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Purpose To support acquisition of accurate, reproducible and high-quality preclinical imaging data, various standardisation resources have been developed over the years. However, it is unclear the impact of those efforts in current preclinical imaging practices. To better understand the status quo in the field of preclinical imaging standardisation, the STANDARD group of the European Society of Molecular Imaging (ESMI) put together a community survey and a forum for discussion at the European Molecular Imaging Meeting (EMIM) 2022. This paper reports on the results from the STANDARD survey and the forum discussions that took place at EMIM2022. Procedures The survey was delivered to the community by the ESMI office and was promoted through the Society channels, email lists and webpages. The survey contained seven sections organised as generic questions and imaging modality-specific questions. The generic questions focused on issues regarding data acquisition, data processing, data storage, publishing and community awareness of international guidelines for animal research. Specific questions on practices in optical imaging, PET, CT, SPECT, MRI and ultrasound were further included. Results Data from the STANDARD survey showed that 47% of survey participants do not have or do not know if they have QC/QA guidelines at their institutes. Additionally, a large variability exists in the ways data are acquired, processed and reported regarding general aspects as well as modality-specific aspects. Moreover, there is limited awareness of the existence of international guidelines on preclinical (imaging) research practices. Conclusions Standardisation of preclinical imaging techniques remains a challenge and hinders the transformative potential of preclinical imaging to augment biomedical research pipelines by serving as an easy vehicle for translation of research findings to the clinic. Data collected in this project show that there is a need to promote and disseminate already available tools to standardise preclinical imaging practices.

Purpose: In this study, we tested the antitumor activity of the dual phosphoinositide 3-kinase (PI3K)/mTOR inhibitor BEZ235 against gastric cancer in vitro and in vivo.Experimental Design: Gastric cancer cell lines (N87, MKN45, and MKN28) were incubated with BEZ235 and assessed for cell viability, cell cycle, and PI3K/mTOR target inhibition. In vivo, athymic nude mice were inoculated with N87, MKN28, or MKN45 cells and treated daily with BEZ235. 3′-Deoxy-3′-[18F]fluorothymidine ([18F]FLT) uptake was measured via small animal positron emission tomography (PET).Results:In vitro, BEZ235 dose dependently decreased the cell viability of gastric cancer cell lines. The antiproliferative activity of BEZ235 was linked to a G1 cell-cycle arrest. In vivo, BEZ235 treatment resulted in PI3K/mTOR target inhibition as shown by dephosphorylation of AKT and S6 protein in all xenograft models. However, BEZ235 treatment only inhibited tumor growth of N87 xenografts, whereas no antitumor effect was observed in the MKN28 and MKN45 xenograft models. Sensitivity to BEZ235 in vivo correlated with downregulation of the proliferation marker thymidine kinase 1. Accordingly, [18F]FLT uptake was only significantly reduced in the BEZ235-sensitive N87 xenograft model as measured by PET.Conclusion: In conclusion, in vivo sensitivity of gastric cancer xenografts to BEZ235 did not correlate with in vitro antiproliferative activity or in vivo PI3K/mTOR target inhibition by BEZ235. In contrast, [18F]FLT uptake was linked to BEZ235 in vivo sensitivity. Noninvasive [18F]FLT PET imaging might qualify as a novel marker for optimizing future clinical testing of dual PI3K/mTOR inhibitors. Clin Cancer Res; 17(16); 5322–32. ©2011 AACR.

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Transporter-mediated drug-drug interactions (DDIs) are of concern in antimicrobial drug development, as they can have serious safety consequences. We used positron emission tomography (PET) imaging-based pharmacokinetic (PK) analysis to assess the effect of different drugs, which may cause transporter-mediated DDIs, on the tissue distribution and excretion of [ 18 F]ciprofloxacin as a radiolabeled model antimicrobial drug.

Nanodiamonds (NDs) represent a new class of nanoparticles and have gained increasing interest in medical applications. Modifying the surface coating by attaching binding ligands or imaging probes can transform NDs into multi-modal targeting probes. This study evaluated the biokinetics and biodistribution ofNDs were coated with an albumin-derived copolymer modified with desferrioxamine to provide a chelator for radiolabeling. In vivo studies were conducted in AR42J tumor-bearing CD1 mice to evaluate biodistribution and tumor accumulation of the NDs.Coated NDs were successfully radiolabeled usingRadiolabeling of coated NDs could be achieved. However, the obtained results indicate these coated NDs' limitations in their biodistribution within the conducted studies.

