Design, synthesis, in vitro characterization and preliminary imaging studies on fluorinated bile acid derivatives as PET tracers to study hepatic transporters.

With the aim of identifying a fluorinated bile acid derivative that could be used as [18F]-labeled Positron Emission Tomography (PET) tracer for imaging the in vivo functioning of liver transporter proteins, and particularly of OATP1B1, three fluorinated bile acid triazole derivatives of cholic, deoxycholic and lithocholic acid (CATD, DCATD and LCATD 4a-c, respectively) were synthesized and labeled with tritium. In vitro transport properties were studied with cell-based assays to identify the best substrate for OATP1B1. In addition, the lead compound, LCATD (4c), was tested as a substrate of other liver uptake transporters OATP1B3, NTCP and efflux transporter BSEP to evaluate its specificity of liver transport. The results suggest that 4c is a good substrate of OATP1B1 and NTCP, whereas it is a poor substrate of OATP1B3. The efflux transporter BSEP also appears to be involved in the excretion of 4c from hepatocytes. The automated radiosynthesis of [18F]-4c was accomplished in a multi-GBq scale and a pilot imaging experiment in a wild type rat was performed after i.v. administration to assess the biodistribution and clearance of the tracer. PET imaging revealed that radioactivity was primarily located in the liver (tmax=75s) and cleared exclusively through the bile, thus allowing to image the hepatobiliary excretion of bile acids in the animal model. These findings suggest that [18F]-LCATD 4c is a promising PET probe for the evaluation of hepatic transporters OATP1B1, NTCP and BSEP activity with potential for studying drug-drug interactions and drug-induced toxicity involving these transporters.

Last-Step Enzymatic [(18) F]-Fluorination of Cysteine-Tethered RGD Peptides Using Modified Barbas Linkers.

We report a last-step fluorinase-catalyzed [(18) F]-fluorination of a cysteine-containing RGD peptide. The peptide was attached through sulfur to a modified and more hydrophilic variant of the recently disclosed Barbas linker which was itself linked to a chloroadenosine moiety via a PEGylated chain. The fluorinase was able to use this construct as a substrate for a transhalogenation reaction to generate [(18) F]-radiolabeled RGD peptides, which retained high affinity to cancer-cell relevant αv ß3 integrins.

Glucose uptake by the brain on chronic high-protein weight-loss diets with either moderate or low amounts of carbohydrate.

Previous work has shown that hunger and food intake are lower in individuals on high-protein (HP) diets when combined with low carbohydrate (LC) intakes rather than with moderate carbohydrate (MC) intakes and where a more ketogenic state occurs. The aim of the present study was to investigate whether the difference between HPLC and HPMC diets was associated with changes in glucose and ketone body metabolism, particularly within key areas of the brain involved in appetite control. A total of twelve men, mean BMI 34·9 kg/m², took part in a randomised cross-over trial, with two 4-week periods when isoenergetic fixed-intake diets (8·3 MJ/d) were given, with 30% of the energy being given as protein and either (1) a very LC (22 g/d; HPLC) or (2) a MC (182 g/d; HPMC) intake. An ¹8fluoro-deoxyglucose positron emission tomography scan of the brain was conducted at the end of each dietary intervention period, following an overnight fast (n 4) or 4 h after consumption of a test meal (n 8). On the next day, whole-body ketone and glucose metabolism was quantified using [1,2,3,4-¹³C]acetoacetate, [2,4-¹³C]3-hydroxybutyrate and [6,6-²H2]glucose. The composite hunger score was 14% lower (P= 0·013) for the HPLC dietary intervention than for the HPMC diet. Whole-body ketone flux was approximately 4-fold greater for the HPLC dietary intervention than for the HPMC diet (P< 0·001). The 9-fold difference in carbohydrate intakes between the HPLC and HPMC dietary interventions led to a 5% lower supply of glucose to the brain. Despite this, the uptake of glucose by the fifty-four regions of the brain analysed remained similar for the two dietary interventions. In conclusion, differences in the composite hunger score observed for the two dietary interventions are not associated with the use of alternative fuels by the brain.

