The precursor 9-O-desmethyl-(+)-DTBZ [(+)-(2R,3R,11bR)-9-O-desmethyl-α-DTBZ] was purchased from ABX GmbH (Radeberg, Germany). The nonradioactive standard FE-(+)-DTBZ-d4 [(2R,3R,11bR)-9-(2-fluoroethoxy)-3-isobutyl-10-methoxy-2,3,4,6,7,11b-hexahydro-1H-pyrido[2,1-a]isoquinolin-2-ol] and the deuterated 2-bromoethyl tosylate (BrCD2CD2OTs) was purchased from PharmaSynth AS (Tartu, Estonia). All other chemicals were obtained from commercial sources with the highest grade and used without any further purification. Solid-phase extraction [SPE] cartridges, Sep-Pak QMA Light and Sep-Pak tC18 Plus, were purchased from Waters Corporation (Milford, MA, USA). The tC18 Plus cartridge was activated using (1) ethanol [EtOH] (10 mL) and (2) water (10 mL, 18 MΩ). The SPE cartridge Sep-Pak QMA Light was activated using (1) potassium carbonate (K2CO3) solution (0.5 M, 10 mL) and (2) water (15 mL, 18 MΩ).
Radiosynthesis of [18F]FE-DTBZ-d4 via 2-[18F]FE bromide-d4 ([18F]FCD2CD2Br, [18F]FEtBr-d4)
Production of [18F]F-
Fluorine-18 fluoride [[18F]F - ] was produced from a PETtrace Cyclotron (GEMS, GE, Uppsala, Sweden) using 16.4 MeV protons via the18O(p,n)18F reaction on18O-enriched water [[18O]H2O]. [18F]F - was isolated from [18O]H2O on a preconditioned Sep-Pak QMA Light anion-exchange cartridge and subsequently eluted from the cartridge with a solution of K2CO3 (1.8 mg, 13 μmol), Kryptofix 2.2.2 (4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo-[8.8.8]hexacosane-K2.2.2) (9.8 mg, 26 μmol) in water (85 μL, 18 MΩ), and MeCN (2 mL) to a reaction vessel (10 mL). The solvents were evaporated at 160°C for 10 to 15 min under continuous nitrogen flow (70 mL/min) to form a dry complex of [18F]F-/K2CO3/K2.2.2, and the residue was cooled to room temperature [RT].
Radiosynthesis of 2-[18F]FE bromide-d4 ([18F]FCD2CD2Br, [18F]FEtBr-d4)
2-Bromoethyl tosylate-d4 (15 μL) in o-dichlorobenzene [o-DCB] (700 μL) was added to the reaction vessel containing dried [18F]F-/K2CO3/K2.2.2 complex at RT. The reaction mixture was heated at 135°C for 10 min to produce [18F]FEtBr-d4. The crude [18F]FEtBr-d4 (boiling point 71.5°C) was purified by distillation at 80°C under nitrogen flow (25 mL/min) and trapped in a second reaction vessel (5 mL) at -15°C containing 9-O-desmethyl-(+)-DTBZ precursor (2.0 to 2.5 mg, 6.55 to 8.19 μmol) and NaOH (15 μL, 5 M in water) in anhydrous N,N-dimethylformamide [DMF] (500 μL).
Radiosynthesis of [18F]FE-DTBZ-d4
The reaction mixture containing [18F]FEtBr-d4, 9-O-desmethyl-(+)-DTBZ precursor, and NaOH in DMF was heated at 110°C for 5 min to produce [18F]FE-DTBZ-d4. The crude reaction mixture was diluted with 200 μL water before injecting into a high-performance liquid chromatography [HPLC] semi-preparative reverse-phase μBondapak column (C18, 7.8 Ø × 300 mm, 10 μm, Waters Corporation) for purification. The column outlet was connected to an UV absorbance detector (λ = 214 nm) in series with a GM tube for radioactivity detection. Elution with mobile phase CH3CN/10 mM H3PO4 (15:85) at a flow rate of 6 mL/min gave a radioactive fraction corresponding to pure [18F]FE-DTBZ-d4 (retention time = 12 min). The fraction was diluted with water (50 mL, 18 MΩ), and the resulting mixture was loaded onto a preconditioned Sep-Pak tC18 Plus cartridge. The cartridge was washed with water (10 mL), and the isolated product, [18F]FE-DTBZ-d4, was eluted with 1 mL of EtOH into a sterile vial containing a phosphate-buffered saline solution (7 mL).
