Preparation and evaluation of 99mTc-labeled cyclic arginine–glycine–aspartate (RGD) peptide for integrin targeting

ARTICLE IN PRESS Applied Radiation and Isotopes 68 (2010) 1896–1902

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Preparation and evaluation of 99mTc-labeled cyclic arginine–glycine–aspartate (RGD) peptide for integrin targeting Dong-Eun Lee, Young-Don Hong, Kang-Hyuk Choi, So-Young Lee, Pil-Hoon Park, Sun-Ju Choi n Radioisotope Research Division, Basic Science and Technology Department, Korea Atomic Energy Research Institute (KAERI), Daejon 305-353, Republic of Korea

a r t i c l e in f o

a b s t r a c t

Article history: Received 29 January 2010 Received in revised form 15 April 2010 Accepted 28 April 2010

Technetium coordination chemistry has been a subject of interest in the development of radiopharmaceuticals, especially imaging radiotracers. Due to the extensive work done on developing chelates for 99mTc, various chelators have been investigated and applied to radiopharmceuticals. Previous studies on the coordination chemistry of the [99mTc ¼ O] core have established peptide-derived sequences as effective chelating ligands. These observations led to the design of tetradentate ligands derived from amino acid sequences. Such amino acid sequences provide a tetradentate coordination site for chelation to the radionuclide and an effective functional group for conjugation to biomolecules using conventional solid-phase synthetic routes. A derivative of a novel tripeptide chelating sequence, Pro–Gly–Cys (PGC) has been developed where it is possible to form stable technetium complexes with the [99mTc ¼O] via N3S1 tetradentate coordination core that serves this function and can be readily incorporated into biomolecules using solid-phase synthesis techniques. As a model system, the RGD peptide was selected which has been well known to target the integrin receptor for angiogenesis and tumor imaging agents. The results of in vivo studies with these novel radiolabeled compounds in tumor xenografts demonstrated a distribution in tumor targeting and other organs, such as kidney, liver and intestines. & 2010 Elsevier Ltd. All rights reserved.

Keywords: 99m Tc N3S1 Cyclic RGD peptide Integrin Tumor targeting

1. Introduction In nuclear medicine, to use a receptor binding or other biological interactions is often called target-specific radiopharmaceuticals. Recent advances in the development of radiopharmaceuticals for imaging and therapy with radionuclides are intimately dependent on the successful application of target-specific biomolecules (Bakker et al. (1991); Liu, 2008). After the successful application of 111In-labeled octreotide to the somatostatin receptor-targeting imaging agent (Bakker et al. (1991)), various researchers have intensively studied to find new target-specific radiopharmaceuticals based on small biomolecules, such as peptides and bioactive molecules. Many small biomolecules have been radiolabeled to evaluate their potential use as new imaging agents and therapy. These include radiolabeled receptor-specific peptides, namely, RGD derivatives, somatostatin analogues, a-MSH and folate receptor

Abbreviations: Fmoc, 9-Fluorenylmethoxycarbonyl; Allyl, Allyl; Pbf, 2,2,4,6, 7-Pentamethyldihydrobenzofuran-5-sulfonyl; Mtt, 4-Methyltrityl; Trt, Trityl; HBTU, O-Benzotriazole-N, N,N0 ,N0 -tetramethyl-uronium-hexafluoro-phosphate; HOBt, N-Hydroxybenzotriazole; DIPEA, Diisopropylethylamine; DMF, Dimethylformamide; DCM, Dichloromethane; DIC, 1,3-Diisopropylcarbodiimide; TFA, Trifluoroacetic acid; TIS, Triisopropylsilane; EDT, Ethanedithiol n Corresponding author. Tel.: + 82 42 868 8449; fax: + 82 42 868 8448. E-mail address: [email protected] (S.-J. Choi). 0969-8043/$ - see front matter & 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.apradiso.2010.04.029

