Ga]Ga-PSMA-11 in Human Plasma and Human Microsomes New Developments in the Field of anti-PSMA Tracers for mCRPC and Bioanalysis of [

7-7-2017

New Developments in the Field of anti-PSMA Tracers for mCRPC and Bioanalysis of [ ⁶⁸ Ga]Ga-PSMA-11 in

Human Plasma and Human Microsomes

Kamp, J

UNIVERSITAIR MEDISCH CENTRUM GRONINGEN

Kamp, J

MASTER THESIS MEDICAL PHARMACEUTICAL SCIENCES

Medical Pharmaceutical Sciences Faculty of Science & Engineering

Master Thesis:

New Developments in the Field of anti-PSMA Tracers for mCRPC and Bioanalysis of

[ ⁶⁸ Ga]Ga-PSMA-11 in Human Plasma and Human Microsomes

Author:

Jasper Kamp (s1853155) July 2017

Supervisors:

Dr. G. Luurtsema (UMCG) Dr. H.H. Boersma (UMCG)

MPS Examiner:

Dr. ir. I.A.M. de Graaf (RuG)

Kamp, J

MASTER THESIS MEDICAL PHARMACEUTICAL SCIENCES

Table of Contents

Prologue ... 6

General introduction ... 7

References ... 8

Chapter I ... 10

Abstract ... 11

1. Introduction... 11

2. Methods ... 12

2.1 Literature acquisition ... 12

2.2 PubMed searches ... 12

2.3 Inclusion and Exclusion criteria ... 12

2.4 Literature selection ... 13

3. Results... 13

4.

Cu-labeled tracers for prostate cancer imaging and therapy ... 13

4.1 Recent developments in

Cu-labeling and Copper-64 anti-PSMA tracers ... 13

4.2 Preclinical studies ... 14

4.3 Clinical studies ... 15

4.4 Comparison with the gold standard: [

Ga]Ga-PSMA-11 ... 15

5.

F-labeled tracers for prostate cancer imaging ... 17

5.1 Recent developments in

F-labeling and Fluorine-18 anti-PSMA tracers ... 17

5.2 Pre-clinical studies ... 18

5.3 Clinical studies ... 20

5.4 Comparison with the gold standard: [

Ga]Ga-PSMA-11 ... 21

6.

Lu-labeled tracers for prostate cancer imaging and therapy ... 22

6.1 Recent developments in

Lu-labeling and

Lu- PSMA tracers ... 22

6.2 Pre-clinical studies ... 23

6.3 Clinical studies ... 24

7. Outcomes and future perspectives ... 25

7.1 Theranostic couples ... 25

7.2 Shortcomings in the current tracer development process ... 25

7.3 Future perspectives ... 28

8. Conclusions ... 28

9. References ... 29

Chapter II ... 31

Abstract ... 32

1. Introduction... 32

2. Methods ... 33

2.1 Software ... 33

2.2 [

Ga]Ga-PSMA-11 preparation ... 33

2.3 Bioanalysis ... 33

2.4 Metabolism and stability ... 36

3. Results... 37

3.1 Bioanalysis ... 37

3.2 Metabolism and Stability ... 47

4. Discussion... 48

4.1 Bioanalysis ... 50

4.2 Metabolism ... 53

4.3 Study limitations and Future studies ... 54

5. Conclusions ... 56

6. References ... 56

Appendices ... I

Appendix I: PSMA-11 calibration chromatograms and calculations ... II

Appendix II: SOP microsome studies ... XI

Appendix III: Overview sample sequence for recovery studies ... XIX

Appendix IV: Raw data recovery studies ... XX

Appendix V: LC/MS-TOF settings ... XXIII

Appendix VI: LC/MS/MS settings ... XXIV

Appendix VII: LC/MS/MS transitions ... XXV

Appendix VIII: LC/MS/MS mass chromatograms: XBridge column ... XXVIII

Appendix IX: UPLC chromatograms PSMA-11 and

Ga-PSMA-11 ... XXXI

Appendix X: Raw data in vitro metabolic stability studies ... XXXII

Appendix XI: Raw data patient studies ... XL

Appendix XII: LC/MS-TOF calibration data with

Ga-PSMA-11 ... XLV

Kamp, J

MASTER THESIS MEDICAL PHARMACEUTICAL SCIENCES

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Prologue

lthough prostate cancer is a disease only seen in (elderly) men, it is responsible for a high dis- ease burden on our society. In 2012, 1,1 million new cases were diagnosed worldwide and – despite several major advances in prostate cancer diagnosis, staging and therapy – an estimated 307 000 men died because of this disease worldwide in 2012.

These numbers indicate the need for improved tools to battle prostate cancer. One of these tools is positron emission tomography (PET) combined with computed tomography (CT). PET/CT is a relative- ly new method that can be used for the diagnosis and staging of prostate cancer. This method relies on the accumulation of a radioactive tracer in target tissues, which can then be detected and localised with the PET scan. The combination of the PET with CT enables medical professionals to obtain de- tailed information about tumour location and/or staging.

In this thesis, we will be discussing several of the most promising new developments in the field of radio tracers for prostate cancer and try to elucidate what makes these new developments different from the current gold standard for prostate cancer PET imaging: [⁶⁸Ga]Ga-PSMA-11.

The second chapter of this thesis will discuss several of the fist studies concerning the bioanalysis and metabolism of [⁶⁸Ga]Ga-PSMA-11. This tracer was first described in 2012, after which it evolved ̶ only within a matter of years ̶ to become the gold standard for metastatic prostate cancer PET imag- ing.

Eventually, the findings discussed in this thesis may serve as a basis for future studies, aimed at de- veloping new PET tracers for prostate cancer or studies that aim to increase our knowledge concerning tracer metabolism.

A

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General introduction

rostate cancer (PCa) is the second most often diagnosed type of cancer worldwide, and it is the most common type of non-skin cancer in males above 70 years of age (1,2) with an estimated 1,1 million new cases and 307 000 deaths in 2012 (3). Geographical and racial/ethnic factors are shown to be important variables for the incidence rates of PCa, with the highest incidence rates in Af- ro-Americans and the lowest incidence rates in Asians (1). The incidental detection of PCa via Trans Urethral Resection of the Prostate (TURP) in patients with benign prostate hyperplasia and later the upcoming of prostate specific antigen (PSA) testing, have both influenced the temporary increase in the detection rates of PCa in high-income countries (1,4,5).

Epidemiological studies have shown strong evidence for the role of genetic predisposition to PCa.

Approximately 9% of the PCa patients have a form of hereditary PCa. Furthermore, patients with he- reditary PCa often show an earlier upset of the disease than patients who do not suffer from hereditary PCa (6). In addition, other, non-genetic factors, such as sexual behaviour, diet and alcohol consumption have shown to influence the disease progression (7,8). However, there is yet insufficient evidence to define certain lifestyle changes that would decrease the risk of developing clinical PCa (2).

