Brain delivery enabled by transient blood-brain barrier disruption induced by regadenoson: a PET imaging study.

BACKGROUND: Regadenoson, an agonist of adenosine A2 receptors, enables transient blood-brain barrier (BBB) disruption. The relevance of regadenoson as a pharmacological strategy for brain delivery was investigated using in vivo PET imaging in rats. RESEARCH DESIGN AND METHODS: Kinetic modeling of brain PET data was performed to estimate the impact of regadenoson (0.05 mg.kg-1, i.v.) on BBB permeation compared with control rats (n = 4-6 per group). Three radiolabeled compounds of different sizes, which do not cross the intact BBB, were tested. RESULTS: Regadenoson significantly increased the BBB penetration (+116 ± 13%, p < 0.001) of [18F]2-deoxy-2-fluoro-D-sorbitol ([18F]FDS, MW = 183 Da), a small-molecule marker of BBB permeability. The magnitude of the effect was different across brain regions, with a maximum increase in the striatum. Recovery of BBB integrity was observed 30 min after regadenoson injection. Regadenoson also increased the brain penetration (+72 ± 45%, p < 0.05) of a radiolabeled nanoparticle [89Zr]AGuIX (MW = 9 kDa). However, the brain kinetics of a monoclonal antibody ([89Zr]mAb, MW = 150 kDa) remained unchanged (p > 0.05). CONCLUSIONS: PET imaging showed the features and limitations of BBB disruption induced by regadenoson in terms of extent, regional distribution, and reversibility. Nevertheless, regadenoson enables the brain delivery of small molecules or nanoparticles in rats.

Brain Glucose Metabolism as a Readout of the Central Nervous System Impact of Cigarette Smoke Exposure and Withdrawal and the Effects of NFL-101, as an Immune-Based Drug Candidate for Smoking Cessation Therapy.

Neuroimaging biomarkers are needed to investigate the impact of smoking withdrawal on brain function. NFL-101 is a denicotinized aqueous extract of tobacco leaves currently investigated as an immune-based smoking cessation therapy in humans. However, the immune response to NFL-101 and its ability to induce significant changes in brain function remain to be demonstrated. Brain glucose metabolism was investigated using [18F]fluoro-deoxy-glucose ([18F]FDG) PET imaging in a mouse model of cigarette smoke exposure (CSE, 4-week whole-body inhalation, twice daily). Compared with control animals, the relative uptake of [18F]FDG in CSE mice was decreased in the thalamus and brain stem (p < 0.001, n = 14 per group) and increased in the hippocampus, cortex, cerebellum, and olfactory bulb (p < 0.001). NFL-101 induced a humoral immune response (specific IgGs) in mice and activated human natural-killer lymphocytes in vitro. In CSE mice, but not in control mice, single-dose NFL-101 significantly increased [18F]FDG uptake in the thalamus (p < 0.01), thus restoring normal brain glucose metabolism after 2-day withdrawal in this nicotinic receptor-rich region. In tobacco research, [18F]FDG PET imaging provides a quantitative method to evaluate changes in the brain function associated with the withdrawal phase. This method also showed the CNS effects of NFL-101, with translational perspectives for future clinical evaluation in smokers.

Successful Prediction of Human Hepatic Concentrations of Transported Drugs Using the Proteomics-Informed Relative Expression Factor Approach.

Tissue drug concentrations determine the efficacy and toxicity of drugs. When a drug is the substrate of transporters that are present at the blood:tissue barrier, the steady-state unbound tissue drug concentrations cannot be predicted from their corresponding plasma concentrations. To accurately predict transporter-modulated tissue drug concentrations, all clearances (CLs) mediating the drug's entry and exit (including metabolism) from the tissue must be accurately predicted. Because primary cells of most tissues are not available, we have proposed an alternative approach to predict such CLs, that is the use of transporter-expressing cells/vesicles (TECs/TEVs) and relative expression factor (REF). The REF represents the abundance of the relevant transporters in the tissue vs. in the TECs/TEVs. Here, we determined the transporter-based intrinsic CL of glyburide (GLB) and pitavastatin (PTV) in OATP1B1, OATP1B3, OATP2B1, and NTCP-expressing cells and MRP3-, BCRP-, P-gp-, and MRP2-expressing vesicles and scaled these CLs to in vivo using REF. These predictions fell within a priori set twofold range of the hepatobiliary CLs of GLB and PTV, estimated from their hepatic positron emission tomography imaging data: 272.3 and 607.8 mL/min for in vivo hepatic sinusoidal uptake CL, 47.8 and 17.4 mL/min for sinusoidal efflux CL, and 0 and 4.20 mL/min for biliary efflux CL, respectively. Moreover, their predicted hepatic concentrations (area under the hepatic concentration-time curve (AUC) and maximum plasma concentration (Cmax )), fell within twofold of their mean observed data. These data, together with our previous findings, confirm that the REF approach can successfully predict transporter-based drug CLs and tissue concentrations to enhance success in drug development.

