Analysis of [⁶⁸Ga]NODAGA-exendin-4 PET data

Exendin-4 is a subcutaneously administered incretin peptide drug, used in the treatment of type 2 diabetes. [⁶⁸Ga]NODAGA-exendin-4 is an high-affinity agonist of glucagon-like peptide 1 receptor (GLP-1R), and has been validated for imaging pancreatic β-cells in rats (Mikkola et al., 2014). [⁶⁸Ga]NODAGA-exendin-4 can be produced with high specific activity, which is especially important for imaging GLP-1 receptors that are usually expressed in low levels; only very low mass of ligand can be administered, especially in small animals, to avoid pharmacological and receptor blocking effects (Brom et al., 2010; Mikkola et al., 2014; Nalin et al., 2014; Rydén et al., 2016; Mikkola et al., 2016). Automated GMP compliant production for [⁶⁸Ga]Ga-DO3A-VS-Cys⁴⁰-exendin-4 has been developed (Velikyan et al., 2017).

Chelators are commonly conjugated to the free amine of a C-terminally attached lysine or cysteine of exendin-4. In addition, exendin-4 possesses naturally two lysines at positions 12 and 27, which could be used as conjugation sites, although that may affect the binding affinity, internalization, metabolism, and excretion rate (Jodal et al., 2014). NODAGA can chelate many radiometals, including ⁶⁸Ga, ¹¹¹In, ⁶⁴Cu, and Al¹⁸F. When the two lysine positions (12 and 27) where used to attach NODAGA, and compared to ligand where NODAGA was attached to terminally added lysine (40), all ligands showed favourable kinetics and specific uptake (Jodal et al., 2014).

Exendin-4 labelled with ⁶⁸Ga and other radiometals show high non-specific uptake in the kidneys, which may lead to to high radiation dose. Iodine-labelled exendin-4 ligands cause lower dose to kidneys, but radiation dose to thyroid may become significant. ¹⁸F-labelled exendin-4 tracers show lower radioactivity in the kidneys (Mikkola et al., 2016; Dialer et al., 2018). Despite of the initial concerns of the absorbed dose to the kidneys, effective radiation doses with [⁶⁸Ga]NODAGA-exendin-4 were found to be low for adults and children, allowing repeated examinations (Boss et al., 2020). Repeated scans in humans are possible also with [⁶⁸Ga]Ga-DO3A-VS-Cys⁴⁰-exendin-4 (Selvaraju et al., 2015).

High ⁶⁸Ga activity in the kidneys hampers image analysis in small animals; especially the pancreas is difficult to locate in the images (Mikkola et al., 2014). Bandara et al (2016) could identify pancreas in rats with the help of the CT (Inveon small animal PET/CT scanner); the tracers were ⁶⁸Ga-labelled DO3A-VS-Cys⁴⁰-exendin-4 and NODA-VS-Cys⁴⁰-exendin-4.

[⁶⁸Ga]NOTA-exendin-4 and [⁶⁸Ga]DOTA-exendin-4 can be used to localize insulinomas (Luo et al., 2016; Antwi et al., 2015 and 2018). Previously, [⁶⁸Ga]Ga-DO3A-VS-Cys⁴⁰-exendin-4 PET had detected a metastatic insulinoma (Eriksson et al., 2014), and [Lys⁴⁰(Ahx-DOTA-⁶⁸Ga)NH₂]-exendin-4 had already shown promise in insulinoma imaging in a mouse model (Bauman et al., 2015). Sensitivity and specificity of preoperative imaging of insulin producing pancreatic neuroendocrine tumours with [⁶⁸Ga]NODAGA-exendin-4 is being studied in a clinical trial.

Ex vivo autoradiography has shown that [⁶⁸Ga]NODAGA-exendin-4 uptake is localized in macrophage-rich, GLP-1R-positive atherosclerotic lesions in the aorta of atherosclerotic and diabetic mice (Ståhle et al., 2017). PET imaging in myocardial infarction model in rats has shown that GLP-1R is upregulated during healing, and autoradiography confirmed that [⁶⁸Ga]NODAGA-exendin-4 uptake correlated with the amount of CD68-positive macrophages in the infarcted region (Ståhle et al., 2018).

Model

[⁶⁸Ga]Ga-DO3A-VS-Cys⁴⁰-exendin-4 PET study has shown striking variations among the species, caused by differences in β-cell mass and GLP-1R expression in β-cells and exocrine pancreas (Eriksson et al., 2017).