To be able to compare different patients and treatment outcomes, several staging systems are pro- posed for PCa. A widely-used method for staging PCa is the Gleason grading. The Gleason grading sys- tem is based solely on the architectural pattern of the PCa tumour and is determined by the most abundant pattern and the second most abundant pattern (9). When more than two types of grades are found in tumour tissue, the Gleason score is determined by the highest grade and by the most abun- dant grade (2).

PCa is most commonly found in patients by a digital rectal examination (DRE) or by elevated pros- tate specific antigen (PSA) levels. Approximately 18% of the PCa cases is diagnosed by DRE only, with- out consideration of PSA levels (10). Since most PCas are situated in the peripheral zone (zone of the prostate located closest to the rectum), these cancers may be detected by DRE when the volume of the cancer is ≥0,2 ml (2). Studies showed that PCas found by DRE are often associated with aggressive forms of PCa (Gleason score ≥ 7)(11,12). Therefore, an abnormal DRE is an indication for a prostate biopsy (2). A prostate biopsy may be indicated when abnormal PSA levels are found or/and when abnormalities are found by DRE (2) to confirm the diagnosis of PCa. In addition, a risk assessment can be performed to reduce the number of unnecessary biopsies (13).

The introduction of PSA as a biomarker for the diagnosis has drastically changed the way of diag- nosing PCa. PSA is an antigen produced by the prostate, which generally reaches blood concentrations of maximum 4 ng/ml in men without PCa (14,15). However, studies have shown that in some cases no PSA elevation is seen, despite the presence of PCa (15,16). In contrast, since PSA is an organ specific marker, but not a cancer specific marker, elevated PSA levels may also be found in patients with non- malignant conditions of the prostate, such as BPH (2).

Another important tool for diagnosis and staging of PCa is imaging. Multiparametric magnetic reso- nance imaging (mpMRI) is a sensitive tool for the detection and localization of high risk PCa (Gleason ≥ 7)(17,18). Although mpMRI currently is an important method for the staging and the localization of specific types of high-risk PCa, the results are still largely influenced by the experience of the reader (2). This is shown by the large inter-reader variability (19). Therefore, new and more sensitive imaging methods are needed, to be able to accurately localize and stage PCa lesions.

A relatively new development in the field of PCa imaging is the upcoming of PET/CT. Examples of PCa tracers for PET/CT are ¹¹C- or ¹⁸F-Choline, ¹⁸F-FDG or ¹⁸F-acetate (20,21), all of which are targeting tumour tissue, based on metabolic changes in these tissues. Although PET/CT imaging with such trac- ers can provide useful information about tumour biology and diversity (21), the sensitivity and speci- ficity of many of these radiotracers are highly variable between studies (2,21). An example of a tracer which shows such a high variability, is choline PET/CT, for which the sensitivity for the detection of nodal metastases varies from 10% to 73%(2).

Targeting the prostate specific membrane antigen (PSMA), also known as glutamate carboxypepti- dase II (22), is shown to be a promising method to specifically target PCa tumours (21). PSMA is a transmembrane protein of which the largest part, which also includes the binding motif, is situated in the extracellular domain (23). Since almost 95% of the PCa cells overexpress PSMA (24), it is a promis- ing target for tracers. Especially the ⁶⁸Ga-labeled small molecule PSMA inhibitor showed promising results with a higher sensitivity than ¹⁸F-choline PET/CT in patients with recurrent PCa (21,25). This small molecule PSMA inhibitor , also known as PSMA-11, consists out of a Glu-urea-Lys motif, com- bined with a N,N′-bis [2-hydroxy-5-(carboxyethyl)benzyl] ethylenediamine- N,N′- diacetic acid (HBED- CC) chelator , to bind the ⁶⁸Ga (fig. 1)(26).

P

Kamp, J

MASTER THESIS MEDICAL PHARMACEUTICAL SCIENCES

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described by M. Eder et. al. in 2012 (26). Only a few years after this first report, [⁶⁸Ga]Ga-PSMA- 11 is considered to be the gold standard as it comes to PET/CT imaging of metastatic castrate resistant prostate cancer (mCRPC). However, no reports addressing the [⁶⁸Ga]Ga-PSMA- 11 metabolism and pharmaco- kinetics (PK) are published.

Furthermore, although [⁶⁸Ga]Ga- PSMA-11 is a significant leap forward compared to the older types of tracers – such as ¹⁸F- choline – a new generation of promising tracers is already on its way.

This thesis is divided in two chapters. In the first chapter of this thesis, we will discuss sev- eral exciting, new developments

in the field of PET radiotracers. In addition, this chapter will be used to address a very important shortcoming in the ‘standard’ way newly developed PET-tracers are being evaluated – namely the lack of decent metabolism and PK studies. As will be discussed in more detail later in this thesis, a broad understanding of tracer metabolism and PK may greatly benefit the interpretation of imaging data.

Therefore, the second chapter in this thesis will discuss the several of the first studies addressing the metabolism of [⁶⁸Ga]Ga-PSMA-11. These studies are the first steps of a larger research project, that is aimed to eventually elucidate the metabolism and PK of the [⁶⁸Ga]Ga-PSMA-11 tracer.

References

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Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012. Int J cancer [Internet]. 2015 Mar 1;136(5):E359-86. Available from: http://arxiv.org/abs/1011.1669 4. Potosky AL, Miller BA, Albertsen PC, Kramer BS. The Role of Increasing

Detection in the Rising Incidence of Prostate Ca ... The Role of Increasing Detection in the Rising Incidence of Prostate Cancer. Jama.

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6. Hemminki K. Familial risk and familial survival in prostate cancer. World J Urol [Internet]. 2012 Apr 25;30(2):143–8. Available from:

http://link.springer.com/10.1007/s00345-011-0801-1

7. Leitzmann M, Rohrmann S. Risk factors for the onset of prostatic cancer:

age, location, and behavioral correlates. Clin Epidemiol [Internet]. 2012 Jan;4(1):1. Available from: http://www.dovepress.com/risk-factors-for- the-onset-of-prostatic-cancer-age-location-and-behavi-peer-reviewed- article-CLEP

8. Attard G, Parker C, Eeles RA, Schröder F, Tomlins SA, Tannock I, et al.

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9. Epstein JI, Allsbrook WCJ, Amin MB, Egevad LL. The 2005 International

Society of Urological Pathology (ISUP) Consensus Conference on Gleason Grading of Prostatic Carcinoma. Am J Surg Pathol [Internet].

