Tumor-Targeted Perfluorinated Micelles as Efficient Theranostic Agents Combining Positron Emission Tomography and Radiosensitization.

We report the synthesis of biocompatible perfluorinated micelles designed to improve radiotherapeutic efficacy in a radioresistant tumor environment. In vitro and in vivo behaviors of perfluorinated micelles were assessed at both cellular and tissular levels. The micellar platform offers key advantages as theranostic tool: (i) small size, allowing deep tissue penetration; (ii) oxygen transport to hypoxic tissues; (iii) negligible toxicity in the absence of ionizing radiation; (iv) internalization into cancer cells; (v) potent radiosensitizing effect; and (vi) excellent tumor-targeting properties, as monitored by positron emission tomography. We have demonstrated strong in vitro radiosensitizing effects of the micelle and in vivo tumor targeting, making this nanometric carrier a promising tool for the potentiation of focused radiotherapy.

AGuIX nanoparticle-nanobody bioconjugates to target immune checkpoint receptors.

This article presents bioconjugates combining nanoparticles (AGuIX) with nanobodies (VHH) targeting Programmed Death-Ligand 1 (PD-L1, A12 VHH) and Cluster of Differentiation 47 (CD47, A4 VHH) for active tumor targeting. AGuIX nanoparticles offer theranostic capabilities and an efficient biodistribution/pharmacokinetic profile (BD/PK), while VHH's reduced size (15 kDa) allows efficient tumor penetration. Site-selective sortagging and click chemistry were compared for bioconjugation. While both methods yielded bioconjugates with similar functionality, click chemistry demonstrated higher yield and could be used for the conjugation of various VHH. The specific targeting of AGuIX@VHH has been demonstrated in both in vitro and ex vivo settings, paving the way for combined targeted immunotherapies, radiotherapy, and cancer imaging.

Efficient PD-L1 imaging of murine glioblastoma with FUS-aided immunoPET by leveraging FcRn-antibody interaction.

Rationale: The passage of antibodies through the blood-brain barrier (BBB) and the blood-tumoral barrier (BTB) is determinant not only to increase the immune checkpoint inhibitors efficacy but also to monitor prognostic and predictive biomarkers such as the programmed death ligand 1 (PD-L1) via immunoPET. Although the involvement of neonatal Fc receptor (FcRn) in antibody distribution has been demonstrated, its function at the BBB remains controversial, while it is unknown at the BTB. In this context, we assessed FcRn's role by pharmacokinetic immunoPET imaging combined with focused ultrasounds (FUS) using unmodified and FcRn low-affinity IgGs targeting PD-L1 in a preclinical orthotopic glioblastoma model. Methods: Transcranial FUS were applied over the whole brain in mice shortly before injecting the anti-PD-L1 IgG 89Zr-DFO-C4 or its FcRn low-affinity mutant 89Zr-DFO-C4Fc-MUT in a syngeneic glioblastoma murine model (GL261-GFP). Brain uptake was measured from PET scans acquired up to 7 days post-injection. Kinetic modeling was performed to compare the brain kinetics of both C4 formats. Results: FUS efficiently enhanced the delivery of both C4 radioligands in the brain with high reproducibility. 89Zr-DFO-C4Fc-MUT mean concentrations in the brain reached a significant uptake of 3.75±0.41%ID/cc with FUS against 1.92±0.45%ID/cc without, at 1h post-injection. A substantial and similar entry of both C4 radioligands was observed at a rate of 0.163±0.071 mL/h/g of tissue during 10.4±4.6min. The impaired interaction with FcRn of 89Zr-DFO-C4Fc-MUT significantly decreased the efflux constant from the healthy brain tissue to plasma compared with non-mutated IgG. Abolishing FcRn interaction allows determining the target engagement related to the specific binding as soon as 12h post-injection. Conclusion: Abolishing Fc-FcRn interaction confers improved kinetic properties to 89Zr-DFO-C4Fc-MUT for immunoPET imaging. FUS-aided BBB/BTB disruption enables quantitative imaging of PD-L1 expression by glioblastoma tumors within the brain.

