biko Academic Hospital, Pretoria, South Africa.
  • 4School of Chemistry, University of KwaZulu-Natal, Durban, South Africa.
  • 5Radiochemistry, The South African Nuclear Energy Corporation, Pelindaba, South Africa.
  • 6Department of Science and Technology, Preclinical Drug Development Platform, North West University, Potchefstroom, South Africa. Electronic address: zeevaart@necsa.co.za.

 

ABSTRACT:

INTRODUCTION:

Human antimicrobial peptides are of interest for the development of positron emission tomography (PET) tracers as they exhibit desirable characteristics that make them good candidates for targeting vectors. Due to their natural role in the innate immune system they selectively bind to pathogenic bacteria and yeast, whilst remaining minimally immunogenic and cytotoxic to humans. Research into ubiquicidin (UBI)-based tracers has focused on (99m)Tc as a radionuclide, however, the use of bi-functional chelators such as 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), in combination with ⁶⁸Ga as a radionuclide, allows for a simple radiolabeling procedure which is preferable in a clinical setting using PET/CT.

METHODS:

The peptides fragments UBI29-41, UBI30-41 were synthesized by standard microwave Fmoc/tert-butyl (tBu)-solid phase synthetic protocols. Characterizations were performed using analytical HPLC and LC/MS. Both NOTA-conjugated peptides were exposed to (nat)Ga³⁺; their complexed form was quantified by direct LC/MS injection. This complexation was utilized to testify bacterial and mammalian cell binding potential of fluorophore-linked NOTA-UBI29-41/30-41. ⁶⁸Ga labeled NOTA-UBI fragments were also tested for competitive interaction to Staphylococcus aureus to proof the binding target. ⁶⁸Ga was eluted from SnO₂- and TiO₂-based ⁶⁸Ge/⁶⁸Ga generators using fractionated elution and anion exchanged-based post-procession. NOTA-peptide radiolabeling was carried out including optimization of buffer molarity, NOTA-peptide concentration(s), incubation temperature and -duration as well as considering various SPE purification cartridges.

RESULTS:

Pure UBI29-41, UBI30-41 and NOTA-UBI30-41 were successfully characterized. Both, NOTA-UBI fragments exhibited complexation rates to (nat)Ga³⁺)≥ 99%. The percentage binding was significantly higher to Staphylococcus aureus bacilli over Mt4 human leucocytes (P>0.05) for NOTA-UBI29-41[Lys(Abz)]<NOTA-UBI30-41[Lys(Abz)]. Significant lower binding was observed for both ⁶⁸Ga-labeled NOTA-UBI fragments (P >0.03) after pre-incubation with excess unlabeled NOTA-UBI. Reproducible ⁶⁸Ga radiolabeling ranged for 51-85% and 46-78% for NOTA-UBI29-41 and NOTA-UBI30-41, respectively.

CONCLUSION:

Aside from successful peptide syntheses the first ever ⁶⁸Ga-radiolabeling method is reported for NOTA-UBI fragments. The NOTA-conjugation didn’t compromise the selective and specific interaction with bacterial cells in vitro. Both tracers are warranting prospective imaging of infection with PET/CT. Copyright © 2014 Elsevier Inc.

KEYWORDS: (68)Ga-NOTA-UBI; (68)Gallium; Infection imaging; NOTA; PET/CT; Ubiquicidin

PMID: 24630816

 

SUPPLEMENT:

Infectious diseases have a major impact on global morbidity and mortality despite advances in both, diagnosis and treatment. The early identification and correct diagnosis of an active infection is crucial to its effective control or adequate treatment. Millions of antibiotic doses are consumed unnecessarily resulting in bacterial multi drug resistance and cost increases for therapy. Innovative diagnostic radiopharmaceuticals for detecting infection are of paramount importance as nuclear medicine techniques for detecting infections have the advantage of non-invasive whole-body imaging, which might be of great value in cases of occult infection. However, there are limitations, mainly the problem of differentiating between infectious and sterile inflammatory processes. In order to determine the correct course of treatment, clinicians need to be able to discriminate between sites of sterile inflammation and those that are infected by pathogenic microorganisms. Sites that are infected appear, at least superficially, similar to sites that are inflamed due to other reasons, such as trauma, ischemia or the presence of foreign bodies such as implants or neoplasm. Radiolabeled leukocytes and 67Ga-citrate were the most commonly administered tracers for infection imaging, but since efficient cGMP 68Ge/68Ga generators became commercially available, 68Ga-citrate and other novel 68Ga-labeled compounds were introduced for specific diagnosis of infected tissues. 68Ga labeled peptides have also become relevant for diagnostic imaging due to their favourable pharmacokinetics. Interestingly, peptides with antimicrobial properties are found in abundance in mammals, amphibians, and plants, as a part of innate immunity against infection. Human cationic anti-microbial peptides such as ubiquicidin (UBI) have now been shown to differentiate between mammalian and bacterial or fungal cells due to differences in electrostatic charges on the cellular envelope. For the present study, the UBI fragment UBI29-41 was conjugated with NOTA, subsequently radiolabeled with 68Ga and investigated in vitro [1] and in a preclinical rabbit infection model [2] by image acquisition with PET/CT.

