Selected ⁶⁸Ga-siderophores versus ⁶⁸Ga-colloid and ⁶⁸Ga-citrate: biodistribution and small animal imaging in mice

Abstract

Background

⁶⁸Ga-triacetylfusarinine C (TAFC) and ⁶⁸Ga-ferrioxamine E (FOXE) show great potential to be used as highly sensitive and selective tracers for Aspergillus infection imaging. Here we report on a comparison of the ex vivo biodistribution and small animal imaging of ⁶⁸Ga-TAFC and ⁶⁸Ga-FOXE versus ⁶⁸Ga-colloid and ⁶⁸Ga-citrate as unspecific control in mice.

Methods

The radiochemical purity of tested ⁶⁸Ga labelled tracers was determined by RP-HPLC or ITLC-SG. Ex vivo biodistribution was studied in normal DBA/2 mice 30 min and 90 min p.i. Static and dynamic imaging were performed using µPET/CT.

Results

⁶⁸Ga-TAFC and ⁶⁸Ga-FOXE showed rapid renal excretion and low blood values even 90 min p.i. ⁶⁸Ga-TAFC showed almost no retention in other organs while ⁶⁸Ga-FOXE displayed some uptake in gastrointestinal tract. ⁶⁸Ga-colloid and ⁶⁸Ga-citrate revealed significantly different ex vivo biodistribution. ⁶⁸Ga-colloid showed pronounced radioactivity retention in the liver, while ⁶⁸Ga-citrate displayed high blood values and significant retention of radioactivity in highly perfused organs.

Conclusions

From the results, both ⁶⁸Ga-TAFC and ⁶⁸Ga-FOXE have excellent and significantly different in vivo behaviour compared to ⁶⁸Ga-colloid and ⁶⁸Ga-citrate. ⁶⁸Ga-TAFC in particular confirmed its great potential use as a specific tracer for Aspergillus infection imaging.

INTRODUCTION

Triacetylfusarinine C (TAFC) and ferrioxamine E (FOXE) are common trihydroxamate-type siderophores. Siderophores are relatively low molecular weight compounds produced by bacteria, fungi and some plants for scavenging iron from the environment to make this vital mineral available to the microbial cell^1,2. It has recently been recognised that iron plays a fundamental role in infection in general³ and in fungal infections in particular. Schrettl et al.⁴ reported that the siderophore system is essential for the virulence of Aspergillus fumigatus, which is the main pathogen responsible for invasive aspergillosis (IA) complications.

IA is a life-threatening infection, hazardous especially for haematopoietic stem cell and solid organ transplant recipients, as well as for patients with solid tumours and haematological malignancies^5,6. Early and accurate diagnosis of IA is crucial for the survival of such affected patients⁷. Unfortunately, currently available methods for the diagnosis of IA lack sufficient specificity and/or sensitivity.

We have recently shown that various siderophores can be labelled with generator produced ⁶⁸Ga (ref.⁸), based on the similarities between iron (III) and gallium (III). Gallium-68 is a positron emitter that has recently attracted great interest for molecular imaging applications using positron emission tomography (PET) (ref.^9–11). It can be produced from a long shelf-life and cost-effective generator system. The physical half-life of ⁶⁸Ga (67.7 min) permits production and application of most low molecular weight radiopharmaceuticals such as peptides, oligonucleotides, antibody fragments and potentially also siderophores.

⁶⁸Ga labelled TAFC and FOXE show great promise as highly selective, highly sensitive tracers for Aspergillus infection imaging^12,13. This could prove indispensable in the early, accurate diagnosis of IA. For translation into clinical practice more data on the pharmacokinetics and biodistribution of these promising new tracers are warranted. Here we report on a comparison of in vivo biodistribution and small animal imaging of selected ⁶⁸Ga-siderophores (⁶⁸Ga-TAFC and ⁶⁸Ga-FOXE) versus radiochemical impurities, which can develop during ⁶⁸Ga labelling (⁶⁸Ga-colloid and ⁶⁸Ga-acetate) and a representative of infection imaging agents (⁶⁸Ga-citrate) as unspecific control in mice.

