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Jul 24, 2008 - among them CPP derived from the native peptide hormone human calcitonin (hCT) could meet these requirements. Therefore, they are ...
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Bioconjugate Chem. 2008, 19, 1596–1603

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 68Ga 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 radiometabolites. 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: * 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: [email protected]. † Institute of Biochemistry. ‡ Institute of Radiopharmacy.

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 pharmacological 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., 68Ga. This isotope has

10.1021/bc800149f CCC: $40.75  2008 American Chemical Society Published on Web 07/24/2008

hCT-Derived Cell-Penetrating Peptides

the advantage of an appropriate half-life of 68 min and can be easily obtained from a 68Ge/68Ga radionuclide generator (20). For the binding of such trivalent metal cations to peptides, DOTA (1,4,7,10-tetraazacyclotetradecane-N,N′,N′′,N′′′ tetraacetic acid) has proven to be a very suitable chelator (21, 22). The aim of the present work was to evaluate the in ViVo biodistribution and the metabolic stability of hCT(9-32) and a 12,16 D-phenylalanine-modified analogue, [f ]-hCT(9-32). For this purpose, both peptides were equipped with the chelator DOTA and subsequently labeled with the radioisotope 68Ga. By analyzing blood and urine fractions, it was shown that the amino acid modifications indeed improve the in ViVo stability of the hCT(9-32) analogue.

MATERIALS AND METHODS Materials. For peptide synthesis, NR-Fmoc-protected amino acids, 1-hydroxybenzotriazole (HOBt) and 4-(2′,4′-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxy (Rink amide) resin were purchased from NovaBiochem (La¨ufelfingen, Switzerland). The side chain protecting groups for the amino acids were tert-butyl (tBu) for Tyr and Thr; tert-butyloxy (tBuO) for Asp; trityl (Trt) for Asn, Gln, and His; and either tert-butyloxycarbonyl (Boc) or 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl (Dde) for Lys according to the synthesis strategy. N,N′-diisopropylcarbodiimide (DIC) was obtained from Sigma-Aldrich (Taufkirchen, Germany). Trifluoroacetic acid (TFA), N,N-diisopropylethylamine (DIEA), thioanisole, p-thiocresole, hydrazine hydrate solution, and piperidine were purchased from Fluka (Taufkirchen, Germany). Acetonitrile (ACN for HPLC) was obtained from Merck (Darmstadt, Germany). Diethyl ether, dichloromethane and N,N-dimethylformamide (DMF, peptide synthesis grade) were obtained from Biosolve (Valkenswaard, Netherlands). DOTA (1,4,7,10-tetraazacyclotetradecaneN,N′,N′′,N′′′ tetraacetic acid) was purchased as t-Bu-ester from Macrocyclics (Dallas, USA). Peptide Synthesis. Peptides were synthesized according to the Fmoc-strategy by using an automated multiple solid-phase peptide synthesizer (Syro, MultiSynTech, Bochum, Germany) on Rink amide resin (30 mg, resin loading 0.5 mmol g-1). The following peptides were synthesized as C-terminal amides: hCT(9-32), LGTYTQDFNKFHTFPQTAIGVGAP; [f12,16]hCT(9-32), LGTfTQDfNKFHTFPQTAIGVGAP; and random (rd)-hCT(9-32), FLTAGQNTIQTPVKTGGHFPFADY. To equip the peptides N-terminally with the chelator DOTA, the preswollen resin was incubated with 3 equiv of DOTA-tris (t-Bu-ester), 3 equiv of HOBt, and 3 equiv of DIC for 3 h at room temperature. Finally, the peptides were cleaved from the resin by using a mixture of TFA/thioanisole/thiocresole (90:5:5, v/v/v) within 3 h at room temperature, simultaneously removing all acid-labile protecting groups. Subsequently, the peptides were precipitated from ice cold diethyl ether, collected by centrifugation, washed four times, and lyophilized from water/tert-butyl alcohol (3:1, v/v). Purification of the peptides was achieved by preparative RPHPLC on a RP C-18 column (Vydac, 250 × 25 mm, 10 µm) with a gradient of 20-60% B in A (A ) 0.1% TFA in water; B ) 0.08% TFA in acetonitrile) over 50 min and a flow of 10 mL min-1. The peptides were analyzed by MALDI-TOF mass spectrometry (Voyager RP, Perseptive Biosystems) and by analytical RP-HPLC on a Vydac RP18-column (4.6 × 250 mm; 5 µm, 300 Å) using a linear gradient of 10-60% B in A over 30 min and a flow rate of 0.6 mL min-1. The experimentally detected masses were in full agreement with calculated masses, and the purity of all peptides was >95% according to analytical RPHPLC.

