68Ga-DOTA-peptides
The DOTA-conjugated peptides were purchased from Almac Sciences (By Gladsmuir, Scotland, UK), ABX advanced biochemical compounds GmbH (Radeberg, Germany) and NeoMPS (Strasbourg, France).
Linear 9-amino acid DOTA-chelated peptide (GGGGKGGGG) with and without a PEG linker (8-amino-3,6-diooxaoctanoyl, PEG derivative, MW 145.16 Da) between the DOTA and the N terminal amino acid was labelled with 68Ga as previously described [8], and named as 68Ga-DOTAVAP-P1 and 68Ga-DOTAVAP-PEG-P1. Briefly, 68Ga was obtained in the form of 68GaCl3 from a 68Ge/68Ga generator (Cyclotron Co., Obninsk, Russia) by elution with 0.1 M HCl. The 68GaCl3 eluate (500 μl) was mixed with sodium acetate (18 mg; Sigma-Aldrich, Seelze, Germany) to give a pH of approximately 5.5. Then, DOTA-peptide (35 nmol) was added and the mixture was incubated at 100°C for 20 min. No further purification was needed.
The radiochemical purity was determined by reversed-phase HPLC (μBondapak C18, 7.8 × 300 mm2, 125 Å, 10 μm; Waters Corporation, Milford, MA, USA). The HPLC conditions for 68Ga-DOTAVAP-P1 have been described previously [9]. The HPLC conditions for 68Ga-DOTAVAP-PEG-P1 were slightly different and as follows: flow rate = 4 ml/min, λ = 215 nm, A = 2.5 mM trifluoroacetic acid, B = acetonitrile and C = 50 mM phosphoric acid. Linear A/B/C gradient was 100/0/0 for 0 to 3 min, 40/60/0 for 3 to 9 min, and 0/0/100 for 9 to 16 min. The radio-HPLC system consisted of LaChrom instruments (Hitachi; Merck, Darmstadt, Germany): pump L7100, UV detector L-7400 and interface D-7000; an on-line radioisotope detector (Radiomatic 150 TR, Packard, Meriden, CT, USA); and a computerised data acquisition system.
In vitro stability and solubility
The in vitro stability of the 68Ga-labelled peptides was evaluated in human and rat plasma. Several samples were taken during the 4-h incubation period at 37°C. Proteins from plasma samples were precipitated with 10% sulphosalicylic acid (1:1 v/v), centrifuged at 3,900 × g for 3 min at 4°C, and filtered through 0.45-μm Minispike filter (Waters Corporation). The filtrate was analysed by radio-HPLC.
The octanol-water distribution coefficient, logD, of the 68Ga-DOTA-peptides was determined using the following procedure. Approximately 5 kBq of 68Ga-labelled peptide in 500 μl of phosphate-buffered saline (PBS, pH 7.4) was added to 500 μl of 1-octanol. After the mixture had been vortexed for 3 min, it was centrifuged at 12,000 × g for 6 min, and 100-μl aliquots of both layers were counted in a gamma counter (1480 Wizard 3″ Gamma Counter; EG&G Wallac, Turku, Finland). The test was repeated three times. The logD was calculated as = log10 (counts in octanol/counts in PBS).
Animals
All animal experiments were approved by the Lab-Animal Care & Use Committee of the State Provincial Office of Southern Finland and carried out in compliance with the Finnish laws relating to the conduct of animal experimentation.
Male Sprague-Dawley rats (n = 14) were purchased from Harlan, Horst, The Netherlands. Twenty-four hours before the PET studies, turpentine oil (Sigma-Aldrich; 0.05 ml per rat) was injected subcutaneously into their neck area in order to induce a sterile inflammation [10]. Six rats were PET imaged and additional eight animals were used for in vivo metabolite analyses.
