Novel DOTA-[alpha]-melanocyte-stimulating hormone analogs for melanoma targeting : the impact of dimerization, carbohydration and negative charges on the in vivo biodistribution

Bapst, Jean-Philippe. Novel DOTA-[alpha]-melanocyte-stimulating hormone analogs for melanoma targeting : the impact of dimerization, carbohydration and negative charges on the in vivo biodistribution. 2008, Doctoral Thesis, University of Basel, Faculty of Science.


Official URL: http://edoc.unibas.ch/diss/DissB_8353

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Various epidemiological surveys have recently demonstrated that the incidence and mortality rates of
cutaneous malignant melanoma are still increasing in western countries. Incidence rates have dramatically
increased during the last 70 years, raising from 1:1,500 in 1935 to 1:68 in 2002. Although its morbidity
rates in certain population groups have slightly declined lately, it remains the most common malignancy
among young adults. Malignant melanoma represents 5% of all skin cancers, but 71% of all skin cancer
deaths in Caucasian populations. Unless primary melanoma tumors are detected early enough and
adequate surgery can be performed, the prognosis of the disease is very poor, particularly, because of its
high metastasizing potential and the difficulty to detect and to treat either the primary or the secondary
Ionizing radiation is one of the major means to kill tumor cells in patients suffering from cancer. Specific
targeting of radioisotopes to the surface of cancer cells, with the purpose of exposing them to in situ
generated radiation, was initially studied with antibodies as vehicles. Radiopeptides for targeting began to
interest radiochemists and nuclear oncologists later, when structural peptide analogues with excellent
biostability and bioactivity became available; in addition, these analogues carried suitable chemical groups
for incorporation of a variety of different isotopes. The idea, however, to apply radioactive peptides to
receptor-mediated targeting of tumor cells dates back to the early 1970s, when peptide hormone receptors
(i.e. binding sites) had been identified on cell membranes. Radiopeptides are attractive tools for cancer
diagnosis and therapy because a variety of human tumors overexpress surface receptors for regulatory
peptides or peptide hormones. The best examples illustrating the rationale of this strategy are radiolabeled
somatostatin (SST) analogs, which are commercially available (OctreoScan® and OctreoTher®) and
routinely used in clinics to image or even treat neuroendocrine tumors and tumors of the nervous system
expressing SST receptors.
Melanocortin type 1 receptors (MC1R) are overexpressed at the surface of melanoma cells. The hormone
!-MSH is the native ligand of MC1R, and !-MSH analogs bind with great affinity as well. Therefore, !-MSH
derivatives with improved in vivo stability and behavior and bearing chelates able to incorporate various
radionuclides have been developed. Nevertheless, the side-effects of nonspecific retention in the kidneys
limits the therapeutic efficacy of most radiopeptides, as nephrotoxicity is the dose-limiting factor.
Simultaneously, diagnosis of tumors localized in the renal region can be markedly compromised.
Elevated renal uptake and prolonged retention of radiolabeled antibody fragments and peptides in this and
other organs represent a major issue for the therapeutic application of such agents. Over recent years, one
of the focuses of research has therefore been to find new methods to reduce renal uptake. Several
strategies have been investigated without sufficient success. Whether variations in the net charge of the
peptide itself, use of chelator complexes other than DOTA, study of different isotopes, the position of the
chelator complexes in the peptide molecule or of various regulating elements within the peptide sequence
such as, e.g., cyclization of the peptide, none of these approaches resulted in substantial satisfaction.
Therefore, new strategies are needed to potentially solve the kidney uptake issue, or at least to improve the
ratio between tumor uptake and kidney uptake of radioactivity.
Dimerization of peptides has been studied in the field of MSH derivatives since 1977, when several MSH
molecules (up to 300) were attached to the tobacco mosaic virus (TMV), yielding a complex that displayed
a 1,500-fold higher potency than !-MSH. Other dimeric !-MSH derivative synthesized later showed
increased in vitro affinity. Finally, various dimeric ACTH derivatives displayed increased potencies
compared to their monomeric equivalents. Thus, this approach was tempting to test new dimeric peptides
derived from known efficient sequences, taking latest findings about peptide sequences and key structural
elements into account. The idea was not to hit simultaneously two receptors, but to increase the
concentration of the binding motif in the vicinity of the receptor in order to potentially improve tumor uptake
of the peptides in vivo. Three dimeric peptides were successfully synthesized, labeled with 111In and tested,
and although they exhibited excellent receptor binding affinities in the subnanomolar range and good
internalization properties, the in vivo data did not match the expectations. Indeed, no increase in tumor