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10. Richie JP, Catalona WJ, Ahmann FR, Hudson MA, Scardino PT, Flanigan RC, et al. Effect of patient age on early detection of prostate cancer with serum prostate-specific antigen and digital rectal examination. Urology.

1993;42(4):365–74.

11. Okotie OT, Roehl KA, Han M, Loeb S, Gashti SN, Catalona WJ.

Characteristics of Prostate Cancer Detected by Digital Rectal Examination Only. Urology. 2007;70(6):1117–20.

12. Gosselaar C, Roobol MJ, Roemeling S, Schröder FH. The Role of the Digital Rectal Examination in Subsequent Screening Visits in the European Randomized Study of Screening for Prostate Cancer (ERSPC), Rotterdam.

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13. Schmid M, Trinh Q-D, Graefen M, Fisch M, Chun FK, Hansen J. The role of biomarkers in the assessment of prostate cancer risk prior to prostate biopsy: Which markers matter and how should they be used? World J Urol [Internet]. 2014 Aug 14;32(4):871–80. Available from:

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14. Society AC. http://www.cancer.org/ [Internet]. 2016. Available from:

http://www.cancer.org/cancer/prostatecancer/moreinformation/prost atecancerearlydetection/prostate-cancer-early-detection-tests 15. Sasaki M, Ishidoya S, Ito A, Saito H, Yamada S, Mitsuzuka K, et al. Low

Percentage of Free Prostate-specific Antigen (PSA) Is a Strong Predictor of Later Detection of Prostate Cancer Among Japanese Men With Serum Levels of Total PSA of 4.0 ng/mL or Less. Urology [Internet]. 2014

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16. Thompson IM, Pauler DK, Goodman PJ, Tangen CM, Lucia SPHM, Parnes HL, et al. Prevalence of Prostate Cancer among Men with a Prostate- Specific Antigen Level ≤ 4.0 ng per Milliliter. N Engl J Med [Internet].

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Fig. 1: Chemical structure of the small molecule PSMA-11. Adopted from M. Eder et.al. (26).

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19. Heijmink SWTPJ, Fütterer JJ, Hambrock T, Takahashi S, Scheenen TWJ, Huisman HJ, et al. Prostate cancer: body-array versus endorectal coil MR imaging at 3 T--comparison of image quality, localization, and staging performance. Radiology. 2007;244(1):184–95.

20. Jadvar H. Molecular Imaging of Prostate Cancer: PET Radiotracers. Natl Inst Heal. 2012;199(2):278–91.

21. Wibner AG, Burger IA, Sala E, Hricak H, Weber WA, Vargas HA. Molecular Imaging of Prostate Cancer. Radiographics. 2016;36(1):142–59.

22. Weineisen M, Schottelius M, Simecek J, Baum RP, Yildiz A, Beykan S, et al.

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Theranostic Concept and First Proof-of-Concept Human Studies. J Nucl Med [Internet]. 2015;56(8):1169–76. Available from:

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Available from: http://dx.doi.org/10.1016/j.urolonc.2013.09.003 24. Liolios C, Schäfer M, Haberkorn U, Eder M, Kopka K. Novel Bispecific

PSMA/GRPr Targeting Radioligands with Optimized Pharmacokinetics for Improved PET Imaging of Prostate Cancer. Bioconjug Chem.

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Chapter I

The Role of Lutetium-177, Copper-64 and Fluorine-18 in state of the art Theranostic

PSMA Targeting Tracers for Prostate

Cancer: A Systematic Review

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Abstract

In this review, we discuss several state-of-the-art, PSMA targeting radiotracers for the imaging and/or radiotherapy of prostate cancer (PCa). We mainly focus on the most recent developments in the field of ¹⁸F-, ⁶⁴Cu- and ¹⁷⁷Lu-labeled anti-PSMA tracers, since these types of tracers have the poten- tial to form new theranostic couples with Gallium-68 tracers (Lutetium-177 and Copper-64) or to ena- ble improved PET image quality, compared to [⁶⁸Ga]Ga-PSMA-11 (Copper-64 and Fluorine-18). Fur- thermore, we aim to compare several of these tracers with the current gold standard for metastatic castration resistant prostate cancer (mCRPC) PET imaging,[⁶⁸Ga]Ga-PSMA-11.

PubMed was systematically searched for relevant articles concerning Fluorine-18, Copper-64, Lute- tium-177, PSMA and prostate cancer. Duplicates, reviews and publications that were not relevant, were excluded. In addition, secondary references were acquired by including relevant references from the initial literature selection.

Initially, 769 articles were acquired from PubMed, including 498 unique articles. After screening for exclusion and inclusion criteria, 37 relevant articles were acquired. In addition, 16 articles were ob- tained by screening the included articles for relevant references.

177Lu-labeled PSMA conjugates showed promising results for radionuclide therapy (RNT) in heavily pre-treated patients. ¹⁸F- and ⁶⁴Cu-labeled PSMA tracers showed promising results for PET imaging of mCRPC. Furthermore, ⁶⁴Cu-labeled PSMA tracers are potential candidates for future RNT. For the fu- ture, radiolabeled PSMA inhibitors are likely to have a large impact on the treatment and diagnosis of prostate cancer. However, more extensive pharmacokinetic and metabolic studies are needed to better understand the behaviour of these tracers in the human body and to be able to optimize their use, pos- sibly in combination with other targeting tracers such as GRPR.

Keywords: PSMA, Lutetium-177, Copper-64, Fluorine-18, prostate cancer, PET

1. Introduction

PET/CT imaging is a relatively new imaging technique, which uses tracers such as ¹¹C- or ¹⁸F- labeled choline,

18F-labeled FDG or ¹⁸F-labeled acetate to image cancer lesions (1,2). This type of imaging is useful for selecting patients with biochemical relapse after previous radio- therapy (3). The mechanism of action of these conven- tional tracers is mainly based on changes in metabolism in cancer cells. However, since these modalities are not only cancer specific, uptake can also be observed in inflammatory tissue, benign tumours or benign pros- tate hyperplasia (2). Therefore, tracers that specifically target cancer tissues are needed. In addition, although PET/CT imaging with these tracers can provide useful information about tumour biology and diversity (2), the sensitivity and specificity of many of these conventional tracers are highly variable between studies (3,2).

Targeting the prostate specific membrane antigen (PSMA), also known as glutamate carboxypeptidase II (4), is shown to be a promising method to target PCa tumours (2). PSMA is overexpressed on PCa cells and in the neovasculature of other types of cancer, such as breast cancer, bladder cancer and glioblastomas (5).

