Background
68Ga-labeled peptides are increasingly used for positron emission tomography (PET), since
68Ga is a readily available PET radionuclide. Because
68Ga is a generator-produced positron emitter, it is widely available and relatively cheap. PET imaging is advantageous over conventional scintigraphy and SPECT because of its excellent sensitivity in combination with its superior spatial resolution. A recent study in patients with neuro-endocrine tumors (NET) showed that PET imaging with the somatostatin analog
68Ga-DOTA-TOC was more sensitive for detecting NET lesions than conventional somatostatin receptor scintigraphy with
111In-octreotide [
1]. Moreover,
68Ga-labeling of peptides conjugated with a chelator (e.g., DOTA, NOTA) is a fast and efficient one-step reaction. Because of all these characteristics, there is an increasing interest in the application of
68Ga-labeled peptides.
It has been shown that for efficient receptor targeting, low peptide doses should be administered, since higher peptide doses could lead to receptor saturation and reduced uptake in the target tissue [
2‐
5], especially in preclinical imaging studies. In rodents, relatively high activity doses (3–10 MBq) have to be administered to acquire PET images with adequate image quality. Therefore,
68Ga-labeled peptides with a high specific activity should be produced to administer high activity doses at a low amount of peptide. However, when producing
68Ga-labeled compounds with a high specific activity, the formation of insoluble
68Ga-species, such as
68Ga(OH)
3 may occur. These insoluble
68Ga-species, generally referred to as “
68Ga-colloid” will accumulate in the liver, spleen, and bone marrow. Indeed, previous studies showed enhanced uptake in liver and spleen of
68Ga-labeled tracers as compared to the
111In-labeled compounds [
3,
6,
7]. This enhanced tracer uptake in liver and spleen might result in decreased target-to-background ratios. We previously showed that insoluble
68Ga-species can be removed from the labeling reaction of
68Ga-labeled DOTA-exendin-3, a tracer targeting the glucagon-like peptide-1 receptor (GLP-1R), using (preparative) reversed-phase high-performance liquid chromatography (RP-HPLC) [
3]. However, this purification method is time consuming and the solution of purified
68Ga-labeled peptide is diluted, making post-purification concentration necessary. Due to the short half-life of
68Ga (68 min), this method is not convenient in clinical practice. Moreover, purification with RP-HPLC requires expensive equipment. Solid phase extraction is an alternative method for purification of radiolabeled compounds and is a fast, simple, and cheap purification method that is now routinely used for
68Ga-tracer purification [
8,
9].
In this study, we examined the effect of the purification method on the in vivo characteristics of
68Ga-DOTA-exendin-3 in BALB/c nude mice.
68Ga-DOTA-exendin-3 was purified by RP-HPLC, gel filtration, or solid phase extraction. Unpurified
68Ga-DOTA-exendin-3 and
111In-DOTA-exendin-3 were used as a reference in this study. In addition, we describe a quality control method, based on instant thin layer chromatography (ITLC), to determine the colloid content of the
68Ga-labeled tracer [
10].
Methods
Peptides and radionuclides
[Lys
40(DOTA)]exendin-3 (DOTA-exendin-3) was purchased from Peptide Specialty Laboratories (Heidelberg, Germany). In this compound, DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) is conjugated to the ε-amino group of the lysine at position 40 (K40) and the C-terminal carboxyl group is amidated [
3].
68GaCl
3 was eluted from a TiO
2-based 1110 MBq
68Ge/
68Ga generator (IGG100, Eckert and Ziegler, Berlin, Germany) with 0.1 N Ultrapure HCl (J.T. Baker, Deventer, The Netherlands).
111InCl
3 was obtained from Covidien (Petten, The Netherlands).
Radiolabeling
DOTA-exendin-3 was labeled with
68Ga and
111In as previously described [
3]. Briefly, 120 MBq
68Ga in 1000 μl Ultrapure 0.1 N HCl was added to 10 μg DOTA-exendin-3 in 120 μl 2.5 M HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, Sigma Aldrich, St. Louis, MO, USA). After 20-min incubation at 95 °C, EDTA was added to a final concentration of 5 mM and the reaction mixture was incubated at room temperature for another 5 min. Subsequently, 10 % Tween-80 (Sigma Aldrich, St. Louis, MO, USA) was added to a final concentration of 0.1 % to prevent sticking of the radiolabeled peptide to the vessel wall and quality control was performed as described below.
DOTA-exendin-3 was labeled with 111In by adding 10 MBq 111InCl3 to 1 μg peptide in 0.1 M 2-(N-morpholino)ethanesulfonic acid (MES), pH 5.5, under similar conditions as described above.