antagonists (Bakker et al. (1991); Leamon et al. (2002); Liu, 2008; Miao and Quinn (2008)). The rational strategy for incorporating radionuclides with favorite characteristics into target molecules has been the most considerable aspect in developing radiopharmaceuticals. For diagnostic radiopharmaceuticals, various gamma-emitting isotopes including 99mTc, 111In, 62/64Cu and 67/68Ga have been used for SPECT or PET imaging (Liu, 2008). While 64Cu and 68Ga are particularly useful for PET, 99mTc is the most widely used for SPECT imaging due to its optimal nuclear properties (T1/2 ¼6 h, 140 keV gamma photons) and its convenient availability from commercial 99Mo/99mTc generator at low costs. This makes it attractive to use of 99mTc for developing a radiotracer for cancer imaging. 99m Tc has diverse oxidation states which may make difficult for its application as a radionuclide for the development of radiopharmaceuticals. Among the core structure of 99mTc, [99mTc(V) ¼O]3 + is the most extensively developed with tetradentate ligand chelates of the NxS4  x system. For successful chelation of 99mTc, the [99mTc(V) ¼O]3 + core with NxS4  x tetradentate ligands have been extensively investigated, including N2S2 (DADS, MAMA and DADT), N3S1 triamidethiols which contain bioactive molecules with target-receptor affinity (Liu, 2008). Various peptide sequences such as, Gly–Gly–Cys (N3S1) and Gly–Ala–Gly–Gly–Gly (N4) have also been proposed as

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chelators for 99mTc-labeling of biomolecules (Cyr et al. (2007); Pearson et al. (1996); Haubner et al. (2004)). There are several advantages of using peptide-based chelators, including easy preparation of biomolecule–chelator conjugates using a solidphase synthesis and convenient alteration by substitution in chelator amino acid sequence during peptide synthesis for tuning chelators (Cyr et al. (2007); Liu and Edwards (1999)). Integrin avß3 receptors have been the target of potential radiopharmaceuticals due to their implication in a wide range of tumor markers, such as angiogenesis and metastasis. Integrin avb3 is highly expressed on proliferating endothelial cells and solid tumor cells, whereas it is not expressed on quiescent endothelial cells (Brooks et al. (1994); Zitzmann et al. (2002)).

P-G-C-(N-ethyl) amide

PGC-c(RGDyK) NH2

HN

NH O

O

O

O O

NH HN

O

N H

NH HN NH HS

NH HS

O

H N

O NH

NH HN

1897

The highly selective expression of avb3 integrins in the neovascular tissue and various tumors make this a suitable target for both diagnostic imaging and target therapy (Chen et al. (2004a); Decristoforo et al. (2006); Liu et al. (2003); Temming et al. (2005)). RGD peptide is a key binding moiety that has been shown to bind specifically to integrin receptors (Zitzmann et al. (2002)). RGD derivatives have been labeled with various radionuclides and these radiotracers have been successfully evaluated for integrin receptor targeting imaging (Liu, 2008; Temming et al. (2005)). In this study, a novel tripeptide chelating sequence, Pro–Gly– Cys (PGC) was developed where it is possible to form stable technetium complexes with the [99mTc ¼O] via N3S1 tetradentate coordination core (Fig. 1). The PGC, which consists of amino acid sequence, can be introduced into bioactive molecules using a conventional solid-phase synthetic strategy for preparing targeted radiopharmaceuticals. As a model system, the RGD peptide derivative, cyclic RGDyK was selected as a targeting moiety for the avb3 integrin receptor. The details of its synthesis are described in Scheme 1. Subsequently, the in vitro affinity and the in vivo tumor targeting of this novel compound to avb3 expressing tumors were determined.

H O

NH

O N H

O

O

OH

OH 99m

Fig. 1. Structure of novel chelator having N3S1 coordination core for Tc and its RGD-conjugate derivative. The novel N3S1 chelator comprised of amino acids, Pro–Gly–Cys and its RGD-conjugate derivative were prepared by solid-phase synthesis method.

