CT evaluation of a small peptide-a potential PET probe for carbonic anhydrase IX

Bioorganic & Medicinal Chemistry 27 (2019) 785–789

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Fluorine-18 click radiosynthesis and microPET/CT evaluation of a small peptide-a potential PET probe for carbonic anhydrase IX

T

Lina Jiaa, Xiao Lib, Dengfeng Chengc, Lan Zhanga,

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a

Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai 201800, China Department of Nuclear Medicine, Changhai Hospital, Shanghai 200433, China c Zhongshan Hospital, Fudan University, Shanghai 200032, China b

ARTICLE INFO

ABSTRACT

Keywords: Carbonic anhydrase IX 18 F-labeling Tumor hypoxia PET imaging Peptide

Carbonic anhydrase IX (CA IX) is the first carbonic anhydrase found to be associated with cancer that is overexpressed in a variety of human solid tumors. As a surrogate marker for hypoxia, the expression of CA IX is strongly upregulated in hypoxic tumors by hypoxia and hypoxia-inducible factor 1a (HIF-1a). In our pursuit of a CA IX-specific PET probe, we designed and synthesized a peptide-based CA IX imaging probe by the efficient click reaction of 1,3-dipolar cycloaddition of terminal alkynes and organic azides. The probe 18F-CA IX-P1-4-10 was obtained with a radiochemical yield of 35–45% (n = 5) and radiochemical purity of > 99% in 70–80 min (HPLC purification time included). 18F-CA IX-P1-4-10 had good stability in phosphate buffered saline (PBS), but about 51% peptide degradation was detected in new-born calf serum (NBCS) after incubation. Preliminary microPET/CT experiments demonstrated a specific uptake of 18F-CA IX-P1-4-10 in HT29 tumor and the uptake of 18 F-CA IX-P1-4-10 was blocked by peptide CA IX-P1-4-10-Yne pretreatment. Immunohistochemical staining and western blotting studies confirmed the HT29 tumor was CA IX-positive which further proved tumor accumulation of 18F-CA IX-P1-4-10 was correlated with CA IX expression. The results suggest that 18F-CA IX-P1-4-10 is a promising PET tracer for the specific imaging of CA IX-expressing tumors at the molecular level.

1. Introduction Carbonic anhydrase IX (CA IX) is a member of a family of carbonic anhydrases (CAs), a group of proteins that catalyze the reversible conversion of carbon dioxide to carbonic acid and which are involved in many crucial physiological processes connected with ion transport, pH and CO2 homeostasis, respiration and gluconeogenesis, tumorigenicity1–3. CA IX expressed on the extracellular surface is the first carbonic anhydrase found to be associated with cancer, and it exhibits a particular expression pattern because of its relatively limited presence in normal tissue (especially the gastrointestinal tract) and over expression in a variety of human solid tumors4–6. Many studies demonstrate that CA IX is upregulated in malignant tissues and is associated with poor prognosis, such as cervical7, head and neck8,9, breast10,11, lung12,13, pancreas14, colorectal15, and soft tissue sarcoma16. CA IX is a surrogate marker for hypoxia. The expression of CA IX is strongly upregulated in hypoxic tumors by hypoxia and hypoxia-inducible factor 1a (HIF-1a)17–19 which may help to maintain the intracellular pH, giving tumors a survival advantage and enhancing resistance to classical chemo- and radiotherapies20,21. The strong

