.2019 Sep 14;11(9):1371.

doi: 10.3390/cancers11091371.

Bispecific GRPR-Antagonistic Anti-PSMA/GRPR Heterodimer for PET and SPECT Diagnostic Imaging of Prostate Cancer

Bogdan Mitran¹, Zohreh Varasteh^{2 3}, Ayman Abouzayed⁴, Sara S Rinne⁵, Emmi Puuvuori⁶, Maria De Rosa^{7 8}, Mats Larhed^{9 10}, Vladimir Tolmachev¹¹, Anna Orlova^{12 13}, Ulrika Rosenström¹⁴

Affiliations

¹ Department of Medicinal Chemistry, Uppsala University, 751 23 Uppsala, Sweden. bogdan.mitran@ilk.uu.se.
² Department of Medicinal Chemistry, Uppsala University, 751 23 Uppsala, Sweden. zohreh.varasteh@tum.de.
³ Department of Nuclear Medicine, Klinikum rechts der Isar der TUM, 81675 Munich, Germany. zohreh.varasteh@tum.de.
⁴ Department of Medicinal Chemistry, Uppsala University, 751 23 Uppsala, Sweden. ayman.abouzayed@ilk.uu.se.
⁵ Department of Medicinal Chemistry, Uppsala University, 751 23 Uppsala, Sweden. sara.rinne@ilk.uu.se.
⁶ Department of Medicinal Chemistry, Uppsala University, 751 23 Uppsala, Sweden. emmi.puuvuori@ilk.se.
⁷ Department of Medicinal Chemistry, Uppsala University, 751 23 Uppsala, Sweden. mderosa@fondazionerimed.com.
⁸ Drug Discovery Unit, Ri.MED Foundation, 90133 Palermo, Italy. mderosa@fondazionerimed.com.
⁹ Department of Medicinal Chemistry, Uppsala University, 751 23 Uppsala, Sweden. mats.larhed@ilk.uu.se.
¹⁰ Science for Life Laboratory, Department of Medicinal Chemistry, Uppsala University, 751 23 Uppsala, Sweden. mats.larhed@ilk.uu.se.
¹¹ Department of Immunology, Genetics and Pathology, Uppsala University, 751 23 Uppsala, Sweden. vladimir.tolmachev@igp.uu.se.
¹² Department of Medicinal Chemistry, Uppsala University, 751 23 Uppsala, Sweden. anna.orlova@ilk.uu.se.
¹³ Science for Life Laboratory, Department of Medicinal Chemistry, Uppsala University, 751 23 Uppsala, Sweden. anna.orlova@ilk.uu.se.
¹⁴ Department of Medicinal Chemistry, Uppsala University, 751 23 Uppsala, Sweden. ulrika.rosenstrom@ilk.uu.se.

PMID:31540122
PMCID: PMC6771040
DOI: 10.3390/cancers11091371

Bispecific GRPR-Antagonistic Anti-PSMA/GRPR Heterodimer for PET and SPECT Diagnostic Imaging of Prostate Cancer

Bogdan Mitran et al. Cancers (Basel).2019.

.2019 Sep 14;11(9):1371.

doi: 10.3390/cancers11091371.

Authors

Affiliations

¹ Department of Medicinal Chemistry, Uppsala University, 751 23 Uppsala, Sweden. bogdan.mitran@ilk.uu.se.
² Department of Medicinal Chemistry, Uppsala University, 751 23 Uppsala, Sweden. zohreh.varasteh@tum.de.
³ Department of Nuclear Medicine, Klinikum rechts der Isar der TUM, 81675 Munich, Germany. zohreh.varasteh@tum.de.
⁴ Department of Medicinal Chemistry, Uppsala University, 751 23 Uppsala, Sweden. ayman.abouzayed@ilk.uu.se.
⁵ Department of Medicinal Chemistry, Uppsala University, 751 23 Uppsala, Sweden. sara.rinne@ilk.uu.se.
⁶ Department of Medicinal Chemistry, Uppsala University, 751 23 Uppsala, Sweden. emmi.puuvuori@ilk.se.
⁷ Department of Medicinal Chemistry, Uppsala University, 751 23 Uppsala, Sweden. mderosa@fondazionerimed.com.
⁸ Drug Discovery Unit, Ri.MED Foundation, 90133 Palermo, Italy. mderosa@fondazionerimed.com.
⁹ Department of Medicinal Chemistry, Uppsala University, 751 23 Uppsala, Sweden. mats.larhed@ilk.uu.se.
¹⁰ Science for Life Laboratory, Department of Medicinal Chemistry, Uppsala University, 751 23 Uppsala, Sweden. mats.larhed@ilk.uu.se.
¹¹ Department of Immunology, Genetics and Pathology, Uppsala University, 751 23 Uppsala, Sweden. vladimir.tolmachev@igp.uu.se.
¹² Department of Medicinal Chemistry, Uppsala University, 751 23 Uppsala, Sweden. anna.orlova@ilk.uu.se.
¹³ Science for Life Laboratory, Department of Medicinal Chemistry, Uppsala University, 751 23 Uppsala, Sweden. anna.orlova@ilk.uu.se.
¹⁴ Department of Medicinal Chemistry, Uppsala University, 751 23 Uppsala, Sweden. ulrika.rosenstrom@ilk.uu.se.

