A New Optical and Nuclear Dual-Labeled Imaging Agent Targeting

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Bioconjugate Chem. 2007, 18, 397−402

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A New Optical and Nuclear Dual-Labeled Imaging Agent Targeting Interleukin 11 Receptor Alpha-Chain Wei Wang,* Shi Ke, Sunkuk Kwon, Sasidhar Yallampalli, Arlin G. Cameron, Kristen E. Adams, Michel E. Mawad, and Eva M. Sevick-Muraca Department of Radiology, Division of Molecular Imaging, Baylor College of Medicine, Houston, Texas 77030 . Received August 30, 2006; Revised Manuscript Received January 5, 2007

Optical imaging has great potential for studying molecular recognitions both in ViVo and in Vitro, yet nuclear imaging is the most effective clinical molecular imaging modality. The combination of optical and nuclear imaging modalities may provide complementary information for improving diagnosis and management of diseases. In this study we developed an optical and nuclear dual-labeled imaging agent, 111In-DTPA-Bz-NH-SA-K(IR-783-S-PhCO)-c(CGRRAGGSC)NH2, called DLIA-IL11RR. 111In-DTPA-Bz-NH-SA is the radiotracer moiety; a near-infrared dye IR-783-S-Ph-COOH serves as the fluorescent emitter; and the cyclic peptide c(CGRRAGGSC), which is known to target interleukin 11 receptor alpha-chain (IL-11RR), delivers the desired imaging agent to its target. Experiments revealed that the cyclic peptide c(CGRRAGGSC) continued to possess the targeting capability to IL-11RR after the conjugation of the optical and nuclear tracers. Furthermore, the presence of the metal isotope chelator did not cause quenching of fluorescence emission. The cross validation and direct comparison of optical and nuclear imaging of a tumor was achieved using a single injection, and the preliminary results show the conjugate has tumor targeting capabilities in ViVo.

INTRODUCTION Near-infrared (NIR) fluorescence imaging is an active and promising area for noninvasive, in ViVo molecular imaging (14). It is used to probe the expression of disease-associated biomarkers in small animals and provide real-time information about molecular events. In the wavelength range of 700-900 nm, NIR light causes minimal autofluorescence and is minimally absorbed by hemoglobin (the principal absorber of visible light) as well as by water and lipids (the principal absorbers of infrared light). Furthermore, since stable fluorophores can be reactivated by propagating excitation light, as many as 109 photons per second per molecule can be generated from a fluorophore with a fluorescent lifetime of one nanosecond. The sensitivity of fluorescence in microscopy is well established, and the correlation between medical imaging and pathology may be offered by optical imaging (5, 6). On the other hand, nuclear imaging techniques such as positron emission tomography (PET) and single photon emission tomography (SPECT) are currently the most effective clinical molecular imaging modalities due to their intrinsically high sensitivity at deep tissue sites and capability of quantitation. However, radionuclide imaging may suffer from low photon counts requiring long scan times as well as from a finite halflife and radiation dosage preventing longitudinal imaging in patients (7). Considering the importance and advantages of both nuclear and fluorescence imaging, a combination of these two techniques provides an attractive approach for enhancing the imaging accuracy and providing complementary information for improving diagnosis and management of diseases. The strategy to achieve this goal is to develop an optical and nuclear duallabeled imaging agent. Although NIR fluorophores and radiotracers possess excellent photophysical properties, none are tumor specific. To address this problem, a targeting moiety must * To whom correspondence should be addressed. E-mail: [email protected]. Phone +1 713 798 6017. Fax +1 713-798-2749.

