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.

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.

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.

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-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.

[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 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.

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.