PET imaging and ex vivo biodistribution
The whole-body distribution and kinetics of 68Ga-DOTAVAP-P1 (n = 3) and 68Ga-DOTAVAP-PEG-P1 (n = 3) in rats harbouring a sterile inflammation were studied with a high-resolution research tomograph (Siemens Medical Solutions, Knoxville, TN, USA). The rats were anaesthetised with isoflurane (induction 3%, maintenance 2.2%). Two rats were imaged at the same time, and they were kept on a warm pallet during the imaging procedure. Following a 6-min transmission for attenuation correction, the rats were intravenously (i.v.) injected with 68Ga-DOTAVAP-P1 (15.8 ± 3.0 MBq, 19.4 ± 0.0 μg, 19.6 ± 0.0 nmol) or with 68Ga-DOTAVAP-PEG-P1 (17.7 ± 1.6 MBq, 21.0 ± 1.3 μg, 18.5 ± 1.1 nmol) as a bolus via a tail vein using a 24-gauge cannula (BD Neoflon, Becton Dickinson Infusion Therapy AB, Helsingborg, Sweden). Dynamic imaging lasting for 60 min started at the time of injection. The data acquired in list mode were iteratively reconstructed with a 3-D ordered subsets expectation maximisation algorithm with 8 iterations, 16 subsets and a 2-mm full-width at half-maximum post-filter into 5 × 60 s and 11 × 300 s frames. Quantitative analyses were performed by drawing regions of interest (ROI) on the inflammatory foci, muscle (hind leg), heart, kidney, liver and urinary bladder. Time-activity curves (TACs) were extracted from the corresponding dynamic images (Vinci software, version 2.37; Max Planck Institute for Neurological Research, Cologne, Germany). The average radioactivity concentrations in the ROIs (kilobecquerels per millilitre) were used for further analyses. The uptake was reported as standardised uptake value (SUV), which was calculated as the radioactivity of the ROI divided by the relative injected radioactivity expressed per animal body weight. The radioactivity remaining in the tail was compensated.
After the PET imaging, the animals were sacrificed. Samples of blood, urine and various organs were collected, weighed and measured for radioactivity using the gamma counter (Wizard, EG&G Wallac). The results were expressed as SUVs.
Blood analyses
Blood samples (0.2 ml of each) were drawn at 5, 10, 15, 30, 45, 60 and 120 min after injection of 68Ga-DOTA-peptides into heparinised tubes (Microvette 100; Sarstedt, Nümbrecht, Germany). Radioactivity of whole blood was measured with the gamma counter (Wizard, EG&G Wallac). Plasma was separated by centrifugation (2,200 × g for 5 min at 4°C), and plasma radioactivity was measured. The ratio of radioactivity in blood versus plasma was calculated. To determine plasma protein binding, proteins were precipitated with 10% sulphosalicylic acid, and the radioactivity in protein precipitate and supernatant was measured. The plasma supernatant was further analysed by radio-HPLC in order to evaluate the in vivo stability of the 68Ga-labelled peptides.
In vivo stability data were used in order to generate metabolite-corrected plasma TACs for 68Ga-DOTAVAP-P1 and 68Ga-DOTAVAP-PEG-P1, which were further used for the calculation of pharmacokinetic parameters. The area under curve (AUC) of the plasma TAC from 0 to infinity was calculated using a non-compartmental analysis employing the trapezoidal rule. The clearance (CL) of the 68Ga-labelled peptides after a single intravenous bolus dose was calculated by dividing the injected dose by the AUC. The plot of the natural logarithm of parent tracer concentration against time after bolus injection became linear in the end phase, as the tracer was eliminated according to the laws of first-order reaction kinetics. The elimination rate constant (k
el) was calculated as the negative slope of the linear part of the plot. The plasma elimination half-life (t
1/2) was calculated as t
1/2 = ln(2)/k
el. The metabolic half-lives of the 68Ga-DOTA-peptides were calculated according to the results of radio-HPLC, i.e. the time point when 50% of the total radioactivity is still bound to the intact peptide.
Statistical analyses
All the results are expressed as means ± standard deviation (SD) and range. The correlations between PET imaging and ex vivo measurement values were evaluated using linear regression analysis. Inter-group comparisons were made using an unpaired t test. Statistical analyses were conducted using Origin 7.5 software (Microcal, Northampton, MA, USA). A P value less than 0.05 was considered as statistically significant.