In Vivo Imaging of Transplanted Islets with 64Cu-DO3A-VS-Cys40

Jun 21, 2011 - was determined using a gamma counter. ... activity in each excised specimen was measured using a gamma counter ... a Pixera 600 camera...
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In Vivo Imaging of Transplanted Islets with 64 Cu-DO3A-VS-Cys40-Exendin-4 by Targeting GLP-1 Receptor Zhanhong Wu,† Ivan Todorov,† Lin Li,‡ James R. Bading,§ Zibo Li,|| Indu Nair,† Kohei Ishiyama,† David Colcher,§ Peter E. Conti,|| Scott E. Fraser,^ John E. Shively,‡ and Fouad Kandeel*,† †

Department of Diabetes, Endocrinology and Metabolism Department of Immunology § Department of Cancer Immunotherapy and Tumor Immunology Beckman Research Institute of City of Hope, Duarte, California 91010, United States Molecular Imaging Center, Department of Radiology, University of Southern California, Los Angeles, California 90033, United States ^ Division of Biology, California Institute of Technology, Pasadena, California 91125, United States

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bS Supporting Information ABSTRACT: Glucagon-like peptide 1 receptor (GLP-1R) is highly expressed in pancreatic islets, especially on β-cells. Therefore, a properly labeled ligand that binds to GLP-1R could be used for in vivo pancreatic islet imaging. Because native GLP-1 is degraded rapidly by dipeptidyl peptidase-IV (DPP-IV), a more stable agonist of GLP-1 such as Exendin-4 is a preferred imaging agent. In this study, DO3A-VSCys40-Exendin-4 was prepared through the conjugation of DO3A-VS with Cys40-Exendin-4. The in vitro binding affinity of DO3A-VSCys40-Exendin-4 was evaluated in INS-1 cells, which overexpress GLP1R. After 64Cu labeling, biodistribution studies and microPET imaging of 64Cu-DO3A-VS-Cys40-Exendin-4 were performed on both subcutaneous INS-1 tumors and islet transplantation models. The subcutaneous INS-1 tumor was clearly visualized with microPET imaging after the injection of 64Cu-DO3A-VS-Cys40-Exendin-4. GLP-1R positive organs, such as pancreas and lung, showed high uptake. Tumor uptake was saturable, reduced dramatically by a 20-fold excess of unlabeled Exendin-4. In the intraportal islet transplantation models, 64Cu-DO3A-VS-Cys40-Exendin-4 demonstrated almost two times higher uptake compared with normal mice. 64Cu-DO3AVS-Cys40-Exendin-4 demonstrated persistent and specific uptake in the mouse pancreas, the subcutaneous insulinoma mouse model, and the intraportal human islet transplantation mouse model. This novel PET probe may be suitable for in vivo pancreatic islets imaging in the human.

’ INTRODUCTION In 2007, 23.6 million children and adults in the United States (7.8% of the population) had diabetes. The pathophysiology of diabetes is complex, but the main clinical features result from impairment of insulin secretion and/or function. In humans, the pancreas is a small, elongated organ that lies behind the stomach and adjacent to the liver, duodenum, and spleen. The insulinproducing β-cells that govern glucose homeostasis are located in the islets of Langerhans, which comprise only 2 3% of all pancreatic tissues. β-cells represent up to 80% of the islets, while other types of cells (mainly R, δ, and pancreatic polypeptide (PP) cells) contribute to the remaining islet cell mass. Islet transplantation is a promising treatment option for Type 1 diabetes (T1D) patients; however, the fate of the graft over time remains difficult to follow, due to the lack of available tools capable of monitoring islet graft loss. Currently, the estimates of islet survival and function are limited to indirect assessments r 2011 American Chemical Society

based on exogenous insulin requirements or metabolic studies. Therefore, the development of noninvasive imaging methods for the assessment of the pancreatic β-cell mass and status is becoming important for monitoring patients after islet transplantation. A variety of currently available imaging techniques, including magnetic resonance imaging (MRI),1 6 bioluminescence imaging (BLI),7 9 and nuclear imaging (positron emission tomography (PET) and single photon emission computed tomography (SPECT)), have been tested for the study of β-cells. In particular, PET is a noninvasive functional imaging technique that provides adequate resolution, high sensitivity, and accurate quantification of physiological, biochemical, and pharmacological processes in living subjects. Received: March 15, 2011 Revised: June 20, 2011 Published: June 21, 2011 1587

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Figure 1. (A) Synthesis and Cu-64 labeling of 64Cu-DO3A-VS-Cys40-Exendin-4. (B) Chemical structure of (C) Sequences of Exendin-4 and GLP-1(7 36)-amide.

