Improving the Theranostic Potential of Exendin 4 ... - ACS Publications

Article Cite This: Bioconjugate Chem. 2019, 30, 1745−1753

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Improving the Theranostic Potential of Exendin 4 by Reducing the Renal Radioactivity through Brush Border Membrane EnzymeMediated Degradation Mingru Zhang,†,‡ Orit Jacobson,‡ Dale O. Kiesewetter,‡ Ying Ma,‡ Zhantong Wang,‡ Lixin Lang,‡ Longguang Tang,‡ Fei Kang,†,‡ Hongzhang Deng,‡ Weijing Yang,‡ Gang Niu,‡ Jing Wang,*,† and Xiaoyuan Chen*,‡ Downloaded via UNIV FRANKFURT on July 22, 2019 at 07:00:15 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

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Department of Nuclear Medicine, Xijing Hospital, Fourth Military Medical University, Xi’an, Shannxi 710032, China Laboratory of Molecular Imaging and Nanomedicine, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, Maryland 20892, United States

‡

S Supporting Information *

ABSTRACT: As highly expressed in insulinomas, the glucagon-like peptide-1 receptor (GLP-1R) is believed to be an attractive target for diagnosis, localization, and treatment with radiolabeled exendin 4. However, the high and persistent radioactivity accumulation of exendin 4 in the kidneys limits accurate diagnosis and safe, as well as effective, radiotherapy in insulinomas. In this study, we intend to reduce the renal accumulation of radiolabeled exendin 4 through degradation mediated by brush border membrane enzymes. A new exendin 4 ligand NOTA-MVK-Cys40-Leu14-Exendin 4 containing MetVal-Lys (MVK) linker between the peptide and 1,4,7triazacyclononane-1,4,7-triacetic acid (NOTA) chelator was synthesized and labeled with 68Ga. The in vitro mouse serum stability and cell binding affinity of the tracer were evaluated. Initial in vitro cleavage of the linker was determined by incubation of a model compound Boc-MVK-Dde with brush border membrane vesicles (BBMVs) with and without the inhibitor of neutral endopeptidase (NEP). Further cleavage studies were performed with the full structure of NOTA-MVK-Cys40-Leu14-Exendin 4. Kidney and urine samples were collected in the in vivo metabolism study after intravenous injection of 68Ga-NOTA-MVKCys40-Leu14-Exendin 4. The microPET images were acquired in INS-1 tumor model at different time points; the radioactivity uptake of 68Ga-NOTA-MVK-Cys40-Leu14-Exendin 4 in tumor and kidneys were determined and compared with the control radiotracer without MVK linker. 68Ga-NOTA-MVK-Cys40-Leu14-Exendin 4 was stable in mouse serum. The MVK modification did not affect the affinity of NOTA-MVK-Cys40-Leu14-Exendin 4 toward GLP-1R. The in vitro cleavage study and in vivo metabolism study confirmed that the MVK sequence can be recognized by BBM enzymes and cleaved at the amide bond between Met and Val, thus releasing the small fragment containing Met. MicroPET images showed that the tumor uptake of 68 Ga-NOTA-MVK-Cys40-Leu14-Exendin 4 was comparable to that of the control, while the kidney uptake was significantly reduced. As a result, more favorable tumor to kidney ratios were achieved. In this study, a novel exendin 4 analogue, NOTAMVK-Cys40-Leu14-Exendin 4, was successfully synthesized and labeled with 68Ga. With the cleavable MVK sequence, this ligand could be cleaved by the enzymes on kidneys, and releasing the fragment of 68Ga-NOTA-Met-OH, which will rapidly excrete from urine. As the high and consistent renal radioactivity accumulation could be significantly reduced, NOTA-MVK-Cys40Leu14-Exendin 4 shows great potential in the diagnosis and radiotherapy for insulinoma.

