Metabolically Stabilized 68Ga-NOTA-Bombesin for PET Imaging of

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Metabolically-stabilized 68Ga-NOTA-bombesin for PET Imaging of prostate cancer and influence of protease inhibitor Phosphoramidon Susan Richter, Melinda Wuest, Cody N. Bergman, Stephanie Krieger, Buck E. Rogers, and Frank Wuest Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.5b00970 • Publication Date (Web): 14 Mar 2016 Downloaded from http://pubs.acs.org on March 22, 2016

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Molecular Pharmaceutics


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Metabolically-stabilized 68Ga-NOTA-bombesin for PET Imaging of prostate

2

cancer and influence of protease inhibitor phosphoramidon

3 4 5 6

Susan Richter1, Melinda Wuest1, Cody N. Bergman1, Stephanie Krieger2,

7

Buck E. Rogers2, Frank Wuest1

8 9 10

1

11

Canada

12

2

13

USA

Department of Oncology, University of Alberta, Cross Cancer Institute, Edmonton, AB T6G 2X4,

Department of Radiation Oncology, Washington University School of Medicine, St. Louis, MO 63108,

14 15 16 17 18

KEYWORDS:

19

phosphoramidon.

Bombesin, gastrin-releasing peptide (GRP) receptor,

68

Ga, prostate cancer, PET,

20 21 22 23

*Corresponding author: Frank Wuest, Department of Oncology, University of Alberta, 11560 University

24

Avenue, Edmonton, AB T6G 1Z2, Canada. Tel.: +1 (780) 989-8150; Fax: +1 (780) 432-8483. E-mail

25

address: [email protected].

26

1

ACS Paragon Plus Environment


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1

ABSTRACT

2

Peptide receptor-based targeted molecular imaging and therapy of cancer is on the current forefront

3

of nuclear medicine preclinical research and clinical practice. The frequent overexpression of gastrin-

4

releasing peptide (GRP) receptors in prostate cancer stimulated the development of radiolabeled

5

bombesin derivatives as high affinity peptide ligands for selective targeting of the GRP receptor. In this

6

study, we have evaluated a novel 68Ga-labeled bombesin derivative for PET imaging of prostate cancer

7

in vivo. In addition, we were interested in testing recently proposed “serve-and-protect” strategy to

8

improve metabolic stability of radiolabeled peptides in vivo and to enhance tumor uptake. GRP

9

receptor targeting peptides NOTA-BBN2 and

nat

Ga-NOTA-BBN2 demonstrated a characteristic

10

antagonistic profile and high binding affinity towards the GRP receptor in PC3 cells (IC50 4.6–8.2 nM).

11

Radiolabeled peptide 68Ga-NOTA-BBN2 was obtained from NOTA-BBN2 in radiochemical yields greater

12

than 62% (decay-corrected). Total synthesis time was 35 min, including purification using solid-phase

13

extraction.

14

peptidases in vivo within the investigated time frame of 60 min. Interestingly, metabolic stability was

15

not further enhanced in the presence of protease inhibitor phosphoramidon. Dynamic PET studies

16

showed high tumor uptake in both, PC3- and LNCaP-bearing BALB/c nude mice (SUV5min >0.6; SUV60min

17

>0.5). Radiotracer 68Ga-NOTA-BBN2 represents a novel radiometal-based bombesin derivative suitable

18

for GRP receptor targeting in PC3 and LNCaP mouse xenografts. Further increase of metabolic stability

19

in vivo and enhanced tumor uptake was not observed upon administration of protease inhibitor

20

phosphoramidon. This led to the conclusion that the recently proposed “serve-and-protect” strategy

21

may not be valid for peptides exhibiting favourable intrinsic metabolic stability in vivo.

68

Ga-NOTA-BBN2 exhibited favorable resistance against metabolic degradation by

22

2


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1

INTRODUCTION

2

Various

3

68

4

neuroendocrine tumors (NETs) through specific targeting of somatostatin receptors (sst2, sst3, and

5

sst5).1 Success of targeting NETs with small radiolabeled peptides for imaging and therapy prompted

6

the development of other peptide-based radiopharmaceuticals for targeting more common

7

malignancies such as prostate cancer.2 Prostate cancer is the most commonly diagnosed form of

8

cancer among men with an estimated 24,000 new cases and 4,100 deaths in Canada in 2015.3

9

A highly promising

68

Ga-octreotide peptide analogues (68Ga-DOTA-TOC,

68

Ga-DOTA-NOC,

68

Ga-DOTA-TATE and

Ga-HA-DOTA-TATE) represent an important breakthrough in the clinical management of patients with

68

Ga-labeled radiotracer for PET imaging of recurrent prostate cancer is 68

Ga-PSMA-HBED-CC.4 Diagnostic

10

peptidometic prostate-specific membrane antigen (PSMA) inhibitor

11

value of 68Ga-PSMA-HBED-CC has been demonstrated in 319 patients so far.4 Gastrin-releasing peptide

12

(GRP) receptors are attractive alternative targets for molecular imaging and peptide receptor

13

radionuclide therapy (PRRT) of prostate cancer. Molecular targeting of GRP receptors in prostate

14

cancer and other human malignancies like breast, colon and small-cell lung cancer (SCLC) is based on

15

elevated overexpression of GRP receptors in cancers compared to their rather low endogenous

16

expression levels in most other tissues and organs.5-7

17

Human prostate cancer cell lines PC3 and LNCaP are suitable models for studying GRP receptor-

18

mediated molecular targeting due to different GRP receptor levels. While PC3 cells express high

19

densities of GRP receptor (47,600 binding sites per cell) representing a late-stage prostate carcinoma,

20

whereas the lymph node metastasis-derived cell line LNCaP is described to possess lower densities of

21

the receptor (100 binding sites per cell) representing an early-stage prostate cancer.8,9 Both prostate

22

cancer cell lines also differ in their hormone sensitivity. PC3 cells are androgen-independent, but

23

LNCaP cells require androgens for growth.

24

Recently, we described metabolically stabilized bombesin analog QWAV-Sar-H-FA01010-Tle-NH2

25

(BBN2) for molecular imaging of GRP receptors in PC3 tumors. Peptide BBN2 was radiolabeled with

26

fluorine-18 via prosthetic group chemistry using [18F]SFB (N-succinimidyl-4-[18F]fluorobenzoate) or

27

glucose analogue [18F]FDG (2-deoxy-2-[18F]fluoro-D-glucose). Radiolabeled BBN2 was studied in PC3

28

tumors, and PET imaging confirmed favorable GRP receptor-specific tumor uptake and

29

radiopharmacological profile as an GRP receptor antagonist.10,11

3



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1

Here we report on the synthesis and evaluation of bombesin-derivative BBN2 decorated with

2

macrocyclic chelator NOTA for radiolabeling with gallium-68. Gallium-68 is a short-lived positron

3

emitter with a half-life of 68 min (Eβ+max = 1.9 MeV, β+ = 89%) that is available through a

4

generator (68Ge: half-life of 271 d).12

5

radiochemistry, and 68Ga provides high quality PET images due to its physical properties.13

6

In the past, several

7

examples include

8

NOTA- and

9

aminobenzoyl

68

68

68

68

68

Ge/68Ga-

Ga is compatible with straightforward and reproducible

Ga-labeled bombesins have been described in preclinical studies. Prominent

Ga-NOTA-P2-RM26 (68Ga-NOTA-PEG2-[DPhe6,Sta13,Leu14]-bombesin(6-14))14,

68

Ga-

Ga-NODAGA-MJ9 containing a 4-amino-1-carboxymethyl-piperidine linker or a Gly-4moiety,15

and

68

(68Ga-DOTA-4-amino-1-carboxymethyl-piperidine-

Ga-DOTA-RM2

10

[DPhe6,Sta13,Leu14]-bombesin(6-14).16 Pan et al. described GRP receptor targeting with

