*Article* **[ <sup>18</sup>F]2-Fluoro-2-deoxy-sorbitol PET Imaging for Quantitative Monitoring of Enhanced Blood-Brain Barrier Permeability Induced by Focused Ultrasound**

**Gaëlle Hugon <sup>1</sup> , Sébastien Goutal <sup>1</sup> , Ambre Dauba <sup>1</sup> , Louise Breuil <sup>1</sup> , Benoit Larrat <sup>2</sup> , Alexandra Winkeler <sup>1</sup> , Anthony Novell <sup>1</sup> and Nicolas Tournier 1,\***


**Abstract:** Focused ultrasound in combination with microbubbles (FUS) provides an effective means to locally enhance the delivery of therapeutics to the brain. Translational and quantitative imaging techniques are needed to noninvasively monitor and optimize the impact of FUS on blood-brain barrier (BBB) permeability in vivo. Positron-emission tomography (PET) imaging using [18F]2 fluoro-2-deoxy-sorbitol ([18F]FDS) was evaluated as a small-molecule (paracellular) marker of bloodbrain barrier (BBB) integrity. [18F]FDS was straightforwardly produced from chemical reduction of commercial [18F]2-deoxy-2-fluoro-D-glucose. [18F]FDS and the invasive BBB integrity marker Evan's blue (EB) were i.v. injected in mice after an optimized FUS protocol designed to generate controlled hemispheric BBB disruption. Quantitative determination of the impact of FUS on the BBB permeability was determined using kinetic modeling. A 2.2 ± 0.5-fold higher PET signal (*n* = 5; *p* < 0.01) was obtained in the sonicated hemisphere and colocalized with EB staining observed post mortem. FUS significantly increased the blood-to-brain distribution of [18F]FDS by 2.4 <sup>±</sup> 0.8-fold (*V*T; *p* < 0.01). Low variability (=10.1%) of *V*<sup>T</sup> values in the sonicated hemisphere suggests reproducibility of the estimation of BBB permeability and FUS method. [18F]FDS PET provides a readily available, sensitive and reproducible marker of BBB permeability to noninvasively monitor the extent of BBB disruption induced by FUS in vivo.

**Keywords:** blood-brain barrier; integrity marker; sorbitol; positron emission tomography; focused ultrasound

### **1. Introduction**

The blood-brain barrier (BBB) plays a critical role in protecting the brain from potentially harmful substances of the circulation while controlling brain homeostasis [1]. Integrity of the BBB is mainly carried out by tight junctions between adjacent endothelial cells forming the brain microvasculature [2]. This considerably limits the paracellular (i.e., between cells) passage of solutes across the BBB which contributes to the sanctuary site property of the brain [2,3]. Many drug molecules and therapeutics cannot naturally permeate the intact BBB into the brain parenchyma. As a consequence, the BBB is a bottleneck in the development of central nervous system (CNS)-targeting therapeutics, which complicates the treatment of brain diseases, notably cerebral malignancies [4].

Numerous strategies and technologies have been proposed to overcome the BBB and achieve sufficient brain delivery of therapeutics [5]. Among these strategies, focused ultrasound in combination with microbubbles (FUS) is emerging as an effective means to locally and temporarily enhance BBB permeability, mainly through the "opening" of

**Citation:** Hugon, G.; Goutal, S.; Dauba, A.; Breuil, L.; Larrat, B.; Winkeler, A.; Novell, A.; Tournier, N. [ <sup>18</sup>F]2-Fluoro-2-deoxy-sorbitol PET Imaging for Quantitative Monitoring of Enhanced Blood-Brain Barrier Permeability Induced by Focused Ultrasound. *Pharmaceutics* **2021**, *13*, 1752. https://doi.org/10.3390/ pharmaceutics13111752

Academic Editors: Vibhuti Agrahari and Prashant Kumar

Received: 17 September 2021 Accepted: 14 October 2021 Published: 20 October 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

the paracellular route by disruption of the tight junctions [6]. A large body of preclinical research has convincingly shown that FUS enhanced the brain exposure of small molecules, biologics or gene therapy [7–11]. More than 30 clinical trials using FUS-induced BBB disruption are ongoing and assessed the translational potential and clinical safety of the technique [12–17]. paracellular route by disruption of the tight junctions [6]. A large body of preclinical research has convincingly shown that FUS enhanced the brain exposure of small molecules, biologics or gene therapy [7–11]. More than 30 clinical trials using FUS-induced BBB disruption are ongoing and assessed the translational potential and clinical safety of the technique [12–17].

