GRPr Targeting Radioligands with Optimized

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Novel bispecific PSMA/GRPr targeting radioligands with optimized pharmacokinetics for improved PET imaging of prostate cancer Christos C. Liolios, Martin Schaefer, Uwe Haberkorn, Matthias Eder, and Klaus Kopka Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.5b00687 • Publication Date (Web): 04 Jan 2016 Downloaded from http://pubs.acs.org on January 12, 2016

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Bioconjugate Chemistry

Revised manuscript

Title page

Novel bispecific PSMA/GRPr targeting radioligands with optimized pharmacokinetics for improved PET imaging of prostate cancer Liolios C.1,*, Schäfer M.1, Haberkorn U. 2,3, Eder M. 1,*, Kopka K. 1,3. 1

Division of Radiopharmaceutical Chemistry, German Cancer Research Center (DKFZ), Heidelberg, Germany. 2 3

Clinical Cooperation Unit Nuclear Medicine, University of Heidelberg, Heidelberg, Germany. German Cancer Consortium (DKTK), Heidelberg, Germany.

Abstract A new series of bispecific radioligands targeting the prostate-specific membrane antigen (PSMA) and the gastrin releasing peptide receptor (GRPr), both expressed on prostate cancer cells, were developed. Their design was based on the bombesin (BN) analogue: H2NPEG2-[D-Tyr6, β-Ala11, Thi13, Nle14]BN(6–14), which binds to GRPr with high affinity and specificity and the peptidomimetic urea-based pseudo-irreversible inhibitor of PSMA: Gluureido-Lys. The two pharmacophores were coupled through Copper(I)-catalyzed AzideAlkyne Cycloaddition (CuAAC) to the bis(tetrafluorophenyl) (TFP) ester of the chelating agent HBED-CC via amino acid-linkers made of positively charged His (H) and negatively charged Glu (E): -(HE)n-, (n=0-3). The BRL were labelled with 68Ga and their preliminary pharmacological properties were evaluated in vitro (competitive and time kinetic binding assays) on prostate cancer cell lines (PC-3, AR42J, LNCaP) and in vivo by biodistribution and small animal PET imaging studies in normal as well as in tumor bearing mice. The IC50 /Ki values determined for all BRL essentially matched the ones of the respective monomers. The maximal cellular uptake of the BLR was already observed between 20 and 30 min. The BRL showed synergistic ability in vivo by targeting both PSMA (LNCaP) and GRPr (PC-3) positive tumors, while the charged -(HE)n-, (n=1-3), linkers significantly reduced the kidney and spleen uptake. The bispecific (PSMA and GRPr) targeting ability and optimised pharmacokinetics of the compounds developed in this study could lead to their future application in clinical practise as more sensitive radiotracers for noninvasive imaging of prostate cancer (PCa) by PET/CT and PET/MRI.

Keywords: 68Ga, PET, prostate cancer diagnosis, PSMA, GRPr, bispecific radioligands, lowmolecular weight heterodimer

