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68Ga-Labeled NODAGA-conjugated ghrelin receptor agonists and inverse agonists

[68Ga]-NODAGA-ghrelin derivatives
, PhD
National Center for Biotechnology Information, NLM, Bethesda, MD 20894

Created: ; Last Update: July 12, 2012.

Chemical name:68Ga-Labeled NODAGA-conjugated ghrelin receptor agonists and inverse agonists
Abbreviated name:[68Ga]-NODAGA-ghrelin derivatives
Synonym:
Agent Category:Peptides
Target:Ghrelin receptor (growth hormone secretagogue receptor (GHS-R1a))
Target Category:Receptor
Method of detection:Positron emission tomography (PET)
Source of signal / contrast:68Ga
Activation:No
Studies:
  • Checkbox In vitro
  • Checkbox Rodents
Structure not available in PubChem.

Background

[PubMed]

Ghrelin is a 28-amino-acid peptide hormone that is produced and secreted mainly by the gastric mucosa and triggers the secretion of growth hormone (GH) from the pituitary by binding to the ghrelin receptor (previously known as the GH secretagogue receptor 1a or GHSR1a) (1). Although ghrelin is known to have several endocrine functions, investigators are interested in this molecule primarily because it is known to stimulate appetite, promote energy storage, and play a role in the development of obesity (1, 2). Therefore, the ghrelin/GHSR pathway is considered to be an important target for the treatment of obesity, cachexia, and anorexia (2, 3). However, ghrelin has a low stability while in circulation, and there is a lack of information regarding its in vivo behavior and biodistribution (2). In order to gain insight into the biological behavior of the ghrelin receptor, radiolabeled peptide agonists and inverse agonists (an inverse agonist binds to the same receptor as an agonist but produces an effect that is opposite to that of the agonist)) were designed and evaluated by Chollet et al. for use with positron emission tomography (PET) (2). The ghrelin agonists and inverse agonists were conjugated to 1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid (NODAGA), a bifunctional chelating agent, and labeled with 68Ga ([68Ga]-ghrelin conjugates designated 1f-4f and 5e). The functional behavior of the tracers was investigated in vitro and the biodistribution and imaging properties of the labeled peptides was studied in rodents (2).

Synthesis

[PubMed]

The various ghrelin peptide derivatives were synthesized on a multiple peptide synthesizer (for the designated name and amino acid sequence of each peptide, see Table 1), and each peptide was labeled with 68Ga as described by Chollet et al. (2). The radiochemical purity and specific activities of the labeled peptides are given in Table 1.

Table 1: Structure and nature of ghrelin derivatives (2).

Designated NumberGhrelin Derivative*Radiochemical Purity (%)Specific Activity (GBq/μmol)
1eNα-NODAGA[68Ga]-ghrelin(1–28)--
2f (2e)a[K16(NODAGA[68Ga]]ghrelin(1–28)96.7 ± 4.023.4 ± 6.6
(631.8 ± 178.2)b
3f (3e)[Dpr3;K16(NODAGA[68Ga])]ghrelin(1–28)99.5 ± 0.512.6 ± 1.1
(340.2 ± 29.7)
4f (4e)[Dpr3;K16(NODAGA[68Ga])]ghrelin(1–16)95.8 ± 4.58.4 ± 6.2
(226.8 ± 167.4)
5e (5d)Nα-NODAGA[68Ga]-KwFwLL-CONH296.2 ± 4.316.1 ± 2.4
(434.7 ± 64.8)

*The amino acid sequence of ghrelin is NH2-GSS3FLSPEHQRVQQRKESKKPPAKLQPR-COOH. The presence of an n-octanoyl group (C8:0) on Ser3 is essential for the biological activity of ghrelin (4).

a: Designated name of peptide containing nonradioactive Ga.

b: Specific activity in mCi/μmol.

The radiochemical yields of the different labeled peptides were not reported. The final preparation of each 68Ga-labeled compound was formulated in electrolyte solution E-153 (for composition, see Chollet et al. (2)) for storage.

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

The biological activity of nonradioactive ghrelin derivatives (1e-4e and 5d) was investigated with an inositol phosphate assay using African green monkey kidney COS-7 cells (2). The EC50 values of the different peptides are presented in Table 2. Peptides 1e (with NODAGA(Ga) on the N-terminus), 3e, and 4e (both with NODAGA(Ga) at Lys16) showed a >1,800-, 3.5-, and 2.6-fold weaker activity compared with ghrelin, respectively. The 5d peptide had a >1,000-fold higher EC50 compared with ghrelin. Only peptide 2e (with NODAGA(Ga) at Lys16) had activity comparable to that of ghrelin. Peptides 1e-4e were reported to show an agonist behavior at the ghrelin receptor, and only 5d behaved as an inverse agonist (Table 2). On the basis of these results, only 68Ga-labeled 2f-4f and 5e were used for the in vivo studies.

Table 2: In vitro activity of nonradioactive ghrelin derivatives in an inositol phosphate turnover assay (2).

PeptideEC50 (nM)Fold EC50 over that of ghrelinNature (A or IA)a
Ghrelin0.55 ± 0.2411A
1e>1,000>1,800A
2e0.72 ± 0.0611.3A
3e1.91 ± 0.4033.5A
4e1.41 ± 0.6022.6A
5d643 ± 9.1>1,000IA

a: A = agonist; IA = inverse agonist.