Local and distant response to intratumoral immunotherapy assessed by immunoPET in mice.

BACKGROUND: Despite the promising efficacy of immune checkpoint blockers (ICB), tumor resistance and immune-related adverse events hinder their success in cancer treatment. To address these challenges, intratumoral delivery of immunotherapies has emerged as a potential solution, aiming to mitigate side effects through reduced systemic exposure while increasing effectiveness by enhancing local bioavailability. However, a comprehensive understanding of the local and systemic distribution of ICBs following intratumoral administration, as well as their impact on distant tumors, remains crucial for optimizing their therapeutic potential.To comprehensively investigate the distribution patterns following the intratumoral and intravenous administration of radiolabeled anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and to assess its corresponding efficacy in both injected and non-injected tumors, we conducted an immunoPET imaging study. METHODS: CT26 and MC38 syngeneic colorectal tumor cells were implanted subcutaneously on both flanks of Balb/c and C57Bl/6 mice, respectively. Hamster anti-mouse CTLA-4 antibody (9H10) labeled with zirconium-89 ([89Zr]9H10) was intratumorally or intravenously administered. Whole-body distribution of the antibody was monitored by immunoPET imaging (n=12 CT26 Balb/c mice, n=10 MC38 C57Bl/6 mice). Tumorous responses to injected doses (1-10 mg/kg) were correlated with specific uptake of [89Zr]9H10 (n=24). Impacts on the tumor microenvironment were assessed by immunofluorescence and flow cytometry. RESULTS: Half of the dose was cleared into the blood 1 hour after intratumoral administration. Despite this, 7 days post-injection, 6-8% of the dose remained in the intratumoral-injected tumors. CT26 tumors with prolonged ICB exposure demonstrated complete responses. Seven days post-injection, the contralateral non-injected tumor uptake of the ICB was comparable to the one achieved through intravenous administration (7.5±1.7% ID.cm-3 and 7.6±2.1% ID.cm-3, respectively) at the same dose in the CT26 model. This observation was confirmed in the MC38 model. Consistent intratumoral pharmacodynamic effects were observed in both intratumoral and intravenous treatment groups, as evidenced by a notable increase in CD8+T cells within the CT26 tumors following treatment. CONCLUSIONS: ImmunoPET-derived pharmacokinetics supports intratumoral injection of ICBs to decrease systemic exposure while maintaining efficacy compared with intravenous. Intratumoral-ICBs lead to high local drug exposure while maintaining significant therapeutic exposure in non-injected tumors. This immunoPET approach is applicable for clinical practice to support evidence-based drug development.

Synthesis and Preclinical Fluorescence Imaging of Dually Functionalized Antibody Conjugates Targeting Endothelin Receptor-Positive Tumors.

For the past two decades, the emerging role of the endothelin (ET) axis in cancer has been extensively investigated, and its involvement in several mechanisms described as "hallmarks of cancer" has clearly highlighted its potential as a therapeutic target. Despite the growing interest in finding effective anticancer drugs, no breakthrough treatment has successfully made its way to the market. Recently, our team reported the development of a new immuno-positron emission tomography probe targeting the ET A receptor (ETA, one of the ET receptors) that allows the successful detection of ETA+ glioblastoma, paving the way for the elaboration of novel antibody-based strategies. In this study, we describe the synthesis of two PET/NIRF (positron emission tomography/near-infrared fluorescence) dually functionalized imaging agents, directed against ETA or ETB, that could be used to detect ET+ tumors and select patients that will be eligible for fluorescence-guided surgery. Both imaging modalities were brought together using a highly versatile tetrazine platform bearing the IRDye800CW fluorophore and desferrioxamine for 89Zr chelation. This so-called monomolecular multimodal imaging probe was then "clicked", via an inverse-electron-demand Diels-Alder reaction, to antibodies conjugated site-specifically with a trans-cyclooctene group. This approach has led to homogeneous and well-defined constructs that retained their high affinity and high specificity for their respective target, as shown by flow cytometry and NIRF in vivo imaging experiments in nude mice bearing CHO-ETA and CHO-ETB tumors. Ultimately, these bimodal immunoconjugates could be used to improve the outcomes of patients with ET+ tumors.