Internalization of GLP-1R tracers, even Exendin-4 tracers, is different between tracers (Jodal et al., 2014), and possibly also in different organs. In vitro, 35% of [Lys⁴⁰(Ahx-DOTA-⁶⁸Ga)NH₂]-exendin-4 was internalized after 2 h (Jodal et al., 2014). In another cell line, ∼5% and ∼10% [Lys⁴⁰(Ahx-DOTA-⁶⁸Ga)NH₂]-exendin-4 was internalized at 2 and 4 h, respectively (Wild et al., 2010). Radioligands can also be slowly released from the cells (Wild et al., 2010).

Semiquantitative SUV and tumour-to-pancreas ratio have been used to analyze insulinoma studies in humans (Luo et al., 2016).

In cynomolgus monkeys, pancreatic [⁶⁸Ga]Ga-DO3A-VS-Cys⁴⁰-exendin-4 TACs were fitted to one-tissue compartment model to estimate the total volume of distribution (V_T = K₁/k₂), fitting also blood volume (V_B) as the third parameter (Selvaraju et al., 2013). The same model was later used to study diabetic (type 1 diabetes model) and non-diabetic pigs: V_T was ∼2.3 and K₁ ∼0.034 in the pancreas of both diabetic and non-diabetic pigs, although perfusion was decreased by almost 50% in diabetic pigs. Blocking with exendin-4 caused over 80% decrease in V_T (Nalin et al., 2014). In occupancy study in cynomolgus monkeys, K₁ decreased with increasing mass, from 0.07 to 0.01, while k₂ remained relatively constant (Selvaraju et al., 2013). V_T decreased from ∼8.4 to ∼0.3 at maximum ligand mass. V_B estimates were in the range 0.15-0.45 (Selvaraju et al., 2013). In the liver, increasing ligand mass led to decrease in both K₁ and k₂, and changes in V_T were negligible (Selvaraju et al., 2013).

Myocardial muscle data from rats can be well fitted to Patlak plot, suggesting marked internalization into cells (Ståhle et al., 2018).

Model input

[Nle¹⁴,Lys⁴⁰(Ahx-NODAGA-⁶⁸Ga)NH₂]-exendin-4 ([⁶⁸Ga]NODAGA-exendin-4) is rapidly cleared from the blood. Uptake in the heart is clearly lower than the radioactivity concentration in the blood even 1 h after administration (Mikkola et al., 2014), suggesting that input function can be derived from VOI placed on heart cavity, without any marked activity spill-over from the heart muscle.

Venous blood sampling has been used to measure PTAC in cynomolgus monkeys and pigs (Selvaraju et al., 2013; Nalin et al., 2014).

Plasma vs blood

Exendin-4 is a relatively large peptide, and it, or its label-carrying metabolites do not pass the membranes of red blood cells, as indicated by the low activity in the blood cell pellet and the stable serum-to-blood ratio in a rat study with [⁶⁴Cu]NODAGA-exendin-4 (Mikkola et al., 2014). Therefore the image-derived blood time-activity curve (TAC) is easy to convert to plasma TAC. Conversion can be based on haematocrit value, preferably measured individually.

In the rat study with [⁶⁴Cu]NODAGA-exendin-4 (Mikkola et al., 2014) the median plasma-to-blood ratio was 1.793 (not published), which, if concentration in RBC is 0, means that haematocrit was ∼0.44.

Metabolite correction

Exendin-4 has an in vivo halflife of about 2.4 h.

[⁶⁸Ga]NODAGA-exendin-4 has been shown to be stable in vitro and in vivo in rats: one hour after administration, 70±5% of the serum radioactivity was due to the intact radioligand (Mikkola et al., 2014; Ståhle et al., 2018). In pancreas, 32±5% of the ⁶⁸Ga radioactivity represented intact tracer. Only minimal amounts of intact tracer were detected in the kidneys and urine (Mikkola et al., 2014). Two radiometabolites of [⁶⁸Ga]NODAGA-exendin-4 can be detected in the urine and plasma (unpublished data). Power function can be used to fit the fractions of parent radioligand in plasma (Fig. 1); function parameters a=0.0452, b=1.5, c=0.218, and e=0.0 were determined from three rats, and parameter d (initial fraction at t=0) can be set to the measured radiochemical purity of each batch.

Analysis

PET images of rats are dominated by the very high uptake in the kidneys and liver (Fig. 3 and 4). Tissue-to-blood ratio (Figure 5) and tissue-to-plasma ratio (Figure 6) is low, as expected in organs with low GLP-1R expression. The uptake in the liver is probably mostly due to radioactive metabolites.