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Introduction In the last decade, receptor binding radiopharmaceuticals attracted intense research attention and became valuable tools in cancer imaging and therapy. Two major types of ligands were used for these applications, peptides and antibodies1–4. Antibodies present high specificity and binding affinity, while their disadvantages are their slow blood clearance, poor tissue penetration, high immunogenicity and the rather high production cost5–8. On the other hand peptides typically show fast clearance, excellent tissue penetration and low immunogenicity and they can easily and inexpensively be produced on a relatively large scale. Their disadvantages are the eventually low tumor uptake and short retention times caused by low in vivo stability and fast dissociation from the target receptor. This putatively makes peptides not as effective as antibodies in tumor targeting5–8. The high tumor retention of antibodies is achieved through multivalent interactions1,5–8, thus the improvement of peptide binding affinity was initially attempted through the multivalent approach9–11. Multivalent interactions significantly reduce the off-rate from the receptor1. Traditionally, this approach involves the use of peptide homodimers or homomultimers in which peptide ligands of the same type are assembled with suitable linkers12. Recently the utilization of bispecific peptides, which we call low-molecular weight heterodimers, has emerged as a novel promising tool for enhanced multi-receptor tumor targeting. In a peptide heterodimer, two different peptidic/non-peptidic ligands, targeting different receptors in one and the same tumor entity are covalently linked by either a flexible or rigid linker with adjustable length. The molecular basis of low-molecular weight heterodimers for tumor targeting is based on the fact that cancer cells co-express multiple biological targets13– 17 . Up to now several approaches considering the design of bispecific peptide structures have been followed, where for example a BN sequence, a known ligand for GRPr18–20, was one part of the heteroligand, and the other part was the cRGD peptide, targeting αvβ3 integrin and tumor angiogenesis21. Several BN–RGD hybrid peptides labeled with PET radionuclides such as 18F, 68Ga and 64Cu have been preclinically evaluated for their ability to image GRPr positive prostate (PC-3) and αvβ3 positive breast cancer (T47D; MDAMB-435) tumors in vivo22–26. The prostate-specific membrane antigen (PSMA) has also been utilized as a target for the design of low-molecular weight heterodimers since nearly 95% of PCa cells overexpress PSMA21,27–29. However tumors often present a high grade of heterogeneity and sometimes PSMA positive tumors contain PSMA negative tissue regions21,29. Recently a bispecific targeting imaging agent, consisting of a low-molecular weight urea-based PSMA inhibitor and the cRGDfK peptide and conjugated either with DOTA for radiometal complexation or with an optical dye, was evaluated in vivo in PC-3 and U87-MG tumors15. At the same time the first bispecific low-molecular weight radioligand able to target both PSMA and GRPr was synthesized and evaluated by our group30. The BN sequence: H2N-PEG2-[D-Tyr6, β-Ala11, Thi13, Nle14]BN(6–14) amide31–33, representing the GRPr binding part was combined with the urea-based PSMA inhibitor Glu-urea-Lys(Ahx) and the HBED-CC chelator 34 for complexation of 68Ga (short-lived positron emitter, β+, 88 %, with a half-life of t1/2 = 68 min). In vitro cell binding and in vivo biodistribution and imaging experiments revealed high binding affinities


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for both GRPr and PSMA30. In comparison with the monomers Glu-NH-CO-NH-Lys(Ahx)HBED-CC (PSMA-11) and HBED-CC-PEG2-[D-Tyr6, β-Ala11, Thi13, Nle14]BN(6–14), (GRPrm) tumor uptake was comparable for LNCaP tumors and almost as high for GRPr expressing AR42J tumors, in any case indicating the functionality of both specificities30,31. The organ distribution profile of our low-molecular weight heterodimer showed PSMA-mediated high uptake in the kidneys and spleen and GRPr-mediated uptake in the pancreas30. In the study presented here we aimed to optimize the pharmacokinetic (PK) properties especially regarding the high kidney and spleen uptake. In the heterodimeric structure hydrophilic linkers were included between the chelator HBED-CC and the BN pharmacophore made from various numbers of charged amino acids (-His-Glu-)0-3, (Scheme 1). For comparative reasons the monomers GRPrm and PSMA-11, were synthesized and evaluated alongside the aimed-at heterodimers in vitro for their cell binding properties in PSMA positive (LNCaP) and GRPr positive (PC-3, AR42J) cell lines and in vivo in normal and in tumor xenografts.

Scheme 1. Synthesis of Glu‐ureido‐Lys‐(HE)n (7a, n=0, 7b, n=1; 7c, n=2; 7d: n=3) (a) Triphosgene, DIPEA, CH2Cl2, 0°C; (b) H-Lys(Alloc)-2CT-Resin, CH2Cl2 (c) Pd[P(C6H5)3]4, morpholine, CH2Cl2; (d) 4 equiv., Fmoc-His(Trt)-OH, Fmoc-Glu(OtBu)-OH, HBTU, DIPEA, DMF; (e) 50% piperidine, DMF; (f) 5-azidopentanoic acid, HBTU, DIPEA, DMF; (g) 95/2.5/2.5 (v/v/v), TFA/TIPS/H2O.