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not suitable for surgery.4−6 Glucagon-like peptide-1 receptor (GLP-1R) is overexpressed at a high incidence and density in insulinomas.7 A prospective cohort study showed that GLP-1R targeting with 68Ga-NOTA-Exendin 4 has a much higher sensitivity than SSTR-2 targeting (97.7% vs 19.5%),7 making it an attractive target for imaging and therapy of the tumors.8−10

INTRODUCTION Insulinomas, characterized by uncontrolled insulin secretion, are rare pancreatic neuroendocrine tumors (pNETs) with an incidence rate of approximately 1 in 250,000 people per year.1 Although about 90% of insulinomas are benign and can be surgically cured, the surgical removal is hampered by difficulties in locating it, since approximately 82% of lesions are less than 2 cm in diameter and 47% are under 1 cm.2,3 There is still no efficacious treatment for the remaining 10% malignant insulinomas which have multiple lesions or metastases that are © 2019 American Chemical Society

Received: April 16, 2019 Revised: May 18, 2019 Published: May 23, 2019 1745

DOI: 10.1021/acs.bioconjchem.9b00280 Bioconjugate Chem. 2019, 30, 1745−1753

Article

Bioconjugate Chemistry

Figure 1. Structures of (A) NOTA-Cys40-Leu14-Exendin 4 and (B) NOTA-MVK-Cys40-Leu14-Exendin 4, where R represents (C)−S∼∼Cys40-Leu14Exendin 4.

without impairing the tumor uptake. Compared with the control, the antibody fragment with MVK linker showed decreased kidney uptake by 62% and 80% at 1 and 3 h p.i., respectively.35 Inspired by this striking result, we introduced this MVK linker to exendin 4 with the intent to improve its theranostic potential. In this study, NOTA-MVK-Cys40-Leu14-Exendin 4 was synthesized and labeled with 68Ga. The recognition and cleavage of the MVK sequence by the enzymes on renal brush border membrane (BBM) was evaluated both in vitro and in vivo. In vivo biodistribution and pharmacokinetics of 68Ga-NOTA-MVKCys40-Leu14-Exendin 4 in tumor and kidneys were evaluated by PET studies.

However, the high and persistent kidney accumulation remains a challenge for the application of GLP-1R agonists.11,12 First, as the tail of the pancreas is close to the left kidney, the high signal from the left kidney may hamper the accurate localization of the lesions.7,13,14 Second, the high kidney accumulation limits the dosage and number of cycles of peptide receptor radionuclide therapy (PRRT).15−17 The mechanism underlying the undesirable renal radioactivity accumulation is the long residence time of radiometabolite(s) generated after lysosomal proteolysis of radiolabeled ligands, following glomerular filtration and subsequent reabsorption in renal cells.18−20 Co-infusion with lysine and arginine has been a clinical routine to protect the kidneys during PRRT. Approximately 40% reduction of renal radioactivity was observed by this method.21−24 However, co-infused amino acids (AA) can cause adverse effects such as vomiting, nausea, and hyperkalemia.25−27 Recently, co-infusion of the plasma expander Gelofusine was described to achieve a similar reduction of renal radioactivity.17,28,29 However, a transient proteinuria was induced probably by disturbance of the effective megalin-mediated reabsorption processes in the kidneys.30,31 Despite great efforts made to reduce the renal radioactivity, further safe ways to reduce the kidney accumulation from early post-injection time are still needed. A strategy reported by Arano et al. using a degradable linker that can be cleaved by renal brush border membrane (BBM) enzymes, especially the one called neutral endopeptidase (NEP), which is abundantly expressed in BBM. The cleavage results in the formation of the metal chelate conjugated to a single amino acid, which is expected to rapidly clear into the urine.32−37 Among all the linkers studied, Met-Val-Lys (MVK) had the best performance on reducing the renal radioactivity

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RESULTS Chemistry and Radiochemistry. NOTA-Cys40-Leu14Exendin 4 and NOTA-MVK-Cys40-Leu14-Exendin 4 were obtained by conjugating Cys40-Leu14-Exendin 4 with Maleimide-NOTA and NOTA-MVK-Maleimide, respectively (Figure 1). Boc-MVK-Dde was synthesized by solid-phase peptide synthesis (SPPS) (Figure S1). NOTA-Met-OH was synthesized by conjugating L-NH2-Met-OH with p-SCN-Bn-NOTA (Figure S2). Nonradioactive Ga standards of NOTA-Cys40Leu14-Exendin 4 and NOTA-MVK-Cys40-Leu14-Exendin 4 were obtained similarly to the published literature.38 More details on the synthesis and analysis are available in the Supporting Information. The 68Ga labeling was very efficient with an overall time of 25 min. The decay corrected radiochemical yield was 58 ± 2.5% (n = 8) for 68Ga-NOTA-Cys40-Leu14-Exendin 4 and 54 ± 3.4% (n = 8) for 68Ga-NOTA-MVK-Cys40-Leu14-Exendin 4. Radiochemical purity was greater than 95% for both compounds. The 1746


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Figure 2. Immunofluorescence staining of NEP expression (green) in (A) INS-1 tumor cells, (B) HEK293T cells, and (C) mouse kidney cortex sections. The nuclei were stained with DAPI (blue).