Glucagon-like peptide 1 (GLP-1) is an incretin peptide released from the intestine in response to nutrient ingestion; its binding to the GLP-1 receptor (GLP-1R) augments glucoseinduced insulin secretion from pancreatic β-cells.10,11 Since GLP-1R is highly expressed in islets, and especially highly expressed on β-cells, ligands of GLP-1R could be ideal probes for pancreatic β-cell imaging.12,13 Exendin-4, a peptide hormone first isolated by Dr. John Eng from the saliva of the gila monster in 1992, is especially attractive as an affinity ligand.14,15 Exendin-4 displays biological properties similar to human GLP-1, with which it shares 53% sequence identity;12 however, it is metabolically much more stable than GLP-1. Previously, Exendin analogues have been labeled with 111In,16 20 125I,21 99mTc,18 and 68 Ga.18,22 However, most tracers were only evaluated in GLP-1 receptor positive insulinoma with experimental models and in humans.19,20 To date, imaging of transplanted human β-cells in humans has been limited to one study of SPECT imaging with Lys40(Ahx-DTPA-111In)NH2]Exendin-4 in one patient who received transplants in muscle.23 Here, we report the development of 64Cu-labeled Exendin-4 for in vivo imaging of GLP-1 receptor. Cu-64 (t1/2 = 12.7 h) decays by β+ (20%) and β emission (37%), as well as electron capture (43%), making it well-suited for radiolabeling proteins, antibodies, and peptides for PET imaging.24 In addition to characterizing the performance of 64Cu-labeled Exendin-4 in a rodent insulinoma model,

64

Cu-DO3A-VS-Cys40-Exendin-4.

we also explored its feasibility for imaging intraportal islet transplantation.

’ EXPERIMENTAL PROCEDURES General. All commercially available chemical reagents were purchased from Aldrich (St. Louis, MO) and used without further purification. Cys40-Exendin-4 was synthesized by standard FMOC chemistry by the Synthetic and Biopolymer Chemistry Core at City of Hope (Duarte, CA). 1,4,7,10-Tetraazacyclododecane-1,4,7-tris(t-butyl acetate) was purchased from Macrocyclics Inc. (Dallas, TX). 64CuCl2 was obtained from Washington University (St. Louis, MO) by the 64Ni(p,n)64 Cu nuclear reaction. 125I-Exendin(9 39) was purchased from Perkin-Elmer (Wellesley, MA). Preparation of DO3A-VS Conjugated Exendin-4 (DO3AVS-Cys40-Exendin-4). DO3A-VS (1,4,7-tris(acetic acid)-10-vinylsulfone-1,4,7,10-tetraazacyclododecane) was prepared as previously reported.25 Cys40-Exendin-4 (0.7 μmol) in 250 μL of water was added to 0.77 μmol of DO3A-VS in 100 μL of water (pH 8.0) (Figure1). The solution was stirred under argon overnight at 25 C and the product was purified by HPLC (Phenomenex Gemini C18, Torrance, CA; flow = 1 mL/min). The product was characterized by LTQ FT mass spectrometer (Thermo, Waltham, MA) (m/z: 4752.30, calcd: 4752.93). 1588

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Bioconjugate Chemistry Radiolabeling of DO3A-VS-Cys40-Exendin-4. DO3A-VS-

Cys40-Exendin-4 (10 μg) was labeled by adding of 74 MBq of 64 Cu2+ in 0.1 M sodium acetate buffer (pH 5.5) followed by 45 min of incubation at 43 C. DTPA (10 mM, 5 μL) was added to chelate unlabeled 64Cu. The radiochemical yield was determined by radio-ITLC and HPLC (flow = 1 mL/min; the mobile phase was changed from 95% solvent A [0.1% trifluoroacetic acid (TFA) in water] and 5% solvent B [0.1% TFA in acetonitrile (ACN)] (0 2 min) to 35% solvent A and 65% solvent B at 32 min). 64Cu-DO3A-VS-Cys40-Exendin-4 was purified by C-18 Sep-Pak cartridge, and the eluant was evaporated and reconstituted in saline for animal studies. Cells. Rat insulinoma cell line INS-1 (GLP-1R positive, kindly donated by Dr. Ian Sweet, University of Washington, Seattle, WA) was maintained under standard conditions: the INS-1 insulinoma cells were grown in RPMI-1640 medium supplemented with 10% fetal bovine serum (Atlanta Laboratories, Atlanta, GA), 20 mM Hepes, 2 mM L-glutamine, 1 mM sodium pyruvate, 1% antibiotic antimycotic solution (Mediatech, Manassas, VA), and 50 mM 2-mercaptoethanol at 37 C in humidified atmosphere containing 5% CO2. The INS-1 insulinoma cells were grown in culture until sufficient cells were available. Human pancreatic islets were isolated by the Southern California Islet Cell Resources Center (SC-ICRC, Duarte, CA) from human pancreata from healthy adult donors with a proper consent for research use and approval by the Institutional Review Board of the City of Hope. Islet isolations were performed using the standard collagenase method for pancreas digestion and islet purification26 following the operating procedures of the SCICRC. Islets cultured for 48 72 h in CMRL1066 based islet culture medium (Mediatech, Manassas, VA) were used in the experiments. In Vitro Cell Binding Assay. The in vitro GLP-1R-binding affinity and specificity of DO3A-VS-Cys40-Exendin-4 and Exendin-4 were assessed via competitive cell binding assays using 125 I-Exendin(9 39) (PerkinElmer, Waltham, MA) as the GLP1R specific radioligand. In detail, INS-1 cells were harvested, washed three times with phosphate buffered saline (1, Mediatech, Manassas, VA), and resuspended (2  106 cells/mL) in the binding buffer.27 Filter multiscreen DV plates (96-well, pore size, 0.65 μm, Millipore, Billerica, MA) were seeded with 105 cells and incubated with 125I-Exendin(9 39) (0.74 kBq/well) in the presence of increasing concentrations of different Exendin-4 analogues (0 1.0 μmol/L). The truncated Exendin(9 39) is an antagonist of GLP-1R and a competitive inhibitor of Exendin-4. The total incubation volume was adjusted to 200 μL. After the cells were incubated for 2 h at room temperature, the plates were filtered through a multiscreen vacuum manifold and washed three times with binding buffer. The hydrophilic polyvinylidenedifluoride (PVDF) filters were collected, and the radioactivity was determined using a gamma counter. The best-fit 50% inhibitory concentration (IC50) values for INS-1 cells were calculated by fitting the data with nonlinear regression using GraphPad Prism (GraphPad Software, La Jolla, CA). Experiments were performed on triplicate samples. Animals. Male, NOD/SCID mice (the City of Hope Animal Resource Center, Duarte, CA), 8 10 weeks old, served as recipients for insulinoma cells and human islet cells. All animal experiments were performed in compliance with the Guidelines for the Care and Use of Research Animals established by the City of Hope/Beckman Research Institute’s Animal Use Committee.

