Liver Imaging with a Novel Hepatitis B Surface ... - ACS Publications

Article pubs.acs.org/molecularpharmaceutics

Liver Imaging with a Novel Hepatitis B Surface Protein Derived SPECT-Tracer Thomas Müller,† Stefan Mehrle,‡ Alexa Schieck,† Uwe Haberkorn,† Stephan Urban,‡ and Walter Mier*,† †

Department of Nuclear Medicine, University Hospital Heidelberg, INF 400, 69120 Heidelberg, Germany Department of Infectious Diseases, Molecular Virology, University Hospital Heidelberg, INF 345, 69120 Heidelberg, Germany

‡

S Supporting Information *

ABSTRACT: Diagnostic imaging of the liver by ultrasound, computed tomography (CT) and magnetic resonance tomography (MRT) is generally limited to the visualization of the morphology. In order to exploit the intriguing liver tropism of the human hepatitis B virus (HBV) for molecular imaging of the liver, peptidic tracers derived from the HBV large envelope protein (L) were studied. An N-terminally stearoylated tracer comprising amino acids 2−48 of the PreS1domain of the L protein was synthesized by solid phase peptide synthesis. Mercaptoacetyltriglycerin (MAG3) was linked to this peptide to enable 99mTc labeling. Biodistribution studies in mice showed an excellent liver accumulation of this novel class of radiotracer with 84%, 84%, 65%, and 16% of the injected dose in the liver after 10 min, 1 h, 4 h, and 24 h, respectively. Imaging studies on a gamma camera showed a clear visualization of the liver already 10 min post intravenous injection. These studies confirmed the exclusive accumulation of the tracer in the liver with negligible background in other organs. Owing to a significant biliary clearance rate of the 99mTc bound via a linker, the tracer enabled imaging of the bile ducts starting 30 min after injection. Analysis of the route of excretion revealed complete clearance within 24 h post injection. Clearance was predominantly via renal secretion. In conclusion, the novel class of tracers shows excellent pharmacokinetic, biodistribution, and clearance kinetics. It provides unique biological information different from the current imaging modalities. Its primary application is likely to be the evaluation of liver cancer patients. Specific indications may include tumor staging, the differentiation of malignant versus benign hepatic lesions, and liver tumors of nonhepatocellular origin. KEYWORDS: molecular imaging, radiopharmaceuticals, solid phase peptide synthesis, hepatitis B, liver

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function and metabolism with tracers such as [ 18 F]fluorodeoxyglucose5 or 99mTc-labeled IDA (iminodiacetic acid)6 for the measurement of hepatobiliary function. Nonetheless, even the known radiotracers do not provide the decisive liver specificity required to visualize hepatocellular characteristics that would allow us to differentiate premalignant cirrhotic nodules from normal hepatic tissue. The success of contrast agents is invariably determined by the target specificity of the tracer. The hepatotropism of hepadnaviridae, that is, the human hepatitis B virus (HBV) might offer the key for such specificity. We have recently discovered that a lipopeptide derived from the large envelope protein of the HBV (Figure 1) shows an extraordinary specificity to bind parenchymal liver cells.7−9 To our knowledge the liver targeting specificity of this peptide outperforms all liver specific tracers known so far. Two recent publications describe the factors considered to mediate HBV hepatotropism through specific binding of the myristoylated N-terminal pre-S1-domain of the HBV L-protein to a hepatocyte specific receptor.10,11

INTRODUCTION The liver is the largest organ of the body with a wide range of functions, including major metabolic processes, detoxification of pharmaceutics, protein synthesis, glycogen storage, and hormone production. The liver is vulnerable to various diseases including different forms of viral and nonviral hepatitis, cirrhosis, alcoholic liver disease, autoimmune liver diseases, nonalcoholic fatty liver disease, and cancers of the liver (i.e., hepatocellular carcinoma). Ultrasound is the first-line imaging modality for the liver; it is followed by computed tomography (CT) and magnetic resonance tomography (MRT) whenever ultrasound imaging yields vague results. Numerous advances of these techniques, i.e. the development of superparamagnetic iron oxide contrast agents1 that accumulate in the reticuloendothelial system (RES) of the liver or gadolinium based hepatobiliary contrast agents2 such as the liver-specific MRI contrast agent gadolinium-ethoxybenzyl-diethylenetriamine pentaacetic acid (Gd-EOB-DTPA-MRI),3,4 approved in 2008, are of particular significance and have improved the value of CT and MRT. However, their predominantly morphology based delineation remains an ultimate limitation. The impact of molecular imaging techniques is to complement the morphological imaging modalities by the unique ability to visualize © 2013 American Chemical Society

