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Development of 99mTc-Labeled Pyridyl Benzofuran Derivatives To Detect Pancreatic Amylin in Islet Amyloid Model Mice Masashi Yoshimura,† Masahiro Ono,*,† Hiroyuki Watanabe,† Hiroyuki Kimura,† and Hideo Saji† †

Department of Patho-Functional Bioanalysis, Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29 Yoshida Shimoadachi-cho, Sakyo-ku, Kyoto 606-8501, Japan S Supporting Information *

ABSTRACT: While islet amyloid deposition comprising amylin is one of pathological hallmarks of type 2 diabetes mellitus (T2DM), no useful amylin-imaging probe has been reported. In this study, we evaluated two 99mTc-labeled pyridyl benzofuran derivatives as novel amylin-imaging probes using the newly established islet amyloid model mouse. Binding experiments in vitro demonstrated that [99mTc]1 displayed a higher affinity for amylin aggregates than [99mTc]2. Autoradiographic studies using human pancreas sections with T2DM revealed that [99mTc]1 clearly labeled islet amyloid in T2DM pancreatic sections, while [99mTc]2 did not. Although the initial uptake of [99mTc]1 by the normal mouse pancreas was low (0.74%ID/g at 2 min post-injection), [99mTc]1 showed higher retention in the model mouse pancreas than that of the normal mouse, and exhibited strong binding to amylin aggregates in the living pancreas of the model mice. These results suggest that [99mTc]1 is a potential imaging probe targeting islet amyloids in the T2DM pancreas.

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INTRODUCTION Diabetes mellitus is one of the most common metabolic disorders characterized by chronic hyperglycemia resulting from defects in insulin secretion, insulin action, or both.1 Owing to changes in lifestyle and dietary habits, the number of the diabetes mellitus patients has been increasing at an alarming rate. The WHO estimated in 2014 that the global prevalence of diabetes was 9% among adults, and it predicts that diabetes will be the seventh leading cause of death in 2030.2 Of note, 90− 95% of patients with diabetes have type 2 diabetes mellitus (T2DM), formerly called non-insulin-dependent diabetes mellitus.1 Even now, the etiology regarding T2DM remains unclear and there is no curative treatment for it. Since T2DM is a progressive disease, early phase therapeutic intervention is essential. However, the current diagnosis of T2DM depends on the blood glucose level, and glucose tolerance means that we are unable to detect T2DM in the early phase.3 Therefore, a novel method for the early diagnosis of T2DM is strongly desired. T2DM is characterized by a progressive decline of the pancreatic β-cell mass (BCM), which is responsible for insulin secretion failure and hyperglycemia.1 In T2DM patients, the BCM can be 40−60% smaller than in healthy controls.4−6 Many radioactive imaging probes targeting pancreatic β-cells have been developed to assess the BCM, such as 11C-DTBZ7,8 and 18F-labeled DTBZ analogues,9−11 18F-FBT,12,13 and 111Inexendin.14 β-Cell imaging can give us critical information on the progression of T2DM. However, β-cell imaging may be unsuitable for evaluation of the severity and staging in the early © XXXX American Chemical Society

phase of T2DM, since the BCM is considered to increase temporarily as an adaptation to increased insulin demand at that time;15,16 that is, the BCM is not reduced steadily according to the T2DM stage. Previous research on BCM expansion was extensively performed in rodents. In both the adult rat and mouse, β-cell replication is induced with a high-fat diet or multiple-day glucose infusion.17 In humans, a BCM increase was reported under insulin-resistant conditions, such as pregnancy and obesity.18 According to these findings, BCM augmentation may compensate for the increased acute glucosestimulated insulin secretion in the earliest stage of T2DM. Therefore, we believe that a new in vivo imaging tracer targeting a biomarker except pancreatic β-cells is needed for the early phase diagnosis of T2DM. The deposition of islet amyloid constitutes one of the marked morphological changes in T2DM pancreatic islets. Islet amyloid is found in more than 90% of T2DM patients at autopsy.19 Islet amyloid is mainly composed of islet amyloid polypeptide, also known as amylin, a 37-amino-acid polypeptide co-expressed and co-secreted by pancreatic β-cells along with insulin.20 The degree of islet amyloid deposition has been reported to be correlated with the disease severity, a BCM decrease, and the development of hyperglycemia.21 Therefore, islet amyloid deposition is considered to have a close relationship with the development and progression of Received: April 4, 2016 Revised: May 9, 2016

