[ 18 F]2-uoro-2-deoxy-sorbitol PET Imaging for Quantitative Estimation of Blood-brain Barrier Permeability in vivo

Purpose The non-transported and non-metabolized sorbitol derivative [ 18 F]2-uoro-2-deoxy-sorbitol ([ 18 F]FDS) can be straightforwardly obtained from chemical reduction of commercial [ 18 F]2-deoxy-2-uoro-D-glucose. [ 18 F]FDS was evaluated as a small-molecule (paracellular) marker of blood-brain barrier (BBB) integrity for PET. Methods Five mice underwent focused ultrasound (FUS) to generate spatially controlled BBB disruption in the right hemisphere. PET kinetics of [ 18 F]FDS in each brain hemisphere were described by a 1-tissue compartment model using an image-derived input function. Results BBB disruption resulted in a 2.4±0.8-fold increase in the brain distribution (V T, p<0.01) of [ 18 F]FDS. Enhanced brain uptake was associated with an increase in the in�ux transfer rate K 1 (+1.4±0.7-fold, p<0.05) and a decrease in the e�ux transfer rate k 2 (-1.7±0.4-fold, p<0.01). Conclusion Thanks to the quantitative performance of PET compared with other neuroimaging techniques, [ 18 F]FDS PET and kinetic modelling provides a readily available and sensitive method for non-invasive determination of different levels of BBB permeability in vivo.


Introduction
The blood-brain barrier (BBB) plays a critical role in protecting the brain from the circulation while controlling brain homeostasis.Integrity of the BBB is mainly carried out by tight junctions between adjacent endothelial cells forming the brain microvasculature [1].A large body of translational research supports that BBB integrity is compromised in many CNS pathological conditions including multiple sclerosis, hypoxic/ischemic insult, traumatic injury, Parkinson's and Alzheimer's diseases, epilepsy and brain tumors [2].Different non-clinical models have shown that BBB leakage is a common feature of the neuroin ammatory cascade and contributes to disease-associated brain damage [3,4].
Imaging techniques enable in vivo determination of BBB integrity with translational perspectives.Dynamic contrast-enhanced (DCE) and dynamic susceptibility contrast MRI using gadolinium (Gd)-based contrast agents such as gadoterate (MW = 558) or Gd-DTPA (Gd-diethylene triamine pentaacetic acid, MW = 546) [7], and brain SPECT using [ 99m Tc]DTPA (MW = 487) are widely used to detect local BBB disruption associated with some situations such as ischemic stroke or brain tumors [8].Despite limited quantitative performance, the predominant use of MRI and SPECT to investigate BBB integrity is likely due to the availability of corresponding imaging probes [9].PET may allow for accurate kinetic modeling and quantitative estimation of the transfer of radiolabeled compounds across the BBB in vivo [10].However, PET using radiolabeled low-or high-MW makers of BBB integrity such as [ 18 F]1-uoro-1-deoxy-D-mannitol (MW = 183) [11] or [ 11 C]inulin (MW = 6179) [12] did not reach mainstream use, probably due to their limited availability in centers not equipped with a cyclotron.Sorbitol, a stereoisomer of mannitol, is a non-transported hydrophilic small molecule (MW = 182) that is poorly metabolized in mammals.The uorinated derivative [ 18 F]2-uoro-2-deoxy-sorbitol ([ 18 F]FDS, MW = 183) can be virtually obtained in all Nuclear Medicine departments from simple chemical reduction of commercial [ 18 F]2-uoro-2-deoxy-D-glucose ([ 18 F]FDG, Fig. 1) [13].[ 18 F]FDS shows non-signi cant brain uptake in healthy rodents and humans [14] and bene ts from favorable pharmacokinetic properties for quantitative PET imaging in vivo [15].
This study aimed at evaluating [ 18 F]FDS PET and kinetic modelling for quantitative determination of BBB permeability in vivo.To this end, a method based on focused ultrasound (FUS) was optimized to induce hemispheric BBB disruption in mice.

Material And Methods
Production of [ 18 F]FDS Synthesis of [ 18 F]FDS from commercial [ 18 F]FDG and quality control was described by Li et al. [13] and is reported as Supplementary material.

Focused Ultrasound
The method for spatially controlled BBB disruption was optimized from previous work [16] to induce reproducible BBB disruption in the right brain hemisphere only.Seven-week-old female NMRI nu/nu mice were anesthetized with 1.5% iso urane in O 2 .A catheter was inserted in the tail vein and the animal was transferred to the sonication system.Microbubbles (50 µL) were intravenously administrated in the tail vein before the beginning of the FUS (n=5) or Sham (no FUS, n=3) session.Detailed FUS protocol is reported as Supplementary material.