Received: May 16, 2012 Accepted: April 19, 2013 Published: April 19, 2013 2230

dx.doi.org/10.1021/mp400038r | Mol. Pharmaceutics 2013, 10, 2230−2236

Molecular Pharmaceutics

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Figure 1. Model of HBV comprising the viral DNA located in the nucleocapsid which is surrounded by the envelope containing the three different surface proteins L, M, and S (framed structures). The large envelope protein L consists of the S, the preS2, and the preS1 moiety. Right: top: domain structure of the HBV L-protein with its N-terminally myristoylated preS1 domain, the preS2 domain, and the S domain; below: structure of the stearoylated N-terminal pre-S1 tracer peptide. The MAG3 sequence, which allows the labeling with 99mTc, is attached to an additional lysine at the C-terminus.

hepatocytes were isolated and cultivated as described by Kreaemer et al.15 Flow cytometry was performed on a BD FACS Calibur II 3 Laser 8 color cytometer (BD Biosciences, Germany) using the BD FACS DIVA software. Briefly staining of either primary cells or cell lines was performed as follows: Cells were trypsinized using a mild trypsinization protocol (0.025% trypsin in phosphate-buffered saline (PBS), incubation for 10 min at 37 °C, neutralization with PBS containing 5% bovine serum albumin (BSA)) and counted, and 1 × 105 cells were resuspended in 200 μL of PBS for staining. 100 nM FITC labeled peptide were added to the cells, and the suspension was incubated for 15 min at 20 °C in the dark on a shaking rotator. Following the staining, cells were washed three times with PBS containing 5% BSA as well as one time with PBS and subsequently resuspended in 200 μL of PBS containing 0.2 μg/ mL Hoechst 3342 (Pierce, Germany). Cells were measured with setting the live gate on nuclei containing cell fraction, and analysis was performed in the FITC channel. C-terminally FITC-conjugated peptides of either wildtype (stearoylHBVpreS2-48) or a corresponding peptide containing two Damino acid substitutions at position 11 and 13, perturbing the essential binding region (stearoyl HBV preS2-48 D 11,13) were used in all experiments. Reversed Phase High-Performance Liquid Chromatography (HPLC). All HPLC runs were carried out on reversed phase columns with a linear gradient from water to acetonitrile, both containing 0.1% trifluoroacetic acid (TFA). The analytical chromatograms were recorded on a HP 1090 series HPLC system supplying a linear gradient from 0 to 100% acetonitrile in 40 min at a flow rate of 0.5 mL/min with a Waters (Eschborn, Germany) Atlantis T3 3 μm column (150 × 4.6 mm). Preparative RP-HPLC of the MAG3 derivatized peptide was carried out on a Gilson 321 HPLC system equipped with a Merck Chromolith SemiPrep RP-18e column (100 × 10 mm), supplying a flow rate of 10 mL/min. Preparative RP-HPLC of the radiolabeled tracer was performed on a Gyncotech P580 instrument equipped with a Shimadzu SPD 6-A multiwavelength detector HPLC system using a

The lipopeptides derived from the large envelope protein of the HBV do neither comprise any structural conformation that might lead to the arise of difficult sequences nor disulfide bridges they are easily accessible by solid phase synthesis.12 Consequently, they may serve as a valuable tool for liver imaging. A stearoylated peptide comprising amino acids 2−48 of the large hepatitis B PreS1 protein was synthesized by solid phase peptide synthesis and labeled with 99mTc. Biodistribution studies in mice revealed the exclusive accumulation of this tracer in the liver. Studies in mice and rats confirmed this specificity and showed the potential for diagnostic imaging of the liver.