Furthermore, it is expressed, although to a smaller ex- tent, in the kidneys, salivary glands and in the small

intestine (6). PSMA is a promising target for tracers, because almost 95% over the PCa cells overexpress PSMA (7). However, PSMA negative areas can someti- mes be observed in PSMA positive tumours, because of the high heterogeneity of the tumours (7). The high heterogeneity of the tumours can complicate PCa imag- ing, since it can affect the image quality. Therefore, targets such as the gastrin-releasing peptide receptor (GRPr) may be used complementary to PSMA, to be able to additionally image the PSMA-negative regions. In addition, the GRPr expression is shown to be increased in early stage PCa, while PSMA expression is mainly enhanced in the later stages of PCa (8).

Only a few years after the introduction of [⁶⁸Ga]Ga- PSMA-11 (9) , this tracer is considered to be the gold standard if it comes to the PET imaging of metastatic castrate resistant prostate cancer (mCRPC) patients.

Although results from studies concerning [⁶⁸Ga]Ga- PSMA-11 are promising, the tracer also has some limi- tations. Examples of these are the relatively short half- life, and the high β⁺ energy in comparison to other iso- topes such as Fluorine-18 and Copper-64. Therefore, new PSMA targeting tracers are being developed (4,10–

12). These new and promising developments include the use of Fluorine-18 for imaging (13) or the use of Copper-64 for both imaging and therapy. Furthermore, Lutetium-177 labeled anti-PSMA tracers have shown to be an exciting new development in the field of mCRPC radio nuclide therapy (RNT) (10,11). In addition, sever-

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imaging and therapy. These types of tracers are also known as theranostics (14).

This systematic review aims to give an overview of several new developments in the field of PSMA target- ing tracers which are based on the same Glu-urea-Lys motif as [⁶⁸Ga]Ga-PSMA-11. The focus will be especially on Fluorine-18 , Copper-64 andLutetium-177 contain- ing tracers, since these types of tracers have the poten- tial to form new theranostic couples with Gallium-68 tracers (Lutetium-177 and Copper-64) or to enable improved PET image quality, compared to [⁶⁸Ga]Ga- PSMA-11 (Copper-64 and Fluorine-18). This review will discuss several important tracer characteristics, such as radiolabeling properties, tracer sensitivity, tracer specificity and the metabolic stability. Further- more, the differences between the gold standard [⁶⁸Ga]Ga-PSMA-11 and new types of anti-PSMA tracers that are currently being developed, will be evaluated.

Finally, this review will briefly discuss the current way of how most tracers are being developed and evaluated, in an attempt to elucidate potential shortcomings of this current development proces.

2. Methods

2.1 Literature acquisition

The PubMed database was used to search for rele- vant publications. Generally, PubMed was screened for publications concerning Copper-64 , Fluorine-18 and Lutetium-177. In addition, since [⁶⁸Ga]-Ga-PSMA-11 is

regarded as the current gold standard in prostate can- cer imaging, several publications about [⁶⁸Ga]Ga-PSMA- 11 were used to evaluate how new tracers, containing one of the isotopes described above, compare to the

gold standard [⁶⁸Ga]Ga-PSMA-11.

2.2 PubMed searches

Several searches were used for each type of isotope.

To search for literature regarding Copper-64, the fol- lowing searches were used: ’64-Cu AND PSMA’, ’64-Cu AND Prostate AND Cancer’, ’64-Cu AND Ligand AND Prostate’, ’64-Cu AND Chelators’, ‘64-Cu AND Labeling’

and ’64-Cu AND PSMA NOT Anti-body’. For the screen- ing for publications regarding Fluorine-18 and PSMA, the searches ’18-F OR 18F OR F-18 AND PSMA’, ’18-F OR 18F OR F-18 AND PSMA AND Labeling’, and ’18-F OR 18F OR F-18 AND Prostate AND Cancer AND Imag- ing AND PSMA’ were used. Finally, PubMed was screened for publications regarding PSMA and Luteti- um-177 by using ‘Lutetium AND PSMA’, ‘Lutetium AND Prostate AND Cancer’, ‘Lutetium AND Therapy AND Prostate’, ‘Lutetium AND Imaging AND PET’, ‘Lutetium AND Ligand AND Prostate AND Cancer’, ‘Lutetium AND Kinetics’ and ‘PSMA AND chelator’. In addition, the

‘PSMA AND Chelator’ search was also relevant for the other isotopes.

2.3 Inclusion and Exclusion criteria

Articles were excluded when none of the words of the PubMed searches were mentioned in the title or in Fig. 1: Schematic overview of the literature acquisition.

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study, only original studies were included for this re- view. Letters to the editor, reports on scientific con- gresses or (systematic) reviews were excluded from the selection. Finally, publications that could not be used to answer the research questions were excluded.

2.4 Literature selection

After PubMed was screened for relevant articles, all titles were exported from PubMed to a database (Mi- crosoft Excel). First, all duplicate articles were removed from the database. After removing the duplicates, the titles were selected according to the inclusion and ex- clusion criteria. In addition, publications that did not concern original studies (e.g. reviews, congress reports, letters to the editor) were removed from the selection.

Articles from the final selection were used for this re- view. In addition, when it deemed necessary for the discussion of this review, relevant articles referred to in the ‘relevant articles’ publications were used. A schematic overview of the literature acquisition in shown in fig. 2.

3. Results

The initial PubMed search resulted in 471, 173 and 125 articles for Lutetium-177, Copper-64 and Fluorine- 18 respectively, with a total of 769 articles. After the first screening, 253 duplicates were removed from the three literature databases (the Lutetium-177, Copper- 64 and Fluorine-18 databases). In addition, pooling of these databases showed another 18 duplicates between the three databases. Screening for the inclusion and exclusion criteria resulted in the exclusion of 467 arti- cles of which two letters to the editor, and 465 articles that were not relevant for this review and/or did not describe an original study (reviews). Articles were con- sidered irrelevant when the article only concerned new developments in the area of PET scan technologies (software, crystals etc.) , when the article did not dis- cuss one of the isotopes mentioned above and when the article did not concern prostate cancer and PSMA tar- geting. Furthermore, since the focus of this review is on the anti-PSMA Glu-urea-Lys peptide, references con- cerning antibodies were excluded. However, we decid- ed to include some references concerning antibodies, when the reference provided valuable background in- formation or when references concerning the anti- PSMA peptide were lacking. Initially, a total of 37 rele- vant articles were acquired from the initial PubMed search (fig. 1). In addition, 16 secondary articles were acquired by screening the references from the ‘relevant articles’ .

4.

Cu-labeled tracers for prostate cancer imaging and therapy

4.1 Recent developments in ⁶⁴Cu-labeling and Cop- per-64 anti-PSMA tracers

The PubMed search for chelators for Copper-64 and PSMA (the Glu-urea-Lys motif) specifically, resulted in a limited number of publications. Therefore, publications about ⁶⁴Cu-chelators for ligands other than the Glu- urea-Lys peptide were also included, to search for che- lators that may also be applicable for PSMA.