Animal Studies

Rodents

[PubMed]

For biodistribution studies, male Wistar rats (n = 4 animals/time point) were injected with 0.3 MBq (8.1 μCi) of each 68Ga-labeled peptide (2f-4f and 5d) through the tail vein, and the rodents were euthanized at 5 min and 60 min postinjection (p.i.) (2). All the major organs and blood were harvested from the animals to determine the amount of radioactivity accumulated in the different tissues. The amount of tracer present in each organ was expressed as a percent of injected dose per gram tissue (% ID/g). At 5 min p.i., the label was present mainly in the kidney with 2f, 3f, and 4f and with accumulation values of 26.6 ± 11.8% ID/g, 33.2 ± 2.2% ID/g, and 27.7 ± 2.9% ID/g, respectively. By 60 min p.i. the amount of tracer in the kidney from these radiochemicals increased to 63.3 ± 25.9% ID/g, 65.3 ± 2.2% ID/g, and 52.3 ± 5.9% ID/g, respectively. The amount of label in the liver with 2f, 3f, and 4f at 60 min p.i. was 8.1 ± 0.8% ID/g, 22.3 ± 2.4% ID/g, and 3.4 ± 0.2% ID/g, respectively. In the intestine, the amount of radioactivity from these tracers at 60 min p.i. was 0.9 ± 0.1% ID/g, 1.5 ± 0.4% ID/g, and 10.7 ± 2.1% ID/g, respectively. All other organs showed an accumulation of <1.0% ID/g at both time points. From these observations the investigators concluded that all the agonist tracers (2f-4f) were eliminated mainly through the urinary and the hepatobiliary system of the animals.

No change in accumulation of radioactivity in the kidney was observed with 5e (inverse agonist) at both time points (4.4 ± 0.2% ID/g and 4.4 ± 0.6% ID/g at 5 min p.i. and 60 min p.i., respectively). However, the accumulation of label from this tracer in the liver was 11.6 ± 1.4% ID/g and and 9.1 ± 2.0% ID/g at 5 min p.i. and 60 min p.i. , respectively. In the intestine the uptake was 9.4 ± 0.7% ID/g and 10.7 ± 2.1% ID/g at 5 min p.i. and 60 min p.i., respectively. . In addition, the concentration of radioactivity from 5e was higher in the blood at both time points (3.78 ± 0.43% ID/g and 1.53 ± 0.25% ID/g at 5 min p.i. and 60 min p.i., respectively) compared with the agonist-labeled compounds (with all these radiolabeled compounds, the amount of radioactivity in the blood was <2.0% ID/g and <0.3% ID/g at 5 min p.i. and 60 min p.i., respectively). No blocking studies were reported so the extent of specific binding could not be determined.

To investigate the metabolic stability of 2f-4f and 5e, the labeled compounds were administered to the rats as before (number of animals not reported), and arterial blood and urine samples were taken from the animals at various time points ranging from 1 min p.i. to 60 min p.i (2). Plasma separated from the whole blood was subjected to radio-high-performance liquid chromatography analysis with a Zorbax C18 300SB column. Among all the radiolabeled ghrelin derivatives, only the inverse agonist 5e was reported to remain stable under in vivo conditions. With this tracer, <5% of the total radioactivity was associated with metabolites in the blood at 60 min p.i., and in urine the label was detected in trace amounts of the metabolites at this time point. In contrast, all the agonists had 40% (2f and 3f) to 100% (4f) of their label associated with at least two metabolites in the blood and the urine at this time point.

For PET imaging, 90 ± 33 MBq (2.4 ± 0.81 mCi) 68Ga-labeled 2f-4f and 5e was infused over 1 min into the tail vein of anesthetized, spontaneously breathing rats (n = 4 animals/time point) (2). With 2f and 4f the radioactivity was detected primarily in the kidney at 5 min p.i., and with 3f the label was observed mainly in the liver and the intestines. However, with 5e the tracer was visible in all the organs with higher concentrations in the kidney, liver, and the intestine. From time-activity curves it was apparent that the accumulation of radioactivity in the kidneys was rapid with 2f and 4f, intermediate for 3f, and slow with 5d. The curves showed that with 3f there was a high accumulation of label in the liver, and there was little change in accumulated radioactivity in the organ for up to 50 min p.i. Rapid accumulation of radioactivity in the hepatic tissue was observed with 2f, 4f, and 5e by ~1.5 min p.i., and the label was gradually lost from this organ by 50 min p.i. Rapid accumulation of label in the heart was observed with all the tracers at ~1.5 min p.i., followed by a rapid loss of radioactivity with the agonists. However, with 5e the loss of label from this organ was gradual but less pronounced.

From these studies based on the in vivo stability of the radiolabeled agonists and the inverse agonist, the investigators concluded that only 5e, the inverse agonist, appeared to be most suitable for the development of a peptide-based therapeutic for the treatment of obesity (2).

Other Non-Primate Mammals

[PubMed]

No publication is currently available.

Non-Human Primates

[PubMed]

No publication is currently available.

Human Studies

[PubMed]

No publication is currently available.

Supplemental Information

[Disclaimers]

No information is currently available.

References

1.
Lim C.T., Kola B., Korbonits M. The ghrelin/GOAT/GHS-R system and energy metabolism. Rev Endocr Metab Disord. 2011;12(3):173–86. [PubMed: 21340583]
2.
Chollet C., Bergmann R., Pietzsch J., Beck-Sickinger A.G. Design, evaluation, and comparison of ghrelin receptor agonists and inverse agonists as suitable radiotracers for PET imaging. Bioconjug Chem. 2012;23(4):771–84. [PubMed: 22372770]
3.
Soares J.B., Roncon-Albuquerque R. Jr, Leite-Moreira A. Ghrelin and ghrelin receptor inhibitors: agents in the treatment of obesity. Expert Opin Ther Targets. 2008;12(9):1177–89. [PubMed: 18694382]
4.
Kojima M., Kangawa K. Ghrelin: structure and function. Physiol Rev. 2005;85(2):495–522. [PubMed: 15788704]

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