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Radiolabeling. Two hundred microliters of 68Ga3+-eluate was added to 60 µL of DOTA-modified peptide (1 mM in 40% propylenglycol). The pH was adjusted to 3.7 - 4.1 by the addition of 0.5 M ammonium acetate. The solution was stirred at 90 °C for 10 min. Then, 10 µL (10 mM) of EDTA was added, the solution was incubated for 10 min at 40 °C, and the success of the 68Ga complexation was controlled by radio-thin layer chromatography (radio-TLC) and radio-HPLC. Analysis by radio-TLC was carried out on Merck-RP-18 sheets using either a 10% aqueous ammonium acetate solution (v/v) or a 4:6 mixture (v/v) of ACN (0.05% TFA) and H2O (0.05% TFA) as mobile phases. One micorliter of the sample was run on the sheet, subsequently the sheet was dried, and the activity spot was detected by using a Fujifilm Imaging Plate and a Bas Reader 2000 (Fuji, Raytest). The reaction mixture was applied to size exclusion chromatography (HiTrap 5 mL column, elution with isotonic sodium chloride containing 1% propylenglycol), and the radioactive peptide fraction was used in the experiments. The HPLC system consisted of a Beckman System Gold 125 solvent module equipped with a UV detector (Hewlett-Packard Series 1100) and an external radiochemical detector (RFT 20 046). Analysis was performed on a Zorbax C18 column with a gradient of 10-90% B (ACN with 0.05% TFA) in A (H2O with 0.05% TFA) over 13 min and a flow of 2 mL min-1. Animals, Feeding, Husbandry, and Biodistribution Studies in Rats. The animal research committee of the Regierungspra¨sidium Dresden approved the animal facilities and the experiments according to institutional guidelines and the German animal welfare regulations. The experimental procedure used conforms to the European ConVention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes (ETS No. 123), to the Deutsches Tierschutzgesetz, and to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (DHEW Publication No. (NIH) 82-23, Revised 1996, Office of Science and Health Reports, DRR/NIH, Bethesda, MD 20205). Wistar rats (Wistar Unilever, HsdCpb:Wu, Harlan Winkelmann GmbH, Borchen, Germany) were housed under standard conditions with free access to standard food and tap water. Animals were between 7 and 9 weeks of age. The rats were housed in an Animal Biosafety Level 1 (ABSL-1) acclimatized facility with a temperature of 23 ( 2 °C and humidity of 55 ( 5%. Animals were under a 12 h light cycle in temperature (27 ( 1 °C)-controlled airflow cabinets. The animals had free access to standard pellet feed and water. Four animals (body weight 191 ( 9 g) for each time point were intravenously injected into a tail vein with approximately 55 µCi (2 MBq) 68Ga-DOTAhCT(9-32), 68Ga-DOTA-[f12,16]-hCT(9-32), or 68Ga-DOTArd-hCT(9-32) in 0.5 mL of electrolyte solution E-153 (Serumwerk Bernburg, Germany) supplemented with 0.1% (w/v) human serum albumin (Serumwerk Bernburg, Germany). Animals were euthanized at 5 and 60 min post injection. Blood and the major organs were collected, wet-weighed, and counted in a Wallac WIZARD Automatic Gamma Counter (PerkinElmer, Germany). The radioactivity of the tissue samples was decaycorrected and calibrated by comparing the counts in tissue with the counts in aliquots of the injected tracer that had been measured in the gamma counter at the same time. The activity in the selected tissues and organs was expressed as percentinjected dose per organ (% ID) or as standardized uptake value (SUV) according to the following equation: tissue _ tracer _ concentration × body _ weight ) injected _ dose (Bq/g) %ID/g ×g) ×g Bq 100% The values are quoted as the means ( standard deviation (mean ( SD) for each group of four animals. SUV )

