As Marion said I am Linda van der Graaf and I work as a research technician at the department of nuclear medicine at the Erasmus MC. This is the first time that I’m doing a talk on a big meeting like this, so I’m very excited . As said, I am going to tell you about a new bombesin receptor antagonist. This project is a collaboration between our group in Rotterdam, the group of Nock and Maina in Athens and the group Martinez in Montpellier.
Bombesin analogues bind to GRP receptors that are overexpressed on prostate cancer and breast cancer tumours. In this animation you see how the peptide binds to the receptor on the cell surface Proof of concept has been obtained clinically with the well known peptides AMBA and MP2248 that have different structures, linkers and radionuclides, but are both targeting the GRP receptor. These peptides are agonists, that have the disadvantages that they have a high bioactivity, causing side effects like vomiting, they have a high pancreatic uptake that gives problems during PRRT, and they have mitogenic effects
On the other hand we have the antagonists that also have a high affinity for the GRP receptor and no bioactivity, but they have less internalization then agonists. We have always believed that antagonists would not work for imaging and radionuclide therapy, because these peptides only bind to the surface of the tumour cells and do not internalize, which makes the retention time very short However, there are groups who found that antagonists DO work, like with Demobesin and RM2 Jean Martinez is a peptide chemist in Montpellier and he has a lot of peptides, but didn’t use them in combination with radio activity. For us, he coupled a DOTA to JMV594 and called that peptide JMV4168.
The aim of this study is to evaluate JMV versus the well known peptide AMBA in the PC3 mouse model. We did this with different techniques, in vitro tests like looking at the receptor affinity, antagonistic properties and internalization. We also did in vivo biodistribution in female SCID and male Nu/Nu mice with the diagnostic radionuclide Indium 111 and Lutetium 177, that can be used for therapy, and we tested this in two tumour models, PC3 that is commonly used in research and PC295 that is a more physiological tumour. We also looked at the in vivo stability and we did in vivo SPECT/CT studies. This is the structure of JMV4168, Here you see the chelator DOTA that is necessary for complexation of the radionuclide and this is coupled to the peptide via 2 bita ala groups
Now we come to the results, in this graph we see the displacement of the compound by increasing concentrations of unlabeled peptide. The IC50 values of JMV and AMBA are in the same nanomolar range, 1.3 nM for JMV and 0.7 for AMBA, so there is a twice better affinity for AMBA, because the lower the value, the better In another experiment we found that JMV is a competitive antagonist, because it binds at the same binding site on the receptor.
(Grafiek van gemiddelden van 10 20 30 60 minuten.) With regard to internalization in PC3 cells for 60 minutes at 37 degrees we see a clear difference between JMV and AMBA. The yellow bar is the fraction that binds to the membrane and the green bar is the fraction that is internalized. We see that the antagonist JMV has a low internalisation and, as shown earlier in literature, the agonist AMBA has a high internalisation.
So now we switch to the biodistribution studies. In the next slides JMV has yellow bars and AMBA has green bars and please note the different axes in the different graphs. We injected indium in male nude mice and 4 hours after injection, we took out the organs and counted them for activity in the gamma counter. First, we see AMBA in nude mice with PC3 tumor. The uptake in the tumor is ok, but in other organs, especially the pancreas and the intestin, the uptake is very high. This is because GRP receptors are also present in the pancreas, which is a disadvantage when we want to do therapy with this peptide. When we now look at JMV, we see a little higher uptake in the tumor, of about 10%, but unlike with AMBA, the uptake in the pancreas is now very little, below 2%! And when we do the same experiment in nude mice with PC295 tumor we see the same pattern in uptake, with AMBA a high uptake in the pancreas and with JMV a low uptake in the pancreas.
Because the group in Athens uses female SCID mice in stead of male nude mice, we wanted to see the difference in uptake in PC3 tumour and pancreas. The first bar is the uptake when the peptide is blocked with an excess of cold peptide, the second is 4 hours after injection without block and de third bar is 24 hours after injection. Now we see that the tumour uptake is twice as high as the uptake we had on the previous slide with nude mice and again the uptake in the pancreas is very high. When we look at JMV in SCID mice, we again see a higher uptake in the tumour than with nude mice and a low uptake in the pancreas.
