Partner und Internationale Organisationen
(Englisch)
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A, B, CZ, DK, FIN, F, D, GR, H, I, NL, N, PL, P, SI, E, S, CH, GB
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Abstract
(Englisch)
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The COST project 'Labeling of small clinically relevant biomolecules with the organometallic aquo-ion [99mTc(OH2)3(CO)3]+ within the COST action B12 has made progress along several branches. It is one of the major objectives of this project to introduce new ligand types in small biomolecules, to label these derivatives and to study their biological behavior in vitro or in vivo. Furthermore, to be of clinical relevance, the technology has to be transferred within the members of the action and, consequently, the labeling process be made as convenient as possible. These restrictions could be achieved for several biomolecules as outlined later in detail, but in particular to peptides, estrogenes, central nervous system (CNS) receptor ligands and to an inhibitor of glucose transporter systems. CNS-receptor, Cyclopentadienyl chemistry: As described in the last report, the introduction of small lipophilic ligands such as cyclopentadienyl (Cp) in CNS receptor ligands has been pursued extensively. After the exploration of introducing acidic acetyl-cyclopentadiene in organic model compounds, we have transferred this labeling strategy to true biomolecules such as the serotonergic receptor ligand WAY. Three different compounds (WAY-1, WAY-2 and WAY-4) were produced by a novel synthetic approach. They could be isolated and fully characterized. Reaction in water with long-lived 99Tc gave the expected half-sandwich complexes [WAY-X-CpTc(CO)3] (X = 1, 2, 4) two of which were structurally characterized. The affinity of the two compounds [WAY-1-CpRe(CO)3] and [WAY-4-CpRe(CO)3] for the serotonergic receptor HT1A were tested in collaboration with the 'Forschungszentrum Rossendorf' (FZR) in Germany. It was found that [WAY-1-CpRe(CO)3] had a low affinity (IC50) of about 300 nM but compound [WAY-4-CpRe(CO)3] showed an affinity of 6 nM which compares well with the native compound and indicates full retention of bioactivity. The low affinity of [WAY-1-CpRe(CO)3] is not surprising since it is known from structure-activity relationships that a C1 spacer is too short and the complex attached to it will sterically interfere with the receptor in any case. However, [WAY-4-CpRe(CO)3] is a very promising compound and was subjected to labeling studies. Quantitative aqueous labeling was established with 99mTc using own and commercially available Isolink® kit developed in a former part of this project. The general versatility of the approach is obvious, since quantitative labeling was achieved with the biomolecules as in case of the model complexes, thus, the technology is adaptable to other small biomolecules. Initial biodistribution studies with mice (FZR, Dr. H.-J. Pietzsch) showed a very good first pass brain uptake of about 1.3%. Further biological and synthetic studies are in progress. This methodology was also combined with estradiol as an important future radiopharmaceutical for the diagnosis and treatment of breast cancer. So far, only little progress could be made in this field since the presence of additional hydroxy-groups in the lead structure makes the chemistry of derivatization more complicated. Instead, as an extension to the original plans, a novel labeling method was introduced to yield the aimed compound [estradiol-CpTc(CO)3]. In collaboration with the 'Ecole National Supérieur de Chimie de Paris' (ENSCP) we found that a ferrocene derivative of estradiol (estradiol-Fc) can be used to introduce the [Cp99(m)Tc(CO)3] moiety. When estradiol-Fc is heated in dmso/water mixture, Fc is decomposed under cleavage of 'CpFe' and the [Tc(CO)3]+ moiety introduced instead at the remaining, estradiol bound Cp ring. This work is under further investigation in order to improve yield and labeling conditions and represents a novel additional possibility for the introduction of [R-Cp99(m)Tc(CO)3] in lipophilic biomolecules. Meanwhile, the use of the cyclopentadienyl method is applied by other groups as well, for the labeling of fatty acids but also for other brain tracers in general. In respect of brain uptake, preliminary results with other brain receptor ligands show exceptional high values which clearly indicates that Cp chemistry is the way to go in the context of CNS receptor ligands. Histidin ligands and coupling to biomolecules: As obvious from the labeling of cyclopentadienyl derivatized biomolecules, the choice of appropriate ligands represents a crucial part in the strategies to label small, clinically relevant biomolecules with the [Tc(CO)3]+ moiety. The studies with tripodal histidin, started during