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DOI: 10.1021/acs.bioconjchem.6b00174 Bioconjugate Chem. XXXX, XXX, XXX−XXX

Bioconjugate Chemistry

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T2DM.21 Based on this hypothesis, many amylin aggregation inhibitors have been developed,22−24 and such inhibitors could improve glucose homeostasis in diabetes model mice.25 From the above, we hypothesized that amylin may be a new potential surrogate biomarker for T2DM, and we believe that amylin imaging in vivo can facilitate a better understanding of the pathophysiology of T2DM and provide important information regarding the development of drugs targeting islet amyloid and the therapeutic outcomes. We previously reported that the 125I-labeled pyridyl benzofuran (PBF) derivative [125I]IPBF could have potential as an amylin-imaging probe under in vitro conditions.26 In this study, we designed novel 99mTc-labeled PBF derivatives to develop more useful single photon emission computed tomography (SPECT) probes for in vivo imaging. 99mTc (t1/2 = 6.01 h, 141 keV) is the most commonly used radionuclide in nuclear medicine with SPECT since it is readily produced by a 99 Mo/99mTc generator and is essentially available at any time. 99m Tc-labeled imaging agents will provide convenient and costeffective diagnostic methods with SPECT. Different from 125I, a chelating structure is necessary for metal 99mTc binding to organic molecules. PBF scaffold as the binding moiety for islet amyloid is connected to the 99mTc-chelating moiety via triethylene glycol as a spacer. We chose two bifunctional cheating ligands, iminodiacetic acid (IDA) and N,N-dipicorylamine (DPA), which can easily form 99mTc tricarbonyl complexes.27 Two 99mTc-labeled PBF derivatives, [99mTc]1 and [99mTc]2 (Figure 1), were synthesized and evaluated as

RESULTS Radiolabeling of [99mTc]1 and [99mTc]2. The radiosynthesis of 99mTc-labeled tricarbonyl complexes is shown in Figure 2. The [99mTc(CO)3(H2O)3]+ core was prepared using Isolink carbonyl kit vials (Paul Scherrer Institute, Villigen, Swizerland) and subsequently used for the preparation of 99m Tc(CO)3 complexes. After adjustment of the pH of the solution (pH 7), the 99mTc-labeled products were obtained under microwave heating at 110 °C for 5 min in the sealed vials with radiochemical yields of 73.5% and 54.3%, respectively. The identities of the 99mTc-labeled tracers were verified by comparative HPLC using the corresponding Re complexes (Figure S1). As there is no stable technetium isotope, rhenium, the congener of technetium, has been widely used as a nonradioactive surrogate for the structural identification of technetium complexes. [99mTc]1 Displayed Higher Affinity for Amylin Aggregates than [99mTc]2. To evaluate the binding properties of [99mTc]1 and [99mTc]2 for islet amyloids, we carried out in vitro binding experiments using amylin aggregates. Figure 3

99m

Figure 3. Binding of [99mTc]1 and [99mTc]2 with amylin aggregates. Values are the mean ± standard error of three independent determinations.