MATERIALS AND METHODS

Reagents

All chemicals obtained commercially were of the highest available purity and were used without further purification. TAFC and FOXE were gained from EMC microcollections GmbH (Tuebingen, Germany) and ⁶⁸Ge/⁶⁸Ga generator from Eckert & Ziegler Eurotope GmbH (Berlin, Germany).

Radiolabelling

⁶⁸GaCl₃ was obtained from the ⁶⁸Ge/⁶⁸Ga generator using 0.1N HCl as eluent. 300 µL of the fractionated generator eluate (20-80 MBq of ⁶⁸GaCl₃) was added to a mixture of 10 µL of TAFC (10 µg in water) or 20 µL of FOXE (20 µg in 10% ethanol) and 30 µL of 1.1M sodium acetate solution. After 15 min at room temperature (TAFC) or 20 min at 80°C (FOXE), 100 µL of 1.1M sodium acetate solution were added to increase the pH to 6-7.

⁶⁸Ga-acetate/colloid was prepared using excess (150 µL) of 1.1M sodium acetate solution mixed with the fractionated ⁶⁸Ge/⁶⁸Ga generator eluate (300 µL) to reach a reaction pH of 6-7 for 15 min at room temperature.

⁶⁸Ga-colloid was prepared by mixing a ‘Monday’ fractionated ⁶⁸Ge/⁶⁸Ga generator eluate (300 µL) with 0.5M sodium hydroxide (55 µL). The reaction mixture was incubated for 15 min at room temperature.

Fractionated ⁶⁸Ge/⁶⁸Ga generator eluate (300 µL) was mixed with 0.5M sodium citrate pH ~ 5 (80 µL) for the preparation of ⁶⁸Ga-citrate. The reaction mixture was incubated for 15 min at room temperature.

Quality control

The radiochemical purity of the ⁶⁸Ga labelled tracers was determined using reversed-phase high-performance liquid chromatography (RP-HPLC) and/or instant thin layer chromatography on silica gel impregnated glass fiber sheets (ITLC-SG). Dionex UltiMate 3000 (Thermo Scientific, Waltham, MA, USA) and GABI Star (Raytest, Straubenhardt, Germany) radiometric detector were used for RP-HPLC analysis of ⁶⁸Ga-siderophores. A Nucleosil 120-5 C18 250 × 40 mm column (WATREX, Prague, Czech Republic) with a flow rate of 1 mL/min was used with the following gradient: acetonitrile (ACN)/0.1% trifluoracetic acid (TFA)/H₂O: 0-2 min, 0% ACN; 2-15 min, 0-36% ACN; 15-18 min, 36-60% ACN; 18-19.5 min, 60% ACN; 19.5-20 min, 60-0% ACN; 20-24 min, 0% ACN.

ITLC-SG (Varian, Lake Forest, CA, USA) was used for ⁶⁸Ga-citrate, ⁶⁸Ga-acetate/colloid and ⁶⁸Ga-colloid quality control. The radiochemical purity of ⁶⁸Ga-citrate was determined by ITLC-SG using methanol/glacial acetic acid (9:1) as mobile phase (free ⁶⁸Ga, R_f = 0 and ⁶⁸Gacitrate, R_f = 1) (ref.¹⁴). Two methods were used for the assessment of ⁶⁸Ga-acetate/colloid and ⁶⁸Ga-colloid using ITLC-SG as previously described¹⁵. The distribution of radioactivity along the ITLC-SG strips was measured on a Cyclone Plus Storage Phosphor system (PerkinElemer, Waltham, MA, USA).

Animal experiments

All animal experiments were conducted in accordance with regulations and guidelines of the Czech Animal Protection Act (No. 246/1992) with the approval of the Czech Ministry of Education Youth and Sports (MSMT-18933/2013-1), and the institutional Animal Welfare Committee of the Faculty of Medicine and Dentistry of Palacky University in Olomouc. The studies were performed using DBA/2 mice (Anlab, Prague, Czech Republic).