11

ATBBN (68Ga-NOTA-DPhe-Gln-Trp-Ala-Val-Gly-His-Leu-NHCH2CH3) in comparison with more hydrophilic

12

68

13

NHCH2CH3).17 GRP receptor agonist AMBA (DO3A-CH2CO-Gly-(4-aminobenzoyl)-bombesin(7-14) amide)

14

was decorated with different chelators such as NOTA and NODAGA and radiolabeled with

15

Radiotracer

16

imaging profile compared to metabolism-based targeting of prostate cancer with 18F-methylcholine.19

17

68

18

DOTA-4-amino-1-carboxymethylpiperidine-DPhe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2 and used in a

19

multicenter study for detection of primary and metastatic prostate carcinoma in 14 patients.20 Safety

20

profile and dosimetry of

21

GRP receptor pan-bombesin derivative

22

bombesin(6-14)amide in patients with gastrointestinal stromal tumors. However, all reported peptide-

23

based radiotracers included various challenges for a successful translation into clinical application. A

24

qualified candidate for clinical translation should possess the following characteristics: (1) high tumor

25

accumulation and retention, which is partially the result of a high affinity and high specific-binding of

26

the radiopeptide probe to its target and sufficient metabolic stability in vivo; (2) high tumor-to-

27

background ratios for favorable image contrast; and (3) fast clearance of radioactivity from non-target

28

organs and tissues to reduce background signal.

Ga-NOTA-

(68Ga-NOTA-Gly-Gly-Gly-Arg-Asp-Asn-DPhe-Gln-Trp-Ala-Val-Gly-His-Leu-

Ga-NOTA-MATBBN

68

68

68

Ga.18

Ga-AMBA was studied in human prostate cancer xenografts showing a superior PET

Ga-labeled bombesin peptides were also used in humans.

68

68

Ga-BAY86-7548 was prepared from

Ga-BAY86-7548 was studied in healthy men.21 Another clinical study used 68

Ga-BZH3 (68Ga-DOTA-PEG2-[DTyr6,βAla11,Thi13,Nle14]-

4


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1

Another special challenge with radiolabeled peptides is their limited metabolic stability in vivo.

2

Recently, the de Jong group has introduced a highly promising “serve-and-protect” strategy to increase

3

metabolic stability, bioavailability, prolonged circulation times and tumor-localization of various

4

radiopeptides containing a natural peptide sequence (somatostatin-based peptide

5

SS14, bombesin and gastrin derivatives

6

stabilized truncated CCK-minigastrin-analog

7

neutral endopeptidase (NEP) inhibitor phosphoramidon (PA).23-26

8

These studies discussed inhibition of neutral endopeptidase (NEP, EC 3.4.24.11), a key enzyme within

9

the metabolic degradation of peptides in general and bombesin in particular (cleavage between His12

10

and Leu13 residue)27,28. Inhibition of neutral endopeptidase through natural product protease inhibitor

11

phosphoramidon led to a significantly reduced metabolic degradation of radiopeptides in vivo.

12

The goal of the present study was the radiopharmacological investigation of novel bombesin-derivative

13

68

14

xenografts. The positive experience with our recently reported

15

high metabolic stability and favorable binding affinity towards the GRP receptor prompted us to extend

16

our research activities to the development of a

17

radiotracer for clinical translation. This work also included analysis of metabolic stability of 68Ga-NOTA-

18

BBN2 in vivo with and without co-administration of the endopeptidase inhibitor phosphoramidon to

19

test the “serve-and-protect” concept with intrinsic metabolically stable radiopeptides like 68Ga-NOTA-

20

BBN2 to enhance tumor uptake.

111

177

In-DOTA-PanSB1 and 111

111

In-DOTA-Ala1-

Lu-DOTA-GRP(13-27)) as well as

In-DOTA-MG11 in tumor sites based on co-injection of

Ga-NOTA-BBN2 as suitable radiopeptide for PET imaging of GRP receptors in prostate cancer

68

18

F-labeled BBN2 analogs in terms of

Ga-labeled BBN2 analog to develop a suitable

21

5



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1

MATERIALS AND METHODS

2

Reagents. All chemicals were obtained from Sigma-Aldrich (St. Louis, MO, U.S.A.). Peptide synthesis

3

reagents were purchased from NovaBioChem. Fmoc-amino acid FA01010 ((4R,5S)-Fmoc-4-amino-5-

4

methylheptanoic acid) and linker Fmoc-Ava-OH were obtained from Polypeptide Inc. (San Diego, CA,

5

U.S.A). Stabilized bombesin peptide amide was synthesized via a combination of manual coupling

6

procedures and automated solid-phase peptide synthesis (SPPS) using the Syro I peptide synthesizer

7

(MultiSynTech/Biotage, Charlotte, NC, U.S.A). A 20 mCi (740 MBq) iThemba LABS 68Ge/68Ga-generator

8

from isoSolutions Inc. (Vancouver, B.C., Canada) was used as 68Ga source. Mass spectra were recorded

9

on an AB Sciex Voyager Elite matrix-assisted laser desorption ionisation mass spectrometer time-of-

10

flight (MALDI-MS TOF, AB Sciex, Foster City, CA, U.S.A.). Analytical HPLC was performed on a Shimadzu

11

system (Mandel Scientific, Guelph, ON, Canada) equipped with a DGU-20A5 degasser, a SIL-20A HT

12

autosampler, a LC-20AT pump, a SPD-M20A photo-diode-array detector and a Ramona Raytest

13

radiodetector using a Phenomenex Luna 10u C18(2) 100A, 250 x 4.6 mm column. Semi-preparative

14

HPLC was performed on a Gilson system (Mandel Scientific, Guelph, ON, Canada) with a 321 pump and

15

a 155 dual wavelength detector installed with a Phenomenex Jupiter 10u Proteo 90A, 250 x 10 mm,

16

4.5 µm C18 column. UV absorbance was monitored at a wavelength of 210 and 254 nm. Mobile phase

17

consisted of water/0.2%TFA as solvent A and acetonitrile as solvent B.

18

Human androgen-independent prostate cancer cell line PC3 (American Type Tissue Culture Centre,

19

Manassas, VA, U.S.A) was cultivated in 45% RPMI1640 Dulbecco’s modified Eagle’s medium (DMEM)

20

supplemented with 45% Ham's F-12 and 10% heat-inactivated fetal bovine serum (FBS) from Invitrogen

21

(Life Technologies Inc., Grand Island, NY, U.S.A). Human androgen-dependent prostate cancer cell line

22

LNCaP was obtained from ATCC (American Type Tissue Culture Centre, Manassas, VA, U.S.A). 125I-Tyr4-

23

BBN was obtained from PerkinElmer (Waltham, MA, U.S.A.). Cell-associated radioactivity was

24

measured on a 2480 automatic gamma counter WIZARD2® (PerkinElmer, Waltham, MA, U.S.A.).