Figure 1. Chemical structure of

Tc-labeled PBF derivatives.

amylin-imaging probes. IDA and DPA have different characteristics, especially regarding their size and net charge,27 so that we can compare the effect of these chelates on the binding affinity for islet amyloid. In the present study, we evaluated [99mTc]1 and [99mTc]2 as amylin-imaging probes not only by in vitro experiments but also by ex vivo experiments using islet amyloid model mice, established by the novel method of orthotopic implantation of amylin aggregates. This is the first study on the development of amylin-imaging probes using islet amyloid model mice.

shows binding properties as amylin aggregate-bound radioactivity (%) at different concentrations of amylin. The percent radioactivity of [99mTc]1 and [99mTc]2 bound to amylin increased dependent on the concentration of aggregates, indicating that both compounds bound to the amylin aggregates. To compare the affinities of [99mTc]1 and [99mTc]2, a competition binding assay was conducted using [125I]IPBF as a radioligand. The results are shown in Table 1. We previously

Figure 2. Radiolabeling of [99mTc]1 and [99mTc]2. B


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Bioconjugate Chemistry Table 1. Inhibition Study with [125I]IPBF compound

Kia (nM)

Re-1 Re-2 IPBFb

146 ± 47.5 3430 ± 447 2.66 ± 0.554

a Values are expressed as the means ± standard error of three independent determinations. bReprinted from ref 26.

Figure 5. Stability in the mouse plasma.

reported that IPBF displayed high binding affinity for amylin aggregates in this assay. In the present study, Re-1 displayed moderate binding affinity (Ki = 146 nM) while Re-2 showed very low affinity (Ki = 3430 nM). [99mTc]1 Demonstrates Binding to Islet Amyloid in Human Pancreas Sections, But Not [99mTc]2. To confirm the affinity for islet amyloid in the human pancreas, in vitro autoradiography experiments with [99mTc]1 and [99mTc]2 were carried out using pancreatic sections from a T2DM patient and a healthy adult. An autoradiographic image of [99mTc]1 showed the intensive accumulation of radioactivity in the T2DM pancreas (Figure 4a). The accumulation corresponded with the

Biodistribution in Normal Mice. To evaluate the pharmacokinetics of [99mTc]1 and [99mTc]2, biodistribution experiments were performed in normal mice (Figure 6, Tables

Figure 6. Biodistribution in normal mice. *Expressed as %ID/organ.

S1 and S2). [99mTc]1 and [99mTc]2 exhibited a low initial uptake by the mouse pancreas (0.74%ID/g and 1.37%ID/g at 2 min post-injection, respectively), which is the target organ. [99mTc]1 intensively accumulated in the liver at the initial timepoint (62.1%ID/g at 2 min post-injection), whereas there was a sequential shift to the intestine over time. [99mTc]2 also displayed a high initial uptake by the liver (35.2%ID/g at 2 min post-injection), but it was also excreted by the kidney (13.5% ID/g at 2 min post-injection). Novel Mouse Model with Amylin Aggregates Transplanted into the Pancreas. To assess the binding affinity to amylin aggregates in the living pancreas tissue, we constructed a novel islet amyloid mouse model. Amylin aggregates were transplanted directly into the nude mouse pancreas. To validate this nude mouse model, we investigated fluorescent staining with ThS and immunohistochemical staining with an antibody against human amylin peptide. ThS staining revealed that amylin aggregates injected into the mouse pancreas were retained (Figure 7a). In the immunohistochemical staining with anti-amylin antibody, the immunoreactive signal of amylin was consistent with ThS staining, demonstrating that the amyloid was made up of the transplanted amylin peptide (Figure 7b). [99mTc]1 Accumulated in the Pancreas of Islet Amyloid Model Mice. To evaluate the binding affinity of [99mTc]1 for amylin aggregates in vivo, biodistribution studies

Figure 4. In vitro autoradiography. (a) Autoradiogram of [99mTc]1 using pancreatic sections from a T2DM patient. (d) Autoradiogram of [99mTc]1 using a healthy adult. (g) Autoradiogram of [99mTc]2 using pancreatic sections from a T2DM patient. (b,e,h) Thioflavin-S staining with the same sections, respectively, indicating the presence of islet amyloid depositions. (c,f,i) Immunohistochemical staining with an anti-amylin antibody of adjacent sections, respectively.