Three types of chelators were identified: (1) acyclic chelators, (2) macrocyclic chelators and (3) cross- bridged macrocylic chelators (15,16) of which the mac- rocyclic DOTA (1,4,7,10-tetraazacyclododecane- 1,4,7,10-tetraacetic acid) and TETA (1,4,8,11- tetraazacyclotetradecane-1,4,8,11-tetraacetic acid) are currently the most used chelators (16,17). The stability of ⁶⁴Cu-labeled tracers mainly depends on the stability of the ⁶⁴Cu-chelator complex (15). Although DOTA and TETA have shown to be more stable in vivo than the acyclic chelators such as EDTA and DTPA, these macro- cyclic chelators only show a moderate in vivo stability (15,18,19). This moderate in vivo stability is thought to cause trans chelation of Copper-64 from the original chelator to endogenous proteins. Superoxide dismutase (SOD) is thought to be the one of the most important copper binding proteins (16,20). This protein removes oxygen radicals from the body, by a reaction catalysed by copper, which is incorporated in the SOD protein (16). Furthermore, transchelation of Copper-64 can occur to metallothionein or albumin in the liver or blood respectively (21).This transchelation leads to a lower image contrast, which is caused by a higher up- take of Copper-64 in non-target tissues(22,23)

.

NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid), a macrocylic chelator, is shown to have a more favoura- ble in vivo stability than the other macrocyclic chelators such as DOTA, OXO-DO3A and PCTA (15,24). Research has shown that the size of the macrocycle influences the complex in vivo stability, which may explain why the relatively compact NOTA forms a more stable complex with Copper-64 than other macrocyclic chelators such as DOTA, OXO-DO3A and PCTA (16,24).

Furthermore, several studies showed high in vivo stability of cross-bridged tetraamine bicyclic polyam- inocarboxylates, especially for CB-TE2A (1,4,8,11- tetraazabicyclo[6.6.2]- hexadecane-4,11-diacetic acid) (15,16). However, the harsh conditions needed for the complexation of Copper-64 with this chelators poses a challenge, since biomolecules –such as antibodies or peptides –are often vulnerable for high temperatures.

To solve this problem, click chemistry might offer a solution (19).

The use of Copper-64 for PET imaging is thoroughly studied. However, our literature research resulted in only two studies concerning ⁶⁴Cu-labeled Glu-urea-Lys peptide specifically (10,15). Because of the lack of pub- lications about this ⁶⁴Cu-labeled peptide, it is hard to

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are suited best to bind Copper-64 to the Glu-urea-Lys peptide. However, we were able to identify several chelator types that showed high in vivo stability when bound to biomolecules other than the Glu-urea-Lys moiety.

Especially the cross-bridged type chelators TE2A and TE1K1P showed promising results when combined with an anti-body and with the Glu-urea-Lys motif (for TE2A). In addition, the NODAGA chelator is shown to have a high stability in vivo. Although NODAGA is slight- ly less stable than the cross-bridged chelators, it is an attractive complexating agent, since the complexation process can be easily performed under mild conditions (17). This enables radiolabeling of relatively instable molecules, such as antibodies. In addition, R. Ferdani et.

al. showed that the CB-TE2A chelator can be easily la- beled under mild conditions (21). This in contrast to the TE2A chelator, which is usually radiolabeled in harsh conditions or by using complicated click chemistry (19,21).

Although we were able to identify several chelating agents for ⁶⁴Cu-labeling, the large heterogeneity of the data causes that direct comparison of characteristics, such as stability or PSMA binding affinity, between chelating agents, is a challenge. However, it is possible to elucidate certain trends. First of all, in general, acy- clic chelators are less stable than the macrocyclic chela- tors. In addition, cross-bridged macrocyclic chelators are, in general, more stable than macrocyclic chelators.

Furthermore, the macrocyclic chelator NODAGA showed almost a similar stability as cross-bridged che- lators. CB-TE2A and CB-TE1K1P showed an excellent stability in comparison with the other chelators.

NODAGA, CB-TE2A and CB-TE1K1P are therefore con- sidered to be currently the best options for the devel- opment of new ⁶⁴Cu-labeled PSMA tracers.

4.2 Preclinical studies

The biodistribution of ⁶⁴Cu-labeled tracer molecules is largely influenced by the charge and lipophilicity of the ⁶⁴Cu-chelator complex (25). Studies have shown that a positive charge of the copper complex leads to a high uptake in the liver and the kidneys resulting in a lower clearance relative to neutral or negatively charged complexes (19,25,26). B. Rogers et. al. exam- ined the biodistribution of several types of ⁶⁴Cu- chelators conjugated with the 1A3-F(ab′)2 antibody, in hamsters and rats (27). This study showed that, in

comparison to neutral or negatively charged complexes, conjugates with positively charged and/or lipophilic complexes showed an increased accumulation in the liver and kidneys and a decreased clearance. Similar results were reported by T. Jones-Wilson et. al. (28).

These studies suggest that neutral or negatively charged ⁶⁴Cu-complexes have most favourable in vivo properties. The exact mechanism which causes accumu- lation of positively charged complexes in the liver and kidneys is still unknown. However, it is suggested that positively charged moieties could be attracted to the negatively charged basal cells in the glomerulus and the negatively charged podocytes in the kidneys (29,30).

S.R. Banerjee et. al. studied the in vitro and in vivo behaviour of several ⁶⁴Cu-chelator complexes, bonded to the Glu-urea-Lys anti-PSMA motif (15). To our knowledge, this is the only report comparing several types of copper chelates bonded to the Glu-urea-Lys motif. The main results are summarized in table 1. All Ki values ranged between 3,98 nM for CB-TE2A, to 13,26 nM for the DOTA conjugate, which is similar to that of ^natGa-PSMA-11 (12,0 ± 2,8 nM)(9). The in vivo evaluation was performed in severe-combined immu- nodeficient (SCID) mice, which were injected with PSMA + PC3 PIP cell and with PSMA – PC3 flu cells.

Micro PET/CT scans were used for the imaging studies.

All tracers were able to clearly show the PSMA+ tu- mours. Furthermore, all tracers showed uptake in the kidneys, which is due to the renal clearance and be- cause of the PSMA expression in the kidneys. A signifi- cant accumulation in the liver was shown by the PCTA, Oxo-DO2A and DOTA copper conjugates. Furthermore, the Oxo-DO3A and DOTA conjugates showed a signifi- cantly higher background uptake, which was likely to be caused by the transchelation of the Copper-64 from the tracer. These findings thus indicate a lower stability of these compounds. However, M.S. Cooper et. al. com- pared several types of ⁶⁴Cu-complexes bonded to ritux- imab (31) and found that ⁶⁴Cu-PCTA and ⁶⁴Cu-DOTA conjugates were stable for 48 hours in human serum.