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MATERIALS AND EXPERIMENTS General Materials and Methods. Chemicals were obtained from Merck (Darmstadt, Germany) and SigmaAldrich (Taufkirchen, Germany) and used without further purification. 4-Methoxybenzyl mercaptoacetic acid was synthesized according to the procedure described by Okarvi et al.13 Amino acid building blocks were from Novabiochem (Bad Soden, Germany). Na99mTcO4 was obtained from a commercial 99 Mo/99mTc generator (IBA Molecular, Schwarzenbruck, Germany). Proton and carbon NMR spectra were recorded at room temperature on a Varian Mercury Plus (300 MHz) or Varian NMR system 500 (500 MHz) spectrometer with DMSO-d6 as internal standard. Chemical shifts (δ) are given in ppm. The signal of the residual protonated solvent [D6]DMSO was used as a reference signal. Cell Culture and Flow Cytometry. Huh-7 cells were cultivated in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen, Germany) containing 10% fetal calf serum (FCS) and penicillin/streptomycin (Invitrogen, Germany). The cells were passaged when reaching 75% confluency. Hep56D cells were cultivated in DMEM media (Invitrogen, Germany) containing 10% FCS as well as NEAA (Invitrogen, Germany) and penicillin/streptomycin (Invitrogen, Germany). The cells were passaged when reaching 75% confluency. HepaRG cells were cultivated as described by Gripon et al.14 Primary murine 2231



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Scheme 1. Synthesis of the Labeling Precursor Stearoyl-HBVpreS2-48-K(S-(4-methoxybenzyl)-MAG3) (1)a

a

(a) Automated Fmoc/tBu solid phase peptide synthesis, HBTU/DIPEA activation, 10 equiv of amino acid in NMP; (b) 4 equiv of stearic acid, 4 equiv of HATU, and 10 equiv of DIPEA in NMP; (c) 2% hydrazine in DMF; (d) repeated cycles of peptide synthesis: coupling, 4 equiv of FmocGly-OH (cycles 1−3) or 4-methoxybenzyl mercaptoacetic acid (cycle 4), 4 equiv of HATU, and 10 equiv of DIPEA in DMF, 50% piperidine in DMF; (e) TFA/TIS/H2O 2 h.

Chromolith Performance RP-18e (100 × 4.6 mm) column (Merck Darmstadt, Germany) with a linear gradient over 10 min, at a flow rate of 2 mL/min. Mass Spectrometry. LC-MS was carried out on an Agilent 1200 series system equipped with a ThermoFisher (Dreieich, Germany) Hypersil GOLD 1.9 μm RP-HPLC column (200 × 2.1 mm) at 50 °C. The HPLC system supplied a flow of 200 μL/min with a linear gradient over 30 min. The peaks were recorded on a Thermo Fisher Exactive mass spectrometer. A mixture of caffeine, Met-Arg-Phe-Ala (MRFA), and Ultramark 1621 was used for mass calibration in the positive-ion mode. Full scan single mass spectra were obtained by scanning from m/z = 200−4000. Synthesis of Stearoyl-HBVpreS2-48-K(S-(4-methoxybenzyl)-MAG3). The peptide synthesis of GQNLSTSNPLGFFPDHQLDPAFRANTANPDWDFNPNKDTWPDANKVGKamide was carried out employing the Fmoc/tBu strategy with HBTU/DIPEA activation on an Applied Biosystems (Darmstadt, Germany) ABI 433A peptide synthesizer (Scheme 1). The synthesis was carried out on a TentaGel R RAM resin (Rapp Polymere, Tübingen, Germany) with a loading of 0.2 mmol/g. Amino acids were used in 10-fold excess. The first amino acid attached was ivDde protected lysine. All amino acids following were side-chain protected by standard protecting groups. N-terminal stearoylation was carried out manually with a 4-fold excess of stearic acid and HATU/DIPEA activation. The subsequent selective on resin cleavage of the ivDde protective group from the C-terminal lysine residue was achieved by repeated treatment with a 2% v/v solution of hydrazine in DMF for 5, 7, 10, and 15 min. Three glycine residues were coupled, also in a 4-fold excess employing HATU/DIPEA activation onto the free amine. Fmoc cleavage was achieved by treatment with 50% v/v piperidine in DMF. Finally, 4-methoxybenzyl mercaptoacetic acid was coupled under the same conditions. After completing the on-resin synthesis, the product was cleaved from the resin with a cocktail consisting of TFA:TIS:H2O 95:2.5:2.5 v/v for 2 h at room temperature. The peptide was precipitated in diethyl ether, washed two times with diethyl ether, and dried. The crude product was purified by semipreparative RP-HPLC as described above and characterized by LC/MS ESI-MS (m/z): 1496.9842 [M + 4 H]4+ (calculated mass for C273H399N71O80S m/z =

