Lutz Schweiger

Monitoring primary breast cancer throughout chemotherapy using FDG-PET.

We have compared 2-deoxy-2-[18F]-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.

Attenuation correction in PET using consistency conditions and a three-dimensional template

Attenuation correction is required in positron emission tomography for accurate quantitation and to reduce artifacts. In previous work, the authors have shown that consistency information can be used to perform attenuation correction in the absence of any transmission measurements. In the previous work, a separate object was used for each slice and was transformed in two dimensions. Here, the authors introduce an extension that performs three-dimensional transformations on a single object over many slices. The aim is to produce a useful attenuation correction in situations where either it is impractical to acquire transmission data or there is significant misregistration between the transmission and emission data. Results are presented for simulated test cases with uniform and nonuniform attenuation and experimental cardiac phantom data. The algorithm as it stands is too unstable to allow for accurate attenuation correction without any transmission measurements. However, it may be useful for correcting misregistration, and may possibly be extended to perform coregistration.

Strategies towards functionalised electronically conducting organic copolymers

Here we describe the synthesis and electrochemical polymerisation of 2,5-di(2-thienyl)-3-(3-cyanopropyl)pyrrole, 2,5-di(2-thienyl)-3-(3-cyanopropyl)furan, and 3′-(3-cyanopropyl)-2,2′:5′,2″-terthiophene. We report a synthetic methodology to these important conducting polymer precursor compounds that is facile, convenient and flexible. The key precursor to this study is the functionalised diketone 1,4-bis(2-thienyl)-2-(3-cyanopropyl)butane-1,4-dione. This molecule undergoes convenient ring closure to the terthiophene and dithienylpyrrole and dithienylfuran derivatives, all of which are, to our knowledge, new compounds. Importantly, this approach provides a flexible route to a range of heterocyclic polymer precursors because the cyanoalkyl functionality is grafted to the diketone before ring closure. Subsequently the nitrile group provides synthetic utility either by reduction to the amine, or hydrolysis to the carboxylic acid. The new compounds described here undergo electrochemical polymerisation leading to fixed ratio copolymers of functionalised pyrrole, thiophene and furan with thiophene itself. We describe the characterisation of these polymers using FT-IR and X-ray photoelectron spectroscopies.

Tumour expression of leptin is associated with chemotherapy resistance and therapy-independent prognosis in gastro-oesophageal adenocarcinomas

Background:Cytotoxic chemotherapy remains the main systemic therapy for gastro-oesophageal adenocarcinoma, but resistance to chemotherapy is common, resulting in ineffective and often toxic treatment for patients. Predictive biomarkers for chemotherapy response would increase the probability of successful therapy, but none are currently recommended for clinical use. We used global gene expression profiling of tumour biopsies to identify novel predictive biomarkers for cytotoxic chemotherapy.Methods:Tumour biopsies from patients (n=14) with TNM stage IB–IV gastro-oesophageal adenocarcinomas receiving platinum-based combination chemotherapy were used as a discovery cohort and profiled with Affymetrix ST1.0 Exon Genechips. An independent cohort of patients (n=154) treated with surgery with or without neoadjuvant platinum combination chemotherapy and gastric adenocarcinoma cell lines (n=22) were used for qualification of gene expression profiling results by immunohistochemistry. A cisplatin-resistant gastric cancer cell line, AGS Cis5, and the oesophageal adenocarcinoma cell line, OE33, were used for in vitro validation investigations.Results:We identified 520 genes with differential expression (Mann–Whitney U, P<0.020) between radiological responding and nonresponding patients. Gene enrichment analysis (DAVID v6.7) was used on this list of 520 genes to identify pathways associated with response and identified the adipocytokine signalling pathway, with higher leptin mRNA associated with lack of radiological response (P=0.011). Similarly, in the independent cohort (n=154), higher leptin protein expression by immunohistochemistry in the tumour cells was associated with lack of histopathological response (P=0.007). Higher leptin protein expression by immunohistochemistry was also associated with improved survival in the absence of neoadjuvant chemotherapy, and patients with low leptin protein-expressing tumours had improved survival when treated by neoadjuvant chemotherapy (P for interaction=0.038). In the gastric adenocarcinoma cell lines, higher leptin protein expression was associated with resistance to cisplatin (P=0.008), but not to oxaliplatin (P=0.988) or 5fluorouracil (P=0.636). The leptin receptor antagonist SHLA increased the sensitivity of AGS Cis5 and OE33 cell lines to cisplatin.Conclusions:In gastro-oesophageal adenocarcinomas, tumour leptin expression is associated with chemoresistance but a better therapy-independent prognosis. Tumour leptin expression determined by immunohistochemistry has potential utility as a predictive marker of resistance to cytotoxic chemotherapy, and a prognostic marker independent of therapy in gastro-oesophageal adenocarcinoma. Leptin antagonists have been developed for clinical use and leptin and its associated pathways may also provide much needed novel therapeutic targets for gastro-oesophageal adenocarcinoma.

