Summary

The growth hormone secretagogue receptor type 1a (GHS-R1a) is the known biological relevant receptor of the endogenous ligand and pleiotropic hormone Ghrelin (acronym growth hormone release inducing), which mediates a broad range of complex biological functions, such as regulation of the body weight, body composition and energy expenditure. Besides GHS-R1a, a truncated form of the receptor exists which is termed GHS-R1b. In contrast to GHS-R1a, GHS-R1b does not bind Ghrelin and is completely inactive. Ghrelin and its receptor GHS-R1a are widely expressed in normal tissues but also in various tumors, including human pituitary adenomas, endocrine neoplasms of the lung, stomach, pancreas, breast, ovarian cancer and prostate carcinomas.
Physiologically, Ghrelin is mainly involved in the positive regulation of energy homeostasis, hunger and body weight gain. The orexigenic mode of action of Ghrelin is well established and occurs via the activation of NPY/AgRP neurons in the hypothalamic arcuate nucleus. The GHS-R1a exhibits unusual high constitutive activity with ∼50% of its maximal capacity in the absence of the agonist (Ghrelin) GHS-R1a induces constant appetite and triggers food intake between meals. In addition, Ghrelin receptors are involved in a series of biological processes including glucose homeostasis, GH-release, gastric motility, regulation of arterial pressure, bone metabolism, heart disease, and immune reactions. Moreover, Ghrelin is a potent anti-inflammatory mediator both in vitro and in vivo and a promising therapeutic agent in the treatment of cachexia, anorexia, age-related disorders, inflammatory diseases and injury. Through the MAPK signaling cascade Ghrelin can induce cell proliferation and could thereby play an important role in cancer. The growing knowledge about the interaction of Ghrelin with tumor cells suggests functional effects of Ghrelin on the tumor itself and the physiology of the body [14–17]. GHS-R1a is expressed in the prostate cancer cell lines PC-3, DU-145 and LnCAP and these cells can also secrete mature Ghrelin. These cells therefore represent a suitable model for in vitro experiments and in vivo studies as xenograft tumor models.
Although widely studied as a promising drug target, the knowledge about Ghrelin signaling, behavior, dynamic interactions with its receptor and functional receptor expression in vivo is still limited and basic bioscientific research is warranted to further evaluate the safety and benefits of Ghrelin drug treatment in patients with cancer. In vivo imaging of the Ghrelin receptor should help to improve the understanding of its mode of action and might become a powerful tool for diagnosis and drug development. So far, only few groups tried to develop probes for PET and optical imaging of Ghrelin receptors. In particular the group of Lewis and colleagues started the research and development of PET radiotracers for imaging of the Ghrelin receptor. The emergence of accessible imaging techniques such as small animal PET could be very valuable to provide in vivo pharmacokinetic information all along the drug discovery process.
In order to develop imaging probes targeting the Ghrelin receptor, the inverse agonist radiotracer ⁶⁸Ga-NODAGA-KwFwLL-NH₂ 1 was previously designed for PET imaging. Despite a high metabolic stability and a broad biodistribution in rats, its poor potency prevented its use for further receptor dynamic studies or therapeutic application. More recently, the hexapeptide K-(D-1-Nal)-FwLL-NH₂ 2 was developed and showed a very high inverse agonist potency toward the Ghrelin receptor as supported by the significantly decreased food intake in rats.
The focus of the present study of the authors was to develop potent inverse agonist radiotracers targeting the Ghrelin receptor as potential imaging and therapeutic agent. The in vivo accumulation of the radiotracer has been used as biomarker in diagnostics and therapy control. The observed agonistic ligand-mediated internalization of the GHSR-1a could help to extend the retention of the small molecular ligand in the target tissue. Therefore, the heptapeptide KK-(D-1-Nal)-FwLL-NH₂ 2 was functionalized on solid support with Palmitic acid and 1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid (NODAGA) and radiolabeled with ⁶⁴Cu, (half-life 12.701 hours, decays by 17.86% positron emission, and 0.653 MeV positron energy). This tracer was chosen, because it is beneficial for high-resolution small animal imaging with longer observation time. Furthermore, ⁶⁸Ga was selected (half-life 67.629 minutes, 89% positron emission, 1.9 MeV positron energy), a generator radionuclide with optimal characteristics for clinical application, but shorter observation time and lower image resolution in the preclinical setting. The corresponding PEGylated and palmitoylated analogues were designed in order to study the influence of such modifications on radionuclide activity distribution and in vivo behavior of the radiotracers, and to improve pharmacokinetics.