be introduced to specifically deliver a dual-labeled imaging agent to diseased sites. Dual-labeled targeting imaging agents, such as the one described herein, allow cross validation and direct comparison between nuclear and fluorescence optical imaging (8-10). By combining optical imaging for enhanced photon signal count without agent half-life concerns and gamma imaging for high sensitivity at deep tissue detection, dual-labeled imaging agents could allow a comprehensive picture of the imaging agent distribution and provide a rapid and efficient imaging method for basic research and clinical diagnosis. Small peptides represent a class of potential targeting unit candidates (11), since they are structurally well defined, easily synthesized, and possess faster circulatory clearance when employed in imaging agents compared to larger antibodies or proteins. Interleukin-11 (IL-11) is a multifunctional cytokine involved in various pathways in blood cells, their precursors, and many other cell types both in Vitro and in ViVo. The effects of IL-11 are largely mediated by the IL-11 receptor alpha-chain (IL-11RR), which is a member of the gp130-dependent receptor group that has been associated with various unique biological actions. Experimental evidence has shown that interleukin (IL11) and its receptor (IL-11RR) are related to breast cancer development and progression and may play a significant role in the bone metastasis of human breast cancer (12-14). IL-11 may also be a predictor of poor prognosis in human breast cancer (15). Consequently, targeting of IL-11RR positive tumors could be a useful strategy for noninvasive imaging. Using phage display technique, Arap et al. (16, 17) identified the cyclic nonapeptide c(CGRRAGGSC), as a mimic motif of IL-11 and showed that it bound specifically to IL-11RR. Herein, we report the development of a new dual-labeled imaging agent, called DLIA-IL11RR [ 111In-DTPA-Bz-NH-SA-K(IR-783-S-PhCO)-c(CGRRAGGSC)NH2] targeting IL-11RR based upon this cyclic nonapeptide. The radioisotope 111In complexes with DTPA-Bz-NH-SA to form a radiotracer and the NIR dye (IR783-S-Ph-CO) functions as an optical signal generator. Both are conjugated via amino functional groups of lysine to the

10.1021/bc0602679 CCC: $37.00 © 2007 American Chemical Society Published on Web 02/22/2007

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Scheme 1. Synthesis of IR783-S-Ph-CONHSa

a

Reagents and conditions: (i) 4-mercaptobenzoic acid in DMF; (ii) HOSu/DIC/DMAP (1.5/1.5/0.5) in DMF.

cyclic peptide K-c(CGRRAGGSC)NH2. The use of this probe enables us to image the interaction between a targeting ligand and an IL-11RR positive tumor in mice by both conventional nuclear imaging and fluorescence-enhanced optical imaging techniques. Herein, we present the chemistry, in Vitro cell binding, and in ViVo imaging of DLIA-IL11RR.

EXPERIMENTAL PROCEDURES Materials. All Fmoc-amino acids, benzotriazol-1-yl-oxy-trispyrrolidino-phosphonium hexafluorophosphate (PyBOP), Obenzotriazole-N,N,N′,N′-tetramethyl-uronium-hexafluoro-phosphate (HBTU), dimethylaminopyridine (DMAP), 1-hydroxybenzotriazole (HOBt), N-hydroxysuccinimide (HOSu), and Fmoc rink linker were purchased from Novabiochem (San Diego, CA); PL-DMA resin was purchased from Polymer Laboratories (Amherst, MA); piperidine, N,N-diisopropylethylamine (DIPEA), ethylenediamine, triethylsilane (TES), 1,3-diisopropylcarbodiimide (DIC), IR-783 and succinic anhydride were purchased from Sigma-Aldrich Chemical (St. Louis, MO); p-aminobenzyl-diethylenetriamine-penta-(acetic acid-t-butyl ester) was purchased from Macrocyclics (Dallas, TX); trifluoroacetic acid (TFA), N-methylmorpholine (NMM), indium chloride, and all other solvents were purchased from VWR (San Dimas, CA); indium111 chloride (111InCl3) was purchased from Perkin-Elmer (Billerica, MA). 10% serum was purchased from Invitrogen Corporate (Carlsbad, CA). Mouse whole blood was purchased from Pel-Freez Biological (Fayetteville, AR). Analyses. Analytical high-performance liquid chromatography (HPLC) was performed on an Agilent 1100 HPLC system equipped with a Varian reverse phase C-18 analytical column (R0086200CG) at a flow rate of 1 mL/min. Samples were eluted with H2O/acetonitrile containing 0.1% TFA varying from three linear gradients (A: 0-40% in 30 min; B: 10-80% in 30 min; C: 30-100% in 30 min). Preparative HPLC was performed on a Varian Prostar 210 HPLC equipped with a 25 × 2.5-cm Varian reverse phase C-18 preparative column (R0080220CB). Matrix-assisted laser desorption ionization mass spectrometry (MALDI) and electrospray ionization mass spectrometry (ESI) were performed by the Protein Chemistry Core Laboratory at Baylor College of Medicine. Fluorescence properties were measured on a Spex Fluorolog III spectrophotometer from HORIBA Jobin Yvon, Inc. (Edison, NJ). Synthesis of p-Succinamidobenzyl-diethylenetriaminepenta-(acetic acid-t-butyl ester) [DTPA(OtBu)5-Bz-NH-SA]. Synthesis of DTPA(OtBu)5-Bz-NH-SA was conducted according to previously published procedures (18). DTPA(OtBu)5Bz-NH2(1.0 mmol) was dissolved in 10 mL of DMF/DCM (1/ 5). Succinic anhydride (1.0 mmol) and DIPEA (0.2 mL) were added to the solution. The mixture was stirred at room temperature for 4 h. After evaporation of the solvents under vacuum, the residue was dissolved in ethyl acetate, washed with 2% KHSO4 and brine, and then dried over MgSO4. After filtration, ethyl acetate was removed to yield a white powder.

