Novel technetium-99m-labeled bivalent PSMA-targeting probe based on hydroxamamide chelate for diagnosis of prostate cancer

Novel technetium-99m-labeled bivalent PSMA-targeting probe based on hydroxamamide chelate for diagnosis of prostate cancer | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Novel technetium-99m-labeled bivalent PSMA-targeting probe based on hydroxamamide chelate for diagnosis of prostate cancer Yoichi Shimizu, Masato Ando, Hiroyuki Watanabe, Masahiro Ono This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4432393/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract Objective: Prostate-specific membrane antigen (PSMA) is a well-known biomarker of prostate cancer. Previously, our group reported that the succinimidyl-cystatin-urea-glutamate (SCUE) moiety has a high affinity for PSMA. In this study, we developed the novel technetium-99m-labeled PSMA-targeting probe “[ 99m Tc]Tc-(Ham-SCUE) 2 ” based on a hydroxamamide chelate with a bivalent SCUE and evaluated its potential as a SPECT imaging probe for the diagnosis of PSMA-expressing prostate cancer. Methods: Ham-SCUE was synthesized by a one-step reaction with Ham-Mal and cysteine-urea-glutamine. Then, Ham-SCUE was reacted with [ 99m Tc]NaTcO 4 for 10 min at room temperature to obtain [ 99m Tc]Tc-(Ham-SCUE) 2 . [ 99m Tc]Tc-(Ham-SCUE) 2 was added to LNCaP (high PSMA expression) cells or PC3 (low PSMA expression) cells, and their radioactivity was measured 60 min after administration. The blocking study was performed by co-incubation of LNCaP cells with various concentrations of 2-PMPA (a PSMA inhibitor) for 15 min before adding [ 99m Tc]Tc-(Ham-SCUE) 2 . The biodistribution of [ 99m Tc]Tc-(Ham-SCUE) 2 in LNCaP/PC3 dual xenografted C.B.-17/Icr scid/scid Jcl mice was evaluated for 120 min after intravenous injection. The blocking study was performed by pretreatment of mice with 2-PMPA (10 mg/kg weight). Results: [ 99m Tc]Tc-(Ham-SCUE) 2 was acquired at radiochemical yields of 56% with a radiochemical purity of over 95%. The cellular uptake level of [ 99m Tc]Tc-(Ham-SCUE) 2 by LNCaP cells was significantly higher than that by PC3 cells (LNCaP: 11.12 ± 0.71 vs. PC3: 1.40 ± 0.13 %dose/mg protein, p<0.01), and the uptake was significantly suppressed by pretreatment with 2-PMPA (2.56 ± 0.37 %dose/mg protein, p<0.05). IC 50 of 2-PMPA was 245 ± 47 nM. In the in vivo study, the radioactivity of LNCaP tumor tissue was significantly higher than that of PC3 tumor tissue at 120 min after the administration of [ 99m Tc]Tc-(Ham-SCUE) 2 (LNCaP: 9.97 ± 2.79, PC3: 1.16 ± 0.23 %ID/g, p<0.01), and was suppressed by pretreatment with 2-PMPA (2.50 ± 0.45 %ID/g, p<0.01). Conclusion: [ 99m Tc]Tc-(Ham-SCUE) 2 has the potential to be a SPECT imaging agent for diagnosing high PSMA-expressing prostate cancer. Technetium-99m PSMA Hydroxamamide 99mTc-labeled bivalent ligand probes Figures Figure 1 Figure 2 Figure 3 Introduction Prostate cancer (PC) is the most common cancer in men, and many cases are associated with a poor prognosis due to residual disease, recurrence, or metastasis after treatment. For appropriate treatment, it is important to distinguish between low- and high-risk tumors that progress slowly and rapidly, respectively, in addition to detecting the site of disease by imaging, early prediction and monitoring of the treatment response, early diagnosis of recurrence, and accurate re-staging in the event of recurrence. Over the past decade, prostate-specific membrane antigen (PSMA), also known as glutamate carboxypeptidase-II, has been focused on as a promising biomarker for the diagnosis of and subsequent therapy for PC by nuclear medical techniques [ 1 , 2 ]. For PSMA-specific drug delivery, asymmetric urea compounds (glutamate/cystatin-urea-glutamate, or E/CUE) have been widely used as targeting ligands owing to their high-level and specific ability to bind to PSMA. Previously, our group reported that the affinity of CUE for PSMA could be enhanced by adding a succinimidyl moiety induced by reacting the thiol group of CUE with a maleimide moiety [ 3 ]. In addition, single-photon emission computerized tomography (SPECT)/positron emission tomography (PET) probes based on that succinimidyl-CUE moiety, known as “SCUE,” showed favorable properties for SPECT/PET-PSMA imaging in preclinical and clinical studies [ 3 – 6 ]. Recently, we developed a novel bifunctional chelate, “Ham-Mal,” composed of a hydroxamamide amide (Ham) and maleimide moiety [ 7 ]. Ham, a thiol-free chelator of oxo-technetium (V) [Tc(V)], is known to form a 99m Tc-complex under moderate reaction conditions, making it possible to rapidly produce 99m Tc-labeled compounds with a bivalent targeting scaffold because it forms 99m Tc-complexes with a metal-to-ligand ratio of 1:2 [ 8 – 11 ]. In this study, we developed a novel 99m Tc-labeled probe, “[ 99m Tc]Tc-(Ham-SCUE) 2 ,” composed of technetium-99m and two SCUEs (Fig. 1 ), and evaluated its property as a SPECT imaging probe targeting high PSMA-expressing PC. Materials and Methods Preparation of Ham-SCUE and [ 99m Tc]Tc-(Ham-SCUE) 2 The precursor “Ham-SCUE” and [ 99m Tc]Tc-(Ham-SCUE) 2 were prepared using our previously reported method with some modifications [ 7 ]. The detailed process is described in Supplementary Materials and Methods. In vitro cell binding assay The in vitro cellular