Biodistribution in mice

Normal DBA/2 mice (female) were retro-orbitally (r.o.) injected with a dose of 1-2 MBq per mouse of the ⁶⁸Ga labelled tracer. Animals were sacrificed by cervical dislocation 30 min and 90 min postinjection (p.i.). The organs (blood, spleen, pancreas, stomach, intestine, kidneys, liver, heart, lung, muscle and femur) were removed and radioactivity was measured in an automatic gamma counter (Wizard², PerkinElmer, Waltham, MA, USA). The results are expressed as percentage of injected dose per gram organ (%ID/g).

Animal imaging

The positron emission tomography (PET) and computed tomography (CT) images were acquired with an Albira PET/SPECT/CT small animal imaging system (Bruker Biospin Corporation, Woodbridge, CT, USA) (ref.¹⁶). Radiolabelled tracers were administered retroorbitally into DBA/2 mice in a dose of 5-10 MBq per mouse. Mice were subsequently anaesthetized with isoflurane (FORANE, Abbott Laboratories, Abbott Park, IL, USA) (2% flow rate) and were kept under anaesthesia during the imaging. Static PET/CT imaging was carried out 5 min, 30 min and 90 min p.i. A 5-min PET scan (axial FOV 148 mm) was performed, followed by a CT scan (axial FOV 65 mm, 45 kVp, 400 µA, at 600 projections). Dynamic imaging was carried out immediately after the injection of ⁶⁸Ga labelled tracer for 90 min (5-min PET scan per frame). Scans were reconstructed with the Albira software (Bruker Biospin Corporation, Woodbridge, CT, USA) using maximum likelihood expectation maximization (MLEM) and filtered backprojection (FBP) algorithms^16,17. After reconstruction, the acquired data were viewed and analyzed with PMOD software (PMOD Technologies Ltd., Zurich, Switzerland). The 3D images were obtained using VolView software (Kitware, Clifton Park, NY, USA).

RESULTS

Radiolabelling and analytics

Both ⁶⁸Ga labelled siderophores displayed high radiochemical purity (≥ 98%) and in vitro stability as previously described⁸. Fig. 1 shows the proposed chemical structures and radiochromatograms of ⁶⁸Ga-TAFC and ⁶⁸Ga-FOXE, with a single main peak for both ⁶⁸Ga-siderophores (⁶⁸Ga-TAFC retention time = 15.8 min and ⁶⁸Ga-FOXE retention time = 13.6 min). Free ⁶⁸Ga analyzed using RP-HPLC showed a single peak at the retention time (Rt) of 3 min as presented in Fig. 1. ⁶⁸Ga-citrate, ⁶⁸Ga-acetate/colloid and ⁶⁸Ga-colloid were determined using ITLC-SG methods. ⁶⁸Ga-citrate and ⁶⁸Ga-colloid were prepared with a radiochemical purity of ≥ 95% and ⁶⁸Ga-acetate/colloid mixture contained ≥ 75% of ⁶⁸Ga-acetate.

Fig. 1.

Proposed chemical structures of ⁶⁸Ga-TAFC (A) and ⁶⁸Ga-FOXE (B) and HPLC-radiochromatograms of ⁶⁸Ga-TAFC, ⁶⁸Ga-FOXE and free ⁶⁸Ga (C).

Ex vivo biodistribution

In normal DBA/2 mice (Table 1), both ⁶⁸Ga-TAFC and ⁶⁸Ga-FOXE showed rapid renal excretion with highest activity retained in kidneys (1.88±0.53 %ID/g for ⁶⁸Ga-TAFC and 0.75±0.24 %ID/g for ⁶⁸Ga-FOXE) and low blood values (0.24±0.10 %ID/g for ⁶⁸Ga-TAFC and 0.04±0.01 %ID/g for ⁶⁸Ga-FOXE) at 90 min after application. ⁶⁸Ga-TAFC showed almost no retention in other organs, while ⁶⁸Ga-FOXE displayed some uptake (0.92 ± 0.09 %ID/g (30 min p.i.) and 0.84±0.05 %ID/g (90 min p.i.)) in the gastrointestinal tract, which was confirmed by µPET imaging. ⁶⁸Ga-acetate/colloid, ⁶⁸Ga-colloid and ⁶⁸Ga-citrate revealed significantly different ex vivo biodistribution compared to ⁶⁸Ga-siderophores (see Table 1). High blood values (17.0±1.0 %ID/g at 90 min p.i.) and pronounced retention of radioactivity in highly perfused organs (4.1±1.1 %ID/g for spleen, 4.3±0.5 %ID/g for liver, 5.2±1.2 %ID/g for heart, 7.2±0.7 %ID/g for lung at 90 min p.i.) were found in mice injected with ⁶⁸Ga-acetate/colloid. ⁶⁸Ga-citrate showed comparable in vivo behaviour as ⁶⁸Ga-acetate/colloid. The highest level of radioactivity was found in the blood (19.9±1.5 %ID/g at 90 min p.i.) and highly perfused organs revealed significant levels of retained activity (5.9±2.7 %ID/g for spleen, 3.8±0.3 %ID/g for liver, 9.0±0.7 %ID/g for heart, 10.9±1.4 %ID/g for lung at 90 min p.i.). In contrast, ⁶⁸Ga-colloid displayed predominant liver uptake (36.2±2.7 %ID/g at 90 min p.i.).

