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Identifier 000451916
Title Radiolabeled compounds in molecular imaging of pancreatic adenocarcinoma
Alternative Title Ραδιοσημασμένες ενώσεις στη μοριακή απεικόνιση του αδενοκαρκινώματος του παγκρέατος
Author Κανελλόπουλος, Παναγιώτης
Thesis advisor Λιαπάκης, Γεώργιος
Reviewer Koukouraki, S.
Maina, Nock T.
Kampa, M.E.
Mavromoustakos, T.
Venihaki, M.
Nock, B. A.
Abstract Summary Pancreatic cancer (PC) was identified as the 10th most common cancer in humans and 4th cause of cancer deaths in USA in 2018. The asymptomatic character of PC at the early stages in combination with the lack of effective diagnostic tools for its early diagnosis contribute in these poor outcomes. Recent advances in nuclear medicine technology (positron emission tomography (PET) and single positron emission computed tomography (SPECT), alongside with the recent advent of novel molecular radiopharmaceuticals, may offer clinicians better diagnostic tools in near future. In this way, the visualization of primary and metastatic PC lesions may become feasible in a non-invasive, fast and convenient way, after interaction of the radiolabeled probe (e.g. a peptide radioligand) with cancer-related “finger-print” biomolecules – “targets” (e.g. a G – protein coupled receptor or GPCR). Currently, there is an urgent need for new radiolabeled probes for effective and reliable diagnosis of PC, and especially of exocrine pancreatic ductal adenocarcinoma (PDAC) representing 95% of PC cases. Interestingly, neurotensin subtype 1 receptor (NTS1R) may serve as a viable biomolecular target in PDAC diagnosis, owing to its overexpression in 95% of PDAC cases in combination with its lack of expression in healthy pancreas and in chronic pancreatitis. The availability of the native NTS1R peptide ligand neurotensin (NT) and numerous synthetic NT-analogs provide the basis for the development of new NT-like radiopharmaceuticals. Like any peptide, NT and its analogs undergo rapid proteolytic degradation; two major peptidases, neprilysin (NEP) and angiotensin converting enzyme (ACE) are implicated in their rapid breakdown in the body. Likewise, NTS1R-targeting radiopeptides are degraded on their way to tumor sites with negative impact on their tumor-targeting capabilities. The aim of the present work was the development of new strategies and/or peptide radioligands based on the C-terminal hexapeptide NT(8-13), to improve in vivo stability, enhanced tumor-targeting and favorable overall pharmacokinetics and eventually suitable for accurate PC/PDAC diagnosis with SPECT. Two major approaches were pursued to achieve this goal: (1) Re-evaluation of previously reported [ 99mTc]Tc-labeled NT(8- 13)-analogs without or during in situ inhibition of NEP and/or ACE. (2) Design of new NT-like radiopeptides after key-structural changes of the NT(8-13) motif; the diagnostic efficacy of these analogs was again evaluated without or during in situ NEP and/or ACE inhibition. To address the first task, previously reported NT analogs, DT1 (N4-Gly7-Arg8-Arg9-Pro10-Tyr11-Ile12- Leu13-OH), DT5 (N4-βAla-Arg-Dab-Pro-Tyr-Ile-Leu-OH) and DT6 (N4-βAla-Arg-Dab-Pro-Tyr-Tle-Leu-OH) were labeled with Tc-99m. Their uptake in colon adenocarcinoma WiDr cells, stability in peripheral mice blood and biodistribution in WiDr tumor-bearing mice were compared, leading to the following conclusions: i) The Dab9/Arg9-substitution resulted in lower cell uptake in vitro, no measurable improvement of metabolic stability, lower uptake in WiDr tumors and elevated renal uptake. ii) the Tle12/Ile12-substitution drastically increased in vivo stability, but led to impaired cell uptake in vitro and tumor uptake in vivo. [99mTc]Tc-DT1 showed superior in vitro performance, combining excellent WiDr tumor targeting and fast pharmacokinetics in vivo during dual NEP and ACE inhibition. Hence [99mTc]Tc-DT1 was chosen as reference for the evaluation of the new structurally modified analogs. To test all analogs in a reliable and convenient NTS1R-positive PC model, the suitability of four different PC cell lines was compared: AsPC-1, PANC-1, MiaPaca-2 and Capan-1. By incubating [99mTc]Tc-DT1 with these cell lines the following rank of cell uptake was established: AsPC-1 >> PANC-1 >>MiaPaca-2 > CAPAN-1 cells. Furthermore, the tumorgenicity of AsPC-1 cells tested in SCID mice was found comparable with that of WiDr cells, with both cell lines requiring a time-span of 3 – 4 weeks for tumors to grow at the inoculation sites. In view of the above, AsPC-1 cells were selected as the PC cell line of choice in this thesis. Overall, the [99mTc]Tc-DT1 reference displayed similar performance both in vitro and in vivo between the WiDr and AsPC-1 cell models. Although phosphoramidon (PA) has been successfully applied for NEP inhibition thus far, a switch to the registered antihypertensive drug Entresto® was decided for such purposes, in support of future translation from animals to patients. Interestingly, Entresto® was proven equally effective as PA, with minor