binding assay of [ 99m Tc]Tc-(Ham-SCUE) 2 was performed with LNCaP prostate adenocarcinoma cells (high PSMA-expressing cells) and PC3 human prostate carcinoma cells (low PSMA-expressing cells) by the method reported previously, with some modifications [ 5 ]. The detailed process is described in Supplementary Materials and Methods. In vivo biodistribution The in vivo biodistribution study of [ 99m Tc]Tc-(Ham-SCUE) 2 was performed with LNCaP/PC3 xenografted mice using the method reported previously with some modifications [ 5 ]. The detailed process is described in Supplementary Materials and Methods. Statistics Data are presented as the mean ± S.E.M. (for the in vitro study) or the mean ± S.D. (for the in vivo study). Student’s t -test was used to assess the significance of differences with JMP 17 software (SAS Institute Inc., Cary, NC, USA). Differences at the 95% confidence level (P < 0.05) were considered significant. Results Synthesis of Ham-SCUE Ham-SCUE was obtained by reacting Ham-Mal with CUE, with a yield of 53% (Supplementary File, Scheme S1). Radiolabeling of [ 99m Tc]Tc-(Ham-SCUE) 2 99m Tc labeling of Ham-SCUE was performed. [ 99m Tc]Tc-(Ham-SCUE) 2 was acquired by reacting Ham-SCUE with 99m Tc pertechnetate and tin(II) tartrate hydrate as a reducing agent in the solvent of phosphate buffer (pH 7.4) (Supplementary File, Scheme S2). [ 99m Tc]Tc-(Ham-SCUE) 2 was acquired with a radiochemical yield of 56.0 ± 6.2% and radiochemical purity of over 95% (Supplementary File, Figure S1 ). In vitro cellular uptake study Cellular uptake of [ 99m Tc]Tc-(Ham-SCUE) 2 is expressed as the %dose/mg protein (Fig. 2 ). The radioactivity of LNCap cells (high PSMA expression) was significantly higher than that of PC3 cells (low PSMA expression) (LNCaP: 11.12 ± 0.71 vs. PC3: 1.40 ± 0.13%dose/mg protein, p < 0.01), and significantly reduced by pretreatment with excess 2-PMPA, a well-known PSMA inhibitor (2.56 ± 0.37%dose/mg protein, p < 0.05). To quantitatively evaluate the binding affinity of [ 99m Tc]Tc-(Ham-SCUE) 2 to PSMA, binding inhibition experiments were performed in the presence of various concentrations of 2-PMPA on LNCaP cells. As a result, the accumulation of [ 99m Tc]Tc-(Ham-SCUE) 2 on LNCaP cells decreased in a concentration-dependent manner with 2-PMPA, and the 50% inhibitory concentration (IC 50 ) of 2-PMPA was 245 ± 47 nM (Supplementary File, Figure S2). In vivo biodistribution The uptake levels of [ 99m Tc]Tc-(Ham-SCUE) 2 in each organ are expressed as %ID/g, except for that in the stomach and thyroid, presented as %ID (Supplementary File, Table S1 ). In the normal organs, high radioactivity was observed in the kidney and spleen. As for tumor tissues, radioactivity of LNCaP was significantly higher than that of PC3 from 5 min after the administration of [ 99m Tc]Tc-(Ham-SCUE) 2 . In addition, its accumulation level in LNCaP tumors was reduced by blocking with excess 2-PMPA at 120 min after administration [9.97 ± 2.79%ID/g (non-blocking) vs. 2.50 ± 0.45%ID/g (blocking) (p < 0.01)], while there was no significant difference in accumulation in PC3 tumors between non-blocking and blocking groups [1.16 ± 0.23%ID/g (non-blocking) vs. 1.17 ± 0.16%ID/g (blocking) (p = 0.97)] (Fig. 3 ). Discussion In this study, we designed and synthesized a novel 99m Tc-labeled bivalent PSMA-targeting probe, “[ 99m Tc]Tc-(Ham-SCUE) 2, ” based on a hydroxamamide chelate. The precursor “Ham-SCUE” was obtained successfully via the thiol-maleimide reaction of Ham-Mal and CUE (Supplementary File, Scheme S1), with a yield of 53%, which was comparable to our previous report [ 7 ]. The radiolabeling of [ 99m Tc]Tc-(Ham-SCUE) 2 was performed using the method we established previously (Supplementary File, Scheme S2)[ 7 ]. In the RP-HPLC analysis after the reaction, the disappearance of the peak derived from [ 99m Tc]NaTcO 4 and the subsequent appearance of two new peaks were observed (Supplementary File, Figure S1 ), suggesting that the chelation of Ham-SCUE and 99m Tc proceeded successfully since the 99m Tc-Ham complex is known to form two tautomers [ 10 ]. The radiochemical yield of [ 99m Tc]Tc-(Ham-SCUE) 2 was 56%, which was also comparable with the other Ham-based 99m Tc labeled probes ([ 99m Tc]Tc-(Ham-RGD) 2 : 49% [ 7 ]). Taking into account that the 99m Tc-labeling reaction proceeded under moderate conditions (simply incubating at room temperature for 10 min), these results suggest that [ 99m Tc]Tc-(Ham-SCUE) 2 can be acquired easily with a favorable chemical yield. In the in vitro study, [ 99m Tc]Tc-(Ham-SCUE) 2 accumulated markedly in LNCaP (high PSMA-expressing) cells rather than in PC3 (low PSMA-expressing) cells, and its accumulation was blocked by 2-PMPA, a PSMA inhibitor (Fig. 2 ). This suggests that [ 99m Tc]Tc-(Ham-SCUE) 2 was taken up specifically in the high PSMA-expressing PC cells. In addition, we evaluated IC 50 of 2-PMPA against [ 99m Tc]Tc-(Ham-SCUE) 2 whose value was 245 nM (Supplementary File, Figure S2), which was about 70 times higher than that of our previously reported 99m Tc-labeled probe “[ 99m Tc]Tc-TMCE” with a monovalent SCUE moiety (3.4 nM)[ 5 ]. The bivalent targeting scaffold is considered to offer higher binding affinity than monovalent counterparts, and some conventional PET/SPECT imaging probes, including PSMA-targeting probes, are designed based on that