In order to simulate infection in mammals Staphylococcus aureus was incubated overnight in nutrient broth shaking at 37˚C and 1cm3 saline solution containing 2 × 108 CFU was injected into the right thigh muscle, and 1cm3 of turpentine oil into the left hind thigh muscle (simulating sterile inflammation) of New Zealand White rabbits. PET/CT imaging was commenced 72 h after inoculation. (Post mortem bacteriological testing of sections of infected or inflamed sites of thighs confirmed bacterial infection). Whole body PET/CT imaging was performed (under anaesthesia) following the administration 68Ga-NOTA-UBI29-41 intravenously. Images were acquired in three-dimensional mode and reconstructed. Blood and urine samples were collected at regular intervals to determine blood clearance and the biological half-life.

A representative PET image, taken at 60 min, showed highest activity accumulation in urinary bladder, liver and kidneys, representing renal excretion (Figure 1A). The spleen, heart and lung showed low uptake with subsequent declining organ activity concentration. Minimal uptake of activity was observed in the musculoskeletal tissue. Steady accumulation in urinary bladder and kidneys dominated the 68Ga-NOTA-UBI29-41 biodistribution, however, it’s acceptable for a PET agent, because it can be easily kept under a hazardous radiation level and will seldom disturb image analysis. Biodistribution of 68Ga-NOTA-UBI29-41 and its accumulation in both the muscular infection site and the inflammation site (Figure 1A; B) was followed up to 90 min after injection of the radiopharmaceutical. The Staphylococcus aureus bearing right thigh muscle was clearly visible in the PET/CT images, showing elevated uptake of 68Ga-NOTA-UBI29-41 compared to the contralateral thigh muscle (bearing sterile inflammation) at 5, 30, 60 and 90 min postinjection (Figure 1C-F) and the infection site showed gradual accumulation of the tracer over the time. The maximum 68Ga-NOTA-UBI29-41 accumulation in an infected muscle was found 5.2 times higher than healthy muscle tissue.

Results of the fluid analysis of 68Ga-NOTA-UBI29-41 shows that the peak blood activity concentration was obtained 1 min after injection, with a fast release of blood-bound activity is indicated by an exponential decline over 60 min with a biological blood half-life of 29 min.

The results support that 68Ga NOTA–UBI29-41 is an efficient and sensitive tracer of in vivo imaging of infection. 68Ga NOTA–UBI29-41 exhibited significant uptake ratios between muscular infection and inflammation. Further clinical evaluation of this novel compound is warranted to investigate its potential use as a first-line PET/CT infection imaging agent in humans.

 

ACKNOWLEDGEMENTS

We would also like to thank Delene van Wyk, Cindy Davis, Roleen Celliers, Brenda Mokaleng and Dr. Daniel Goosen for their excellent support of this study. The authors would like to thank the Nuclear Technologies in Medicine and the Biosciences Initiative (NTeMBI), a national technology platform developed and managed by the South African Nuclear Energy Corporation (Necsa) and funded by the Department of Science and Technology.

 

REFERENCES

[1] Thomas Ebenhan, Nicholas Chadwick, Mike M. Sathekge, Patrick Govender, Thavendran Govender, Hendrik G. Kruger, Biljana Marjanovic-Painter and Jan Rijn Zeevaart. Peptide synthesis, characterization and 68Ga-radiolabeling of NOTA-conjugated ubiquicidin fragments for prospective infection imaging with PET/CT. Nuclear Medicine and Biology. 41 (2014) 390-400.

[2] Thomas Ebenhan, Jan Rijn Zeevaart, Jacobus D.Venter, Thavendrian Govender, Gert H. Kruger and Mike M. Sathekge. Preclinical Evaluation of a Promising Infection Imaging PET/CT Biomarker: Feasibility Study of 68Ga-labeled UBI29-41-1,4,7-triazacyclononane-1,4,7-triacetic Acid in a New Zealand Rabbit Model. Journal of Nuclear Medicine. 55 (2014) 308-314.

 

fig-1-for-biomedfrontiers
FIGURE 1.  Coronal PET images acquired at 60 min after intravenous injection of (A) 48 to 155 MBq 68Ga-NOTA-UBI29-41 into healthy rabbits and (B) 37-174 MBq 68Ga-NOTA-UBI29-41 into rabbits with muscular infection (right thigh) and with sterile muscular inflammation muscle (left thigh).  Axial PET/CT images displaying hind thighs (C) 5 min, (D) 30 min, (E) 60 min, and (F) 90 min after injection.

 

Contacts:
Jan Rijn Zeevaart, PhD
a) Radiochemistry, Necsa, PO Box 582, Pretoria, South Africa
b) Department of Science and Technology, Preclinical Drug Development Platform, North West University, Potchefstroom, 2520, North West Province, South Africa. E-mail: zeevaart@necsa.co.za

Thomas Ebenhan, PhD
University of Pretoria & Steve Biko Academic Hospital
Private Bag X169, Pretoria, 0001, South Africa
E-mail: thomas.ebenhan@gmail.com

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