ESI for C45H74N4O13 calcd. [M + H]+ 879. 53; found 879. 4; HPLC (gradient C) retention time 23.98 min. Synthesis of 2-(2-(2-(4-Carboxyphenylthio)-3-(2-(3,3-dimethyl-1-(4-sulfobutyl)indolin-2-ylidene)ethylidene)cyclohex1-enyl)vinyl)-3,3-dimethyl-1-(4-sulfobutyl)-3H-indolium [IR783-S-Ph-COOH] (2) (Scheme 1). IR-783 (1) (250 mg, 0.33 mmol) and 4-mercaptobenzoic acid (156 mg, 1 mmol) were dissolved in 5 mL of DMF and stirred overnight at room temperature. After the solvent was removed, the residue was dissolved in MeOH and then precipitated in ether. The solid was filtered out and further purified with flash chromatography with ethyl acetate and methanol. MALDI for C45H53N2O8S3+ calcd. [M]+ 845. 30; found 845. 30; HPLC (gradient B) retention time 18.56 min; Ex/Em 794/810 in MeOH. Synthesis of 2-(2-(3-(2-(3,3-dimethyl-1-(4-sulfobutyl)indolin-2-ylidene)ethylidene)-2-(4-((2,5-dioxopyrrolidin-1-yloxy)carbonyl)-phenylthio)cyclohex-1-enyl)vinyl)-3,3-dimethyl-1(4-sulfobutyl)-3H-indolium [IR-783-S-Ph-CO-NHS] (3) (Scheme 1). IR-783-S-Ph-COOH (2) (200 mg, 0.24 mmol), DIC (56 µL, 0.36 mmol), HOSu (42 mg, 0.36 mmol), and DMAP (15 mg, 0.12 mmol) were dissolved in 5 mL of DMF and stirred overnight at room temperature. After the solvent was removed, the residue was dissolved in MeOH and then precipitated in ether. The solid was filtered out and purified with reverse phase HPLC. MALDI for C49H56N3O10S3+ calcd. [M]+ 942. 31; found 942. 30; HPLC (gradient B) retention time 20.27 min. Synthesis of cyclic peptide c(Cys-Gly-Arg-Arg-Ala-GlyGly-Ser-Cys)NH2 (4) and its DTPA chelating conjugate DTPA-Bz-NH-SA-Lys-c(Cys-Gly-Arg-Arg-Ala-Gly-Gly-SerCys)NH2 (8) (Scheme 2). First, Fmoc-rink linker was attached to PL-DMA resin, which had been treated with ethylenediamine overnight. The linear peptide Cys-Gly-Arg-Arg-Ala-Gly-GlySer-Cys-NH2 and its conjugate DTPA-Bz-SA-Lys-Cys-Gly-ArgArg-Ala-Gly-Gly-Ser-Cys-NH2 were synthesized using Symphony synthesizer from Protein Technologies, Inc. (Tucson, AZ) following Fmoc standard procedure. The protecting groups of amino acid side chains were t-butyloxycarbonyl (Boc) for Lys, trityl (Trt) for Cys, 2,2,4,6,7-pentamethyl-dihydrobenzofurane5-sulfonyl (Pbf) for Arg, t-butyl (tBu) for Ser and t-butoxy (tBuO) for DTPA. Each amino acid was sequentially coupled on solid support using HBTU, HOBt, and NMM as coupling reagents. The conjugation of DTPA(OtBu)5-Bz-NH-SA to the linear peptide Lys(Boc)-Cys(Trt)-Gly-Arg(Pbf)-Arg(Pbf)-AlaGly-Gly-Ser(But)-Cys(Trt)-resin was achieved in the presence of PyBOP, HOBt, and DIPEA. Cyclization between Cys-Cys for peptide 4 and 8 was achieved in NH4OH solution (pH 9. 5) after linear peptides were cleaved from the support using TFA/ H2O/TES (94/2/4). The products were purified by reverse phase HPLC. MALDI for c(CGRRAGGSC)NH2 (4) (C30H54N16O10S2) calcd. [M + H]+ 863. 37; found 863. 37; HPLC (gradient A) retention time 8.71 min. MALDI for DTPA-Bz-NH-SA-K-