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.

Dissimilar Effect of P-Glycoprotein and Breast Cancer Resistance Protein Inhibition on the Distribution of Erlotinib to the Retina and Brain in Humans and Mice.

P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) are two ATP-binding cassette efflux transporters that are coexpressed at the human blood-brain barrier (BBB) and blood-retina barrier (BRB). While pharmacological inhibition of P-gp and/or BCRP results in increased brain distribution of dual P-gp/BCRP substrate drugs, such as the tyrosine kinase inhibitor erlotinib, the effect of P-gp and/or BCRP inhibition on the retinal distribution of such drugs has hardly been investigated. In this study, we used positron emission tomography (PET) imaging to assess the effect of transporter inhibition on the distribution of [11C]erlotinib to the human retina and brain. Twenty two healthy volunteers underwent two PET scans after intravenous (i.v.) injection of a microdose (<5 µg) of [11C]erlotinib, a baseline scan, and a second scan either with concurrent i.v. infusion of tariquidar to inhibit P-gp (n = 5) or after oral intake of single ascending doses of erlotinib (300 mg, 650 mg, or 1000 mg, n = 17) to saturate erlotinib transport. In addition, transport of [3H]erlotinib to the retina and brain was assessed in mice by in situ carotid perfusion under various drug transporter inhibition settings. In comparison to the baseline PET scan, coadministration of tariquidar or erlotinib led to a significant decrease of [11C]erlotinib total volume of distribution (VT) in the human retina by -25 ± 8% (p ≤ 0.05) and -41 ± 16% (p ≤ 0.001), respectively. In contrast, erlotinib intake led to a significant increase in [11C]erlotinib VT in the human brain (+20 ± 16%, p ≤ 0.001), while administration of tariquidar did not result in any significant changes. In situ carotid perfusion experiments showed that both P-gp and BCRP significantly limit the distribution of erlotinib to the mouse retina and brain but revealed a similar discordant effect at the mouse BRB and BBB following co-perfusion with tariquidar and erlotinib as in humans. Co-perfusion with prototypical inhibitors of solute carrier transporters did not reveal a significant contribution of organic cation transporters (e.g., OCTs and OCTNs) and organic anion-transporting polypeptides (e.g., OATP2B1) to the retinal and cerebral distribution of erlotinib. In conclusion, we observed a dissimilar effect after P-gp and/or BCRP inhibition on the retinal and cerebral distribution of [11C]erlotinib. The exact mechanism for this discrepancy remains unclear but may be related to the function of an unidentified erlotinib uptake carrier sensitive to tariquidar inhibition at the BRB. Our study highlights the great potential of PET to study drug distribution to the human retina and to assess the functional impact of membrane transporters on ocular drug distribution.

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.

Isotopic Radiolabeling of Crizotinib with Fluorine-18 for In Vivo Pet Imaging.

Crizotinib is a tyrosine kinase inhibitor approved for the treatment of non-small-cell lung cancer, but it is inefficient on brain metastases. Crizotinib is a substrate of the P-glycoprotein, and non-invasive nuclear imaging can be used to assess the brain penetration of crizotinib. Positron emission tomography (PET) imaging using fluorine-18-labeled crizotinib would be a powerful tool for investigating new strategies to enhance the brain distribution of crizotinib. We have synthesized a spirocyclic hypervalent iodine precursor for the isotopic labeling of crizotinib in a 2.4% yield. Because crizotinib is an enantiomerically pure drug, a chiral separation was performed to afford the (R)-precursor. A two-step radiolabeling process was optimized and automated using the racemic precursor to afford [18F](R,S)-crizotinib in 15 ± 2 radiochemical yield and 103 ± 18 GBq/µmol molar activity. The same radiolabeling process was applied to the (R)-precursor to afford [18F](R)-crizotinib with comparable results. As a proof-of-concept, PET was realized in a single non-human primate to demonstrate the feasibility of [18F](R)-crizotinib in in vivo imaging. Whole-body PET highlighted the elimination routes of crizotinib with negligible penetration in the brain (SUVmean = 0.1). This proof-of-concept paves the way for further studies using [18F](R)-crizotinib to enhance its brain penetration depending on the P-glycoprotein function.