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relationships of CA IX expression between with hypoxia and treatment outcome in the clinic, as well as its easily accessible extracellular active site, make CA IX a promising target for cancer diagnosis and tumors hypoxia identification by noninvasive nuclear imaging. Efforts have been mainly devoted to developing anti-CA IX antibodies and low-molecular-weight agents-based CA IX imaging probes more recently. I-124 labeled anti-CA IX antibody cG250 is the most clinically investigated radiotracer22,23. The multicenter Phase III study has been completed to demonstrate the diagnostic utility of 124I-cG250 PET/CT pre-surgical imaging in patients with operable renal masses24. Imaging study results exhibited superior specificity and sensitivity in accurately and noninvasively identifying clear cell renal cell carcinoma (ccRCC). However, although encouraging results were obtained, the poor in vivo pharmacokinetics of antibodies is still a major obstacle to further development. The labeled sulfonamides-based CA IX imaging agents with radioisotopes including 11C25, 18F26,27, 64Cu28, 68Ga29,30, 99m Tc31–33, and 111In34 were extensively developed in recent years. 18FVM4-037, an ethoxzolamide derivative, has entered into phase II pilot study for PET/CT imaging of renal cell carcinoma35. Study results demonstrated moderate uptake in primary tumors (SUVmean 3.04) and

Corresponding author. E-mail address: [email protected] (L. Zhang).

https://doi.org/10.1016/j.bmc.2019.01.014 Received 19 October 2018; Received in revised form 12 January 2019; Accepted 16 January 2019 Available online 17 January 2019 0968-0896/ © 2019 Elsevier Ltd. All rights reserved.

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excellent visualization of CA IX positive metastases (SUVmax 5.92). While visualizing of primary ccRCC lesions is challenging due to high uptake in the normal kidney parenchyma and may limit its application. [64Cu]XYIMSR-06 is a quite promising CA-IX imaging agents derived from sulfonamide inhibitors for PET imaging of ccRCC28. Radiotracer uptake within the tumor reached a maximum of 19.3 %ID/g at 4 h, and the tumor-to-kidney ratio was 7.1 at 24 h. However, the usefulness of this tracer needs to be evaluated in hypoxia models further. Despite the potential advantages of peptides to antibodies for tumor imaging, such as improved tumor penetration, rapid blood clearance and less toxicity, there was extremely rare peptides-based CA IX imaging probes reported in the literatures. That's probably because there was few specific CA IX targeting peptide drugs developed. Rana and her co-authors reported a peptide CA IX-P1-4-10 screened using the technology of phage display and it has high binding affinity and specificity for CA IX by the optimization of properties of the parent peptide36. Here, we report the synthesis and evaluation of the peptide CA IX-P1-410 labeled with fluorine-18 using click chemistry for imaging the expression of CA IX with PET.

readily to be degraded in new-born calf serum (NBCS). After 3 h, 48.5% of the parent tracer (tR = 14.6 min) remained intact. Peptide degradation was occurred to the probe of 18F-CA IX-P1-4-10, however, no defluorination (the retention time of 18F− was about 2.8 min) was detected (Fig. 2b). 2.3. In vivo PET imaging studies MicroPET/CT scans were performed on HT29 xenograft model and selected coronal and transversal images at 60 min after injecting 18F-CA IX-P1-4-10 were shown in Fig. 3. 18F-CA IX-P1-4-10 was stable against defluorination as negligible tracer uptake was found in the bone. Very small amount of radioactivity entered the brain demonstrated that this probe could not pass through the blood-brain barrier. There was not obvious uptake in the lungs, either. Most of the injected radioactivity was mainly metabolized through liver (SUVmean = 14.21 ± 3.68) and kidneys (SUVmean = 2.08 ± 0.09). Tumor was clearly visible near the right shoulder. A specific uptake of 18F-CA IX-P1-4-10 in the outer core of HT29 tumor (high-count density regions in the tumor as indicated by the arrows) at 1 h was observed (the SUVmean was 0.38 ± 0.03) and displayed a heterogeneous intratumoral distribution. Tumor hypoxia arises in regions with impaired oxygen delivery. The further away the tumor cells are from the blood vessels, the less and even no oxygen and nutrients they will get (Fig. 4)38. As the expression of CA IX is regulated by hypoxia and HIF-1a, so as can be seen from Fig. 3, tracer uptake was predominant in the outer core of the tumors (HIF-1(+) area) where the expression of CA IX was relatively high. Radioactivity uptake of the inner core (The tumor showed no necrosis in our experiment) and rim (aerobic area) of the tumors were relatively low, for tumor cells having relatively low expression of CA IX. The CA IX specificity of 18F-CA IX-P1-4-10 in vivo was confirmed by a blocking experiment that the probe was co-injected with CA IX-P1-4-10-Yne (10 mg/kg), and the SUV of tumor was 0.09 ± 0.04.

