Radiolabelling of the exendin-4 derivatives
Ex4NOD12, Ex4NOD27 and Ex4NOD40, shown in Figure 1, were synthetised by Peptide Specialty Laboratories (Heidelberg, Germany). [Lys40(Ahx-DTPA)NH2]-exendin-4 (HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSK(DTPA-Ahx)-NH2) and [Lys40(DTPA)]exendin-3 (HSDGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSK(DTPA)-NH2), synthetised by Peptide Specialty Laboratories were used as already established standards. The chelators NODAGA and diethylene triamine pentaacetic acid (DTPA) were conjugated to the ε-amino group of the lysine either at position 12 or 27 with aminocaproic acid (Ahx) as spacer or attached to a lysine, which was linked to the C-terminal end of the peptide leading to a lysine in position 40. In either case, the C-terminal carboxyl group was amidated. In order to improve oxidative stability, the methionine in position 14 of Ex4NOD40 was replaced with norleucine in both Ex4NOD12 and Ex4NOD27.
68GaCl3 was eluted from a TiO2-based 1.85 GBq 68Ge/68Ga generator (Eckert & Ziegler, Berlin, Germany) with 0.1 N Ultrapure HCl (Merck Millipore, Switzerland).
The generator was eluted drop by drop, fractions of 200 μL each were collected, and the fractions with the highest activity were used for labelling.
Labelling of all peptides was performed in 4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid (HEPES) buffer in a final concentration of 0.25 M and pH 4.5. 0.23 to 0.41 nmol peptide was added to 60 to 77 MBq 68GaCl3, followed by a 15-min incubation of the reaction mixture at 90°C. For quality control, the sample was diluted in 0.1 mM sodium DTPA and analysed using reversed-phase high-performance liquid chromatography (RP-HPLC) on a C18 reversed-phase column (Dr. Maisch Reprospher 300 C18-TN, 46 mm × 150 mm; 5 μm). The column was eluted with water containing 0.1% trifluoroacetic acid (TFA), with a linear gradient from 15% to 95% acetonitrile for 10 min followed by an isocratic elution at 95% acetonitrile for additional 5 min with a flow rate of 1 mL/min.
67Ga labellings were performed using 55 to 65 MBq 67GaCl3 (Mallinckrodt, Netherlands) and 1 to 6.75 nmol Ex4NOD40, Ex4NOD12 and Ex4NOD27 in 10 μL 0.4 M ammonium acetate pH 5.5 and 20 μL Milli-Q water followed by 30 min incubation at 90°C. Quality control was performed as described above.
Cell culture
Chinese hamster lung cell line stably transfected with the GLP-1 receptor gene (CHL-GLP-1R positive cells) were cultured in Dulbecco's modified Eagle's medium (DMEM) with 4.5 g/L d-glucose and GlutaMax. In addition, the media contained 10% fetal calf serum, 100 IU/mL penicillin G, 10 mg/mL streptomycine, 500 μg/mL geneticin sulfate, 1 mM sodium pyruvate and 0.1 mM non-essential amino acids. The cells were maintained in a humidified 5% CO2 atmosphere at 37°C and were harvested by trypsinisation with trypsin/EDTA.
Labelling of exendin-4 derivatives with stable isotopes
All peptides were labelled by adding 40 μL of the respective 0.25 mM peptide solution and 2 μL of a 10 mM natGaCl3 (Sigma-Aldrich, Switzerland) solution in 60 μL 0.4 M ammonium acetate buffer (pH 5.5) followed by a 15-min incubation at 90°C. The labelling was verified by liquid chromatography-mass spectrometry (LC/MS) on an Atlantis C18 (25 cm × 4.6 mm; 5 μm) column.
IC50 determination
The half-maximal inhibitory concentrations (IC50) of natGa-labelled Ex4NOD12, Ex4NOD27 and Ex4NOD40 were determined using CHL-GLP-1R positive cells grown on six well plates (approximately 0.8 × 106 cells/well grown overnight). The two already published peptides natIn-labelled [Lys40(DTPA)]exendin-3 and [Lys40(Ahx-DTPA)NH2]-exendin-4 were used to determine if the cell line has an influence on the results. A 4-kBq (120 fmol)-67Ga-labelled Ex4NOD40 was used for detection of the binding. The cells were washed twice with PBS and incubated for 60 min on ice with 100 μL (4 kBq; 120 fmol) 67Ga-labelled Ex4NOD40 in the presence of increasing concentrations of non-radioactive-labelled exendin-4 derivative (10−11 to 10−6 M). The total volume was adjusted with media (DMEM with 0.1% BSA) to 1 mL. For the total binding, no natGa-labelled peptide was added. After incubation with the labelled peptides, the cells were washed twice with phosphate buffered saline (PBS), solubilised with 1 mL sodium hydroxide (NaOH) and collected and the activity was quantified using a γ-counter (Packard Cobra II Auto Gamma, PerkinElmer, Switzerland). The IC50 values were calculated by fitting the data with non-linear regression using least squares fit with GraphPad Prism (GraphPad Software, La Jolla, CA, USA). Experiments were performed on triplicate samples.