Because Lutetium is a good therapeutic radionuclide, we wanted to see if this gave the same uptake as indium and also now, we see that with JMV, the uptake in the tumour is high, around 18%, and the uptake in the pancreas is below 2%. Even after 96 hours there is activity measured in the tumour, so this can be an excellent peptide for therapy.
Now we come to the NanoSPECT/CT studies. In agreement with the biodistribution, there is a high uptake in the tumour 1 hour after injection, after 4 hours it is a little less and after 24 hours there is still a little activity left in the tumour.
Besides that we also performed a dynamic nanoSPECT/CT scan, in 60 minutes, we made 12 scans of 5 minutes each. As you can also see in the time activity curve, because of the rapid clearance of small peptides in the kidneys, the activity in the kidneys is high in the first 10 minutes. The tumor uptake is than low, but this will change overtime. From 15 to 35 minutes the activity in the kidneys is getting less and the activity in the tumour is getting higher and at 35 minutes, it’s about the same in tumour and kidneys. From 40 to 60 minutes you see that the uptake in the tumour is still getting a little higher and the activity in the kidneys is getting lower. When we put the 12 scans together, you get this nice movie, where you can clearly see the activity overtime.
To conclude, antagonists are a very interesting approach, with less side effects. JMV has a high tumor uptake. The retention of JMV in the tumour is not as good as for agonists, but the way it is now, is already excellent for radionuclides with a short halflife. The uptake in the panceas is low, which makes the tumor pancreas ratio much better than for agonists. In the future, we want to look at more stabilized compounds. Maybe dimers are an option, maybe they have a better retention in the tumor. And we want to try radionuclide therapy to erradicate tumors. Thank you for your attention.
Emim 20 juni 2011 5
Molecular Radiopharmacy SPECT/CT imaging of prostate cancer xenografts using a new bombesin receptor antagonist L.M. van der Graaf1, T. Maina2, P.J. Marsouvanidis2, M. Melis1, S.C. Berndsen1, E.P. Krenning1, J. Martinez3, L. Brunel3, J. Fehrentz3, B.A. Nock2, M. de Jong11 Erasmus MC, Rotterdam, Netherlands, 2NCSR “Demokritos”, Athens, Greece, 3Faculté de Pharmacie, CNRS-Universités de Montpellier 1 et 2, Montpellier, France
Introduction Bombesin analogues bind GRP-receptors overexpressed on prostate cancer and breast cancer Proof of concept obtained clinically: AMBA, MP2248 Different structures/linkers/radionuclides: All targeting GRPR Agonists, disadvantages: High bioactivity (side effects) High pancreatic uptake (problems during PRRT) Mitogenic effects
Introduction Antagonists: affinity, no bioactivity, less internalization We have always believed, antagonists would not work for imaging and radionuclide therapy However: - [99mTc]Demobesin 1: Maina et al, Eur J Nucl Med Mol Imaging, 2003; Reubi et al, J Nucl Med,2008 - RM2: Maecke et al, Eur J Nucl Med Mol Imaging, 2011 J Martinez et al. compounds (JMV): J. Med. Chem. 2000, 43, 2356-2361, Peptides, 1998, 19, 57-63 - DOTA coupled to JMV594: JMV4168 (JMV)
AimEvaluate JMV versus AMBA in the PC3-model• In vitro: receptor affinity, antagonistic properties, internalization• In vivo biodistribution in SCID and Nu/Nu mice, 111In vs. 177Lu, PC-3: commonly used PC-295: more physiological• In vivo stability• In vivo nanoSPECT/CT JMV4168
Results: GRP receptor affinity IC50 JMV & AMBA 150 111 In-JMV+JMV% binding 100 111 In-AMBA+AMBA Competitive antagonist 50 Binds the same binding site on the receptor 0 -14 -12 -10 -8 -6 -4 Log block (M) IC50 JMV: 1.3 nM versus 0.7 nM for AMBA
Conclusions Antagonists: very interesting approach, less side effects JMV: High tumor uptake JMV: retention in tumor not as good as agonist, excellent for short-lived radionuclides Pancreas low: tumor/pancreas ratio much better than for agonist!Future outlook: Stabilized compounds Dimers: better retention in tumor? Radionuclide therapy to erradicate tumors