Erik De Blois

There are many reports on the determination of RCP by HPLC, including accuracy, linearity, precision, repeatability, detection limits [61-63]. To our knowledge, there are no criteria to qualify a radiodetection based HPLC separation method. With differences in eluens, gradient, flow, column type and length there might be an increase variation in finding impurities, and thus variation in RCP. Standardisation of separation method includes HPLC-eluens, gradient or isocratic, flow, column type and length, detection and interpretation of chromatograms. Therefore, in our opinion, RCP is actually expressed in percentages of arbitrary units. Therefore we suggested minimal requirement for RCP by HPLC and radiodetection for radiolabeled DTPA or DOTA-peptides which should include base-to-base separation of: a. At least 2 peaks of the DTPA conformations of a radiolabeled DTPA-peptide [64] b. Metal-peptide vs. peptide for DTPA and DOTA-peptide [65, 66] c. For Met-containing regulatory peptides: Met-sulfoxide radiopeptide vs intact radiolabeled peptide [46] These requirements are important to standardize RCP measurements for reliable comparisons of RCP quantifications between different systems/laboratories. Ready-for-use liquid formulation For safety reasons it would be desirable to store and transport the ready-for-use liquid formulation (diagnostics and therapeutics) of radiolabeled peptides. In chapter 5 we describe the presence of quenchers in a single-vial liquid pharmaceutical formulation of a radiolabeled peptide, in a quantity sufficient to prevent radiolysis of the formulation. Radiolabeled peptides are generally stored and transported in the form of multi-vial kit formulations. Usually the contents of these vials are lyophilized or frozen and should be brought into solution subsequently in a mutual reaction to produce the intended radiolabeled peptide. For health physics reasons it would be desirable to be able to store and transport the ready-for-use liquid formulation of the radiolabeled peptide. This could avoid similar labeling of radiopharmaceuticals in different hospitals. So, a physician could administer the labeled peptide without performing extra handling like a radiochemical reaction by local radiochemist, but simply by diluting the contents of the vial in a radiopharmaceutical liquid that can be administered by injection or by infusion [48]. Here we describe how to maintain RCP and thus prolong storage time. As a result this increases the availability of the ready-for-use radiolabeled SS-analogs to worldwide. In this thesis, stability and radiolysis of radiolabeled SS-analogs were monitored by HPLC. HPLC methods were optimized to distinguish between non-labeled and radiolabeled peptides vs. the radiolysed peptides. The effect of quenchers on the stability of radiolabeled SS-analogs, even under therapeutic conditions was optimized and monitored up to 7-9 days after radiolabeling. Pharmacokinetics The focus for improvement of BN-based imaging (by increasing tumor-to-background ratio) has predominantly relied on development of new BN-based analogs with enhanced affinity for the GRPR [10, 67-69]. Instead, in chapter 6 we investigated the impact of various experimental factors in order to further improve uptake in target and target background ratio of peptide receptor-mediated imaging. To ensure purity of the labeled peptide many study groups perform purification of BN-based analogs by HPLC between labeling and administration. In order to determine the impact of this purification step, we studied the effect of HPLC-purification mass dependently by performing biodistribution studies. An essential parameter to evaluate the performance of targeted imaging probes is the ratio between activity in tumor vs. background organs. Contrast between the two is dependent on the amount of administered peptide. BN-based analogs have already pharmacological effects after administration in nanomole quantities. The high affinity GRPR have a low capacity in tumor and other tissues and also the expression varies between tissues [10, 70, 71]. Therefore, in the present study we investigated GRPR saturation by injecting an increasing amount of peptide (bellshape) and measuring target vs. non-target organs. From this bellshape an optimal peptide dose was calculated for further applications [72]. Furthermore we studied the impact of specific activity (MBq/nmol) on tumor-to-background ratio by varying injected peptide amount while injecting a constant amount of activity (MBq). GRPR-positive tumor tissue showed a significant 2 to 3-fold increase in absolute uptake after HPLC-purification while a stable tumor-to-pancreas ratio remained. Low peptide amounts resulted in a decline in uptake in tumor, kidney and pancreas. Tumor-to-pancreas ratio improved six-fold. GRPR saturation 4h prior to injection of 111In-DOTA-AMBA resulted in improvement of the tumor-to-pancreas ratio. All these findings increased knowledge of how to improve tumor uptake and to perform a proper BN-based image. Nevertheless it also shows the increase importance of labeling of BN analogs with high SA. Conclusion With all the above-mentioned options in mind and although radiolabeled regulatory peptides have been investigated and successfully applied for peptide receptor radionuclide scintigraphy and therapy for nearly 2 decades, there are still many options to improve and fully exploit peptide receptor radionuclide scintigraphy and therapy. Outlook for future concepts and research It can be concluded from literature and the above-mentioned arguments that peptide receptor radionuclide scintigraphy can be performed with either 111In- or 177Lu-labeled DTPA- or DOTA-peptides. However, although DOTA-peptides can also be radiolabeled at high specific activity, this specific activity might be not high enough for PRS and PRRT with ligands which have pharmalogical side effects using small amounts (nmoles), the delivery of sufficient amounts of activity (MBq) to these regions may be too low for scintigraphy and therapy. However, there may be several other ways to circumvent these limitations. Since Maximum SA is directly related to half-life of isotope i.e. for imaging the use of PET radionuclides (Ga) and for therapy a-emitters could be good alternative. a-emitting radionuclides have a high Linear Energy Transfer (high energy deposition within a short range), consequently the cell kill probability is high, but only if the target (e.g. DNA) is within range. Also improvements of the characteristics of the radioligand might be achieved by investigating the role of linkers and chelators [73-76], and by studies of the pharmacokinetics of the peptide derivatives [77]. The tools in analytical chemistry are constantly improving, e.g. as recently reported by Asti et al. [65] for a base-to-base chromatographic separation by UPLC of a DOTA-peptide labeled with different not radioactive metal-ions. How this new technique will affect the radiodetection, including sensitivity of radiopeptides is currently under investigation and is already proven to be very promising. To increase SA, purity and content of the used peptides is very important. Therefore we developed an alternative and indirect method to quantify DOTA-peptide, which include titration of the DOTA-moiety with non-radioactive metal ions. This titration is a indirect and alternative method to quantify purity and content of DOTA-peptides, even in the presence of UV-absorbing additions and/or impurities. Optimization and maintaining RCP with quenchers for 111In- or 177Lu-labeled SS-, MG- or BN-peptides can be used as a model bombesin or 111In- or 177Lu-labeled regulatory peptides and other radiopeptides or proteins containing amino acids like Met, Trp and Tyr. However, maintaining RCP with described mixtures of quenchers is not a guarantee for protection of other peptides or proteins. Addition of quencher mixtures to maintain RCP must be investigated nuclide and peptide dependently. PRS or PRRT with DOTA-conjugated Met-containing CCK or bombesin analogs at high RCP is a challenge, therefore further research for chelators are required. Moreover, chelators should include the following characteristics: high kinetic and thermodynamic stability, preferably also with a therapeutic isotope like 177Lu, and without the need of a heating procedure, this might help to maintain RCP [78]. The receptor affinity of radiolysed peptide decrease dramatically [10]. There is a lack of knowledge about affinity of other radiolysed radiopeptides, i.e. for damaged Trp or Tyr. Unfortunately these specific radiolysed radiopeptides were not identified and characterised in this study and the influence on receptor affinity is currently unknown.