Fluorine-18 and Radiometal Labeling of Biomolecules via Disulfide Rebridging.

Biomolecules labeled with positron-emitting radionuclides like fluorine-18 or radiometals like copper-64 and zirconium-89 are increasingly employed in nuclear medicine for diagnosis purposes. Given the fragility and complexity of these compounds, their labeling requires mild conditions. Besides, it is essential to develop methods inducing minimal modification of the tertiary structure, as it is fundamental for the biological activity of such complex entities. Given these requirements, disulfide rebridging represents a promising possibility since it allows protein modification as well as conservation of the tertiary structure. In this context, we have developed an original radiofluorinated dibromopyridazine dione prosthetic group for labeling of disulfide-containing biomolecules via rebridging. We employed it to radiolabel octreotide, a somatostatin analogue, and to radiolabel fragment antigen binding (Fab) targeting programmed death-ligand 1 (PD-L1), whose properties were then evaluated in vitro and in vivo by positron emission tomography (PET) imaging. We next extended our strategy to the radiolabeling of cetuximab, a monoclonal antibody, with various radiometals commonly used in PET imaging (zirconium-89, copper-64) by developing various rebridging molecules bearing the appropriate chelators. The stabilities of the radiolabeled antibody conjugates were assessed in biological conditions.

Anticancer drug delivery by focused ultrasound-mediated blood-brain/tumor barrier disruption for glioma therapy: From benchside to bedside.

The therapeutic management of gliomas remains particularly challenging. Brain tumors present multiple obstacles that make therapeutic innovation complex, mainly due to the presence of blood-tumor and blood-brain barriers (BTB and BBB, respectively) which prevent penetration of anticancer agents into the brain parenchyma. Focused ultrasound-mediated BBB disruption (FUS-BBBD) provides a physical method for non-invasive, local, and reversible BBB disruption. The safety of this technique has been demonstrated in small and large animal models. This approach promises to enhance drug delivery into the brain tumor and therefore to improve survival outcomes by repurposing existing drugs. Several clinical trials continue to be initiated in the last decade. In this review, we provide an overview of the rationale behind the use of FUS-BBBD in gliomas and summarize the preclinical studies investigating different approaches (free drugs, drug-loaded microbubbles and drug-loaded nanocarriers) in combination with this technology in in vivo glioma models. Furthermore, we discuss the current state of clinical trials and devices developed and review the challenges to overcome for clinical use of FUS-BBBD in glioma therapy.

Imaging the impact of blood-brain barrier disruption induced by focused ultrasound on P-glycoprotein function.