differences observed in the stabilization effects of the two agents attributed to: i) the administration routes (PA – iv, Entresto® - per os), ii) individual animal status and iii) the minor ACE-inhibition ability of PA at higher doses. In the second part of this thesis, structural modifications of the DT1 motif were adopted comprising two major interventions. C-terminal modification (1st group), or introduction of pendant groups at the ε-amine of Lys7 replacing Gly7 (2nd group) in the original DT1 template. The first group comprises the analogs DT7 (N4-Gly-Arg- Arg-Pro-Tyr-Ile-Leu-D-Asn-OH) and DT13 (N4-Gly-Arg-Arg-Pro-Tyr-Ile-β3hLeu-OH), both retaining high binding affinity for NTS1R. The respective [99mTc]Tc-radioligands showed increased in vivo stability in peripheral mice blood, although they achieved maximum stability only after treatment of mice with the Entresto®+Lisinopril (Lis) combination. Both [99mTc]Tc-DT7 and [99mTc]Tc-DT13 displayed poor uptake in AsPC-1 cells in vitro, thereby failing to qualifying for further study of their biodistribution profiles in AsPC-1 tumor-bearing mice. In the second Lys7-modified set of compounds, a pendant palmitoyl group was first attached: DT8 (N4- Lys(palmitoyl)-Arg-Arg-Pro-Tyr-Ile-Leu-OH), mimicking analogs of the invertebrate NT-counterpart contulakin-G. DT8 displayed an excellent affinity for NTS1R and [99mTc]Tc-DT8 high uptake in AsPC-1 cells. The radioligand stability in mice peripheral blood was excellent as well. These results translated into a very promising tumor uptake of [99mTc]Tc-DT8 in AsPC-1 xenografts bearing SCID mice which however was compromised by a very unfavorable and persistently high background radioactivity. Thus, the strong binding of the palmitoyl pendant group of [99mTc]Tc-DT8 to albumin and its high lipophilicity resulted in poor endpharmacokinetics. In the 2nd generation of compounds with a pendant group attached at the ε-amine of Lys 7: a 4-(4- methylphenyl)butyric acid (MPBA) moiety was attached either directly – DT9 (N4-Lys(MPBA)-Arg-Arg-Pro- Tyr-Ile-Leu-OH) or via a polyethylenoglycol PEG4-spacer – DT10 (N4-Lys(PEG4-MPBA)-Arg-Arg-Pro-Tyr-Ile- Leu-OH); in a third analog a PEG6 (Met-PEG5-CH2-COOH) was introduced – DT11 (N4-Lys(PEG6)-Arg-Arg- Pro-Tyr-Ile-Leu-OH). These modifications were excellently tolerated by the NTS1R, with DT9, DT10 and DT11 displaying sub-nanomolar receptor affinities. [99mTc]Tc-DT9 and [99mTc]Tc-DT10 were taken up by AsPC-1 cells equally well with [99mTc]Tc-DT1, while [99mTc]Tc-DT11 showed lower cell uptake. As expected, the radioligands lacking an albumin binding domain (ABD) -functionality, [99mTc]Tc-DT1 and [99mTc]Tc-DT11 demonstrated poor binding to albumin. In contrast, [99mTc]Tc-DT9 and [99mTc]Tc-DT10 were well bound to albumin, with their binding being significantly reduced by the albumin-binding pain-killer ibuprofen. While [99mTc]Tc-DT9 displayed similar in vivo stability with the [99mTc]Tc-DT1 reference in peripheral mice blood, [99mTc]Tc-DT10 and [99mTc]Tc-DT11 were significantly more stable, revealing the impact of steric factors. All radiotracers reached their maximum stability by treatment of animals with the Entresto®+Lis combination. It should be noted however that [99mTc]Tc-DT10 could achieve similar stability levels by Entresto® alone with [99mTc]Tc-DT9 and [99mTc]Tc-DT11 during Entresto®+Lis treatment. In SCID mice bearing AsPC-1 xenografts the three radioligands displayed by far a faster clearance from the background compared with [99mTc]Tc-DT8. In the control animals the uptake of the three new radioligands was higher than the [99mTc]Tc-DT1 reference, further increasing in the animals treated with the Entresto®+Lis combination ([99mTc]Tc-DT9 and [99mTc]Tc-DT11) or with Entresto® only ([99mTc]Tc-DT10). In conclusion, [99mTc]Tc-DT10, outperformed all analogs of the thesis in terms of stability, tumor targeting and background clearance, especially after treatment of animals with a single inhibitor, the registered drug Entresto®. These results emphasize the validity and importance of the in situ stabilization approach, leading to notable improvements in performance of biodegradable NTS1R-targeting radioligands. They have also shown that the introduction of pendant groups at Lys7 introduced in the NT(8-13) chain, was well tolerated by the NTS1R. The performance of resulting radioligands was found highly dependent on pendant group features, such as lipophilicity, albumin binding capability and steric factors. The performance of [99mTc]Tc-DT10 was very promising at the preclinical level, especially in combination with Entresto®, fairly competing with other NTS1Rtargeting SPECT radiotracers already in clinical tests. Further studies are warranted to assess this option in PC patients.
Language English
Subject Radiopharmaceuticals
Παγκρεατικό αδενοκαρκίνωμα
Υποδοχέας νευροτενσίνης
Issue date 2022-12-07
Collection   School/Department--School of Medicine--Department of Medicine--Doctoral theses
  Type of Work--Doctoral theses
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