Detailed Analysis Concerning the Biodistribution and Metabolism of Human Calcitonin-Derived Cell-Penetrating Peptides I. Neundorf,* ,† R. Rennert, † J. Franke, † I. Közle, ‡ and R. Bergmann ‡ Faculty of Life Science, Pharmacy and Psychology, Institute of Biochemistry, Bru ¨derstr 34, D-04103 Leipzig, Germany, and Research Center Dresden-Rossendorf, Institute of Radiopharmacy, Bautzner Landstrasse 128, D-01328 Dresden, Germany. Received April 10, 2008; Revised Manuscript Received June 17, 2008 The interest in using small peptides for therapeutic and diagnostic in ViVo applications is based on several advantageous features such as good penetration into tissues and rapid clearance from the body. Because of their size, they can easily be synthesized chemically. The recently discovered cell-penetrating peptides (CPP) and among them CPP derived from the native peptide hormone human calcitonin (hCT) could meet these requirements. Therefore, they are nowadays widely used as delivery vectors for a variety of bioactive molecules. However, the knowledge about the distribution and metabolism of CPP in ViVo is very limited. Hence, evaluation of the pharmacological features of any promising peptide is a crucial challenge in its development process. Herein, we studied the in ViVo radiopharmacology of 68 Ga radiolabeled DOTA-modified, hCT-derived CPP in rats using small animal PET. Furthermore, the arterial blood at different time points and urine were analyzed for radio- metabolites. It was shown that D-amino acid modifications of the sequence hCT(9-32) resulted in an increased in ViVo stability and lower retention in the kidney cortex of this peptide. INTRODUCTION During the past decade, the interest in using cell-penetrating peptides (CPP) as vectors for drug delivery has dramatically increased (1, 2). By using CPP, it is possible to introduce membrane-impermeable substances into mammalian cells. There is a growing demand for such delivery strategies since many pharmacologically interesting molecules provide on the one hand promising therapeutic properties in Vitro but are on the other hand often poorly bioavailable in ViVo. Because of their physicochemical properties, the transport of such substances into cells and to their biological target is often restricted. That is the reason why during the last few years delivery systems using CPP as drug carriers have been under intense investigation. Meanwhile, a number of different cargos have been transported successfully into various cells, e.g., peptide nucleic acids (PNA) (3), proteins (4), oligonucleotides (5), or nanoparticles (6). Cell-penetrating properties have been described also for the peptide hormone human calcitonin (hCT). Human calcitonin consists of 32 amino acids and is C-terminally amidated and secreted in the thyroid gland. Because of a high permeability through the nasal epithelium, it is applied as an intranasal therapeutic in the treatment of several bone diseases (7). Recently, the truncated sequence hCT(9-32) was shown to translocate through cell membranes, although its N-terminal part (residues 1-8) responsible for receptor activation was lacking (8). By using hCT(9-32), it was possible to efficiently shuttle different cargos such as the antiproliferative drug daunorubicin, fluorophores, or even large molecules such as the enhanced green fluorescent protein (eGFP) into cells (9–11). The interest in using cell-penetrating peptides for therapeutic in ViVo applications is based on some advantageous features: these small peptides penetrate more readily into tissues, are distributed more uniformly, and disappear more rapidly from the body than larger molecules such as antibodies. However, major drawbacks in using such peptides as in ViVo therapeutics are their low resistance against proteolytic enzymes and their rapid clearance by the kidneys. The enzymatic degradation can effectively be inhibited by molecular modifications such as the introduction of D-amino acids or amino-alcohols and the insertion of unnatural amino acids or side chains (12). Recently, we modified the N-terminally truncated hCT fragment hCT(9-32) by the introduction of either D-phenylalanine or N-methylphenylalanine at positions 12 and 16, which were previously shown to be the initial cleavage sites for an enzymatic digestion in Vitro (13). We could demonstrate that the half-lives of these modified peptides were increased in Vitro in human blood plasma as well as in the cell culture supernatant (14). Although several examples for successful drug delivery in ViVo exist (15–18), so far a careful analysis of the in ViVo behavior of these hCT-derived peptides as well as for most of the CPP described in the literature is missing. Moreover, in most studies the therapeutic potential of CPP is evaluated by using tissue culture models. However, the evaluation of pharmacologi- cal features is a crucial challenge not only in drug discovery but also in the drug development process. Therefore, by detailed studies, it should be possible to design molecules possessing on the one hand the desired biological activity and on the other hand the required pharmacological potency and metabolic stability. Furthermore, the results of such studies could help to decide whether or not a further development of a compound makes sense. An elegant possibility to investigate the three-dimensional distribution of radiolabeled compounds in ViVo is the positron emission tomography (PET). PET provides noninvasive and nondestructive quantitative measurements of the time course of biomolecules within living subjects (19). The prerequisite for PET studies is the labeling of the compound of interest with a positron-emitting radionuclide, e.g., 68 Ga. This isotope has * Corresponding author. Dr. Ines Neundorf, Faculty of Life Sciences, Pharmacy and Psychology, Institute of Biochemistry, Bru ¨derstr 34, D-04103 Leipzig, Germany. Phone: +49 (0) 341 97 36 832. Fax: +49 (0) 341 97 36 909. E-mail: neundorf@uni-leipzig.de. † Institute of Biochemistry. ‡ Institute of Radiopharmacy. Bioconjugate Chem. 2008, 19, 1596–1603 1596 10.1021/bc800149f CCC: $40.75  2008 American Chemical Society Published on Web 07/24/2008