Figure 3. Serum stability of (A) 68Ga-NOTA-Cys40-Leu14-Exendin 4 and (B) 68Ga-NOTA-MVK-Cys40-Leu14-Exendin 4. (C) Cell competitive binding assay of NOTA-Cys40-Leu14-Exendin 4 (red) and NOTA-MVK-Cys40-Leu14-Exendin 4 (blue) in INS-1 cell.

specific activity corrected to the start of the reaction was 95.6 ± 22.4 GBq/μmol (n = 8; range 18.5 to 137 GBq/μmol) for 68GaNOTA-Cys40-Leu14-Exendin 4, and 102.3 ± 30.8 GBq/μmol (n = 8; range 25.7 to 146 GBq/μmol) for 68Ga-NOTA-MVKCys40-Leu14-Exendin 4. Immunofluorescence Staining. The result of immunofluorescence staining showed positive NEP expression in INS-1

cells (Figure 2A), HEK293T cells (Figure 2B), and mouse kidney cortex (Figure 2C). Mouse Serum Stability and Cell Binding Assay. Extraction efficiency from mouse serum was around 85% for both 68Ga-NOTA-Cys40-Leu14-Exendin 4 and 68Ga-NOTAMVK-Cys40-Leu14-Exendin 4. Both compounds were stable in mouse serum at 37 °C for at least 1 h (Figure 3A and B). The IC50 values for NOTA-Cys40-Leu14-Exendin 4 and NOTA1747


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Figure 4. (A) Cleavage of model compound of Boc-MVK-Dde. (B) Cleavage efficiency of Boc-MVK-Dde with and without the presence of NEP inhibitor phosphoramidon. (C) In vitro metabolism study of NOTA-MVK-Cys40-Leu14-Exendin 4 incubated with BBMVs. (D) In vivo metabolism study of 68Ga-NOTA-MVK-Cys40-Leu14-Exendin 4 from kidney and urine samples.

MVK-Cys40-Leu14-Exendin 4 were 0.46 ± 0.02 and 0.80 ± 0.03 nM, respectively (Figure 3C), indicating that MVK modification did not affect the binding affinity of the peptide. In Vitro Enzyme Mediated Cleavage Study. We evaluated the metabolic cleavage of a model compound, BocMVK-Dde, in order to evaluate the specificity of the cleavage site. The major metabolite exhibited the ion 248.09 [M-H]− by ESI-LC-MS (Figure S3), which was consistent with the structure Boc-Met-OH (FW 249.10). Metabolic degradation was significantly inhibited upon incubation with phosphoramidon, a specific NEP inhibitor. After a 3 h incubation, about 70% of Boc-Met-Dde was degraded, while incubation in the presence of the inhibitor showed only 10% degradation (Figure 4B). These same studies with NOTA-MVK-Cys40-Leu14-Exendin 4 as substrate showed that, after 3 h incubation, NOTA-Met-OH was released in the incubation mixture, as confirmed by chromatogram of the authentic NOTA-Met-OH (Figure 4C). In Vivo Metabolism Study. The radioactivity extraction efficiency of urine and kidney samples were 86 ± 0.6% and 77 ± 3.0%, respectively. In the radiochromatogram of kidney extracts (Figure 4D), 64.8% of the radioactivity appeared at 16.2 min, which was 68Ga-NOTA-Met-OH, as confirmed by the nonradioactive standard of Ga-NOTA-Met-OH. About 23% eluted with the retention time of the parent compound (27.8 min). As for the urine sample (Figure 4D), about 46% of the radioactivity was 68Ga-NOTA-Met-OH, followed by 43.7% metabolites distributed between 17.5 and 18.5 min. There was no parent compound remaining in the urine. MicroPET Imaging. Representative projection microPET images of INS-1 tumor-bearing mice at different time points after intravenous injection of 3.7−5.5 MBq (100−150 μCi, 0.2 nmol) of 68Ga-NOTA-Cys40-Leu14-Exendin 4 and 68Ga-NOTAMVK-Cys40-Leu14-Exendin 4 were shown in Figure 5A. The INS-1 tumors were clearly visible after intravenous administration of both compounds without much difference in the tumor uptake (Figure 6B), and when subjected to co-injection with excess amount of Cys40-Leu14-Exendin 4, the tumor uptake can be completely blocked (Figure 5A), indicating that these two compounds were both specific ligands to GLP-1R and the affinities to the receptor were very similar, which was in line with