Numerous strategies and technologies have been proposed to overcome the BBB and achieve sufficient brain delivery of therapeutics [5]. Among these strategies, focused ultrasound in combination with microbubbles (FUS) is emerging as an effective means to locally and temporarily enhance BBB permeability, mainly through the "opening" of the

*Pharmaceutics* **2021**, *13*, x FOR PEER REVIEW 2 of 15

The development of FUS is tightly linked to the availability of markers of BBB integrity, with the aim to quantitatively assess the impact of various FUS conditions in enhancing BBB permeability [18]. In the literature, many different BBB integrity markers have been described for nonclinical in vitro and ex vivo (terminal) experiments [19]. Low molecular weight (MW, g/mol) hydrophilic molecules such as fluorescein (MW = 332, fluorescent detection), (radio)labeled analogues of sucrose (MW = 342) or mannitol (MW = 182) (Figure 1A) are usually preferred as quantitative "paracellular" markers of membrane integrity (Figure 1) [1,20]. Low MW is associated with enhanced sensitivity to subtle change in barrier permeability compared with higher MW compounds such as Evan's blue (EB, MW = 961, highly bound to plasma proteins), radiolabeled proteins (albumin MW = 65,000), dextrans (MW = 1500–70,000) or inulin (MW = 6179) [1,21]. The development of FUS is tightly linked to the availability of markers of BBB integrity, with the aim to quantitatively assess the impact of various FUS conditions in enhancing BBB permeability [18]. In the literature, many different BBB integrity markers have been described for nonclinical in vitro and ex vivo (terminal) experiments [19]. Low molecular weight (MW, g/mol) hydrophilic molecules such as fluorescein (MW = 332, fluorescent detection), (radio)labeled analogues of sucrose (MW = 342) or mannitol (MW = 182) (Figure 1A) are usually preferred as quantitative "paracellular" markers of membrane integrity (Figure 1) [1,20]. Low MW is associated with enhanced sensitivity to subtle change in barrier permeability compared with higher MW compounds such as Evan's blue (EB, MW = 961, highly bound to plasma proteins), radiolabeled proteins (albumin MW = 65,000), dextrans (MW = 1500–70,000) or inulin (MW = 6179) [1,21].

**Figure 1.** Chemical structures of mannitol (**A**), sorbitol (**B**). Chemical reduction of [18F]2-fluoro-2 deoxy-glucose ([18F]FDG) to [18F]2-fluoro-2-deoxy-sorbitol ([18F]FDS) using sodium borohydride (NaBH4) is shown in (**C**). **Figure 1.** Chemical structures of mannitol (**A**), sorbitol (**B**). Chemical reduction of [18F]2-fluoro-2 deoxy-glucose ([18F]FDG) to [18F]2-fluoro-2-deoxy-sorbitol ([18F]FDS) using sodium borohydride (NaBH<sup>4</sup> ) is shown in (**C**).

