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An 18 F-labelled human epidermal growth factor receptor (HER2) receptor binding radiotracer is a potential tool to non-invasively identify HER2 positive tumour lesions in subjects with recurrent metastatic breast cancer. Having explored the manual radiochemistry to conjugate the Affibody molecule ZHER2:2891 with [18 F]4-fluorobenzaldehyde, we have developed and optimised a full protocol for the automated GE FASTlab synthesiser. Our chemometric model predicted the best radiochemical purity for a short conjugation time (2.8 minutes), a low temperature (65°C), and a medium Affibody molecule precursor amount (5.5 mg). Under these optimised conditions, [18 F]GE-226 was produced after solid-phase extraction purification with activity yield of 30% ± 7 (n = 18) and a radiochemical purity of 94% ± 2 (n = 18). The synthesis and purification was complete after 43 minutes and provided apparent molar activities of 12 to 30 GBq/μmol (n = 12) at the end of synthesis.

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Gonadotropin releasing hormone (GnRH) is recognized as an important neuromodulator affecting behavior and has been associated with the progression of Alzheimer's disease. The peptide has been shown to have a bidirectional transport through the blood–brain-barrier (BBB), which may account for the cognitive effects of systemically administered GnRH. In this study, four novel 18F-GnRH peptide analogues were synthesized and their in vitro and in vivo characteristics studied in male rats. GnRH peptides were assembled by solid-phase peptide synthesis, either as the full length D-Lys6-GnRH (pyroGlu1-His2-Trp3-Ser4-Tyr5-D-Lys6-Leu7-Arg8-Pro9-Gly10-NH2) or as D-Lys6-desGly10-GnRH-NHEt. In all, four GnRH peptide analogues were synthesized and reacted with N-succinimidyl-4-fluorobenzoate (SFB) to yield the fluorinated versions. Binding affinities of the analogues were determined in a competitive binding assay for both human and rat GnRH receptors. Ki-values for the GnRH peptides were found to be subnanomolar, with D-Lys6(FBA)-desGly10-GnRH-NHEt (7) being most potent with a Ki-value of around 50 pM for GnRH receptor species. Radiolabeling was performed using N-succinimidyl-4-[18F]fluorobenzoate ([18F]SFB) in 33.3 ± 12.8% isolated decay corrected (d.c.) yield within 1.5–2 h. Rat serum stability over 2 h revealed minor degradation (≤5%). For in vivo studies, 18F-peptides (4–30 MBq) were injected intravenously via the tail vein into rats and brain uptake was evaluated by means of dynamic PET (2 h) followed by biodistribution studies. PET showed limited or no uptake in brain for the 18F-peptides which predominantly cleared rapidly by renal excretion. Specific binding in the pituitary gland was confirmed for the 18F-peptide, 7, by blocking with the GnRH agonist buserelin.

Human epidermal growth factor receptor (HER2)–targeted Affibody molecules radiolabeled with 18F allow the noninvasive assessment of HER2 status in vivo through PET imaging. Such agents have the potential to improve patient management by selecting individuals for HER2-targeted therapies and allowing therapy monitoring. The aim of this study was to assess different 18F radiolabeling strategies of the HER2-specific Affibody molecule ZHER2:2891, preclinically determine the biologic efficacy of the different radiolabel molecules, and select a preferred radiolabeling strategy to progress for automated manufacture. Methods: Cysteine was added to the C terminus of the Affibody molecule for the coupling of maleimide linkers, and 3 radiolabeling strategies were assessed: silicon-fluoride acceptor approach (18F-SiFA), 18F-AlF-NOTA, and 4-18F-fluorobenzaldehyde (18F-FBA). The biodistributions of the radiolabeled Affibody molecules were then determined in naive CD-1 nude mice, and tumor targeting was assessed in CD-1 nude mice bearing high-HER2-expressing NCI-N87 tumors and low-HER2-expressing A431 tumors. The 111In-ABY-025 compound, which has demonstrable clinical utility, served as a reference tracer. Results: The non–decay-corrected radiochemical yields based on starting 18F-fluoride using the 18F-FBA, 18F-SiFA, and 18F-AlF-NOTA methods were 13% ± 3% (n = 5), 38% ± 2% (n = 3), and 11% ± 4% (n = 6), respectively. In naive mice, both the 18F-AlF-NOTA-ZHER2:2891 and the 111In-ABY-025 compounds showed a significant kidney retention (70.3 ± 1.3 and 73.8 ± 3.0 percentage injected dose [%ID], respectively, at 90 min after injection), which was not observed for 18F-FBA-ZHER2:2891 or 18F-SiFA-ZHER2:2891 (4.8 ± 0.6 and 10.1 ± 0.7 %ID, respectively, at 90 min). The 18F-SiFA-ZHER2:2891 conjugate was compromised by increasing bone retention over time (5.3 ± 1.0 %ID/g at 90 min after injection), indicating defluorination. All the radiolabeled Affibody molecules assessed showed significantly higher retention in NCI-N87 tumors than A431 tumors at all time points (P