Figure 5. (A) Projection microPET images of INS-1 tumor mice at different time points after intravenous injection of 68Ga-NOTA-Cys40Leu14-Exendin 4 and 68Ga-NOTA-MVK-Cys40-Leu14-Exendin 4. (B) Tumor-to-kidney ratios of 68Ga-NOTA-Cys40-Leu14-Exendin 4 and 68 Ga-NOTA-MVK-Cys40-Leu14-Exendin 4 at different time points.

the result of competitive binding assay. Although the two tracers showed similar renal radioactivity levels before 20 min p.i., the kidney uptake of 68Ga-NOTA-MVK-Cys40-Leu14-Exendin 4 quickly achieved the peak value of around 40%ID/g between 30 and 45 min, then decreased gradually over time and maintained at the level of 31%ID/g at 3 h p.i., while the values of the other one just rose over time, reaching 82%ID/g at 3 h p.i. and still 1748


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Figure 6. Quantification of (A) kidney and (B) tumor uptakes of 68Ga-NOTA-Cys40-Leu14-Exendin 4 and 68Ga-NOTA-MVK-Cys40-Leu14-Exendin 4 in INS-1 tumor mice at different time points. Non-decay-corrected (C) kidney and (D) tumor time-activity curves of 68Ga-NOTA-Cys40-Leu14Exendin 4 and 68Ga-NOTA-MVK-Cys40-Leu14-Exendin 4.

Table 1. Biodistribution of 68Ga-NOTA-Cys40-Leu14-Exendin 4 and 68Ga-NOTA-MVK -Cys40-Leu14-Exendin 4 in INS-1 tumor model (n = 4/group) 68

Ga-NOTA-Cys40-Leu14-Exendin 4

tumor heart lung liver spleen stomach intestine pancreas kidney muscle bone blood

68

Ga-NOTA-MVK-Cys40-Leu14-Exendin 4

1h

2h

1 h blocking

1h

2h

1 h blocking

25.55 ± 1.73 0.19 ± 0.03 7.27 ± 1.88 0.54 ± 0.03 0.41 ± 0.10 1.30 ± 0.43 0.53 ± 0.16 6.01 ± 0.41 70.81 ± 8.05 0.11 ± 0.08 0.09 ± 0.01 0.19 ± 0.02

25.87 ± 1.93 0.19 ± 0.02 7.59 ± 1.05 0.58 ± 0.09 0.38 ± 0.12 1.68 ± 0.51 0.68 ± 0.12 6.75 ± 1.05 94.24 ± 9.76 0.04 ± 0.01 0.10 ± 0.01 0.12 ± 0.02

0.85 ± 0.03 0.25 ± 0.01 2.33 ± 0.51 0.54 ± 0.02 0.37 ± 0.04 0.46 ± 0.02 0.28 ± 0.03 0.21 ± 0.01 78.87 ± 13.54 0.25 ± 0.07 0.18 ± 0.01 0.68 ± 0.08

24.65 ± 1.37 0.64 ± 0.12 9.56 ± 0.21 1.55 ± 0.18 1.27 ± 0.29 2.04 ± 0.56 0.84 ± 0.32 7.59 ± 1.07 43.31 ± 3.81 0.22 ± 0.15 0.15 ± 0.05 0.34 ± 0.07

24.86 ± 1.13 0.17 ± 0.03 10.29 ± 0.58 0.53 ± 0.14 0.33 ± 0.12 0.94 ± 0.34 0.61 ± 0.12 5.61 ± 1.45 30.76 ± 3.77 0.17 ± 0.04 0.08 ± 0.01 0.18 ± 0.04

1.00 ± 0.08 0.31 ± 0.07 1.07 ± 0.22 0.49 ± 0.11 0.31 ± 0.03 0.66 ± 0.22 0.47 ± 0.18 0.23 ± 0.01 45.88 ± 5.30 0.12 ± 0.01 0.38 ± 0.11 0.57 ± 0.03