uptake could be observed, and the dimeric derivatives suffered from very high kidney uptake, making them
unsuitable for diagnostic or therapeutic purposes.
A new type of 111In-labeled !-MSH glycopeptide analogs was then investigated. Glycopeptides were initially
introduced to improve drug delivery to target tissues, either by taking advantage of specific uptake
mechanisms or by enhancing the bioavailability of peptides. Glycopeptides were shown to exhibit
prolonged effects (glycosylated enkephalin peptides) due to better delivery to the target tissue, enhanced
renal peptide uptake from blood (glycosylated Arg8-vasopressin), an improved stability toward enzymatic
degradation in vivo, or a better intestinal absorption, thus enhancing the bioavailability of the peptides.
Other effects of glycation on peptide properties appeared later, including higher or lower accumulation in
the proximal tubules of the renal cortex, depending on the coupled sugar. Structure-activity relationship
studies could describe structural features to exploit or avoid in order to target the kidney. It was observed
that the affinity of peptides for kidney membrane cells could be modulated by attaching different types of
sugars to the molecules, and this led to systematic SAR studies confirming the observations. Other studies
also mention that after carbohydration of somatostatin derivatives, a switch in the excretion way could be
observed. It appeared that the glycopeptides tended to exhibit a switch from the hepato-biliary towards the
renal excretion way, without affecting the uptake in targeted tissues.
This was the basis for the development of carbohydrated !-MSH derivatives in this thesis. Six glycated !-
MSH derivatives, based on the sequence of DOTA-NAPamide (one of the peptides exhibiting the best
pharmacokinetic profile to date) were synthesized and tested. Various carbohydrate moieties were coupled
at different positions along the peptide sequence, in order to determine the influence of the type of sugar or
its position on the in vitro and in vivo properties of !-MSH analogs. Competitive binding assays displayed
results in accordance with the data obtained for the reference peptide, indicating that carbohydration does
not affect target receptor affinity. Biodistribution experiments with melanoma-bearing mice delivered
interesting results. While C-terminal glycation enhanced kidney uptake and retention time, side-chainglycation
seemed only to increase kidney uptake. The N-terminal end, on the other hand, is apparently the
best position for carbohydration. Indeed, two of the three peptides displayed promising results. Introduction
of a galactose moiety was particularly favorable, as it delivered a better tumor-to-kidney ratio of the area
under the curve (4-48h) than the reference peptide DOTA-NAPamide. Thus, carbohydration was shown to
exhibit a high impact on the pharmacokinetics of !-MSH analogs. Some major tendencies on the biological
characteristics after glycation could be drawn, and a new candidate with good potential as lead for further
derivation could be developed.
Finally, novel analogs of negatively charged !-MSH were investigated in this work. It has been shown in
the past that the surface of tubular cells is negatively charged and that anionic molecules are excreted
more easily than cationic molecules, probably because of repulsive electrostatic effects. Therefore,
derivatives carrying an overall negative charge were synthesized and tested. One of them bore two
negatively-charged D-Asp and the chelate at its C-terminal end, in order to enhance the renal excretion of a
potential metabolite. The peptide yielded poor results, both in vitro and in vivo. Affinity of the peptide for the
receptor was lost. In another peptide, DOTA was coupled over a Gly-spacer to a phosphorylated Tyr
located at the N-terminal end of the peptide. While the new derivative displayed average results in vitro, its
in vivo data were excellent. Indeed, an even better tumor-to-kidney ratio of the area under the curve (4-48h)
than DOTA-Gal-NAPamide was reached, delivering the best linear 111In-labeled !-MSH analog to date.
Although this study encompasses only a limited panel of derivatives from each group of peptides
investigated, some important trends for the development of further derivatives could be established.
Complete sets of in vitro and in vivo data were collected for all the peptides, providing valuable information
for the elucidation of structural features required to improve the pharmacokinetic behavior of peptides for
targeting the MC1R. Two new lead candidates provided excellent data; they could be further optimized by
combining features of both of them. Indeed, no linear 111In-labeled !-MSH analog exhibited such interesting
tumor-to-kidney ratios as DOTA-Gal-NAPamide and DOTA-phospho-MSH2-9 to date.
Advisors:Eberle, Alex N.
Committee Members:Schubiger, P. August and Itin, Hans-Peter
Faculties and Departments:03 Faculty of Medicine > Departement Biomedizin > Former Units at DBM > Endocrinology (Eberle)
UniBasel Contributors:Eberle, Alex N.
Item Type:Thesis
Thesis Subtype:Doctoral Thesis
Thesis no:8353
Thesis status:Complete
Number of Pages:185
Identification Number:
edoc DOI:
Last Modified:22 Jan 2018 15:50
Deposited On:13 Feb 2009 16:42

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