1496.9867) and 1H NMR (Supplemental Figure 2 in the Supporting Information). Radiolabeling. A sample of 10 μL of a 1 mM peptide solution per 10 MBq generator eluate was added to an equal amount of 0.5 M phosphate buffer at pH 9.0. Then 2 μL of dimethyl sulfoxide (DMSO), 4 μL of sodium tartrate solution (100 mg/mL), 2 μL of lactose monohydrate (100 mg/mL), and 1 μL of SnCl2 solution (2.0 mg/mL) were added. The mixture was incubated at 95 °C for 15 min and subsequently purified on a RP-HPLC as described above. The solvent of the fraction collected was evaporated at room temperature under reduced pressure and the residue dissolved in H2O/DMSO. The typical decay-corrected yield of the radiolabeling reactions was >60%. Biodistribution and Imaging Studies. Animal experiments were conducted in accordance with German laws for animal protection. For imaging studies, animals were anesthetized with Sevoflurane (Baxter, Unterschleißheim, Germany) and placed on a gamma-imager (Biospace, France) equipped with a high energy collimator. The radiolabeled tracer was injected into the tail vein of either female NMRI mice or male Wistar rats and images were recorded at the indicated points in time with 5 min acquisition time. Each mouse received a dose of approximately 8 MBq; rats were injected with tracer amounts of approximately 15 MBq per animal. For the biodistribution studies, the radiolabeled tracer was injected into the tail vein of female NMRI mice (n = 12). Each mouse received a dose of approximately 1 MBq. After the time period indicated, the animals were scarified (n = 3 per time period after tracer administration), dissected, and the radioactivity in each organ was measured with a Berthold LB 951G gamma counter. The results were displayed as percentage of the injected radioactivity.

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RESULTS Despite its length of 48 amino acids, the novel SPECT-tracer stearoyl-HBVpreS2-48-K(S-(4-methoxybenzyl)-MAG3) could be synthesized in high yields by standard Fmoc-chemistry. The ease of the synthesis was in agreement with the prediction of the Peptide Companion software.16 The relatively low propensity to aggregation can be attributed to the fact that the preS domain can be predicted an intrinsically unstructured 2232



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protein.12 This can be explained by the high proline content of its amino acid sequence. Consequently, the tracer was obtained in good yield and high purity after preparative HPLC. The carrier peptide was characterized by RP-HPLC and mass spectrometry. First, the peptide was C-terminally conjugated to FITC, and its binding capacity to primary human hepatocytes was assessed by flow cytometry. It could be shown that the wild type FITC conjugated peptide efficiently binds to purified primary human hepatocytes (98% ± 3%), while a modification of the peptide with two amino acid substitutions in the essential binding motif leads to a complete abrogation of the binding capacity (0.3% ± 1%). See Supplemental Figure 4 in the Supporting Information. Binding capacity of proliferating hepatocytes as well as carcinoma cells lines of hepatic origin has been analyzed by Meier et al., demonstrating the high specificity of the peptides for well-differentiated healthy hepatocytes.10 The peptide was derivatized with a S-4-methoxy protected MAG3 moiety17 for labeling with 99mTc. The radiolabeling of this chelator was accomplished by heating to 95 °C in the presence of sodium tartrate, lactose, and tin chloride for 15 min. Under the optimized conditions this exchange labeling gave a decay corrected yield of 60% which represents a reasonable yield considering the complexity of this tracer (see Supplemental Figure 1 in the Supporting Information). Subsequently, the labeled tracer was purified by preparative HPLC. The biodistribution studies in mice revealed an almost exclusive hepatic accumulation. As shown in Figure 2, 84% of

the tracer is distributed but does not accumulate significantly anywhere else than in the liver.

Figure 3. Static planar images of a female NMRI mouse at different points in time after intravenous injection of 8 MBq of 99mTc-stearoylHBVpreS2-48-K(MAG3). The acquisition time of each image was 5 min.

Planar imaging in male Wistar rats, showed an uptake kinetics comparable to the results obtained in mice (Figure 4). Due to the larger size of the rats it was possible to obtain images of the abdomen with the liver clearly resolved. After 30 min, the intestines became visible in these images. The tracer is transiently trapped in the liver and slowly processed via the biliary route. In these images it could also be observed that a minor fraction of the tracer is cleared via the kidneys. However, because of the previously determined strong retention of iodinated tracers derived from stearoylated peptides comprising amino acids 2−48 of the PreS1-domain of the L protein,8 it can be concluded that the signal observed in the intestines represents is a cleaved species containing 99mTc and not the full length peptide.