Internalisation assay
The internalisation kinetics of 67Ga-Ex4NOD12, 67Ga-Ex4NOD27 and 67Ga-Ex4NOD40 were determined using CHL-GLP-1 receptor positive cells as described above. A 4 kBq (120 fmol) of the respective 67Ga-labelled peptide was used as a probe. Cells were incubated for specific time points (5, 15, 30, 60 and 120 min, respectively) at 37°C; non-specific binding was determined by adding an additional 100 μL of natGa-labelled tracer to a final concentration of 1 μM of the corresponding peptide. After incubation, the supernatant was aspirated and the wells were washed with 1 mL PBS. Both the supernatant and wash fractions were pooled and used to determine the non-bound fraction. In order to dislodge the surface-bound peptide, the cells were incubated at room temperature with 1 mL glycine buffer pH 2.6 for 5 min. The glycine wash was collected separately. The internalised fraction was identified by adding 1 mL 1 M NaOH to the cells with subsequent collection of the lysates. The activity in all three fractions was measured in a γ-counter (Packard Cobra II Auto Gamma, Perkin Elmer, Switzerland). Experiments were performed on triplicate samples.
Plasma stability
A 5-MBq (0.2 nmol)-labelled peptide was added into 250 μL fresh human blood plasma and incubated at 37°C for 48 h. Forty-microlitre samples were taken at specific time points (0, 1, 4, 24, and 48 h) and mixed with 200 μL precipitation solution (methanol/water, 0.1% TFA 1:1). The sample was then filtered through a Thomson Single StEP Filter vial 0.45 μm PVDF (Thomson Instrument Company, Oceanside, CA, USA) and analysed via RP-HPLC on a Discovery BioWide Pore C18 (15 cm × 2.1 mm; 3 μm) column. The column was eluted with water containing 0.1% TFA, with a linear gradient from 15% to 95% acetonitrile for 10 min followed by an isocratic elution at 95% acetonitrile for an additional 5 min with a flow rate of 1 mL/min.
Biodistribution
All in vivo experiments were approved by the local veterinarian department and conducted in accordance with the Swiss law of animal protection. Biodistribution studies of 68Ga-Ex4NOD12, 68Ga-Ex4NOD27 and 68Ga-Ex4NOD40 were compared. Six-week-old female CD1 nu/nu mice were subcutaneously inoculated in both shoulder regions with 8 × 106 CHL-GLP-1R positive cells suspended in PBS pH 7.4. After 4 weeks, the mice were randomly divided into groups of four mice each. The mice were injected with approximately 750 kBq (3 to 8 pmol) of the respective 68Ga-labelled peptide via the tail vein. The mice were sacrificed at specific time points (0.5, 1, 2 h). In order to determine GLP-1 receptor-mediated uptake, one group per peptide was co-injected with excess (100 μg each) unlabelled peptide and euthanised 2 h after injection. Blood, heart, lungs, spleen, kidneys, pancreas, stomach, intestine, liver, muscle, bone and tumour were removed and weighed and activity was determined in a γ-counter (Packard Cobra II Auto Gamma, Perkin Elmer, Switzerland). The percentage injected activity per gram tissue (%iA/g) was calculated for each tissue. The statistical significance was determined using Student's unpaired t test.
Circular dichroism
Three sets of spectra were recorded for each peptide with a concentration of 20 μM in Milli-Q water using a Chirascan spectropolarimeter (Applied Photophysics, UK) with a 0.1-cm path length in the range of 190 to 280 nm. First, a baseline CD spectrum was recorded at 20°C followed by thermal denaturation at 94°C for 30 min. After the denaturation, a spectrum was recorded to determine the linearisation of the peptide. Subsequently, the sample was cooled to 20°C and another set of spectra was recorded to evaluate the refolding of the peptide.