2. Results and discussion 2.1. Chemistry and radiochemistry Due to the advantages of the click reaction of 1,3-dipolar cycloaddition of terminal alkynes and organic azides in the radiopharmaceutical preparation, such as high labeling yield, mild reaction conditions, tolerance of solvents and pH, high chemoselectivity and perfect regioselectivity, this method was applied to the F-18 labeling of the peptide CA IX-P1-4-10 in this study. The terminal alkynyl group was equipped to the peptide of CA IX-P1-4-10 for F-18 click labeling. Synthesis of the cold standard 19F-CA IX-P1-4-10 was conducted as shown in Scheme 1 by four-step conversion using 2-fluoroethanol (1) as starting material. The cold standard was characterized by MS (m/z 1110.00 for [M]+, 555.80 for [1/2M + H]+ (C50H72O13N15F, calculated molecular weight [MW] = 1110.19)). It was applied for establishment of HPLC analytical and purifying methods. Reagents and conditions: (a) TsCl, TEA, DMAP, CH2Cl2, rt; (b) NaN3, DMF, rt; (c) CuSO4, Sodium L-ascorbate, DMF, sodium phosphate buffer, pH 6.0. The click-labeling synthon [18F]3 was prepared according to our reported protocol37.The peptide CA IX-P1-4-10-Yne equipping a terminal alkynyl group was labeled with [18F]3 using the efficient click reaction (Scheme 2). 18F-CA IX-P1-4-10 was obtained in an overall radiochemical yield of 35–45% (n = 5) from aqueous [18F]fluoride (non-decay corrected) and > 99% radiochemical purity in 70–80 min including HPLC purification time. The specific activity of the probe was > 54 GBq/μmol at the end of synthesis (EOS). Radiotracer 18F-CA IX-P1-4-10 was identified by co-elution with reference compound 19FCA IX-P1-4-10 by HPLC (Fig. 1).

2.4. Immunohistochemistry and western blotting To assess the specificity of tumor uptake, immunohistochemistry and western blotting were conducted with tumor tissues extracted from the HT29 mice that completed the PET imaging. Immunohistochemistry (Fig. 5) and western blotting (Fig. 6) analysis demonstrated that CA IX protein is expressed in the HT29 tumor. Fig. 5a–b shows the immunohistochemical staining of the tumor sections incubated with antiCA IX antibody and anti-CD31 antibody. Strong staining was observed around the blood vessels wall of the tumor section stained with CD31, which proved the presence of adequate tumor vasculature in the tumor. As the further away the tumor cells are from the blood vessels, the higher degree of hypoxia is, and the more the CA IX is expressed. This point was further validated by the heterogeneous distribution of radioactivity within the tumor in imaging results. 3. Conclusions

2.2. In vitro stability

We have synthesized 18F-CA IX-P1-4-10 as a potential PET probe for non-invasive imaging of CA IX positive tumor at the molecular level, opening up an application of novel peptide-based PET probe for tumor imaging with CA IX over-expression.

The in vitro stability of 18F-CA IX-P1-4-10 was evaluated by radioHPLC. The tracer displayed good stability after incubation in phosphate buffered saline (PBS) at 37 °C for 3 h. Defluorination or radioactive metabolic degradation products were not observed, revealing 100% of the parent tracer remained intact (Fig. 2a). However, the tracer was

4. Experimental 4.1. General information

Scheme 1. Synthesis of cold standard

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CA IX-P1-4-10-Yne (NHVPLSPy-Yne) was purchased from ChinaPeptides Co., Ltd. Sodium L-ascorbate and Acetonitrile (AcroSeal) were purchased from Acros. Sodium azide (NaN3) was purchased from Jingyan Chemicals (Shanghai) Co., Ltd. [18F−] was purchased from Shanghai Atom Kexing Pharmaceuticals Co., Ltd. 2-Fluoroethyl-4-

F-CA IX-P1-4-10. 786

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Scheme 2. Radiosynthesis of 18F-CA IX-P1-4-10 via the Cu(I)-Catalyzed 1, 3-Dipolar Cycloaddition.