concept to enhance their specificity for target biomolecules [ 12 ]. Therefore, [ 99m Tc]Tc-(Ham-SCUE) 2 also matched the concept and thus could enhance the affinity for PSMA compared with probes we reported previously. In the in vivo study, [ 99m Tc]Tc-(Ham-SCUE) 2 showed high accumulation in LNCaP tumors compared with PC3 tumors from 5 min after i.v. administration (Table S1 ), and its accumulation level was reduced by blocking with excess 2-PMPA at 120 min after administration (Fig. 3 ), which suggests that [ 99m Tc]Tc-(Ham-SCUE) 2 accumulated specifically in tumors with expression levels of PSMA. High-level radioactivity was also observed in the kidneys and spleen. Considering previous reports [ 4 , 6 ] and the suppression of radioactivity by pretreatment with 2-PMPA (Fig. 3 ), this might be due to the high expression of PSMA in those organs. Regarding the stomach and thyroid, where 99m Tc ions abundantly accumulate, relatively low radioactivities were observed, which suggests that [ 99m Tc]Tc-(Ham-SCUE) 2 did not release free 99m Tc in mice. Conclusions In this study, we successfully developed a novel 99m Tc-labeled bivalent PSMA-targeting probe, “[ 99m Tc]Tc-(Ham-SCUE) 2 ”, and confirmed its potential as a SPECT imaging probe for the diagnosis of high PSMA-expressing PC. Declarations Acknowledgments This study was supported by grants from the Japan Agency for Medical Research and Development (Numbers: 20ck0106426h0003 and JP24ama221604). References Lütje S, Heskamp S, Cornelissen AS, Poeppel TD, van den Broek SA, Rosenbaum-Krumme S, et al. PSMA Ligands for Radionuclide Imaging and Therapy of Prostate Cancer: Clinical Status. Theranostics. 2015;5(12):1388–401. Maurer T, Eiber M, Schwaiger M, Gschwend JE. Current use of PSMA-PET in prostate cancer management. Nat Rev Urol. 2016;13(4):226–35. Harada N, Kimura H, Ono M, Saji H. Preparation of asymmetric urea derivatives that target prostate-specific membrane antigen for SPECT imaging. J Med Chem. 2013;56(20):7890–901. Harada N, Kimura H, Onoe S, Watanabe H, Matsuoka D, Arimitsu K, et al. Synthesis and Biologic Evaluation of Novel 18F-Labeled Probes Targeting Prostate-Specific Membrane Antigen for PET of Prostate Cancer. J Nucl Med. 2016;57(12):1978–84. Kimura H, Sampei S, Matsuoka D, Harada N, Watanabe H, Arimitsu K, et al. Development of (99m)Tc-labeled asymmetric urea derivatives that target prostate-specific membrane antigen for single-photon emission computed tomography imaging. Bioorg Med Chem. 2016;24(10):2251–6. Saga T, Nakamoto Y, Ishimori T, Inoue T, Shimizu Y, Kimura H, et al. Initial evaluation of PET/CT with (18) F-FSU-880 targeting prostate-specific membrane antigen in prostate cancer patients. Cancer Sci. 2019;110(2):742–50. Shimizu Y, Ando M, Iikuni S, Watanabe H, Ono M. Development of a hydroxamamide-based bifunctional chelating agent to prepare technetium-99m-labeled bivalent ligand probes. Sci Rep. 2021;11(1):18714. Nakayama M, Saigo H, Kai E, Koda A, Ozeki H, Harada K, et al. Hydroxamamide as a chelating moiety for the preparation of 99m Tc radiopharmaceuticals (I). Nucl Med Commun. 1992;13(6):445–9. Nakayama M, Saigo H, Koda A, Ozeki K, Harada K, Sugii A, et al. Hydroxamamide as a chelating moiety for the preparation of 99m Tc radiopharmaceuticals—II. The 99m Tc complexes of hydroxamanide derivatives. Appl Radiat Isot. 1994;45(6):735–40. Nakayama M, Xu LC, Koga Y, Harada K, Sugii A, Nakayama H, et al. Hydroxamamide as a chelating moiety for the preparation of 99mTc-radiopharmaceuticals III. Characterization of various 99mTc-hydroxamamides. Appl Radiat Isot. 1997;48(5):571–7. Thipyapong K, Uehara T, Tooyama Y, Braband H, Alberto R, Arano Y. Insight into technetium amidoxime complex: oxo technetium(V) complex of N-substituted benzamidoxime as new basic structure for molecular imaging. Inorg Chem. 2011;50(3):992–8. Dai R, Cai Z, Hu R, Huang Y, Fu L, Yang J, et al. (177)Lu-Labeled Bivalent Ligands of Prostate-Specific Membrane Antigen for Endoradiotherapy of Prostate Cancer. Mol Pharm. 2024;21(2):883–94. Supplementary Files 99mTcHamSCUE2supplementarydatafinal.doc Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 21 May, 2024 Reviewers invited by journal 19 May, 2024 Editor assigned by journal 16 May, 2024 First submitted to journal 16 May, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4432393","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":304324576,"identity":"651931c8-0409-4224-9dbf-ea80820f2e5f","order_by":0,"name":"Yoichi Shimizu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABDUlEQVRIie2QvWrDMBCATwic5eisElD6CDKGtFBIX0Umg5e4zQMU42JIltBZIaXPoiKwFz+Ax3TvYAhkKqXqD61D7GQtVB8cnDi+O90BOBx/EpJSEMAZoH3oncJhJfhR8LgCQG2Eak/pQhTZ3WY6TaKlivM1lIZf9VKPwe0I6Kp9jCifsr4SJl6x60hAZQJEbZV8DORBtytVOKModPzIJkMGtQkX7GbLwNNAlDykJBH/VaSd8nZUobL/qVTfCpl1K6cfu6Aw/nLxci5kGQVYanoR3o+xa5eTYv68wddkwIrJcF3nl7w3T0lVb0fc77jYWaOTBxKyr9R+CX3VasCg0cmzkTRKrF1xOByOf8c7n+Na9oy2jYgAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0003-2831-3597","institution":"Kyoto University","correspondingAuthor":true,"prefix":"","firstName":"Yoichi","middleName":"","lastName":"Shimizu","suffix":""},{"id":304324577,"identity":"55c88c23-7677-41cc-af8b-217c18018ef4","order_by":1,"name":"Masato Ando","email":"","orcid":"","institution":"Department of Patho-Functional Bioanalysis, Graduate School of Pharmaceutical Sciences, Kyoto