PMID:31540122
PMCID: PMC6771040
DOI: 10.3390/cancers11091371

Abstract

Simultaneous targeting of the prostate-specific membrane antigen (PSMA) and gastrin-releasing peptide receptor (GRPR) could improve the diagnostic accuracy in prostate cancer (PCa). The aim of this study was to develop a PSMA/GRPR-targeting bispecific heterodimer for SPECT and positron emission tomography (PET) diagnostic imaging of PCa. The heterodimer NOTA-DUPA-RM26 was produced by manual solid-phase peptide synthesis. NOTA-DUPA-RM26 was labeled with¹¹¹In and⁶⁸Ga, with yields >98%, and demonstrated a high stability and binding specificity to PSMA and GRPR. IC₅₀ values for^natIn-NOTA-DUPA-RM26 were 4 ± 1 nM towards GRPR and 824 ± 230 nM towards PSMA. An in vivo binding specificity 1 h pi of¹¹¹In-NOTA-DUPA-RM26 in PC3-PIP-xenografted mice demonstrated partially blockable tumor uptake when co-injected with an excess of PSMA- or GRPR-targeting agents. Simultaneous co-injection of both agents induced pronounced blocking. The biodistribution of¹¹¹In-NOTA-DUPA-RM26 and⁶⁸Ga-NOTA-DUPA-RM26 revealed fast activity clearance from the blood and normal organs via the kidneys. Tumor uptake exceeded normal organ uptake for both analogs 1 h pi.⁶⁸Ga-NOTA-DUPA-RM26 had a significantly lower tumor uptake (8 ± 2%ID/g) compared to¹¹¹In-NOTA-DUPA-RM26 (12 ± 2%ID/g) 1 h pi. Tumor-to-organ ratios increased 3 h pi, but decreased 24 h pi, for¹¹¹In-NOTA-DUPA-RM26. MicroPET/CT and microSPECT/CT scans confirmed biodistribution data, suggesting that⁶⁸Ga-NOTA-DUPA-RM26 and¹¹¹In-NOTA-DUPA-RM26 are suitable candidates for the imaging of GRPR and PSMA expression in PCa shortly after administration.

Keywords: GRPR; PET; PSMA; SPECT; heterodimer; molecular imaging; prostate cancer.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

**Figure 1**
(A) Binding specificity of¹¹¹In-6 (left) and⁶⁸Ga-6 (right) to PC3-PIP cells (prostate-specific membrane antigen (PSMA) positive/gastrin-releasing peptide receptor (GRPR) positive). Cell containing dishes (n = 3) were incubated with radiolabeled conjugates (1 nM). One set of dishes was pre-saturated with an excess amount (300 nM) of non-labeled PSMA-617 (+PSMA-617), or RM26 (+RM26), or both of them (+both). Cell-associated activity was normalized to control samples. Data are presented as the average ± standard deviation. (B) Binding inhibition of¹¹¹In-NOTA-PEG₆-RM26 (left, PSMA was blocked with 1 mM of PSMA-11 in all samples) and¹¹¹In-PSMA-617 (right, GRPR was blocked with 1 mM solution of RM26 in all samples). Data are presented as the mean value ± standard deviation. (C) Cellular processing of¹¹¹In-6 on PC3-PIP cells under continuous incubation with 2 nM of labeled heterodimer at 37 °C. Cell-associated activity was normalized to the maximum uptake. Data are presented as the average ± standard deviation.