The radiochemical purity, identity, and stability of [18F]FE-DTBZ-d4 were determined by analytical HPLC using a reverse-phase μBondapak column (C18, 3.9 Ø × 300 mm, 10 μm, Waters Corporation) with mobile phase CH3CN/10 mM H3PO4 (15:85) and a flow rate of 3 mL/min. The effluent was monitored with an UV absorbance detector (λ = 214 nm) coupled with a radioactive detector (β-flow, Beckman Coulter, Inc., Fullerton, CA, USA). The identity of [18F]FE-DTBZ-d4 was confirmed by co-injection with the authentic nonradioactive FE-DTBZ-d4.
Specific radioactivity determination
The specific radioactivity [SRA] of the product was measured by the same analytical HPLC method for quality control. SRA was calibrated for UV absorbance (λ = 214 nm) response per mass of ligand and calculated as the radioactivity of the radioligand (in gigabecquerels) divided by the amount of the associated carrier substance (in micromoles). Each sample was analyzed three times and compared to a reference standard analyzed three times.
Liquid chromatography-mass spectrometry [LC-MS/MS] analysis of the purified labeled product, [18F]FE-DTBZ-d4, and of the reference standard FE-DTBZ-d4 was performed using a Waters Acquity™ Ultra Performance LC system connected with a Micromass Premier™ Quadrupole Time-of-Flight [TOF] mass spectrometer (Waters Corporation). LC was performed using a Waters Acquity UPLC™ BEH column (C18, 2.1 Ø × 50 mm, 1.7 μm; Waters Corporation) kept at 40°C. The mobile phase consisted of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). Samples were analyzed using a linear gradient (0% to 50% B, from 0 to 4 min; 50% to 100% B, from 4 to 4.50 min and then kept at 100% B to 5 min). The flow rate was 0.5 mL/min. The MS was operated in positive-mode electrospray ionization [ESI], with the following settings: capillary voltage 3.0 kV, cone voltage 45 V, source temperature 120°C, dissolvation temperature 350°C, and collision-energy ramp ranging from 20 to 30 eV. [18F]FE-DTBZ-d4 was analyzed after radioactive decay without further dilution. FE-DTBZ-d4 (1 mg/mL) was diluted 200 times with water.
In vitro homogenate saturation binding
Endocrine (n = 4, 80% to 85% islets) or exocrine (n = 4) tissue preparations were isolated from human pancreata and homogenized in 50 mM tris(hydroxymethyl)aminomethane [TRIS] by a Polytron PT3000 homogenator (Kinematica AG, Littau, Switzerland). The tissue homogenates (0.5 to 6 mg/mL) were incubated in 1 mL of 50 mM TRIS with different concentrations of tracer around an expected dissociation constant K
d of 3 nM. The non-displaceable binding was determined by addition of 10 to 20 μM tetrabenazine (BIOTREND Chemikalien GmbH, Cologne, Germany).
All samples were incubated at RT for 60 min to reach an equilibrium and then moved onto a 1.2 μm Whatman filter (Brandel, Gaithersburg, MD, USA; pretreated with 0.05% polyethylenimine for > 1 h) by a C-48 cell harvester (Brandel, Gaithersburg, MD, USA). The filter components associated to each group were pooled and measured in a well counter (Uppsala Imanet AB, GE Healthcare, Uppsala, Sweden). Tissue samples and references were prepared in triplicates, and filter binding controls, in duplicates.
Tissue protein content (in milligram protein per sample) was assessed by a Bio-Rad Protein Assay (Bio-Rad, Hercules, CA, USA), and absorbance was measured with an EL808 microplate reader (BioTek, Winooski, VT, USA). The BP, defined as the ratio between the receptor density [B
max] and dissociation constant [K
d], was determined by nonlinear regression.
Male Swedish landrace piglets (n = 4, 15 to 17 kg, 11 to 14 weeks old) were anesthetized by Zoletil forte (Virbac, Carros Cedex, France). The animals were intubated and placed on a ventilator, and the anesthesia was maintained by 2.5% sevofluran. Normoglycemia was confirmed by determining blood glucose content (plasma glucose concentration 4.4 to 7.6 mM). The animal experiments were approved by the local ethics committee for animal research and performed in accordance with local institutional and Swedish national rules and regulations.