The P-glycoprotein (P-gp/ABCB1) is a major efflux transporter which impedes the brain delivery of many drugs across the blood-brain barrier (BBB). Focused ultrasound with microbubbles (FUS) enables BBB disruption, which immediate and delayed impact on P-gp function remains unclear. Positron emission tomography (PET) imaging using the radiolabeled substrate [11C]metoclopramide provides a sensitive and translational method to study P-gp function at the living BBB. A FUS protocol was devised in rats to induce a substantial and targeted disruption of the BBB in the left hemisphere. BBB disruption was confirmed by the Evan's Blue extravasation test or the minimally-invasive contrast-enhanced MRI. The expression of P-gp was measured 24 h or 48 h after FUS using immunostaining and fluorescence microscopy. The brain kinetics of [11C]metoclopramide was studied by PET at baseline, and both immediately or 24 h after FUS, with or without half-maximum P-gp inhibition (tariquidar 1 mg/kg). In each condition (n = 4-5 rats per group), brain exposure of [11C]metoclopramide was estimated as the area-under-the-curve (AUC) in regions corresponding to the sonicated volume in the left hemisphere, and the contralateral volume. Kinetic modeling was performed to estimate the uptake clearance ratio (R1) of [11C]metoclopramide in the sonicated volume relative to the contralateral volume. In the absence of FUS, half-maximum P-gp inhibition increased brain exposure (+135.0 ± 12.9%, p < 0.05) but did not impact R1 (p > 0.05). Immediately after FUS, BBB integrity was selectively disrupted in the left hemisphere without any detectable impact on the brain kinetics of [11C]metoclopramide compared with the baseline group (p > 0.05) or the contralateral volume (p > 0.05). 24 h after FUS, BBB integrity was fully restored while P-gp expression was maximally down-regulated (-45.0 ± 4.5%, p < 0.001) in the sonicated volume. This neither impacted AUC nor R1 in the FUS + 24 h group (p > 0.05). Only when P-gp was inhibited with tariquidar were the brain exposure (+130 ± 70%) and R1(+29.1 ± 15.4%) significantly increased in the FUS + 24 h/tariquidar group, relative to the baseline group (p < 0.001). We conclude that the brain kinetics of [11C]metoclopramide specifically depends on P-gp function rather than BBB integrity. Delayed FUS-induced down-regulation of P-gp function can be detected. Our results suggest that almost complete down-regulation is required to substantially enhance the brain delivery of P-gp substrates.

Sonoporation-assisted micelle delivery in subcutaneous glioma-bearing mice evaluated by PET/fluorescent bi-modal imaging.

Tumor-specific drug delivery is a major challenge for the pharmaceutical industry. Nanocarrier systems have been widely investigated to increase and control drug delivery to the heterogeneous tumor microenvironment. Classically, the uptake of nanocarriers by solid tumor tissues is mainly mediated by the enhanced permeability and retention effect (EPR). This EPR effect depends on the tumor type, its location, the physicochemical properties of the carriers, and the blood perfusion of the tumoral lesions. The main goal of this study was to evaluate in vivo tumor uptake of micelle carriers, assisted by microbubble/ultrasound sonoporation. Micelles were tracked using bi-modal imaging techniques to precisely localize both the nanocarrier and its payload. Micelles were loaded with a near infrared fluorophore and radiolabeled with zirconium-89. Their pharmacokinetics, biodistribution and passive tumor targeting properties were evaluated in a subcutaneous glioblastoma (U-87 MG) mouse model using optical and PET imaging. Finally, accumulation and diffusion into the tumor micro-environment was investigated under microbubble-assisted sonoporation, which helped homogenize the delivery of the micelles. The in vivo experiments showed a good correlation between optical and PET images and demonstrated the stability of the micelles in biological media, their high and long-term retention in the tumors and their clearance through the hepato-biliary pathway. This study demonstrates that bi-modal imaging techniques are powerful tools for the development of new nanocarriers and that sonoporation is a promising method to homogenize nanomedicine delivery to tumors.

ImmunoPET imaging-based pharmacokinetic profiles of an antibody and its Fab targeting endothelin A receptors on glioblastoma stem cells in a preclinical orthotopic model.

BACKGROUND: The resistance of glioblastoma stem cells (GSCs) to treatment is one of the causes of glioblastoma (GBM) recurrence. Endothelin A receptor (ETA) overexpression in GSCs constitutes an attractive biomarker for targeting this cell subpopulation, as illustrated by several clinical trials evaluating the therapeutic efficacy of endothelin receptor antagonists against GBM. In this context, we have designed an immunoPET radioligand combining the chimeric antibody targeting ETA, chimeric-Rendomab A63 (xiRA63), with 89Zr isotope and evaluated the abilities of xiRA63 and its Fab (ThioFab-xiRA63) to detect ETA+ tumors in a mouse model xenografted orthotopically with patient-derived Gli7 GSCs. RESULTS: Radioligands were intravenously injected and imaged over time by µPET-CT imaging. Tissue biodistribution and pharmacokinetic parameters were analyzed, highlighting the ability of [89Zr]Zr-xiRA63 to pass across the brain tumor barrier and achieve better tumor uptake than [89Zr]Zr-ThioFab-xiRA63. CONCLUSIONS: This study shows the high potential of [89Zr]Zr-xiRA63 in specifically targeting ETA+ tumors, thus raising the possibility of detecting and treating ETA+ GSCs, which could improve the management of GBM patients.