Dual-Labeled Imaging Agent Targeting Interleukin 11 RR Scheme 2. Synthesis of

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111

In-DTPA-Bz-NH-SA-Lys(IR783-S-Ph-CO)-c(CGRRAGGSC)NH2a

a Reagents and conditions: (i) Fmoc solid-phase peptide synthesis; (ii) DTPA(But)5-Bz-NH-SA/PyBOP/HOBt/DIPEA (3:3:3:6 Eq) in DMF; (iii) TFA/H2O/TES (95:1:4); (iv) H4NOH basic solution (pH 9. 5); (v) IR-783-S-Ph_CONHS in DMF with 10% DIPEA; (vi) 111InCl3 or InCl3 in NaOAc (0. 1 N).

c(CGRRAGGSC)NH2 (8) (C61H98N22O23S2) calcd [M + H]+ 1571. 66; found 1571. 70; HPLC (gradient A) retention time 13.24 min. Synthesis of DTPA-Bz-NH-SA-K(IR783-S-Ph-CO)-c(CGRRAGGSC)NH2 (9) (Scheme 2). Conjugation of IR783-S-PhCONHS (3) to ω-NH2 of Lys in DTPA-Bz-NH-SA-Kc(CGRRAGGSC)NH2 was carried out in DIPEA/DMF(1/9) solution overnight. After the solvent was removed, the residue was washed with ether several times. The product was purified by reversed phase HPLC. MALDI for C106H149N24O30S5 calcd. [M]+ 2397. 95; found 2397. 94; HPLC (gradient B) retention time 18.19 min; Ex/Em 794/810 in MeOH. Cold and Radioactive Indium Labeling of DTPA-Bz-NHSA-Lys(IR783-S-Ph-CO)-c(CGRRAGGSC)NH2 to Yield DLIA-IL11RR (10, 11) (Scheme 2). DTPA-Bz-NH-SA-K(IR783-S-Ph-CO)-c(CGRRAGGSC)NH2 (9) in 0.1 N sodium acetate solution was mixed with an aqueous solution of cold InCl3 or radioactive 111InCl3 for 30 min. The purity of the labeled

compound was analyzed using an HPLC system equipped with a radiometric detector from Bioscan (Washington, DC). MALDI for C106H146InN24O30S5 calcd. [M]+ 2509. 83; found 2509.9; HPLC (gradient B) retention time 17.39 min; Ex/Em 794/810 in MeOH. Peptide Stability Test in Serum. DTPA-Bz-NH-SA-Kc(CGRRAGGSC)NH2 (8) (1 mg) was incubated in 1 mL of culture medium containing 10% of fetal serum albumin at 37 °C. At various time intervals, 50 µL aliquot was injected into an HPLC system. The samples were eluted with water and acetonitrile containing 0.1% TFA varying from 0 to 40% over 30 min and detected with a diode array UV/vis detector at 230 and 275 nm. Peptide Stability Test in Mouse Whole Blood. DTPA-BzNH-SA-K-c(CGRRAGGSC)-NH2 (8) (1 mg) was incubated in 200 µL of mouse whole blood at 37 °C. At various time intervals, 20 µL of the mixture was added into 100 µL of PBS and then centrifuged. 50 µL of the supernatant was injected