30.76 ± 3.77%ID/g at 1 and 2 h p.i., which were 61% and 33% of those of the control, respectively.

lacked the sign of plateau (Figure 6A). As a result, the tumor to kidney ratios of 68Ga-NOTA-MVK-Cys40-Leu14-Exendin 4 were significantly higher than those of 68Ga-NOTA-Cys40-Leu14Exendin 4 (P < 0.0001), specifically with 1.82, 2.28, 3.16, and 3.22 times of those of control at 0.5, 1, 2, and 3 h p.i., respectively (Figure 5B). Biodistribution Studies. In order to further confirm the PET imaging quantification, the biodistribution of the two exendin 4 analogues were evaluated in INS-1 tumor-bearing mice immediately after 1 and 2 h PET imaging. As shown in Table 1, the INS-1 tumor uptake of the two tracers were much similar, both around 25%ID/g at 1 and 2 h p.i. When co-injected with excess amount of Cys40-Leu14-Exendin 4, uptakes in tumors and other GLP-1R expressing organs, like pancreas and lung, could be significantly blocked. The renal accumulations of 68GaNOTA-MVK-Cys40-Leu14-Exendin 4 were 43.31 ± 3.81 and

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DISCUSSION The main purpose of this study is to find a way to safely and effectively reduce the renal activity of exendin 4 analogues at early time points. An attractive strategy reported by Arano et al. involves the liberation and urinary excretion of radiometal chelated compounds from the covalently conjugated antibody fragments cleaved by enzymes on the BBM, among which NEP plays the most important role.35 A great deal of effort has been made on the design and development of the cleavable linkage, and so far MVK performed best with the most effective reduction of renal activity. However, it remains uncertain whether this MVK linker will work with exendin 4 analogues, therefore careful design and verification has been made in this study. 1749


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this MVK strategy. Thereby, a higher initial dosage or more rounds of treatment in radiotherapy can be achieved.

First, in order to maintain the ability of tumor targeting, the affinity should not be affected after MVK modification of exendin 4 analogues. The competitive cell binding results showed that there was no difference in the IC50 values between NOTA-Cys40-Leu14-Exendin 4 and NOTA-MVK-Cys40-Leu14Exendin 4, which confirmed that the affinity was not affected by the introduction of MVK. Second, the whole conjugate should remain stable in plasma so as not to impair the radioactivity levels during circulation. As a result, the in vitro plasma stability showed that both tracers remain intact in plasma at 37 °C, which is consistent with the in vivo PET images and the ex vivo biodistribution studies, that both of which had similar tumor uptakes to our previously reported studies.39,40 The immunofluorescence result showed that the NEP was extensively expressed in mouse kidney cortex and HEK293T cells, which laid the foundation for the enzyme-mediated degradation in the kidney. Although INS-1 cells also showed NEP expression, the radioactivity accumulation in tumor was not affected at all. The reason could be that exendin 4 is a very poor substrate for NEP and cannot be digested by NEP,41,42 which was also confirmed by our mouse serum stability study (Figure 3B), as NEP is abundant in blood.43 According to the cleavage mechanism, the enzymes on the BBM will recognize the MVK motif and cleave it at the amide bond between Met and Val.35 To confirm this mechanism, an in vitro cleavage study was performed firstly with a model compound Boc-MVK-Dde. This compound was chosen based on the following considerations: first, it was an intermediate during the SPPS of MVK sequence; second, the Dde group had an intense UV absorption which could be used for the tracking of the parent compound. After the incubation of Boc-MVK-Dde with BBMVs, the fragment of Boc-Met-OH was found, indicating that MVK sequence could be recognized by the enzyme and cleaved at the position expected. NEP inhibitor was able to effectively block the cleavage of Boc-MVK-Dde, suggesting the important role NEP played in the cleavage of MVK sequence. Further cleavage study performed using the full structure of NOTA-MVK-Cys40-Leu14-Exendin 4 also detected NOTA-Met-OH after incubation with BBMVs. Although exendin 4 was resistant to NEP, its metabolic fragments could be digested effectively by NEP.42 As shown in Figure 4C, when NOTA-MVK-Exendin 4 was incubated with the enzyme mixture from BBM, NOTA-MVK-Exendin 4 could be first cleaved into MVK containing fragments by the enzyme mixture, and then release NOTA-Met-OH under the digestion of NEP. In the in vivo metabolism study, the main metabolite in kidneys and urine was 68Ga-NOTA-Met-OH, which was in line with the mechanism that with MVK inserted, the 68Ga-NOTA-Met-OH fragment could be liberated and excreted rapidly. PET images and ex vivo biodistribution studies indicated that with the linker MVK, the renal accumulation was significantly reduced without impairing the tumor uptake. One thing worth noting is that the signals of bladder in 68Ga-NOTA-MVK-Cys40Leu14-Exendin 4 PET images were much higher than those of control, which was consistent with the hypothesis that the cleaved 68Ga-NOTA-Met-OH was not reabsorbed in the kidneys, but rapidly drained to the bladder. The non-decaycorrected time-activity curves of kidney showed that the area under curve (AUC) of 68Ga-NOTA-MVK-Cys40-Leu14-Exendin 4 was almost half that of 68Ga-NOTA-Cys40-Leu14-Exendin 4 (Figure 6C), while for tumor no difference was found between the two radiotracers (Figure 5D), which means that the radiation dose to kidneys can be decreased by 50% by using