Design, Radiosynthesis, and Evaluation of New Fluorinated Analogs of MeDAS for Myelin PET Imaging.

Positron emission tomography (PET) imaging of the myelin sheath is a powerful tool to investigate multiple sclerosis, monitor its evolution, and support drug development. Radiotracers based on N,N-dimethylaminostilbene (MeDAS) fluorinated analogs have been designed for myelin PET imaging but were never translated to humans. We have synthesized three original fluorinated analogs of MeDAS with low metabolic rates for which binding to myelin in a healthy rat brain was demonstrated by fluorescence microscopy. A tosyl precursor was synthesized for the lead compound PEGMeDAS and automated fluorine-18 radiolabeling afforded [18F]PEGMeDAS in 25 ± 5% radiochemical yield and 102 ± 15 GBq/µmol molar activity. Biodistribution in healthy rats demonstrated the brain penetration with low penetration of radiometabolites. However, E to Z isomerization observed in plasma hampers further investigations of this family of molecules and requires complementary data on the in vivo behavior of the Z isomer.

Refining the delivery and therapeutic efficacy of cetuximab using focused ultrasound in a mouse model of glioblastoma: An 89Zr-cetuximab immunoPET study.

INTRODUCTION: Glioblastoma (GBM) is the most common and deadly form of primary brain tumor. Between 30 % and 60 % of GBM are characterized by overexpression of the Epidermal Growth Factor Receptor (EGFR). The anti-EGFR antibody Cetuximab (CTX) showed a favorable effect for EGFR+ colorectal cancer but failed to demonstrate efficacy for GBM. Insufficient CTX passage through the blood-brain barrier (BBB) and the blood-tumor barrier (BTB) is assumed to be the primary determinant of the limited efficacy of this immunotherapy. OBJECTIVE: Using positron emission tomography (PET) imaging, we have previously demonstrated that focused ultrasound (FUS) combined with microbubbles (µB) allowed significant and persistent delivery of CTX across the BBB in healthy mice. In the current study, we investigated by PET imaging the combination impact of CTX and FUS on orthotopic GBM preclinical model. METHODS: After radiolabeling CTX with the long half-life isotope 89Zr, PET images have been acquired overtime in mice bearing U251 (EGFR+) with or without FUS treatment. Autoradiography combined with immunofluorescence staining was used to corroborate CTX delivery with EGFR expression. A survival study was conducted simultaneously to evaluate the therapeutic benefit of repeated CTX monotherapy associated or not with FUS. RESULTS: Ex vivo analysis confirmed that FUS enhanced and homogenized the delivery of CTX into all the FUS exposure area, including the tumor and the contralateral hemisphere at the early-time-point. Interestingly, FUS did not improve the long-term accumulation and retention of CTX in the tumor compared with the control group (no FUS). No significant difference in the CTX treatment efficacy, determined by the survival between FUS and non-FUS groups, has been either observed. This result is consistent with the absence of change in the CTX distribution through the GBM tumor after FUS. The neuroinflammation induced by FUS is not significant enough to explain the failure of the CTX delivery improvement. CONCLUSION: All together, these data suggest that the role of FUS combined with µB on the CTX distribution, even after multiple therapeutic sessions and glial cell activation is insufficient to improve survival of GBM mice compared with CTX treatment alone in this model.

Impact of Cytochrome Induction or Inhibition on the Plasma and Brain Kinetics of [11C]metoclopramide, a PET Probe for P-Glycoprotein Function at the Blood-Brain Barrier.