Results Chemistry The synthesis of PSMA-11 was according to previously published methods35. The H2N-PEG2BN(6-14) part of GRPrm was synthesized according to standard Fmoc peptide protocols on a



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rink amide resin cleaved from the resin and purified. The BRL synthesis started with the isocyanate of the glutamyl moiety (2) and with standard fmoc protocols resulted in Gluureido-Lys (7a) and its -HEn- derivatives 7b-c (7b: n=1; 7c: n=2; 7d: n=3) (Scheme 1). In parallel the Fe-protected mono TFP-ester of HBED-CC (10a), reacted with the H2N-PEG2BN(6-14) pharmacophore to 11, and after the Fe removal to 16, GRPrm. The monomer GRPrm was purified and identified with mass spectrometry (Table 1).

Table 1 MALDI mass spectrometry data of the free ligands [M+H]+. +

+

Compound

m/z calculated [M+H]

m/z experimental [M+H]

GRPrm

1800.0

1800.8

PSMA-11

947.4

947.4

HE0

2101.3

2100.5

HE1

2547.8

2547.3

HE2

2814.1

2814.0

HE3

3080.3

3080.3

The bis-TFP activated ester (10b) was reacted initially with the BN pharmacophore to give 13 and then with 7a to give HE0 (Scheme 2 and 3). For the synthesis of -HEn- (n = 1-3) derivatives, intermediate 13 was initially reacted with 2-propynylamine to give compound 14, which then reacted with compounds 7b-c, using CuAAC, to the Fe-protected heterodimeric structures (17) HEn (n=1-3) (Scheme 3). The final synthesis step for all heterodimers HEn (n=0-3) included the iron removal from the HBED-CC complex by trapping the compounds on a Sep-Pak C-18 cartridge and then washing the cartridge with HCl (1 M). All final products (18, 19 and 20) were further purified with semipreparative RP-HPLC and their molecular mass was analyzed with MALDI-MS (Table 1).


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O O HO

HO

HO

O

O O

N

N

O O

OH

c

OH HO

HO

F

O

8

O

O

O

F

O

O

O O

HO

HO

10a

O

HO O

O

b

Fe

OH F

O F

OH

O

O

O

F O

F

O O

O

9 [Fe(HBED-CC)]-

N

PEG 2-[D-Tyr 6, β-Ala 11, Thi13

O

O

F

OH N

O

H N

N

O

O

N

d

11

O

F

N O

10 min, RT O HO

PEG 2-[D-Tyr 6, β-Ala11, Thi13, Nle14] BN(6-14)

O

O

N

a

N

F

Fe

H N

Fe N

O

HO

HBED-CC

O

O

F

F

F

Fe

F

O

N

F

N O

F

O

O

O

O

F O

O

c

O

O

10b

H N

Fe N

N

PEG 2-[D-Tyr 6, β-Ala

O O O

e

13

H 2N 16 h

excess O N H

O O O

O Fe N

N

H N PEG -[D-Tyr 6, β-Ala11, Thi13, Nle14] BN(6-14) 2 O

O O

14

Scheme 2. Synthesis of Fe‐protected HBED‐CC (9), its mono‐ (10a) and bis‐TFP esters (10b)1 and of monomer GRPrm (12). (a) 1.2 equiv. FeCl3, 2-5% (v/v) DIPEA, MeOH/H2O (1:1, v/v); (b) 10 equiv. TFP, 4 equiv. DIPC, DMF, 5 d, RT; (c) 0.95 equiv., H2N-PEG2-[D-Tyr6, β-Ala11, Thi13, Nle14]BN(6–14), 2 equiv. DIPEA, DMF (1 mL), 4 h, RT. (d) Fe decomplexation from HBED-CC with 1 M HCl acid solution and peptide separation with Sep-Pak C-18.

1

The presented coordination sphere for Fe protected HBED-CC is only representative.



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Scheme 3. Synthesis of the low‐molecular weight heterodimeric target compounds HEn (n=0‐3) (16, 18, 19 and 20). (a) 7a and 1 equiv. of 12 for 16 h in DMF, RT; (b) Fe decomplexation from HBED-CC with 1 M HCl acid solution and peptide separation with SepPak C-18; (c) 4 equiv. CuSO4, 4 equiv. Na-Ascorbate, THF/H2O (1:1, v/v), 16 h, RT; (b) 5 equiv. Na2S, ACN/H2O (1:1, v/v), 0.2 % (v/v) HCl.