results of the amyloid-specific dye thioflavin-S (ThS) staining using the same section (Figure 4b) and immunohistochemical staining with an anti-amylin antibody using an adjacent section (Figure 4c). Furthermore, [99mTc]1 showed almost no accumulation in the healthy adult pancreas (Figure 4d). On the other hand, [99mTc]2 did not show any marked labeling of islet amyloid in the T2DM pancreatic sections (Figure 4g). [99mTc]1 and [99mTc]2 Were Stable in the Mouse Plasma. We investigated the stability of [99mTc]1 and [99mTc]2 in the mouse plasma. Since each compound generated no degradation products after 2 h incubation, they were sufficiently stable in the mouse plasma (Figure 5). C


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Figure 7. Orthotopic amylin aggregate implantation. (a) Thioflavin-S staining with a pancreatic section from a mouse with transplanted amylin aggregates. (b) Immunohistochemical staining with an antiamylin antibody of an adjacent section. These data indicate that transplanted amylin aggregates are maintained in the mouse pancreas.

were carried out using the nude mouse model with amylin aggregates transplanted into the pancreas and control mice which received Tris-NaCl buffer without amylin peptide, and then the radioactivity of the pancreas of each group was compared. Whereas there was no significant difference between islet amyloid model and control mice, [99mTc]1 displayed a 1.6fold higher accumulation in the pancreas of the model mice than that of the control mice at 1 h post-injection (Figure 8, 0.470%ID/g vs 0.296%ID/g, respectively).

Figure 9. Ex vivo autoradiography with the islet amyloid model and control mice. (a,e) Autoradiograms of [99mTc]1 and [99mTc]2 using the model mice with amylin aggregates transplanted into the pancreas, respectively. (c) Autoradiogram of [99mTc]1 using the control mouse. (b,d,f) Thioflavin-S staining with the same sections, respectively.

pathogenesis is comparable with that of other diseases such as Alzheimer’s or Parkinson’s disease.28 The development of a useful amylin-imaging probe will promote research on islet amyloid in T2DM and provide valuable in vivo information. Although both [99mTc]1 and [99mTc]2 displayed binding affinity for amylin aggregates in binding experiments, the level of radioactivity which bound to amylin aggregates was markedly different (Figure 3). Since the binding radioactivity of [99mTc]1 was almost two times greater than that of [99mTc]2, the binding affinity of [99mTc]1 should be much higher than [99mTc]2. Furthermore, we carried out inhibition assay using each Re complex as a nonradioactive surrogate of 99mTc complexes. Re1 displayed more than 20-fold higher affinity than Re-2 in binding inhibition experiments (Table 1). These differences in the binding affinity may be caused by the size of metaltricarbonyl complexes. Since the DPA (the chelate part of [99mTc]2 and Re-2) is bulkier than IDA (the chelate part of [99mTc]1 and Re-1), the steric hindrance of [99mTc]2 and Re-2 may crucially decrease the affinity for amylin aggregates. Although IDA is the compact core of metal-tricarbonyl complexes, Re-1 displayed moderate binding affinity (146 nM) while IPBF showed high affinity (2.66 nM). This indicated that even the compact IDA core markedly interfered with binding to amylin. The chelates are structurally distant from the pyridyl benzofuran serving as a binding moiety; however, they had a marked influence on the affinity. Several studies revealed that the steric hindrance of the metal-chelate complexes disturbed binding to amyloid aggregates, and a longer linker between chelates and binding moieties reduced the influence of the chelate.29−31 Moreover, IDA is well-known to form negatively charged complexes with 99mTc while DPA forms positively charged complexes.27 Therefore, the difference in the net charge between both 99mTc complexes may affect the binding affinity for amylin aggregates. The results obtained from the present study suggest the strong effect of bulky chelates on affinity. These results on the binding affinity may be meaningful for the molecular design of amyloid probes conjugated with radioactive metal in the future. In vitro autoradiography experiments demonstrated that [99mTc]1 specifically binds to islet amyloid in the T2DM pancreatic tissue section, while little nonspecific accumulation

Figure 8. [99mTc]1 accumulation in the pancreas of islet amyloid model and control mice at 1 h post-injection.