[11C]metoclopramide PET imaging provides a sensitive and translational tool to explore P-glycoprotein (P-gp) function at the blood-brain barrier (BBB). Patients with neurological diseases are often treated with cytochrome (CYP) modulators which may impact the plasma and brain kinetics of [11C]metoclopramide. The impact of the CYP inducer carbamazepine or the CYP inhibitor ritonavir on the brain and plasma kinetics of [11C]metoclopramide was investigated in rats. Data obtained in a control group were compared with groups that were either orally pretreated with carbamazepine (45 mg/kg twice a day for 7 days before PET) or ritonavir (20 mg/kg, 3 h before PET) (n = 4 per condition). Kinetic modelling was performed to estimate the brain penetration (VT) of [11C]metoclopramide. CYP induction or inhibition had negligible impact on the plasma kinetics and metabolism of [11C]metoclopramide. Moreover, carbamazepine neither impacted the brain kinetics nor VT of [11C]metoclopramide (p > 0.05). However, ritonavir significantly increased VT (p < 0.001), apparently behaving as an inhibitor of P-gp at the BBB. Our data suggest that treatment with potent CYP inducers such as carbamazepine does not bias the estimation of P-gp function at the BBB with [11C]metoclopramide PET. This supports further use of [11C]metoclopramide for studies in animals and patients treated with CYP inducers.

TSPO PET Imaging as a Potent Non-Invasive Biomarker for Diffuse Intrinsic Pontine Glioma in a Patient-Derived Orthotopic Rat Model.

Diffuse intrinsic pontine gliomas (DIPG), the first cause of cerebral pediatric cancer death, will greatly benefit from specific and non-invasive biomarkers for patient follow-up and monitoring of drug efficacy. Since biopsies are challenging for brain tumors, molecular imaging may be a technique of choice to target and follow tumor evolution. So far, MR remains the imaging technique of reference for DIPG, although it often fails to define the extent of tumors, an essential parameter for therapeutic efficacy assessment. Thanks to its high sensitivity, positron emission tomography (PET) offers a unique way to target specific biomarkers in vivo. We demonstrated in a patient-derived orthotopic xenograft (PDOX) model in the rat that the translocator protein of 18 kDa (TSPO) may be a promising biomarker for monitoring DIPG tumors. We studied the distribution of 18F-DPA-714, a TSPO radioligand, in rats inoculated with HSJD-DIPG-007 cells. The primary DIPG human cell line HSJD-DIPG-007 highly represents this pediatric tumor, displaying the most prevalent DIPG mutations, H3F3A (K27M) and ACVR1 (R206H). Kinetic modeling and parametric imaging using the brain 18F-DPA-714 PET data enabled specific delineation of the DIPG tumor area, which is crucial for radiotherapy dose management.

Pharmacokinetics derived from PET imaging of inspiring radio-enhancer platinum nanoparticles.

Personalized medicine approach in radiotherapy requires the delivery of precise dose to the tumor. The concept is to increase the effectiveness of radiotherapy while sparing the surrounding heathy tissue. This can be achieved by the use of high-Z metal-based nanoparticles (NPs) as radio-enhancers and PET imaging for mapping NPs distribution to guide the irradiation. In the present study, radio-enhancing platinum NPs were radiolabeled and imaged to assess their pharmacokinetics over time. PET imaging of these NPs revealed high enhanced permeation and retention effect. The maximal tumor accumulation (4.8 ± 0.8 %ID/cc) was observed at 24 h post-injection along with persistent accumulation of the NPs, especially at the tumor ring, even after several days. These properties positively suggest the potential clinical use of these NPs.

Impact of Radiolabeling Strategies on the Pharmacokinetics and Distribution of an Anti-PD-L1 PET Ligand.