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DISCUSSION The liver is the primary site of nutrient metabolization and degradation of toxic substances. The selective targeting of hepatocytes, the primary parenchymal cells of the liver, has been an important goal. Various approaches have been undertaken to resolve this issue. One approach is to exploit the active phagocytosis of the reticuloendothelial system (RES) in the liver. This led to the development of liver imaging drugs consisting of radiolabeled particles of a defined size. These particles target the RES of the spleen, the bone marrow, and the liver. The size of the particles used determines the respective organ preference. Particles of 200−1000 nm are preferentially taken up into the liver.18 Selective internal radiation therapy (SIRT)19 is an alternative approach to use particles for the targeting of the liver. The achievements of SIRT, a highly efficient modality that has gradually been introduced over the recent years,20 are based on infusion of radiolabeled microspheres into the hepatic arteries that supply the hepatic tumors. As the majority of the tumor blood supply is derived from the hepatic artery, the spheres that contain a beta emitting isotope are trapped in the tumor’s vascular bed and cause an efficient palliative effect. However, liver targeting with particles does neither yield comprehensive morphological and anatomical information nor provide any functional information of the biliary system. An alternative to particulate tracers are radiolabeled derivatives of acetanilido-N-imino-diacetic acid (hepatobiliary IDA = HIDA) such as 2,6-diisopropyl-HIDA (DISIDA). 21 These compounds are taken up by the hepatocytes via a carrier-mediated organic-anion pathway and subsequently excreted via the bile. This allows the assessment

Figure 2. Percentage of the injected dose values obtained in the biodistribution study of 99mTc-stearoyl-HBVpreS2-48-K(MAG3) in female NMRI mice, n = 3 per point in time. The activity concentration in the liver approximates a first order kinetics with a half-life of approximately 9.8 h in the liver. Insert: linear regression of the accumulation data to determine the half live in the liver.

the injected dose of the tracer is trapped in the liver already 10 min after intravenous injection (Supplementary Table 1 in the Supporting Information). The radionuclide vanishes from the liver with first-order kinetics with a biological half-life of approximately 9.8 h as determined by linear regression of the accumulation data (Figure 2, insert). This study was complemented by a determination of the route of excretion of the peptide by measuring urinary and fecal elimination of the peptide over a time frame of 24 h. See Supplemental Figure 3 in the Supporting Information. Planar imaging studies were performed in rats and mice. As the mice were about of the size of the collimator, the distribution of the tracer can be clearly depicted in the whole body (Figure 3). Following the uptake for the first five minutes, 2233



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Figure 4. Static planar images of a male Wistar rat at different points in time after intravenous injection of 15 MBq of 99mTc-stearoyl-HBVpreS2−48K(MAG3). The image of the rat overlaid by the scintigraphy obtained at 10 min p.i. demonstrates the field of view. The acquisition time of each image was five minutes.

of hepatobiliary function. While the novel tracer 99mTc-stearoylHBVpreS2-48-K(MAG3) shows an uptake characteristics similar to the HIDA derivatives, its hepatocyte binding is exclusive resulting in the enormous liver specificity and the low background values shown in Figure 2. As shown by measurements of the relative fractions of tracer excreted via the biliary and the renal route, most of the 99mTcstearoyl-HBVpreS2-48-K(MAG3) injected is excreted within 24 h. This shows that it provides the properties required for the development of molecular imaging agents: a fast enrichment, highly specific uptake, as well as fast clearance from the nontarget organs. Even though the novel peptidic tracer comprises a fatty acid residue, its pharmacokinetic parameters are highly different from the lipopeptides clinically used such as the 13 amino acid, cyclic lipopeptide antibiotics daptomycin (16), and the longacting glucagon-like peptide-1 (GLP-1) analogue liraglutide.22,23 In these peptides the fatty acid is incorporated to allow for protraction of action as the albumin binding of lipopeptides causes an extended retention time in the circulation.24 The FDA-approved peptide liraglutide, a GLP-1 analogue that is 97% homologous to native human GLP-1, is the prime example for this approach. Its 16-carbon fatty acid chain causes noncovalent binding to albumin, which slows down the absorption from the injection site. In addition the fatty acid modification preserves the molecule from degradation, primarily by the enzyme dipeptidyl peptidase-4. Due to its albumin binding and increased enzymatic stability an elimination half-life of 13 h allows for once-daily dosing of liraglutide. The stearoylated peptides, comprising amino acids 2−48 of the PreS1 domain of the large hepatitis B envelope protein, show a fatty acid dependent albumin binding capacity (C. Gähler, unpublished results). The rapid extraction from the circulation to the liver reveals the high binding specificity of the novel tracer. It has to be pointed out that the albumin binding is a required but not sufficient prerequisite for the hepatotropism observedsingle amino acid mutants of the