2. Materials and methods 2.1. Generals All the chemicals and reagents used in this study were analytically graded purchased from a commercial company and used without any further purification. Fmoc-protected amino acid, O-Benzotriazole-N,N,N0 ,N0 -tetramethyl-uronium-hexafluorophosphate (HBTU) and N-Hydroxybenzotriazole (HOBt) were

Coupling Fmoc-Amino acid Allyl

Allyl

HBTU, HOBt, DIPEA, DMF

Asp-NH2 Trityl Resin

Asp-Gly-Arg-Lys-D-Tyr-NH2

Fmoc Deprotection

Pbf MTT

20% Piperidine in DMF COOH

NH2

Asp-Gly-Arg-Lys-D-Tyr Tetrakis(triphenylphosphine)Paladium(0) 1,3-Dimethylbarbituric acid in DCM

Pbf MTT

CO

NH

Asp-Gly-Arg-Lys-D-Tyr DIC, DIC HOBt, HOBt DMF Pbf MTT

Coupling Fmoc-Amino acid HBTU, HOBt, DIPEA, DMF 1% TFA

CO

NH

Asp-Gly-Arg-Lys-D-Tyr p y g y y

Fmoc Deprotection

Pbf Cys-Gly-Pro

20% Piperidine in DMF Trt CO TFA:TIS:EDT:Thioanisole:H2O = 90:2.5:2.5:2.5:2.5

NH

Asp-Gly-Arg-Lys-D-Tyr Cys-Gly-Pro

Scheme 1. Solid-phase synthetic route to the PGC derivative of RGD, Pro–Gly–Cys–cyclic(RGDyK).

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purchased from Anaspec (Frenont, CA, USA). Automated solidphase synthesis was accomplished by the use of a Multiple Biomolecular Synthesizer (Peptron, Daejeon, Republic of Korea). Analytical and preparative RP-HPLC was performed on a SHIMAZU prominence HPLC by using Shiseido capcell pak C-18 column. The wavelength used for UV detection was 220 nm for analytical RP-HPLC. The LC/MS was performed by using HP 1100 series. The labeling yield and radiochemical purity (RCP) for radiolabeled compound were analyzed by HPLC equipped with Waters 2695 pump, UV detector (Waters 2487), RI detector (In/US g-detector system) and a X-terra C-18 column (5 mm, 4.6  250 mm) at a flow rate of 1.0 ml/min with a gradient mobile phase. The mobile phase consisted of 0.1% trifluoroacetic acid (TFA) in water (A) and 0.1% TFA in acetonitrile (B). The elution profile was like that, solvent (A) 100–90% for 2 min, 90–60% for 10 min, 60–30% for 2 min, 30% for 3 min and 30–100% for 3 min. The instant thin layer chromatography (ITLC) used silica-gel strips and a saline and MEK as a mobile phase, respectively. Scintillation counting was performed on a Wallac 1470 Wizard automated gamma counter (PerkinElmer Life Sciences). Sodium pertechnetate (Na99mTcO4) was obtained from a 99Mo/99mTc generator (Samyoung Unitech, Daejeon, Republic of Korea). All radioactivities were measured by using an ionizing chamber (Atomlab 200, Bio-dex).