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CONCLUSION In this study, a novel exendin 4 analogue, NOTA-MVK-Cys40Leu14-Exendin 4, was successfully synthesized and labeled with 68 Ga. With the cleavable MVK sequence, this ligand could be cleaved by the enzymes on kidneys, and releasing the fragment of 68Ga-NOTA-Met-OH, which would rapidly excrete from urine. As the high and consistent renal radioactivity accumulation could be significantly reduced, NOTA-MVK-Cys40-Leu14Exendin 4 shows great potential in the diagnosis and radiotherapy for insulinoma.

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MATERIALS AND METHODS General. NOTA-MVK-maleimide, Cys40-Leu14-Exendin 4, Boc-Met-OH, Fmoc-Val-OH were purchased from CS Bio Inc. (CA, USA). 2-Chlorotrityl chloride resin (1.3 mmol/g) and Fmoc-Lys(Dde)-OH were purchased from AAPPTec LLC (KY, USA). Maleimide-NOTA was purchased from CheMatech (Dijon, France) and p-SCN-Bn-NOTA was obtained from Macrocyclics (TX, USA). Anti-neutral endopeptidase monoclonal antibody (SN75−07) was purchased from Invitrogen (Thermo Fisher, MA, USA). All other chemicals were purchased from Sigma-Aldrich (MO, USA). Solid-phase peptide synthesis was performed on a CS336X Automated Peptides Synthesizer from CS Bio Inc. Mass spectra (MS) were acquired from a Waters Acquity UPLC system coupled with Waters Qtof Premier MS (LC-MS). The 1H NMR and 13C spectra were collected on Bruker AVANCE300 spectrometer at 300 MHz. Fluorescence images were acquired with a Zeiss LSM 780 confocal microscope. Sep-Pak light cartridge (Waters) was used for solid phase extraction of labeled compounds. 68GaCl3 was eluted from a 68Ge−68Ga generator (iThemba, South Africa) with 0.6 M HCl. Product purification was performed on Waters 600 gradient system with a Waters 996 photodiode array (PDA) detector using a Higgins PROTO 300 C-18 column (5 μm, 250 mm × 20 mm). Semi-preparative HPLC was performed on an Agilent’s 1100 series HPLC system with a Phenomenex Luna C18 column (5 μm, 250 mm × 10 mm). Analytical HPLC was performed on the same Agilent’s 1100 system with a Bioscan radioisotope detector using an Agilent Zorbax 300SB-C18 column (5 μm, 250 mm × 4.6 mm). The flow rate for preparative, semi-preparative, and analytical HPLC was 12, 5, and 1 mL/min, respectively, running with the same mobile phase A (0.1% TFA in water) and B (0.1% TFA in acetonitrile). The linear gradient of method A starts from 5% A for 5 min and increases to 65% within 30 min, while the linear gradient of method B starts from 15% A for 3 min and increases to 70% within 17 min. Chemistry. Details of the organic syntheses and characterization of NOTA-Cys40-Leu14-Exendin 4, NOTA-MVK-Cys40Leu14-Exendin 4, NOTA-Met-OH, and the nonradioactive Ga standards of NOTA-MVK-Cys40-Leu14-Exendin 4 and NOTAMet-OH were described in the Supporting Information. Radiochemistry. Freshly eluted 68GaCl3 (200 μL, 5−6 mCi) was added to either NOTA-Cys40-Leu14-Exendin 4 or NOTA-MVK-Cys40-Leu14-Exendin 4 (2 nmol, 10 μL) solution, and the pH was adjusted to 3−4 by adding 200 μL 1 M 4-(2hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES). The mixture was heated at 37 °C for 15 min, and then loaded onto a 1750