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Xenograft Tumor Model. NOD/SCID mice were injected with INS-1 rat insulinoma cells (5  106, suspended in 0.1 mL of 1 phosphate buffered saline) subcutaneously in the thigh. Tumors were established within 18 21 days postinjection. Blood glucose values were monitored daily and 6% glucose solution was fed ad libitum once the blood glucose level was lower than 3.3 mmol/L. Islet Transplantation. Islets with a diameter of less than 250 μm were used for transplantation. Islets were transplanted into the livers of NOD/SCID mice via the portal vein. Immediately before intraportal infusion, 1000 islet equivalents were suspended in 200 μL of Hank’s balanced salt solution (Mediatech, Manassas, VA), and loaded in a 1 mL syringe. Under general anesthesia, the portal vein was exposed by extra-abdominal repositioning of the bowel and the islets were infused into the portal vein via a 27-gauge needle. Biodistribution and MicroPET Imaging. Biodistribution and microPET imaging of 64Cu-DO3A-VS-Cys40-Exendin-4 were performed in mice bearing INS-1 tumor or islet grafts to evaluate the in vivo specific binding to GLP-1R. For blocking studies, Exendin-4 (20 μg) was coinjected with the radiotracer. For biodistribution studies, NOD/SCID mice bearing INS-1 tumor (n = 3, for each time point) were injected intravenously with 64 Cu-DO3A-VS-Cys40-Exendin-4 (0.05 μg, 0.37 MBq, 100 μL). Activity injected into each mouse was measured in a dose calibrator (Capintec, Ramsey, NJ). Animals were sacrificed under inhalation anesthesia at 1 h, 4 h, and 22 h postinjection (p.i.). Tissues and organs of interest were excised and weighed. Radioactivity in each excised specimen was measured using a gamma counter; radioactivity uptake was expressed as percent injected dose per gram (%ID/g). Mean uptake (%ID/g) and corresponding standard deviation was calculated for each group of animals. For microPET imaging, mice were injected via the tail vein with 3.7 7.4 MBq of 64Cu-DO3A-VS-Cys40-Exendin-4 (0.5 1 μg). Serial imaging of INS-1 tumor (at 1, 4, and 22 h; scan duration 20, 20, and 45 min, respectively) and islet transplantation model (at 2 and 5 h; scan duration 10 and 20 min, respectively) was performed using a microPET R4 scanner (Concorde Microsystems, Knoxville, TN; 8 cm axial field of view, spatial resolution 2.0 mm). Images were reconstructed with a MAP iterative algorithm using the microPET Manager software (Concorde Microsystems, Knoxville, TN). Images were then analyzed using the Acquisition Sinogram Image Processing (ASIPro) software (Concorde Microsystems, Knoxville, TN). Average radioactivity accumulation within liver was obtained from a three-dimensional region of interest (ROI) drawn within the central portion of the PET liver image. Image intensities were converted to units of activity concentration (Bq/cm3) using a calibration factor obtained by scanning a cylindrical phantom filled with a known activity concentration of 64Cu. Assuming a tissue density of 1 g/mL, measured tissue activity concentrations were converted to units of Bq/g, then multiplied by 100 and divided by the injected dose to obtain an image-derived percent injected dose per gram of tissue (% ID/g). Histology Study. Livers of mice transplanted with human islets and human pancreas were fixed with 10% formalin and processed for paraffin embedding. 5 μm parallel sections were stained with hematoxylin and eosin or immunostained for insulin and GLP-1R, as described previously.28,29 Guinea pig antihuman insulin (DAKO, Carpinteria, CA) and rabbit antihuman GLP-1R (Novus Biologicals, Littleton, CO) were used as primary antibodies. The corresponding secondary antibodies were conjugated 1589

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Bioconjugate Chemistry with FITC or Texas Red (Jackson Immunoresearch, West Grove, PA). The slices were counter-stained for DNA with diamidinophenylindole (DAPI) (Sigma, St. Louis, MO) and visualized with an Olympus BX51 fluorescent microscope equipped with a Pixera 600 camera. Statistical Analysis. Quantitative data were expressed as mean ( SD. Means were compared using one-way ANOVA and Student’s t test. P values of 99%. 64Cu-DO3A-VS-Cys40Exendin-4 was analyzed by ITLC and HPLC. The HPLC retention time of 64Cu-DO3A-VS-Cys40-Exendin-4 was 23 min. For radio-TLC analysis, 64Cu-DO3A-VS-Cys40-Exendin-4 remained at the origin of the TLC plate, while the retention factor (Rf) value for free 64Cu-DTPA was 0.9 1.0. The specific activity of 64 Cu-DO3A-VS-Cys40-Exendin-4 was around 3.3  1015 Bq/mol. In Vitro Cell Binding Affinity. We compared the receptorbinding affinity of DO3A-VS-Cys40-Exendin-4 with that of Exendin-4 using a competitive cell-binding assay (Figure 2). Both peptides inhibited the binding of 125I-Exendin(9 39) to GLP-1R positive INS-1 cells in a dose-dependent manner.