25 26

Peptide Synthesis. Bombesin peptide Ava-Gln7-Trp8-Ala9-Val10-Sar11-His12-FA0101013-Tle14-NH2 (BBN2)

27

was synthesized prior to modification with bifunctional chelator pSCN-Bn-NOTA (S-2-(4-

28

Isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid, Macrocyclics, Dallas, TX, U.S.A.) to

29

provide NOTA-BBN2 for radiolabeling with

68

Ga and the synthesis of non-radioactive reference 6


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nat

1

compound

Ga-NOTA-BBN2. Peptide synthesis of BBN2 was performed on an automated peptide

2

synthesizer (Syro I, MultiSynTech/Biotage, Charlotte, NC, U.S.A.) using the Fmoc-orthogonal solid phase

3

peptide synthesis starting from the Rink-amide MBHA resin (loading: 0.6 mmol/g). Amino acid

4

components of BBN2 are tert.-leucine (Tle), (4R,5S)-4-amino-5-methylheptanoic acid (FA01010),

5

histidine (His), sarcosine (Sar), valine (Val), alanine (Ala), tryptophan (Trp), glutamine (Gln) and linker

6

moiety 5-aminovaleric acid (Ava). Detailed description on the synthesis procedure, cleavage conditions

7

and analytical characterization of the bombesin sequence Ava-Gln7-Trp8-Ala9-Val10-Sar11-His12-

8

FA0101013-Tle14-NH2 (BBN2) can be found in a previously published manuscript.9

9 10

Synthesis of labeling precursor NOTA-BBN2. 5 mg (1 eq., 4.7 µmol) of Ava-Gln7-Trp8-Ala9-Val10-Sar11-

11

His12-FA0101013-Tle14-NH2 (BBN2) was dissolved in 200 µL of DMF in a LoBind Eppendorf tube before

12

3.5 mg (1.3 eq., 6.3 µmol) of pSCN-Bn-NOTA in DMF (150 µL) was added. The pH was adjusted to 9 by

13

the addition of 2.5 µL (4 eq., 18.8 µmol) of triethylamine (TEA). The reaction mixture was incubated at

14

50 oC for 3 h before it was subjected to semi-preparative HPLC purification. HPLC purification was

15

performed using a Phenomenex Jupiter 10u Proteo 90A, 250 x 10 mm, 4.5 µm C18 column at a flow

16

rate of 2 mL/min and a gradient of water/0.2% TFA as solvent A and acetonitrile as solvent B: 0-10 min

17

10% B, 25 min 50% B, 30-40 min 80% B, 40-45 min 90% B (tR = 30.8 min). HPLC solvent was reduced

18

under vacuum using a rotary evaporator and lyophilisation gave the chelator-modified peptide NOTA-

19

BBN2 as a white powder (6.2 mg, 4.1 µmol, 87% isolated yield). MW C72H108N18O16S 1512.8, measured

20

MALDI-MS (positive) m/z 1513.6 [M+H]+, 1535.5 [M+Na]+, 1551.5 [M+K]+.

21

Quality control was performed on an analytical Shimadzu HPLC system using a Phenomenex Luna 10u

22

C18(2) 100A, 250 × 4.6 mm column at a constant flow rate of 1 mL/min and the following gradient with

23

water/0.2% TFA as solvent A and acetonitrile as solvent B: 0-3 min 10% B, 10 min 30% B, 17 min 50% B,

24

23 min 70% B, 27-30 min 90% B (tR = 17.3 min, purity >99%).

25 nat

Ga-NOTA-BBN2. 1.5 mg (1 eq., 1.0 µmol) of NOTA-BBN2 was

26

Synthesis of reference compound

27

incubated with an excess (21 eq.) of natGaCl3 as Ga source in 100 mM NH4OAc buffer (pH 5.5). Prior to

28

the reaction with peptide NOTA-BBN2, natGaCl3 was challenged with EDTA each dissolved in 100 µL of

29

100 mM NH4OAc buffer (pH 5.5) for a 5 min incubation time at room temperature. 7



Page 8 of 37


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1

After adjusting the pH to 5.5 with 100 mM NH4OAc buffer the peptide Ga-EDTA mixture was allowed to

2

react for 48 h at ambient temperature. The reaction batch was purified via semi-preparative HPLC

3

performed on a Phenomenex Luna C18(2) 100A, 250 x 10 mm column using a gradient of water/0.2%

4

TFA as solvent A and acetonitrile as solvent B: 0-10 min 20% B, 25 min 50% B, 30-40 min 80% B and a

5

flow rate of 2 mL/min. The fractions containing the Ga-complex were collected, and the solvent was

6

reduced under vacuum using a rotary evaporator. Subsequent lyophilisation gave peptide natGa-NOTA-

7

BBN2 as white powder (1.2 mg, 0.8 µmol, 80% isolated yield). MW C72H105GaN18O16S 1578.7, measured

8

MALDI-MS (positive) m/z 1579.5 [M+H]+, 1601.5 [M+Na]+. Purity of natGa-NOTA-BBN2 was assessed on

9

an analytical Shimadzu HPLC system using a Phenomenex Luna 10u C18(2) 100A, 250 × 4.6 mm column

10

at a constant flow rate of 1 mL/min and the following gradient with water/0.2% TFA as solvent A and

11

acetonitrile as solvent B: 0-3 min 10% B, 10 min 30% B, 17 min 50% B, 23 min 70% B, 27-30 min 90% B

12

(tR = 17.0 min, purity > 96%).

13 14

Competitive Binding Assay. In vitro competitive binding of peptides was analyzed in human prostate

15

adenocarcinoma PC3 cells in triplicate as described before.9 Briefly, determination of the concentration

16

of half-maximum inhibition (IC50 values) was carried out as a competition against

17

(0.05 nM final concentration) using increasing concentrations of NOTA-BBN2 or

18

the range of 80 pM to 10,000 nM. After incubation for 2 h at 4 oC and several washing steps, cells were

19

harvested. Counts per minute (cpm) of cell-associated radioactivity was measured in a Wizard gamma

20

counter, decay-corrected and plotted versus log of peptide concentration to give the typical sigmoidal

21

dose-response curves.

125

I-Tyr4-bombesin

nat

Ga-NOTA-BBN2 in

22 23

Calcium Release Assay. Cells were plated on coverslips (Warner Instruments Cat #64-0701) treated

24

with 2 M HCl and 100 µg/mL Poly-D-lysine at 80% confluence 24 h before imaging. Media was removed

25

and cells were washed twice with calcium imaging buffer (140 mM NaCl, 4 mM KCl, 10 mM HEPES,

26

5 mM glucose, 1.3 mM MgSO4, 2.4 mM CaCl2) and incubated with 1 mL of calcium imaging buffer and

27

10 µL of Fura-2/Pluronic F-127 (Life Technologies Ltd., Invitrogen, Paisley, UK) for 30 min at 37 °C. Cells

28

were then washed 3 times with calcium imaging buffer and incubated with calcium imaging buffer at

29

37 °C for 30 min.

8


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1

Cells were then positioned on the stage of an inverted fluorescence microscope and flushed with

2

calcium imaging buffer using a perfusion system. Images were obtained using a ORCA-R2 CCD camera

3

and Aquacosmos software (Hamamatsu Photonics, Japan) with alternating illumination at 340 and 380

4

nm. Fluorescence images were collected at 2 sec intervals through an objective lens (S Flour x 10/0.50

5

numerical aperture, Nikon) and an emission filter (470-550 nm). To test NOTA-BBN2, cells were flushed

6

with control agonist (1 nM Tyr4-BBN(1-14) for 30 sec to 2 min, then flushed with buffer and allowed to

7

return to baseline for 30 min. Cells were then perfused with NOTA-BBN2 (0.1 nM-100 nM) for 2 min

8

and then stimulated with 1 nM Tyr4-BBN(1-14). Cells were then allowed to return to baseline. Regions

9

of interest (ROIs) were selected based on cells with the best response. Changes in intracellular calcium

10

levels were determined by the Fura-2 ratio (F340/380) and changed to [Ca2+] using the formula Y=((R-

11

Rmin)/(Rmax-R)*(F380max/F380min))*Kd and the data were plotted using Prism v.6 (GraphPad Inc.)

