| 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.
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