Figure 2. Competitive binding assays of 125I-Exendin(9 39) and DO3A-VS-Cys40-Exendin-4 or Exendin-4 in INS-1 cells . Data are mean ( SE (n = 3). X axis reflects concentration of nonradolabeled competitor. IC50 values for DO3A-VS-Cys40-Exendin-4 and Exendin-4 were 190.6 ( 3.5 and 131.07 ( 8.3 pM, respectively.

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The IC50 value for DO3A-VS-Cys40-Exendin-4 (190.6 ( 3.5 pmol/L) was comparable to that of Exendin-4 (130.7 ( 8.3 pmol/L). The results showed that DO3A conjugation had a minimal effect on the receptor-binding affinity. Biodistribution Studies and MicroPET Imaging. The GLP1R targeting efficacy and biodistribution patterns of 64CuDO3A-VS-Cys40-Exendin-4 in NOD/SCID mice bearing INS1 rat insulinoma xenografts (GLP-1R positive) were evaluated at multiple time points (1, 4, and 22 h) with static microPET scans. Representative sliced decay-corrected coronal images at different time points are shown in Figure 3A. The INS-1 tumors were clearly visualized with high tumor-to-background contrast for 64 Cu-DO3A-VS-Cys40-Exendin-4. To obtain quantitative biodistribution data, we performed dissection and direct assay of tissue activity concentration in groups of insulnoma-bearing mice sacrificed at 1, 4, and 22 h. The data are shown in Figure 3B. Uptake of 64Cu-DO3A-VS-Cys40Exendin-4 in INS-1 insulinomas was 12.7 ( 2.9, 14.2 ( 2.6, and 9.2 ( 4.0% ID/g at 1, 4, and 22 h p.i., respectively (Figure 3B). This result is consistent with the excretion pattern from the microPET imaging results. Similar to the microPET imaging result, 64Cu-DO3A-VS-Cys40-Exendin-4 mainly accumulated in the kidneys, which remained at 46.8 ( 11.4%ID/g at 23 h p.i.. The GLP-1 receptor specificity of 64Cu-DO3A-VS-Cys40Exendin-4 was confirmed by a blocking experiment where the radiotracer was coinjected with excess amount of cold Exendin-4 (20 μg). As can be seen from Figure 4A, in the presence of unlabeled Exendin-4, the INS-1 tumor uptake was significantly lower (2.7 ( 2.0% ID/g) than that without Exendin-4 blocking (14.2 ( 2.6% ID/g, P < 0.01) at 4 h p.i. In the blocking experiment, the uptake of 64Cu-DO3A-VS-Cys40-Exendin-4 in other GLP-1 positive organs (pancreas, lungs, and duodenum) was significantly decreased (Figure 4B). To test the tracer in an islet transplant model, we implanted 1000 IEQ human islets into mouse liver via portal vein. Nine days after implantation, mice were imaged with 64Cu-DO3AVS-Cys40-Exendin-4 (Figure 5A). Liver uptake of the radiotracer was significantly higher (P < 0.01) in transplanted mice (4.0 ( 0.2 and 5.3 ( 0.7%ID/g at 2 and 5 h p.i., respectively) than in control mice (1.7 ( 0.6 and 2.4 ( 0.8%ID/g at 2 and 5 h p.i., respectively) (Figure 5B). Histology studies (Figure 6) clearly demonstrated that human islet grafts were present in mouse liver and that most insulin-positive cells are also GLP-1Rpositive, which are similar to those for native human islets in the pancreas. This study confirmed that the higher liver uptake of

Figure 3. (A) Representative coronal microPET images of a NOD/SCID mouse bearing an INS-1 xenograft and injected with 64Cu-DO3A-VS-Cys40Exendin-4 (arrows point to the tumor; image is saturated if uptake g20%ID/g). (B) Biodistribution of 64Cu-DO3A-VS-Cys40-Exendin-4 in NOD/ SCID mice bearing INS-1 xenograft at 1 h, 4 h, and 23 h p.i.. The data are mean ( SD (n = 3). 1590

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Figure 4. (A) Representative coronal microPET images of NOD/SCID mice bearing INS-1 xenograft 4 h after injection of 64Cu-DO3A-VS-Cys40Exendin-4 in the absence/presence of excess Exendin-4 (arrows point to the tumors, image is saturated if uptake g20%ID/g). (B) Biodistribution of 64 Cu-DO3A-VS-Cys40-Exendin-4 in NOD/SCID mice bearing INS-1 xenograft in the absence/presence of excess Exendin-4 at 4 h p.i. The data are mean ( SD (n = 3; *P < 0.01, **P < 0.015).