12

software.

13 68

14

Radiolabeling of NOTA-BBN2 with Gallium-68. Radionuclide

15

(trace-metal grade) from the

16

Fuerstenfeldbruck, Germany). The high activity fraction of the 68Ga-eluate (2.5-2.7 mL) was collected in

17

the plastic reaction vessel while the rest of radioactivity was dispensed in the 20 mL syringe of the GRP

18

module dispenser unit. 600-900 µL (65-85 MBq) of the 68Ga-generator eluate was removed from the

19

plastic reaction vessel, diluted with 300-500 µL of 4 M NaOAc buffer (pH 9.4) to adjust pH to 5 and

20

reacted with 20 µg of NOTA-BBN2 in 20 µL of DI water for 8 min at room temperature in a LoBind

21

Eppendorf tube.

22

An EDTA challenge step for 10 min at room temperature followed by the addition of 12 µL of 10 mM

23

EDTA. The reaction mixture was diluted with 4 mL of PBS (pH 7.4) before purification via solid-phase

24

extraction (SPE) using a Sep-Pak tC18 Plus cartridge (Waters Corporation, Milford, MA, U.S.A.) (pre-

25

conditioned with 10 mL of MeCN and 10 mL of PBS). The cartridge-trapped

26

with 1.1 mL of EtOH. EtOH was evaporated at 85 oC in a stream of nitrogen before 68Ga-NOTA-BBN2

27

was re-dissolved in 100 µL of saline (0.9% w/v of NaCl). Typically, 22-52 MBq of

28

(radiochemical yield: 62±14% (decay-corrected)) was synthesized within 35±4 min (n=11).

68

Ga was eluted with ~5 mL of 1 N HCl

Ge/68Ga-generator via automated GRP module (Scintomics GmbH,

9


68

Ga-peptide was eluted

68

Ga-NOTA-BBN2


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1

The 68Ga-labeled peptide was analyzed for radiochemical purity with radio-TLC on RP18 plates using 1

2

M NH4OAc/MeOH 10/90 (Rf = 0.1-0.2) and radio-HPLC using the Shimadzu HPLC system equipped with

3

a Phenomenex Luna 10u C18(2) 100A, 250 x 4.6 mm column at a constant flow rate of 1 mL/min and

4

the following gradient with water/0.2% TFA as solvent A and acetonitrile as solvent B: 0-3 min 10% B,

5

10 min 30% B, 17 min 50% B, 23 min 70% B, 27-30 min 90% B (tR = 17.4 min, radiochemical purity

6

>96%).

7 8

Determination of lipophilicity (68Ga-NOTA-BBN2). Lipophilicity was determined according to the

9

shake-flask method by determining the partition coefficient of the

68

Ga-labelled peptide in n-octanol

10

and PBS buffer (pH 7.4) as aqueous phase.29 The organic and the aqueous phase were pre-saturated

11

24 h before the actual start of the experiment. 500 µL of each layer were added to 2 MBq of

12

NOTA-BBN2 in a LoBind Eppendorf tube and the mixture was mixed vigorously for 3 min. The layers

13

were allowed to separate by centrifugation at 2000 rpm for 5 min. Aliquots of 100 µL were removed

14

from each phase and measured in a Wizard gamma counter (Wallac 1480 Wizard-3, Perkin-Elmer,

15

Woodbridge, Ontario, Canada). Calculated logD7.4 values are expressed as mean±SD from

16

3 experiments each performed in triplicate.

68

Ga-

17 18

Animal studies. All animal studies were carried out according to the guidelines of the Canadian Council

19

on Animal Care (CCAC) and approved by the Cross Cancer Institute Animal-Care Committee. In vivo

20

studies were done using normal BALB/c and male PC3 and LNCaP tumour-bearing BALB/c nude mice

21

(body weight: 20 - 24 g, Charles River Laboratories, Saint-Constant, QC, Canada).

22

For tumor xenografts, about 5-6x106 of PC3 cells in 100 µL of PBS or 20-25x106 of LNCaP cells in 200 µL

23

of PBS/Matrigel (50/50) were injected into the shoulder of male nude BALB/c mice subcutaneously.

24

Androgen-dependency of LNCaP tumors required addition of a 1.5 mg pellet containing

25

dehydroepiandrosterone DHEA (60 day release; Innovative Research of America, Sarasota, FL, U.S.A.),

26

which was implanted subcutaneously into the upper right flank at the same time when LNCaP cells

27

were injected. After 3-5 weeks PC3 tumors and after 6-8 weeks LNCaP tumors reached sizes of ∼ 300 -

28

500 mm3 and were used for the experiments described.

29

10


Page 11 of 37



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

1

Metabolic stability studies in vivo in the presence and absence of phosphoramidon. For metabolic

2

stability studies in vivo, normal BALB/c mice were anesthetized through inhalation of isoflurane in 40%

3

oxygen/ 60% nitrogen (gas flow 1 L/min) prior to i.v. radiotracer injection via the tail vein. Mice were

4

injected with 12-25 MBq of 68Ga-NOTA-BBN2. For metabolic stability in vivo in the presence of enzyme

5

inhibitor, normal BALB/c mice were co-injected with 300 µg of phosphoramidon (disodium salt, Sigma-

6

Aldrich, USA) in 50 µL of saline (10%EtOH) and the radiopeptide 68Ga-NOTA-BBN2 in 50 µL of saline.

7

Venous blood samples were collected at 5, 15, 30, and 60 min post injection via the mouse tail vein and

8

further processed. Blood cells were separated by centrifugation (13,000 rpm x 5 min). Supernatant was

9

removed and containing proteins were precipitated by addition of 2 volume parts of methanol (2v

10

MeOH/1v sample). Another centrifugation step (13,000 rpm x 5 min) was performed to obtain the

11

plasma in the supernatant. Fractions of blood cells, proteins and plasma were measured in a Wizard

12

gamma counter to determine radioactivity per sample. The clear plasma supernatant was injected onto

13

a Shimadzu HPLC system. The samples were analyzed using a Phenomenex Luna 10u C18(2) 100A, 250

14

x 4.6 mm column at a constant flow rate of 1 mL/min and the following gradient with water/0.2% TFA

15

as solvent A and acetonitrile as solvent B: 0-3 min 10% B, 10 min 30% B, 17 min 50% B, 23 min 70% B,

16

27-30 min 90% B.

17 68

18

Dynamic PET imaging studies. PET imaging of radiopeptide

Ga-NOTA-BBN2 was performed on a

19

INVEON PET/CT scanner (Siemens Preclinical Solutions, Knoxville, U.S.A.). Prior to radiotracer injection,

20

mice were anesthetized through inhalation of isoflurane in 40% oxygen/ 60% nitrogen (gas flow 1

21

L/min), and body temperature was kept constant at 37 oC. Mice were placed in a prone position into

22

the centre of the field of view.

23

A transmission scan for attenuation correction was not acquired. Mice were injected with 4-13 MBq of

24

68

25

For blocking studies, PC3 tumor-bearing BALB/c mice were pre-dosed with 300 µg of NOTA-BBN2 in

26

50 µL of saline 10 min prior to radiotracer injection. For enzyme inhibitor studies, PC3 tumor-bearing

27

BALB/c mice were co-injected with 300 µg of phosphoramidon (disodium salt, Sigma-Aldrich, U.S.A.) in

28

50 µL of saline (10% EtOH) and

29

over 60 min in 3D list mode.