University","correspondingAuthor":false,"prefix":"","firstName":"Masato","middleName":"","lastName":"Ando","suffix":""},{"id":304324578,"identity":"8c44302e-28df-48fb-9429-6f6ec2e8cf0c","order_by":2,"name":"Hiroyuki Watanabe","email":"","orcid":"","institution":"Department of Patho-Functional Bioanalysis, Graduate School of Pharmaceutical Sciences, Kyoto University","correspondingAuthor":false,"prefix":"","firstName":"Hiroyuki","middleName":"","lastName":"Watanabe","suffix":""},{"id":304324579,"identity":"a54fea28-9966-4a96-a425-3dc0fc35cba5","order_by":3,"name":"Masahiro Ono","email":"","orcid":"","institution":"Department of Patho-Functional Bioanalysis, Graduate School of Pharmaceutical Sciences, Kyoto University","correspondingAuthor":false,"prefix":"","firstName":"Masahiro","middleName":"","lastName":"Ono","suffix":""}],"badges":[],"createdAt":"2024-05-16 16:37:02","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4432393/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4432393/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":57789985,"identity":"bd09fa5e-f506-464f-ab3f-22280abcdf59","added_by":"auto","created_at":"2024-06-05 17:19:18","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":7471,"visible":true,"origin":"","legend":"\u003cp\u003eChemical structure of [\u003csup\u003e99m\u003c/sup\u003eTc]Tc-(Ham-SCUE)\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e","description":"","filename":"OnlineFigure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4432393/v1/a80c2abd1e4a048f270d8f0a.png"},{"id":57789984,"identity":"456c38b9-1f6e-4ec0-b8fd-7fbd6b9f5930","added_by":"auto","created_at":"2024-06-05 17:19:18","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":5063,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e cellular binding of [\u003csup\u003e99m\u003c/sup\u003eTc]Tc-(Ham-SCUE)\u003csub\u003e2\u003c/sub\u003e in LNCaP and PC3 cells. Results are expressed as the mean ± standard error of six independent experiments.\u003c/p\u003e","description":"","filename":"OnlineFigure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4432393/v1/556e4778599d732318eff8bc.png"},{"id":57789987,"identity":"2783a413-78ad-484d-98dc-3b1c319ca5e5","added_by":"auto","created_at":"2024-06-05 17:19:18","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":11523,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eIn vivo\u003c/em\u003e blocking study of [\u003csup\u003e99m\u003c/sup\u003eTc]Tc-(Ham-SCUE)\u003csub\u003e2\u003c/sub\u003e. All mice were sacrificed at 120 min post-injection of [\u003csup\u003e99m\u003c/sup\u003eTc]Tc-(Ham-SCUE)\u003csub\u003e2\u003c/sub\u003e. Results are expressed as the mean ± standard deviation of five mice. *Values are expressed as the % injected dose (%ID). Student’s \u003cem\u003et\u003c/em\u003e-test was performed to assess the significance of differences between 2-PMPA pretreatment [2-PMPA(+)] and nontreatment [2-PMPA(-)] groups. #: p\u0026lt;0.05, ##: p\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"OnlineFigure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4432393/v1/fe2401bdc8975480bae33942.png"},{"id":57790830,"identity":"1fa7f209-60e7-44a0-b1f1-7a9031fe2912","added_by":"auto","created_at":"2024-06-05 17:27:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":380790,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4432393/v1/b5d0388e-0f1d-4c67-9fbe-795fef2d9a60.pdf"},{"id":57790829,"identity":"f0d4b168-69e1-4d89-a6dc-a1f48078acdc","added_by":"auto","created_at":"2024-06-05 17:27:18","extension":"doc","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":1598976,"visible":true,"origin":"","legend":"","description":"","filename":"99mTcHamSCUE2supplementarydatafinal.doc","url":"https://assets-eu.researchsquare.com/files/rs-4432393/v1/bf33739974248d8ec9f6f28e.doc"}],"financialInterests":"","formattedTitle":"Novel technetium-99m-labeled bivalent PSMA-targeting probe based on hydroxamamide chelate for diagnosis of prostate cancer","fulltext":[{"header":"Introduction","content":"\u003cp\u003eProstate cancer (PC) is the most common cancer in men, and many cases are associated with a poor prognosis due to residual disease, recurrence, or metastasis after treatment. For appropriate treatment, it is important to distinguish between low- and high-risk tumors that progress slowly and rapidly, respectively, in addition to detecting the site of disease by imaging, early prediction and monitoring of the treatment response, early diagnosis of recurrence, and accurate re-staging in the event of recurrence. Over the past decade, prostate-specific membrane antigen (PSMA), also known as glutamate carboxypeptidase-II, has been focused on as a promising biomarker for the diagnosis of and subsequent therapy for PC by nuclear medical techniques [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. For PSMA-specific drug delivery, asymmetric urea compounds (glutamate/cystatin-urea-glutamate, or E/CUE) have been widely used as targeting ligands owing to their high-level and specific ability to bind to PSMA. Previously, our group reported that the affinity of CUE for PSMA could be enhanced by adding a succinimidyl moiety induced by reacting the thiol group of CUE with a maleimide moiety [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. In addition, single-photon emission computerized tomography (SPECT)/positron emission tomography (PET) probes based on that succinimidyl-CUE moiety, known as \u0026ldquo;SCUE,\u0026rdquo; showed favorable properties for SPECT/PET-PSMA imaging in preclinical and clinical studies [\u003cspan additionalcitationids=\"CR4 CR5\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRecently, we developed a novel bifunctional chelate, \u0026ldquo;Ham-Mal,\u0026rdquo; composed of a hydroxamamide amide (Ham) and maleimide moiety [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Ham, a thiol-free chelator of oxo-technetium (V) [Tc(V)], is known to form a \u003csup\u003e99m\u003c/sup\u003eTc-complex under moderate reaction conditions, making it possible to rapidly produce \u003csup\u003e99m\u003c/sup\u003eTc-labeled compounds with a bivalent targeting scaffold because it forms \u003csup\u003e99m\u003c/sup\u003eTc-complexes with a metal-to-ligand ratio of 1:2 [\u003cspan additionalcitationids=\"CR9 CR10\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. In this study, we developed a novel \u003csup\u003e99m\u003c/sup\u003eTc-labeled probe, \u0026ldquo;[\u003csup\u003e99m\u003c/sup\u003eTc]Tc-(Ham-SCUE)\u003csub\u003e2\u003c/sub\u003e,\u0026rdquo; composed of technetium-99m and two SCUEs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), and evaluated its property as a SPECT imaging probe targeting high PSMA-expressing PC.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of Ham-SCUE and [\u003csup\u003e99m\u003c/sup\u003eTc]Tc-(Ham-SCUE)\u003csub\u003e2\u003c/sub\u003e\u003c/h2\u003e \u003cp\u003eThe precursor \u0026ldquo;Ham-SCUE\u0026rdquo; and [\u003csup\u003e99m\u003c/sup\u003eTc]Tc-(Ham-SCUE)\u003csub\u003e2\u003c/sub\u003e were prepared using our previously reported method with some modifications [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The detailed process is described in Supplementary Materials and Methods.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eIn vitro cell binding assay\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003ein vitro\u003c/em\u003e cellular binding assay of [\u003csup\u003e99m\u003c/sup\u003eTc]Tc-(Ham-SCUE)\u003csub\u003e2\u003c/sub\u003e was performed with LNCaP prostate adenocarcinoma cells (high PSMA-expressing cells) and PC3 human prostate carcinoma cells (low PSMA-expressing cells) by the method reported previously, with some modifications [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The detailed process is described in Supplementary Materials and Methods.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eIn vivo biodistribution\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003ein vivo\u003c/em\u003e biodistribution study of [\u003csup\u003e99m\u003c/sup\u003eTc]Tc-(Ham-SCUE)\u003csub\u003e2\u003c/sub\u003e was performed with LNCaP/PC3 xenografted mice using the method reported previously with some modifications [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The detailed process is described in Supplementary Materials and Methods.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eStatistics\u003c/h2\u003e \u003cp\u003eData are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;S.E.M. (for the \u003cem\u003ein vitro\u003c/em\u003e study) or the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;S.D. (for the \u003cem\u003ein vivo\u003c/em\u003e study). Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test was used to assess the significance of differences with JMP 17 software (SAS Institute Inc., Cary, NC, USA). Differences at the 95% confidence level (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) were considered significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eSynthesis of Ham-SCUE\u003c/h2\u003e \u003cp\u003eHam-SCUE was obtained by reacting Ham-Mal with CUE, with a yield of 53% (Supplementary File, Scheme S1).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eRadiolabeling of [\u003csup\u003e99m\u003c/sup\u003eTc]Tc-(Ham-SCUE)\u003csub\u003e2\u003c/sub\u003e\u003c/h2\u003e \u003cp\u003e \u003csup\u003e99m\u003c/sup\u003eTc labeling of Ham-SCUE was performed. [\u003csup\u003e99m\u003c/sup\u003eTc]Tc-(Ham-SCUE)\u003csub\u003e2\u003c/sub\u003e was acquired by reacting Ham-SCUE with \u003csup\u003e99m\u003c/sup\u003eTc pertechnetate and tin(II) tartrate hydrate as a reducing agent in the solvent of phosphate buffer (pH 7.4) (Supplementary File, Scheme S2). [\u003csup\u003e99m\u003c/sup\u003eTc]Tc-(Ham-SCUE)\u003csub\u003e2\u003c/sub\u003e was acquired with a radiochemical yield of 56.0\u0026thinsp;\u0026plusmn;\u0026thinsp;6.2% and radiochemical purity of over 95% (Supplementary File, Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eIn vitro cellular uptake study\u003c/h2\u003e \u003cp\u003eCellular uptake of [\u003csup\u003e99m\u003c/sup\u003eTc]Tc-(Ham-SCUE)\u003csub\u003e2\u003c/sub\u003e is expressed as the %dose/mg protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The radioactivity of LNCap cells (high PSMA expression) was significantly higher than that of PC3 cells (low PSMA expression) (LNCaP: 11.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.71 vs. PC3: 1.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13%dose/mg protein, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and significantly reduced by pretreatment with excess 2-PMPA, a well-known PSMA inhibitor (2.