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into an HPLC system. The samples were eluted with water and acetonitrile containing 0.1% TFA varying from 0 to 40% over 30 min and detected with a diode array UV/vis detector at 230 and 275 nm. Cell Binding Study of Imaging Agent. Human breast cancer that expresses IL-11RR (MDA-MB-231) was purchased from American Type Culture Collection (Manassas, VA). A highly metastatic subline MDA-MB-231 (1833) was a kindly donated by Dr. Massaque at the Memorial Sloan-Kettering Cancer Center. The cells were cultured in MDEM DMEM/F12 (Invitrogen, Carlsbad, CA) with 10% FBS (Hyclone, Logan, UT) in a humidified incubator maintained at 37 °C with 5% CO2. In vitro cell binding was performed on both cell lines. A solution (1 µM) of dual-labeled imaging agent was incubated with cells for 10 min at 37 °C. The cells were fixed and stained with 1 µM of Sytox Green in 95% ethanol for 15 min at 4 °C. Images were recorded by a Leica fluorescent microscope (Model: DM6000 B, Leica Microsystems GmbH, Ernst-Leitz-Strasse, Germany) equipped with 100 W xenon lamp and fluorescent filters. Autocrine Stimulation. MDA-MB-231 cells that express IL11 RR were first co-incubated and stimulated by 0.5 mg/mL of IL-11 (Neumega, Oprelvekin, Wyeth Pharmaceuticals Inc. Philadelphia, PA) for 18 h. DLIA-IL11RR was then added to achieve a final concentration of 1 µM for 10 min at 37 C. Cells were washed 3 times with PBS. The autocrine stimulatory effect of IL-11 was determined by measuring the intensity of bound DLIA-IL11RR normalized by cell number for both stimulated and untreated cells. Statistical analysis was performed using SAS software version 9.0 (SAS Institute Inc., Cary, NC) for Microsoft Windows. The difference between normalized intensities of stimulated and unstimulated cells was compared using the oneway ANOVA. The significant value (p) was set at 0.5. In ViWo NIR Optical and Nuclear Imaging Studies of Imaging Agent. Human breast cancer xenografts were injected s.c. with 1 × 106 MDA-MB-231 cells on the hindlimb. Tumor growth occurred over 3-4 weeks reaching 7-8 mm in diameter. At this size, 2 nmol of DLIA-IL11RR was injected intravenously into the tail vein of the mice. Mice were imaged on a Siemens Preclinical microCAT II for SPECT/CT. The integration time for SPECT was 20 min and CT required 8 min. In ViVo fluorescence-based optical imaging was accomplished by illuminating the animal with light from a laser diode (80 mW at 785 nm light) expanded to an approximately 8-cm-diameter circular area. The re-emitted fluorescent light was collected by an electron-multiplying charge-coupled device (EMCCD) camera (model PhotoMAX:512B, Princeton Instruments, Trenton, NJ). The resolution of the EMCCD camera was 16 × 16 µm in a 512 × 512 pixels square frame-transfer format. Filter sets used in this study included a bandpass filter (830 nm center wavelength) to transmit fluorescent emission and holographic filter (785 nm center wavelength) to reject reflected excitation light. Image acquisition was accomplished using V++ software from Digital Optics (Auckland, New Zealand). Data processing and analysis were accomplished using Matlab software from The MathWorks, Inc. (Natick, MA). The integration time for each image was 800 ms. The whole body images were taken at 24 h after the injection and 48 h after the injection, mice were sacrificed and images of the excised organs were taken. Threedimension SPECT/CT and images/movies were reconstructed by Amira (version 3.1, Konrad-Zuse-Zentrum fur Informationstechnik Berlin, Germany).

RESULTS AND DISCUSSION Chemistry. In this study, the cyclic nonapeptide c(CGRRAGGSC)NH2 was selected as targeting molecule. To develop an optical and nuclear dual-labeled targeting imaging agent