Table 1.

Biodistribution of ⁶⁸Ga-TAFC, ⁶⁸Ga-FOXE, ⁶⁸Ga-acetate/colloid, ⁶⁸Ga-colloid and ⁶⁸Ga-citrate in normal DBA/2 mice 30 min and 90 min p.i.

Organ	68Ga-TAFC		68Ga-FOXE		68Ga-acetate/colloid		68Ga-colloid		68Ga-citrate
	30 min	90 min	30 min	90 min	30 min	90 min	30 min	90 min	30 min	90 min
Blood	2.92 ± 1.48	0.24 ± 0.09	1.04 ± 0.10	0.04 ± 0.01	21.7 ± 2.64	17.0 ± 1.00	10.8 ± 0.32	8.40 ± 1.04	20.8 ± 2.60	19.9 ± 1.53
Spleen	0.68 ± 0.12	0.09 ± 0.04	0.35 ± 0.13	0.05 ± 0.01	5.01 ± 0.06	4.05 ± 1.08	9.72 ± 2.68	5.91 ± 2.67	3.49 ± 0.70	4.16 ± 0.64
Pancreas	1.04 ± 0.39	0.13 ± 0.01	0.52 ± 0.23	0.06 ± 0.02	3.54 ± 0.45	2.57 ± 0.47	1.85 ± 0.42	1.51 ± 0.30	4.16 ± 0.16	3.70 ± 0.28
Stomach	1.64 ± 0.48	0.35 ± 0.41	0.63 ± 0.16	0.06 ± 0.01	2.35 ± 0.21	2.84 ± 0.45	1.38 ± 0.13	1.32 ± 0.01	3.21 ± 0.54	3.62 ± 0.29
Intestine	1.37 ± 0.35	0.49 ± 0.25	0.92 ± 0.09	0.84 ± 0.05	2.78 ± 0.42	3.85 ± 0.30	1.29 ± 0.15	1.55 ± 0.07	3.30 ± 0.38	4.29 ± 0.23
Kidneys	11.8 ± 3.60	1.88 ± 0.53	3.12 ± 0.29	0.75 ± 0.24	5.59 ± 0.27	4.44 ± 0.31	2.46 ± 0.63	2.73 ± 0.53	5.46 ± 1.40	5.79 ± 0.32
Liver	0.79 ± 0.30	0.11 ± 0.02	0.86 ± 0.02	0.13 ± 0.01	4.84 ± 0.85	4.34 ± 0.49	36.7 ± 5.92	36.2 ± 2.67	3.38 ± 0.34	3.86 ± 0.35
Heart	1.10 ± 0.36	0.08 ± 0.02	0.46 ± 0.12	0.05 ± 0.01	7.06 ± 0.71	5.25 ± 1.16	3.35 ± 0.31	2.65 ± 0.50	8.12 ± 1.28	9.05 ± 0.77
Lung	2.51 ± 0.97	0.27 ± 0.12	0.95 ± 0.15	0.10 ± 0.01	8.45 ± 0.33	7.24 ± 0.66	4.76 ± 0.89	5.57 ± 1.25	10.6 ± 3.76	10.9 ± 1.46
Muscle	0.65 ± 0.31	0.35 ± 0.15	0.22 ± 0.06	0.06 ± 0.03	1.22 ± 0.23	1.51 ± 0.51	0.68 ± 0.10	0.63 ± 0.06	1.79 ± 0.23	1.93 ± 0.19
Femur	0.55 ± 0.06	0.76 ± 0.49	0.15 ± 0.05	0.12 ± 0.09	1.64 ± 0.32	1.84 ± 0.24	1.20 ± 0.05	1.17 ± 0.07	2.51 ± 0.37	2.81 ± 0.50