A novel class of alkyne linked [Tyr(3)]octreotate analogues have been labelled by a copper catalysed azide-alkyne cycloaddition reaction (CuAAC) to form a 1,4-substituted triazole using the reagent [(18)F]2-fluoroethyl azide. An unexpected variability in reactivity during the CuAAC reaction was observed for each alkyne analogue which has been investigated. Two lead alkyne linked [Tyr(3)]octreotate analogues, G-TOCA (3a) and βAG-TOCA (5a) have been identified to be highly reactive in the click reaction showing complete conversion to the [(18)F]2-fluoroethyl triazole linked [Tyr(3)]octreotate analogues FET-G-TOCA (3b) and FET-βAG-TOCA (5b) under mild conditions and with short synthesis times (5 min at 20 °C). As well as ease of synthesis, in vitro binding to the pancreatic tumour AR42J cells showed that both FET-G-TOCA and FET-βAG-TOCA have high affinity for the somatostatin receptor with IC(50) of 4.0±1.4, and 1.6±0.2 nM, respectively.

Maltacines comprise a family of cyclic peptide lactone antibiotics produced by a strain of Bacillus subtilis. The previously proposed amino acid sequences of the linear ring-opened molecules show similarity to the lipopeptide antibiotic Fengycin IX that is also produced by a strain of B. subtilis. There were some discrepancies in the Maltacin data that could not be explained. To address this and gain more information into the structure of the linear ring-opened Maltacines, the two members D1c, E1b and Fengycin IX acid were synthesised and their MS2, MS3 and MS4 spectra compared. The similarity of the product ion spectra of Maltacin and Fengycin IX acid revealed that proline occupies an internal position in Maltacin. This finding led to revision of the interpretation of the amino acid sequences of the Maltacines. The proposed new structures of the Maltacines shows that the cyclic part of the molecules is the same as in Fengycin IX acid and Fengycin XII acid, but they have unique N-terminal sequences not found in Fengycins, and thus represent novel lipopeptide antibiotics.

Colon cancer prevention currently relies on colonoscopy using white light to detect and remove polyps, but small and flat polyps are difficult to detect and frequently missed when using this technique. Fluorescence colonoscopy combined with a fluorescent probe specific for a polyp biomarker may improve polyp detection. Here we describe GE-137, a water-soluble probe consisting of a 26-amino acid cyclic peptide that binds the human tyrosine kinase c-Met conjugated to a fluorescent cyanine dye. Intravenous administration of GE-137 leads to its accumulation specifically in c-Met-expressing tumors in mice, and it is safe and well tolerated in humans. Fluorescence colonoscopy in patients receiving intravenous GE-137 enabled visualization of all neoplastic polyps that were visible with white light (38), as well as an additional nine polyps that were not visible with white light. This first-in-human pilot study shows that molecular imaging using an intravenous fluorescent agent specific for c-Met is feasible and safe, and that it may enable the detection of polyps missed by other techniques.

