68Ga-Sienna+ for PET-MRI Guided Sentinel Lymph Node Biopsy: Synthesis and Preclinical Evaluation in a Metastatic Breast Cancer Model.

Sentinel lymph node biopsy (SLNB) is commonly performed in cancers that metastasise via the lymphatic system. It involves excision and histology of sentinel lymph nodes (SLNs) and presents two main challenges: (i) sensitive whole-body localisation of SLNs, and (ii) lack of pre-operative knowledge of their metastatic status, resulting in a high number (>70%) of healthy SLN excisions. To improve SLNB, whole-body imaging could improve detection and potentially prevent unnecessary surgery by identifying healthy and metastatic SLNs. In this context, radiolabelled SPIOs and PET-MRI could find applications to locate SLNs with high sensitivity at the whole-body level (using PET) and guide high-resolution MRI to evaluate their metastatic status. Here we evaluate this approach by synthesising a GMP-compatible 68Ga-SPIO (68Ga-Sienna+) followed by PET-MR imaging and histology studies in a metastatic breast cancer mouse model. Methods. A clinically approved SPIO for SLN localisation (Sienna+) was radiolabelled with 68Ga without a chelator. Radiochemical stability was tested in human serum. In vitro cell uptake was compared between 3E.Δ.NT breast cancer cells, expressing the hNIS reporter gene, and macrophage cell lines (J774A.1; RAW264.7.GFP). NSG-mice were inoculated with 3E.Δ.NT cells. Left axillary SLN metastasis was monitored by hNIS/SPECT-CT and compared to the healthy right axillary SLN. 68Ga-Sienna+ was injected into front paws and followed by PET-MRI. Imaging results were confirmed by histology. Results.68Ga-Sienna+ was produced in high radiochemical purity (>93%) without the need for purification and was stable in vitro. In vitro uptake of 68Ga-Sienna+ in macrophage cells (J774A.1) was significantly higher (12 ± 1%) than in cancer cells (2.0 ± 0.1%; P < 0.001). SPECT-CT confirmed metastasis in the left axillary SLNs of tumour mice. In PET, significantly higher 68Ga-Sienna+ uptake was measured in healthy axillary SLNs (2.2 ± 0.9 %ID/mL), than in metastatic SLNs (1.1 ± 0.2 %ID/mL; P = 0.006). In MRI, 68Ga-Sienna+ uptake in healthy SLNs was observed by decreased MR signal in T2/T2*-weighted sequences, whereas fully metastatic SLNs appeared unchanged. Conclusion.68Ga-Sienna+ in combination with PET-MRI can locate and distinguish healthy from metastatic SLNs and could be a useful preoperative imaging tool to guide SLN biopsy and prevent unnecessary excisions.

An innovative small molecule for promoting neuroreparative strategies.

In this study, a new regenerative strategy to treat several neurodegenerative diseases is suggested by the use of a multitarget approach induced by our small molecule, MC111. Considering the importance of P-gp and BCRP expression on stem cell differentiation and the involvement of TLR4 on neurodegeneration processes, we investigated the effect of MC111, belonging to our library of P-gp active compounds on: (i) TLR4 signaling; (ii) P-gp and BCRP activity and expression; (iii) neurite sprouting. The observed findings exerted by MC111, open a new scenario for a multitarget and regenerative approach in neurodegenerative diseases encouraging the in vivo evaluation of MC111 as new tool in neuroreparative medicine.

Comparison of In Vitro Assays in Selecting Radiotracers for In Vivo P-Glycoprotein PET Imaging.

Positron emission tomography (PET) imaging of P-glycoprotein (P-gp) in the blood-brain barrier can be important in neurological diseases where P-gp is affected, such as Alzheimer´s disease. Radiotracers used in the imaging studies are present at very small, nanomolar, concentration, whereas in vitro assays where these tracers are characterized, are usually performed at micromolar concentration, causing often discrepant in vivo and in vitro data. We had in vivo rodent PET data of [11C]verapamil, (R)-N-[18F]fluoroethylverapamil, (R)-O-[18F]fluoroethyl-norverapamil, [18F]MC225 and [18F]MC224 and we included also two new molecules [18F]MC198 and [18F]KE64 in this study. To improve the predictive value of in vitro assays, we labeled all the tracers with tritium and performed bidirectional substrate transport assay in MDCKII-MDR1 cells at three different concentrations (0.01, 1 and 50 µM) and also inhibition assay with P-gp inhibitors. As a comparison, we used non-radioactive molecules in transport assay in Caco-2 cells at a concentration of 10 µM and in calcein-AM inhibition assay in MDCKII-MDR1 cells. All the P-gp substrates were transported dose-dependently. At the highest concentration (50 µM), P-gp was saturated in a similar way as after treatment with P-gp inhibitors. Best in vivo correlation was obtained with the bidirectional transport assay at a concentration of 0.01 µM. One micromolar concentration in a transport assay or calcein-AM assay alone is not sufficient for correct in vivo prediction of substrate P-gp PET ligands.