Fig. 1. HPLC chromatograms of 18F-CA IX-P1-4-10 (γ-trace, red) with co-injection of the non-radioactive reference compound 19F-CA IX-P1-4-10 (UVtrace, 254 nm, black).

Fig. 2. HPLC chromatograms of 18F-CA IX-P1-4-10 (γ-trace) after incubation in PBS (a) and NBCS (b) for 3 h.

toluenesulfonate (2) and 2-Fluoroethylazide (3) were synthesized according to our previous report37. Goat anti-human CAIX polyclonal antibody , rat anti-mouse CD31 antibody and HRP-conjugated rabbit anti-goat IgG were purchased from Santa Cruz Biotechnology, Inc. 10% Bis-Tris Gel for SDS-PAGE was purchased from Thermo Fisher Scientific Inc. BeyoECL Plus Kit was purchased from Beyotime Institute of Biotechnology. All other chemicals and solvents were purchased from Sinopharm Chemical Reagent Co., Ltd. All of the reagents and solvents were used without further purification unless noted otherwise. Reversed-phase extraction C18 Sep-Pak cartridges were obtained from Waters Co., Ltd. and pretreated with methanol and water before use. The syringe filter (0.22 μm) was obtained from Sinopharm Chemical Reagent Co., Ltd. Mass spectra (MS) were obtained on a Shimadzu (LC-MS2010) TOFESI-MS Spectrometer. Semi-preparative and analytical HPLC were performed on an Agilent 1100 System equipped with a variable-wavelength UV detector and radiodetector connected in series. A semi-preparative column (Agilent XDB C18, 9.4 × 300 mm, with a flow rate of 2 mL/min, UV at 254 nm) was used for radiolabeling yields, stability experiments, and final purification of 18F-CA IX-P1-4-10. HPLC Solvent: solvent A: water/ 0.1% TFA; solvent B: acetonitrile/0.1% TFA. Gradient details: 0–20 − 24–25 min, 20–25 − 75–20% B.

(4.0 μL, 1.5 M) was added a solution of CA IX-P1-4-10-Yne (1 mg, 0.98 μmol) in sodium phosphate buffer (200 μL) followed by addition of 2-fluoroethylazide (3) (4.9 μmol) in DMF (30 μL) at ambient temperature. The mixture was then stirring continued at 50 °C until there was no CA IX-P1-4-10-Yne analyzed by HPLC. After cooling, the reaction was quenched with H2O (5 mL), and passed through a C18 cartridge. The trapped compound was eluted off the cartridge using 1 mL methanol with a retention time of 14.8 min on the HPLC. TOF-ESI-MS: m/z 1110.00 for [M]+, 555.80 for [1/2M + H]+ (C50H72O13N15F, calculated molecular weight [MW] = 1110.19) 4.3. Radiochemistry 4.3.1. 2-[18F]Fluoroethylazide ([18F]3) 2-[18F]Fluoroethylazide ([18F]3) was synthesized according to our previously described protocol37 with minor modifications. 2-Azidoethyl-4-toluenesulfonate (4) (5 mg, 0.02 mmol) in 0.4 mL of acetonitrile was added to the obtained no-carrier-added dry K[18F]F-K2.2.2 complex (370–1000 MBq). The reaction vessel was heated for 10 min at 95 °C. [18F]3 was distilled at 85 °C with a flow of nitrogen (60 mL/min) into a trapping vial containing 0.1 mL of acetonitrile. 4.3.2. Synthesis of 18F-CA IX-P1-4-10 To the reactor vial with copper-(II) sulfate (25 μL, 0.45 M), sodium L-ascorbate (25 μL, 1.5 M), acetonitrile (100 μL) were added successively. After mixing with a vortex, the peptide CAIX-P1-4-10-Yne (2.0 mg, 1.96 μmol) in sodium phosphate buffer (300 μL, pH 6.0, 0.2 M) was added. The solution was then mixed with the vortex for 1 min.