Ga-NOTA-BBN2 in 150 µL of isotonic saline solution (0.9% w/v of NaCl) through a tail vein catheter.

68

Ga-NOTA-BBN2 in 50 µL of saline. Data acquisition was performed

11



Page 12 of 37


1 2 3 1 4 5 2 6 7 3 8 9 4 10 11 5 12 13 6 14 15 7 16 17 8 18 19 9 20 10 21 22 11 23 24 12 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

The dynamic list mode data were sorted into sinograms with 54 time frames (10x2, 8x5, 6x10, 6x20, 8x60, 10x120, 6x300s). The frames were reconstructed using maximum a posteriori (MAP) as reconstruction mode. No correction for partial volume effects was applied. The image files were processed using the ROVER v2.0.51 software (ABX GmbH, Radeberg, Germany). Masks defining 3D regions of interest (ROI) were set, and the ROIs were defined by thresholding. ROIs covered all visible tumor mass of the subcutaneous tumors, and the thresholds were defined by 50% of the maximum radioactivity uptake level. Mean standardized uptake values [SUVmean = (activity/mL tissue)/(injected activity/body weight), mL/g] were calculated for each ROI and time-activity curves (TAC) were generated. All semi-quantified PET data are presented as means ± SEM. Statistical differences were tested by Student's t test and were considered significant for P< 0.05.

12


Page 13 of 37



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

1

RESULTS

2

Radiosynthesis of

3

BBN2 functionalized with macrocyclic chelator NOTA (NOTA-BBN2) was prepared in high yields (87%)

4

and chemical purity (>99%) suitable for radiolabeling with gallium-68. Gallium-68 was obtained from a

5

20 mCi (740 MBq)

6

module. However, the labeling reaction of NOTA-BBN2, purification and isolation of 68Ga-NOTA-BBN2

7

was conducted manually. An outline of the radiosynthesis of 68Ga-NOTA-BBN2 is depicted in Figure 1.

68

Ga-NOTA-BBN2 and lipophilicity determination. Stabilized bombesin derivative

68

Ge/68Ga-generator that was eluted remotely controlled via the Scintomics GRP

8

((Figure 1))

9 10 11

NOTA-BBN2 (20 µg) was labeled with the 68Ga eluate adjusted to pH 5 with 4 M sodium actetate buffer

12

at room temperature. The reaction time was 18 min, including the ligand challenge step using acyclic

13

ligand EDTA (ethylenediaminetetraacetic acid) in excess. Labeling was quantitative after an 8 min

14

reaction time and the formed radiolabeled peptide remained stable after ligand challenge with EDTA.

15

Purification of the reaction mixture was performed using solid-phase extraction via a tC18 Plus

16

cartridge.

17

dissolving in saline for subsequent radiopharmaceutical studies.

18

decay-corrected radiochemical yields 62±14% (n=11) with >96% radiochemical purity. The total

19

synthesis time was 35±4 min. As starting activity was typically low (in the range of 60 - 90 MBq),

20

effective specific activity of 4 GBq/µmol was generated considering the present amount of 20 µg of

21

precursor peptide (NOTA-BBN2). Partition coefficient of

22

value of -2.10±0.01 in n-octanol and PBS (pH 7.4) which represents the lipophilicity of the peptide

23

radiotracer.

68

Ga-labeled peptide was eluted with EtOH and isolated by evaporation of EtOH and re-

68

68

Ga-NOTA-BBN2 was obtained in

Ga-NOTA-BBN2 was determined as logD

24 25

In vitro competitive binding assay and Ca2+ release assay. A radiometric competitive binding assay

26

was performed to test the inhibitory potency of natGa-NOTA-BBN2 and labeling precursor NOTA-BBN2

27

GRP receptor-expressing PC3 prostate cancer cells.

13



Page 14 of 37


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

1

Both peptides competed for binding to GRP receptors in a concentration-dependent manner at 4 oC for

2

2 h against GRP receptor-binding radioligand

3

peptide Tyr-BBN(1-14) served as internal reference.

4

Figure 2 displays generated sigmoidal concentration-response curves and derived IC50 values (half

5

maximum inhibition). Both peptides retained high affinity to the GRP receptor, and their inhibitory

6

potencies were in the same order of magnitude as endogenous-derived ligand Tyr4-BBN(1-14) with an

7

IC50 value of 2.4 nM. natGa-NOTA-BBN2 (IC50 value of 4.6 ± 1.2 nM) binds with somewhat higher affinity

8

to the GRP receptor compared to NOTA-BBN2 (IC50 value of 8.2 ± 0.2 nM).

125

I-Tyr-BBN(1-14) as tracer. GRP receptor-binding

9

((Figure 2))

10 11 12

Figure 3 shows the results for the Fura-2 based calcium release assay. This assay was used to elucidate

13

agonistic or antagonistic pharmacological profile NOTA-BBN2. The assay protocol included flushing of

14

the PC3 cells with control agonist (1 nM Tyr4-BBN(1-14)) for 30 s to 2 min in a first step to stimulate

15

intracellular calcium response (first peak) and re-adjustment to baseline level before cells were

16

perfused with NOTA-BBN2 using different concentrations (0.1 nM, 1 nM, 10 nM, 100 nM). Upon

17

subsequent addition of Tyr4-BBN(1-14), inhibition of calcium response (second peak) was observed at

18

10 and 100 nM of NOTA-BBN2, while a 1 nM concentration only partially inhibited the response. A

19

concentration of 0.1 nM had no effect at all. This result demonstrated that NOTA-BBN2 acts as an

20

antagonist on Tyr4-BBN(1-14) induced intracellular calcium release through GRP receptors. NOTA-

21

BBN2 inhibited the calcium response to 50% at a concentration of approximately 1 nM.

22

((Figure 3))

23 24 68

25

Dynamic PET imaging of

Ga-NOTA-BBN2 in PC3 and LNCaP tumor-bearing mice. Tumor-targeting

26

property of 68Ga-NOTA-BBN2 was studied in two different male prostate cancer xenografts (hormone-

27

dependent LNCaP and hormone-independent PC3) with dynamic PET imaging.

14


Page 15 of 37



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

1

Figure 4 displays PET images of PC3 and LNCaP tumor-bearing BALB/c mouse at 60 min after injection

2

of

3

tumor and muscle tissue.

68

Ga-NOTA-BBN2 as well as corresponding time-activity-curves (TACs) for radioactivity levels in

4

((Figure 4))

5 6 7

Radioactivity accumulation in PC3 tumor tissue resulted in a SUV of 0.56±0.05 and a SUV of 0.47±0.09

8

(both n=3) in LNCaP tumors after 60 min post injection. Tumor-to-muscle ratios in both xenograft

9

models were relatively high with 5.86±0.37 for PC3 and 5.79±1.02 for LNCaP. The elimination pathway 68

Ga-labeled peptide

68

10

of

Ga-NOTA-BBN2 was observed as predominately via kidneys towards final

11

radioactivity accumulation in the bladder. In addition, a negligible amount of radioactivity accumulated

12

in hepatobiliary organs with minor activity retention in liver and intestine. The visible ‘hot spot’ in close

13

proximity to the liver can be identified as the filled gallbladder.