2.2. Synthesis of PGC-ethylamide and PGC-RGD derivative PGC-c(RGDyK) is an RGD peptide derivative containing tripeptide chelator moiety consisting of Pro–Gly–Cyc (PGC) and cyclic [Arg, Gly, Asp, D-Tyr and Lys], c(RGDyK). PGC-c(RGDyK) was synthesized by applying standard Fmoc strategy in Peptron Inc. (Daejeon, Republic of Korea), as detailed in Scheme 1. Briefly, trityl resin conjugated FmocAspartic acid allylester was used as a solid support of the reaction. The linear sequence peptide was prepared by applying the consecutive method of coupling between amine and carboxylic acid. As an activating reagent to ensure efficient coupling, O-benzotriazoleN,N,N0 ,N0 -tetramethyl-uronium-hexafluoro-phosphate (HBTU) and N-hydroxybenzotriazole (HOBt) were applied. The Fmoc-protecting group was removed, followed by the sequential addition of Fmoc-GlyOH, Fmoc-Arg (Pbf)-OH, Fmoc-Lys (MTT)-OH, Fmoc-D-Tyr-OH. Fmoc protecting groups were removed after every coupling step under standard conditions (20% piperidine in DMF). Cyclization of RGDyK was accomplished by the reaction between amine of tyrosine and carboxylic acid of aspartic acid by applying a conventional amide coupling method after deprotection of an allyl group of aspartic acid using tetrakis (triphenylphosphine) palladium (0) and 1, 3-dimethylbarbituric acid in dichloromethane (DCM). To introduce the N3S1 chelating moiety (PGC), the c(RGDyK) sequence was elongated by the sequential additions of Fmoc-Cys (Trt)-OH, Fmoc-Gly-OH and FmocPro-OH in e-amine of lysine. After the last assembly step, the resulting peptide, PGC-c(RGDyK) was cleaved from the polymeric support by treatment with 90% trifluoroacetic acid (TFA) containing 2.5% triisopropylsilane (TIS), 2.5% ethanedithiol (EDT), 2.5% thioanisole and 2.5% deionized water. (TFA:TIS:EDT:Thioanisole:H2O¼90:2.5: 2.5:2.5:2.5). This reaction also resulted in the simultaneous removal of the Pbf and trityl protecting groups. The crude PGC-c(RGDyK) product was purified by Shimadzu HPLC using capcell pak C18 column, mobile phase with 0.1% TFA/water (A) and 0.1% TFA/acetonitrile (B) and gradient conditions with 0–10% B in 2 min, 10–40% B in 10 min and 40–70% B in 2 min at a flow rate 1 ml/min. Under these conditions, PGC-c(RGDyK) typically eluted at 5.9 min. The purified PGC-c(RGDyK) was analyzed by LC/MS (HP 1100). Major ion peak M+1 (m/z): 877.4. The PGC-ethylamide was also prepared using similar solid-phase synthesis described above and a major peak for LC/MS was found at 303.

2.3. Radiolabeling of

99m

Tc-PGC-c(RGDyK)

PGC-ethylamide and PGC-c(RGDyK) was dissolved in 1 ml of distilled water to give a concentration of 10  3 M (10  6 mol/ml). The labeling of compounds with 99mTc was performed by transchelation with 99mTc-glucoheptonate (99mTc-GH) using a kit vial formulation. Briefly, the glucoheptonate (GH) kit vial containing 20 mg GH and 0.45 mg stannous chloride (SnCl2) was dissolved with 400 ul of sodium 99mTc pertechnetate (Na99mTcO4) from generator containing up to 370 MBq, and was allowed to stand for 10 min at room temperature for preparing 99mTc-GH. The reaction solution was analyzed by ITLC on silica gel strips with saline or MEK as the mobile phase to check the amount of colloid formation. In the saline as a mobile phase, free 99mTcO4  and 99mTc-GH migrate at Rf 1.0, and colloid remains at Rf 0.0. In the MEK, free 99mTcO4  migrates at Rf 1.0 and 99mTc-GH and colloid remains at Rf 0.0, respectively. A 200 ml aliquot of 99m Tc-GH from this vial (5 mCi or 185 MBq) was then added to various concentrations of the peptide solution (10  7 mol, 10  8 mol and 10  9 mol) in 400 ml of 50 mM sodium acetate buffer (pH 5.5) and the reaction was allowed to stand for 20 min at room temperature. The radiochemical purity (RCP) and the efficiency of radiolabeling yield of compounds were determined by RP-HPLC on Waters HPLC system with g-detector. The in vitro stability study was performed by incubation in 37 1C for 24 h. The aliquot was analyzed by RP-HPLC with g-detector to determine the stability of this radiolabeled compound. No decomposition was observed. 2.4. Log P (n-octanol/saline) The log P value was determined by using a slight modification of the method described elsewhere (Decristoforo et al. (2006)). A 100 ml of aliquot of radiolabeled compound was added to 1 ml of octanol/water in an eppendorf tube. The solution was mixed for 5 min and centrifuged for 5 min. About 100 ml aliquots of both the n-octanol phase and the saline phase, respectively, were transferred and the radioactivity was measured using a gammacounter. The log value of the partition coefficient P (n-octanol/ saline) was calculated. The experiments were performed in triplicates. 2.5. Cell uptake assay Cell uptake experiments with radiolabled c(RGDyK) were performed according to the previously reported procedure with slight modifications (Decristoforo et al. (2006); Shi et al. (2008)). Briefly, avb3 positive U87MG and avb3 negative MCF-7 cells were grown in 75 T-flask until 80–90% confluence. Cells were harvested with trypsin and washed with PBS buffer and 106 cells were transferred to eppendorf tubes. After incubation with radiolabeled PGC-c(RGDyK) ( 4105 cpm, 2 nM) in 1 ml of the binding buffer [cell binding buffer composition; 20 mM 2-amino-2-(hydroxymethyl)-1,3-propanediol, hydrochloride (Tris–HCl), pH 7.4, 150 mM NaCl, 2 mM CaCl2, 1 mM MgCl2, 1 mM MnCl2 and 0.1% (w/v) BSA] at 37 1C for 1 h, incubation was interrupted by centrifugation followed by removal of the supernatant and rapid washing twice with cell binding buffer. Thereafter, the cells were washed with an acid wash buffer (50 mM glycine–HCl buffer, pH 2.4) to remove the membrane bound radioligand, and the supernatant was collected to measure the membrane bound fraction of radioactivity. Cells were lysed with 1 N NaOH, transferred and the internalized fraction of radioactivity was measured with g-counter. Counted radioactivity in a membrane bound fraction and internalized fraction could be ascribed to