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PET Imaging. General anesthesia of rats was induced with inhalation of desflurane 9% (v/v) (Suprane, Baxter, Germany) in 40% oxygen/air (gas flow 1 L/min), and was maintained with desflurane 6% (v/v). For arterial blood sampling, a 3.5 Fr umbilical vessel catheter prefilled with heparinized saline was inserted into the right carotid artery, and 200 µL blood samples were taken at 1.5, 3, 5, 10, 20, 30, and 60 min after tracer application. The volume was substituted by E-153. In the PET experiments, 0.5 mCi (18 MBq) of 68Ga-DOTA-hCT(9-32), 68 Ga-DOTA-[f12,16]-hCT(9-32), or 68Ga-DOTA-rd-hCT(9-32) in 0.5 mL was administered intravenously over 1 min into a tail vein. PET imaging in rat was performed over 60 min with a microPET P4 scanner (Siemens CTI Molecular Imaging Inc., Knoxville). Data acquisition was performed in 3D list mode. A transmission scan was carried out prior to the injection of the radiolabeled peptides using a 57Co point source. Emission data were collected continuously for 60 min after injection of the 68 Ga-labeled peptides. The list mode data were sorted into sinograms using a framing scheme of 15 × 10 s, 5 × 30 s, 5 × 60 s, 4 × 300 s, and 4 × 600 s frames. The data were attenuation corrected, and frames were reconstructed by Ordered Subset Expectation Maximization applied to 3D sinograms (OSEM3D) with 14 subsets, 6 OSEM3D iterations, 2 maximum a posteriori (MAP) iterations, and 0.05 β-value for smoothing. The pixel size was 0.3 by 0.3 by 1.21 mm in a matrix of 256 × 256 × 63, and the resolution in the center of field of view was 1.85 mm. No correction for recovery and partial volume effects was applied. The image files were processed using the ROVER software (ABX GmbH, Radeberg, Germany). Summed frames from 30 to 90 s post injection (p.i.) and 30 to 90 min p.i. were used to define the regions of interest (ROI). The data were normalized to the injected radioactivity by using standards from the injection solution measured in a γ-well counter (Isomed 2000, Dresden, Germany) cross-calibrated to the PET scanner and expressed in %ID or SUV. Statistical Analysis. The data are expressed as the means ( SD. One-way analysis of variance (ANOVA) was used for statistical evaluation. Means were compared using Student’s t-test. A P value of 95%) and identity was controlled by RP-HPLC and MALDITOF mass spectrometry, respectively. Subsequently, the DOTA-labeled peptides were complexed with 68Ga in 0.5 ammonium acetate (containing approximately 230 MBq), at pH 3.5-4.2 for 10 min at 90 °C. The reaction mixtures were analyzed by radio-TLC and radio-HPLC (see Figure 1 for 68Ga-DOTA-hCT(9-32) and 68Ga-DOTA-[f12,16]hCT(9-32)). The radiochemical yield of 32% ( 5 (decaycorrected) was obtained with a radiochemical purity of >90% for all preparations according to the radio-HPLC measurements. Biodistribution. 68Ga-DOTA-hCT(9-32), 68Ga-DOTA12,16 [f ]-hCT(9-32), and the 68Ga-labeled random peptide were injected intravenously into a tail vein in male Wistar rats. After 5 and 60 min postinjection, the rats were euthanized, and the radioactivity of the blood and the tissue samples were determined. The results of the biodistribution studies are summarized in Figure 2 (see Supporting Information for detailed data Tables 1-4). The activity in the selected tissues and organs was expressed as percent-injected dose (%ID) (see Figure 2A) or as standardized uptake value (SUV) (see Figure 2B). It can be seen that the peptides left the blood stream immediately after injection and that the general retention in the body was low. However,

hCT-Derived Cell-Penetrating Peptides

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Figure 2. Biodistribution of 68Ga-DOTA-hCT(9-32), 68Ga-DOTA-[f12,16]-hCT(9-32), 68Ga-DOTA-rd-hCT(9-32) in male Wistar rats 5 and 60 min after single intravenous application represented as %ID ( SD (A) and as SUV ( SD (B) (4 animals per time point).