Regulation of P-glycoprotein and Breast Cancer Resistance Protein Expression Induced by Focused Ultrasound-Mediated Blood-Brain Barrier Disruption: A Pilot Study.

The blood-brain barrier (BBB) controls brain homeostasis; it is formed by vascular endothelial cells that are physically connected by tight junctions (TJs). The BBB expresses efflux transporters such as P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), which limit the passage of substrate molecules from blood circulation to the brain. Focused ultrasound (FUS) with microbubbles can create a local and reversible detachment of the TJs. However, very little is known about the effect of FUS on the expression of efflux transporters. We investigated the in vivo effects of moderate acoustic pressures on both P-gp and BCRP expression for up to two weeks after sonication. Magnetic resonance-guided FUS was applied in the striatum of 12 rats. P-gp and BCRP expression were determined by immunohistochemistry at 1, 3, 7, and 14 days postFUS. Our results indicate that FUS-induced BBB opening is capable of (i) decreasing P-gp expression up to 3 days after sonication in both the treated and in the contralateral brain regions and is capable of (ii) overexpressing BCRP up to 7 days after FUS in the sonicated regions only. Our findings may help improve FUS-aided drug delivery strategies by considering both the mechanical effect on the TJs and the regulation of P-gp and BCRP.

[11C]glyburide PET imaging for quantitative determination of the importance of Organic Anion-Transporting Polypeptide transporter function in the human liver and whole-body.

Organic Anion-Transporting Polypeptides (OATPs) are known to control the liver uptake of many drugs. Non-hepatic expression of OATPs has been reported although functional importance for whole-body pharmacokinetics (WBPK) remains unknown. Glyburide is a well described substrate of several hepatic and non-hepatic OATPs. Dynamic whole-body positron emission tomography (DWB-PET) with [11C]glyburide was performed in humans for determination of the importance of OATPs for liver uptake and WBPK. Seven healthy male subjects (24.7 ± 3.2 years) underwent [11C]glyburide PET scan with concomitant blood sampling. All subjects underwent baseline [11C]glyburide PET scan. Five subjects underwent a subsequent [11C]glyburide PET scan after infusion of the potent OATP inhibitor rifampicin (9 mg/kg i.v.). The transfer constant (kuptake) of [11C]glyburide from blood to the liver was estimated using the integration plot method. The tissue exposure of [11C]glyburide was described by the area under the time-activity curve (AUC) and corresponding tissue/blood ratio (AUCR). [11C]glyburide was barely metabolized in both the baseline and rifampicin conditions. Parent (unmetabolized) [11C]glyburide accounted for > 90 % of the plasma radioactivity. Excellent correlation was found between radioactive counting in arterial blood samples and in the image-derived input function, in both the baseline and rifampicin conditions (R2 = 97.9 %, p < 0.01). [11C]glyburide predominantly accumulated in the liver. Rifampicin decreased liver kuptake by 77.3 ± 7.3 %, which increased exposure in blood, kidneys, spleen, myocardium and brain (p < 0.05). No significant change in AUCR was observed except in the liver (p < 0.01). [11C]glyburide benefits from metabolic stability and high sensitivity to OATP inhibition which enables quantitative determination of OATP function. DWB-PET suggests negligible role for non-hepatic OATPs in controlling the tissue distribution of [11C]glyburide.

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.

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.

ABCB1 and ABCG2 Together Limit the Distribution of ABCB1/ABCG2 Substrates to the Human Retina and the ABCG2 Single Nucleotide Polymorphism Q141K (c.421C> A) May Lead to Increased Drug Exposure.