Targeting the molecular pathways associated with angiogenesis offers great potential in detecting disease pathology using in vivo imaging technologies. Initiation of angiogenesis requires activation and migration of endothelial cells in order for neovascularization to proceed. Endothelial cells associate with the extracellular matrix through specific interactions with a variety of cell adhesion receptors known as integrins. Peptides containing the tripeptide sequence RGD are known to bind with high affinity to the alphavbeta3 and alphavbeta5 integrins associated with angiogenesis. We present herein the synthesis and in vitro binding affinity of the RGD-containing peptide NC-100717 and a range of molecular probes derived from this intermediate.

[structure: see text] The combination of the cysteine thiol protecting groups Trt, Acm, tBu, and MeBzl were used for the regioselective formation of an alpha-conotoxin dimer peptide containing four disulfide bridges. Additionally, a protocol is described whereby two one-pot oxidations were employed in order to improve the efficiency of the folding process. The target compound was produced in good yield.

UNLABELLED The incidence and prevalence of gastroenteropancreatic neuroendocrine tumors has been increasing over the past 3 decades. Because of high densities of somatostatin receptors (sstr)--mainly sstr-2--on the cell surface of these tumors, (111)In-diethylenetriaminepentaacetic acid-octreotide scintigraphy has become an important part of clinical management. (18)F-radiolabeled analogs with suitable pharmacokinetics would permit PET with more rapid clinical protocols. METHODS We compared the affinity in vitro and tissue pharmacokinetics by PET of 5 structurally related (19)F/(18)F-fluoroethyltriazole-Tyr(3)-octreotate (FET-TOCA) analogs: FET-G-polyethylene glycol (PEG)-TOCA, FETE-PEG-TOCA, FET-G-TOCA, FETE-TOCA, and FET-βAG-TOCA to the recently described (18)F-aluminum fluoride NOTA-octreotide ((18)F-AIF-NOTA-OC) and the clinical radiotracer (68)Ga-DOTATATE. RESULTS All (19)F-fluoroethyltriazole-Tyr(3)-octreotate compounds retained high agonist binding affinity to sstr-2 in vitro (half-maximal effective concentration, 4-19 nM vs. somatostatin at 5.6 nM). Dynamic PET showed that incorporation of PEG linkers, exemplified by (18)F-FET-G-PEG-TOCA and (18)F-FETE-PEG-TOCA, reduced uptake in high sstr-2-expressing AR42J pancreatic cancer xenografts. (18)F-FET-βAG-TOCA showed the lowest nonspecific uptake in the liver. Tumor uptake increased in the order (68)Ga-DOTATATE < (18)F-AIF-NOTA ≤ (18)F-FET-βAG-TOCA < (18)F-FET-G-TOCA. The uptake of (18)F-FET-βAG-TOCA was specific: a radiolabeled scrambled peptide, (18)F-FET-βAG-[W-c-(CTFTYC)K], did not show tumor uptake; there was lower uptake of (18)F-FET-βAG-TOCA in AR42J xenografts when mice were pretreated with 10 mg of unlabeled octreotide per kilogram; and there was low uptake of (18)F-FET-βAG-TOCA in low sstr-2-expressing HCT116 xenografts. CONCLUSION We have developed novel fluoroethyltriazole-Tyr(3)-octreotate radioligands that combine high specific binding with rapid target localization and rapid pharmacokinetics for high-contrast PET. (18)F-FET-βAG-TOCA and (18)F-FET-G-TOCA are candidates for future clinical evaluation.

The present study demonstrates the usefulness of whole body section fluorescence imaging, a novel technique used in optical imaging drug discovery. This method is in principle an analog of whole body autoradiography, except that fluorescence is measured instead of radioactivity. The method was shown to have a linear concentration-response relationship over a 1000-fold concentration range. Densitometric image analysis allowed semiquantitative studies of drug disposition and selective tumor retention of an optical imaging drug candidate.