they were rapidly taken up by kidneys and liver and substantially excreted into the intestine. High activity was observed after 5 min in the kidneys (4.90 ( 2.55%ID per gram tissue for 68GaDOTA-hCT(9-32), 6.99 ( 1.09%ID g-1 for 68Ga-DOTA[f12,16]-hCT(9-32) and 6.03 ( 0.60%ID g-1 for 68Ga-DOTArd-hCT(9-32)). Interestingly, 68Ga-DOTA-[f12,16]-hCT(9-32) had a lower retention in the kidney cortex, and the activity of the parent 68Ga-DOTA-hCT(9-32) peptide increased here after 60 min i. v. injection to 7.17 ( 1.30%ID g-1. Some amount of the peptides was metabolized by the hepatobiliary tract too, as was indicated by the detected activities in the liver (1.91 ( 0.52%ID g-1 for 68Ga-DOTA-hCT(9-32), 2.67 ( 0.39%ID g-1 for 68Ga-DOTA-[f12,16]-hCT(9-32), and 2.98 ( 0.14%ID g-1 for 68Ga-DOTA-rd-hCT(9-32)). No relevant accumulation in other tissues was observed. Furthermore, as detected by the low activities in the brain none of the tested peptides was able to cross the blood-brain barrier. PET Imaging Studies. Dynamic PET studies were carried out to validate the data from the biodistribution experiments and to study the kinetics of the radiolabeled hCT-derived peptides. Therefore, whole body PET scans were carried out over 60 min postinjection of 68Ga-DOTA-hCT(9-32), 68GaDOTA-[f12,16]-hCT(9-32), and 68Ga-DOTA-rd-hCT(9-32). A fast clearance out of the body was observed (see Supporting Information, Figure 1 for PET maximum intensity projections

of 68Ga-DOTA-[f12,16]-hCT(9-32) distribution at different time points). Figure 3A and B show coronal PET images of the three peptides distribution in male Wistar rats 45 min after single intravenous application. High activity accumulations were observed in the kidneys. Furthermore, the in ViVo kinetic of the labeled peptides was analyzed within regions-of-interest (ROI). Figure 3C displays time-activity curves of 68Ga-DOTA-hCT(9-32), 68Ga-DOTA[f12,16]-hCT(9-32), and 68Ga-DOTA-rd-hCT(9-32) in the kidneys of male Wistar rats after single intravenous application in SUV as calculated from kidney ROI. These data were in agreement with the corresponding results obtained from biodistribution experiments and show again the activity retention in the kidneys. The increasing activity in the kidneys of 68GaDOTA-[f12,16]-hCT(9-32) in the second half of the measurement was a result of low urine flow in this rat and not a sign of changed metabolism. Additionally, we determined metabolite corrected blood time-activity curves of the three radiolabeled injected peptides. The data were calculated from heart ROI and the metabolite fraction in the blood at seven different time points after tracer application (see Figure 4). We could estimate that the blood clearance of the original peptides reached terminal half-lives of 68Ga-DOTA-hCT(9-32) 15.9 min, 68Ga-DOTA-[f12,16]hCT(9-32) 20.9 min, and 68Ga-DOTA-rd-hCT(9-32) 15.8 min.

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hCT(9-32) displayed fewer radio-metabolites. After 30 min postinjection, three major peaks arose (in which one could be related to the parent compound) but to different extents, indicating the higher metabolic stability of the D-Phe-modified hCT peptide. Analysis of the Metabolites in the Urine. We also analyzed the urine for metabolite content by using radio-HPLC. The metabolite patterns found in the urine samples of rats injected with 68Ga-DOTA-hCT(9-32) allowed a differentiation of eight peaks with retention times from 5.5 min-11.2 min (data not shown), whereas no unaffected peptide could be found (12.8 min, see Figure 1). The main peak was detected at 7.0 min. Unfortunately, the fractionated peptide concentrations were too low for mass spectrometric metabolite analysis. In contrast, the radio-HPLC of urine samples derived from rats injected with 68Ga-DOTA-[f12,16]-hCT(9-32) showed only three peaks, eluting from 12.0 to 13.1 min. These peaks were collected and analyzed by MALDI-TOF mass spectrometry. As presented in Figure 5, three major metabolites were identified: 68 68 Ga-DOTA-[f 12,16 ]-hCT(9-17), Ga-DOTA-[f 12,16 ]68 12,16 hCT(9-18), and Ga-DOTA-[f ]-hCT(9-19) (see also Table 1).