Radiolabelling In all cases, radiolabelling with 68Ga resulted in one single species as determined by analytical RP-HPLC, while the radiochemical yield was over 99% (Fig. 1). In addition the retention times (tR) were decreased along with the number of charged amino acids inserted in the flexible spacer analysis (Table 1). All compounds were proved stable at room temperature even after 3 h post labelling.


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Fig. 1 Comparative RP-HPLC analysis studies of the ligands HEn, n=0-3, after labeling with 68Ga. The gamma-trace is presented. The RP-HPLC analysis was performed on a Chromolith RP-18e, 100 × 4.6 mm; Merck, Darmstadt, Germany). Runs were performed using a linear A−B gradient (0 % B to 100 % B in 6 min) at a flow rate of 4 mL/min. Solvent A consisted of 0.1 % aqueous TFA and solvent B was 0.1 % TFA in CH3CN.

Determination of binding affinity for PSMA and GRPr. An in vitro competitive cell binding assay was performed in order to determine the binding potential of the heterodimers for both PSMA and GRPr, with LNCaP cells, PC-3 and AR42J cells, respectively. The results, expressed as IC50 /Ki values, for all compounds under study, heterodimers: HEn (n=0-3) (16, 18, 19 and 20) and monomers: PSMA-11 (Glu-urea-Lys-HBEDCC) and GRPrm (12), are presented in Table 2. The statistical comparison (ANOVA) between the values of the monomers and the heterodimers revealed that there was no significant difference between their IC50 values. The IC50 values determined for the heterodimers essential matched the ones of GRPrm in the case of the PC-3 cell line, while there was a slight increase in the case of AR42J and LNCaP cells.

Table 2



Competition binding assay for GRPr on PC-3 cells (106), AR42J (106) and PSMA on LNCaP cells (106). ANOVA vs compound

IC50 (nM)

log IC50(Std.Er)

1

Ki (nM)

Log Ki (Std.Er)

AR42J

PC-3

monomer

LNCaP

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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GRPPrm

3.65

0.56 (0.05)

-

3.52

0.55 (0.06)

-

HE0

7.72

0.88 (0.08)

NS

7.45

0.87 (0.11)

NS

HE1

7.28

0.86 (0.07)

NS

7.04

0.85 (0.08)

NS

HE2

4.40

0.64 (0,11)

NS

4.25

0.63 (0.11)

NS

HE3

7.09

0.85 (0.09)

NS

6.85

0.84 (0.09)

NS

GRPrm

1.29

0.11 (0.09)

-

1.25

0.10 (0.08)

-

HE0

3.33

0.52 (0.07)

**

3.42

0.53 (0.05)

**

HE1

2.58

0.42 (0.06)

*

2.74

0.44 (0.05)

**

HE2

5.06

0.70 (0.08)

****

3.69

0.57 (0.06)

***

HE3

3.68

0.57 (0.07)

***

4.08

0.61 (0.06)

***

PSMA-11

7.5

0.87 (0.11)

-

12.0

1.08 (0.09)

-

HE0

25.4

1.40 (0.04)

**

23.8

1.38 (0.04)

*

HE1

17.4

1.23 (0.03)

*

16.0

1.20 (0.03)

NS

HE2

25.2

1.40 (0.09)

**

24.6

1.39 (0.10)

*

42.4

1.63 (0.04)

****

40.9

1.61 (0.02)

***

HE3 1

2

Standard Error, NS: no statistically significant difference to respective monomer GRPrm or PSMA-11

Binding studies on cancer cell lines over time. To study the specific binding over time for 68Ga-labelled GRPrm (12), PSMA-11 and HEn (n=03) (16, 18, 19 and 20) a solution (30 nM) of each compound was added to a cell suspension of LNCaP or PC-3 cells and then an aliquot was extracted at specific time points, in which the cell bound activity was measured. The results expressed as the percentage of the radioactivity added to the cell suspension (normalised for 106 cells) are presented in Fig. 2. All PSMA-targeted radioligands tested on the LNCaP androgen sensitive cells presented similar binding kinetics with a plateau of maximal binding after 20 min. The monomer PSMA11 presented slightly higher total binding (55-60 % IA/106 cells) in comparison with the bispecific heterodimers (20-40 % IA/106 cells). The plateau of maximal binding for the GRPr positive PC-3 cells was reached after 40 min for both the monomer GRPrm (12) and the heterodimers HEn (16, 18, 19 and 20). Similarly with PSMA-11 on LNCaP cells, the monomer GRPrm presented slightly higher uptake values on PC-3 than the heterodimers.