[99mTc]1 Shows Binding to the Transplanted Amylin Aggregates in the Living Mouse Pancreas. To further characterize [99mTc]1 as an amylin imaging probe, ex vivo autoradiography studies using islet amyloid model and control mice were performed. The pancreas was removed at 30 min after the injection of [99mTc]1 for autoradiography. Autoradiographic analysis revealed the intensive accumulation of [99mTc] 1 at the site of amyloid in the transplanted model mice (Figure 9a), but not in control mice (Figure 9c). The same pancreatic sections were stained with ThS, and the localizations of transplanted amyloid corresponded with the accumulation of radioactivity (Figure 9b), while there was no marked staining in the sections of control mice (Figure 9d). However, both showed moderate radioactivity accumulation collided with areas of ThS staining, indicating nonspecific binding. Conversely, an autoradiographic image of [99mTc]2 obtained using the same method of [99mTc]1 revealed little accumulation of radioactivity at sites of transplanted islet amyloids (Figure 9e).

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DISCUSSION Although T2DM is often considered one of the protein misfolding disorders, a group of diseases characterized by the accumulation of abnormally folded proteins, few studies have investigated the involvement of islet amyloid in the T2DM pathogenesis. The subject has been mostly neglected in the diabetes field despite the fact that the evidence implicating the accumulation of misfolded protein aggregates in T2DM D


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the novel model mouse with transplanted amylin aggregates should help accelerate the development of amylin-imaging probes. In conclusion, we developed a novel amylin-imaging probe and conducted its in vivo evaluation using islet amyloid model mice. As already mentioned, [99mTc]1 showed marked labeling of islet amyloid in the model mouse pancreas. This is the first report showing that amylin-imaging probe binds to amylin aggregates in the living mouse pancreas. Although the pharmacokinetics should be improved for in vivo imaging, [99mTc]1 is a potentially useful tracer targeting amylin. The results obtained in the study using islet amyloid model mice suggest the feasibility of in vivo amylin-imaging in the future.