**Figure 2**
(A) In vivo biodistribution of¹¹¹In-6 (left) and⁶⁸Ga-6 (right) (50 pmol/animal) over time in PC3-PIP-xenografted BALB/c nu/nu mice. Activity uptake in tissues was calculated as the percent of injected dose per tissue weight (%ID/g). (B) Tumor-to-normal-tissues ratios for¹¹¹In-6 (left) and⁶⁸Ga-6 (right). Data are presented as the average ± standard deviation.

**Figure 3**
In vivo specificity of¹¹¹In-6 (A) and⁶⁸Ga-6 (B) tested in BALB/c nu/nu mice bearing PC3-PIP-xenografts, 1 h pi. Mice were intravenously injected with 50 pmol of¹¹¹In-6 or⁶⁸Ga-6 alone or together with 1.5 nmol non-labeled RM26, 1.5 nmol non-labeled PSMA-617, or both. Activity uptake in tissues was calculated as the percent of injected dose per tissue weight (%ID/g). Data are presented as the average ± standard deviation.

**Figure 4**
(A) micro positron emission tomography (microPET)/CT and (B) microSPECT/CT MIP images of PC3-PIP-xenografted mice (PSMA positive/GRPR positive) after an iv injection of⁶⁸Ga-6 and¹¹¹In-6. Blocked animals were co-injected with PSMA-617 for the blocking of PSMA and RM26 for the blocking of GRPR, or both.

**Scheme 1**
Synthesis of the bispecific heterodimer6. (I) triphosgene, TEA, DCM, −78 °C to rt; (II) L-Glu(OBn)-OtBu⋅HCl, TEA, −78 °C to rt; (III) H₂, Pd/C, MeOH, (IV) Fmoc-NH-dPEG(6)-COOH, PyBOP, DIEA, DMF; (V) Fmoc-Lys(Alloc)-OH, PyBOP, DIEA, DMF; (VI) 20% (v/v) piperidine in DMF; (VII) Fmoc-8-Aoc-OH, PyBOP, DIEA, DMF; (VIII) 1, PyBOP, DIEA, DMF; (IX) Pd(PPh₃)₄, PhSiH₃, DCM; (X) NOTA-bis(tBu) ester, PyBOP, DIEA, DMF; (XI) TFA, H₂O, thioanisole, TES.

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References

1. Litwin M.S., Tan H.J. The Diagnosis and Treatment of Prostate Cancer: A Review. JAMA. 2017;317:2532–2542. doi: 10.1001/jama.2017.7248. - DOI - PubMed
1. Spick C., Herrmann K., Czernin J. Evaluation of Prostate Cancer with 11C-Acetate PET/CT. J. Nucl. Med. 2016;57:30–37. doi: 10.2967/jnumed.115.169599. - DOI - PubMed
1. Mansi R., Fleischmann A., Mäcke H.R., Reubi J.C. Targeting GRPR in urological cancers--from basic research to clinical application. Nat. Rev. Urol. 2013;10:235–244. doi: 10.1038/nrurol.2013.42. - DOI - PubMed
1. Schwarzenboeck S.M., Rauscher I., Bluemel C., Fendler W.P., Rowe S.P., Pomper M.G., Asfhar-Oromieh A., Herrmann K., Eiber M. PSMA Ligands for PET Imaging of Prostate Cancer. J. Nucl. Med. 2017;58:1545–1552. doi: 10.2967/jnumed.117.191031. - DOI - PubMed
1. Czarniecki M., Mena E., Lindenberg L., Cacko M., Harmon S., Radtke J.P., Giesel F., Turkbey B., Choyke P.L. Keeping up with the prostate-specific membrane antigens (PSMAs): An introduction to a new class of positron emission tomography (PET) imaging agents. Transl. Androl. Urol. 2018;7:831–843. doi: 10.21037/tau.2018.08.03. - DOI - PMC - PubMed

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Bispecific GRPR-Antagonistic Anti-PSMA/GRPR Heterodimer for PET and SPECT Diagnostic Imaging of Prostate Cancer

Affiliations

Bispecific GRPR-Antagonistic Anti-PSMA/GRPR Heterodimer for PET and SPECT Diagnostic Imaging of Prostate Cancer

Authors

Affiliations

Abstract

Conflict of interest statement

Figures

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References

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