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Radiolabeled Peptides for Cancer Diagnosis and Therapy

Friday, 19 May, 2006 - 16:00
Campus: Brussels Humanities, Sciences & Engineering campus
Faculty: Science and Bio-engineering Sciences
Veronique Maes
phd defence

Since many tumors overexpress receptors for neuropeptides, such as neurotensin and
bombesin, the use of radiolabeled peptides for receptor targeting has become a powerful
tool in nuclear oncology. Internalization of the receptor-bound ligands can result in the
accumulation of radioactivity within the target cells. Incorporation of g-emitting
radionuclides allows the visualization of the tumor using the single-photon emission
computed tomography (SPECT) or positron emission tomography (PET) techniques,
whereas a- and b--radiations are able to destroy the tumor cells. Although many
advantages have been associated with the use of peptides as targeting molecules,
strategies to overcome their rapid metabolic degradation and poor biodistribution
properties have to be evaluated. This thesis deals with the synthesis of
radiopharmaceuticals based on stable analogs of neurotensin(8-13) (Arg-Arg-Pro-Tyr-Ile-
Leu) and bombesin(7-14) (Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2) for the diagosis and
treatment of exocrine ductal pancreatic carcinoma and breast- and prostate carcinoma
respectively. An important aspect in the design of new radiopharmaceuticals is the
improvement of the pharmacokinetic properties of the targeting peptides in order to obtain
better tumor-to-background ratios for diagnosis and to meet the more stringent
requirements for biodistribution for therapeutic applications. Different strategies have been
applied in this project to optimize the biodistribution profile of the labeled peptides. These
modifications should not have a negative influence on the binding affinity.

The synthesis of the neurotensin (NT) analogs was performed on a Merrifield polystyrene
resin using the Boc/OBn solid-phase peptide synthesis protocol. For radiolabeling with
99mTc, the retro-Na-carboxymethyl histidine ((NaHis)Ac) was attached to the N-terminus
of the peptides, whereas DTPA was coupled to the neurotensin analogs for labeling with
111In. The construction of the peptides containing the (NaHis)Ac chelator was achieved on
solid support. For the conjugation of DTPA to the peptides, different strategies in solution
and on solid-phase have been evaluated. The half-life of the native NT(8-13) sequence of
only a few minutes in plasma could be increased to more than 20 days by N-methylation of
the Arg9 and substitution of the Ile12 by the voluminous Tle, resulting in the NT-XII
analog ((NaHis)Ac-Arg-MeArg-Pro-Tyr-Tle-Leu). This analog already made it into clinical
trials. With the purpose to reduce the accumulation of the radiopharmaceuticals in liver
and kidneys, the peptides were made more hydrophilic. The introduction of the hydrophilic
shikimic acid through a Lys spacer between the (NaHis)Ac chelator and the double
stabilized NT sequence, leading to NT-XVIII ((NaHis)Ac-Lys(shikimyl)-Arg-MeArg-Pro-Tyr-
Tle-Leu), did result in the expected lower kidney and liver accumulation. However, the
receptor affinity and especially tumor uptake were also reduced. The introduction of a
bAlabAla spacer between the NT sequence and the chelator-shikimic acid moiety did not
improve the receptor affinity. The replacement of the Tyr11 residue by the bulky 2’,6’-
dimethyltyrosine (Dmt) in NT-XIX ((NaHis)Ac-Arg-MeArg-Pro-Dmt-Tle-Leu), despite
resulting in a decreased receptor affinity, maintained the accumulation in the tumor at the
same level as in NT-XII. This analog has the best characteristics of all published NTanalogs
so far and it will soon be evaluated in clinical trials. This research was performed
in collaboration with the group of Dr. P. Bläuenstein and Dr. E. Garcia-Garayoa from the
Paul Scherrer Institute (Villigen, Switzerland), who carried out the radiolabeling and all
biological assays.

For the labeling of the neurotensin analogs with 111In, the best strategy for the
conjugation of the DTPA chelator to the peptides had to be found. The coupling of tri-tBu-
DTPA to the resin bound peptides is the most general method. However, the desired
peptides were not obtained in high yields. The major problem during the synthesis is the
formation of complexes with metallic impurities. Great attention should be paid in the
future to avoid these impurities or to find better procedures to extract the metal from the
complex. The stabilized 111In-DTPA-NT analogs have been studied by the group of Dr. A.
Gruaz-Guyon (University Paris 7) for their interaction with the NTR1 receptor in the
presence of a bispecific antibody in order to enhance the tumor selectivity. However, the
double (MeArg9, Tle12) and triple stabilized (MeArg9, Dmt11, Tle12) DTPA-NT analogs
showed, contrary our expectations, a reduced affinity for the receptor on HT-29 cells.

In addition to the synthesis of stable neurotensin analogs for radiolabeling, fluorescein
labeled stable NT(8-13) analogs were synthesized. The fluorescent dye FITC was coupled
to the N-terminus of the peptide on solid-support. The introduction of the FITC-group had
a negative influence on the receptor binding affinity. This reduced receptor affinity was
limited by the introduction of an aminovaleroyl spacer. It was shown that for the best
analog the fluorescently labeled neurotensin is internalized in the cells.

The bombesin (BN) analogs were synthesized on a Rink amide resin using the Fmoc/OtBu
solid-phase peptide synthesis strategy. All synthesized bombesin analogs were used for
99mTc-labeling and therefore contain the (NaHis)Ac chelator. Since the His12-Leu13
peptide bond is very susceptible to enzymatic hydrolysis, new BN(7-14) analogs have been
synthesized in which the Leu in position 13 was replaced by b2hLeu, b3hLeu and Cha.
These changes led to the expected improved plasma stability, but only the analog
containing the Cha instead of Leu retained affinity for the GRP receptor. To prevent
oxidation of the sulfur of the Met residue it was replaced by Nle. The effect of the
introduction of different spacer as well as the introduction of the hydrophilic shikimic acid
through a Lys spacer between the BN(7-14) sequence ((NaHis)Ac-Gln-Trp-Ala-Val-Gly-His-
Cha-Nle-NH2) and the (NaHis)Ac chelator on the biodistribution profile of the peptide has
been studied. The introduction of the shikimic acid in BBS-42 resulted in an enormous
decrease in liver and kidney accumulation while maintaining high affinity for the GRP
receptor. This analog, together with the bombesin analog BBS-43 in which the b3hSerbAla-
bAla spacer was introduced, are the first analogs which showed increasing target-tonon
target ratios, indicating the much faster clearance from the non-target organs
compared to the clearance from the target tissues. SPECT images have already been made
with BBS-42 in nude mice bearing PC-3 tumor xenografts and showed a clear visualization
of the tumor. These results look very promising for the further evaluation of these

In a last part of this thesis, the carbohydration of bombesin analogs containing the
(NaHis)Ac chelator via the Maillard reaction followed by Amadori rearrangement has been
studied. Since the (NaHis)Ac moiety has an unusually reactive secondary a-amine, various
chemical strategies have been explored to perform the Maillard reaction on peptides
containing this chelator. The best results were obtained when the Maillard reaction was
carried out on the resin bound peptides after construction of the chelator. However,
difficult reaction control and poor RP-HPLC separation gave the peptide in only low yields.
An alternative method, based on chemoselective oxime ligation, has already been tried on
a bombesin sequence. The first experiments showed a selective reaction on the
hydroxylamine function and not on the secondary a-amine of the chelator. Although still
some work is left in optimizing this strategy the first results look promising.