Molecular imaging with PET offers an alternative method to quantify programmed-death-ligand 1 (PD-L1) to accurately select patients for immunotherapies. More and more clinical and preclinical trials involve radiolabeling of antibody fragments for their desirably fast clearance and high tumor penetration. As the radiolabeling strategy can significantly impact pharmacokinetics and biodistribution, we explored in this work a site-specific radiofluorination strategy on an anti-PD-L1 fragment antigen-binding (Fab) and compared the pharmacokinetic and biodistribution properties with the same Fab labeled using stochastic radiolabeling chemistry. We applied an enzymatic bioconjugation mediated by a variant of the lipoic acid ligase (LplA) that promotes the formation of an amide bond between a short peptide cloned onto the C terminus of the Fab. A synthetic analogue of the enzyme natural substrate, lipoic acid, was radiolabeled with fluorine-18 for site-specific conjugation by LplA. We compared the biodistribution of the site-specifically labeled Fab with a stochastically labeled Fab on lysine side chains in tumor-bearing mice. The two methods of fluorination demonstrate a comparable whole-body biodistribution. The 89Zr-labeled Fab had different biodistribution compared to either 18F-labeled Fab. We attribute the difference to [89Zr] metabolism. Fab-LAP-[18F]FPyOctA therefore reflects better the true pharmacokinetic profile of the Fab.

Pharmacokinetic Imaging Using 99mTc-Mebrofenin to Untangle the Pattern of Hepatocyte Transporter Disruptions Induced by Endotoxemia in Rats.

Endotoxemia-induced inflammation may impact the activity of hepatocyte transporters, which control the hepatobiliary elimination of drugs and bile acids. 99mTc-mebrofenin is a non-metabolized substrate of transporters expressed at the different poles of hepatocytes. 99mTc-mebrofenin imaging was performed in rats after the injection of lipopolysaccharide (LPS). Changes in transporter expression were assessed using quantitative polymerase chain reaction of resected liver samples. Moreover, the particular impact of pharmacokinetic drug-drug interactions in the context of endotoxemia was investigated using rifampicin (40 mg/kg), a potent inhibitor of hepatocyte transporters. LPS increased 99mTc-mebrofenin exposure in the liver (1.7 ± 0.4-fold). Kinetic modeling revealed that endotoxemia did not impact the blood-to-liver uptake of 99mTc-mebrofenin, which is mediated by organic anion-transporting polypeptide (Oatp) transporters. However, liver-to-bile and liver-to-blood efflux rates were dramatically decreased, leading to liver accumulation. The transcriptomic profile of hepatocyte transporters consistently showed a downregulation of multidrug resistance-associated proteins 2 and 3 (Mrp2 and Mrp3), which mediate the canalicular and sinusoidal efflux of 99mTc-mebrofenin in hepatocytes, respectively. Rifampicin effectively blocked both the Oatp-mediated influx and the Mrp2/3-related efflux of 99mTc-mebrofenin. The additive impact of endotoxemia and rifampicin led to a 3.0 ± 1.3-fold increase in blood exposure compared with healthy non-treated animals. 99mTc-mebrofenin imaging is useful to investigate disease-associated change in hepatocyte transporter function.

Optimizing Immuno-PET Imaging of Tumor PD-L1 Expression: Pharmacokinetic, Biodistribution, and Dosimetric Comparisons of 89Zr-Labeled Anti-PD-L1 Antibody Formats.