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37%dose/mg protein, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo quantitatively evaluate the binding affinity of [\u003csup\u003e99m\u003c/sup\u003eTc]Tc-(Ham-SCUE)\u003csub\u003e2\u003c/sub\u003e to PSMA, binding inhibition experiments were performed in the presence of various concentrations of 2-PMPA on LNCaP cells. As a result, the accumulation of [\u003csup\u003e99m\u003c/sup\u003eTc]Tc-(Ham-SCUE)\u003csub\u003e2\u003c/sub\u003e on LNCaP cells decreased in a concentration-dependent manner with 2-PMPA, and the 50% inhibitory concentration (IC\u003csub\u003e50\u003c/sub\u003e) of 2-PMPA was 245\u0026thinsp;\u0026plusmn;\u0026thinsp;47 nM (Supplementary File, Figure S2).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eIn vivo biodistribution\u003c/h2\u003e \u003cp\u003eThe uptake levels of [\u003csup\u003e99m\u003c/sup\u003eTc]Tc-(Ham-SCUE)\u003csub\u003e2\u003c/sub\u003e in each organ are expressed as %ID/g, except for that in the stomach and thyroid, presented as %ID (Supplementary File, Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). In the normal organs, high radioactivity was observed in the kidney and spleen. As for tumor tissues, radioactivity of LNCaP was significantly higher than that of PC3 from 5 min after the administration of [\u003csup\u003e99m\u003c/sup\u003eTc]Tc-(Ham-SCUE)\u003csub\u003e2\u003c/sub\u003e. In addition, its accumulation level in LNCaP tumors was reduced by blocking with excess 2-PMPA at 120 min after administration [9.97\u0026thinsp;\u0026plusmn;\u0026thinsp;2.79%ID/g (non-blocking) vs. 2.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45%ID/g (blocking) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01)], while there was no significant difference in accumulation in PC3 tumors between non-blocking and blocking groups [1.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23%ID/g (non-blocking) vs. 1.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16%ID/g (blocking) (p\u0026thinsp;=\u0026thinsp;0.97)] (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we designed and synthesized a novel \u003csup\u003e99m\u003c/sup\u003eTc-labeled bivalent PSMA-targeting probe, \u0026ldquo;[\u003csup\u003e99m\u003c/sup\u003eTc]Tc-(Ham-SCUE)\u003csub\u003e2,\u003c/sub\u003e\u0026rdquo; based on a hydroxamamide chelate. The precursor \u0026ldquo;Ham-SCUE\u0026rdquo; was obtained successfully via the thiol-maleimide reaction of Ham-Mal and CUE (Supplementary File, Scheme S1), with a yield of 53%, which was comparable to our previous report [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The radiolabeling of [\u003csup\u003e99m\u003c/sup\u003eTc]Tc-(Ham-SCUE)\u003csub\u003e2\u003c/sub\u003e was performed using the method we established previously (Supplementary File, Scheme S2)[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. In the RP-HPLC analysis after the reaction, the disappearance of the peak derived from [\u003csup\u003e99m\u003c/sup\u003eTc]NaTcO\u003csub\u003e4\u003c/sub\u003e and the subsequent appearance of two new peaks were observed (Supplementary File, Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), suggesting that the chelation of Ham-SCUE and \u003csup\u003e99m\u003c/sup\u003eTc proceeded successfully since the \u003csup\u003e99m\u003c/sup\u003eTc-Ham complex is known to form two tautomers [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The radiochemical yield of [\u003csup\u003e99m\u003c/sup\u003eTc]Tc-(Ham-SCUE)\u003csub\u003e2\u003c/sub\u003e was 56%, which was also comparable with the other Ham-based \u003csup\u003e99m\u003c/sup\u003eTc labeled probes ([\u003csup\u003e99m\u003c/sup\u003eTc]Tc-(Ham-RGD)\u003csub\u003e2\u003c/sub\u003e: 49% [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]). Taking into account that the \u003csup\u003e99m\u003c/sup\u003eTc-labeling reaction proceeded under moderate conditions (simply incubating at room temperature for 10 min), these results suggest that [\u003csup\u003e99m\u003c/sup\u003eTc]Tc-(Ham-SCUE)\u003csub\u003e2\u003c/sub\u003e can be acquired easily with a favorable chemical yield.\u003c/p\u003e \u003cp\u003eIn the \u003cem\u003ein vitro\u003c/em\u003e study, [\u003csup\u003e99m\u003c/sup\u003eTc]Tc-(Ham-SCUE)\u003csub\u003e2\u003c/sub\u003e accumulated markedly in LNCaP (high PSMA-expressing) cells rather than in PC3 (low PSMA-expressing) cells, and its accumulation was blocked by 2-PMPA, a PSMA inhibitor (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). This suggests that [\u003csup\u003e99m\u003c/sup\u003eTc]Tc-(Ham-SCUE)\u003csub\u003e2\u003c/sub\u003e was taken up specifically in the high PSMA-expressing PC cells. In addition, we evaluated IC\u003csub\u003e50\u003c/sub\u003e of 2-PMPA against [\u003csup\u003e99m\u003c/sup\u003eTc]Tc-(Ham-SCUE)\u003csub\u003e2\u003c/sub\u003e whose value was 245 nM (Supplementary File, Figure S2), which was about 70 times higher than that of our previously reported \u003csup\u003e99m\u003c/sup\u003eTc-labeled probe \u0026ldquo;[\u003csup\u003e99m\u003c/sup\u003eTc]Tc-TMCE\u0026rdquo; with a monovalent SCUE moiety (3.4 nM)[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The bivalent targeting scaffold is considered to offer higher binding affinity than monovalent counterparts, and some conventional PET/SPECT imaging probes, including PSMA-targeting probes, are designed based on that concept to enhance their specificity for target biomolecules [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Therefore, [\u003csup\u003e99m\u003c/sup\u003eTc]Tc-(Ham-SCUE)\u003csub\u003e2\u003c/sub\u003e also matched the concept and thus could enhance the affinity for PSMA compared with probes we reported previously.