This difference can be explained by the absence of ac- tive metabolic enzymes, such as Superoxide Dismutase (SOD) in the in vitro model. This example clearly illus- trates the additional value of in vivo metabolic studies, since, in the human body, tracers are subjected to a more complex array of metabolic pathways, than can be simulated in a simple in vitro stability study.

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jugate showed a high uptake in the PSMA+ tumour and in the kidneys. Furthermore, both CB-TE2 conjugates showed a low liver uptake, which is likely to be the result of a high complex stability. However, the NOTA conjugate showed some accumulation was observed in the liver, which indicates a lower in vivo stability than CB-TE2A. Furthermore, the CB-TE2A diasteriomers showed a higher renal clearance than the NOTA conju- gates. Because of the higher renal clearance and higher stability CB-TE2A was considered to be the most suita- ble chelator (15).

4.3 Clinical studies

Although the application of Copper-64 for the PET imaging of cancer has been studied by many research groups, to our knowledge, only one human study with

64Cu-labeled PSMA has been published so far. In this study from B. Grubmüller et. al . (10), a total of 29 pa- tients with high suspicion for recurrent PCa or who were planned for surgery or PSMA RNT, was included.

In 23 of these patients, PET/CT imaging with ⁶⁴Cu- labeled PSMA showed at least 1 lesion suspicious for PCa. The images showed excellent contrast at 1 hour p.i.

(fig. 2), even at low PSA levels. Because the small num- ber of patients included in this study, more clinical studies will be needed to evaluate the use and the po- tential benefits of ⁶⁴Cu-labeled PSMA in patients. How-

ever, this study showed that ⁶⁴Cu-labeled PSMA is a promising tracer for PET/CT imaging for PCa, because of its favourable nuclear properties resulting in high image resolution due to the favourable β⁺ energy of and delayed imaging due to the half-life of 12,7 hours.

4.4 Comparison with the gold standard: [⁶⁸Ga]Ga- PSMA-11

Since the development of [⁶⁸Ga]Ga-PSMA-11 in 2012 (9)and the introduction as the gold standard in the clinic, the search continues for new anti-PSMA tracers with improved imaging characteristics and with an improved availability compared to [⁶⁸Ga]Ga-PSMA-11.

Because of the favourable nuclear properties of Copper- 64 (T1/2 = 12,7 h; Eβ+max = 656KeV), Copper-64 is of high interest for the development of tracers for PET imaging. Although the possibilities for Copper-64 in the field of cancer imaging are being thoroughly studied, the use of Copper-64 in anti-PSMA tracers is still lim- ited. A ⁶⁴Cu-containing anti-PSMA tracer would, howev- er, have several advantages compared to the current gold standard.

Image resolution

Because of the relatively low Eβ+max of Copper-64, a higher spatial image resolution would be expected for

64Cu-containing tracers, compared to those labeled with Gallium-68 (Eβ+max 1899,1 KeV). In addition, because of

Fig. 2: In Human ⁶⁴Cu-labeled PSMA PET/CT imaging showed nodal and skeletal metastases. (A) ⁶⁴Cu- labeled PSMA PET; (B,C) Axial PET images; (D,E) Axial CT images; (F,G) corresponding PET/CT images.

Images are adopted from B. Grubmüller et. al. (10).

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formed at later time points than when using shorter lived isotopes such as Gallium-68. Therefore, more activity can be cleared from the blood, which has a posi- tive effect on the image contrast. The increase in image resolution and image contrast, compared to that of images obtained with Gallium-68, will improve the diagnostic value of the scans, because smaller lesions could be detected. A report from J. Johnbeck et. al. dis- cussed a head-to-head comparison between [⁶⁴Cu]Cu- DOTATATE and [⁶⁸Ga]Ga-DOTATOC in patients with neuroendocrine tumours (fig. 3) (32). The [⁶⁴Cu]Cu- DOTATATE showed substantially higher lesion detec- tion rates than [⁶⁸Ga]Ga-DOTATOC. Although the trac- ers were not identical, the authors mention that [⁶⁸Ga]Ga-DOTATOC and [⁶⁸Ga]Ga-DOTATATE are both known to show similar sensitivities. Furthermore, the authors state that no evidence for the superiority of one of these tracers has been published, and that it is there-

fore possible to use these two different tracers to eval- uate the spatial resolution.

Theranostics

The development of so called Theranostics is a relative- ly new and exciting development in the field of PET imaging and RNT. Theranostic tracers enable both im- aging and therapy with one single tracer. Because of the types of radiation emitted by Copper-64 (β⁺ 17,8%; β^- 38,4%; Auger electron 43%), tracers labeled with this isotope are also suggested for RNT (15,33). However, to our knowledge, no reports concerning Copper-64 for RNT of PCa are published. Furthermore, Copper-64 could be exchanged for RNT by Copper-67, which is a promising candidate for RNT because the pure β^- emis- sion. Copper-64 and Copper-67 could therefore make up an interesting theranostic couple. A recent study, in which the transmembrane cell adhesion receptor αVβ3 integrin was targeted with a ⁶⁴Cu-containing tracer

Fig. 3: Comparison between PET/CT scans (left) and CT scans (right) with [⁶⁴Cu]Cu-DOTATATE and [⁶⁸Ga]Ga- DOTATOC in a patient with neuroendocrine tumours and metastases. Adopted from J. Johnbeck et. al. (32).

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ising results for the application of Copper-64 for RNT against cancer (33). The ⁶⁴Cu-tracer used in this study showed a delay in tumour growth in tumours express- ing the αVβ3 receptor. Therefore, it would be interest- ing to investigate the possibilities of ⁶⁴Cu-labeled trac- ers for targeting PSMA.

Availability

The relatively long T1/2 of 12,7 hours, makes that

64Cu-tracers are suitable for distribution from a nuclear facility to hospitals that do not have a facility for the production of tracers. This is a large advantage com- pared to Gallium-68, because the area to which ⁶⁸Ga- tracers can be distributed is substantially smaller than that of ⁶⁴Cu-tracers, due to the shorter half-life of Galli- um-68 (68 minutes). In addition, only a few patient doses per elution are obtained by ⁶⁸Ga-generators, which makes the wide-spread distribution even more complicated. However, the fact that Gallium-68 can be produced in a generator can also be an advantage, since generators can be relatively easily installed in almost every hospital. This is not the case with a cyclotron, which needs highly specialized personnel to operate the cyclotron and much more strict safety regulations. Fur- thermore, currently there are only a few nuclear facili- ties that are able to produce high quality Copper-64.