Quality control
Quality control was performed using RP-HPLC on a C18 reversed-phase column (Zorbax Rx-C18; 4.6 mm × 25 cm; Agilent Technologies, Palo Alto, CA, USA) and instant thin layer chromatography (ITLC). The column was eluted with mixture of water containing 0.1 % trifluoroacetic acid (TFA) and acetonitrile with a linear gradient from 3 to 100 % acetonitrile in 10 min (flow rate 1 ml/min). ITLC was performed on silica gel ITLC (Pall Corporation Life Sciences, New York, NY, USA). Two mobile phases were used: 0.1 M EDTA in 0.25 M NH4Ac, pH 5.5 (R
f = 0: 68Ga-labeled exendin and 68Ga-colloid, R
f = 1: 68Ga-EDTA) and 1.25 M NH4Ac, pH 5.5: dimethylformamide (DMF) (1:1) (R
f = 0: 68Ga-colloid, R
f = 1: 68Ga-DOTA-exendin-3 and 68Ga-EDTA).
Validation of the quality control by ITLC for the detection of 68Ga-colloid
68Ga-colloid was prepared by adding 1250 μl 2.5 M HEPES to 500 μl 68GaCl3 in 0.1 M HCl. The final pH of this mixture was approximately 6. The mixture was incubated at 95 °C for 15 min, and EDTA was added to a final concentration of 5 mM. The amount of 68Ga-colloid was determined by TLC with 0.1 M EDTA in 0.25 M NH4Ac, pH 5.5 as a mobile phase (R
f = 0: 68Ga-colloid, R
f = 1 68Ga-EDTA) and 1.25 M NH4Ac, pH 5.5: DMF (1:1) as a mobile phase (R
f = 0: 68Ga-colloid, R
f = 1: 68Ga-EDTA). The reaction mixture was applied on a disposable PD-10 desalting column, containing SephadexTM G-25 medium (GE Life Sciences, Diegem, Belgium) and was eluted with 6 ml phosphate-buffered saline (PBS) containing 5 mM EDTA. The fraction containing the majority of the radioactivity was collected (from 3–4 ml), representing 68Ga-colloid, and quality control was performed by ITLC as described above.
68Ga-DOTA-exendin-3 was purified by RP-HPLC as described below, and various amounts of the 68Ga-colloid were added to obtain final 68Ga-colloid concentrations of 1, 2, 3, 4, 5, 10, 20, 40, and 80 % (n = 4). The amount of 68Ga-colloid was determined by ITLC with 1.25 M NH4Ac, pH 5.5: DMF (1:1) as the mobile phase (R
f = 0: 68Ga-colloid, R
f = 1: 68Ga-DOTA-exendin-3 and 68Ga-EDTA). RP-HPLC-purified 68Ga-DOTA-exendin-3, 68Ga-EDTA, and 68Ga-colloid were used as controls. ITLC strips were exposed to an imaging plate (Fuji Film BAS-SR 2025, Raytest, Straubenhardt, Germany) for 1 min. Images were acquired with a radioluminography laser imager (Fuji Film BAS 1800 II system, Raytest, Straubenhardt, Germany) and analyzed with Aida Image Analyzer software (Raytest). Correlation between the measured 68Ga-colloid fraction and the added 68Ga-colloid content was determined by linear regression using GraphPad Prism 5. The detection limit of the ITLC method was defined by the Y-intercept as determined by linear regression analysis.
RP-HPLC purification of 68Ga-DOTA-exendin-3
After radiolabeling, the reaction mixture was purified by HPLC, using a C8 reversed-phase column (Zorbax eclipse XDB C8 4.6 mm × 150 mm, 5 μm, Agilent Technologies). The column was eluted with water containing 0.1 % TFA (0–5 min), 40 % ethanol (5–10 min) followed by a linear gradient from 40 to 90 % ethanol in 5 min (flow rate 1 ml/min). The fractions containing 68Ga-DOTA-exendin-3 (retention time 14–15 min) were collected and diluted with PBS containing 0.5 % bovine serum albumin (BSA) to a final ethanol concentration of less than 10 % before injection into mice (injection volume 0.2 ml). The radiochemical purity of the purified 68Ga-labeled DOTA-exendin-3 preparations was determined using ITLC as described above.
Purification of 68Ga-DOTA-exendin-3 by gel filtration
Purification by gel filtration was performed on disposable PD-10 desalting columns, containing SephadexTM G-25 medium (GE Life Sciences, Diegem, Belgium). The column was preconditioned by eluting with 10 ml PBS containing 0.5 % (v/w) BSA, and the reaction mixture was loaded onto the column. The column was eluted with PBS-BSA (0.5 %), and 0.5 ml fractions were collected. The majority of the radioactivity, representing 68Ga-DOTA-exendin-3, was collected in fractions 5 and 6 (2-3 ml). The radiochemical purity of the tracer in the fractions was analyzed by RP-HPLC and ITLC as described above.