stearoylated peptides, comprising amino acids 2−48 of the PreS1 domain of the large hepatitis B envelope protein do not show specific accumulation in the liver any longer but still retain the capability to associate to serum albumin.9 The radiotracer described in this paper allows a completely novel approach for the imaging of the liver. In contrast to targeting the RES which is not exclusively a liver tissue, this novel tracer specifically targets hepatocytes. As a consequence the tracer is not found at significant levels in tissues other than liver tissue. Its applications may include the assessment of cirrhosis and its complications, that is, the visualization of dysplastic nodules in the cirrhotic liver, the scintigraphic cholangiopancreatography, the determination of gallstones lodged in the ducts surrounding the gallbladder, and intrahepatic cysts. An eventual differentiation of the binding to hepatocytes with various differentiation states was tested by FACS analysis. The specific binding of a fluorescently labeled lipopeptide possessing the hepatocyte binding characteristics of 99mTcstearoyl-HBVpreS2-48-K(MAG3) was assessed on a panel of different cancer cell lines as well as primary hepatocytesit confirmed that the interaction is specific for hepatocytesand more importantly, the experiment revealed a clear correlation of the binding capacity and the differentiation state of the cells (these data will be comprehensively presented in a manuscript in preparation). Based on these findings the main clinical application of the novel tracer is likely to be the differentiation of malignant versus benign hepatic lesions and liver tumors of nonhepatocellular origin. It may further be hypothesized that the tracer might be used to identify patients who would benefit from drugs with specific action in HCC25 and to separate treatment responders from nonresponders early. The mechanism of uptake of HBV has not yet been revealed.26 Hence, the molecular mechanisms behind the process of hepatocellular uptake cannot be understood in detail.27 The use of this tracer might allow further elucidation of the structural determinants 2234