The PET/CT studies were performed using a Discovery ST scanner (General Electric, Milwaukee, WI, USA). All scans were acquired in 2D mode and reconstructed by OSEM iterative algorithm. Four piglets were administered 3.4 to 16.3 MBq/kg [18F]FE-DTBZ-d4 as an intravenous [i.v.] bolus, and the biodistribution as well as the kinetics of the tracer in the abdomen was studied with a dynamic sequence over 90 min (the head first, prone, 4 frames × 30 s, 3 × 1 min, 5 × 3 min and 14 × 5 min). Arterial blood samples were acquired during the first 90 min, and both the plasma- and whole blood volume-corrected tracer contents were measured using the well counter. A whole-body PET/CT examination was performed 100 to 120 min post-administration.
[18F]F- (9.2 to 12.5 MBq/kg) was administered as an i.v. bolus to three piglets to assess the bone uptake PK/PD. Arterial blood and plasma samples were measured using the well-counter. The animals were positioned with a field of view as in the [18F]FE-DTBZ-d4 dynamic scan.
The data in this study was compared to previously published PK/PD porcine data on non-deuterated [18F]-FE-DTBZ. For details on the methodologies used in this study, see Eriksson et al. .
PET and CT images were analyzed by PMOD (PMOD Technologies Ltd., Zürich, Switzerland). Values are given as means ± standard error of the mean [SEM] unless otherwise stated, and statistics are based on Student's t test.
Tissue volumes of interest [VOIs] and regions of interest [ROIs] were delineated on CT images or partially summed PET images. The kinetic uptake in the cortical bone tissue for all three tracers was assessed by generating isocontour VOIs over the lumbar vertrebral bodies in CT images.
Estimation of tracer defluorination
Since the studies on [18F]FE-DTBZ had been performed previously without metabolite correction analysis, we decided to quantify the defluorination retroactively by kinetic modeling of the cortical bone uptake. When modeling the PK/PD of a PET tracer, we generally study the relationship between three different functions (Equation 1) which we can express in a vector form; the input function [C
input], the transfer function [H
TR], and the output function [C
tissue] were defined as follows:
The gradual accumulation of radioactivity in the cortical bone tissue starting around 5 min after administration was assumed to consist entirely of [18F]F- uptake. It is difficult to delineate the cortical bone structures in the spinal column due to its relative small thickness even if isocontour thresholding is used. Partial volume effects [PVEs] will still yield kinetics containing contributions from both the cortical bone and bone marrow. Compounding this problem is the observation that the bone marrow expresses VMAT2 and subsequently, will exhibit some degree of specific [18F]FE-DTBZ and [18F]FE-DTBZ-d4 uptake. To reduce the contribution from non-cortical sources, VOIs over the bone marrow were drawn, and the associated kinetics was subtracted from the cortical isocontour VOIs. The resulting kinetics after this operation was assessed to describe [18F]F- PK/PD in the cortical bone tissue and used as the output curve C
tissue for the quantification of tracer defluorination.
Cortical bone uptake of [18F]F- in pigs is best described by a two-tissue compartment model. The H
TR is given in Equation 2, where φ
1, and θ
2 are macro-parameters determined by the rate constants K
3, and k
T and V
S denote the total and specific binding distribution volumes, respectively:
TR in the cortical bone was determined by the kinetic modeling module PKIN for PMOD.
input of [18F]F- is in this specific case not created by an instantaneous bolus, but rather from a gradual reaction where [18F]FE-DTBZ or [18F]FE-DTBZ-d4 is the substrate and [18F]F- is the product. The rate of defluorination can then be expressed in a single parameter k
defluorination if we assume that the reaction is concentration-dependent and unchanging over time (or during the time frame investigated here). C
in is then uniquely determined from the arterial blood plasma curve [C
p] and k
defluorination (Equation 3):
The defluorination rate constant in plasma can then be determined by minimizing Equation 4:
All optimization was performed using MatLab 7.8.0 (MathWorks, Inc., Natick, MA, USA).
Quantification of pancreatic uptake by kinetic modeling
A one-tissue compartment model was used to estimate the compound parameter V
T as well as the tissue specific rate constants K
1 to k
2 in the pancreas. The C
p was used as an input function, corrected for tracer metabolism by defluorination parameter k
defluorination determined from cortical bone uptake. The kinetic parameters were estimated by the PMOD modeling module PKIN.