Purification of 68Ga-DOTA-exendin-3 by solid phase extraction
Solid phase extraction was performed using a hydrophilic-lipophilic balance (HLB) reversed-phase sorbent cartridge (Waters Oasis©, Milford, MA, USA). The cartridge was activated by elution with 1 ml ethanol, the residual ethanol was removed with 1 ml water (Versol, Lyon, France), and the column was conditioned with 1 ml 0.1 N HCl:2.5 M HEPES (8:1, similar to the reaction mixture). The reaction mixture was loaded, and 68Ga-EDTA was washed from the column with 2 ml 0.1 N HCl:2.5 M HEPES (8:1). After removal of the residual HCl-HEPES mixture with 1 ml water, 68Ga-DOTA-exendin-3 was eluted with 200 μl ethanol. The radiochemical purity of the tracer in the fractions was analyzed by RP-HPLC and ITLC as described above. Before injection into mice, the eluate containing 68Ga-DOTA-exendin-3 was diluted with PBS containing 0.5 % BSA to a final ethanol concentration of less than 10 %.
Biodistribution studies
Animal experiments were performed after approval of the local ethical committee (RUDEC) for animal experiments. The biodistribution of unpurified, gel filtration-, RP-HPLC-, and SPE-purified 68Ga-DOTA-exendin-3 was determined in BALB/c nude mice. Mice (n = 5) were injected intravenously with 3 MBq 68Ga-labeled exendin-3 at a peptide dose of 0.3 μg (60 pmol). As a control, another group of mice received 370 kBq 111In-DTPA-exendin-3 (60 pmol). The mice were euthanized 1 h post-injection by CO2/O2 suffocation, a blood sample was taken, and samples of relevant tissues were dissected, weighed, and counted.
MicroPET
Mice were injected intravenously with 3 MBq (0.3 μg) unpurified, gel filtratrion, RP-HPLC- or SPE-purified 68Ga-DOTA-exendin-3. Mice were euthanized 1 h p.i. by CO2/O2 suffocation and PET images were acquired during 45 min using a small-animal PET/CT scanner (Inveon™; Preclinical Solutions, Siemens Medical Solutions USA, Inc., Knoxville, TN, USA). Images were reconstructed by OSEM3D/MAP reconstruction with the following parameters: 256 × 256 matrix, 2 OSEM3D iterations, 18 MAP iterations, and a resolution of 0.075 mm uniform variance. CT images were acquired for anatomical correlation directly after PET imaging (spatial resolution 113 μm, 80 kV, 500 μA, exposure time 300 ms).
Statistical analysis
All mean values are expressed as mean ± standard deviation (SD). Statistical analysis was performed using unpaired two-tailed t test using GraphPad Prism (version 5). In order to determine whether there was a overall difference in blood and kidney accumulation of the various preparations, a one-way ANOVA was performed. The level of significance was set at p < 0.05.
Discussion
During the radiolabeling procedure of DOTA-conjugated compounds with
68Ga, in most cases, insoluble colloidal
68Ga species are formed, especially when compounds are labeled at a high specific activity. This insoluble
68Ga-colloid results in enhanced accumulation in the liver and spleen when injected in laboratory animals or patients, resulting in reduced image quality. Therefore, prevention of formation or removal of
68Ga-colloid is required. We evaluated three purification methods for the removal of
68Ga-colloid: solid phase extraction (SPE), preparative HPLC, and gel filtration. Solid phase extraction was a fast and simple method that effectively removed
68Ga-colloid from a labeling mixture of
68Ga-DOTA-exendin-3, resulting in negligible liver and spleen accumulation similar to that of
111In-DOTA-exendin-3. The major advantage of this technique is that purification of the labeling mixture can be performed within 5 min. Purification by preparative RP-HPLC resulted in similar spleen and liver accumulation. However, this technique is more time-consuming and less suited for purification of
68Ga-labeled tracers, due to the short half-life of
68Ga. Remarkably, the uptake of RP-HLPC-purified
68Ga-DOTA-exendin-3 in the pancreas was significantly higher. This is probably due to a slightly lower peptide dose, which results in higher pancreatic uptake as previously described [
3]. Gel filtration removed
68Ga-colloid less efficiently, resulting in higher liver and spleen uptake than SPE and RP-HPLC purification. It should be noted that gel filtration could be more appropriate for the purification of larger compounds such as proteins. The higher molecular weight of proteins combined with the low molecular weight of
68Ga-colloid could result in better separation. However, the feasibility to separate
68Ga-colloid from high molecular weight compounds by gel filtration should be evaluated.
