Cell-penetrating peptides (CPPs) are able to penetrate the cell membrane carrying cargoes such as peptides, proteins, oligonucleotides, siRNAs, radioisotopes, liposomes, and nanoparticles. Consequently, many delivery approaches have been developed to use CPPs as tools for drug delivery. However, until now a systematic analysis of their in vivo properties including potential tumor binding specificity for drug targeting purposes has not been conducted. Ten of the most commonly applied CPPs were obtained by solid phase peptide synthesis and labeled with (111)In or (68)Ga. Uptake studies were conducted using a panel of six tumor cell lines of different origin. The stability of the peptides was examined in human serum. Biodistribution experiments were conducted in nude mice bearing human prostate carcinoma. Finally, positron emission tomography (PET) measurements were performed in male Wistar rats. The in vitro uptake studies revealed high cellular uptake values, but no specificity toward any of the cell lines. The biodistribution in PC-3 tumor-bearing nude mice showed a high transient accumulation in well-perfused organs and a rapid clearance from the blood. All of the CPPs revealed a relatively low accumulation rate in the brain. The highest uptake values were observed in the liver (with a maximal uptake of 51 %ID/g observed for oligoarginine (R(9))) and the kidneys (with a maximal uptake of 94 %ID/g observed for NLS). The uptake values in the PC-3 tumor were low at all time points, indicating a lack of tumor specific accumulation for all peptides studied. A micro-PET imaging study with (68)Ga-labeled penetratin, Tat and transportan(10) (TP(10)) confirmed the organ distribution data. These data reveal that CPPs do not show evidence for application in tumor targeting purposes in vivo. However, CPPs readily penetrate into most organs and show rapid clearance from the circulation. The high uptake rates observed in vitro and the relatively low specificity in vivo imply that CPPs would be better suited for topical application in combination with cargoes which show passive targeting and dominate the pharmacokinetic behavior. In conclusion, CPPs are suitable as drug carriers for in vivo application provided that their pharmacokinetic properties are also considered in design of CPP drug delivery systems.

Combination therapy has improved the quality of life for patients with squamous cell carcinomas of the head and neck (HNSCCs) but has not decisively changed prognosis. Targeted therapies, which enhance accumulation of the drug in the tumor, may be realized using tumor-specific binding peptides. This paper identifies and characterizes an HNSCC affine peptide. Methods: From a phage library comprising 10 9 different displayed peptides, 1 peptide was enriched after 5 in vitro selection rounds on HNO223 tumor cells. Subsequently, the gained peptide sequence H 2 N-SPRGDLAVLGHKY-CONH 2 (HBP-1) was synthesized as an amide and labeled with 125 I. In vitro studies for binding kinetics and competition were performed with 5 different HNSCC cell lines. Furthermore, the stability of the peptide was evaluated in human serum. The in vivo biodistribution of 131 Ilabeled peptide was determined in HNSCC tumor-bearing nude mice. The results were further validated in human HNSCC tumor tissue sections using fluorescence-labeled HBP-1. Competition experiments were performed to determine the binding sequence and validate the target. Results: The HBP-1 motif was enriched in 62% of all phages sequenced. Labeled 125 I-HBP-1 showed binding to 5 different HNSCC cell lines and a maximum binding to HNO97 cells, with 11% of the applied dose per 10 6 cells and an inhibitory concentration of 50% of 38.9 nM. Stability experiments in human serum showed a half-life of 55 min. In 2 different HNSCC tumor xenografts, 131 I-HBP-1 accumulated rapidly, with stable uptake until 45 min after intravenous application. Peptide immunohistochemistry of HNSCC tissue sections exhibited tumor staining by HBP-1, whereas normal tissue remained negative. Sequence mutation and competition experiments revealed that the intrinsic RGD motif in combination with the intrinsic LXXL motif is responsible for the binding ability of HBP1. The RGDLXXL sequence within this peptide is known and indicates that binding occurs via the a v b 6 rather than the a v b 3 integrin. Conclusion: Within the sequence of HBP-1 is a RGDLXXL motif, and most likely it is targeting the a v b 6 receptor of the integrin family of cell adhesion receptors. HBP-1 represents a promising lead structure for the development of targeted therapies or diagnostic procedures in patients with HNSCC. More than 5% of all malignant tumors worldwide are head and neck cancer, with more than 100,000 cases diagnosed in Europe each year. In the Western world, squamous cell carcinoma of the head and neck (HNSCC) accounts for more than 90% of all head and neck cancers (1). Most cases are diagnosed in patients who are between 50 and 70 y old, with a 5 times higher rate for men than for women. The 5-y survival rate of less than 30% is due to a high lymphogenic metastatic tendency, a high recurrence rate, and an increased occurrence of secondary tumors. Treatment strategies for advanced HNSCC have evolved from less effective monotherapy to an integrated, more effective multidisciplinary approach. A new field under investigati...

SFTI derivatives are small stable molecules readily accessible by solid-phase synthesis. The trypsin inhibition was not influenced by the cyclization, and addition of a chelator had no significant influence. The exceptional rigidity and stability allow the use of SFTI derivatives as scaffolds for the introduction of tumor-specific peptide motifs which could be used to increase cell-binding affinities and thus their use as diagnostic and/or therapeutic tools.