Figure 5. (A) Coronal microPET images of 64Cu-DO3A-VS-Cys40-Exendin-4 in NOD/SCID mice transplanted with human islets into liver or native NOD/SCID mice (white arrows point to the livers, red arrows point to kidneys, and yellow arrows point to bladders). Images are normalized to reflect radiodecay-corrected relative image intensity per unit injected activity (image is saturated if uptake g10%ID/g). (B) Image-derived liver uptake compared between the islet transplant model and normal mice. The data are the mean ( SD (n = 3). Uptake was higher in transplanted mice at both time points (P < 0.01).

the islet transplantation model was due to a GLP-1R targeting effect.

’ DISCUSSION A major challenge for both experimental diabetes research and clinical diabetes care is to obtain spatial and quantitative information about islet or β-cell mass (BCM). Imaging technology has advanced rapidly in recent years, making it possible to noninvasively image small and/or deep structures within the body. It would be of great benefit to the diabetes community to be able to image the pancreatic BCM noninvasively. Such a method would not only facilitate early clinical diagnosis of betacell loss during development of T1D, but also provide an approach with which to evaluate antidiabetic drugs with the potential to stimulate β-cell regeneration. Noninvasive imaging could also be very important for monitoring the fate of transplanted islets.

It is difficult to quantify β-cell mass noninvasively due to the small size (50 500 μm in diameter) of islets and their scattered distribution pattern throughout the pancreas. Therefore, a ligand with high affinity and specificity for pancreatic β-cells is required. Several receptors and their ligands have been reported for β-cell imaging, including sulfonylurea receptor 1 (SUR1),30 32 the vesicular acetylcholine transporter (VAChT),33 and monoamine transporter 2 (VMAT2);34 38 however, most of the imaging results are suboptimal, and an ideal probe for accurate and noninvasive imaging of pancreatic β-cells has not yet been developed. GLP-1R is highly expressed in islets, especially on β-cells in the pancreas. Therefore, the ligands of GLP-1R could be ideal probes for pancreatic β-cell imaging. In this study, 64Cu-DO3AVS-Cys40-Exendin-4 was prepared and shown to have high binding affinity to GLP-1R in vitro. The high and specific expression of GLP-1R on INS-1 cells makes the rodent insulinoma model an 1591

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Figure 6. Human islet grafts in the liver of NOD/SCID mice and native human pancres. Parallel mice liver and human pancreas sections were stained with hematoxylin/eosin and immunostained for insulin (green), GLP-1R (red), and DNA (blue). Scale bar, 100 μm.

ideal platform to test the GLP-1R targeting efficacy and specificity. As expected, the GLP-1R positive INS-1 tumor demonstrated high and persistent uptake of 64Cu-DO3A-VS-Cys40Exendin-4. Receptor specificity was confirmed by a blocking experiment. The pancreas and lung, which have high GLP-1R expression, showed relatively high specific uptake. The uptake of pancreas was reduced by unlabeled Exendin-4 from 5.9 ( 0.8% ID/g to 1.1 ( 0.1%ID/g at 4 h p.i. (P < 0.01), and the lung uptake was reduced from 6.4 ( 2.2%ID/g to 1.8 ( 0.9%ID/g (P < 0.015). It needs to be pointed out that the human lung expressed significantly smaller amounts of GLP-1 receptor compared with rodent lung.39 High kidney uptake of 64Cu-DO3A-VS-Cys40-Exendin-4 was observed in both the microPET imaging and biodistribution studies, which was similar with other radiometal-labeled Exendin analogues.16 18,22 Previously, other researchers have demonstrated that GLP-1R has low expression in kidney.39 41 It has been shown that the kidneys are of great importance for the removal of 64Cu-DO3A-VS-Cys40-Exendin-4 and/or its metabolites from the circulation.42,43 In our research, we have found 64 Cu-labeled Exendin-4 was cleared rapidly from blood (Supporting Information Figure S1) and the high kidney uptake could not be reduced by the blocking study (coinjection with nonradiolabeled Exendin-4). L-Polyglutamic acid (PGA), Gelofusine, or the combination of PGA and Gelofusine pretreatment could significant reduce kidney uptake of Ga-68 labeled Exendin4, while the tumor uptake was not affected.18,44 Although 64CuDO3A-VS-Cys40-Exendin-4 is stable in vitro (Supporting Information Figure S2-A), metabolites was observed in kidney homogenate (Supporting Information Figure S2-C). Therefore, the most likely reason for the high kidney uptake is that the peptide is filtered through glomerulus and subsequently degraded by enzymes in the tubuli brush border membrane.42 Human islets were transplanted into NOD/SCID mice livers through the portal vein, and a subsequent histological study

clearly showed the presence of functional human islets at the time of imaging. Higher liver uptake was observed with microPET imaging on day 10 after transplantation compared with that of normal mice. As shown in Figure 5B, liver uptake in the islet transplantation model was almost twice that in controls. Thus, 64 Cu-DO3A-VS-Cys40-Exendin-4 PET can noninvasively detect transplanted islets in the liver. It is noted that liver uptake increased between 2 and 5 h, which may be caused by metabolism and transchelation of Cu2+ to superoxide dismutase.45 48 This is a principle proof study and only a single number of human islets were transplanted into mice liver and a single time point microPET scan was performed post islet transplantation. Further studies are needed to test the feasibility of long-term monitoring of transplanted islets with 64Cu-DO3A-VS-Cys40Exendin-4. In future studies, we will transplant different numbers of islets into mice liver and perform microPET scan at different time points post-transplantation to correlate the β-cell mass and uptake of mice livers.