For a broader application of this radionuclide in nuclear medicine and imaging, an increase in production capac- ities is warranted.

5.

F-labeled tracers for prostate cancer imaging

5.1 Recent developments in ¹⁸F-labeling and Flu- orine-18 anti-PSMA tracers

In contrast to Copper-64 , Fluorine-18 can be both directly bound to a spacer moiety by covalent bonding or it can be complexated by using aluminium monofluo- ride [Al¹⁸F]²⁺ (12,34–37). Literature search showed 4 reports in which Fluorine-18 was covalently bonded to a Glu-urea-Lys anti-PSMA motif via a spacer molecule.

The Heidelberg research group developed and eval- uated the [¹⁸F]F-PSMA-1007 tracer, which showed a very similar structure and in vivo behaviour as PSMA- 617 (12,34). PSMA-617 was originally developed to link Lutetium-177 to the Glu-urea-Lys PSMA binding motif (38). A Fluorine-18 tracer with similar properties as PSMA-617, was developed in order to form a theranostic couple with [¹⁷⁷Lu]Lu-PSMA-617 (12). The PSMA-1007 precursor was labeled with Fluorine-18 by a reaction of 6-(18)F-F-Py-TFP (6-(18)F-fluoronicotinic acid 2,3,5,6-tetrafluorophenyl ester) with the PSMA- 1007 precursor (2 mg/ml in DSMO). Furthermore, in vitro stability testing, showed that [¹⁸F]F-PSMA-1007 was stable for 4 hours in human serum.

In another study, from J. Kelly et. al., click chemistry was used for ¹⁸F-labeling in a pre-clinical study (35) . In

this study, two new classes of

[¹⁸F]fluoroethyltriazolylphenyl urea-based PSMA lig- ands were developed and evaluated. Six types of trazol- ylphenyl urea compounds were synthesized by derivat- ization with [¹⁸F]triazoles, by using a Cu(I) catalysed click-reaction (100°C, 20 min). Unfortunately, no data concerning the in vitro or in vivo stability of the tracers was reported.

Y. Chen et. al. reported about several types of Glu- urea-Lys PSMA inhibitors for PET or SPECT, containing Fluorine-18 or iodine-123 (36). The ¹⁸F-containing tracer [¹⁸F]6 (2-[3-[1-carboxy-5-(4-[18F]fluoro- benzoylamino)-pentyl]-ureido]-pentanedioic acid) was synthesized by the reaction of a tri-PBM Gly-C(O)-Lys ester with N- hydroxysuccinimidyl-4- [¹⁸F]fluorobenzoate, after which the PBM groups were removed by a reaction with trifluoroacetic acid and anisole. The radiochemical yield varied between 30%- 35%.

18F-SFB (succinimidyl 4-18F-fluorobenzoate) is a commonly used method ¹⁸F-labeling, which has shown to produce a high radiochemical yield (39). However, the literature acquisition showed only one report about this type of labeling of an anti-PSMA binding motif (Cys- urea-Glu) (39). This research group previously report- ed about the development of a high affinity anti-PSMA tracer, containing Iodine-123. In the current study, this

Chelator¹ Ki (nM) Imaging results Tumor visualitation

NOTA 6,23 High tracer concentrations in both tumor and kidneys. Low liver uptake. Yes PCTA 10,76 Moderate/High tumor uptake. Significant uptake in the liver at 2,5h p.i. Yes Oxo-DO3A 5,47 Moderate/High tumor uptake. Significant uptake in the liver at 2,5h p.i.. Significantly

higher background uptake. Yes

CB-TE2A² 3,98 High tracer concentrations in both tumor and kidneys. Clear tumor delineation at

early timepoints. Low liver uptake. Yes

CB-TE2A² 4,65 High tracer concentrations in both tumor and kidneys. Clear tumor delineation at

early timepoints. Low liver uptake. Yes

DOTA 13,26 Moderate/High tumor uptake. Significant uptake in the liver at 2,5h p.i.. Significantly

higher background uptake. Yes

1All chelators were bonded to the Glu-urea-Lys PSMA binding moiety. All tracers were labeled with ^63/65Cu . ²Diasteriomers of CB- TE2A.

Table 1: Comparison of different types of chelators for copper labeling. ⁴Cu-labeled inhibitors of prostate-specific membrane antigen for PET imaging of prostate cancer (15).

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((R)-5-carboxy-5-(2-(3-123I-

iodobenzamido)acetamido)pentyl)-2,5-

dioxopyrrolidin-3yl)thio)ethyl) ureido)pentanedioic acid), was used to synthesize a similar, ¹⁸F-containing tracers. The ¹⁸F-containing tracers were synthesized by substituting the iodobenzamido group of ¹²³I-IGLCE by a fluorobenzamido group, using ¹⁸F-SFB. Several types of tracers, with slight modifications, were pre- pared by the reaction of ¹⁸F-SFB with maleimide based precursor molecules at room temperature for 5 minutes. After this, the Cys-CO-Glu peptide was added to the reaction mixture to acquire the final tracer mole- cule. The four tracers that were tested for in vitro stabil- ity in mouse serum, showed no decomposition of the tracers after 1 hour incubation.

Another approach to link Fluorine-18 to the Glu- urea-Lys motif is by complexation of [Al¹⁸F]²⁺ with a chelator. However, a disadvantage of this approach is that the complexation process often needs high tem- peratures, which can cause damage to molecules, espe- cially biological molecules such as peptides or antibod- ies. Although the Glu-urea-Lys PSMA inhibitor is able to sustain high temperature labeling (37), many other biomolecules may be damaged by the heating. There- fore F. Cleeren et. al. developed and evaluated several new polydentate chelators which could be labeled with [Al¹⁸F]²⁺ at moderate temperatures (37). F. Cleeren et.

al. synthesized 8 chelators, of which 4 were evaluated.

To evaluate the influence of the temperature on the radiochemical yield, labeling was performed at 5 differ- ent temperatures. Compound H3L2 ((2,2'-(ethane-1,2- diylbis((2-hydroxybenzyl)azanediyl))diacetic acid tri- fluoroacetate salt) and H3L3 ((2-(benzyl(2- ((carboxymethyl)(2-

hydroxybenzyl)amino)ethyl)amino)acetic acid tri- fluoroacetate salt) showed a substantial increase in radiochemical yield when labeling at 40°C. However, increasing the temperature above 40°C did not result in a higher radiochemical yield. Furthermore, com- pound H3L1 showed similar radiochemical yields for all temperatures. Compound H2L4 (2-(benzyl(2- ((carboxymethyl)(pyridin-2-

ylmethyl)amino)ethyl)amino)acetic acid trifluoroace- tate salt) showed an increase in radiochemical yield with increasing temperatures.