14 15

Blocking studies with NOTA-BBN2 in PC3 xenografts. Blocking studies were performed to demonstrate

16

specific binding of 68Ga-NOTA-BBN2 to the GRP receptor. Figure 5 shows results of radioactivity uptake

17

in PC3 tumors (and muscle uptake) after injection of 68Ga-NOTA-BBN2 in the presence and absence of

18

300 µg of NOTA-BBN2. Presence of 300 µg of NOTA-BBN2 resulted in significantly reduced tumor

19

uptake at 60 min p.i. which was visible in the PET image as well as in the TACs generated from tumor

20

ROI’s. A 40% blocking effect was demonstrated by measuring the SUV60min of 0.37±0.01 under control

21

conditions and the SUV60min of 0.23±0.01 (n=3) after injection of a pharmacological dose of NOTA-

22

BBN2. Tumor-to-muscle ratio was reduced from 4.22±0.57 to 2.99±0.34 (n=3) under blocking

23

conditions.

24 25

((Figure 5))

26 27

Effect of phosphoramidon on tumor uptake of 68Ga-NOTA-BBN2. Another focus of this study was to

28

investigate the influence of protease inhibitor phosphoramidon on metabolic stability and tumor

29

uptake according to the recently proposed “serve-and-protect” strategy.23

15



Page 16 of 37


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68

1

Interestingly, in our study PC3 tumor uptake of

Ga-NOTA-BBN2 after 60 min p.i. was not further

2

increased in the presence of 300 µg phosphoramidon (Figure 6).

3

((Figure 6))

4 5 6

Conversely, analysis of tumor uptake revealed an opposite trend towards a reduced tumor uptake in

7

the presence of the enzyme blocker. Determined SUV values after 60 min p.i. were reduced by ∼25%

8

from 0.50±0.06 (control) to 0.38±0.05 (n=3) under phosphoramidon treatment. This observation was

9

tumor specific since muscle clearance pattern was not impaired. Also the elimination pathway of 68Ga-

10

NOTA-BBN2 remained the same in phosphoramidon treated PC3-BALB/c mice.

11 68

Ga-NOTA-BBN2. In vivo metabolic stability of

12

Effect of phosphoramidon on metabolic stability of

13

68

14

radiopeptide and collection of blood samples at representative time points of 5, 15, 30 and 60 min.

15

Figure 7 summarizes the results of metabolic stability analysis of

16

samples in the presence and absence of 300 µg of phosphoramidon. Approximately 50% of

17

radiopeptide

18

remained stable over the remaining time course of the experiment resulting in ∼ 40% of intact 68Ga-

19

NOTA-BBN2 after 60 min p.i.. Surprisingly, presence of phosphoramidon did not increased plasma

20

stability of 68Ga-NOTA-BBN2 neither after 5 min nor after 60 min p.i.. This finding was in contradiction

21

to observed positive stability enhancing effects from recent literature studies.22 All detected

22

radiometabolites were of more hydrophilic nature than parent compound 68Ga-NOTA-BBN2. No 68Ga-

23

NOTA species was found which was indicative of a stable NOTA chelator-peptide linkage. However,

24

exact chemical nature of detected radiometabolites was not further determined.

Ga-NOTA-BBN2 was studied in normal BALB/c mice by intravenous administration of the

Ga-NOTA-BBN2 in mouse plasma

68

Ga-NOTA-BBN2 was metabolized within the first 10 min, while

25 26

68

((Figure 7))

27 28

16


68

Ga-labeled peptide

Page 17 of 37



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

1

DISCUSSION

2

The introduction of radiopeptides as high affinity and specific-targeting radiopharmaceuticals in

3

oncologic nuclear medicine has led to significant improvements and advancements in diagnosis and

4

therapy to enhance cancer patient care. Among many other radiopeptides, current research activities

5

on radiopeptides are directed towards the development of GRP receptor-targeting radiolabeled

6

bombesin derivatives possessing high metabolic stability, high tumor uptake and favorable

7

pharmacological profile like reduced hepatobiliary uptake and low background accumulation in vivo.

8

The present study introduced 68Ga-labeled bombesin derivative 68Ga-NOTA-BBN2 as novel radiotracer

9

for PET imaging of GRP receptors in prostate cancer. Our study described the radiosynthesis and 68

10

radiopharmacological evaluation of

Ga-NOTA-BBN2 in two prostate cancer xenograft models.

11

Moreover, we tested the influence of recently proposed protease inhibitor concept (“serve-and-

12

protect” strategy)23 on 68Ga-NOTA-BBN2 as a radiopeptide with intrinsic metabolic stability.

13

In a first step, in vitro pharmacology studies were performed with novel peptides NOTA-BBN2 and

14

nat

15

range (IC50 = 8.2 nM for NOTA-BBN2 and IC50 = 4.6 nM for

16

high affinity binding to the receptor. High binding affinity to the target (GRP receptor) is required for

17

high tumor uptake and retention of the radiopeptide. Modification with macrocyclic chelator NOTA

18

and the complexation with

19

binding affinities compared to earlier introduced fluorine-containing BBN2 peptides FBz-Ava-BBN2

20

(IC50 = 8.7 nM) and FDG-AOAc-BBN2 (IC50 = 16.5 nM)10,11 using the same competitive binding assay.

21

Furthermore, pharmacology of peptide interaction with Gαq protein-coupled GRP receptor was

22

determined with calcium release assay. Peptide agonists are characterized by internalization of the

23

formed ligand-receptor complex stimulating intracellular Ca2+ release resulting in Ca2+mediated second

24

messenger signaling. Antagonists bind to the receptor, but no internalization of the formed ligand-

25

receptor complex and hence to second messenger signaling cascade occurs. As observed during the

26

Fura-2 based intracellular calcium release assay, NOTA-BBN2 was able to reduce the stimulated

27

calcium signal using increasing concentrations of the peptide, which confirms antagonist properties of

28

studied bombesin derivatives NOTA-BBN2 and natGa-NOTA-BBN2.

Ga-NOTA-BBN2. Binding affinities towards the GRP receptor in PC3 cells were in the low nanomolar

nat

Ga3+ afforded compound

nat

Ga-NOTA-BBN2) which is important for

nat

Ga-NOTA-BBN2 which displayed higher

17



Page 18 of 37


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

1

It was shown that GRP receptor antagonists demonstrated superior pharmacokinetic performance

2

compared to agonists based on higher receptor avidities and the absence of internalization giving the

3

benefit of avoiding side effects for the future development of peptide-based radiotherapeutics.15,30

4

Labeling precursor NOTA-BBN2 was prepared by solid-phase peptide synthesis and chelator

5

attachment in solution in high yields of over 80%. Reference compound

6

synthesized for identification purposes of the corresponding radiotracer

7

vitro pharmacology studies. Complexation of NOTA-BBN2 with gallium-68 was achieved using very low

8

peptide amounts of 20 µg, which equals 13 nmol of NOTA-BBN2. The pH of the generator eluate

9

([68Ga]Ga3+ in 1 N HCl) was adjusted with sodium acetate buffer to a pH of 5 to generate a reaction 68

68

nat

Ga-NOTA-BBN2 was

Ga-NOTA-BBN2 and for in

Ga3+ incorporation. Reaction at room temperature gave quantitative

10

medium preferable for

11

incorporation of the radiometal 68Ga3+. The radiosynthesis afforded radiochemically pure 68Ga-NOTA-

12

BBN2 within ∼35 min, in high reproducible radiochemical yields (decay-corrected) of ∼62% after

13

evaporation of EtOH and reformulation in saline. Radiosynthesis procedure is in alignment with the

14

rather short physical half-life of gallium-68 (68 min) and comparable to that of other

15

peptide syntheses described in the literature. A major difference of our described radiosynthesis in this

16

study is the performance of the 68Ga3+ incorporation into NOTA-BBN2 at room temperature compared

17

to the use of elevated temperatures as reported for various 68Ga-complexation reactions with NOTA.14-

18

17,31

19

BBN2 was further decreased to -2.1 compared to lipophilicity of [18F]SFB-labelled BBN2 (logP = +1.22)10

20

and [18F]FDG-labelled BBN2 (logP = -0.73).11

21

The more hydrophilic nature of 68Ga-NOTA-BBN2 directs the radiopeptide towards more favourable in

22

vivo radiopharmacokinetics as demonstrated by the more profound renal elimination route of

23

NOTA-BBN2 with only very little radioactivity in the liver.