To support acquisition of accurate, reproducible and high-quality preclinical imaging data, various standardisation resources have been developed over the years. However, it is unclear the impact of those efforts in current preclinical imaging practices. To better understand the status quo in the field of preclinical imaging standardisation, the STANDARD group of the European Society of Molecular Imaging (ESMI) put together a community survey and a forum for discussion at the European Molecular Imaging Meeting (EMIM) 2022. This paper reports on the results from the STANDARD survey and the forum discussions that took place at EMIM2022.The survey was delivered to the community by the ESMI office and was promoted through the Society channels, email lists and webpages. The survey contained seven sections organised as generic questions and imaging modality-specific questions. The generic questions focused on issues regarding data acquisition, data processing, data storage, publishing and community awareness of international guidelines for animal research. Specific questions on practices in optical imaging, PET, CT, SPECT, MRI and ultrasound were further included.Data from the STANDARD survey showed that 47% of survey participants do not have or do not know if they have QC/QA guidelines at their institutes. Additionally, a large variability exists in the ways data are acquired, processed and reported regarding general aspects as well as modality-specific aspects. Moreover, there is limited awareness of the existence of international guidelines on preclinical (imaging) research practices.Standardisation of preclinical imaging techniques remains a challenge and hinders the transformative potential of preclinical imaging to augment biomedical research pipelines by serving as an easy vehicle for translation of research findings to the clinic. Data collected in this project show that there is a need to promote and disseminate already available tools to standardise preclinical imaging practices.

Multidrug resistance-associated protein 1 (MRP1, encoded by the ABCC1 gene) may contribute to the clearance of amyloid-beta (Aβ) peptides from the brain into the blood and stimulation of MRP1 transport activity may be a therapeutic approach to enhance brain Aβ clearance. In this study, we assessed the effect of thiethylperazine, an antiemetic drug which was shown to stimulate MRP1 activity in vitro and to decrease Aβ load in a rapid β-amyloidosis mouse model (APP/PS1-21), on MRP1 transport activity by means of positron emission tomography (PET) imaging with the MRP1 tracer 6-bromo-7-[11C]methylpurine. Groups of wild-type, APP/PS1-21 and Abcc1(−/−) mice underwent PET scans before and after a 5-day oral treatment period with thiethylperazine (15 mg/kg, once daily). The elimination rate constant of radioactivity (kelim) was calculated from time–activity curves in the brain and the lungs as a measure of tissue MRP1 activity. Treatment with thiethylperazine had no significant effect on MRP1 activity in the brain and the lungs of wild-type and APP/PS1-21 mice. This may either be related to a lack of an MRP1-stimulating effect of thiethylperazine in vivo or to other factors, such as substrate-dependent MRP1 stimulation, insufficient target tissue exposure to thiethylperazine or limited sensitivity of the PET tracer to measure MRP1 stimulation.

Introduction Tau deposition is one of the hallmarks of Alzheimer's disease (AD) and can be visualized and quantified using [18F]THK-5317 together with kinetic modeling. To determine the feasibility of this approach, we measured blood/plasma pharmacokinetics and radiotracer metabolism in female and male rats. Methods Female and male rats (n = 11–12) were cannulated via the femoral artery for continuous blood sampling. Blood sampling was performed at regular intervals after intravenous injection of [18F]THK-5317. After collection of the last blood sample, animals were sacrificed, and organs were excised. Blood from minute 5, 20 and 60 was centrifuged to obtain plasma. Radiolabeled metabolites in plasma, brain, liver and urine were analyzed by radio-thin-layer chromatography (radio-TLC). Results Plasma pharmacokinetics and metabolism were significantly different between female and male rats. [18F]THK-5317 plasma clearance was faster in female (0.66 ± 0.08 mL/h/kg BW) than in male (0.52 ± 0.11 mL/h/kg BW) rats (p = .005). The percentage of unmetabolized parent was significantly different between both sexes at 20 min and 60 min p.i. In the liver, a 1.6-fold higher radioactivity concentration was found in male versus female animals and in addition also the percentage of unmetabolized parent was different. Conclusion Our results show pronounced sex differences in blood/plasma pharmacokinetics and metabolism of [18F]THK-5317 in rats. Female animals showed a faster plasma clearance compared to males. These results underline the importance of investigating both sexes and also support the notion that individual input functions or sex-specific population-based input functions are needed for kinetic modeling analyses. Advances in knowledge First preclinical study in rats showing pronounced sex differences in blood/plasma pharmacokinetics and metabolism of [18F]THK-5317. Implications for patient care Sex-specific differences might also be present in humans and thus clinical trials should have adequate sample size to account for effects in men and women separately.