The widely expressed and poly-specific ABC transporters breast cancer resistance protein (ABCG2) and P-glycoprotein (ABCB1) are co-localized at the blood-brain barrier (BBB) and have shown to limit the brain distribution of several clinically used ABCB1/ABCG2 substrate drugs. It is currently not known to which extent these transporters, which are also expressed at the blood-retinal barrier (BRB), may limit drug distribution to the human eye and whether the ABCG2 reduced-function single-nucleotide polymorphism (SNP) Q141K (c.421C > A) has an impact on retinal drug distribution. Ten healthy male volunteers (five subjects with the c.421CC and c.421CA genotype, respectively) underwent two consecutive positron emission tomography (PET) scans after intravenous injection of the model ABCB1/ABCG2 substrate [11C]tariquidar. The second PET scan was performed with concurrent intravenous infusion of unlabelled tariquidar to inhibit ABCB1 in order to specifically reveal ABCG2 function.In response to ABCB1 inhibition with unlabelled tariquidar, ABCG2 c.421C > A genotype carriers showed significant increases (as compared to the baseline scan) in retinal radiotracer influx K 1 (+62 ± 57%, p = 0.043) and volume of distribution V T (+86 ± 131%, p = 0.043), but no significant changes were observed in subjects with the c.421C > C genotype. Our results provide the first evidence that ABCB1 and ABCG2 may together limit the distribution of systemically administered ABCB1/ABCG2 substrate drugs to the human retina. Functional redundancy between ABCB1 and ABCG2 appears to be compromised in carriers of the c.421C > A SNP who may therefore be more susceptible to transporter-mediated drug-drug interactions at the BRB than non-carriers.

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.

Imaging-Based Characterization of a Slco2b1(-/-) Mouse Model Using [11C]Erlotinib and [99mTc]Mebrofenin as Probe Substrates.

Organic anion-transporting polypeptide 2B1 (OATP2B1) is co-localized with OATP1B1 and OATP1B3 in the basolateral hepatocyte membrane, where it is thought to contribute to the hepatic uptake of drugs. We characterized a novel Slco2b1(-/-) mouse model using positron emission tomography (PET) imaging with [11C]erlotinib (a putative OATP2B1-selective substrate) and planar scintigraphic imaging with [99mTc]mebrofenin (an OATP1B1/1B3 substrate, which is not transported by OATP2B1). Dynamic 40-min scans were performed after intravenous injection of either [11C]erlotinib or [99mTc]mebrofenin in wild-type and Slco2b1(-/-) mice. A pharmacokinetic model was used to estimate the hepatic uptake clearance (CL1) and the rate constants for transfer of radioactivity from the liver to the blood (k2) and excreted bile (k3). CL1 was significantly reduced in Slco2b1(-/-) mice for both radiotracers (p < 0.05), and k2 was significantly lower (p < 0.01) in Slco2b1(-/-) mice for [11C]erlotinib, but not for [99mTc]mebrofenin. Our data support previous evidence that OATP transporters may contribute to the hepatic uptake of [11C]erlotinib. However, the decreased hepatic uptake of the OATP1B1/1B3 substrate [99mTc]mebrofenin in Slco2b1(-/-) mice questions the utility of this mouse model to assess the relative contribution of OATP2B1 to the liver uptake of drugs which are substrates of multiple OATPs.

Repurposing 99mTc-Mebrofenin as a Probe for Molecular Imaging of Hepatocyte Transporters.

Hepatocyte transporters control the hepatobiliary elimination of many drugs, metabolites, and endogenous substances. Hepatocyte transporter function is altered in several pathophysiologic situations and can be modulated by certain drugs, with a potential impact for pharmacokinetics and drug-induced liver injury. The development of substrate probes with optimal properties for selective and quantitative imaging of hepatic transporters remains a challenge. 99mTc-mebrofenin has been used for decades for hepatobiliary scintigraphy, but the specific transporters controlling its liver kinetics have not been characterized until recently. These include sinusoidal influx transporters (organic anion-transporting polypeptides) responsible for hepatic uptake of 99mTc-mebrofenin, and efflux transporters (multidrug resistance-associated proteins) mediating its canalicular (liver-to-bile) and sinusoidal (liver-to-blood) excretion. Pharmacokinetic modeling enables molecular interpretation of 99mTc-mebrofenin scintigraphy data, thus offering a widely available translational method to investigate transporter-mediated drug-drug interactions in vivo. 99mTc-mebrofenin allows for phenotyping transporter function at the different poles of hepatocytes as a biomarker of liver function.