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the sum of cell uptake. The concentration of proteins in 1 N NaOH fraction was determined by using Bradford protein assay kit in order to normalize the radioactivity value with respect to the averaged sample protein content. The cell uptake data was expressed as % uptake (cell uptake/total added activity) per mg protein. The specificity of cell binding was also evaluated by competitive binding experiments in the presence of 10 nM–100 mM of the cold c(RGDyK). Each data represents a result of the average of triplicate wells. 2.6. In vivo biodistribution studies The biodistribution of the radiolabeled PGC-c(RGDyK) was determined in female Balb/C athymic (nu/nu) mice (provided by OrientBio, Republic of Korea) with Calu6 tumors. Briefly, Calu6 cells derived from a human lung carcinoma were maintained in an eagle’s minimum essential medium (EMEM) supplemented with 10% FBS, 2 mM L-glutamine, 100 unit of penicillin per milliliter and 100 mg of streptomycin per liter and incubated at 37 1C in 5% CO2. About 1  106 cells of Calu6 were injected subcutaneously (s.c.) in the lateral aspect of the right leg and allowed to grow to an approximately 300–400 mg for further biodistribution studies for 2–3weeks. The stock solution of the 99m Tc-labeled peptide used in the animal studies was prepared for 2  10  8 mol/ml with an activity of 370 MBq/ml. Solutions for injection were prepared by the dilution of the stock solution to obtain a solution of 3.7 MBq/ml. The mice were administered 99m Tc-labeled peptides intravenously via the tail vein at an injection dose of 370 kBq/100 ml. The animals were sacrificed 2 and 24 h injection. Blood, tumor and other tissues were subsequently dissected and counted in a g-counter. In blocking test, Calu6 tumor bearing mice received intravenous injection of 60 mg c(RGDyK) (3 mg/kg body weight), and 30 min later, 370 kBq of the radiolabeled peptide was administered to each mouse. Percent injected dose (%ID) and percent injected dose per gram of organ (%ID/g organ) were calculated from referred appropriate decay corrections. All groups consisted of four or five mice. All mean values are given as 7SD.

3. Results 3.1. Preparation of PGC-ethylamide and PGC-cRGDyK Peptide sequences, Pro–Gly–Cys (PGC) designed to serve as a chelator were synthesized by Fmoc-based solid-phase synthesis. To prepare the RGD derivative containing a novel tripeptide N3S1 chelator (PGC), an RGD peptide was incorporated with PGC at the e-amino group of lysine in c(RGDyK) during peptide synthesis (Scheme 1). The final peptides were cleaved from the resin using TFA/water with simultaneous removal of the all protecting groups. The crude PGC-c(RGDyK) product was purified by Shimadzu HPLC using capcell pak C18 column in which PGCc(RGDyK) typically eluted at 5.9 min. The purified PGC-c(RGDyK) and PGC-ethylamide were analyzed by LC/MS (HP 1100). Major ion peak for LC/MS was found at M+ 1 (m/z): 877.4 and 303, respectively. 3.2. Radiolabeling and characterization In this investigation, the labeling yield and radiochemistry for the preparation of 99mTc-PGC-c(RGDyK) and 99mTc-PGC were analyzed by RP-HPLC equipped RI detector. All the radiolabeled compounds revealed a high labeling yield ( Z98%) at 10  8 mol. As shown in Fig. 2, 99mTc-PGC-c(RGDyK) and 99mTc-PGC were