Evaluation of [18F]MC225 as a PET radiotracer for measuring P-glycoprotein function at the blood-brain barrier in rats: Kinetics, metabolism, and selectivity.

P-glycoprotein is a protective efflux transporter at the blood-brain barrier showing altered function in many neurological disorders. The purpose of this study was to validate [18F]MC225 as a radiotracer for measuring P-glycoprotein function with positron emission tomography. Three groups of Sprague-Dawley rats were used to assess tracer uptake at baseline (group 1), after inhibition of P-glycoprotein (group 2), and after inhibition of both P-glycoprotein and breast cancer resistance protein (Bcrp, group 3). A two-tissue compartment model with a metabolite-corrected plasma input function provided the best fit to the positron emission tomography data, but parameter estimates were more reliable in a one-tissue compartment model, which was selected as the preferred model. Regional distribution volumes ( VT) in the control group ranged from 6 to 11, which is higher than for other radiotracers. [18F]MC225 showed transporter selectivity, since inhibition of P-glycoprotein caused a two to fourfold increase in the cerebral VT values, but additional inhibition of Bcrp did not cause any further increase. Metabolic stability of [18F]MC225 was moderate (at 1 h post-injection 15% of plasma radioactivity and 76% of brain radioactivity represented intact parent). Thus, [18F]MC225 may be a useful radiotracer to measure especially increases of P-glycoprotein function at the blood-brain barrier.

P-glycoprotein Function in the Rodent Brain Displays a Daily Rhythm, a Quantitative In Vivo PET Study.

The blood-brain barrier (BBB) contributes to brain homeostasis by protecting the brain from harmful compounds. P-glycoprotein (P-gp) is one of the major efflux transporters at the BBB. In the present study, we assessed whether (1) P-gp function in the brain is constant or fluctuates across the day and (2) if it is affected by sleep deprivation. Four groups of rats were PET scanned with a radiolabeled P-gp substrate [18F]MC225, each at a different moment of the 12-h light-dark cycle to study diurnal variations: early sleep phase (ZT3), late sleep phase (ZT9), early active phase (ZT15), and late active phase (ZT21). In two additional groups, controls were allowed to sleep normally while experimental animals were sleep-deprived for 10 h in a slowly rotating drum during the sleep phase. Kinetic modeling with a one-tissue compartment model fit resulted for all brain regions in 1.2-1.8-fold higher distribution volumes (V T ) at ZT15 than at other time points. V T -values at ZT3, ZT9, and ZT21 were not significantly different from each other. Regional tracer distribution volumes in controls and sleep-deprived animals were also not significantly different. Our results indicate that P-gp function in rats displays a daily rhythm with reduced function at the beginning of the active phase. This rhythm is not dependent on sleep since acute sleep deprivation had no effect. Knowing the diurnal variation of P-gp function could be important for the design of PET studies and for choosing the correct administration time for P-gp-dependent drugs.

Synthesis and Preclinical Evaluation of Three Novel Fluorine-18 Labeled Radiopharmaceuticals for P-Glycoprotein PET Imaging at the Blood-Brain Barrier.

P-Glycoprotein (P-gp), along with other transporter proteins at the blood-brain barrier (BBB), limits the entry of many pharmaceuticals into the brain. Altered P-gp function has been found in several neurological diseases. To study the P-gp function, many positron emission tomography (PET) radiopharmaceuticals have been developed. Most P-gp radiopharmaceuticals are labeled with carbon-11, while labeling with fluorine-18 would increase their applicability due to longer half-life. Here we present the synthesis and in vivo evaluation of three novel fluorine-18 labeled radiopharmaceuticals: 4-((6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)methyl)-2-(4-fluorophenyl)oxazole (1a), 2-biphenyl-4-yl-2-fluoroethoxy-6,7-dimethoxy-1,2,3,4-tetrahydro-isoquinoline (2), and 5-(1-(2-fluoroethoxy))-[3-(6,7-dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)-propyl]-5,6,7,8-tetrahydronaphthalen (3). Compounds were characterized as P-gp substrates in vitro, and Mdr1a/b((-/-))Bcrp1((-/-)) and wild-type mice were used to assess the substrate potential in vivo. Comparison was made to (R)-[(11)C]verapamil, which is currently the most frequently used P-gp substrate. Compound [(18)F]3 was performing the best out of the new radiopharmaceuticals; it had 2-fold higher brain uptake in the Mdr1a/b((-/-))Bcrp1((-/-)) mice compared to wild-type and was metabolically quite stable. In the plasma, 69% of the parent compound was intact after 45 min and 96% in the brain. Selectivity of [(18)F]3 to P-gp was tested by comparing the uptake in Mdr1a/b((-/-)) mice to uptake in Mdr1a/b((-/-))Bcrp1((-/-)) mice, which was statistically not significantly different. Hence, [(18)F]3 was found to be selective for P-gp and is a promising new radiopharmaceutical for P-gp PET imaging at the BBB.