Human Biodistribution and Radiation Dosimetry of the P-Glycoprotein Radiotracer [11C]Metoclopramide.

PURPOSE: To assess in healthy volunteers the whole-body distribution and dosimetry of [11C]metoclopramide, a new positron emission tomography (PET) tracer to measure P-glycoprotein activity at the blood-brain barrier. PROCEDURES: Ten healthy volunteers (five women, five men) were intravenously injected with 387 ± 49 MBq of [11C]metoclopramide after low dose CT scans and were then imaged by whole-body PET scans from head to upper thigh over approximately 70 min. Ten source organs (brain, thyroid gland, right lung, myocardium, liver, gall bladder, left kidney, red bone marrow, muscle and the contents of the urinary bladder) were manually delineated on whole-body images. Absorbed doses were calculated with QDOSE (ABX-CRO) using the integrated IDAC-Dose 2.1 module. RESULTS: The majority of the administered dose of [11C]metoclopramide was taken up into the liver followed by urinary excretion and, to a smaller extent, biliary excretion of radioactivity. The mean effective dose of [11C]metoclopramide was 1.69 ± 0.26 µSv/MBq for female subjects and 1.55 ± 0.07 µSv/MBq for male subjects. The two organs receiving the highest radiation doses were the urinary bladder (10.81 ± 0.23 µGy/MBq and 8.78 ± 0.89 µGy/MBq) and the liver (6.80 ± 0.78 µGy/MBq and 4.91 ± 0.74 µGy/MBq) for female and male subjects, respectively. CONCLUSIONS: [11C]Metoclopramide showed predominantly renal excretion, and is safe and well tolerated in healthy adults. The effective dose of [11C]metoclopramide was comparable to other 11C-labeled PET tracers.

Complete inhibition of ABCB1 and ABCG2 at the blood-brain barrier by co-infusion of erlotinib and tariquidar to improve brain delivery of the model ABCB1/ABCG2 substrate [11C]erlotinib.

P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2) restrict at the blood-brain barrier (BBB) the brain distribution of the majority of currently known molecularly targeted anticancer drugs. To improve brain delivery of dual ABCB1/ABCG2 substrates, both ABCB1 and ABCG2 need to be inhibited simultaneously at the BBB. We examined the feasibility of simultaneous ABCB1/ABCG2 inhibition with i.v. co-infusion of erlotinib and tariquidar by studying brain distribution of the model ABCB1/ABCG2 substrate [11C]erlotinib in mice and rhesus macaques with PET. Tolerability of the erlotinib/tariquidar combination was assessed in human embryonic stem cell-derived cerebral organoids. In mice and macaques, baseline brain distribution of [11C]erlotinib was low (brain distribution volume, VT,brain < 0.3 mL/cm3). Co-infusion of erlotinib and tariquidar increased VT,brain in mice by 3.0-fold and in macaques by 3.4- to 5.0-fold, while infusion of erlotinib alone or tariquidar alone led to less pronounced VT,brain increases in both species. Treatment of cerebral organoids with erlotinib/tariquidar led to an induction of Caspase-3-dependent apoptosis. Co-infusion of erlotinib/tariquidar may potentially allow for complete ABCB1/ABCG2 inhibition at the BBB, while simultaneously achieving brain-targeted EGFR inhibition. Our protocol may be applicable to enhance brain delivery of molecularly targeted anticancer drugs for a more effective treatment of brain tumors.

An original radio-biomimetic approach to synthesize radiometabolites for PET imaging.