1899

identified at 12 and 10 min, respectively. The RP-HPLC analysis also indicated that the labeling compound was stable for up to 24 h when it was stored at room temperature. The log P values of the 99mTc-PGC-c(RGDyK) and 99mTc-PGC were  2.90 and  0.35, respectively. 3.3. In vitro cell uptake experiment The cell binding studies of 99mTc-PGC-c(RGDyK) were performed using U87MG glioblastoma cells which highly express avb3 integrin receptors (Zhang et al. (2005)). The cell binding specificity of radiolabeled RGD was compared by performing competitive binding experiments with nonradiolabeled c(RGDyK). The results were shown in Fig. 3 c(RGDyK) inhibited the binding of 99mTc-PGC-c(RGDyK) to U87MG cells in a dose-dependent manner. In the cell uptake experiment, specific cell uptake for radiolabeled RGD was found to be 1.1670.05% uptake/mgprotein. Cell uptake could be reduced to o0.15% uptake/mgprotein in the presence of excess nonradiolabeled c(RGDyK), indicating a specific binding for integrin receptor in U87MG cells (Fig. 3). Uptake in receptor-negative MCF-7 cells was comparably low ( o0.15%/mg-protein). 3.4. In vivo biodistribution studies The biodistribution of the 99mTc-PGC-c(RGDyK) was measured at 2 and 24 h after administration, and at 2 h with co-injection of excess c(RGDyK). The results of the biodistribution studies in Calu6 tumor xenografts are summarized in Table 1 and Fig. 4. Calu6 tumor uptake was 1.38 70.30%ID/g at 2 h and 0.78 7 0.19%ID/g at 24 h after administration. 99mTc-PGC-c(RGDyK) was rapidly cleared from the blood and major organs, except those related with the excretion route. Uptake in liver and intestine revealed high: 22.16 75.66%ID/g and 1.2570.86%ID/g at 2 h and 2.4771.29%ID/g and 0.3970.02%ID/g at 24 h, respectively. These data indicate that 99mTc-PGC-c(RGDyK) clears through the hepatobiliary excretion to intestine. Uptake in the remaining organs was below 1%ID/g at 2 h. The ratio of tumor to blood was 10.6573.11 at 2 h and 17.3570.33 at 24 h. In vivo blocking experiments with an excess c(RGDyK) resulted in a significantly reduced uptake of 99mTc-PGC-c(RGDyK) in tumor. Tumor uptake was decreased from 1.3870.30%ID/g to 0.5370.06%ID/g (Fig. 4). A comparison of biodistribution for 99mTc-PGC-c(RGDyK) and 99m Tc-PGC was also performed. As shown in Table 1, clearance of radioactivity in blood was more rapid for 99mTc-PGC-c(RGDyK). 99m Tc-PGC showed a substantially higher liver and intestine uptake (3.5270.71%ID/g and 80 78.87%ID/g, respectively) indicating hepatobiliary excretion without significant retention in the blood.

4. Discussion Radiolabeled receptor-specific peptides for use in nuclear medicine have been greatly interested in the development of radiopharmaceuticals. Among them, RGD peptide for integrin receptors targeting has been investigated as an attractive potential target moiety for various cancer cells imaging and therapy (Haubner et al. (1999); Liu, 2008; Temming et al. (2005)). The avb3 integrin is expressed on the endothelial cells concerned in neovasculature and various tumor cells and has received the most recent attention for a successful radiolabeled receptor targeting moiety. For radiolabeling biomolecules with 99mTc, there has been an interest in using small peptides, as NxS4  x derivative chelators. Some investigators have reported that

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Fig. 2. g-HPLC profile of

99m

Tc-PGC and

99m

Tc-PGC-c(RGDyK) (a)

1.6 1.4

PGC-c(RGDyK) only PGC-c(RGDyK) + Block

99m

Tc-PGC (10 min) and (b)

99m

Tc-PGC-c(RGDyK) (12 min).