2a

70 60 % total radioactivity added

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


% of total radioactivity added

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50 40

68

Ga-GRPr Ga-HE0 68 Ga-HE1 68 Ga-HE2 68 Ga-HE3 nonspecific 68

30 20 10 0 0

20

40 60 Time (min)

80

100

2b Fig. 2 Total cell related radioactivity over time for

68

Ga-labelled versions of monomers PSMA-11



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

and GRPrm and heterodimers HEn, n=0-3 (30 nM) on LNCaP (a) and PC-3 cells (b). Aliquots were taken at: 15, 30, 45, 60 and 90 min, cell related activity was counted in a gamma counter and the results were expressed as % of the total activity added in 106 cells. Nonspecific binding was determined by adding a blocking solution of 2-PMPA or native BN (1000fold concentration as compared with the respective radioligand, 30 μM).

Biodistribution studies in normal mice The in vivo behaviors of the 68Ga-labelled versions of HEn (n=0-3) in comparison with those of the 68Ga-labelled monomers GRPrm and PSMA-11 were studied in mice after i.v. administration of each compound (100 μL, 1-2 MBq, 0.1-0.2 nmol/mouse). Initially a comparative pharmacokinetic study was conducted between the first generation heterodimer, 68Ga-HE0, (68Ga-16) and the two monomers: 68Ga-PSMA-11 and 68Ga-GRPrm (68Ga-12). The results of biodistributions (1 h p.i.) and blocking studies with coadministration of 2-PMPA and native BN are summarized in Fig. 3. 68Ga-PSMA-11 resented high spleen (17.9 ± 2.87 % ID/g) and kidney (139.4 ± 21 % ID/g) uptake, while for 68Ga-GRPrm the highest uptake was for pancreas (8.5 ± 2.2 %ID/g) and intestines (4.2 ± 1.8 % ID/g).

Fig. 3 Comparative biodistribution studies 1h p.i. between the 68Ga-labelled HE0 and the respective monomers (PSMA-11 and GRPrm) in Balb nu/nu male mice (average weight 20 ± 2.0 g) by


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injecting a solution (in saline, pH 7) of each compound (100 μL, 1-2 MBq/mouse; 0.1-0.2 nmol) into the tail vein of the animals. The results are expressed as percentage of the injected dose per g (% ID/g) for each organ or tissue. Blocking experiments were conducted by co-injecting an amount of native BN (1 μL of a 100 mM solution) or 2-PMPA (15 μL of a 100 mM solution) along with the radiolabelled ligand.

The heterodimer, 68Ga-HE0, (68Ga-16) presented high kidney uptake (164.4 ± 8.9 % ID/g), similar to 68Ga-PSMA-11, and a rather high spleen uptake (4.4 ± 0.5 % ID/g), which was less than the one of 68Ga-PSMA-11 Its uptake in pancreas (8.1 ± 1.0 % ID/g) was similar to 68GaGRPrm (68Ga-12), while intestinal uptake was less (1.30 ± 0.16 % ID/g). In vivo blocking experiments were conducted by co-injecting an excess of native BN or 2-PMPA, respectively. When BN was co-administered a decrease in the spleen (from 4.41 to 0.5 % ID/g), kidney (from 164.38 to 29.69 % ID/g) and in the pancreatic uptake (from 8.05 to 0.57 % ID/g) was observed, while with 2-PMPA spleen, kidney and pancreatic uptake were also decreased from their initial values to 0.47, 2.87 and 3.24 % ID/g, respectively. The pharmacokinetics of 68Ga-HE0 (68Ga-16) in comparison with 68Ga-HEn (n = 1-3) were investigated with biodistribution experiments in mice, and the results are summarized in Table 3. In most cases, the incorporation of the HE spacer improved the biodistribution profile compared with 68Ga-HE0 (68Ga-16) due to the reduction of radioactivity accumulation in critical non-targeted organs like kidneys, liver and spleen (Table 3). The area under the curve AUC0-90 min was also calculated for these organs and the results are summarized in Table 3. The AUC values representing the total exposure of those organs to the administered compound were much lower in case of the HE spacer incorporation. Especially for kidneys and liver a reduction of more than 50 % was observed. A statistically significant reduction in other non-targeted organs was observed for HEn (n = 1-3) compared with 68Ga-HE0, i.e. in blood and heart, muscle, intestines, lungs, while, in most cases, for the GRPr expressing pancreas it was increased.