4.2. Chemistry Cold standard – 19F-CA IX-P1-4-10 To a stirred solution of sodium phosphate buffer (0.2 M, pH 6.0, 100 μL), copper-(II) sulfate (6.5 μL, 0.45 M) and sodium L-ascorbate 787

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Fig. 3. MicroPET/CT images acquired at 1 h postinjection of 18F-CA IX-P1-4-10 in HT-29 colorectal cancer xenograft-bearing mice. (a), 1 h post-injection of 18F-CA IX-P1-4-10. Arrows indicate high-count density regions in the tumor. (b), 1 h post-injection of 18 F-CA IX-P1-4-10 and CA IX-P1-4-10-Yne. Arrows indicate location of tumor. The different scale was used.

Fig. 6. Western blotting analysis of CA IX expression in a HT-29 tumor.

samples were filtered through a 0.22 μm membrane and then analyzed by Radio-HPLC. 4.5. Micro PET/CT imaging studies Fig. 4. Tumor microenvironment. HIF-1-active regions (red in the left photograph) locate closer to blood vessels than pimonidazole-positive regions (green in the left photograph). Pimo positive regions locate next to necrotic regions and hardly express HIF-1α, thus have little HIF-1 activity38.

All animal studies followed the specifications for laboratory animal studies provided by Zhongshan Hospital, Fudan University. When tumors reached 6–8 mm in diameter, PET imaging studies were performed using an Inveon micro-PET/CT scanner (Siemens Multimodality Inveon). Mice bearing HT29 tumors were randomly divided into the control group and the blocking group (n = 3/group). For the control group, about 1.85 MBq (50 μCi) of 18F-CA IX-P1-4-10 was injected through tail vein under isoflurane anesthesia and mice were anesthetized throughout the period of imaging. For the blocking group, mice was injected with CA IX-P1-4-10-Yne (500 μg, 0.49 μmol, in PBS) 30 min before injection of 1.85 MBq (50 μCi) of 18F-CA IX-P1-4-10. For both groups, before PET, small-animal CT imaging was performed for anatomical reference and then a 10 min acquisition was performed at 30, 60 and 90 min after tracer injection. The decay-corrected dynamic PET data were reconstructed using an ordered subset expectation maximization 3D (OSEM3D). The mean standardized uptake values (SUVmean) of the tumor were automatically obtained from the Invecon Acquisition Workplace.

Subsequently, a solution of [18F]3 (75–370 MBq) in acetonitrile (200 μL) was added. The reaction mixture was vibrated for 15 min at 50 °C. After filtering through a syringe filter (0.22 μm), the product was isolated via semi-preparative radio-HPLC. The product peak was collected (tR = 14.5 min), and the solvents were removed under reduced pressure. The dry product was formulated with 0.9% NaCl for further use. 4.4. Stability experiments in vitro The in vitro stability of 18F-CA IX-P1-4-10 was investigated in NBCS and PBS. The formulated radiolabeled peptide 18F-CA IX-P1-4-10 (∼3.7 MBq, 50 μL) was added to 1 mL of NBCS or 1 mL of PBS, and the mixture was gently shaken at 37 °C for 3 h. During the incubation, aliquots (200 μL) were collected at 30 min, 90 min and 180 min. The

Fig. 5. Immunohistochemical staining of the HT29 tumor with anti-CA IX antibody (a) and anti-CD31 antibody (b) (Arrows show the areas of tumor vascular walls). Original magnification, 200 (a and b). 788

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4.6. Immunohistochemistry and western blotting