Background To better understand the etiology and pathomechanisms of Alzheimer’s disease, several transgenic animal models that overexpress human tau or human amyloid-beta (Aβ) have been developed. In the present study, we generated a novel transgenic rat model by cross-breeding amyloid precursor protein (APP) rats with tau rats. We characterized this model by performing positron emission tomography scans combined with immunofluorescent labeling and cerebrospinal fluid analyses. Methods APP/Tau rats were generated by cross-breeding male McGill-R-Thy1-APP transgenic rats with female hTau-40/P301L transgenic rats. APP/Tau double transgenic rats and non-transgenic (ntg) littermates aged 7, 13, and 21 months were subjected to dynamic [11C] PiB scan and dynamic [18F]THK-5317 scans. For regional brain analysis, a template was generated from anatomical MR images of selected animals, which was co-registered with the PET images. Regional analysis was performed by application of the simplified reference tissue model ([11C]PiB data), whereas [18F]THK-5317 data were analyzed using a 2-tissue compartment model and Logan graphical analysis. In addition, immunofluorescent labeling (tau, amyloid) and cerebrospinal fluid analyses were performed. Results [11C]PiB binding potential (BPND) and [18F]THK-5317 volume of distribution (VT) showed an increase with age in several brain regions in the APP/Tau group but not in the ntg control group. Immunohistochemical analysis of brain slices of PET-scanned animals revealed a positive correlation between Aβ labeling and [11C]PiB regional BPND. Tau staining yielded a trend towards higher levels in the cortex and hippocampus of APP/Tau rats compared with ntg littermates, but without reaching statistical significance. No correlation was found between tau immunofluorescence labeling results and the respective [18F]THK-5317 VT values. Conclusions We thoroughly characterized a novel APP/Tau rat model using combined PET imaging and immunofluorescence analysis. We observed an age-related increase in [11C]PiB and [18F]THK-5317 binding in several brain regions in the APP/Tau group but not in the ntg group. Although we were able to reveal a positive correlation between amyloid labeling and [11C]PiB regional brain uptake, we observed relatively low human tau and amyloid fibril expression levels and a somewhat unstable brain pathology which questions the utility of this animal model for further studies.

The standardization of preclinical imaging is a key factor to ensure the reliability, reproducibility, validity, and translatability of preclinical data. Preclinical standardization has been slowly progressing in recent years and has mainly been performed within a single institution, whereas little has been done in regards to multicenter standardization between facilities. This study aimed to investigate the comparability among preclinical imaging facilities in terms of PET data acquisition and analysis. In the first step, basic PET scans were obtained in 4 different preclinical imaging facilities to compare their standard imaging protocol for 18F-FDG. In the second step, the influence of the personnel performing the experiments and the experimental equipment used in the experiment were compared. In the third step, the influence of the image analysis on the reproducibility and comparability of the acquired data was determined. Distinct differences in the uptake behavior of the 4 standard imaging protocols were determined for the investigated organs (brain, left ventricle, liver, and muscle) due to different animal handling procedures before and during the scans (e.g., fasting vs. nonfasting, glucose levels, temperature regulation vs. constant temperature warming). Significant differences in the uptake behavior in the brain were detected when the same imaging protocol was used but executed by different personnel and using different experimental animal handling equipment. An influence of the person analyzing the data was detected for most of the organs, when the volumes of interest were manually drawn by the investigators. Coregistration of the PET to an MR image and drawing the volume of interest based on anatomic information yielded reproducible results among investigators. It has been demonstrated that there is a huge demand for standardization among multiple institutions.

Background: Whole-body PET images can be obtained by using the “step-and-shoot” (SaS) method (using multiple bed positions) or continuous bed motion (CBM). As transmission scans are not always feasible, an alternative method where attenuation data can be generated via emission-based attenuation correction (AC) maps is of interest. The aim of this preclinical study was to investigate the influence of the acquisition method and AC on the quantitation accuracy of [18F]FDG-PET.Methods: [18F]FDG-PET phantom images were acquired using either SaS or CBM. Transmission scans were recorded for the SaS method using a 57Co-point source. Emission-based attenuation sinograms were obtained from the images after segmentation and inverse Fourier rebinning. PET images were reconstructed without AC, transmission based (TX-AC) and emission-based (EM-AC) attenuation correction. Moreover, [18F]FDG-PET scans of rats bearing mammary carcinomas acquired using either SaS or CBM were analyzed retrospectively and quantification in tissues was compared.Results: Phantom recovery coefficients (RC) varied greatly, ranging from 0.49 ± 0.01 to 1.15 ± 0.07, dependent on acquisition method, reconstruction algorithm and AC method. In CBM acquired images, EM-AC improved quantification accuracy when compared to no-AC images in the phantom studies (RC 0.79 ± 0.02 vs. 0.49 ± 0.01, respectively) and in tumors of rats (DMBA model: 1.16±0.42 SUV vs. 0.86±0.28 SUV, respectively).Conclusion: The method of AC has a strong influence on the quantification of [18F]FDG. Our data indicates that EM-AC improves quantification in images obtained by CBM and SaS. However, the obtained values were still underestimated when compared to TX-AC corrected images.