DISCUSSION Figure 3. (A and B) Coronal PET images of 68Ga-DOTA-hCT(9-32) (1), 68Ga-DOTA-[f12,16]-hCT(9-32) (2), and 68Ga-DOTA-rd-hCT(932) (3) distribution in male Wistar rats 45 min after single intravenous application. The color scales of the upper images (A) are normalized to the maximum, and the color scales of the lower images (B) are expanded to show the background activity distribution. (C) Time-activity curves of 68Ga-DOTA-hCT(9-32) (9), 68Ga-DOTA-[f12,16]hCT(9-32) (2), and 68Ga-DOTA-rd-hCT(9-32) (() in the kidneys of male Wistar rats after single intravenous application in SUV. The data were calculated from kidney ROI.

Figure 4. Metabolite corrected blood time-activity curves of 68GaDOTA-hCT(9-32) (9), 68Ga-DOTA-[f12,16]-hCT(9-32) (2), and 68 Ga-DOTA-rd-hCT(9-32) (() in male Wistar rats after single intravenous application in SUV. The data were calculated from heart ROI and metabolite fraction in the blood at 1, 3, 5, 10, 20, 30, and 60 min after tracer application.

Metabolite Analysis in Blood Samples. Metabolic stability of the parent radiometal-labeled peptides was determined in arterial blood samples at seven different time points (1.5, 3, 5, 10, 20, 30, and 60 min postinjection). Analysis by radio-HPLC showed different cleavage patterns for the distinct peptides (see Supporting Information, Figure 2). Unfortunately, the concentrations of the collected peaks from the radio-HPLC were too low for further mass spectrometric analysis of the evolved metabolites. However, the random peptide was cleaved very rapidly in a set of metabolites. After 30 min postinjection, the HPLCchromatograph revealed seven different peaks, whereas one could be attributed to the original compound 68Ga-DOTA-rdhCT(9-32). In contrast to this, the cleavage patterns of the two peptides 68Ga-DOTA-hCT(9-32) and 68Ga-DOTA-[f12,16]-

During the past decade, the successful application of a number of peptides in in Vitro studies has raised the hope of their therapeutic and diagnostic application in ViVo. Indeed, the demand on such small peptides as therapeutic or diagnostic agents is increasing, for instance as delivery vectors for membrane-impermeable drugs (1) or as radiopharmaceuticals in nuclear medicine (23). However, even if a peptide is well characterized in Vitro, the prediction of its in ViVo behavior is difficult because of the countless factors that may influence the route of the peptide inside the body, its structural integrity, or its clearance. Therefore, it is likely that a careful investigation of the pharmacological features of a promising peptide is one major prerequisite for its possible future application in ViVo. Concerning the family of cell-penetrating peptides, until now only few studies exist that investigated these features (2, 24, 32). Thus, the true potential of CPP in ViVo remains to be elucidated. One of the major problems in the application of peptides is often their short biological half-life due to proteolytic cleavage. Also for the parent hormone human calcitonin, poor absorption and rapid proteolytic degradation are described. Therefore, the oral route is limited by extensive proteolytic degradation in the GI lumen and low intrinsic intestinal membrane permeability (25). However, it was shown that enzymatic degradation can be inhibited by modifications of the peptide sequence, e.g., by replacement of L-amino acids by D-amino acids (26) or the insertion of unnatural amino acids or side chains (12). Accordingly, we modified recently the cell-penetrating peptide hCT(9-32) by the introduction of D-phenylalanine at amino acid positions 12 and 16. In Vitro stability tests in heparinised human blood plasma revealed a significantly increased metabolic stability of the resulting novel peptide [f12,16]-hCT(9-32) (14). But although the in ViVo stability of peptides in blood might be well modeled by in Vitro stability tests in heparinised plasma (27, 28), this approach neglects for instance the renal and hepatic clearance that will occur in ViVo. Therefore, the aim of this work was a systematic examination of the in ViVo stability and biodistribution of the hCT-derived cell-penetrating peptides hCT(9-32) and [f12,16]-hCT(9-32). To exclude any artifacts, we also investigated the tissue distribution of a radiolabeled random hCT(9-32) sequence. Therefore, the two peptides as well as the random sequence were N-terminally labeled with DOTA and subsequently complexed with 68Ga. We recently showed for the peptides DOTA-