\u003c/p\u003e \u003cp\u003eIn the \u003cem\u003ein vivo\u003c/em\u003e study, [\u003csup\u003e99m\u003c/sup\u003eTc]Tc-(Ham-SCUE)\u003csub\u003e2\u003c/sub\u003e showed high accumulation in LNCaP tumors compared with PC3 tumors from 5 min after i.v. administration (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), and its accumulation level was reduced by blocking with excess 2-PMPA at 120 min after administration (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), which suggests that [\u003csup\u003e99m\u003c/sup\u003eTc]Tc-(Ham-SCUE)\u003csub\u003e2\u003c/sub\u003e accumulated specifically in tumors with expression levels of PSMA. High-level radioactivity was also observed in the kidneys and spleen. Considering previous reports [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] and the suppression of radioactivity by pretreatment with 2-PMPA (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), this might be due to the high expression of PSMA in those organs. Regarding the stomach and thyroid, where \u003csup\u003e99m\u003c/sup\u003eTc ions abundantly accumulate, relatively low radioactivities were observed, which suggests that [\u003csup\u003e99m\u003c/sup\u003eTc]Tc-(Ham-SCUE)\u003csub\u003e2\u003c/sub\u003e did not release free \u003csup\u003e99m\u003c/sup\u003eTc in mice.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn this study, we successfully developed a novel \u003csup\u003e99m\u003c/sup\u003eTc-labeled bivalent PSMA-targeting probe, \u0026ldquo;[\u003csup\u003e99m\u003c/sup\u003eTc]Tc-(Ham-SCUE)\u003csub\u003e2\u003c/sub\u003e\u0026rdquo;, and confirmed its potential as a SPECT imaging probe for the diagnosis of high PSMA-expressing PC.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eThis study was supported by grants from the Japan Agency for Medical Research and Development (Numbers: 20ck0106426h0003 and JP24ama221604).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eL\u0026uuml;tje S, Heskamp S, Cornelissen AS, Poeppel TD, van den Broek SA, Rosenbaum-Krumme S, et al. PSMA Ligands for Radionuclide Imaging and Therapy of Prostate Cancer: Clinical Status. Theranostics. 2015;5(12):1388\u0026ndash;401.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMaurer T, Eiber M, Schwaiger M, Gschwend JE. Current use of PSMA-PET in prostate cancer management. Nat Rev Urol. 2016;13(4):226\u0026ndash;35.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHarada N, Kimura H, Ono M, Saji H. Preparation of asymmetric urea derivatives that target prostate-specific membrane antigen for SPECT imaging. J Med Chem. 2013;56(20):7890\u0026ndash;901.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHarada N, Kimura H, Onoe S, Watanabe H, Matsuoka D, Arimitsu K, et al. Synthesis and Biologic Evaluation of Novel 18F-Labeled Probes Targeting Prostate-Specific Membrane Antigen for PET of Prostate Cancer. J Nucl Med. 2016;57(12):1978\u0026ndash;84.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKimura H, Sampei S, Matsuoka D, Harada N, Watanabe H, Arimitsu K, et al. Development of (99m)Tc-labeled asymmetric urea derivatives that target prostate-specific membrane antigen for single-photon emission computed tomography imaging. Bioorg Med Chem. 2016;24(10):2251\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSaga T, Nakamoto Y, Ishimori T, Inoue T, Shimizu Y, Kimura H, et al. Initial evaluation of PET/CT with (18) F-FSU-880 targeting prostate-specific membrane antigen in prostate cancer patients. Cancer Sci. 2019;110(2):742\u0026ndash;50.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShimizu Y, Ando M, Iikuni S, Watanabe H, Ono M. Development of a hydroxamamide-based bifunctional chelating agent to prepare technetium-99m-labeled bivalent ligand probes. Sci Rep. 2021;11(1):18714.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNakayama M, Saigo H, Kai E, Koda A, Ozeki H, Harada K, et al. Hydroxamamide as a chelating moiety for the preparation of \u003csup\u003e99m\u003c/sup\u003eTc radiopharmaceuticals (I). Nucl Med Commun. 1992;13(6):445\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNakayama M, Saigo H, Koda A, Ozeki K, Harada K, Sugii A, et al. Hydroxamamide as a chelating moiety for the preparation of \u003csup\u003e99m\u003c/sup\u003eTc radiopharmaceuticals\u0026mdash;II. The \u003csup\u003e99m\u003c/sup\u003eTc complexes of hydroxamanide derivatives. Appl Radiat Isot. 1994;45(6):735\u0026ndash;40.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNakayama M, Xu LC, Koga Y, Harada K, Sugii A, Nakayama H, et al. Hydroxamamide as a chelating moiety for the preparation of 99mTc-radiopharmaceuticals III. Characterization of various 99mTc-hydroxamamides. Appl Radiat Isot. 1997;48(5):571\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eThipyapong K, Uehara T, Tooyama Y, Braband H, Alberto R, Arano Y. Insight into technetium amidoxime complex: oxo technetium(V) complex of N-substituted benzamidoxime as new basic structure for molecular imaging. Inorg Chem. 2011;50(3):992\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDai R, Cai Z, Hu R, Huang Y, Fu L, Yang J, et al. (177)Lu-Labeled Bivalent Ligands of Prostate-Specific Membrane Antigen for Endoradiotherapy of Prostate Cancer. Mol Pharm. 