24

profile comparable to that of various PEGylated bombesin derivatives 68Ga-NOTA-PEG(n=2,3,4,6)-RM26

25

by Varasteh et al. (logD= -2.2, -2.4, -2.4, -2.5 for n = 2, 3, 4, 6). However, increase of the PEG linker

26

length had only a minor influence on a more favorable pharmacological profile of the radiopeptide.31

27

Dynamic PET studies of

28

results for preclinical molecular imaging of GRP receptors in prostate cancer. LNCaP tumors represent a

29

frequently used human prostate carcinoma model with functional androgen receptor and PSMA

Compared to our previously reported

68

18

F-labeled BBN2 derivatives, lipophilicity of

68

68

Ga-NOTA-

68

Ga-NOTA-

68

Ga-

Ga-NOTA-BBN2 exhibits a lipophilicity

Ga-NOTA-BBN2 in PC3 and LNCaP tumor-bearing mice showed promising

18


Page 19 of 37



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1

expression which is a suitable model for metastatic and androgen-sensitive human prostate cancer.32

2

The small molecule size of

3

radioactivity from the blood and non-target tissues resulting in high tumor-to-muscle ratios of ∼5.8 to

4

5.9 after 60 min p.i.. The PC3 and LNCaP tumor is clearly visible on the left shoulder flank of each

5

animal after administration of radiopeptide 68Ga-NOTA-BBN2.

6

The obtained time-activity-curves are comparable for PC3 and LNCaP tumors with a slightly higher SUV

7

in the case of PC3 tumors. Washout of

8

compared to recently reported [18F]FDG-AOAc-BBN2.11

9

Our PET imaging results were comparable to the in vivo profile of

68

Ga-NOTA-BBN2 and its hydrophilic nature enables fast clearance of

68

Ga-NOTA-BBN2 from tumor tissue was less pronounced

68

Ga-labelled statine-bombesin

10

analog 68Ga-NOTA-MJ9 in PC3 mice.15 Furthermore, by using the two different tumor models for PET

11

imaging experiments, we were able to discuss GRP receptor expression with androgen receptor

12

expression in vivo. Our PET imaging data indicated that

13

independent of hormone receptor status of the tumor. Tumor uptake and tumor-to-muscle ratios are

14

comparable in both xenograft models. The observed slightly lower uptake in LNCaP-xenografts can be

15

attributed to the lower number of binding sites (GRP receptors) per cell in LNCaP cells in comparison to

16

PC3 cells.9 However, our result differed from the study recently described by Mansi et al. The authors

17

observed a 25% decreased tumor-to-muscle ratio in LNCaP compared to PC3 xenografts using GRP

18

receptor antagonist

19

Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2) after 60 min p.i..16

20

Moreover, tumor uptake of 68Ga-NOTA-BBN2 in PC3 BALB/c mice was 2-3 times higher (SUV60min 0.56)

21

compared to our recent results using various 18F-labeled BBN2 derivatives (SUV60min = 0.15 for [18F]FBz-

22

Ava-BBN2 and SUV60min = 0.27 for [18F]FDG-AOAc-BBN2).12 One possible explanation is the observed

23

higher binding affinity of

24

(16.5 nM).11 Excretion of 68Ga-NOTA-BBN2 from the body as determined by PET imaging experiments

25

occurred predominantly via the renal pathway as visible by radioactivity clearance through the kidneys

26

and radioactivity accumulation in the bladder. Only little radioactivity levels were found in the liver and

27

intestines.

68

68

Ga-NOTA-BBN2 uptake seems to be

Ga-RM2 (bombesin-derived, DOTA-4-amino-1-carboxymethyl-piperidine-DPhe-

nat

Ga-NOTA-BBN2 (4.6 nM) compared to FBz-BBN2 (8.7 nM) and FDG-BBN2

19



Page 20 of 37


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

68

1

Specificity of

Ga-NOTA-BBN2 uptake in PC3 tumors through binding to GRP receptors was

2

demonstrated by blocking studies with NOTA-BBN2 (300 µg dose/animal). Tumor uptake was

3

significantly reduced in animals pre-dosed with NOTA-BBN2 in comparison to control animals.

4

An important possible limitation of using radiopeptides as radiopharmaceuticals for molecular imaging

5

and therapy is their frequently observed low metabolic stability in vivo based on their degradation by

6

various proteases present in the blood plasma and digestive system.

7

This problem has been tackled in the past by means of various chemical modifications (cyclization,

8

formation of pseudo-peptide bonds and peptide bond modification, incorporation of unnatural amino

9

acids). However, this chemistry-driven optimization requires particular fine-tuning as modifications

10

often result in the loss of binding affinity, reduced tumor uptake and unfavourable pharmacokinetics.

11

A simple and highly innovative approach was recently reported by the introduction of the “serve-and-

12

protect” strategy to address radiopeptide degradation through proteases. The “serve-and-protect”

13

concept was particularly focused on inhibition of neutral endopeptidase with natural product inhibitor

14

phosphoramidon. This led to impressive enhancement of in vivo metabolic stability and tumor uptake

15

of various radiopeptides labeled with long-lived radionuclides like In-111 and Lu-177. 23-25

16

Using GRP receptor antagonist

17

presence of phosphoramidon from 12 to 80% after 5 min p.i.. In addition, PC3 tumor uptake increased

18

from ∼4 to 21% ID/g after 4 h p.i. of

19

investigated native and stabilized truncated CCK2-receptor-targeting gastrin peptides labeled with In-

20

111 in the presence and absence of phosphoramidon. Furthermore, they demonstrated in situ

21

inhibition of neutral endopeptidase with phosphoramidon as promising tool to enhance diagnostic

22

efficacy through improved stability and tumor uptake.26

23

In our study, we observed that the majority of the enzymatic degradation of

24

occurs within the first 10 min p.i. and that ∼40% of the radiolabeled peptide remained intact after

25

60 min p.i.. This confirms the intrinsic stability of the BBN2 sequence.10,11 NOTA chelator-peptide

26

linkage via a thiourea bond also remained stable as no 68Ga-NOTA species was found in the HPLC traces

27

of plasma samples. However, co-injection of phosphoramidon with 68Ga-NOTA-BBN2 did not lead to an

28

increase in metabolic stability at any investigated time point resulting in ∼40% of intact radiopeptide

29

68

111

In-PanSB1, Nock et al. found an increase of plasma stability in the

111

In-PanSB1.23 In a very recent publication the same group also

Ga-NOTA-BBN2 after 60 min p.i..

20


68

Ga-NOTA-BBN2 itself

Page 21 of 37



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

1

Evidently, in our PET study it seems that the application of protease inhibitor phosphoramidon did not

2

have a beneficial effect on a further stabilization of 68Ga-NOTA-BBN2 and hence an enhanced tumor

3

uptake.