Colorectal cancer is a major cause of cancer death. Morbidity, mortality and healthcare costs can be reduced if the disease can be detected at an early stage. Screening is a viable approach as there is a clear link to risk factors such as age. We have developed a fluorescent contrast agent for use during colonoscopy. The agent is administered intravenously and is targeted to an early stage molecular marker for colorectal cancer. The agent consists of a targeting section comprising a peptide, and a fluorescent reporter molecule. Clinical imaging of the agent is to be performed with a far red fluorescence imaging channel (635 nm excitation/660-700 nm emission) as an adjunct to white light colonoscopy. Preclinical proof of mechanism results are presented. The compound has a Kd of ~3nM. Two human xenograft tumour models were used. Tumour cells were implanted and grown subcutaneously in nude mice. Imaging using a fluorescence reflectance imaging system and quantitative biodistribution studies were performed. Substances tested include the targeted agent, and a scrambled sequence of the peptide (no binding) used as a negative control. Competition studies were also performed by co-administration of 180 times excess unlabelled peptide. Positive imaging contrast was shown in the tumours, with a clear relationship to expression levels (confirmed with quantitative biodistribution data). There was a significant difference between the positive and negative control substances, and a significant reduction in contrast in the competition experiment.© (2008) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

Objectives: The purpose of this study was to evaluate the feasibility of noninvasive imaging of angiotensin II (AT) receptor upregulation in a mouse model of post-myocardial infarction (MI) heart failure (HF). Background: Circulating AT levels do not reflect the status of upregulation of renin-angiotensin axis in the myocardium, which plays a central role in ventricular remodeling and evolution of HF after MI. Appropriately labeled AT or AT receptor blocking agents should be able to specifically target AT receptors by molecular imaging techniques. Methods: AT receptor imaging was performed in 29 mice at various time points after permanent coronary artery ligation or in controls using a fluoresceinated angiotensin peptide analog (APA) and radiolabeled losartan. The APA was used in 19 animals for intravital fluorescence microscopy on a beating mouse heart. Tc-99m losartan was used for in vivo radionuclide imaging and quantitative assessment of AT receptor expression in 10 mice. After imaging, hearts were harvested for pathological characterization using confocal and 2-photon microscopy. Results: No or little APA uptake was observed in control animals or within infarct regions on days 0 and 1. Distinct uptake occurred in the infarct area at 1 to 12 weeks after MI; the uptake was at maximum at 3 weeks and reduced markedly at 12 weeks after MI. Ultrasonographic examination demonstrated left ventricular remodeling, and pathologic characterization revealed localization of the APA tracer with collagen-producing myofibroblasts. Tc-99m losartan uptake in the infarct region (0.524 ± 0.212% injected dose/g) increased 2.4-fold as compared to uptake in the control animals (0.215 ± 0.129%; p < 0.05). Conclusions: The present study demonstrates the feasibility of in vivo molecular imaging of AT receptors in the remodeling myocardium. Noninvasive imaging studies aimed at AT receptor expression could play a role in identification of subjects likely to develop heart failure. In addition, such a strategy could allow for optimization of anti-angiotensin therapy in patients after MI. © 2008 American College of Cardiology Foundation.

Antimicrobial peptides (AMPs) are considered to play an important role as host-defense molecules in both vertebrates and invertebrates. This work was undertaken to characterize AMPs from hemocyte extracts of the small spider crab, Hyas araneus. A novel proline-arginine-rich AMP of 37 amino acids was isolated and characterized. The peptide, named arasin 1, has a chimeric structure with an N-terminal domain rich in proline and arginine and a C-terminal domain containing two disulfide linkages. The peptide precursor of 64 amino acids, deduced from a cDNA library, contained a hydrophobic pre-region of 25 amino acids, directly followed by the mature peptide. C-terminally, this precursor had two additional amino acids, which seem to be cleaved off post-translationally. Synthetic arasin 1 showed antibacterial activity. A putative isoform of arasin 1, named arasin 2, was found at the genetic level, and both transcripts were shown by real-time RT-PCR to be expressed mainly in hemocytes.