Purpose Multidrug resistance-associated proteins (MRPs) mediate the hepatobiliary and renal excretion of many drugs and drug conjugates. The positron emission tomography (PET) tracer 6-bromo-7-[11C]methylpurine is rapidly converted in tissues by glutathione-S-transferases into its glutathione conjugate, and has been used to measure the activity of Abcc1 in the brain and the lungs of mice. Aim of this work was to investigate if the activity of MRPs in excretory organs can be measured with 6-bromo-7-[11C]methylpurine. Procedures We performed PET scans with 6-bromo-7-[11C]methylpurine in groups of wild-type, Abcc4(−/−) and Abcc1(−/−) mice, with and without pre-treatment with the prototypical MRP inhibitor MK571. Results 6-Bromo-7-[11C]methylpurine-derived radioactivity predominantly underwent renal excretion. In blood, MK571 treatment led to a significant increase in the AUC and a decrease in the elimination rate constant of radioactivity (kelimination,blood). In the kidneys, there were significant decreases in the rate constant for radioactivity uptake from the blood (kuptake,kidney), kelimination,kidney, and the rate constant for tubular secretion of radioactivity (kurine). Experiments in Abcc4(−/−) mice indicated that Abcc4 contributed to renal excretion of 6-bromo-7-[11C]methylpurine-derived radioactivity. Conclusions Our data suggest that 6-bromo-7-[11C]methylpurine may be useful to assess the activity of MRPs in the kidneys as well as in other organs (brain, lungs), although further work is needed to identify the MRP subtypes involved in the disposition of 6-bromo-7-[11C]methylpurine-derived radioactivity. Electronic supplementary material The online version of this article (10.1007/s11307-018-1230-y) contains supplementary material, which is available to authorized users.

Ko143 is a reference inhibitor of the adenosine triphosphate-binding cassette (ABC) transporter breast cancer resistance protein (humans: ABCG2, rodents: Abcg2) for in vitro and in vivo use. Previous in vitro data indicate that Ko143 binds specifically to ABCG2/Abcg2, suggesting a potential utility of Ko143 as a positron emission tomography (PET) tracer to assess the density (abundance) of ABCG2 in different tissues. In this work we radiolabeled Ko143 with carbon-11 (11C) and performed small-animal PET experiments with [11C]Ko143 in wild-type, Abcg2(−/−), Abcb1a/b(−/−) and Abcb1a/b(−/−)Abcg2(−/−) mice to assess the influence of Abcg2 and Abcb1a/b on tissue distribution and excretion of [11C]Ko143. [11C]Ko143 was extensively metabolized in vivo and unidentified radiolabeled metabolites were found in all investigated tissues. We detected no significant differences between wild-type and Abcg2(−/−) mice in the distribution of [11C]Ko143-derived radioactivity to Abcg2-expressing organs (brain, liver and kidney). [11C]Ko143 and possibly its radiolabeled metabolites were transported by Abcb1a and not by Abcg2 at the mouse blood-brain barrier. [11C]Ko143-derived radioactivity underwent both hepatobiliary and urinary excretion, with Abcg2 playing a possible role in mediating the transport of radiolabeled metabolites of [11C]Ko143 from the kidney into urine. Experiments in which a pharmacologic dose of unlabeled Ko143 (10 mg/kg) was co-administered with [11C]Ko143 revealed pronounced effects of the vehicle used for Ko143 formulation (containing polyethylene glycol 300 and polysorbate 80) on radioactivity distribution to the brain and the liver, as well as on hepatobiliary and urinary excretion of radioactivity. Our results highlight the challenges associated with the development of PET tracers for ABC transporters and emphasize that inhibitory effects of pharmaceutical excipients on membrane transporters need to be considered when performing in vivo drug-drug interaction studies. Finally, our study illustrates the power of small-animal PET to assess the interaction of drug molecules with membrane transporters on a whole body level.