Efficient bioconjugation of 5-fluoro-5-deoxy-ribose (FDR) to RGD peptides for positron emission tomography (PET) imaging of α(v)ß(3) integrin receptor.

The utility of 5-fluoro-5-deoxyribose (FDR) as an efficient bioconjugation agent for radiolabelling of the RGD peptides c(RGDfK) and c(RGDfC) is demonstrated. The bioconjugation is significantly superior to that achieved with 2-fluoro-2-deoxyglucose (FDG) and benefits from the location of the fluorine at C-5, and that ribose is a 5-membered ring sugar rather than a 6-membered ring. Both features favour ring opening to the aldehydic form of the sugar to promote smooth oxime ligation with aminooxy ether functionalised peptides. [(18)F]FDR was prepared in this study by synthesis from fluoride-18 using an automated synthesis protocol adapting that used routinely for [(18)F]FDG. c(RGDfK) was functionalised with an aminooxyacetyl group (Aoa) via its lysine terminus, while c(RGDfC) was functionalised with an aminooxyhexylmaleimide (Ahm) through a cysteine-maleimide conjugation. Bioconjugation of [(18)F]FDR to c(RGDfC)-Ahm proved to be more efficient than c(RGDfK)-Aoa (92% versus 65%). The unlabelled ((19)F) bioconjugates c(RGDfK)-Aoa-FDR and c(RGDfC)-Ahm-FDR were prepared and their in vitro affinity to purified integrin αvß3 was determined. c(RGDfK)-Aoa-FDR showed the greater affinity. Purified "hot" bioconjugates c(RGDfK)-Aoa-[(18)F]FDR and c(RGDfC)-Ahm-[(18)F]FDR were assayed by incubation with MCF7, LNCaP and PC3 cell lines. In both cases the conjugated RGD peptides showed selectivity for PC3 cells, which express αvß3 integrin, with the c(RGDfK)-Aoa-[(18)F]FDR demonstrating better binding, consistent with its higher in vitro affinity. The study demonstrates that [(18)F]FDR is an efficient bioconjugation ligand for RGD bioactive peptides.

Metabolic alterations in a rat model of takotsubo syndrome.

AIMS: Cardiac energetic impairment is a major finding in takotsubo patients. We investigate specific metabolic adaptations to direct future therapies. METHODS AND RESULTS: An isoprenaline-injection female rat model (vs. sham) was studied at Day 3; recovery assessed at Day 7. Substrate uptake, metabolism, inflammation, and remodelling were investigated by 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography, metabolomics, quantitative PCR, and western blot (WB). Isolated cardiomyocytes were patch-clamped during stress protocols for redox states of NAD(P)H/FAD or [Ca2+]c, [Ca2+]m, and sarcomere length. Mitochondrial respiration was assessed by seahorse/Clark electrode (glycolytic and ß-oxidation substrates). Cardiac 18F-FDG metabolic rate was increased in takotsubo (P = 0.006), as was the expression of GLUT4-RNA/GLUT1/HK2-RNA and HK activity (all P < 0.05), with concomitant accumulation of glucose- and fructose-6-phosphates (P > 0.0001). Both lactate and pyruvate were lower (P < 0.05) despite increases in LDH-RNA and PDH (P < 0.05 both). ß-Oxidation enzymes CPT1b-RNA and 3-ketoacyl-CoA thiolase were increased (P < 0.01) but malonyl-CoA (CPT-1 regulator) was upregulated (P = 0.01) with decreased fatty acids and acyl-carnitines levels (P = 0.0001-0.02). Krebs cycle intermediates α-ketoglutarate and succinyl-carnitine were reduced (P < 0.05) as was cellular ATP reporter dihydroorotate (P = 0.003). Mitochondrial Ca2+ uptake during high workload was impaired on Day 3 (P < 0.0001), inducing the oxidation of NAD(P)H and FAD (P = 0.03) but resolved by Day 7. There were no differences in mitochondrial respiratory function, sarcomere shortening, or [Ca2+] transients of isolated cardiomyocytes, implying preserved integrity of both mitochondria and cardiomyocyte. Inflammation and remodelling were upregulated-increased CD68-RNA, collagen RNA/protein, and skeletal actin RNA (all P < 0.05). CONCLUSION: Dysregulation of glucose and lipid metabolic pathways with decreases in final glycolytic and ß-oxidation metabolites and reduced availability of Krebs intermediates characterizes takotsubo myocardium. The energetic deficit accompanies defective Ca2+ handling, inflammation, and upregulation of remodelling pathways, with the preservation of sarcomeric and mitochondrial integrity.