It is difficult to determine from the shapes of the TTACs and tissue-to-plasma ratios whether the tracer kinetics is strictly reversible or if there is a tiny component of irreversible uptake. Model-independent graphical analyses, Logan plot and Patlak plot, are both linear (Figures 7 and 8), and thus do not help in deciding between reversible and irreversible model. Small part of the uptake in muscles and lungs may also be due to the radioactive metabolites. The very low slope in the Patlak plot demonstrates that the impact of active metabolites or irreversible uptake component during the 60-min study is small.

Reversible one-tissue and irreversible two-tissue compartmental models (1TCM and 2TCM, respectively) were fitted to the regional TTACs, both providing good fits (Figures 9 and 10). In skeletal and myocardial muscles the k₃ was zero, suggesting that reversible models are appropriate. Estimates of the V_B were reasonable, 5% for the skeletal muscle and 12% for the myocardium. The volume of distribution (V_T), calculated as K₁/k₂, was 0.055 for the muscle and 0.31 for myocardium. The sum of V_T and V_B from the compartmental model fit is close to the V_T computed using the Logan plot.

Although fits for the liver and lungs were good, the results are not expected to be physiologically valid, because of the metabolites in the liver, and because of the unknown air/tissue fraction in the lungs; additionally, the arterial input function is not fully valid for the lungs since it represents activity in plasma that has already circulated through lungs.

Kinetic analysis does not tell in what extent the calculated V_Ts represent specific binding of [⁶⁸Ga]NODAGA-exendin-4 to the GLP-1 receptors, or non-specific binding to other targets. However, blocking studies have shown that specific binding component is substantial.

In conclusion, reversible 1TCM seems to be feasible method for assessment of V_T. Logan plot gives overestimated values because of the relatively high contribution from the vascular activity, but could be used to assess relative changes in V_T if simultaneous changes in V_B are not expected.

In infarcted myocardial muscle the GLP-1R expression is increased during healing process. Internalization of [⁶⁸Ga]NODAGA-exendin-4 leads to marked increase in the irreversible uptake component, allowing the usage of Patlak plot for the analysis (Ståhle et al., 2018).

See also:

• GLP-1R
• Analysis of [¹⁸F]exendin-4 PET data
• Pancreas
• VMAT2

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References:

Boss M, Buitinga M, Jansen TJP, Brom M, Visser EP, Gotthardt M. PET-based human dosimetry of ⁶⁸Ga-NODAGA-exendin-4, a tracer for β-cell imaging. J Nucl Med. 2020; 61(1): 112-116. doi: 10.2967/jnumed.119.228627.

Gao H, Kiesewetter DO, Zhang X, Huang X, Guo N, Lang L, Hida N, Wang H, Wang H, Cao F, Niu G, Chen X. PET of glucagonlike peptide receptor upregulation after myocardial ischemia or reperfusion injury. J Nucl Med. 2012; 53: 1960-1968. doi: 10.2967/jnumed.112.109413.

Jodal A, Lankat-Buttgereit B, Brom M, Schibli R, Béhé M. A comparison of three ^67/68Ga-labelled exendin-4 derivatives for β-cell imaging on the GLP-1 receptor: the influence of the conjugation site of NODAGA as chelator. EJNMMI Res. 2014; 4:31. doi: 10.1186/s13550-014-0031-9.

Mikkola K, Yim C-B, Fagerholm V, Ishizu T, Elomaa V-V, Rajander J, Jurttila J, Saanijoki T, Tolvanen T, Tirri M, Gourni E, Béhé M, Gotthardt M, Reubi JC, Mäcke H, Roivainen A, Solin O, Nuutila P. ⁶⁴Cu- and ⁶⁸Ga-labelled [Nle¹⁴,Lys⁴⁰(Ahx-NODAGA)NH₂]-exendin-4 for pancreatic beta cell imaging in rats. Mol Imaging Biol. 2014; 16: 255-263. doi: 10.1007/s11307-013-0691-2.

Mikkola K, Yim C-B, Lehtiniemi P, Kauhanen S, Tarkia M, Tolvanen T, Nuutila P, Solin O. Low kidney uptake of GLP-1R-targeting, beta cell-specific PET tracer, ¹⁸F-labeled [Nl1¹⁴,Lys⁴⁰]-exendin-4 analog, shows promise for clinical imaging. EJNMMI Res. 2016; 6:91. doi: 10.1186/s13550-016-0243-2.

Tags: GLP-1R, Exendin-4, Pancreas, Beta-cell, Incretin

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Updated at: 2020-01-09
Created at: 2018-08-15
Written by: Vesa Oikonen