Translational imaging techniques such as magnetic resonance imaging (MRI), singlephoton emitting computed tomography (SPECT) or positron emission tomography imaging (PET) are increasingly regarded as methods to noninvasively monitor the impact of FUS on BBB permeability [18]. So far, dynamic contrast-enhanced (DCE), T1 relaxometry and dynamic susceptibility contrast MRI using gadolinium (Gd)-based contrast agents such as gadoterate (MW = 558), Gd-DTPA (Gd-diethylene triamine pentaacetic acid, MW = 546) [22,23] or large iron nanoparticles [24] have been used to detect BBB disruption in vivo. Brain SPECT using [99mTc]DTPA (MW = 487) is also widely used as a BBB integrity marker [25]. The predominant use of MRI and SPECT to investigate BBB integrity is likely due to the wide availability of corresponding imaging probes [26]. In humans, the clinical proof-of-concept of FUS-induced BBB disruption has been achieved using DCE-MRI to assess and localize BBB disruption in vivo [12]. However, accurate and absolute quantification of the brain penetration of contrast agent using MRI is very challenging [22]. More quantitative neuroimaging techniques are needed to accurately estimate the impact of Translational imaging techniques such as magnetic resonance imaging (MRI), singlephoton emitting computed tomography (SPECT) or positron emission tomography imaging (PET) are increasingly regarded as methods to noninvasively monitor the impact of FUS on BBB permeability [18]. So far, dynamic contrast-enhanced (DCE), T1 relaxometry and dynamic susceptibility contrast MRI using gadolinium (Gd)-based contrast agents such as gadoterate (MW = 558), Gd-DTPA (Gd-diethylene triamine pentaacetic acid, MW = 546) [22,23] or large iron nanoparticles [24] have been used to detect BBB disruption in vivo. Brain SPECT using [99mTc]DTPA (MW = 487) is also widely used as a BBB integrity marker [25]. The predominant use of MRI and SPECT to investigate BBB integrity is likely due to the wide availability of corresponding imaging probes [26]. In humans, the clinical proof-of-concept of FUS-induced BBB disruption has been achieved using DCE-MRI to assess and localize BBB disruption in vivo [12]. However, accurate and absolute quantification of the brain penetration of contrast agent using MRI is very challenging [22]. More quantitative neuroimaging techniques are needed to accurately estimate the impact of FUS on BBB integrity.

FUS on BBB integrity. Compared with MRI or SPECT, PET imaging benefits from absolute quantitative performances and high sensitivity so that quantitative determination of the concentration of microdose radiolabeled compounds in the brain is possible. Furthermore, high frame rate allows for kinetic modeling and estimation of transfer rate across the BBB [18]. For most imaging centers, the main limitation of PET is the limited availability of compounds other than commercial and daily produced radiopharmaceuticals such as [18F]2-fluoro-2-deoxy-D-glucose ([18F]FDG, Figure 1). Consistently, PET using radiolabeled low- or high-MW Compared with MRI or SPECT, PET imaging benefits from absolute quantitative performances and high sensitivity so that quantitative determination of the concentration of microdose radiolabeled compounds in the brain is possible. Furthermore, high frame rate allows for kinetic modeling and estimation of transfer rate across the BBB [18]. For most imaging centers, the main limitation of PET is the limited availability of compounds other than commercial and daily produced radiopharmaceuticals such as [18F]2-fluoro-2-deoxy-D-glucose ([18F]FDG, Figure 1). Consistently, PET using radiolabeled low- or high-MW makers of BBB integrity such as [18F]1-fluoro-1-deoxy-D-mannitol (MW = 183) [27] or [ <sup>11</sup>C]inulin (MW = 6179) [28], which synthesis requires production of radioisotope by a cyclotron and dedicated radiochemistry facilities, did not reach mainstream use.

Sorbitol (Figure 1B), a stereoisomer of mannitol, is a non-transported hydrophilic small molecule (MW = 182) that is poorly metabolized in mammals. The fluorinated derivative [ <sup>18</sup>F]2-fluoro-2-deoxy-sorbitol ([18F]FDS, MW = 183, Figure 1C) can be virtually obtained in all nuclear medicine departments and molecular imaging laboratory from simple chemical reduction of commercial [18F]FDG [29]. [18F]FDS shows nonsignificant brain uptake in healthy rodents and humans [30] and benefits from favorable pharmacokinetic properties for quantitative PET imaging in vivo [31].

This study aimed at evaluating [18F]FDS PET for quantitative monitoring of enhanced BBB permeability induced by FUS in vivo. To this end, transcranial FUS conditions were optimized to induce hemispheric BBB disruption in mice. The kinetics of [18F]FDS across the BBB were compared in the sonicated and nonsonicated hemispheres.

#### **2. Materials and Methods**