Introduction [ 11 C]Erlotinib PET has shown promise to distinguish non-small cell lung cancer (NSCLC) tumors harboring the activating epidermal growth factor receptor (EGFR) mutation delE746-A750 from tumors with wild-type EGFR. To assess the suitability of [ 11 C]erlotinib PET to detect the emergence of acquired erlotinib resistance in initially erlotinib-responsive tumors, we performed in vitro binding and PET experiments in mice bearing tumor xenografts using a range of different cancer cells, which were erlotinib-sensitive or exhibited clinically relevant resistance mechanisms to erlotinib. Methods The following cell lines were used for in vitro binding and PET experiments: the epidermoid carcinoma cell line A-431 (erlotinib-sensitive, wild-type EGFR) and the three NSCLC cell lines HCC827 (erlotinib-sensitive, delE746-A750), HCC827 EPR (erlotinib-resistant, delE746-A750 and T790M) and HCC827 ERLO (erlotinib-resistant, delE746-A750 and MET amplification). BALB/c nude mice with subcutaneous tumor xenografts underwent two consecutive [ 11 C]erlotinib PET scans, a baseline scan and a second scan in which unlabeled erlotinib (10mg/kg) was co-injected. Logan graphical analysis was used to estimate total distribution volume (V T ) of [ 11 C]erlotinib in tumors. Results In vitro experiments revealed significantly higher uptake of [ 11 C]erlotinib (5.2-fold) in the three NSCLC cell lines as compared to A-431 cells. In all four cell lines co-incubation with unlabeled erlotinib (1μM) led to significant reductions in [ 11 C]erlotinib uptake (−19% to −66%). In both PET scans and for all four studied cell lines there were no significant differences in tumoral [ 11 C]erlotinib V T values. For all three NSCLC cell lines, but not for the A-431 cell line, tumoral V T was significantly reduced following co-injection of unlabeled erlotinib (−20% to −35%). Conclusions We found no significant differences in the in vitro and in vivo binding of [ 11 C]erlotinib between erlotinib-sensitive and erlotinib-resistant NSCLC cells. Our findings suggest that [ 11 C]erlotinib PET will not be suitable to distinguish erlotinib-sensitive NSCLC tumors from tumors with acquired resistance to erlotinib.

In recent years, radiofluorinated alkyl azides have been reported for click radiolabeling and pretargeted PET imaging, but only little is known about the biodistribution and metabolism of these compounds. In this work, we present a significantly improved procedure for the synthesis of [18F]fluoroethyl azide and reinvestigated this radiolabeled probe in detail showing poor stability and very restricted suitability for in vivo application. Therefore, modified low-molecular-weight [18F]fluoroalkyl azides were developed. Propargyl-tagged endomorphin-1 (as model compound) was successfully radiolabeled in high yield and short reaction time making these probes useful and efficient bioorthogonal tools for rapid radiolabeling. Biodistribution, pharmacokinetics and in vivo stability were studied by preclinical PET/MR scanning and metabolite analysis. The results of this study revealed only limited applicability of [18F]fluoroalkyl azides for in vivo application.

Tau deposition is one of the hallmarks of Alzheimer's disease (AD) and can be visualized and quantified using [Female and male rats (n = 11-12) were cannulated via the femoral artery for continuous blood sampling. Blood sampling was performed at regular intervals after intravenous injection of [Plasma pharmacokinetics and metabolism were significantly different between female and male rats. [Our results show pronounced sex differences in blood/plasma pharmacokinetics and metabolism of [First preclinical study in rats showing pronounced sex differences in blood/plasma pharmacokinetics and metabolism of [Sex-specific differences might also be present in humans and thus clinical trials should have adequate sample size to account for effects in men and women separately.

Aim of this study was to investigate if cellular [18F]FBPA uptake can be increased upon preloading with amino acids. [18F]FBPA uptake was assessed in HuH-7, CaCo-2 and B16-F1 cells pretreated with different concentrations or incubation times of L-BPA, L-tyrosine or L-DOPA. Without preloading, highest uptake of [18F]FBPA was observed in B16-F1 cells, followed by CaCo-2 cells and HuH-7 cells. In all cell lines higher [18F]FBPA accumulation (up to 1.65-fold) was obtained with increasing L-BPA, L-DOPA and L-tyrosine concentrations.