Table 1 Biodistribution of 99mTc-PGC-c(RGDyK) in nude mice bearing human Calu6 at 2 and 24 h postinjection, respectively, and of 99mTc-PGC at 2 h. Data are expressed as %ID/g organ. (Block: Preinjection of c(RGDyK),  3 mg/kg before 99mTc-PGCc(RGDyK) administration).

% uptake/mg-protein

1.2 99m

1

2h

99m

Tc-PGC-c(RGDyK) 24 h

2 h + Block

Tc-PGC

2h

Blood 0.14 7 0.04 0.057 0.01 0.117 0.04 0.42 7 0.10 Liver 1.25 7 0.86 0.39 7 0.02 0.757 0.20 3.52 7 0.71 Kidney 0.58 7 0.32 0.34 7 0.14 0.667 0.23 1.64 7 0.80 Spleen 0.08 7 0.01 0.12 7 0.05 0.207 0.12 0.35 7 0.13 Heart 0.40 7 0.24 0.037 0.02 0.107 0.03 0.26 7 0.05 Intestine 22.16 7 5.66 2.47 7 1.29 111.627 11.74 80.00 7 8.87 Lung 0.33 7 0.27 0.077 0.04 0.217 0.16 0.21 7 0.12 Stomach 0.74 7 0.18 0.53 7 0.18 0.307 0.13 0.91 7 0.35 Tumor 1.38 7 0.30 0.78 7 0.19 0.537 0.06 0.57 7 0.21 Tumor-to-blood 10.65 7 3.11 17.35 7 0.33 5.047 1.30 1.43 7 0.73

0.8 0.6 0.4 0.2 0 U87MG

MCF-7

Fig. 3. Cell uptake of 99mTc-PGC-c(RGDyK) in avb3-positive U87MG and negative MCF-7 cells. (block: co-incubation with 10 uM c(RGDyK)).

peptide sequences form a metal coordination core for radionuclide 99mTc (Cyr et al. (2007); Liu, 2008). For this purpose, a novel tripeptide N3S1 chelator system, Pro–Gly–Cys (PGC) for

radiolabeling with 99mTc was designed and prepared. In this system, it is postulated that the cyclic amine of proline becomes part of the coordination core for 99mTc (Fig. 1). In this study, as a model system, an attempt was made to label an RGD peptide targeting the avß3 integrin with 99mTc for tumor imaging and tumor-induced angiogenesis. To prepare the RGD derivative containing chelator for 99mTc, an RGD peptide with PGC

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3.00 PGC-c(RGDyK) + Block PGC-c(RGDyK) 2hr PGC-c(RGDyK) 24hr

2.50

%ID/g

2.00 1.50 1.00 0.50 0.00 Blood

Liver

Kidney Spleen

Heart

Lung Stomach Tumor

Fig. 4. Biodistribution of 99mTc-PGC-c(RGDyK) in athymic nude mice bearing Calu6 tumor at 2 and 24 h p.i., and of 99mTc-PGC-c(RGDyK) in the presence of an excess unlabeled c(RGDyK) 2 h after injection.

