Edvin Wilhelm Johannesen

In vivo pH imaging with (99m)Tc-pHLIP.

PurposeA novel molecular imaging agent has been developed recently, which stains tissues of low extracellular pH [pH (low) insertion peptide, pHLIP®]. A pH-dependent process of peptide folding and insertion into cell membranes has been found in vitro. Targeting of acidic solid tumours has been demonstrated in vivo using fluorescence and PET labels. Here, we present proof of feasibility studies of pHLIP with a single-photon emission computed tomography (SPECT) label, 99mTc-AH114567, with focus on preclinical efficacy and imageability.ProceduresLewis lung carcinoma, lymph node carcinoma of the prostate and prostate adenocarcinoma tumour xenografts were grown in mice and characterised by the angiogenesis marker 99mTc-NC100692 and by extracellular pH measurements with 31P-MRS of 3-aminopropyl phosphonate. Biodistribution was assessed and CT/SPECT imaging performed. Oral administration of bicarbonate served as control.Results and ConclusionTc-AH114567 can be obtained via a robust synthesis with good radiolabelling profile and improved formulation. The tracer retains the pH-dependent ability to insert into membranes and to target tumours with similar pharmacokinetics and efficacy that had been demonstrated earlier for pHLIP with optical or 64Cu PET labels. Despite the inherent challenges of SPECT compared to optical and PET imaging, e.g., in terms of lower sensitivity, 99mTc-AH114567 shows adequate image quality and contrast. The main development need for transitioning SPECT labelled pHLIP into the clinic is more rapid background signal reduction, which will be the focus of a subsequent optimisation study.

A targeted molecular probe for colorectal cancer imaging

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.

Abstract 357: Nonclinical tumor efficacy studies of [18F]AH113804, a novel PET imaging agent with high affinity for the human c-Met receptor

c-Met, the tyrosine-kinase receptor for hepatocyte growth factor (HGF)/scatter factor, is involved in tumour growth, invasion and metastasis in many human cancers of epithelial origin. Various tumour types are reported to overexpress c-Met, whilst expression in most normal tissues is relatively low. [18F]AH113804 is a peptide-based molecular imaging agent being developed by GE Healthcare for the in vivo evaluation of tumour and metastatic c-Met expression by PET imaging. In vitro affinity studies with fluorescently labelled analogues confirmed that whilst the AH113804 peptide had high affinity for human c-Met (hc-Met), there was limited or no affinity for dog or rodent c-Met, respectively. In mice, relatively high uptake of [18F]AH113804 was observed in human xenograft tumours known to express high levels of hc-Met, with rapid clearance from key background tissues such as muscle (tumour to muscle ratio of >5 achieved by 30 minutes post-injection). This biodistribution profile allowed the tumour to be clearly visualised by micro-PET imaging. Tumour uptake was significantly reduced by co-administration of excess non-radioactive peptide, confirming tumour uptake was specific. Tumour uptake of [18F]AH113804 was also shown to correlate with expression of hcMet, with a relative retention of 2.0±0.1, 1.2±0.2 and 0.7±0.2% retained dose per gram normalised for body weight (% rd/g/100g bw) 60 minutes post-injection in xenograft tumours with relatively high (HT-29 tumours), lower (U87 tumours) and no (LLC tumours) expression of hc-Met respectively (as assayed by ELISA). Finally, 111InCl3 was used in a dual tumour model as a non-specific marker of blood pool to confirm differences in tumour uptake were not related to differences in tumour blood pool or delivery. There was a significant difference in [18F]AH113804 uptake between HT-29 (2.2±0.8% rd/g/100 g bw) and LLC (0.9±0.2% rd/g/100 g bw) tumours (p 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 357. doi:1538-7445.AM2012-357

Imaging efficacy of a targeted imaging agent for fluorescence endoscopy

Colorectal cancer is a major cause of cancer death. A significant unmet clinical need exists in the area of screening for earlier and more accurate diagnosis and treatment. We have identified a fluorescence imaging agent targeted to an early stage molecular marker for colorectal cancer. The agent is administered intravenously and imaged in a far red imaging channel as an adjunct to white light endoscopy. There is experimental evidence of preclinical proof of mechanism for the agent. In order to assess potential clinical efficacy, imaging was performed with a prototype fluorescence endoscope system designed to produce clinically relevant images. A clinical laparoscope system was modified for fluorescence imaging. The system was optimised for sensitivity. Images were recorded at settings matching those expected with a clinical endoscope implementation (at video frame rate operation). The animal model was comprised of a HCT-15 xenograft tumour expressing the target at concentration levels expected in early stage colorectal cancer. Tumours were grown subcutaneously. The imaging agent was administered intravenously at a dose of 50nmol/kg body weight. The animals were killed 2 hours post administration and prepared for imaging. A 3-4mm diameter, 1.6mm thick slice of viable tumour was placed over the opened colon and imaged with the laparoscope system. A receiver operator characteristic analysis was applied to imaging results. An area under the curve of 0.98 and a sensitivity of 87% [73, 96] and specificity of 100% [93, 100] were obtained.

Molecular targeting as a contrast agent mechanism for fluorescence endoscopy

Several mechanisms of action can be employed for a molecular imaging contrast agent for use with endoscopy. Targeting of cell surface molecules that are up regulated at an early disease stage, with a fluorescent labelled vector is one attractive approach. However, it suffers from the inherent limitation that the concentration of agent available is fundamentally limited by the concentration of receptor molecules available. Simple models indicate that for successful imaging with a targeting approach, the imaging system should be able to adequately image concentrations in the nanomolar region. Such low reporter molecule concentrations have implications for the choice of contrast agent. Target tissue size and location, the tissue native fluorescence contribution, the brightness of the reporter molecule, and photobleaching thresholds are all factors which contribute to the choice of reporter. For endoscopic imaging of millimetre sized target tissue volumes close to the surface Cy5TM (650-700nm) wavelengths are preferable to Cy3TM (550-600nm) and Cy7TM (750-800nm). We have constructed a system optimised for sensitivity by tailoring light delivery, collection, filtering and detection, in order to address the fundamental technical performance limits for endoscopic applications. It is demonstrated through imaging system calibration, phantom based measurement and animal imaging data that low nanomolar concentrations of Cy5 based fluorescent contrast agent in millimetre sized superficial lesions are adequately imaged with a clinically relevant endoscope system in real time. It is concluded that targeting is a technically viable approach for endoscopic applications.

Abstract 2430: PET imaging of c-Met expression in non-human primates using [18F]AH113804

[ 18 F]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 [ 18 F]AH113804 in the non-human primate. Cynomolgus monkey (Macaca fascicularis) was selected since [ 18 F]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-[ 18 F]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 [ 18 F]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 [ 18 F]AH113804 imaging was performed (GE Discovery 16 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 [ 18 F]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. [ 18 F]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 18 F]AH113804 liver uptake and retention observed after competition with non-radioactive AH113804 reflects specific binding of [ 18 F]AH113804. Dynamic distribution studies showed a rapid uptake in liver and kidney and mainly urinary excretion. There were no obvious effects on body temperature, blood pressure or heart rate following co-injection of 0.15 mg/kg of AH113804. Overall, these results confirmed that [ 18 F]AH113804 has desirable properties for a c-Met targeted molecular imaging agent. Since there are a number of anti-c-Met therapies in clinical development, [ 18 F]AH113804 has potential to be used as an aid in therapy selection and/or monitoring. 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