Positron Emission Tomography Imaging of Prostate Cancer with Ga

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PET Imaging of Prostate Cancer with Ga-68 Labeled GRPR Agonist BBN7-14 and Antagonist RM26 Siyuan Cheng, Lixin Lang, Zhantong Wang, Orit Jacobson, Bryant C. Yung, Guizhi Zhu, Dongyu Gu, Ying Ma, Xiaohua Zhu, Gang Niu, and Xiaoyuan Chen Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.7b00726 • Publication Date (Web): 18 Dec 2017 Downloaded from http://pubs.acs.org on December 20, 2017

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Bioconjugate Chemistry

PET Imaging of Prostate Cancer with Ga-68 Labeled GRPR Agonist BBN7-14 and Antagonist RM26 Siyuan Cheng†, ‡, Lixin Lang‡, Zhantong Wang ‡, Orit Jacobson‡, Bryant Yung‡, Guizhi Zhu‡, Dongyu Gu‡, Ying Ma‡, Xiaohua Zhu†*, Gang Niu‡*, and Xiaoyuan Chen‡*

†

Department of Nuclear Medicine and PET, Tongji Hospital, Tongji Medical College, Huazhong

University of Science and Technology, Wuhan 430000, P. R. China

‡

Laboratory of Molecular Imaging and Nanomedicine, National Institute of Biomedical Imaging and

Bioengineering (NIBIB), National Institutes of Health (NIH), Bethesda, Maryland (MD) 20892, United States of America (USA)

*

For correspondence or reprint contact either of the following:

Dr. Xiaohua Zhu ([email protected]); Dr. Gang Niu ([email protected]); Dr. Xiaoyuan Chen ([email protected])

ACS Paragon Plus Environment

Bioconjugate Chemistry 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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ABSTRACT

2 3

Radiolabeled bombesin (BBN) analogs have long been used for developing

4

gastrin-releasing peptide receptor (GRPR) targeted imaging probes, and tracers with

5

excellent in vivo performance including high tumor uptake, high contrast, and

6

favorable pharmacokinetics are highly desired. In this study, we compared

7

68

8

antagonist (D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2, RM26) for PET imaging

9

of prostate cancer. The in vitro stabilities, receptor binding, cell uptake, internalization,

Ga-labeled GRPR agonist (Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2, BBN7-14) and

10

and

11

68

12

abilities and kinetics were investigated using PC-3 tumor xenografted mice. BBN7-14,

13

P3-RM26, NOTA-Aca-BBN7-14, and NOTA-PEG3-RM26 showed similar binding

14

affinity

15

68

efflux

properties

of

the

probes

68

Ga-NOTA-Aca-BBN7-14

and

Ga-NOTA-PEG3-RM26 were studied in PC-3 cells, and the in vivo GRPR targeting

to

GRPR.

In

PC-3

tumor-bearing

mice,

the

tumor

uptake

of

Ga-NOTA-PEG3-RM26 remained at around 3.00 %ID/g within 1 h after injection,


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1

in contrast with 68Ga-NOTA-Aca-BBN7-14, which demonstrated rapid elimination and

2

high background signal. Additionally, the majority of

3

remained intact in mouse serum at 5 min after injection, while almost all of

4

68

5

more favorable in vivo pharmacokinetic properties and metabolic stabilities of the

6

antagonist probe relative to its agonist counterpart. Overall, the antagonistic GRPR

7

targeted probe

8

agonist 68Ga-NOTA-Aca-BBN7-14 for PET imaging of prostate cancer patients.

68

Ga-NOTA-PEG3-RM26

Ga-NOTA-Aca-BBN7-14 was degraded under the same conditions, demonstrating

68

Ga-NOTA-PEG3-RM26 is a more promising candidate than the

9



1

INTRODUCTION

2

Prostate cancer (PCa) accounts for almost 20% of the newly diagnosed

3

cancers among men in the United States in 2017 and remains the third leading cause

4

of cancer related male death.1 Typical diagnosis of PCa relies on histopathological

5

examination of suspected prostate biopsy tissues or specimens from benign prostatic

6

enlargement surgeries or transurethral resection of the prostate following detection of

7

elevated prostate-specific antigen (PSA) levels, and/or abnormal digital rectal

8

examination (DRE), and bone scanning. X-ray computed tomography and magnetic

9

resonance imaging (MRI) are currently the major imaging techniques for further

10

identification of PCa.2 However, the capacity of conventional diagnostic techniques