Positron emission tomography (PET) using fluorine-18 (18F)-labeled 2-nitroimidazole radiotracers has proven useful for assessment of tumor oxygenation. However, the passive diffusion-driven cellular uptake of currently available radiotracers results in slow kinetics and low tumor-to-background ratios. With the aim to develop a compound that is actively transported into cells, 1-(6'-deoxy-6'-[18F]fluoro-β-d-allofuranosyl)-2-nitroimidazole (β-[18F]1), a putative nucleoside transporter substrate, was synthetized by nucleophilic [18F]fluoride substitution of an acetyl protected labeling precursor with a tosylate leaving group (β-6) in a final radiochemical yield of 12±8% (n=10, based on [18F]fluoride starting activity) in a total synthesis time of 60min with a specific activity at end of synthesis of 218±58GBq/μmol (n=10). Both radiolabeling precursor β-6 and unlabeled reference compound β-1 were prepared in multistep syntheses starting from 1,2:5,6-di-O-isopropylidene-α-d-allofuranose. In vitro experiments demonstrated an interaction of β-1 with SLC29A1 and SLC28A1/2/3 nucleoside transporter as well as hypoxia specific retention of β-[18F]1 in tumor cell lines. In biodistribution studies in healthy mice β-[18F]1 showed homogenous tissue distribution and excellent metabolic stability, which was unaffected by tissue oxygenation. PET studies in tumor bearing mice showed tumor-to-muscle ratios of 2.13±0.22 (n=4) at 2h after administration of β-[18F]1. In ex vivo autoradiography experiments β-[18F]1 distribution closely matched staining with the hypoxia marker pimonidazole. In conclusion, β-[18F]1 shows potential as PET hypoxia radiotracer which merits further investigation.

24-nor-ursodeoxycholic acid (norUDCA) is a novel therapeutic approach to cholestatic liver diseases. In mouse models of cholestasis, norUDCA induces basolateral multidrug resistance-associated proteins 4 (Mrp4) and 3 in hepatocytes, which provide alternative escape routes for bile acids accumulating during cholestasis but could also result in altered hepatic disposition of concomitantly administered substrate drugs. We used positron emission tomography imaging to study the influence of norUDCA on hepatic disposition of the model Mrp4 substrate [(18)F]ciprofloxacin in wild-type and Mdr2((-/-)) mice, a model of cholestasis. Animals underwent [(18)F]ciprofloxacin positron emission tomography at baseline and after norUDCA treatment. After norUDCA treatment, liver-to-blood area under the curve ratio of [(18)F]ciprofloxacin was significantly decreased compared to baseline, both in wild-type (-34.0 ± 2.1%) and Mdr2((-/-)) mice (-20.5 ± 6.0%). [(18)F]Ciprofloxacin uptake clearance from blood into liver was reduced by -17.1 ± 9.0% in wild-type and by -20.1 ± 7.3% in Mdr2((-/-)) mice. Real-time PCR analysis showed significant increases in hepatic Mrp4 and multidrug resistance-associated protein 3 mRNA after norUDCA. Transport experiments in organic anion transporting polypeptide (OATP)1B1-, OATP1B3-, and OATP2B1-transfected cells revealed weak transport of [(14)C]ciprofloxacin by OATP1B3 and OATP2B1 and no inhibition by norUDCA. In conclusion, our data suggest that changes in hepatic [(18)F]ciprofloxacin disposition in mice after norUDCA treatment were caused by induction of basolateral Mrp4 in hepatocytes.

Epigenetics plays a vital role in regulating gene expression and determining the specific phenotypes of eukaryotic cells. Histone deacetylases (HDACs) are important epigenetic regulatory proteins effecting multiple biological functions. Particularly, HDAC6 has become a promising anti-cancer drug target because of its regulation of cell mobility, protein trafficking, degradation of misfolded proteins, cell growth, apoptosis, and metastasis. In this study, we identified one out of six vitamin K3 derivatives, VKT-2, as HDAC6 inhibitor using molecular docking and cell viability assays in HDAC6-overexpressing HuH-7 cancer cells. Microscale thermophoresis and HDAC6 enzymatic assays revealed that VKT-2 bound to HDAC6 and inhibited its function. We further identified its cytotoxic activity. VKT-2 hyperacetylated HDAC6 substrates and disturbed tubulin integrity leading to significant inhibition of tumor migration in both HuH-7 spheroids and U2OS-GFP-α-tubulin cells. Moreover, VKT-2 induced autophagic and apoptotic cell death in HuH-7, while aggresome formation was restrained after VKT-2 treatment. A HuH-7 cell-xenograft model in zebrafish larvae provided evidence that VKT-2 inhibited the tumor growth in vivo. To best of our knowledge, it is the first time to demonstrate that vitamin k3 derivatives (VKT-2) inhibits HDAC6 in solid tumor cells. These unique findings suggested that VKT-2 is a promising anti-cancer agent targeting HDAC6.