Stability testing showed a relatively high stability for the [Al¹⁸F]L3 complex, relative to that of [Al¹⁸F]L1, [Al¹⁸F]L2 and [Al¹⁸F]L4. [Al¹⁸F]L3 showed stable con- centrations in vitro in rat serum up to 60 minutes. After 60 minutes, slow decomposition was seen for [Al¹⁸F]L3.

After 4 hours, 66% of the [Al¹⁸F]L3 was still intact. In contrast, [Al¹⁸F]L1, [Al¹⁸F]L2 and [Al¹⁸F]L4 showed a high degradation between 10-30 minutes of incubation.

This indicates a high instability of the L1, L2 and L4 compounds.

Another promising method for Fluorine-18 labeling was reported by N. Malik et. al.(40) . This study showed that the precursor PSMA-11, which is already used for Gallium-68 labeling (41,42), can also be used for the

labeling of [Al¹⁸F]²⁺. In vitro stability testing of the [Al¹⁸F] PSMA-11 complex in human serum showed no degradation after 4 hours. Similar results were found in a more recent study, which reported 91% and 94%

intact [Al¹⁸F]PSMA-11 after 2 hours in mouse and hu- man serum respectively (43) .

[¹⁸F]DCFPyL (2-(3-{1-Carboxy-5-[(6-[18F]Fluoro- Pyridine-3-Carbonyl)-Amino]-Pentyl}-Ureido)-

Pentanedioic Acid) is a new PSMA based PCa tracer, which was developed by Y. Chen et. al. (44).

[¹⁸F]DCFPyL was synthesized by a reaction of [¹⁸F]Py- TFP ([¹⁸F]6-Fluoro-nicotinic acid-2,3,5,6- tetrafluoro- phenyl ester) with a lys-C(O)-glu urea precursor pro- tected by a p-methoxybenzyl (PMB) group (44). In a more recent study, the radio labeling process was au- tomated by using an automated synthesis unit. This method enables GPM production and direct ¹⁸F-labeling with Fluorine-18 produced in a cyclotron (45).

The literature that is discussed above, shows that there are several ways –click chemistry, [Al¹⁸F]²⁺ com- plexation and ¹⁸F-SFBlabeling –for labeling tracers with Fluorine-18. Although the labeling with [Al¹⁸F]²⁺ with chelators such as NOTA and NODA usually require high temperatures, new methods are being developed to enable labeling at lower temperatures (37). However, as shown by F. Cleeren et. al., ensuring sufficient radio- chemical stability can be challenging. Therefore, click chemistry might produce more stable tracers, because of the covalently bonded Fluorine-18. However, N. Ma- lik et. al. showed that the, already available PSMA-11, is able to form highly stable complex with [Al¹⁸F]²⁺, which makes [Al¹⁸F]PSMA-11 a very attractive ¹⁸F-containing PET tracer. Furthermore, the development of the auto- mated, GMP [¹⁸F]DCFPyL radiolabeling process (45), will greatly benefit the availability of this tracer.

5.2 Pre-clinical studies

J. Cardinale et. al. recently published a preclinical evaluation of [¹⁸F]F-PSMA-1007 (12). In this study, the tracer binding affinity and tumour internalization were evaluated in vitro. In addition, biodistribution studies were performed in mice. The studies showed that [¹⁸F]F-PSMA-1007 had a very high in vitro internaliza- tion ratio (67±13%) and showed a high affinity for PSMA (Ki = 6.7±1.7nM) . Furthermore, a low uptake in non-target tissue was observed in, which resulted in a clear image of the tumour in mice 40 minutes p.i. (12).

The ligands developed by J. Kelly et. al. (35) showed high tumour uptake in mice bearing LNCaP tumour cells, with a peak uptake at 2 hours p.i.. Uptake values remained stable up to 4 hours p.i.. In contrast, uptake in non-target tissue started to decrease as early as 1 hour p.i., which resulted in high contrast images. Further- more, all compounds were mainly renally cleared. Es- pecially [¹⁸F] RPS-040 ((((S)-1-Carboxy-5-(3-(3-(1-(2- fluoroethyl)-1H-1,2,3-triazol-4-

yl)phenyl)ureido)pentyl) carbamoyl) -L-glutamic acid) and [¹⁸F]RPS-041 ((((S)-1-Carboxy-5-(3-(4-(1-(2- fluoroethyl)-1H-1,2,3-triazol-4-

yl)phenyl)ureido)pentyl)carbamoyl)-L-glutamic acid)

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high tumour uptake and a high tumour to kidney and tumour to background ratios. Unfortunately, no data was reported concerning the in vitro or in vivo stability of the tracers.

The [¹⁸F]6 compound developed by Y. Chen et.

al.(36) showed a high affinity for PSMA with a Ki of 0,256 nM with a NAALADase inhibition assay and 0,194 nM with a fluorescence based inhibition assay. In addi- tion, a specific and rapid uptake was observed in PSMA positive tumour cells in mice. Furthermore, the authors stated that [¹⁸F]6 showed a similar tumour uptake, but slower clearance from non-target tissue, compared to [¹⁸F]DCFBC, which was previously reported by this

research group.

The [Al¹⁸F]L3 devel- oped by F. Cleeren et. al.

was conjugated to Glu- urea-Lys PSMA and evaluated in healthy mice. The low activity uptake in the bones indicates a high in vivo stability of [Al¹⁸F]L3.

However, the authors mention that the fast clearance of PSMA from the blood and healthy tissues dictates the need for in vivo stability studies with conjugates that circulate for a longer period in the blood.

In vitro Studies with [Al¹⁸F]PSMA-11 showed a simi- lar binding affinity as [⁶⁸Ga]Ga-PSMA-11 (KD 10,3±2,2 nM vs. 12,58±1,09 nM for [Al¹⁸F]PSMA-11 and [⁶⁸Ga]Ga-PSMA-11 respectively) (40). In the more re- cent pre-clinical study in mice, a significantly higher activity uptake was observed in tumours with high PSMA expression than in tumours with a low PSMA expression (55,7 ± 11,8 %ID/g and 3,1 ± 0.9 %ID/g respectively) (43). Furthermore, some bone uptake was observed, which indicates some defluorination. Since the hydroxyl ions of the hydroxyapatite in the bones are exchanged with free ¹⁸F-ions, free fluorine tends to Fig. 4: Radio-HPLC evaluation of the metabolic stability of [¹⁸F]DCFPyL in mice. Adopted from V.

Bouvet et. al. (45).

Fig. 5: HPLC chromatograms of the DCFPyL tracer. UV chromatogram from the DCFPyL reference (up- per) and the activity chromatogram from a patient sample 173 min p.i.. Adopted from Z. Szabo et. al. (47).