PET imaging of programmed cell death ligand 1 (PD-L1) may help to noninvasively predict and monitor responses to anti-programmed cell death 1/anti-PD-L1 immunotherapies. In this study, we compared the imaging characteristics of 3 radioligands derived from the anti-PD-L1 IgG1 complement 4 (C4). In addition to the IgG C4, we produced a fragment antigen-binding (Fab) C4, as well as a double-mutant IgG C4 (H310A/H435Q) with minimal affinity for the murine neonatal Fc receptor. Methods: The pharmacokinetics, biodistribution, and dosimetry of the 3 89Zr-labeled C4 ligands were compared by longitudinal PET/CT imaging in nude mice bearing subcutaneous human non-small cell lung cancer xenografts with positive (H1975 model) or negative (A549 model) endogenous PD-L1 expression. Results: The C4 radioligands substantially accumulated in PD-L1-positive tumors but not in PD-L1-negative tumors or in blocked PD-L1-positive tumors, confirming their PD-L1-specific tumor targeting. 89Zr-Fab C4 and 89Zr-IgG C4 (H310A/H435Q) were rapidly eliminated compared with 89Zr-IgG C4. Consequently, maximal tumor-to-muscle ratios were obtained earlier, at 4 h after injection for 89Zr-Fab C4 (ratio, ∼6) and 24 h after injection for 89Zr-IgG C4 (H310A/H435Q) (ratio, ∼9), versus 48 h after injection for 89Zr-IgG C4 (ratio, ∼8). Background activity in nontumor tissues was low, except for high kidney retention of 89Zr-Fab C4 and persistent liver accumulation of 89Zr-IgG C4 (H310A/H435Q) compared with 89Zr-IgG C4. Dosimetry estimates suggested that the C4 radioligands would yield organ-absorbed doses tolerable for repeated clinical PET imaging studies. Conclusion: This study highlights the potential of designing radioligands with shorter pharmacokinetics for PD-L1 immuno-PET imaging in a preclinical model and encourages further clinical translation of such radioligands.

Comparative vulnerability of PET radioligands to partial inhibition of P-glycoprotein at the blood-brain barrier: A criterion of choice?

Only partial deficiency/inhibition of P-glycoprotein (P-gp, ABCB1) function at the blood-brain barrier (BBB) is likely to occur in pathophysiological situations or drug-drug interactions. This raises questions regarding the sensitivity of available PET imaging probes to detect moderate changes in P-gp function at the living BBB. In vitro, the half-maximum inhibitory concentration (IC50) of the potent P-gp inhibitor tariquidar in P-gp-overexpressing cells was significantly different using either [11C]verapamil (44 nM), [11C]N-desmethyl-loperamide (19 nM) or [11C]metoclopramide (4 nM) as substrate probes. In vivo PET imaging in rats showed that the half-maximum inhibition of P-gp-mediated efflux of [11C]metoclopramide, achieved using 1 mg/kg tariquidar (in vivo IC50 = 82 nM in plasma), increased brain exposure by 2.1-fold for [11C]metoclopramide (p < 0.05, n = 4) and 2.4-fold for [11C]verapamil (p < 0.05, n = 4), whereby cerebral uptake of the "avid" substrate [11C]N-desmethyl-loperamide was unaffected (p > 0.05, n = 4). This comparative study points to differences in the "vulnerability" to P-gp inhibition among radiolabeled substrates, which were apparently unrelated to their "avidity" (maximal response to P-gp inhibition). Herein, we advocate that partial inhibition of transporter function, in addition to complete inhibition, should be a primary criterion of evaluation regarding the sensitivity of radiolabeled substrates to detect moderate but physiologically-relevant changes in transporter function in vivo.

Quantitative Tissue Pharmacokinetics and EPR Effect of AGuIX Nanoparticles: A Multimodal Imaging Study in an Orthotopic Glioblastoma Rat Model and Healthy Macaque.

AGuIX are emerging radiosensitizing nanoparticles (NPs) for precision radiotherapy (RT) under clinical evaluation (Phase 2). Despite being accompanied by MRI thanks to the presence of gadolinium (Gd) at its surface, more sensitive and quantifiable imaging technique should further leverage the full potential of this technology. In this study, it is shown that 89 Zr can be labeled on such NPs directly for positron emission tomography (PET) imaging with a simple and scalable method. The stability of such complexes is remarkable in vitro and in vivo. Using a glioblastoma orthotopic rat model, it is shown that injected 89 Zr-AGuIX is detectable inside the tumor for at least 1 week. Interestingly, the particles seem to efficiently infiltrate the tumor even in necrotic areas, which places great hope for the treatment of radioresistant tumor. Lastly, the first PET/MR whole-body imaging is performed in non-human primate (NHP), which further demonstrates the translational potential of these bimodal NP.