Preclinical Evaluation of [18F]LCATD as a PET Tracer to Study Drug-Drug Interactions Caused by Inhibition of Hepatic Transporters.

The bile acid analogue [18F]LCATD (LithoCholic Acid Triazole Derivative) is transported in vitro by hepatic uptake transporters such as OATP1B1 and NTCP and efflux transporter BSEP. In this in vivo "proof of principle" study, we tested if [18F]LCATD may be used to evaluate drug-drug interactions (DDIs) caused by inhibition of liver transporters. Hepatic clearance of [18F]LCATD in rats was significantly modified upon coadministration of rifamycin SV or sodium fusidate, which are known to inhibit clinically relevant uptake transporters (OATP1B1, NTCP) and canalicular hepatic transporters (BSEP) in humans. Treatment with rifamycin SV (total dose 62.5 mg·Kg-1) reduced the maximum radioactivity of [18F]LCATD recorded in the liver from 14.2 ± 0.8% to 10.2 ± 0.9% and delayed t_max by 90 seconds relative to control rats. AUCliver 0-5 min, AUCbile 0-10 min and hepatic uptake clearance CLuptake,in vivo of rifamycin SV treated rats were significantly reduced, whereas AUCliver 0-30 min was higher than in control rats. Administration of sodium fusidate (30 mg·Kg-1) inhibited the liver uptake of [18F]LCATD, although to a lesser extent, reducing the maximum radioactivity in the liver to 11.5 ± 0.3%. These preliminary results indicate that [18F]LCATD may be a good candidate for future applications as an investigational tracer to evaluate altered hepatobiliary excretion as a result of drug-induced inhibition of hepatic transporters.

Decreased [18F]fluoro-2-deoxy-d-glucose incorporation and increased glucose transport are associated with resistance to 5FU in MCF7 cells in vitro.

INTRODUCTION: Tumor refractoriness to chemotherapy is frequently due to the acquisition of resistance. Resistant cells selected by exposure to chemotherapy agents may exhibit differences in [18F]fluoro-2-deoxy-d-glucose (FDG) incorporation, as compared with sensitive cells. METHODS: FDG incorporation, hexokinase (HK) activity, glucose transport and ATP content were determined in clones of 5-fluorouracil (5FU)-resistant MCF7 cells, established by long-term exposure to increasing 5FU concentrations, and in parental MCF7 cells. RESULTS: FDG incorporation was decreased in MCF7 cells resistant to 5FU; HK activity was similar in the resistant and sensitive cells, while glucose transport was increased, as compared with sensitive cells. Treatment of cells with the glucose efflux inhibitor phloretin increased FDG incorporation to similar levels in the resistant and sensitive cells. Analysis of microarray data demonstrated the expression of GLUT1, 8 and 10 transporters in MCF7 cells. GLUT8 and 10 expression was decreased in the resistant cells, while GLUT1 was only increased in cells resistant to the lowest 5FU concentration. CONCLUSION: FDG incorporation in 5FU-resistant MCF7 cells is decreased, as compared with sensitive cells. Our findings also suggest that this may be due to high rates of membrane glucose transport in the resistant cells resulting in enhanced efflux of FDG. We believe that this is the first demonstration that facilitative glucose transporters can actually decrease the incorporation of FDG.