was observed in the healthy pancreas tissue (Figure 4a,d). On the other hand, [99mTc]2 did not display complete binding to islet amyloids (Figure 4g). Furthermore, in ex vivo autoradiography experiments using the nude mouse model with amylin aggregates transplanted into the pancreas, [99mTc]1 clearly labeled the aggregates in the living pancreas, although [99mTc]2 only weakly labeled them (Figure 9). These results reflect the ones obtained in the direct binding and binding inhibition experiments. Both [99mTc]1 and [99mTc]2 were stable for 2 h in mouse plasma (Figure 5). Therefore, these findings suggest that the difference in binding characteristics was caused by the binding affinity for amylin aggregates, and not by the stability. Although [99mTc]1 showed almost no nonspecific binding to normal pancreas tissue based on in vitro autoradiography (Figure 4d), some nonspecific binding was observed in ex vivo autoradiography (Figure 9c), suggesting that [99mTc]1 bound to normal tissue in the living pancreas more easily than to pancreatic tissue sections. The reason that this phenomenon occurred cannot be clearly explained at this time. A high initial uptake by the target organ is generally required for in vivo imaging; however, [99mTc]1 showed very low uptake by the pancreas in normal mice (0.74%ID/g at 2 min postinjection) (Figure 6). Despite the disadvantage of the pharmacokinetics, [99mTc]1 showed a 1.6-fold higher accumulation in the pancreas of the model mice than that of the control mice (Figure 8), and the localization of the radioactivity in the model mouse pancreas corresponded with implanted amylin aggregates (Figure 9). These results reveal that [99mTc]1 has a sufficient potential to function as an amylin-imaging probe. Moreover, it is also important that amylin-imaging probes do not accumulate in organs located near the pancreas. However, [99mTc]1 showed high initial uptake by the liver, and sequential accumulation in the intestine (Figure 6). In general, small and highly lipophilic molecules are prone to excretion mainly by the liver and then they shift to the intestine. Since the LogP value of [99mTc]1 was moderate (LogP 1.74), and a more lipophilic molecule, [99mTc]2 (LogP 2.18), displayed modest uptake by the liver, the high [99mTc]1 uptake by the liver was possibly caused by the character of the IDA core. Several imaging probes with an IDA core reported previously showed high uptake by the liver despite their low-to-moderate lipophilicity.32,33 Although [99mTc]1 had a high enough affinity to bind strongly to amylin aggregates in the living pancreas, the characteristics of biodistribution may make it difficult to visualize islet amyloids in vivo. This is the first attempt to evaluate the binding characteristics in vivo of imaging probes targeting islet amyloid deposition. In terms of the evaluation of amyloid-imaging agents, almost all previous studies were performed with transgenic animals, such as tg257634,35 and P301S36 for amyloid-β and tau aggregates, respectively. Of course, transgenic animals expressing human amylin peptide should reflect T2DM pathogenesis more closely; however, amyloid dye-positive plaque was observed at over 10 months old.37,38 The late-onset islet amyloid-forming model mice are not suitable for the in vivo screening of amyloid ligand since long-term bleeding is inevitable. On the other hand, our novel islet amyloid model mouse was easy to prepare and ready to use (Figure 7). Moreover, the high to moderate affinity tracer ([99mTc]1) clearly displayed the labeling of amylin aggregates in the model mouse pancreas, and vice versa, indicating that evaluation using the novel model mouse could reflect the in vitro binding affinity for amylin. For these reasons,

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EXPERIMENTAL PROCEDURES In Vitro Binding Experiments. Preparation of amylin aggregates was carried out according to the methods reported previously (ref 26). A mixture containing 50 μL of [99mTc]1 or [99mTc]2 (4.8 kBq), 50 μL of amylin aggregates (final conc. 6.25−100 nM), 50 μL of DMSO, and 850 μL of PBS(−) containing 10% DMSO and 0.1% BSA, was incubated at room temperature for 3 h. The mixture was then filtered through GF/ B filters (Whatman, Maidstone, Kent, UK) using an M-24 cell harvester (Brandel Inc., Gauthersburg, MD, USA), and the radioactivity of the filters containing the bound 99mTc ligand were measured in a γ counter (Wallac 1470 Wizard; PerkinElmer, Boston, MA, U.S.A.). In Vitro Binding Inhibition. The same method previously reported in ref 26 was used. In Vitro Autoradiography Using Human Pancreas Sections. Paraffin-embedded pancreas tissue sections from a T2DM patient (69-year-old woman) and a healthy adult (28year-old man) were purchased from BioChain Institute Inc. (Hayward, CA, USA). The sections were subjected to two 15 min incubations in xylene, two 1 min incubations in 100% EtOH, two 1 min incubations in 90% EtOH, and one 1 min incubation in 70% EtOH to completely deparaffinize them, followed by two 2.5 min washes in water. The sections were incubated with [99mTc]1 or [99mTc]2 (555 kBq/1 mL) for 1 h at room temperature. They were then washed with water (two 1 min washes). After drying, the 99mTc-labeled sections were exposed to a BAS imaging plate (Fuji Film, Tokyo, Japan) for 1 h. Autoradiographic images were obtained using a BAS5000 scanner system (Fuji Film). ThS Staining of Islet Amyloids in Human Pancreas Sections. ThS staining was carried out after the autoradiographic study using the same pancreas tissue sections. The sections were immersed in 100 μM ThS solution containing 50% EtOH and washed with water. After drying, the sections were observed with the Keyence system (excitation filter, 450− 490 nm; emission filter, 510−560 nm; DM filter; 495 nm). This experiment was performed under a fluorescence microscope (BIOREVO BZ-9000, Keyence Corp., Osaka, Japan). Immunohistochemical Staining Using Human Pancreas Sections. Paraffin-embedded pancreas tissue sections from a T2DM patient (69-year-old woman) and a healthy adult (28-year-old man) were purchased from BioChain Institute Inc. After deparaffinization of the sections according to the same method as described in the in vitro autoradiography experiments, they were then autoclaved for 15 min in 0.01 M citric acid buffer (pH 6.0) for antigen retrieval, followed by two 5 min incubations in PBS-Tween20. The sections were incubated with rabbit polyclonal amylin primary antibody (1:300; E