Drug disposition consists of absorption, distribution, metabolism and excretion (ADME). For these processes, drugs and drug metabolites have to cross cellular membranes in different organs and tissues (e.g. intestine, liver, kidney, etc.). In many cases, movement of drugs and drug metabolites across cellular membranes is not just a passive diffusion-mediated process but occurs via saturable transmembrane transporters belonging either to the solute carrier (SLC) or adenosine triphosphate-binding cassette (ABC) families. When two drugs, which are recognized by the same SLC or ABC transporters, are co-administered, one drug may induce transporter inhibition/saturation and thereby change the disposition of the other drug as compared to when the drugs are administered alone. This phenomenon has been termed transporter-mediated drug-drug interaction (DDI), which is of great concern in drug development as this may impact drug safety and efficacy [1]. In many cases, changes in the activity of transmembrane transporters may lead to pronounced changes in drug tissue distribution (e.g. liver, kidneys, brain) without changes in drug plasma pharmacokinetics [2]. To assess such tissue DDIs in vivo, a methodology is needed to measure drug tissue concentration levels. The non-invasive nuclear imaging method positron emission tomography (PET) is a very powerful tool for measuring tissue distribution of drugs radiolabelled with positron-emitting radionuclides, such as carbon-11 (11C, half-life: 20.4 min) or fluorine-18 (18F, half-life: 109.8 min), in animals or humans [3, 4]. Dedicated small-animal PET systems allow to measure rodents (mice, rats) with high sensitivity and good spatial resolution. As PET does not provide anatomical information, it needs to be combined with anatomical imaging to allow for better definition of organs or tissues of interest. In this chapter, we provide a case report of how PET in combination with magnetic resonance imaging (MRI) can be used to assess transporter-mediated DDIs in different organs of the mouse.

We modified a commercially available synthesis module for nucleophilic [(18)F]fluorinations (TRACERlab(TM) FXFDG, GE Healthcare) to enable the reliable synthesis of 2-[(18)F]fluoro-4-borono-L-phenylalanine ([(18)F]FBPA) via direct electrophilic substitution of 4-borono-L-phenylalanine with [(18)F]F2 gas. [(18)F]FBPA was obtained with a RCY of 8.5±2.0% and a radiochemical purity of 98±1% in a total synthesis time of 72±7 min (n=22). The modified synthesis module might also be useful for the synthesis of other [(18)F]radiopharmaceuticals via electrophilic substitution reactions while still being suitable for nucleophilic substitution reactions.

Pre-clinical positron emission tomography (PET) has evolved in the last few years from pure visualization of radiotracer uptake and distribution towards quantification of the physiological parameters. For reliable and reproducible quantification the kinetic modeling methods used to obtain relevant parameters of radiotracer tissue interaction are important. Here we present different kinetic modeling techniques with a focus on compartmental models including plasma input models and reference tissue input models. The experimental challenges off deriving the plasma input function in rodents and the effect of anesthesia are discussed. Finally, in vivo application of kinetic modeling in various areas of pre-clinical research is presented and compared to human data.

Purpose The application of radiolabelled inhibitors of cytochrome P450 enzymes is a novel approach for molecular imaging of adrenocortical masses to detect adrenal tumours. One potential tracer is radiolabelled iodometomidate (IMTO) with a common option for scintigraphic diagnosis and therapeutic applications. The aim of this study was to radiolabel iodometomidate with the positron-emitting radionuclide iodine-124 ((124)I) for the investigation of the biological behaviour and pharmacokinetics with positron emission tomography (PET). Procedures [(124)I]IMTO has been synthesized by oxidative radioiodo-destannylation, purified via semi-preparative HPLC and formulated in acetate-buffered saline, which contained ascorbic acid and ethanol to avoid radiolytic decomposition. Biological evaluation was performed in rats which received 5.5 ± 0.7 MBq [(124)I]IMTO in vivo. The radioactivity distribution (n = 3) has been dynamically imaged from 0-120 min after intravenous (i.v.) injection by small-animal PET. Regions of interest have been defined manually in the reconstructed PET images, and the activity concentration was expressed as percent injected dose per gram tissue (%ID/g). Results [(124)I]IMTO was prepared with a radiochemical yield of 83 ± 5 % (n = 3) and a radiochemical purity of >97 %. The final formulation of [(124)I]IMTO was stable for up to 48 h at room temperature. Two hours after i.v. administration in rats, radioactivity concentration in the adrenal glands were 2.1 ± 0.3 %ID/g, which was sufficient to achieve highest-contrast adrenal PET images. Conclusions In the present study, the biological characteristics of radioiodinated metomidate were evaluated. [(124)I]IMTO appears as an attractive PET tracer for imaging of adrenals.