Fully-automated radiosynthesis and in vitro uptake investigation of [N-methyl-¹¹C]methylene blue.

Malignant melanoma is a type of skin cancer which can spread rapidly if not detected early and left untreated. Positron Emission Tomography (PET) is a powerful imaging technique for detecting cancer but with only a limited number of radiotracers available the development of novel PET probes for detection and prevention of cancer is imperative. In the present study we present the fully-automated radiosynthesis of [N-methyl-(11)C]methylene blue and an in vitro uptake study in metastasic melanoma cell lines. Using the GE TRACERlab FXc Pro module [N-methyl-(11)C]methylene blue was isolated via solid-phase extraction in an average time of 36 min after end of bombardment and formulated with a radiochemical purity greater than 95%. The in vitro uptake study of [N-methyl-(11)C]methylene blue in SK-MEL28 melanin-expressing melanoma cell line demonstrated in site-specific binding of 51% promoting it as a promising melanoma PET imaging agent.

18F-barbiturates are PET tracers with diagnostic potential in Alzheimer's disease.

Three fluoro-barbiturates were synthesised, showing in vivo sedative efficacy. One of them, [(18)F], was synthesised in radiofluorinated form. PET/CT Imaging with [(18)F] identified ß-amyloid over-expressing transgenic mice (ßA mice) compared to wild type and tau lines. The fluorescent barbiturate 9 was able to label ßA plaques in brain sections of ßA mice, and co-localise with a fluorescent Zn(II) indicator.

Tumour imaging by Positron Emission Tomography using fluorinase generated 5-[¹8F]fluoro-5-deoxyribose as a novel tracer.

INTRODUCTION: 5-[(18)F]Fluoro-5-deoxyribose ([(18)F]FDR) 3 was prepared as a novel monosaccharide radiotracer in a two-step synthesis using the fluorinase, a C-F bond forming enzyme, and a nucleoside hydrolase. The resulting [(18)F]FDR 3 was then explored as a radiotracer for imaging tumours (A431 human epithelial carcinoma) by positron emission tomography in a mice model. METHODS: 5-[(18)F]Fluoro-5-deoxyribose ([(18)F]FDR) 3, was prepared by incubating S-adenosyl-L-methionine (SAM) and [(18)F]fluoride with the fluorinase enzyme, and then incubating the product of this reaction, [(18)F]-5'-fluoro-5'-deoxadenosine ([(18)F]FDA) 2, with an adenosine hydrolase to generate [(18)F]FDR 3. The enzymes were freeze-dried and were used on demand by dissolution in buffer solution. The resulting [(18)F]FDR 3 was then administered to four mice that had tumours induced from the A431 human epithelial carcinoma cell line. RESULTS: The tumour (A431 human epithelial carcinoma) bearing mice were successfully imaged with [(18)F]FDR 3. The radiotracer displayed good tumour imaging resolution. A direct comparison of the uptake and efflux of [(18)F]FDR 3 with 2-[(18)F]fluoro-2-deoxyglucose ([(18)F]FDG) was made, revealing comparative tumour uptake and imaging potential over the first 10-20min. The study revealed however that [(18)F]FDR 3 does not accumulate in the tumour as efficiently as [(18)F]FDG over longer time periods. CONCLUSIONS: [(18)F]FDR 3 can be rapidly synthesised in a two enzyme protocol and used to image tumours in small animal models.

[18F]-5-Fluoro-5-deoxyribose, an efficient peptide bioconjugation ligand for positron emission tomography (PET) imaging.

[(18)F]-5-Fluoro-5-deoxyribose ([(18)F]-FDR) conjugates much more rapidly than [(18)F]-FDG under mild reaction conditions to peptides and offers new prospects for mild and rapid bioconjugation for fluorine-18 labelling in PET imaging.