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was calculated by comparing the sample counts with the count of the diluted initial dose. Immunohistochemical Staining Using Pancreas Sections from Mice with Transplanted Amylin Aggregates. Mice were sacrificed by decapitation and then the pancreas was immediately removed, embedded in Super Cryoembedding Medium (SCEM) compound (SECTION-LAB Co. Ltd., Hiroshima, Japan), and then frozen in a dry ice/hexane bath. Frozen sections were prepared at a 30 μm thickness. Immunohistochemical staining using mouse pancreas sections was performed according to the same method as described in the Immunohistochemical Staining Using Human Pancreas sections except for the washing conditions (PBS was used instead of PBS-Tween20 and the wash time was 3 instead of 5 min). Immunohistochemical staining using mouse pancreas sections was carried out with mouse monoclonal amylin primary antibody (1:200; R10/99, GeneTex Inc., San Antonio, TX, USA). Ex Vivo Autoradiography Using Mice with Amylin Aggregates Transplanted into the Pancreas. A saline solution (200 μL) of [99mTc]1 (66.6 MBq) containing ethanol (20 μL) was injected through the tail vein. The animals were sacrificed by decapitation at 30 min post-injection. Frozen pancreatic tissue sections were prepared according to the same method as described in immunohistochemical staining using mouse pancreas sections. The sections were exposed to a BAS imaging plate (Fuji Film) overnight. Autoradiographic images were obtained using a BAS5000 scanner system (Fuji Film). After autoradiographic examination, the same sections were stained with ThS to confirm the presence of amylin aggregates. Statistics. The Mann−Whitney U test was used to assess statistical differences in the evaluation of [99mTc]1 accumulation in the pancreas of islet amyloid model and control mice. Differences at the 95% confidence level (P < 0.05) were considered significant.