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was then incubated at 37 °C for 3 h, followed by the addition of the same volume of acetonitrile and centrifuged at 13,000 rpm for 5 min. 10 μL of the supernatants were removed and analyzed by HPLC with method B. The cleavage fragments observed on the HPLC chromatograms were collected and analyzed by LCMS. The same procedure was performed with the full structure of NOTA-MVK-Cys40-Leu14-Exendin 4 (10 nmol) to test the liberation of NOTA-Met-OH. Animal Models. All animal studies were conducted under a protocol approved by the NIH Clinical Center Animal Care and Use Committee. Female FVB mice (age, 5−6 weeks; weight, 18−20 g) and female athymic nude/nude mice (age, 5−6 weeks; weight, 18−20 g) were purchased from Envigo. The INS-1 tumor model was developed by subcutaneous injection of 5 × 106 cells into the right shoulder of nude mice. Tumor size was measured using a digital caliper. The mice underwent microPET studies when the tumor volume reached 100−300 mm3 (3 weeks after inoculation). In Vivo Metabolism Study. Each healthy FVB mouse was injected intravenously with 14.8−18.5 MBq of 68Ga-NOTAMVK-Cys40-Leu14-Exendin 4 (389−486 MBq/μmol). Urine samples were collected at 20 min post-injection, mixed with an equal volume of acetonitrile, and centrifuged at 14,000 rpm for 10 min. After the collection of urine, the mice were sacrificed under anesthesia, and the kidneys were removed. The kidneys were added into 1 mL pre-chilled acid wash buffer (0.2 M glycine-0.15 M NaCl, pH 3.0), homogenized on ice, mixed with an equal volume of acetonitrile, and then centrifuged at 14,000 rpm for 10 min. A total of 10 μL of the supernatant from the urine and kidney samples were removed separately for HPLC analysis. The radioactivity of the supernatant and precipitate of both urine and kidney samples were measured with a dose calibrator to calculate the extraction efficiency. All samples were collected in pre-chilled vials, and all further manipulations were conducted on ice or at 4 °C for centrifugation to prevent further degradation during sample workup. In Vivo PET and Biodistribution Studies. PET scans were acquired using an Inveon small animal PET scanner (Siemens Medical Solutions). Five min static PET scans were acquired at 5, 20, 30, 45, 60, 120, and 180 min p.i. of 68Ga-labeled Cys40Leu14-Exendin 4 analogs (0.2 nmol/mouse, 3.7−5.6 MBq) into INS-1 tumor-bearing mice (n = 4/group). Blocking PET scans were acquired at 60 min after coinjection of 68Ga-labeled Cys40Leu14-Exendin 4 analogs (0.2 nmol/mouse) and 50-fold excess Cys40-Leu14-Exendin 4 (n = 4/group). Regions of interest (ROIs) were drawn on decay-corrected whole-body coronal images using ASI Pro software (ASI Pro 5.2.4.0; Siemens Medical Solutions). Biodistribution studies were conducted at 1 and 2 h p.i. (n = 4/group), tissues of tumor, heart, lung, liver, spleen, stomach, intestine, pancreas, kidneys, muscle, bone, and blood were collected, wet-weighed, and measured in a gamma counter. Results were normalized as percentages of the injected dose per gram of tissue (%ID/g). Statistical Analysis. All quantitative data were presented as mean ± SD. Two-tailed paired and unpaired Student’s t tests were used to determine differences within groups and between groups, respectively. P < 0.05 was considered statistically significant while P < 0.01 was considered extremely significant.