An effective technique for the storage of short lived radioactive gaseous waste.

An effective technique is described to deal with volatile, short lived radioactive waste generated as a result of the routinely produced positron emission tomography (PET) radiopharmaceutical 2-deoxy-2-[(18)F]fluoro-D-glucose (FDG). All radioactive gases and aerosols created during the synthesis are collected and stored safely in commercially available TEDLAR gas sampling bags. Once these collected PET by-products decay, the TEDLAR gas bags can be easily emptied and reused. This improved technique is effective, safe, reliable and economical.

F-18-FDG and C-11-Choline Positron Emission Tomography in Human Esophago-Gastric Cancer: Prediction of Response to Therapy.

BACKGROUND: To determine the utility of F-18-FDG and C-11-Choline uptake, in patients with esophageal and esophago-gastric junction tumors who are to undergo either neo-adjuvant or palliative chemotherapy, in predicting response (pathological and survival). METHODS: Eighteen patients with biopsy proven cancer were recruited prospectively. Patients underwent PET imaging before and during the first cycle of chemotherapy (seven and 14 days) with both F-18-FDG and C-11-Choline. Tracer uptake was quantified using Standardized Uptake Values. Pathological tumor response was determined using the Mandard criteria. Cellular proliferation was determined using ki-67 immunohistochemistry. Relationships between tracer uptake and response, one-year survival and cellular proliferation were determined. RESULTS: All 18 tumors were imaged by F-18-FDG PET compared to 16/18 with C-11-Choline. Change in uptake of either tracer did not correlate with pathological response. Pathological response did not influence survival (median-survival, responders = 16.1 months; non-responders = 19.0 months, p = 0.978). There was no significant correlation of change in tracer uptake with survival. C-11-Choline tumor uptake did not correlate with cellular proliferation. CONCLUSION: F-18-FDG PET is superior for imaging of the primary tumor. Neither F-18-FDG nor C-11-Choline PET was able to predict response accurately.

An enzymatic route to 5-deoxy-5-[18F]fluoro-D-ribose, a [18F]-fluorinated sugar for PET imaging.

An efficient two-step, one-pot, biotransformation involving the fluorinase enzyme is described for the synthesis of 5-deoxy-5-[(18)F]fluororibose, a novel [(18)F]-fluorinated sugar suitable for positron emission tomography (PET) applications.

Monitoring primary breast cancer throughout chemotherapy using FDG-PET.

UNLABELLED: We have compared 2-deoxy-2-[(18)F]-fluoro-D-glucose positron emission tomography (FDG-PET) images of large or locally advanced breast cancers (LABC) acquired during Anthracycline-based chemotherapy. The purpose was to determine whether there is an optimal method for defining tumour volume and an optimal imaging time for predicting pathologic chemotherapy response. METHOD: PET data were acquired before the first and second cycles, at the midpoint and at the endpoint of neoadjuvant chemotherapy. FDG uptake was quantified using the mean and maximum standardized uptake values (SUV) and the coefficient of variation within a region of interest. Receiver-operator characteristic (ROC) analysis was used to determine the discrimination between tumours demonstrating a high pathological response (i.e. those with greater than 90% reduction in viable tumour cells) and low pathological response. RESULTS: Only tumours with an initial tumour to background ratio (TBR) of greater than five showed a difference between response categories. In terms of response discrimination, there was no statistically significant advantage of any of the methods used for image quantification or any of the time points. The best discrimination was measured for mean SUV at the midpoint of therapy, which identified 77% of low responding tumours whilst correctly identifying 100% of high responding tumours and had an ROC area of 0.93. CONCLUSION: FDG-PET is efficacious for predicting the pathologic response of most primary breast tumours throughout the duration of a neoadjuvant chemotherapy regimen. However, this technique is ineffective for tumours with low image contrast on pre-therapy PET scans.