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Figure 5. (A) Radio-HPLC of urine samples derived from rats injected with collected peaks.

68

Ga-DOTA-[f12,16]-hCT(9-32). (B) MALDI-TOF analysis of the

Table 1. Peptide Sequences as Well as Calculated and Experimentally Determined Molecular Weights of the Cleavage Products of 68 Ga-DOTA-[f12,16]-hCT(9-32) Found in Urine Samples by MALDI-TOF Analysis peak

name

sequence

MWexp.

MWcalc.

1 2 3

Ga-DOTA-[f12,16]-hCT(9-18) Ga-DOTA-[f12,16]-hCT(9-17) Ga-DOTA-[f12,16]-hCT(9-19)

Ga-DOTA-LGTfTQDfNK Ga-DOTA-LGTfTQDfN Ga-DOTA-LGTfTQDfNKF

1622.8 1494.7 1769.7

1621.9 1493.8 1769.0

hCT(9-32) and DOTA-[f12,16]-hCT(9-32) complexed with cold gallium that they are still internalized in HEK 293 and HeLa cells, respectively (29). Hence, we conclude that the labeling did not influence the cell-penetrating properties of the two peptides when applied in ex ViVo studies. The radiolabeled peptides were then injected in male Wistar rats, and their tissue distribution was determined. A relatively high kidney uptake is typical for all radioconjugates, which are small enough to pass through glomerular membranes and hydrophilic enough to undergo renal clearance (30). This was the case for 68Ga-DOTA-hCT(9-32) and for the modified analogue 68Ga-DOTA-[f12,16]-hCT(9-32). Also, the random sequence was excreted mostly by the renal pathway. All peptides were metabolized to some extent by the hepatobiliary tract too and showed some excretion into the intestine. It was important for us to demonstrate that our CPP are not nonselectively accumulated in a specific tissue. Accumulation in the reticuloendothelial system is often observed for large or aggregated structures and can cause side effects in future in ViVo applications (31). Recently, studies with a fusion protein of TAT and β-galactosidase showed distribution to all tissues in mice, including the brain (2, 24). In our experiments, no delivery over the blood-brain barrier was observed. Another preliminary study concerning the in ViVo behavior of the sweet arrow peptide demonstrated uptake in blood cells (32). Whether our peptides have been indeed taken up in blood cells or in liver or kidney cells has not been elucidated yet. From in Vitro studies with HT-29 colon carcinoma cells, we know that hCT(9-32) can transfer the 68Ga-DOTA label effectively into the cell interior (data not shown). However, the transfer of bioactive cargos in ViVo will be the focus of future studies. All the results observed in the biodistribution studies could be confirmed by using small animal PET imaging. Strong 68Ga activities in the kidneys and the bladder were detected that indicate renal peptide elimination. The corresponding kinetic supports this picture and demonstrates a very rapid peptide clearance from the blood and the kidneys. Furthermore, with the calculated time-activity curves in blood we could demonstrate the higher stability of the 68Ga-DOTA-[f12,16]-hCT(9-32) peptide. The estimated terminal half-life of 20.9 min was clearly higher than that of 68Ga-DOTA-hCT(9-32) (15.9 min). To analyze the metabolic stability of the peptides, radio-HPLC was used to identify the labeled compounds in blood as well as in urine samples. Because of the low peptide content, the blood samples could not be further characterized by MALDI-TOF mass spectrometry. But this is not surprising since a very rapid