To fully exploit the potential of positron emission tomography (PET) imaging to assess drug distribution and pharmacokinetics in the central nervous system, the contribution of radiometabolites to the PET signal has to be determined for correct interpretation of data. However, radiosynthesis and extensive study of radiometabolites are rarely investigated and very challenging for complex drugs. Therefore, an original radio-biomimetic (RBM) approach was developed to rapidly synthesize radiometabolites and non-invasively investigate their kinetics with PET imaging. This method enabled the challenging radiosynthesis of [11C]nor-buprenorphine ([11C]nor-BUP), the main metabolite of buprenorphine (BUP) which has been identified as a substrate of the P-glycoprotein (P-gp) transport function at the blood-brain barrier (BBB). Biomimetic conditions using cytochromes P450 3A4 to convert BUP into nor-BUP were optimized taking into account the short half-life of carbon-11 (t1/2 = 20.4 min). Those conditions afforded 32% of conversion within 20 min and were applied to the biomimetic radiosynthesis of [11C]nor-BUP from [11C]BUP. Automated radiosynthesis of [11C]BUP according to a procedure described in the literature followed by optimized RBM conditions afforded [11C]nor-BUP in 1.5% decay-corrected radiochemical yield within 90 min and 90 ± 15 GBq/µmol molar activity. HPLC quality control showed chemical and radiochemical purities above 98%. To demonstrate the applicability of the RBM approach to preclinical studies, brain PET images in rats showed a drastic lower uptake of [11C]nor-BUP (0.067 ± 0.023%ID/cm-3) compared to [11C]BUP (0.436 ± 0.054%ID/cm-3). P-gp inhibition using Tariquidar increased the brain uptake of [11C]nor-BUP (0.557 ± 0.077%ID/cm-3).

Impact of blood-brain barrier permeabilization induced by ultrasound associated to microbubbles on the brain delivery and kinetics of cetuximab: An immunoPET study using 89Zr-cetuximab.

Epidermal growth factor receptor (EGFR), involved in cell proliferation and migration, is overexpressed in ~50% of glioblastomas. Anti-EGFR based strategies using monoclonal antibodies (mAb) such as cetuximab (CTX) have been proposed for central nervous system (CNS) cancer therapy. However, the blood-brain barrier (BBB) drastically restricts their brain penetration which limits their efficacy for the treatment of glioblastomas. Herein, a longitudinal PET imaging study was performed to assess the relevance and the impact of focused ultrasound (FUS)-mediated BBB permeabilization on the brain exposure to the anti-EGFR mAb CTX over time. For this purpose, FUS permeabilization process with microbubbles was applied on intact BBB mouse brain before the injection of 89Zr-labeled CTX for longitudinal imaging monitoring. FUS induced a dramatic increase in mAb penetration to the brain, 2 times higher compared to the intact BBB. The transfer of 89Zr-CTX from blood to the brain was rendered significant by FUS (kuptake = 1.3 ± 0.23 min-1 with FUS versus kuptake = 0 ± 0.006 min-1 without FUS). FUS allowed significant and prolonged exposure to mAb in the brain parenchyma. This study confirms the potential of FUS as a target delivery method for mAb in CNS.

Validation of Pharmacological Protocols for Targeted Inhibition of Canalicular MRP2 Activity in Hepatocytes Using [99mTc]mebrofenin Imaging in Rats.

The multidrug resistance-associated protein 2 (MRP2) mediates the biliary excretion of drugs and metabolites. [99mTc]mebrofenin may be employed as a probe for hepatic MRP2 activity because its biliary excretion is predominantly mediated by this transporter. As the liver uptake of [99mTc]mebrofenin depends on organic anion-transporting polypeptide (OATP) activity, a safe protocol for targeted inhibition of hepatic MRP2 is needed to study the intrinsic role of each transporter system. Diltiazem (DTZ) and cyclosporin A (CsA) were first confirmed to be potent MRP2 inhibitors in vitro. Dynamic acquisitions were performed in rats (n = 5-6 per group) to assess the kinetics of [99mTc]mebrofenin in the liver, intestine and heart-blood pool after increasing doses of inhibitors. Their impact on hepatic blood flow was assessed using Doppler ultrasound (n = 4). DTZ (s.c., 10 mg/kg) and low-dose CsA (i.v., 0.01 mg/kg) selectively decreased the transfer of [99mTc]mebrofenin from the liver to the bile (k3). Higher doses of DTZ and CsA did not further decrease k3 but dose-dependently decreased the uptake (k1) and backflux (k2) rate constants between blood and liver. High dose of DTZ (i.v., 3 mg/kg) but not CsA (i.v., 5 mg/kg) significantly decreased the blood flow in the portal vein and hepatic artery. Targeted pharmacological inhibition of hepatic MRP2 activity can be achieved in vivo without impacting OATP activity and liver blood flow. Clinical studies are warranted to validate [99mTc]mebrofenin in combination with low-dose CsA as a novel substrate/inhibitor pair to untangle the role of OATP and MRP2 activity in liver diseases.