Results from the nanoScan PET/CT

Anesthetized, spontaneously breathing animals were allowed to stabilize for 10 min after preparation. The animals were positioned on a heated bed to maintain the body temperature at 37°C. Some of the PET studies were carried out with the nanoScanPET/CT. The activity of the injection solution was measured in a well counter (Isomed 2000, Dresden, Germany) cross-calibrated to the PET scanners. The PET acquisition of 60 or 120 min emission scan was started and the infusion of the ⁶⁸Ga-/⁶⁴Cu-labeled compound was initiated with a delay of 10 s. 0.5 mL (rats) or 0.1 mL (mice) of solutions of [⁶⁸Ga]- or [⁶⁴Cu]-peptides were infused over 1 min (with a Harvard apparatus 44 syringe pump) into a lateral tail vein. In blocking experiments the radiotracers were simultaneously injected with 1 mg/kg body weight of Ghrelin or KKD. At the end of the experiment, the animals were deeply anesthetized and sacrificed by an intravenous injection of potassium chloride.
Acquisition was performed in 3D list mode. Emission data were collected continuously. The list mode data were sorted into sinograms with 32 or 38 frames (15 × 10 s, 5 × 30 s, 5 × 60 s, 4 × 300 s, 3 × 600 s, or 9 × 600 s). The data were decay-, scatter-, and attenuation-corrected. The PET images measured with the nanoScanPET/CT were reconstructed using a three-dimensional Ordered Subsets Expectation Maximization (3D-OSEM) algorithm into dynamic frames as described and with a voxel size of 0.05 cm. No correction for partial volume effects was applied. The image volume data were converted to Siemens ECAT7 format for further processing and were then analyzed using the ROVER software (ABX GmbH, Radeberg, Germany). Masks for defining three-dimensional regions of interest (ROI) were set and the ROI’s were defined by thresholding and ROI time activity curves (TAC) were derived for the subsequent data analysis. The time activity curves over the vena cava were derived from ROI determined in the first two minutes after 1 min long infusion of the radiotracers. The ROIs were so determined that no surrounding tissue was included. The ROI data and TAC were further analyzed using R and especially developed program packages.
After different compund studies, The ⁶⁴Cu-labeled 10c was selected for the further biopharmaceutical evaluations because of the high molar activity of the ⁶⁴Cu-compounds, the best imaging properties of ⁶⁴Cu, the lower efficacy, awaiting lower pharmacodynamic side effects, and the simpler synthesis without Palm conjugation, and the resulting lower lipophilicity.
Figure 9-11. shows the10c biodistribution and -kinetics, which was also studied by small animal PET. The tumors (PC-3, DU-145) were clearly visible in these representative images (Control). However, the activity distribution in the tumors was relative heterogeneous following the heterogeneity of the tumor tissue. An additional reason is the high interstitial pressure in the tumor center decreasing the perfusion. The highest levels of 10c were in the liver and kidneys. In the most PET studies, also the thyroid gland was visible, which could not be measured in the extractive biodistribution experiments. This was surprising because of the very small size of the thyroid. The simultaneous injection of KKD with the 10c (lower part of Figure 9.) reduced the activity concentration also in this tissue. In contrast to the biodistribution, the 10c uptake in stomach could not be studied, because of the close anatomical position of the stomach to the liver and kidneys with its high activity uptake. The stomachs relative thin wall caused a spill with the surrounding tissues that didn´t allow clear visualizing of the stomach in the PET images.

Figure 11. shows exemplarily parametric metabolic trapping rate images (Patlak K_m), which were calculated from the blood curves and the dynamic PET images. This shows how the PET study with 10c allows for the modelling of whole-body tracer kinetics by directly estimating the metabolic rate constant based on a common irreversible two-compart kinetic model. In this case, the Patlak K_m, image allows for the demonstration of the metabolic trapping that is dependent on the GHS-R1a density of the tissue and the heterogeneity of the distribution in the tumor. The blocking effect on the thyroid K_m also demonstrated the presence and the GHS-R1a dependent radiotracer accumulation in this organ. A limitation of the parametric imaging was the use of the whole blood activity and not of the arterial blood. However, the slow tissue extraction of the 10c lowered this effect. The other limitation of this method in our example is that the tracer accumulation was not irreversible. It could be demonstrated that the 10c allows quantitative imaging of the Ghrelin receptor distribution within the organism.

• The data support that the studied radiometal Ghrelin inverse agonists could be used for the imaging of functional expression of Ghrelin receptors in vivo that these radiotracers have the potential to gain a deeper understanding on Ghrelin and its receptors in vivo in health and disease, in particular, in cancer.
• The radiotracers were specifically accumulated in xenotransplanted human prostate tumor models (PC-3, DU-145) in mice. The tumors were clearly delineated by PET.
• The [⁶⁴Cu]Cu- or [⁶⁸Ga]Ga-NODAGA-NH-K-K-(D-1-NaI)-F-w-L-L-NH₂ radiolabeled inverse agonists with respect to its relative simple synthesis, high affinity and medium efficacy, high metabolic stability, and the suitable pharmacokinetic profiles, seem to be potent, and safe leads of future imaging agents for visualization and quantitation of GHS-R1a expression in normal and cancer tissues by PET. The GHS-R1a in vivo imaging and quantitation has the potential to serve as biomarkers of the Ghrelin system in precision medicine.

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