Table 3 Comparative biodistribution studies in balb/c nu/nu mice at selected time points 15, 30, 60 and 90 min p.i. between the 68Ga-labelled HEn (n = 0-1). The values are expressed as % ID/g (mean ± SD, n = 3-4). Last column includes the area under the curve (AUC0-90) for selected organs (% ID/g*min). 15 min

30 min

60 min

AUC0

90 min

68

Ga-HE0

-90 min

Blood

0.18

±

0.03

0.67

±

0.04

0.63

±

0.05

0.35

±

0.07

Heart

0.79

±

0.07

0.55

±

0.04

0.64

±

0.08

0.34

±

0.09

Lungs

1.30

±

0.92

1.72

±

0.47

1.21

±

0.03

1.38

±

0.19

Spleen

5.77

±

0.86

5.25

±

1.00

4.41

±

0.51

3.31

±

0.50

386. 6

Liver

3.48

±

0.21

3.06

±

0.21

2.91

±

0.09

2.39

±

0.55

244, 2

Kidneys

95.3 1

±

8.32

107.5 7

±

6.65

164.3 8

±

8.96

166.4 9

±

15.3 9

1127 9

Muscle

0.62

±

0.08

0.30

±

0.11

0.30

±

0.03

0.21

±

0.08

Intestin es

1.35

±

0.20

1.17

±

0.18

1.30

±

0.16

0.96

±

0.56

Brain

0.15

±

0.02

0.22

±

0.08

0.26

±

0.11

0.35

±

0.23

Pancrea s

11.7 1

±

2.59

7.68

±

0.64

8.05

±

1.00

8.49

±

1.21

Ga HE1

15 min

30 min

60 min

90 min

Blood

0.13

±

0.01

0.44

±

0.06

0.36

±

0.04

0.30

±

0.03

Heart

0.66

±

0.07

0.35

±

0.03

0.28

±

0.01

0.29

±

0.02

Lungs

1.55

±

0.10

1.21

±

0.50

0.66

±

0.02

0.69

±

0.03

Spleen

2.97

±

0.20

3.04

±

0.68

2.55

±

0.25

2.77

±

0.63

231. 0

Liver

1.12

±

0.07

0.81

±

0.10

0.65

±

0.02

1.06

±

0.07

70,4 3

Kidneys

83.3 1

±

7.34

80.56

±

7.77

87.57

±

10.3 7

130.2 4

±

2.96

6940

Muscle

0.56

±

0.02

0.21

±

0.08

0.37

±

0.24

0.25

±

0.05

Intestin es

1.91

±

0.43

1.15

±

0.21

0.63

±

0.24

1.26

±

0.16

Brain

0.11

±

0.02

0.15

±

0.05

0.19

±

0.07

0.33

±

0.22

68

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 43


Page 13 of 43

Pancrea s

17.6 1

±

0.17

68

Ga HE2

15 min

Ga HE3

9.95

±

2.21

30 min

5.48

±

1.55

60 min

9.71

±

1.91

90 min

Blood

0.32

±

0.05

0.72

±

0.15

0.51

±

0.16

0.35

±

0.02

Heart

1.43

±

0.14

0.61

±

0.09

0.47

±

0.15

0.35

±

0.01

Lungs

2.73

±

0.26

1.27

±

0.16

0.95

±

0.29

0.76

±

0.08

Spleen

1.89

±

0.14

1.85

±

0.37

1.24

±

0.25

2.13

±

0.70

139. 1

Liver

1.73

±

0.15

0.88

±

0.12

0.87

±

0.13

1.02

±

0.08

87,1 5

Kidneys

89.8 7

±

19.9 2

89.81

±

12.6 8

73.25

±

26.5 6

83.91

±

5.94

6825

Muscle

0.94

±

0.04

0.25

±

0.16

0.23

±

0.08

0.29

±

0.18

Intestin es

2.39

±

0.38

1.77

±

0.35

2.75

±

1.79

1.74

±

0.51

Brain

0.16

±

0.04

0.21

±

0.06

0.32

±

0.09

0.18

±

0.07

Pancrea s

23.8 9

±

4.01

13.33

±

0.82

13.59

±

1.22

17.29

±

2.74

15 min

68

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


30 min

60 min

90 min

Blood

0.13

±

0.01

0.72

±

0.15

0.51

±

0.16

0.35

±

0.02

Heart

0.95

±

0.14

0.61

±

0.09

0.47

±

0.15

0.35

±

0.01