High specific activities of
68Ga-labeled tracers are required to administer high activity doses in combination with low peptide doses. It was shown that high peptide doses lead to reduced uptake in the target tissues due to (partly) saturation of the receptors [
2‐
5], which is of particular importance in preclinical imaging. Moreover, high peptide doses can lead to (toxic) side effects, especially when biologically active compounds are used as the ligand. The need for a high specific activity generally enhances
68Ga-colloid formation, since incorporation of
68Ga present in the labeling mixture in the presence of low amounts of metal binding ligand might be incomplete. Since
68Ga(OH)
3 could be formed at pH 3 in the absence of chelating agents (such as DOTA, DTPA, and EDTA) [
11],
68Ga(OH)
3 is formed in a labeling mixture at a pH between 3.5 and 4 when
68Ga-incorporation is incomplete. Interestingly, in the pH range generally used for
68Ga-labeling procedures (pH 3.5–4), Ga(III) is predominantly present as polymeric Ga(III) hydroxides, while Ga(OH)
3 is the predominant form at pH 7 [
12]. Furthermore, at higher pH, the formation of insoluble GaO(OH) can occur [
13]. GaO(OH) is particularly formed at high temperature [
14], which is of importance since most
68Ga-labeling procedures are carried out at high temperatures (80–100 °C). In contrast to Ga(III) hydroxides, GaO(OH) is only slowly redissolved even at low pH [
14].
Wild et al. showed a similar biodistribution of
68Ga-labeled DOTA-exendin-4 [
15] as
68Ga-DOTA-exendin-3 in our study. In the former study, no purification was performed of the
68Ga-labeled compound, and this lead to slightly higher uptake in the spleen as compared to the SPE and RP-HPLC purified tracer used in our study. However, the splenic uptake of
68Ga-DOTA-exendin-4 was lower than that of the unpurified
68Ga-DOTA-exendin-3 in our study. This lower uptake in the spleen of
68Ga-DOTA-exendin-4 reported by Wild et al. is probably due to a lower
68Ga-colloid content in the labeling mixture. The lower
68Ga-colloid content might be due to a lower specific activity of
68Ga-DOTA-exendin-4 and a different labeling protocol. In the study performed by Wild et al., the
68Ga-eluate was purified and the labeling was performed in a microwave for 5 min. The shorter labeling time in combination with the lower specific activity may lead to faster and more complete incorporation of
68Ga, reducing the risk of
68Ga-colloid formation [
11]. Several other labeling methods [
16,
17] and chelators [
18‐
21] for labeling compounds with
68Ga are described with more efficient labeling yields and faster labeling kinetics. These new strategies for
68Ga-labeling of compounds might also reduce the risk of
68Ga-colloid formation.
We validated a TLC method for the detection of
68Ga-colloid in labeling mixtures of
68Ga-DOTA-exendin-3 and showed a clear linear correlation between added
68Ga-colloid to the labeling mixtures (free of
68Ga-colloid) and the measured
68Ga-colloid content. These results suggest accurate quantification of
68Ga-colloid in the labeling mixture. Determination of the colloid content is of great importance to calculate the specific activity. Neglecting the presence of
68Ga-colloid might result in overestimation of the specific activity and thus administration of a higher peptide dose as initially planned. This is especially true for exendin, where very low peptide doses need to be administered to prevent receptor saturation resulting in lower uptake in GLP-1R-positive tissues [
3]. The detection limit of this method is approximately 3 % making detecting of very low concentrations of colloid difficult. The rather low sensitivity is due to tailing of the radiolabeling peptide hampering the clear delineation between the peak representing the
68Ga-colloid and the peak representing
68Ga-DOTA-exendin-3. The sensitivity of this method for the determination of
68Ga-colloid for other peptides might be different due to different migratory characteristics of the
68Ga-peptide on this TLC system compared to exendin.
Enhanced liver and spleen uptake will hamper the detection of lesions in the upper abdomen. Radiolabeled exendin was successfully used for detection of insulinomas [
22] and could potentially be used for detection of the pancreatic beta cell mass. Enhanced liver and spleen uptake might reduce the sensitivity of these methods. Previous studies explored the feasibility of tumor imaging and atherosclerotic plaques with
68GaCl
3 in mouse models for pancreatic adenocarcinoma and artherosclerosis, respectively [
23,
24]. Although the tumor and atherosclerotic plaques could be detected, the PET images suffered from high background signal in the liver and lungs as well as high concentration in the blood. The binding of
68Ga to transferrin (prolonging the circulation time) and the formation of
68Ga-colloid could explain the enhanced background and is in line with our study.