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After the completion of PET imaging, mice bearing HT29 tumors were sacrificed. Tumor tissues were extracted for further use. 4.6.1. Immunohistochemistry This study was performed following our published procedures39. Formalin-fixed, paraffin-embedded tumor tissue sections (thickness, 5 μm) were deparaffinized in xylene and rehydrated in gradient ethanol. Heat-induced antigen retrieval was conducted in 10 mmol/L citrate buffer. Endogenous peroxidase was blocked by incubation with 3% hydrogen peroxidase for 10 min. After being blocked with 5% BSA for 30 min, the slides were then incubated with primary antibodies rat anti-mouse CD31 antibody and goat anti-human CA IX polyclonal antibody (1:50) for 1 h at 37 °C, followed by peroxidase conjugated secondary antibodies for 0.5 h at 37 °C. Immunostaining was visualized after incubation with 3, 3′-diaminobenzidine tetrahydrochloride solution. Counterstaining was performed with hematoxylin. 4.6.2. Western blotting Tumor tissues were chopping on ice and homogenized with Western Blot tissue lysis solution. The protein was extracted and concentration was determined with BCA protein assay. Proteins (25 μg) were separated by SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were then incubated with goat anti-human CA IX polyclonal antibody (1:200) for 2 h at room temperature, followed by incubation with HRP-conjugated rabbit anti-goat IgG (1:1000) for 1 h at room temperature. Protein bands were visualized with BeyoECL Plus Kit. Research support This work was supported by grants from the National Natural Science Foundation of China (NSFC) under Contract NO.11505269. The authors also acknowledge Shanghai Atom KeXing pharmaceutical Co., Ltd. for providing 18F−. References 1. Swietach P, Wigfield S, Cobden P, et al. Tumor-associated carbonic anhydrase 9 spatially coordinates intracellular pH in three-dimensional multicellular growths. J Biol Chem. 2008;283(29):20473–20483. 2. Chiche J, Ilc K, Laferriere J, et al. Hypoxia-inducible carbonic anhydrase IX and XII promote tumor cell growth by counteracting acidosis through the regulation of the intracellular pH. Cancer Res. 2009;69(1):358–368. 3. Breton S. The cellular physiology of carbonic anhydrases. Jop. 2001;2(4 Suppl):159–164. 4. Clare BW, Supuran CT. A perspective on quantitative structure-activity relationships and carbonic anhydrase inhibitors. Expert Opin Drug Metab Toxicol. 2006;2(1):113–137. 5. Krishnamurthy VM, Kaufman GK, Urbach AR, et al. Carbonic anhydrase as a model for biophysical and physical-organic studies of proteins and protein-ligand binding. Chem Rev. 2008;108(3):946–1051. 6. McDonald PC, Dedhar S. Carbonic anhydrase IX (CAIX) as a mediator of hypoxiainduced stress response in cancer cells. Subcell Biochem. 2014;75:255–269. 7. Loncaster JA, Harris AL, Davidson SE, et al. Carbonic anhydrase (CA IX) expression, a potential new intrinsic marker of hypoxia: correlations with tumor oxygen measurements and prognosis in locally advanced carcinoma of the cervix. Cancer Res. 2001;61(17):6394–6399. 8. Le QT, Kong C, Lavori PW, et al. Expression and prognostic significance of a panel of tissue hypoxia markers in head and neck squamous cell carcinomas. Int J Radiat Oncol Biol Phys. 2007;69(1):167–175. 9. Koukourakis MI, Giatromanolaki A, Sivridis E, et al. Hypoxia-regulated carbonic anhydrase-9 (CA9) relates to poor vascularization and resistance of squamous cell head and neck cancer to chemoradiotherapy. Clin Cancer Res. 2001;7(11):3399–3403. 10. Trastour C, Benizri E, Ettore F, et al. HIF-1 alpha and CA IX staining in invasive breast carcinomas: prognosis and treatment outcome. Int J Cancer. 2007;120(7):1451–1458.

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