’ CONCLUSION In summary, GLP-1R targeted 64Cu-DO3A-VS-Cys40-Exendin-4 showed high and specific uptake in INS-1 tumors. Moreover, we also demonstrated the feasibility of 64Cu-DO3A-VSCys40-Exendin-4 for in vivo imaging intraportally transplanted islets in mice. These results suggest that 64Cu-DO3A-VS-Cys40Exendin-4 is a very promising PET probe for transplanted islets imaging and may be useful for clinical translation. ’ ASSOCIATED CONTENT

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Supporting Information. Additional figures as described in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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’ AUTHOR INFORMATION Corresponding Author

*Fouad Kandeel, M.D., Ph.D. Address: Department of Diabetes, Endocrinology and Metabolism City of Hope, 1500 East Duarte Road, Duarte, CA 91010. Phone: (626)301-8282. Fax: (626)301-8489. E-mail: [email protected].

’ ACKNOWLEDGMENT This work was supported by the grant from the Juvenile Diabetes Research Foundation International (JDRF-37-200920). The authors thank Dr. Ismail Al-Abdullah and the islet isolation team of the Southern California Islet Cell Resource Center at City of Hope for providing the human islets. ’ REFERENCES (1) Leoni, L., and Roman, B. B. (2010) MR imaging of pancreatic islets: tracking isolation, transplantation and function. Curr. Pharm. Des. 16, 1582–94. (2) Medarova, Z., and Moore, A. (2009) MRI as a tool to monitor islet transplantation. Nat. Rev. Endocrinol. 5, 444–52. (3) Medarova, Z., and Moore, A. (2009) MRI in diabetes: first results. Am. J. Roentgenol. 193, 295–303. (4) Marzola, P., Longoni, B., Szilagyi, E., Merigo, F., Nicolato, E., Fiorini, S., Paoli, G. T., Benati, D., Mosca, F., and Sbarbati, A. (2009) In vivo visualization of transplanted pancreatic islets by MRI: comparison between in vivo, histological and electron microscopy findings. Contr. Media Mol. Imaging 4, 135–42. (5) Tai, J. H., Foster, P., Rosales, A., Feng, B., Hasilo, C., Martinez, V., Ramadan, S., Snir, J., Melling, C. W., Dhanvantari, S., Rutt, B., and White, D. J. (2006) Imaging islets labeled with magnetic nanoparticles at 1.5 T. Diabetes 55, 2931–8. (6) Evgenov, N. V., Medarova, Z., Dai, G., Bonner-Weir, S., and Moore, A. (2006) In vivo imaging of islet transplantation. Nat. Med. 12, 144–8. (7) Virostko, J., Radhika, A., Poffenberger, G., Chen, Z., Brissova, M., Gilchrist, J., Coleman, B., Gannon, M., Jansen, E. D., and Powers, A. C. (2010) Bioluminescence imaging in mouse models quantifies beta cell mass in the pancreas and after islet transplantation. Mol. Imaging Biol. 12, 42–53. (8) Roth, D. J., Jansen, E. D., Powers, A. C., and Wang, T. G. (2006) A novel method of monitoring response to islet transplantation: bioluminescent imaging of an NF-kB transgenic mouse model. Transplantation 81, 1185–90. (9) Villalobos, C., Nadal, A., Nunez, L., Quesada, I., Chamero, P., Alonso, M. T., and Garcia-Sancho, J. (2005) Bioluminescence imaging of nuclear calcium oscillations in intact pancreatic islets of Langerhans from the mouse. Cell Calcium 38, 131–9. (10) Holst, J. J. (2007) The physiology of glucagon-like peptide 1. Physiol. Rev. 87, 1409–39. (11) Baggio, L. L., and Drucker, D. J. (2007) Biology of incretins: GLP-1 and GIP. Gastroenterology 132, 2131–57. (12) Goke, R., Fehmann, H. C., Linn, T., Schmidt, H., Krause, M., Eng, J., and Goke, B. (1993) Exendin-4 is a high potency agonist and truncated exendin-(9 39)-amide an antagonist at the glucagon-like peptide 1-(7 36)-amide receptor of insulin-secreting beta-cells. J. Biol. Chem. 268, 19650–5. (13) Malhotra, R., Singh, L., Eng, J., and Raufman, J. P. (1992) Exendin-4, a new peptide from Heloderma suspectum venom, potentiates cholecystokinin-induced amylase release from rat pancreatic acini. Regul. Pept. 41, 149–56. (14) Eng, J., Kleinman, W. A., Singh, L., Singh, G., and Raufman, J. P. (1992) Isolation and characterization of exendin-4, an exendin-3 analogue, from Heloderma suspectum venom. Further evidence for an