4

In contrast, PC3 tumor uptake was lower in the phosphoramidon-treated mice. This is an interesting

5

finding for stabilized

6

uptake of 68Ga-NOTA-BBN2 is not consistent with the proposed “serve-and-protect” concept that has

7

also been successfully applied to stabilized truncated gastrin analogs for targeting cholecystokinin

8

subtype-2 (CCK2) receptor.26

9

In addition, we have also analyzed metabolic stability of

68

Ga-labeled bombesin derivative

68

Ga-NOTA-BBN2. The observed lower tumor

18

F-labeled BBN2 derivative [18F]FDG-AOAc68

10

BBN2 in vivo in the presence and absence of phosphoramidon. As for

11

increase of metabolic stability and tumor uptake was found (data not shown). We assume that no

12

further increase in metabolic stability and tumor uptake occurs due to the intrinsic high metabolic

13

stability of radiopeptides containing the BBN2 motif. This is in agreement with the authors’ report of

14

no measurable effect of phosphoramidon with metabolically-stabilized

15

Moreover, only longer-lived radionuclides like In-111 have been tested for the “serve-and-protect”

16

concept. Radiopeptides labeled with short-lived PET radionuclide such gallium-68 and fluorine-18 may

17

act differently to phosphoramidon inhibiton of proteases. However, this assumption has to be further

18

elucidated in the future.

19

Very recently, an article was published describing

20

bombesin(Sta13-Leu14) derivative, which was also tested towards the "serve-and-protect" concept.36

21

Most experiments presented in the article involved

22

stability data was reported for

23

(which does not possess the natural His-Leu segment) revealed slightly higher tumor uptake (2 fold)

24

upon PA treatment compared to non-treated controls after 60 min p.i.. However, the observed 2-fold

25

increase in tumor uptake in the case of stabilized radiopeptide 68Ga-JMV4168 is lower compared to the

26

data described in the original work describing the "serve-and-protect" concept when a 10 to 15 fold

27

increase in tumor uptake was reported.23

68

177

68

Ga-NOTA-BBN2, no further

Ga-JMV4168 as a

111

68

In-DTPA-octreotide.23

Ga-labeled stabilized

Lu-labeled radiopeptides. No in vivo metabolic

Ga-JMV4168. Biodistribution studies with stabilized

28

21


68

Ga-JMV4168


Page 22 of 37


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68

1

In summary,

Ga-NOTA-BBN2 represents a versatile peptide-based radiotracer to image GRP

2

receptors in prostate cancer in mice. The high intrinsic metabolic stability of the peptide backbone in

3

NOTA-BBN2 holds promise to be used within the theranostic concept by exchanging the diagnostic

4

radionuclide gallium-68 with the therapeutic radionuclide such as lutetium-177 for future peptide-

5

receptor radionuclide therapy of prostate cancer patients.37

6

22


Page 23 of 37



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1

CONCLUSION

2

Gallium-68 labeled radiopeptide 68Ga-NOTA-BBN2 is a metabolically stabilized, antagonistic bombesin

3

analog which shows favorable pharmacokinetics and excellent tumor targeting properties for PET

4

imaging of GRP receptors in hormone dependent and independent prostate cancer models. The use of

5

the “serve-and-protect” concept through protease inhibition with phosphoramidon was applied to

6

radiopeptides labeled with short-lived positron emitters for the first time. However, no further

7

increase of metabolic stability and tumor uptake was found.

8

The possible convenient switch to lutetium-177 for radiotherapy offers the advantage to develop a

9

bombesin-based radiotheranostic probe for translation into the clinic. Clinical translation of bombesin-

10

based radiotheranostics would significantly contribute to the management of prostate cancer patients

11

to improve early diagnosis and therapy.

12 13 14 15 16

AUTHOR INFORMATION

17

Corresponding Author: Dr. Frank Wuest ([email protected])

18 19 20

ACKNOWLEDGEMENTS

21

F.W. thanks the Dianne and Irving Kipnes Foundation for supporting this work. The authors like to

22

thank Gail Hipperson and Dan McGinn from the Vivarium of the Cross Cancer Institute (CCI) for general

23

animal handling.

24

23



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1

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1

Figure legends

2 3

Figure 1. Structure of stabilized bombesin derivative NOTA-BBN2 and radiosynthesis of 68Ga-NOTA-

4

BBN2.

5 nat

6

Figure 2. Binding of NOTA-BBN2 and

Ga-NOTA-BBN2 towards gastrin-releasing peptide (GRP)

7

receptor was determined in a competitive binding assay in prostate cancer cell line PC3 using 125I-Tyr4-

8

BBN as a radioligand. Sigmoidal concentration-response curves and IC50 values obtained from the

9

competitive binding assay are presented on top as mean ± SEM from n=3 (triplicates).

10 11

Figure 3. Results from the intracellular calcium-release assay are shown on the bottom. Increasing

12

concentrations (0.1-100 nM) of NOTA-BBN2 resulted in a reduced intracellular calcium release induced

13

by stimulation with the GRP receptor agonist 125I-Tyr4-BBN.

14 15

Figure 4. Representative PET images (maximum intensity projection) of PC3 tumor (left) and LNCaP

16

tumor-bearing BALB/c mouse (right) 60 min after injection of 68Ga-NOTA-BBN2. Corresponding time-

17

activity curves (TACs) show radioactivity levels in both tumors (PC3: black, LNCaP: gray) compared to

18

muscle tissue as SUV and mean ± SEM from n = 3 experiments.

19 68

20

Figure 5. Representative PET images (maximum intensity projection) of

Ga-NOTA-BBN2 in PC3

21

tumor-bearing BALB/c mouse in the absence (left image) and in the presence (right image) of blocking

22

agent NOTA-BBN2 (300 µg). Time-activity curves (TACs) on the right show the blocking effect in PC3

23

tumor compared to muscle tissue over time as SUV and mean ± SEM from n =3 experiments.

24 25

Figure 6. Effect of protease inhibitor phosphoramidon (PA, 300 µg) on 68Ga-NOTA-BBN2 tumor uptake

26

in PC3 BALB/c nude miceobtained with PET. Left: control and right: in the presence of PA - PET as

27

maximum intensity projections. Corresponding time-activity curves (TACs) on the right show

28

radioactivity levels in PC3 tumors and muscle tissue over time as SUV and mean ± SEM from n = 3-4

29

experiments. 28


Page 29 of 37



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68

1

Figure 7. In vivo metabolic stability of

Ga-NOTA-BBN2 in the presence (white bars)and absence of

2

protease inhibitor phosphoramidon (gray bars) over 60 min p.i.. Data was obtained from n = 3

3

experiments and is displayed as percentage of intact 68Ga-labeled peptide (mean±SEM) for every time

4

point.

5 6

29


Molecular Pharmaceutics Molecular Pharmaceutics

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

1

Figure 1

2

3 4

30


Page 30 of 37

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1

Figure 2

2

3 4

31



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

1

Figure 3

2

3 4

32


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1

Figure 4

2

3 4

33



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

1

Figure 5

2

3 4

34


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1

Figure 6

2

3 4

35



Page 36 of 37


1

Figure 7

% of intact Ga-labelled peptide

2

100

68

Ga-NOTA-BBN2 (n = 3)

68

Ga-NOTA-BBN2 + 300 µ g PA (n = 3)

75 50 25

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

0 5

15

30

time p.i. [min] 3 4

36


60

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Metabolically-stabilized 68Ga-NOTA-bombesin for PET Imaging of prostate cancer and influence of protease inhibitor phosphoramidon Susan Richter, Melinda Wuest, Cody N. Bergman, Stephanie Krieger, Buck E. Rogers, Frank Wuest


Metabolically Stabilized 68Ga-NOTA-Bombesin for PET Imaging of

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