11

for primary lesion detection, staging, or relapse monitoring of PCa is limited.3 For

12

example, the PSA test can be interfered by noncancerous factors such as prostate

13

enlargement, old age, and prostatitis, and low levels of PSA do not necessarily rule

14

out the incidence of PCa.4 The sensitivity and specificity of either ultrasound or MRI

15

is also limited by abnormal signals confounded by prostatitis or benign prostatic

16

hyperplasia (BPH).5,6 The notable multiparametric MRI (MP-MRI) remains imperfect

17

as well with a pooled sensitivity up to 89% and a specificity up to 73%.7

18

Interest in applying molecular imaging to PET has grown and a plethora of

19

radiotracers have been developed and investigated actively for PCa. The classical

20

2-deoxy-2-18F-fluoro-D-glucose (18F-FDG) has been used for evaluating late-stage or

21

recurrent PCa, but is not particularly avid.8,9 Other promising agents targeting


Page 4 of 35

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1

metabolites like fatty acids and amino acids (e.g.

2

18

3

antigens such as prostate-specific membrane antigen (PSMA).11,12 These tracers are

4

proven beneficial for recurrent PC diagnosis and staging. The PSMA targeted tracers

5

have also been applied specially for predicting the optimal timing of PSMA-based

6

therapies.13 However, almost all these tracers show limited diagnostic accuracy for

7

primary lesions,3,10,14 and few of those tracers have been sufficiently investigated and

8

clinically validated to date.

11

C/18F-choline,

11

C-acetate,

F-FACBC) have been further introduced,3,10 as well as agents targeting specific PCa

9

The gastrin-releasing peptide receptor (GRPR) is a G protein-coupled receptor

10

expressed in various organs of mammals, especially in the gastrointestinal tract and

11

the pancreas. Upon binding with the ligand gastrin-releasing peptide (GRP), GRPR

12

can be activated and elicit certain exocrine or endocrine secretions to regulate

13

multiple physiological processes.15 Notably, GRPR overexpression is presented in

14

several types of tumors such as prostate, urinary tract, gastrointestinal stromal, breast,

15

and lung, and related to proliferation and growth of these malignancies.16,17 Especially,

16

GRPR is almost 100% expressed in clinical PCa samples investigated by PCR,

17

immunohistochemistry, or radionuclide binding assay,16 which makes GRPR an

18

attractive target for PCa imaging and therapy.

19

As an amphibian homolog of GRP, bombesin (BBN) was found to bind to

20

GRPR with a high affinity. For decades, the BBN motifs have been used extensively

21

in radioactive imaging or in radionuclide therapy for GRPR overexpressing



Page 6 of 35

1

cancers.18,19 For example, the GRPR agonist BBN7-14, a truncated form of BBN with

2

the sequence of Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2, has been studied as PET or

3

single photon emission computed tomography (SPECT) tracers in both preclinical and

4

clinical research.20-23 In the meantime, numerous clinical trials have been performed

5

using

6

68

7

Recently,

8

D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2 (RM26) has been developed as an

9

antagonist against GRPR and has been applied actively in preclinical studies.29-32

10

Based on these observations, both BBN7-14 and RM26 are considered as high-quality

11

candidates for further clinical translation.

antagonistic

Ga-RM2,24,25 a

68

GRPR

Ga-SB3,26

targeting 68

PET

radiopharmaceuticals

Ga-BAY86-7458,27 and

nine-amino-acid

analog

of

64

including

Cu-CB-TE2A-AR06.28

nonapeptide

BBN6-14,

12

Despite the outstanding tumor targeting potential, BBN related research is

13

accompanied by a debate on the superiority of GRPR antagonist- versus agonist-based

14

tracers.33-36 It is generally claimed that even though antagonists are not internalized,

15

radiolabeled antagonists may depict clearer images and pharmacokinetic profiles than

16

agonists. More data are expected to emerge for direct comparison of specific

17

radiolabeled agonist and antagonist tracers to address this controversy, especially

18

among tracers that are promising for clinical translation.

19

Herein we would like to establish the distinction of GRPR targeted agonist

20

and antagonist with similar sequences by applying

21

for side-by-side comparative studies, including in vitro receptor binding, cell uptake,

68

Ga-labeled BBN7-14 and RM26


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1

internalization, and efflux studies on PC-3 cells, and in vivo microPET imaging study

2

of PC-3 tumor-bearing mice. The in vitro and in vivo stabilities of both radio

3

conjugates were presented and compared as well.