Lungs

1.79

±

0.33

1.27

±

0.16

0.95

±

0.29

0.76

±

0.08

Spleen

2.09

±

0.91

1.85

±

0.37

1.24

±

0.25

2.13

±

0.70

137. 0

Liver

0.83

±

0.10

0.88

±

0.12

0.87

±

0.13

1.02

±

0.08

52,8 8

Kidneys

66.1 0

±

12.3 3

89.81

±

12.6 8

73.25

±

26.5 6

83.91

±

5.94

5807

Muscle

0.58

±

0.12

0.25

±

0.16

0.23

±

0.08

0.29

±

0.18

Intestin es

1.67

±

0.22

1.77

±

0.35

2.75

±

1.79

1.74

±

0.51

Brain

0.10

±

0.02

0.21

±

0.06

0.32

±

0.09

0.18

±

0.07

Pancrea s

13.8 7

±

3.15

13.33

±

0.82

13.59

±

1.22

17.29

±

2.74

Biodistribution studies in PC-3 and LNCaP tumor bearing mice



The in vivo behaviors of the 68Ga-labelled heterodimers HEn (n=0-3) along with the monomers GRPrm and PSMA-11 were also evaluated in balb/c nu/nu male mice, bearing PSMA positive LNCaP and GRPr positive PC-3 tumors. The results of the respective tumor uptakes for both tumor models are presented in Fig. 4 (for the full organ distribution tables see supplementary data), while Table 4 summarizes the tumor-to-normal tissue ratios for muscle, kidney, spleen and liver. As compared to the monomer 68Ga-GRPrm (30 min: 3.06 ± 0.28; 60 min: 1.75 ± 0.3 %ID/g), the 68Ga-labelled HE1, HE2 and HE3 presented similar tumor uptake in PC-3 tumor bearing mice, while for 68Ga-HE0 the tumor uptake was significantly lower (30 min: 1.0 ± 0.16; 60 min: 0.84 ± 0.18 %ID/g). Regarding LNCaP tumor bearing mice,68Ga-HE2 and 68GaHE3 showed a comparably high tumor uptake like the monomeric reference PSMA-11 (30 min: 7.5 ± 3.4; 60 min: 5.9 ± 2.8 %ID/g), while 68Ga-HE0 and 68Ga-HE1 presented lower tumor uptake (30 min: 2.2 ± 0.1 and 2.1 ± 0.17; 60 min: 2.4 ± 0.1 and 2.4 ± 1.2 %ID/g). Regarding the comparison of tumor-to-normal tissue ratios, the incorporation of the HE spacer in all cases resulted in improved contrast in comparison with 68Ga-HE0 and in some cases also compared to the monomers. For example compound 68Ga-HE2 presented higher tumor-toliver ratios in both PC-3 and LNCaP tumor bearing mice in comparison with the monomers.

*** ***

**

m in

****

60

m in

**

30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 43

a


**

Page 15 of 43

% ID/g tumor LNCap 22 20

**

**

18

NS

NS

NS

NS

NS

*

*

**

NS

NS

****

NS

16

68

Ga-PSMAm

14

68

Ga--HE0

68

Ga-HE1

12

68

Ga-HE2

10

68

Ga-HE3

8 6 4 2

60

m in

m in

0

30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


b Fig. 4 Tumor uptake, expressed as % ID/g (mean ± SD, n=3-4), determined from biodistribution studies (30, 60 min p.i.) in balb/c nu/nu mice bearing: (a) LNCaP and (b) PC-3 tumors, after i.v. administration of the 68Ga-labelled monomers (PSMA-11 and GRPrm) and heterodimers HEn (n=0-3). Significant differences are presented with stars above the bars that were compared (P

GRPr Targeting Radioligands with Optimized

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