Data are presented as % injected dose per gram organ (%ID/g ± Sd) (n = 3).

Animal imaging

µPET imaging of normal DBA/2 mice confirmed rapid renal excretion of ⁶⁸Ga-TAFC showing almost all injected activity in bladder at 45 min p.i. (Fig. 2 A and 3 A). ⁶⁸Ga-FOXE revealed similar biodistribution compared to ⁶⁸Ga-TAFC. The only relevant exception was an evident uptake of ⁶⁸Ga-FOXE in gastrointestinal tract, conspicuous from 50 min frame of dynamic imaging (Fig. 2 B and 3 B). In contrast, both ⁶⁸Ga-acetate/colloid and ⁶⁸Gacitrate showed very slow in vivo kinetics with high retention of radioactivity in the blood pool (Fig. 2 C and E). ⁶⁸Ga-colloid displayed rapid liver uptake slowly increasing over time (Fig. 2 D). Static imaging (Fig. 4) confirmed and supported the results gained from ex vivo biodistribution and dynamic imaging.

Fig. 2.

Dynamic µPET imaging of ⁶⁸Ga-TAFC (A), ⁶⁸Ga-FOXE (B), ⁶⁸Ga-acetate/colloid (C), ⁶⁸Ga-colloid (D) and ⁶⁸Ga-citrate (E) up to 90 min p.i. (sagittal slices; injected dose: 5-10MBq; anaesthesia: 2% isoflurane; scan duration: 5-minute PET scan per frame (18 frames); B – bladder, E – eye, G – gastrointectinal tract, H – heart, K – kidney, L - liver).

Fig. 3.

Time activity curves of selected regions of interest (heart, kidneys, bladder) of ⁶⁸Ga-TAFC (A) and ⁶⁸Ga-FOXE (B).

Fig. 4.

Static µPET/CT imaging of ⁶⁸Ga-TAFC (A), ⁶⁸Ga-FOXE (B), ⁶⁸Ga-acetate/colloid (C), ⁶⁸Ga-colloid (D) and ⁶⁸Ga-citrate (E) 5 min, 30 min and 90 min p.i. (supine position; injected dose 5-10MBq; anaesthesia: 2% isoflurane; scan duration: 5-minute PET scan followed by 15-minute CT scan).

DISCUSSION

The development of ⁶⁸Ga-radiopharmaceuticals has increased enormously in the last five years. The renewed popularity of ⁶⁸Ga was initiated mainly by the development of new, simple to use ⁶⁸Ge/⁶⁸Ga generator systems, by the fact that positron emission tomography (PET) has become a routine clinical and preclinical imaging modality and owing to the favourable chemical properties of ⁶⁸Ga and developed ⁶⁸Ga labelling strategies including automation^11,18.

The commercially available ⁶⁸Ge/⁶⁸Ga generator from Eckert & Ziegler Eurotope GmbH (Berlin, Germany) used for our study provides ⁶⁸Ga in its ionic form as ⁶⁸Ga³⁺. Elution is performed using hydrochloric acid solution, since acidic conditions are required to inhibit hydrolysis of the ⁶⁸Ga³⁺ ion (pH < 3), which is a requirement for chemical processing and successful radiolabelling¹⁰.