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(8) Petersen, J.; Dandri, M.; Mier, W.; Lutgehetmann, M.; Volz, T.; von Weizsacker, F.; Haberkorn, U.; Fischer, L.; Pollok, J. M.; Erbes, B.; Seitz, S.; Urban, S. Prevention of hepatitis B virus infection in vivo by entry inhibitors derived from the large envelope protein. Nat. Biotechnol. 2008, 26 (3), 335−41. (9) Schulze, A.; Schieck, A.; Ni, Y.; Mier, W.; Urban, S. Fine mapping of pre-S sequence requirements for hepatitis B virus large envelope protein-mediated receptor interaction. J. Virol. 2010, 84 (4), 1989− 2000. (10) Meier, A.; Mehrle, S.; Weiss, T. S.; Mier, W.; Urban, S. The myristoylated pre-S1-domain of the hepatitis B virus L-protein mediates specific binding todifferentiated hepatocytes. Hepatology 2012, DOI: 10.1002/hep.26181. (11) Schieck, A.; Schulze, A.; Gahler, C.; Muller, T.; Haberkorn, U.; Alexandrov, A.; Urban, S.; Mier, W. Hepatitis B virus hepatotropism is mediated by specific receptor recognition in the liver and not restricted to susceptible hosts. Hepatology 2013, DOI: 10.1002/hep.26211. (12) Schieck, A.; Muller, T.; Schulze, A.; Haberkorn, U.; Urban, S.; Mier, W. Solid-phase synthesis of the lipopeptide Myr-HBVpreS/2-78, a hepatitis B virus entry inhibitor. Molecules 2010, 15 (7), 4773−83. (13) Okarvi, S. M.; Adriaens, P.; A., V. Comparison of the labelling characteristics of mercaptoacetyltriglycine (MAG3) with different Sprotective groups. J. Labelled Compounds Radiopharm. 1997, 39 (10), 853−74. (14) Gripon, P.; Rumin, S.; Urban, S.; Le Seyec, J.; Glaise, D.; Cannie, I.; Guyomard, C.; Lucas, J.; Trepo, C.; Guguen-Guillouzo, C. Infection of a human hepatoma cell line by hepatitis B virus. Proc. Natl. Acad. Sci. U.S.A. 2002, 99 (24), 15655−60. (15) Kreamer, B. L.; Staecker, J. L.; Sawada, N.; Sattler, G. L.; Hsia, M. T.; Pitot, H. C. Use of a low-speed, iso-density Percoll centrifugation method to increase the viability of isolated rat hepatocyte preparations. In Vitro Cell Dev. Biol. 1986, 22 (4), 201−11. (16) Lebl, M.; Krchnak, V.; Lebl, G. Peptide Companion; CSPS Pharmaceuticals: San Diego, CA, 1995. (17) Muhlhausen, U.; Schirrmacher, R.; Piel, M.; Lecher, B.; Briegert, M.; Piee-Staffa, A.; Kaina, B.; Rosch, F. Synthesis of 131I-labeled glucose-conjugated inhibitors of O6-methylguanine-DNA methyltransferase (MGMT) and comparison with nonconjugated inhibitors as potential tools for in vivo MGMT imaging. J. Med. Chem. 2006, 49 (1), 263−72. (18) Holder, L. E.; Saenger, E. L. The use of nuclear medicine in evaluating liver disease. Semin. Roentgenol. 1975, 10 (3), 215−22. (19) Popperl, G.; Helmberger, T.; Munzing, W.; Schmid, R.; Jacobs, T. F.; Tatsch, K. Selective internal radiation therapy with SIR-Spheres in patients with nonresectable liver tumors. Cancer Biother. Radiopharm. 2005, 20 (2), 200−8. (20) Haug, A. R.; Tiega Donfack, B. P.; Trumm, C.; Zech, C. J.; Michl, M.; Laubender, R.; Uebleis, C.; Bartenstein, P.; Heinemann, V.; Hacker, M. 18F-FDG PET/CT Predicts Survival After Radioembolization of Hepatic Metastases from Breast Cancer. J. Nucl. Med. 2012, 53, 371−7. (21) Schwarzrock, R.; Kotzerke, J.; Hundeshagen, H.; Bocker, K.; Ringe, B. 99mTc-diethyl-iodo-HIDA (JODIDA): a new hepatobiliary agent in clinical comparison with 99mTc-diisopropyl-HIDA (DISIDA) in jaundiced patients. Eur. J. Nucl. Med. 1986, 12 (7), 346−50. (22) Russell-Jones, D. Molecular, pharmacological and clinical aspects of liraglutide, a once-daily human GLP-1 analogue. Mol. Cell. Endocrinol. 2009, 297 (1−2), 137−40. (23) Ryan, G. J.; Foster, K. T.; Jobe, L. J. Review of the therapeutic uses of liraglutide. Clin. Ther. 2011, 33 (7), 793−811. (24) Wang, Q.; Graham, K.; Schauer, T.; Fietz, T.; Mohammed, A.; Liu, X.; Hoffend, J.; Haberkorn, U.; Eisenhut, M.; Mier, W. Pharmacological properties of hydrophilic and lipophilic derivatives of octreotate. Nucl. Med. Biol. 2004, 31 (1), 21−30. (25) Jimenez, R. H.; Boylan, J. M.; Lee, J. S.; Francesconi, M.; Castellani, G.; Sanders, J. A.; Gruppuso, P. A. Rapamycin response in tumorigenic and non-tumorigenic hepatic cell lines. PLoS One 2009, 4 (10), e7373.

for the liver tropism as well as the yet unknown entry mechanism of hepadnaviridae. Despite enormous scientific and technological advances, tumor detection still lags behind the angiogenic switch of tumors.28 This results in the known relatively moderate treatment success. The possibility to image neoplasia at the very early stages of disease would represent a decisive step ahead. However, it is still not clear what kinds of hepatocyte, that is, liver cell dysplasia, are premalignant cells. As primary hepatocellular carcinoma are probably derived from chronic hepatitis and liver cirrhosis, the novel tracer represents a potential imaging agent for the detection of premalignant cells.

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ASSOCIATED CONTENT

S Supporting Information *

Additional analytical data, FACS binding data and assessment of the excretion characteristics. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +49-6221-567720, e-mail: [email protected]. Author Contributions

T.M. and S.M. contributed equally to this work. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors gratefully acknowledge the assistance of Karin Leotta for the imaging experiments and the Bundesministerium für Bildung und Forschung (BMBF) for financial support (grant number 01GU0701).

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