for chelator moiety at the e-amine site of lysine in c(RGDyK) using a solid-phase synthesis was incorporated. The PGC sequence served as metal coordination site that could be subjected to radiolabeling using a standard kit formulation. In the case of PGC-c(RGDyK), RGDyK moiety would not be affected by the radiolabeling process. The radiochemical purity (RCP) of 99m Tc-labeled compound was greater than 98% (Fig. 2), which is sufficient for further investigation such as in vitro and in vivo evaluation. When the labeling compound was incubated at 37 1C for 24 h, above 98% of 99mTc remained chelated with PGC-c(RGDyK). 99mTc-PGC-c(RGDyK) showed a specific receptor binding for integrin receptors in vitro in U87MG cells overexpressing the integrin receptors. The 99mTc-PGC-c(RGDyK) was able to specifically bind to the integrin receptor by competition binding experiments with U87MG cells (Fig. 3). The tumor uptake of 99mTc-PGC-c(RGDyK) was 1.3870.30%ID/g at 2 h and 0.7870.19%ID/g at 24 h, respectively. After 24 h postinjection, this radioactivity decreased in all organs. However, compared with decreasing in other organs, it was shown to retain radioactivity in the tumor. The tumor uptake was similar to a previously reported result (0.8570.20%ID/g at 1 h) (Decristoforo et al. (2006)), in which the EDDA/HYNIC-RGD was labeled with 99mTc for an uptake study in integrin negative M21L melanoma cancer, and was similar to the results observed using other radiolabeled peptides as probes in integrin negative LLC tumor model (0.89%ID/g at 4 h) (Jung et al. (2006)). Compared with the previously reported results, the tumor uptake of this 99mTc-PGC-c(RGDyK) is lower in Calu6 than was reported in integrin avb3-positive tumors, such as M21 and U87MG (Decristoforo et al. (2006); Shi et al. (2008)). This has resulted from the fact that the density of receptors in Calu6 cells is lower as compared with 105 binding sites per cell in U87MG cancer cells (Morrison et al. (2009); Zhang et al. (2005)). However, biodistribution results in nude mice bearing human Calu6 tumor suggested that the good tumor-to-blood ratio at 24 h p.i. can still be retained. 99m Tc-PGC-c(RGDyK) showed a predominant intestinal uptake (22.16% ID/g at 2 h) compared to other organs, and the result was similar to the results previously reported (Cyr et al. (2007); Liu and Edwards (1999)). The retention of radioactivity in an intestine would be more likely to interfere with tumor imaging in the abdominal region. However, even though a substantial amount of total radioactivity had been processed thorough the liver into the intestine, even after 24 h, a modest quantity of radioactivity remained in the tumor. Actually, one major disadvantage of using NxS4  x chelators is the high lipophilicity of their chelating core, which often leads to high liver uptake and hepatobiliary excretion of the radiolabeled compound (Liu and Edwards (1999)). Therefore, for the favorable pharmacokinetics as well as metabolic stability, 99mTc-PGC-c(RGDyK) is needed to be improved by

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introducing various modifying groups, such as hydrophilic amino acids and glucose moiety (Chen et al. (2004b); Cyr et al. (2007); Haubner et al. (2001)). Blocking experiments were also performed to determine whether an uptake in the tumor was caused by receptor-mediated specific binding through biodistribution at 2 h in mouse bearing tumor xenografts. The preinjection of excess unlabeled c(RGDyK) significantly decreased an uptake of 99mTc-PGC-c(RGDyK) in the tumor, in which tumor uptake was reduced to 0.5370.06%ID/g in the blockage group, compared to 1.3870.30%ID/g in the nonblock group. Therefore, the result of blocking experiments for biodistribution with excess unlabeled c(RGDyK) in nude mice bearing Calu6 tumors showed that the target specificity could be obtained. In the presence of an excess RGD, the uptake was reduced in most organs (tumor, liver and stomach), but was increased in intestine (111.62711.74%ID/g), and the result was similar to that of 99mTc-PGC (80.0078.87%ID/g), PGC chelator derivative without containing c(RGDyK). These results are similar with the result of log P value. The more lipophilic compound PGC-ethylamide (log P: 0.35) results in the more liver and intestine accumulation while the less lipophilic compound PGC-c(RGDyK) (log P:  2.90) results in less accumulation for these organs.

5. Conclusion In this study, a novel tripeptide N3S1 chelator for 99mTc was designed and evaluated. Results revealed a high labeling yield using kit vial formulation. 99mTc-labeled PGC-c(RGDyK) was also evaluated both in vitro and in vivo as integrin avb3 targeting agent. The results presented in this study demonstrate that 99mTc-PGC-c(RGDyK) has a specific binding for integrin avb3 receptor and showed a modest accumulation of radioactivity in Calu6 tumor. Further modification of PGC-c(RGDyK) introducing alteration of lipophilicity and hydrophilicity may improve biokinetics for use as an imaging agent.

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