Background: The assessment of HER2 expression in biopsies from primary lesions of breast cancer patients is a standard procedure to select patients for HER2-targeted therapies. However, in metastatic disease in which HER2 status can change, determination of HER2 expression is not standard procedure, which complicates patient management. Herein we report evidence for GE226 ([18F]FBA-ZHER2:2891), a highly specific HER2 targeted imaging agent that can determine HER2 expression levels in preclinical tumour models. We propose that GE226 can be progressed to clinical development for non-invasive determination of the HER2 status in recurrent breast cancer patients to improve the clinical management and therapy selection. Methods: The highly selective HER2 targeted 18F-labelled Affibody molecule GE226 was characterized in a murine dual tumour breast cancer model bearing NCI-N87 (high HER2 status) and A431 (low HER2 status) xenografts in separate flanks. Tumour-bearing mice were injected with 3 to 10 MBq of GE226, followed by biodistribution or positron emission tomography (PET) imaging analysis. Results: Biodistribution analysis demonstrated good differentiation of GE226 retention between high and low HER2 expressing tumours (8.4% ID/g and 3.4% ID/g respectively, 30 min post injection). GE226 cleared quickly from background tissue, including kidneys, with excellent ratios for tumour-to-muscle (8.9 for high HER2 status tumours and 3.6 for low HER2 status tumours, 30 min post injection) and tumour-to-blood (2.5 for high HER2 status tumours and 1.0 for low HER2 status tumours, 30 min post injection). PET imaging of GE226 in the dual tumour mouse model showed a marked difference in signal intensity between the two tumour types. Conclusions: The highly selective HER2 targeted Affibody molecule GE226 can image different levels of HER2 expression in a dual-tumour preclinical model of breast cancer with good target-to-background ratios. These data compare favourably with previous patient SPECT and PET studies using the 111In- or 68Ga-labelled HER2-binding Affibody molecule ABY-002 (Baum et al., J Nucl Med. 2010;51(6):892-7) which supports the efficacy of this class of Affibody tracer for visualization of HER2 expressing metastases. We plan to progress GE226 further to assess HER2 status in metastatic breast cancer patients in clinical PET studies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 353. doi:1538-7445.AM2012-353

[18F]AH113804 is a peptide based molecular imaging agent with high affinity for the human c-Met receptor. The objective of this study was to investigate the biodistribution, specific uptake and elimination of [18F]AH113804 in the non-human primate. Cynomolgus monkey (Macaca fascicularis) was selected since [18F]AH113804 retains high affinity to c-Met in this species. The route of administration was intravenous as this is the route of administration in humans. The target compound was prepared in a two-step synthesis route starting with synthesis of 4-[18F]fluorobenzaldehyde which was reacted with the peptide precursor to produce the corresponding oxime. Following semipreparative HPLC purification the pure product was obtained. Frozen section autoradiography using Rhesus monkey (Macaca mulatta) liver (relatively high c-Met expression) and muscle (relatively low c-Met) was performed in PBS buffer with [18F]AH113804 (2.5 kBq/ml) and with and without 0.5 μg/ml of AH113804 to establish specific binding in vitro. In vivo dynamic and repeated whole body PET/CT [18F]AH113804 imaging was performed (GE Discovery16 ST scanner, General Electric, USA), in female Cynomolgus monkeys (n=3) before and after administration of 0.15 mg/ml AH113804 or AH112361 (a scrambled peptide i.e. a negative control). The binding of [18F]AH113804 in Rhesus monkey liver sections was blocked by 40% following co-incubation of unlabelled AH113804 (>1000-fold excess) whereas binding in muscle was unaffected. This result demonstrated specific binding in vitro in Rhesus monkey liver with a certain amount of unspecific binding. In vivo PET imaging studies in the Cynomolgus monkey showed rapid uptake in liver which was reduced by 42% after co-injection of 0.15 mg/kg of AH113804. Consistent with in vitro results, there was no noteworthy muscle retention in vivo. In liver the individual SUV varied but the magnitude of effect by blockade was similar. The ratio of baseline to blockade scans in liver was 1.7, suggesting the presence of specific binding. [18F]AH113804 uptake in liver or muscle was however not significantly reduced after co-injection of 0.15 mg/kg of AH112361 (negative control), the ratio of baseline and blockade scans in liver was Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2430. doi:1538-7445.AM2012-2430