the previous period and preliminarily described in the last report, are now successfully established and already transferred to a number of other groups from within and outside the COST action. We have studied the basic derivatization -, coordination and coupling chemistry of histidin derivatized at the Ne position. Two strategies were followed the same time to achieve the introduction of tripodal histidin derivatives in biologically active molecules. In a first one we used the organometallic fragment [Re(CO)3]+ as a protecting group for the reactive positions in histidin since it occupies all three positions in one single step. This metal based protecting group can conveniently be removed by smooth oxidation after the complex has been coupled to a biologically active molecule. The derivative is than ready to be labeled with [99mTc(CO)3]+. We have applied this strategy to amino acids and peptides. The second pathway comprises full organic protection of histidin. The two (three) protecting groups are released after the coupling to the biomolecule. This approach is applied to biomolecules by several groups i.e. in Basel and Munich to assess the viability in comparison with other methods. So far, labeling of the biomolecules synthesized through both approaches yielded the identical product. The biomolecules tested so far can be labeled at very low concentrations and in quantitative yield, both characteristics are crucial factors for the development of clinically relevant small biomolecules. Since histidin represents a naturally occurring molecule, it can be expected that the biological behavior of the labeled biomolecule will remain essentially unchanged compared to the native one. Preliminary biological studies clearly point in this direction. Other small, biologically active molecules: Among the clinically most relevant molecules are those related to the glucose metabolism of living organism. The major problem in this field is still the introduction of appropriate ligands (complexes). Since the bioactive molecules are in general small, derivatizations will cause significant physico-chemical changes. To principally prove or disprove the possibility of labeling very small biomolecules with the [99(m)Tc(CO)3]+ moiety, we have introduced histidin in phloretin to extend into a new branch of the project. Phloretin is known to inhibit the glucose transporter GLUT 1. The synthesis of the first derivative is multistep and difficult but finally the desired product could be produced in sufficient amounts. The compound was labeled and a preliminary series of cell tests performed at the Free University of Brussels VUB (Prof. J. Mertens). The compound shows a clear but in its extent not sufficient inhibition of the glucose transporter. Currently the lead structure is optimized to improve the inhibition capacity. In particular a longer spacer between the binding and the coordinating part was introduced. Next biological testings will be performed in march. Nucleobases: The labeling of oligonucleotides (antisense strategy) is the research topic of one working group in the COST B12 action. We have therefore directly labeled nucleobases, nucleosides and nucleotides since the labeling of oligonucleotides ( a polymer of nucleotides) still represents a central problem in the field of the antisense strategy. Due to the high affinity of the [Tc(CO)3]+ moiety to aromatic amines, guanine (derivatives) are conveniently labeled through direct coordination to N7 and N9 of guanine. We have structurally characterized several derivatives and have performed preliminary biological cell tests at the 'Klinikum Rechts der Isar, Munich' (Dr. H.-J. Wester). The results show, that the compounds are taken up by the cells but it remains to be proven that the complexes end up in the nucleus. Experiments with different types of cancer cell lines gave a more differentiated picture. The starting compound [Re(OH2)3(CO)3]+ was cytotoxic in all cell lines. Cell uptake studies with the corresponding complex [99mTc(OH2)3(CO)3]+ showed uptake of the radioactivity in the nucleus, thus, further rationalizing that the cytotoxicity is based on binding of the complex to DNA. Since the cells are in a medium containing BSA amino acids and other biomolecules, a significant part of [99mTc(OH2)3(CO)3]+ is not biologically available. Therefore two water ligands have been subsituted with weak bidentate ligands in order to prevent binding to e.g. HSA. The cytotoxicity results with these compounds are variable. Although the show plasmid binding, they were not cytotoxic in all cases. The structure-activity relationship in respect of cytotoxicity is under further study and actually the topic of a Ph.D. thesis. It is planned to file a provisional patent application of these results.
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