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based upon this targeting molecule, it is necessary to introduce a fluorophore, which serves as an optical imaging component, and to select a suitable bifunctional chelator, which is able to be coupled to a targeting compound and capable of forming a stable complex with a radiometal ion. Cyanine dyes are most commonly used as NIR fluorophores. IR-783-S-Ph-CONHS (3) was selected as NIR fluorescence reaction agent for this study, due to its increased chemical stability over IR-783-S-(CH2)2-CONHS (19). Compound 3 was prepared from a commercially available cyanine dye IR-783 (1). First, the chlorine on IR-783 was replaced with S-Ph-COOH (Scheme 1) using a similar method as Strekowski et al. described (20) to form compound 2. Next, the reaction of compound 2 with HOSu yielded the desired compound 3, which can be directly applied for conjugation with an amino functional group. Diethylenetriaminepenta-acetic acid (DTPA) is one of the well-known radiometal chelating agents for such radionuclides as 111In and 90Y. The DTPA derivative DTPA(OtBu)5-Bz-NHSA (18) was selected as a chelating compound. The advantage of this reagent is that it can be easily conjugated to peptides on a solid support and still possesses five carboxylates for radiometal chelation. The reaction scheme and the structure of dual-labeled targeting imaging agent [In-DTPA-Bz-NH-SA-K(IR-783-S-PhCO)-c(CGRRAGGSC)NH2] are summarized in Scheme 2. The synthesis procedure can be divided into six major parts. The first three parts were carried out on solid support. The latter three parts were performed in solution phase. The linear protective peptides on resin, Cys(Trt)-Gly-Arg(Pbf)-Arg(Pbf)Ala-Gly-Gly-Ser(But)-Cys(Trt)-resin (4) and Lys(Boc)-Cys(Trt)Gly-Arg(Pbf)-Arg(Pbf)-Ala-Gly-Gly-Ser(But)-Cys(Trt)-resin (5), were synthesized using Fmoc solid-phase chemistry (part 1). DTPA(OtBu)5-Bz-NH-SA was coupled to the linear peptide Lys(Boc)-Cys(Trt)-Gly-Arg(Pbf)-Arg(Pbf)-Ala-Gly-Gly-Ser(But)-Cys(Trt)-resin (6) through R-amino functional group of lysine (part 2). After deprotection and cleavage of the compounds from the solid support (part 3), the side chain-side chain cyclization between cysteine and cysteine of peptide CGRRAGGSCNH2 or its conjugate DTPA-Bz-NH-SA-K-CGRRAGGSCNH2 (8) was achieved through disulfide bridge formation (part 4). An important consideration in selecting cyclic peptide c(CGRRAGGSC)NH2 as the targeting agent to IL-11RR was the peptide stability, since it may be degraded by enzymes in vivo. Consequently, the stability test of DTPA-Bz-SA-Kc(CGRRAGGSC)NH2 (8) was carried out in two different media: 10% fetal serum albumin serum and mouse whole blood. The peak areas from HPLC analysis of the conjugate (8) were quantified, and the stability was expressed as a percentage of individual peak area at different incubation times divided by the peak area at the initial incubation time. The results indicated that compound 8 was 20% degraded when incubated in a serumcontaining culture medium at 37 °C over 24 h, while 50% degradation was observed when incubated in mouse whole blood. Although modification of the peptide can be carried out later to improve the stability, the data demonstrated that the stability of the peptide chelator complex is sufficient for this study, since imaging was to be accomplished within 24 h. After validating the stability of DTPA-Bz-SA-K-c(CGRRAGGSC)NH2, conjugation of dye (IR-783-S-Ph-CO-NHS) to compound 8 through -amino functional group of lysine was performed in a basic solution (part 5). In the last step (part 6), DTPA-Bz-SA-K(IRdye783-S-Ph-CO)-c(CGRRAGGSC)NH2 (9) was separately labeled with cold indium or with radioactive isotope. The non-radioactive compound 10 was selected for

Dual-Labeled Imaging Agent Targeting Interleukin 11 RR

Figure 1. Excitation and emission fluorescence spectra of compound 2 (IR-783-S-Ph-COOH), compound 9 [DTPA-Bz-SA-K(IR-783-S-PhCO)-c(CGRRAGGGSC)NH2] and compound 10 [In-DTPA-Bz-SAK(IR-783-S-Ph-CO)-c(CGRRAGGSC)NH2] at 1 µM in MeOH at wavelength Ex/Em 795/810 nm.

examination of fluorescence properties and cell studies, while the radiolabeled conjugate 11 was used for the in ViVo imaging study. The fluorescence properties of compounds IR783-S-PhCOOH (2), DTPA-Bz-NH-SA-Lys(IR783-S-Ph-CO)-c(CGRRAGGSC)NH2 (9) and In-DTPA-Bz-NH-SA-Lys(IR783-S-PhCO)-c(CGRRAGGSC)NH2 (10) were evaluated. Upon comparing compounds 2 and 9, we found that the excitation and emission spectra of conjugate 9 remained unchanged (Figure 1). However, after the conjugation of dye to DTPA-cyclopeptide the signal intensity was reduced to 84%. Nevertheless, the spectra in Figure 1 demonstrate that the metal chelation of compound 9 to form compound 10 did not impact fluorescence spectra.