Sep-Pak C18 light Cartridge pre-activated by washing with 5 mL of ethanol and 5 mL of water. The cartridge was rinsed by 10 mL of water, and the product remained on the cartridge was eluted with 100 μL 10 mM HCl in ethanol. The radio chemical purity was tested by analytic radio-HPLC using method B. Cell Culture. The INS-1 rat insulinoma cells and HEK293T cells were purchased from the American Type Culture Collection (MD, USA) and grown in RPMI-1640 medium (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (Gibco), penicillin (100 IU/mL), and streptomycin (100 mg/mL), and in a humidified atmosphere containing 5% CO2 at 37 °C. 50 μM 2-mercaptoethanol and 10 mM HEPES were added to the medium specifically for INS-1cell culture. All the cells were passaged three times per week. Immunofluorescence Staining. Kidneys were collected from mouse and imbedded in O.C.T. compound and kept frozen at −80 °C. 10-μm-thick slices of the frozen sections were prepared with cryostat. The slices were fixed with Z-fix (Anatec Ltd.) for 10−15 min at room temperature and washed with PBS three times, then followed by blocking with 3% BSA in PBS at room temperature for 1 h. Anti-neutral endopeptidase antibody (SN75−07, 1:100, 3% BSA in PBS) was added onto the slices and incubated at 4 °C overnight followed by 0.1% PBST wash for 3 times (5 min each time). Goat anti-rabbit secondary antibody (R&D, 1:100 in PBS) was added and slices were incubated for 1 h at 37 °C. After washing with PBS three times, the slices were mounted with mounting solution containing DAPI. For the cell staining, INS-1 and HEK293T cells were seeded into the 4 well chamber slide (Thermo Scientific) one night before staining. Then the cell slide underwent the same protocol for kidney tissue slides. Fluorescence images were acquired by a Zeiss LSM 780 confocal microscope. Images were processed with the ImageJ software (NIH). Stability in Mouse Serum. 20 μL 68Ga-NOTA-Cys40Leu14-Exendin 4 or 68Ga-NOTA-MVK-Cys40-Leu14-Exendin 4 was mixed with 200 μL mouse serum and incubated at 37 °C. 50 μL aliquots were removed at 0.5 and 1 h, respectively, and mixed with an equal volume of CH3CN. The mixture was centrifuged at 13,000 rpm for 5 min, and 10 μL of the supernatant was subjected to HPLC analysis with method B. The radioactivity of the supernatant and precipitate were measured with a dose calibrator for the calculation of the extraction efficiency. Competitive Cell Binding Assay. The competitive cell binding assay was performed following our previously described procedure.39 INS-1 cells were trypsinized and resuspended in binding buffer (PBS containing 1 mM CaCl2, 5 mM MgCl2, 0.5% (w/v) BSA, and 0.3 mM NaN3), and incubated with 20 nCi (0.74 kBq) (around 0.01 nM) of 125I-GLP-1(7−36) (PerkinElmer, MA, USA) and 0−1000 nM of unlabeled NOTA-Cys40-Leu14-Exendin 4 or NOTA-MVK-Cys40-Leu14Exendin 4. Experiments were performed on triple samples and binding results were expressed as percent of total counts, IC50 values were calculated using Prism software (GraphPad Software Inc., CA, USA). In Vitro Enzyme Mediated Cleavage Study. The brush border membrane vesicles (BBMVs) were isolated from the renal cortex of female nude mice by a Mg/EGTA precipitation method.44 The enzyme-mediated cleavage of the peptide linkage was performed using the following procedure: 50 μL of the model compound Boc-MVK-Dde (0.27 μmol) was added to 50 μL BBMVs solution (diluted by PBS, the protein concentration was 3.4 μg/μL as determined by Nanodrop) with and without the presence of NEP inhibitor phosphoramidon. The mixture 1751


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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.9b00280.

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Materials and Methods (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Phone: 301-451-4246. *E-mail: [email protected]. Phone: 029-84775449. ORCID

Longguang Tang: 0000-0002-9517-1325 Xiaoyuan Chen: 0000-0002-9622-0870 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported in part, by the National Natural Science Foundation of China (Grant No. 81801730, 81871379), the International Cooperation Program of Xijing Hospital (Grant No. XJZT15G01), Shaanxi Science & Technology Co-ordination & Innovation Project (Grant No. 2016KTCQ03-09), Shaanxi Innovation capability support plan (Grant No. 2018PT-08), and the Intramural Research Program of the National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health. We are grateful to Vincent Schram and Lynne Holtzclaw from NICHD of NIH, for their support in the immunofluorescence study.

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REFERENCES

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