Peninsula Laboratories, Belmont, CA, USA) overnight. After three 5 min incubations in PBS-Tween20, they were incubated with biotinylated goat anti-rabbit IgG (Vectastain Elite kit, Vector Laboratories, Burlingame, CA, USA) at room temperature for 1.5 h. After three 5 min incubations in PBS-Tween20, the sections were incubated with the Streptavidin-Peroxidase complex at room temperature for 1 h. After two 5 min incubations in PBS-Tween20 and one 5 min incubation in TBS, they were incubated with DAB as a chromogen for 5 min. After washing with water, the sections were observed under a microscope (BIOREVO BZ-9000; Keyence Corp.). In Vitro Stability in Mouse Plasma. ddY mice (5 weeks old, male) were anesthetized with isoflurane. Blood samples were collected from the hearts and centrifuged at 1000 g for 30 min. The supernatant was collected and stored at −80 °C until use. Then, saline solution (50 μL) of [99mTc]1 or [99mTc]2 containing EtOH (5 μL) was added to 250 μL of mouse plasma. The sample was incubated at 37 °C for 2 h. After incubation, 600 μL of MeOH was added to the sample, and it was centrifuged at 10 000 g for 5 min. The supernatant was collected and filtered with a Cosmonice Filter (S) (0.45 μm, 4 mm) (Nacalai Tesque, Kyoto, Japan), and the filtrate was analyzed by HPLC. In Vivo Biodistribution in Normal Mice. A saline solution (100 μL) of [99mTc]1 or [99mTc]2 (15.1 or 20.2 kBq) containing ethanol (10 μL) was directly injected intravenously into the tail of ddY mice (5 weeks old, male). The mice (n = 5 for each time-point) were sacrificed at 2, 10, 30, and 60 min post-injection. The organs of interest were removed and weighed, and the radioactivity was measured with a γ counter (Wallac 1480 Wizard 3 or Wallac 1470 Wizard; PerkinElmer, Boston, MA, USA). The percentage dose per organ was calculated by a comparison of the tissue counts to suitably diluted aliquots of the injected material. The %dose/g of samples was calculated by comparing the sample counts with the count of the diluted initial dose. Orthotopic Amylin Aggregates for Implantation. A solid form of amylin was purchased from the Peptide Institute (Osaka, Japan). The amylin aggregates were prepared by diluting the peptide solution in DMSO (10 μL) (containing 0.55 mg of peptides) with 20 mM Tris/HCl, 100 mM NaCl, pH 7.5 (90 μL). Orthotopic amylin aggregate xenografts were established in BALB/c-nu/nu mice (8 to 10 weeks old, male) with a surgical method. The tail of the pancreas was delivered through a nearly 10 mm transverse incision made on the left flank of the mouse. Fifty microliters of the aggregate suspension was injected into two to three parts of the pancreas with 29gauge needle syringes. Control mice received the same volume of solvent (mixture of 5 μL of DMSO and 45 μL of 20 mM Tris/HCl). Upon completion, the pancreas was returned to the abdomen and the incision was closed using silk nonabsorbable sutures. Immunohistochemical staining, a biodistribution study, and ex vivo autoradiography using mice with amylin aggregates transplanted into the pancreas were performed on the day following orthotopic amylin aggregate implantation. Evaluation of [99mTc]1 Accumulation in the Pancreas of Islet Amyloid Model and Control Mice. A saline solution (100 μL) of [99mTc]1 (26.1 kBq) containing ethanol (10 μL) was directly injected intravenously into the tail of islet amyloid model and control mice. The mice (n = 5 for each group) were sacrificed at 1 h post-injection. The pancreas was removed and weighed, and the radioactivity was measured with a γ counter (Wallac 1470 Wizard; PerkinElmer). The %dose/g of samples

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.6b00174. Experimental procedures. Synthesis of precursors for radiolabeling and Re complexes. Detailed method for radiolabeling. Detailed method for measurement of LogP values. Figure S1. Representative HPLC profiles of [99mTc]1, [99mTc]2, Re-1, and Re-2. Table S1. In vivo biodistribution of [99mTc]1 in normal mice. Table S2. In vivo biodistribution of [99mTc]2 in normal mice. (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +81-75-753-4608. Fax: +81-75-753-4568. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work supported by a grant from the Japan Society for the Promotion of Science (JSPS) through the “Funding Program for Next Generation World-Leading Researchers (LS060)”, initiated by the Council for Science and Technology Policy F


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(CSTP), and JSPS Research Fellowships for Young Scientists (14J05961).

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ABBREVIATIONS T2DM, type 2 diabetes mellitus; BCM, β-cell mass; SPECT, single photon emission computed tomography; IDA, iminodiacetic acid, DPA, N,N-dipicorylamine; ThS, thioflavin-S; DMSO, dimethyl sulfoxide; PBS, phosphate buffered saline; DAB, 3,3′-diaminobenzidine; TBS, tris-buffered saline

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