peptide clearance from the blood was observed. Nevertheless, from the radio-HPLC chromatographs it was concluded that the random peptide was relatively fast degraded in blood in a lot of metabolites. In contrast to this, the original CPP 68Ga-DOTA[f12,16]-hCT(9-32) and 68Ga-DOTA-hCT(9-32) were metabolically more stable, whereas the D-Phe modified hCT-analogue had the highest stability. These results confirm well our recently performed in Vitro studies (14). More results about the cleavage pattern were obtained from urine fractions that were collected 60 min postinjection. By using radio-HPLC, in the case of 68Ga-DOTA-hCT(9-32) eight radiolabeled peaks were detected with retention times between 5.5-11.2 min. Since the retention time of 12.8 min of the unaffected peptide was lacking, this result indicates the complete proteolytic degradation of 68Ga-DOTA-hCT(9-32). The obviously low metabolic stability of this peptide is in good agreement with previous in Vitro studies (13, 14). Unfortunately, it was not possible to identify any cleavage products of 68Ga-DOTAhCT(9-32) by using MALDI-TOF mass spectrometry. Therefore, an enzymatic cleavage at the former in Vitro identified labile positions 12 and 16 can neither be proven nor excluded. However, in the case of the D-amino acid-modified 68GaDOTA-[f12,16]-hCT(9-32), radio-HPLC revealed three 68Galabeled compounds in urine. The smallest peak (retention time 13.0 min) might be the uncleaved parent compound, but because of its low peptide content, this could not be confirmed by MALDI-TOF mass spectrometry. Nevertheless, HPLC analysis indicated that the parent compound was proteolytically degraded resulting in at least three 68Ga-labeled metabolites. By analyzing these metabolites with MALDI-TOF mass spectrometry, three cleavage products were identified: 68Ga-DOTA-[f12,16]hCT(9-17), 68Ga-DOTA-[f12,16]-hCT(9-18), and 68Ga-DOTA[f12,16]-hCT(9-19). As the major cleavage product, 68GaDOTA-[f12,16]-hCT(9-17) was found. Interestingly, the same metabolite was recently detected by our group in preliminary in Vitro studies (14). However, the in ViVo identified cleavage sites after Lys18 and Phe19 were not observed in the in Vitro study. Probably 68Ga-DOTA-[f12,16]-hCT(9-32) is proteolytically cleaved in ViVo between Asn17-Lys18 by an endopeptidase resulting in the fragment 68Ga-DOTA-[f12,16]-hCT(9-17). In addition, two cleavage sites in the C-terminal part of the peptide (Gln24-Thr25 and Ala26-Ile27) that were found in Vitro were not detected in the present in ViVo study. But more interestingly, no further N-terminal cleavage site was observed, probably indicating the increased stability of the modified hCT(9-32) analogue, which would be in good agreement with our in Vitro

1602 Bioconjugate Chem., Vol. 19, No. 8, 2008

data. It is obvious that a peptide modification with D-amino acids can provide protection against metabolic degradation not only in Vitro but also in ViVo.

CONCLUSIONS The results of the presented in ViVo examination show that Ga-DOTA-hCT(9-32) and 68Ga-DOTA-[f12,16]-hCT(9-32) do not accumulate in a specific tissue. As other investigated radiolabeled peptides, they were relatively rapidly excreted out of the body mostly by the renal system. The blood clearance and the elimination of the 68Ga-DOTA-peptides were relatively high and may be decreased by further structural changes. This will be investigated in more detail in future studies. In addition, we could prove the increased metabolic stability of the modified peptide [f12,16]-hCT(9-32) peptide. Furthermore, the results of this in ViVo degradation study supported very well the results we gained in previous in Vitro experiments. All in all, these studies demonstrate that it is very important to define the pharmacokinetic characteristics of an assumed biologically active compound. The future application of CPP in ViVo will hardly depend on such pharmacological issues. 68

ACKNOWLEDGMENT We gratefully thank R. Herrlich for performing the biodistribution studies, A. Suhr for performing the analysis of the metabolites, and R. Reppich-Sacher for the MALDI-TOF measurements. I.N. thanks Professor Dr. A. G. Beck-Sickinger for generous access to all facilities of the institute. Supporting Information Available: Supplementary tables and figures including biodistribution data, representative PET images, and radio-HPLC elution profiles. This material is available free of charge via the Internet at http://pubs.acs.org.

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