Fluorinase-Coupled Base Swaps: Synthesis of [18F]-5′-Deoxy-5′-fluorouridines†

The application of positron emission tomography (PET) for medical imaging and diagnostics is a rapidly growing area, and cyclotrons are being increasingly commissioned in major hospitals. Developing methods for the introduction of the appropriate isotopes (C, N, F, and O) into organic structural motifs is a major research activity at present. The relatively long half-life of F (t1/2 = 110 min) renders it an attractive radioisotope for PET, and synthetic protocols employing [F]fluoride ion are particularly attractive as this form of the isotope is generated in a very high specific activity in which a cold carrier ([F]fluoride) is not added. Chemical strategies for the incorporation of F into organic compounds are being widely explored, but enzymatic approaches offer a unique, mild, and selective approach for fluorination. We have been interested in using the fluorinase enzyme (E.C. 2.5.1.63, Streptomyces cattleya) as a catalyst for F C bond formation. The enzyme catalyzes the nucleophilic attack of fluoride ion to the C5’ center of (S)-adenosyl-l-methionine (SAM) to generate 5’-deoxy-5’-fluoroadenosine (5’-FDA) and l-methionine. Although the fluorinase is a relatively slow enzyme, it is readily available by overexpression from E. coli. It is easily obtained in milligram per milliliter quantities, is stable for long periods, and can be used at micromolar concentrations. [F]Fluoride is generated by the cyclotron at nanomolar concentrations; therefore the enzyme is usually in excess which can overcome sluggish reaction rates. We recently demonstrated the synthesis of 5’-[F]FDA in high radiochemical yield (RCY) using fluorinase in this manner. More generally, radiolabeled nucleosides are being extensively studied as possible tracers for the assessment of tumor biochemistry. The presence of adenosine receptors and specific uridine receptors in the brain increases the significance of these compounds as tracers for neurological studies. Combining fluorinase with nucleoside-converting enzymes offered an attractive strategy for the preparation of radiolabeled nucleosides. Herein we report that 5’-deoxy5’-fluoronucleosides can be prepared using fluorinase combined with appropriate nucleoside phosphorylases. The reaction of 5’-FDA with a purine nucleoside phosphorylase (PNP, E.C. 2.4.2.1) generates 5-deoxy-5-fluoro-a,d-ribose-1phosphate (5-FDRP) in situ. The reversibility of this reaction offers the potential for swapping adenine for another purine base. PNPs from various sources have been identified but only a few catalyze the depurination of adenosine. In this study the PNP from S. cattleya was used because 5’-FDA is its natural substrate. Accordingly, incubation of the fluorinase and fluoride ion with PNP and 2,6-diaminopurine resulted in the accumulation of the fluorinated nucleoside 1 (Scheme 1). The substrate specificity of this PNP is restricted to adenine analogues having an amine at C6 of the purine. To extend the versatility of the base-swap protocol, reactions with a pyrimidine nucleoside phosphorylase (PyNP, EC 2.4.2.2 Bacillus stearothermophilus) were explored. This enzyme catalyses the reversible phosphorolysis of both uridine and thymidine by displacing the pyrimidine

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.

Synthesis and biological evaluation of novel 2,3-dihydro-1H-1,5-benzodiazepin-2-ones; potential imaging agents of the metabotropic glutamate 2 receptor

A focused library of novel 2,3-dihydro-1H-1,5-benzodiazepin-2-ones containing sites for 11C-, 18F- and 123I-labelling have been prepared and evaluated against membrane expressing human recombinant metabotropic glutamate 2 receptor (mGluR2). The compounds were found to be non-competitive antagonists with nanomolar affinity. HPLC evaluation of the physiochemical properties of these compounds identified two candidates for PET and SPECT imaging of mGluR2.

Last-step enzymatic [18F]-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 β3 integrins.

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.

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.