The in-house accessibility, favourable radiochemical properties of ⁶⁸Ga³⁺ and chemical similarities with Fe³⁺, which is chelated by siderophores, led us to attempt ⁶⁸Ga labelling of selected siderophores. These attempts were successful and we have shown that various siderophores can be labelled with ⁶⁸Ga (ref.⁸). Moreover, we have demonstrated that ⁶⁸Ga-TAFC and ⁶⁸Ga-FOXE are highly selectively accumulated in the infected tissue in Aspergillus fumigatus rat infection model^12,13 and ⁶⁸Ga-TAFC revealed high in vitro specificity towards Aspergillus fumigatus¹⁹. Our studies showed, that ⁶⁸Ga-TAFC in particular appears to be promising candidate for the noninvasive detection of Aspergillus infections by PET.

In this work, we studied in vivo kinetics and biodistribution of different ⁶⁸Ga labelled tracers: ⁶⁸Ga-siderophores (TAFC and FOXE), ⁶⁸Ga-citrate, ⁶⁸Ga-colloid and ⁶⁸Ga-acetate/colloid. ⁶⁸Ga-citrate was chosen as a representative of infection imaging tracer due to successful application of ⁶⁷Ga-citrate for SPECT imaging in the past²⁰ and recent reports on the clinical use of ⁶⁸Ga-Citrate²¹. ⁶⁸Ga-colloid is a radiochemical impurity, which can occur during ⁶⁸Ga labelling at higher pH and could influence the biodistribution of improperly prepared ⁶⁸Ga-siderophores. ⁶⁸Ga-acetate/colloid mixture was studied to compare the in vivo behaviour of ⁶⁸Ga-TAFC and ⁶⁸Ga-FOXE versus reaction mixture used for ⁶⁸Ga labelling of these siderophores.

⁶⁸Ga-TAFC and ⁶⁸Ga-FOXE showed high radiochemical purity and similar retention times using previously described RP-HPLC method¹², while ⁶⁸Ga-citrate, ⁶⁸Ga-colloid and ⁶⁸Ga-acetate/colloid displayed completely different analytical behaviour using ITLC-SG. Subsequent in vivo small animal imaging showed rapid elimination of both ⁶⁸Ga-siderophores mainly via kidneys. This supports the findings of high in vivo stability of these complexes with only intact ⁶⁸Ga-TAFC and ⁶⁸Ga-FOXE found as urinary excretion products¹². The only significant difference in the biodistribution of ⁶⁸Ga-TAFC and ⁶⁸Ga-FOXE was found in certain uptake of ⁶⁸Ga-FOXE in gastrointestinal tract in later time points, which could be caused by the binding of ⁶⁸Ga-FOXE to intestinal microflora. This hypothesis is supported by our previous study, testing of uptake specificity of ⁶⁸Ga-TAFC and ⁶⁸Ga-FOXE in various microorganisms¹⁹, which showed clear uptake of ⁶⁸Ga-FOXE not only in Aspergillus fumigatus, but also in another tested fungal and bacterial species, indicating higher specificity of ⁶⁸Ga-TAFC for imaging Aspergillus fumigatus infections. µPET/CT imaging of ⁶⁸Ga-citrate in mice revealed slow excretion of the radioactivity with high blood pool values, which is in accordance with the investigations of Kumar et al.²², who have studied ⁶⁸Ga-citrate for diagnostic imaging of infection in rats. ⁶⁸Ga-acetate/colloid displayed comparable in vivo behaviour to ⁶⁸Ga-citrate. Both citrate and acetate are weak chelators in vivo. ⁶⁸Ga is rapidly released from the weak complex and bound to transferrin, ferritin and other iron-binding proteins²³, which explain the high blood pool values for both tracers. The in vivo imaging of ⁶⁸Ga-colloid showed clear rapid liver uptake slowly increasing in time. The ex vivo biodistribution data showed perfect correlation with in vivo dynamic and static imaging.

CONCLUSIONS

Both studied ⁶⁸Ga labelled siderophores displayed excellent and significantly different in vivo behaviour compared to ⁶⁸Ga-citrate, ⁶⁸Ga-colloid and ⁶⁸Ga-acetate/colloid, and especially ⁶⁸Ga-TAFC confirmed its great potential to be used as specific tracer for Aspergillus infection imaging. These data on normal biodistribution and pharmacokinetics of ⁶⁸Ga-TAFC and ⁶⁸Ga-FOXE are essential for further translation of these promising compounds into the clinic.