2024;21(2):883\u0026ndash;94.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"annals-of-nuclear-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"anme","sideBox":"Learn more about [Annals of Nuclear Medicine](http://link.springer.com/journal/12149)","snPcode":"12149","submissionUrl":"https://www.editorialmanager.com/anme/default2.aspx","title":"Annals of Nuclear Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Technetium-99m, PSMA, Hydroxamamide, 99mTc-labeled bivalent ligand probes","lastPublishedDoi":"10.21203/rs.3.rs-4432393/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4432393/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eObjective: \u003c/strong\u003eProstate-specific membrane antigen (PSMA) is a well-known biomarker of prostate cancer. Previously, our group reported that the succinimidyl-cystatin-urea-glutamate (SCUE) moiety has a high affinity for PSMA. In this study, we developed the novel technetium-99m-labeled PSMA-targeting probe “[\u003csup\u003e99m\u003c/sup\u003eTc]Tc-(Ham-SCUE)\u003csub\u003e2\u003c/sub\u003e” based on a hydroxamamide chelate with a bivalent SCUE and evaluated its potential as a SPECT imaging probe for the diagnosis of PSMA-expressing prostate cancer.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e Ham-SCUE was synthesized by a one-step reaction with Ham-Mal and cysteine-urea-glutamine. Then, Ham-SCUE was reacted with [\u003csup\u003e99m\u003c/sup\u003eTc]NaTcO\u003csub\u003e4\u003c/sub\u003e for 10 min at room temperature to obtain [\u003csup\u003e99m\u003c/sup\u003eTc]Tc-(Ham-SCUE)\u003csub\u003e2\u003c/sub\u003e. [\u003csup\u003e99m\u003c/sup\u003eTc]Tc-(Ham-SCUE)\u003csub\u003e2 \u003c/sub\u003ewas added to LNCaP (high PSMA expression) cells or PC3 (low PSMA expression) cells, and their radioactivity was measured 60 min after administration. The blocking study was performed by co-incubation of LNCaP cells with various concentrations of 2-PMPA (a PSMA inhibitor) for 15 min before adding [\u003csup\u003e99m\u003c/sup\u003eTc]Tc-(Ham-SCUE)\u003csub\u003e2\u003c/sub\u003e. The biodistribution of [\u003csup\u003e99m\u003c/sup\u003eTc]Tc-(Ham-SCUE)\u003csub\u003e2\u003c/sub\u003e in LNCaP/PC3 dual xenografted C.B.-17/Icr scid/scid Jcl mice was evaluated for 120 min after intravenous injection. The blocking study was performed by pretreatment of mice with 2-PMPA (10 mg/kg weight).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003e[\u003csup\u003e99m\u003c/sup\u003eTc]Tc-(Ham-SCUE)\u003csub\u003e2\u003c/sub\u003e was acquired at radiochemical yields of 56% with a radiochemical purity of over 95%. The cellular uptake level of [\u003csup\u003e99m\u003c/sup\u003eTc]Tc-(Ham-SCUE)\u003csub\u003e2\u003c/sub\u003e by LNCaP cells was significantly higher than that by PC3 cells (LNCaP: 11.12 ± 0.71 vs. PC3: 1.40 ± 0.13 %dose/mg protein, p\u0026lt;0.01), and the uptake was significantly suppressed by pretreatment with 2-PMPA (2.56 ± 0.37 %dose/mg protein, p\u0026lt;0.05). IC\u003csub\u003e50\u003c/sub\u003e of 2-PMPA was 245 ± 47 nM. In the \u003cem\u003ein vivo\u003c/em\u003e study, the radioactivity of LNCaP tumor tissue was significantly higher than that of PC3 tumor tissue at 120 min after the administration of [\u003csup\u003e99m\u003c/sup\u003eTc]Tc-(Ham-SCUE)\u003csub\u003e2\u003c/sub\u003e (LNCaP: 9.97 ± 2.79, PC3: 1.16 ± 0.23 %ID/g, p\u0026lt;0.01), and was suppressed by pretreatment with 2-PMPA (2.50 ± 0.45 %ID/g, p\u0026lt;0.01).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion: \u003c/strong\u003e[\u003csup\u003e99m\u003c/sup\u003eTc]Tc-(Ham-SCUE)\u003csub\u003e2\u003c/sub\u003e has the potential to be a SPECT imaging agent for diagnosing high PSMA-expressing prostate cancer.\u003c/p\u003e","manuscriptTitle":"Novel technetium-99m-labeled bivalent PSMA-targeting probe based on hydroxamamide chelate for diagnosis of prostate cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-05 17:19:13","doi":"10.21203/rs.3.rs-4432393/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2024-05-21T22:33:54+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-05-20T03:24:47+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-05-17T00:07:11+00:00","index":"","fulltext":""},{"type":"submitted","content":"Annals of Nuclear Medicine","date":"2024-05-16T12:36:49+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"annals-of-nuclear-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"anme","sideBox":"Learn more about [Annals of Nuclear Medicine](http://link.springer.com/journal/12149)","snPcode":"12149","submissionUrl":"https://www.editorialmanager.com/anme/default2.aspx","title":"Annals of Nuclear Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"95507ba4-8af9-4acb-9020-cc8cb3ed4eb7","owner":[],"postedDate":"June 5th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-07-03T00:39:58+00:00","versionOfRecord":[],"versionCreatedAt":"2024-06-05 17:19:13","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4432393","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4432393","identity":"rs-4432393","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}