ARTICLE

exendin receptor on dispersed acini from guinea pig pancreas. J. Biol. Chem. 267, 7402–5. (15) Edwards, C. M., Stanley, S. A., Davis, R., Brynes, A. E., Frost, G. S., Seal, L. J., Ghatei, M. A., and Bloom, S. R. (2001) Exendin-4 reduces fasting and postprandial glucose and decreases energy intake in healthy volunteers. Am. J. Physiol. Endocrinol. Metab. 281, E155–61. (16) Gotthardt, M., Lalyko, G., van Eerd-Vismale, J., Keil, B., Schurrat, T., Hower, M., Laverman, P., Behr, T. M., Boerman, O. C., Goke, B., and Behe, M. (2006) A new technique for in vivo imaging of specific GLP-1 binding sites: first results in small rodents. Regul. Pept. 137, 162–7. (17) Wild, D., Behe, M., Wicki, A., Storch, D., Waser, B., Gotthardt, M., Keil, B., Christofori, G., Reubi, J. C., and Macke, H. R. (2006) [Lys40(AhxDTPA-111In)NH2]exendin-4, a very promising ligand for glucagon-like peptide-1 (GLP-1) receptor targeting. J. Nucl. Med. 47, 2025–33. (18) Wild, D., Wicki, A., Mansi, R., Behe, M., Keil, B., Bernhardt, P., Christofori, G., Ell, P. J., and Macke, H. R. (2010) Exendin-4-based radiopharmaceuticals for glucagonlike peptide-1 receptor PET/CT and SPECT/CT. J. Nucl. Med. 51, 1059–67. (19) Christ, E., Wild, D., Forrer, F., Brandle, M., Sahli, R., Clerici, T., Gloor, B., Martius, F., Maecke, H., and Reubi, J. C. (2009) Glucagon-like peptide-1 receptor imaging for localization of insulinomas. J. Clin. Endocrinol. Metab. 94, 4398–405. (20) Wild, D., Macke, H., Christ, E., Gloor, B., and Reubi, J. C. (2008) Glucagon-like peptide 1-receptor scans to localize occult insulinomas. N. Engl. J. Med. 359, 766–8. (21) Mukai, E., Toyoda, K., Kimura, H., Kawashima, H., Fujimoto, H., Ueda, M., Temma, T., Hirao, K., Nagakawa, K., Saji, H., and Inagaki, N. (2009) GLP-1 receptor antagonist as a potential probe for pancreatic beta-cell imaging. Biochem. Biophys. Res. Commun. 389, 523–6. (22) Brom, M., Oyen, W. J., Joosten, L., Gotthardt, M., and Boerman, O. C. (2010) 68Ga-labelled exendin-3, a new agent for the detection of insulinomas with PET. Eur. J. Nucl. Med. Mol. Imaging 37, 1345–55. (23) Pattou, F., Kerr-Conte, J., and Wild, D. (2010) GLP-1-receptor scanning for imaging of human beta cells transplanted in muscle. N. Engl. J. Med. 363, 1289–90. (24) Kumar, R., Dhanpathi, H., Basu, S., Rubello, D., Fanti, S., and Alavi, A. (2008) Oncologic PET tracers beyond [18F]FDG and the novel quantitative approaches in PET imaging. Q. J. Nucl. Med. Mol. Imaging 52, 50–65. (25) Li, L., Tsai, S. W., Anderson, A. L., Keire, D. A., Raubitschek, A. A., and Shively, J. E. (2002) Vinyl sulfone bifunctional derivatives of DOTA allow sulfhydryl- or amino-directed coupling to antibodies. Conjugates retain immunoreactivity and have similar biodistributions. Bioconjugate Chem. 13, 110–5. (26) Ricordi, C., Lacy, P. E., and Scharp, D. W. (1989) Automated islet isolation from human pancreas. Diabetes 38 (Suppl 1), 140–2. (27) Gallwitz, B., Witt, M., Folsch, U. R., Creutzfeldt, W., and Schmidt, W. E. (1993) Binding specificity and signal transduction of receptors for glucagon-like peptide-1(7 36)amide and gastric inhibitory polypeptide on RINm5F insulinoma cells. J. Mol. Endocrinol. 10, 259–68. (28) Todorov, I., Nair, I., Avakian-Mansoorian, A., Rawson, J., Omori, K., Ito, T., Valiente, L., Iglesias-Meza, I., Orr, C., Shiang, K. D., Ferreri, K., Al-Abdullah, I. H., Mullen, Y., and Kandeel, F. (2010) Quantitative assessment of beta-cell apoptosis and cell composition of isolated, undisrupted human islets by laser scanning cytometry. Transplantation 90, 836–42. (29) Tornehave, D., Kristensen, P., Romer, J., Knudsen, L. B., and Heller, R. S. (2008) Expression of the GLP-1 receptor in mouse, rat, and human pancreas. J. Histochem. Cytochem. 56, 841–51. (30) Amartey, J. K., Esguerra, C., Al-Jammaz, I., Parhar, R. S., and Al-Otaibi, B. (2006) Synthesis and evaluation of radioiodinated substituted beta-naphthylalanine as a potential probe for pancreatic betacells imaging. Appl. Radiat. Isot. 64, 769–77. (31) Wangler, B., Schneider, S., Thews, O., Schirrmacher, E., Comagic, S., Feilen, P., Schwanstecher, C., Schwanstecher, M., Shiue, 1593