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Cell Binding Study of Imaging Agent. To evaluate DLIAIL11RR as a potential targeting molecule for in ViVo imaging, the binding ability to IL-11 receptor was examined in Vitro. Figure 2 illustrates cell binding of the imaging agent 10 to IL11RR positive MDA MB-231 and its subline 1833. The cell nuclei were stained with Sytox green, while the red halo arose from the imaging agent associated with the membrane expression of IL-11RR. Furthermore, cell stimulation study was carried out using the MDA-MB-231 cell line (21). The results show that conjugate 10 binding to stimulated cells was statistically significant higher than binding to nonstimulated cells. These preliminary data reveal that the targeting ability of cyclic peptide c(CGRRAGGSC)NH2 to IL-11RR was retained after introducing the nuclear and optical tracers. In ViWo NIR Optical and Nuclear Imaging. Further demonstration of the targeting feasibility of DLIA-IL11RR as an imaging agent was performed in ViVo. Figure 3 demonstrates white light, SPECT/CT, and NIR optical images of the same mouse bearing subcutaneous MDA-MB-231 tumor 24 h after administration of the imaging agent. The white light shows the location of the tumor in the hind leg of the animal. The SPECT/ CT and NIR optical imaging illustrate the uptake of the imaging agent in the region of the tumor. In addition, each of the images display uptake of the agent in the abdominal area, presumably within the kidneys as indicated from the NIR planar imaging. It is important to note that the NIR imaging is not tomographic and therefore is directly comparable to the gamma scintigraphy. SPECT/CT on the other hand provides the three-dimensional image of the location of the imaging agent based upon the indium signature. The tomographic SPECT/CT has resolution on the order of 2 mm and consequently may not provide the same information as the NIR images. Furthermore, since light is attenuated in blood born tissues such as the liver, the NIR

Figure 2. Cell binding study: Two human breast cancer cell lines (MDA-MB-231 and MDA-MB-231-1833) that express IL-11RR receptor were incubated with 1 µM of DLIA-IL11RR. Green (Sytox green) represents cell nuclei, and red (NIR imaging agent) represents conjugate bound to the cells.

Figure 3. SPECT/CT and NIR imaging. Mice bearing human breast cancer xenograft (MDA-MB-231) were injected with 2 nmol of the conjugate DLIA-IL-11RR i.v. Representative images show results 24 h after injection. Tumors (arrows) show more conjugate uptake than muscle in both SPECT/CT and NIR images. High signal intensity in kidneys showed the excretion route of this agent even at very low injection dose.

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imaging may not provide the same depth specificity of the indium label. When tissues are excised and counted for gamma emission, we found that the tumor to muscle, liver to muscle, and kidney to muscle ratios were 2.12, 2.62, 10.42. To date, we have not conducted biodistribution or pharmacological studies to determine the optimal dose or timing for imaging. The high kidney signal counts may reflect excretion routes and therefore suggest that a lower dose/longer post administration imaging time is warranted. While these in Vitro scintigraphy counts provide an absolute measure of the deposition of the imaging agent within the body, there is no validated method for quantifying the uptake on the basis of NIR fluorescence. Development of quantitative methods for NIR imaging and tomography depend upon the development of dual-labeled imaging agents to further improve optical imaging techniques.

CONCLUSION We successfully developed and deployed a new optical and nuclear dual-labeled targeting imaging agent DLIA-IL11RR [In-DTPA-Bz-NH-SA-K(IR-783-S-Ph-CO)c(CGRRAGGSC)NH2] against IL-11RR expressed in cancer as well as in other processes associated with disease. Our data indicate that the cyclic peptide c(CGRRAGGSC)NH2 is a feasible targeting agent of IL-11RR for diagnostic cancer imaging. As shown in our in vitro cell study, introduction of two imaging tracers to cyclic peptide c(CGRRAGGSC)NH2 did not alter the targeting capability of the peptide to IL-11RR. Furthermore, indium isotope chelation did not cause any quenching on fluorescence emission of imaging agent 10 relative to the nonchelated compound 9. Even through peptides usually have lower binding affinity than antibodies, we found that injection of 2 nmol of DLIA-IL11RR was sufficient to observe specific binding in tumor region. Furthermore, we found that the preliminary in ViVo imaging by optical and radionuclear methods were consistent. Before DLIAIL11RR can be validated as a potential imaging agent, the optimal dose and biodistribution remain to be determined.

ACKNOWLEDGMENT This research was supported, in part, by the Department of Radiology at Baylor College of Medicine and National Institutes of Health Grant R01 EB003132.

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