Evan's blue extravasation test
Evan's blue (EB) (Sigma-Aldrich, Saint-Quentin Fallavier, France) at 4% in NaCl 0.9% was freshly prepared as previously described [17].Mice received 100 µL EB i.v immediately after FUS.One hour after injection, i.e. at the end of PET acquisition, animals were euthanized and brains were removed to visually assess EB extravasation (Fig. 2).
PET images were reconstructed by the 3D OSEM/MAP algorithm and corrected for attenuation, random coincidences and scatter.Volumes of interests (VOI s ) were manually delimited using Pmod software (version 3.8, PMOD Technologies Ltd, Zurich, Switzerland).In the FUS group, extravasation of 18 F-FDS was obvious in the sonicated area on late PET images (Fig. 2A).The region with disrupted BBB was delineated and mirrored to the contralateral hemisphere.In Sham animals, VOI s were drawn in each brain hemisphere.Another VOI was drawn on the aorta (blood-pool), obvious on early time-frames, to generate an image derived input function (IDIF).Time Activity Curves (TACs) were corrected for radioactive decay and expressed as standard uptake value (SUV) vs time.Area Under the TAC (AUC) was calculated from 0 to 60 min.A 1-tissue compartment (1-TC) model using IDIF was tested to describe the transport of 18 F-FDS across the BBB and generate parametric mapping of the total volume of distribution (V T ) of [ 18 F]FDS (Fig. 2B).Data are reported as mean ± standard deviation (S.D.) and were statistically compared using either a One-way ANOVA or a paired ttest.(GraphPad Prism, La Jolla, CA, USA).

Results
In hemispheres with intact BBB (Sham animals or contralateral hemisphere), brain PET signal increased rapidly with maximal uptake at T max ~2.0 ± 0.9 min.FUS enhanced the PET signal in the sonicated volume consistent with EB extravasation (Fig. 2) and T max was achieved later at 5.5 ± 2.3 min.Brain uptake of [ 18 F]FDS in the sonicated brain was signi cantly higher compared with the contralateral area (2.12 ± 1.5-fold increase, p < 0.01) or the Sham group (1.84 ± 1.09-fold increase, p < 0.05) (Fig. 3).Brain TACs were accurately described by the 1-TC model which demonstrated a signi cant 2.43 ± 0.8-fold increase in the V T of [ 18 F]FDS in the sonicated area (p < 0.001).Enhanced brain distribution was associated with a 1.4 ± 0.7-fold increase in K 1 (p < 0.05) and a 1.7 ± 0.4-fold decrease in k 2 (p < 0.01) (Fig. 4).

Discussion
[ 18 F]FDS bene ts from the characteristics of an "ideal" marker of BBB integrity [5].This includes clinical safety, metabolic stability, low binding to plasma proteins (< 0.1%) and low baseline brain uptake when the BBB is intact [14,18].Simple production of 18 F-FDS from commercial [ 18 F]FDG makes it an appealing radiopharmaceutical candidate for determination of BBB integrity using quantitative PET.
Unlike [ 18 F]FDG, [ 18 F]FDS is poorly taken up by mammalian cells because it does not undergo facilitated transport [13].[ 18 F]FDS PET has been validated in animals and humans to study renal function [15] or detect/estimate bacterial burden in tissues because sorbitol is a speci c metabolic substrate of some strains of gram-negative bacteria [19].Interestingly, [ 18 F]FDS was shown to visually accumulate in orthotopic brain tumor xenografts in mice, despite negligible uptake by implanted glioma cells in vitro [13].Our results suggest that enhanced [ 18 F]FDS PET signal in the tumor area may be attributed to local BBB leakage.
Pharmacokinetic modelling of brain [ 18 F]FDS PET data is relatively simple.The impact of BBB disruption did not restrict to enhanced in ux (blood-to-brain) transfer.This suggests that considering the uptake phase of the brain distribution only may underestimate the overall impact of BBB integrity on brain exposure of solutes.
FUS offers unique perspectives to induce safe, reproducible and localized BBB disruption in vivo [20].
Interestingly, mapping of [ 18 F]FDS brain distribution within the sonicated area displayed a gradient from the center to the periphery (Fig. 2).It may be hypothesized that heterogeneity in intensity of delivered ultrasound may occur within the sonicated area as a consequence of loss of ultrasound transmission relative to the transducer angulation at the skull surface.Interestingly, such phenomenon could not be detected using high-MW markers of BBB integrity such as gadoterate ou EB [17].
There is a critical need for PET imaging markers to offer more quantitative insight into BBB integrity in pathophysiological conditions [11,[21][22][23].Outcome parameters and parametric images describing BBB transport of [ 18 F]FDS provide absolute quantitative whole-brain mapping of BBB permeability.This may be useful for group comparison or to investigate the dynamics of disease-associated change in BBB permeability in longitudinal studies.In a neuropharmacokinetic perspective, [ 18 F]FDS provides a convenient alternative to EB, DCE-MRI or [ 99m Tc]DTPA SPECT, with better quantitative performance, to interpret the brain penetration of CNS-targeting radioligands with respect to the local permeability of the BBB [24].

DeclarationsFundingGaelle
Hugon received a PhD grant from the CEA.Louise Breuil received a grant from CEA/AP-HP.Con icts of interest/Competing interestsNone.

Figures
Figures

Figure 3 Brain
Figure 3