dx.doi.org/10.1021/bc200132t |Bioconjugate Chem. 2011, 22, 1587–1594

Bioconjugate Chemistry C. Y., Alavi, A., Hohnemann, S., Piel, M., Rosch, F., and Schirrmacher, R. (2004) Synthesis and evaluation of (S)-2-(2-[18F]fluoroethoxy)-4-([3methyl-1-(2-piperidin-1-yl-phenyl)-butyl -carbamoyl]-methyl)-benzoic acid ([18F]repaglinide): a promising radioligand for quantification of pancreatic beta-cell mass with positron emission tomography (PET). Nucl. Med. Biol. 31, 639–47. (32) Wangler, B., Beck, C., Shiue, C. Y., Schneider, S., Schwanstecher, C., Schwanstecher, M., Feilen, P. J., Alavi, A., Rosch, F., and Schirrmacher, R. (2004) Synthesis and in vitro evaluation of (S)-2-([11C]methoxy)4-[3-methyl-1-(2-piperidine-1-yl-phenyl)-butyl-carbam oyl]-benzoic acid ([11C]methoxy-repaglinide): a potential beta-cell imaging agent. Bioorg. Med. Chem. Lett. 14, 5205–9. (33) Clark, P. B., Gage, H. D., Brown-Proctor, C., Buchheimer, N., Calles-Escandon, J., Mach, R. H., and Morton, K. A. (2004) Neurofunctional imaging of the pancreas utilizing the cholinergic PET radioligand [18F]4-fluorobenzyltrozamicol. Eur. J. Nucl. Med. Mol. Imaging 31, 258–260. (34) Inabnet, W. B., Milone, L., Harris, P., Durak, E., Freeby, M. J., Ahmed, L., Sebastian, M., Lifante, J. C., Bessler, M., and Korner, J. (2010) The utility of [11C] dihydrotetrabenazine positron emission tomography scanning in assessing beta-cell performance after sleeve gastrectomy and duodenal-jejunal bypass. Surgery 147, 303–9. (35) Eriksson, O., Jahan, M., Johnstr€om, P., Korsgren, O., Sundin, A., Halldin, C., and Johansson, L. (2010) In vivo and in vitro characterization of [18F]-FE-(+)-DTBZ as a tracer for beta-cell mass. Nucl. Med. Biol. 37, 357–363. (36) Kung, M. P., Hou, C., Lieberman, B. P., Oya, S., Ponde, D. E., Blankemeyer, E., Skovronsky, D., Kilbourn, M. R., and Kung, H. F. (2008) In vivo imaging of beta-cell mass in rats using 18F-FP-(+)-DTBZ: a potential PET ligand for studying diabetes mellitus. J. Nucl. Med. 49, 1171–6. (37) Freeby, M., Goland, R., Ichise, M., Maffei, A., Leibel, R., and Harris, P. (2008) VMAT2 quantitation by PET as a biomarker for betacell mass in health and disease. Diabetes Obes. Metab. 10 (Suppl 4), 98–108. (38) Simpson, N. R., Souza, F., Witkowski, P., Maffei, A., Raffo, A., Herron, A., Kilbourn, M., Jurewicz, A., Herold, K., Liu, E., Hardy, M. A., Van Heertum, R., and Harris, P. E. (2006) Visualizing pancreatic betacell mass with [11C]DTBZ. Nucl. Med. Biol. 33, 855–64. (39) Korner, M., Stockli, M., Waser, B., and Reubi, J. C. (2007) GLP-1 receptor expression in human tumors and human normal tissues: potential for in vivo targeting. J. Nucl. Med. 48, 736–43. (40) Bullock, B. P., Heller, R. S., and Habener, J. F. (1996) Tissue distribution of messenger ribonucleic acid encoding the rat glucagon-like peptide-1 receptor. Endocrinology 137, 2968–78. (41) Orskov, C., and Poulsen, S. S. (1991) Glucagonlike peptideI-(7 36)-amide receptors only in islets of Langerhans. Autoradiographic survey of extracerebral tissues in rats. Diabetes 40, 1292–6. (42) Ruiz-Grande, C., Alarcon, C., Alcantara, A., Castilla, C., Lopez Novoa, J. M., Villanueva-Penacarrillo, M. L., and Valverde, I. (1993) Renal catabolism of truncated glucagon-like peptide 1. Horm. Metab. Res. 25, 612–6. (43) Hassan, M., Eskilsson, A., Nilsson, C., Jonsson, C., Jacobsson, H., Refai, E., Larsson, S., and Efendic, S. (1999) In vivo dynamic distribution of 131I-glucagon-like peptide-1 (7 36) amide in the rat studied by gamma camera. Nucl. Med. Biol. 26, 413–20. (44) Gotthardt, M., van Eerd-Vismale, J., Oyen, W. J., de Jong, M., Zhang, H., Rolleman, E., Maecke, H. R., Behe, M., and Boerman, O. (2007) Indication for different mechanisms of kidney uptake of radiolabeled peptides. J. Nucl. Med. 48, 596–601. (45) Boswell, C. A., Sun, X., Niu, W., Weisman, G. R., Wong, E. H., Rheingold, A. L., and Anderson, C. J. (2004) Comparative in vivo stability of copper-64-labeled cross-bridged and conventional tetraazamacrocyclic complexes. J. Med. Chem. 47, 1465–74. (46) Bass, L. A., Wang, M., Welch, M. J., and Anderson, C. J. (2000) In vivo transchelation of copper-64 from TETA-octreotide to superoxide dismutase in rat liver. Bioconjugate Chem. 11, 527–32. (47) Rogers, B. E., Anderson, C. J., Connett, J. M., Guo, L. W., Edwards, W. B., Sherman, E. L., Zinn, K. R., and Welch, M. J. (1996)

ARTICLE

Comparison of four bifunctional chelates for radiolabeling monoclonal antibodies with copper radioisotopes: biodistribution and metabolism. Bioconjugate Chem. 7, 511–22. (48) Anderson, C. J., and Ferdani, R. (2009) Copper-64 radiopharmaceuticals for PET imaging of cancer: advances in preclinical and clinical research. Cancer Biother. Radiopharm. 24, 379–93.

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