Elacridar (ELC) and tariquidar (TQD) are generally thought to be nontransported inhibitors of P-glycoprotein (Pgp) and breast cancer resistance protein (BCRP), but recent data indicate that they may also be substrates of these multidrug transporters (MDTs). The present study was designed to investigate potential transport of ELC and TQD by MDTs at the blood-brain barrier at tracer doses as used in positron emission tomography (PET) studies. We performed PET scans with carbon-11-labeled ELC and TQD before and after MDT inhibition in wild-type and transporter-knockout mice as well as in in vitro transport assays in MDT-overexpressing cells. Brain entrance of [(11)C]ELC and [(11)C]TQD administered in nanomolar tracer doses was found to be limited by Pgp- and Bcrp1-mediated efflux at the mouse blood-brain barrier. At higher, MDT-inhibitory doses, i.e., 15 mg/kg for TQD and 5 mg/kg for ELC, brain activity uptake of [(11)C]ELC at 25 minutes after tracer injection was 5.8 ± 0.3, 2.1 ± 0.2, and 7.5 ± 1.0-fold higher in wild-type, Mdr1a/b((-/-),()) and Bcrp1((-/-)) mice, respectively, but remained unchanged in Mdr1a/b((-/-))Bcrp1((-/-)) mice. Activity uptake of [(11)C]TQD was 2.8 ± 0.2 and 6.8 ± 0.4-fold higher in wild-type and Bcrp1((-/-)) mice, but remained unchanged in Mdr1a/b((-/-)) and Mdr1a/b((-/-))Bcrp1((-/-)) mice. Consistent with the in vivo findings, in vitro uptake assays in Pgp- and Bcrp1-overexpressing cell lines confirmed low intracellular accumulation of ELC and TQD at nanomolar concentrations and increased uptake at micromolar concentrations. As this study shows that microdoses can behave pharmacokinetically differently from MDT-inhibitory doses if a compound interacts with MDTs, conclusions from microdose studies should be drawn carefully.

Summary Aim: The partial volume effect (PVE) significantly affects quantitative accuracy in PET. In this study we used a micro-hollow sphere phantom filled with 18F, 11C or 68Ga to evaluate different partial volume correction methods (PVC). Additionally, phantom data were applied on rat brain scans to evaluate PVC methods on in vivo datasets. Methods: The four spheres (7.81, 6.17, 5.02, 3.90 mm inner diameter) and the background region were filled to give sphere-to-background (sph/bg) activity ratios of 20 : 1, 10 : 1, 5 : 1 and 2 : 1. Two different acquisition and reconstruction protocols and three radionuclides were evaluated using a small animal PET scanner. From the obtained images the recovery coefficients (RC) and contrast recovery coefficients (CRC) for the different sph/bg ratios were calculated. Three methods for PVC were evaluated: a RC based, a CRC based and a volume of interest (VOI) based method. The most suitable PVC methods were applied to in vivo rat brain data. Results: RCs were shown to be dependent on the radionuclide used, with the highest values for 18F, followed by 11C and 68Ga. The calculated mean CRCs were generally lower than the corresponding mean RCs. Application of the different PVC methods to rat brain data led to a strong increase in time-activity curves for the smallest brain region (entorhinal cortex), whereas the lowest increase was obtained for the largest brain region (cerebellum). Conclusion: This study was able to show the importance and impact of PVE and the limitations of several PVC methods when performing quantitative measurements in small structures.

A low-molecular-weight tetrazine labeled with the short-lived positron emitter carbon-11 was developed as a bioorthogonal PET probe for pretargeted imaging. A method for efficient and fast synthesis of this imaging agent is presented using radiolabeling of a readily available precursor. High reactivity with trans-cyclooctenes was observed and in vivo investigations including PET/MR scanning showed homogeneous biodistribution, good metabolic stability, and rapid excretion in naive mice. These properties are key to the success of bioorthogonal (11)C-PET imaging, which has been shown in a simple pretargeting experiment using TCO-modified mesoporous silica nanoparticles. Overall, this (11)C-labeled tetrazine represents a highly versatile and advantageous chemical tool for bioorthogonal PET imaging and enables pretargeting approaches using carbon-11 for the first time.