4

RESULTS

5

Synthesis and Radiolabeling

6

With excess amounts of NOTA-NHS, the NOTA-Aca-BBN7-14 and

7

NOTA-PEG3-RM26 conjugate were produced in > 95% yield. A m/z of 1338 for

8

[M+H+] was identified for NOTA-Aca-BBN7-14 using matrix-assisted laser desorption

9

ionization–time of flight mass spectrometry (MALDI-TOF MS). NOTA-PEG3-RM26

10

was synthesized and characterized by the same method (m/z = 1601 for [M+H+]).

11

Both conjugates were labeled with 68Ga within 20 min, with the specific activities of

12

21.6 ~ 40.01 MBq/nmol and 26.7 ~ 53.33 MBq/nmol respectively for

13

68

14

yield was > 90-95 %, radiochemical purity was > 98 %. The chemical structures of

15

68

Ga-NOTA-Aca-BBN7-14 and

68

Ga-NOTA-PEG3-RM26, and both radiochemical

Ga-NOTA-Aca-BBN7-14 and 68Ga-NOTA-PEG3-RM26 were presented in Figure 1.



Page 8 of 35

1 2

Figure 1. Schematic structures of GRPR agonist

3

antagonist 68Ga-NOTA-PEG3-RM26 (B).

4

In vitro Stability

5

68

In vitro stabilities of

68

Ga-NOTA-Aca-BBN7-14 (A) and


68

Ga-NOTA-PEG3-RM26

6

in saline and non-heat-inactivated fetal bovine serum (FBS) (Gibco) were determined

7

according to peak integration of analytical high-performance liquid chromatography

8

(HPLC).

9

68

At

0

min

of


the 68

incubation,

the

radiochemical

purities

of

Ga-NOTA-PEG3-RM26 were all > 95 % in both


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1

saline and FBS (Figure 2). After 2 h incubation, the parent compound of

2

68

3

peak of 11.58%, while this metabolism for

4

FBS was not as obvious. Metabolites represented by radio peaks of slightly higher

5

lipophilicity than the parent compounds were observed for both after 2 h incubation in

6

FBS, accompanied by the percentages of the parent compounds dropping to 89.24%

7

and

8

68

Ga-NOTA-Aca-BBN7-14 in saline dropped to 88.42% along with a more hydrophilic

80.58%,

respectively,

68

Ga-NOTA-Aca-BBN7-14 incubated in

for

68

stabilities

of

Ga-NOTA-Aca-BBN7-14

and

Ga-NOTA-PEG3-RM26.

9 10

Figure

11

68

12

after incubation. (A) In vitro radioactive stabilities of 68Ga-NOTA-Aca-BBN7-14 in saline

13

at 0 and 120 min after incubation. (B) In vitro radioactive stabilities of

2.

In

vitro

radioactive

68

Ga-NOTA-Aca-BBN7-14

and

Ga-NOTA-PEG3-RM26 in saline and Fetal Bovine Serum (FBS) for 0 and 120 min



1

68

2

radioactive stabilities of

3

incubation.

4

and 120 min after incubation.

5

Competitive Binding Assay

Page 10 of 35

Ga-NOTA-PEG3-RM26 in saline at 0 and 120 min after incubation. (C) In vitro

6

68

Ga-NOTA-Aca-BBN7-14 in FBS at 0 and 120 min after

(D) In vitro radioactive stabilities of

68

Ga-NOTA-PEG3-RM26 in FBS at 0

The GRPR-binding affinities of BBN7-14, P3-RM26, NOTA-Aca-BBN7-14 and

7

NOTA-PEG3-RM26

8

125

9

Binding of 125I-[Tyr4]BBN to GRPR was displaced by the cold analogs in a

10

concentration-dependent manner. The half maximal inhibitory concentration

11

(IC50) values of BBN7-14, P3-RM26, NOTA-Aca-BBN7-14 and NOTA-PEG3-RM26

12

were 0.32 ± 0.10, 0. 41 ± 0.13, 1.80 ± 0.67 and 2.05 ± 0.50 nM, respectively. The

13

results indicated that the intermolecular targeting abilities of BBN7-14 and P3-RM26

14

for GRPR were comparable. After the NOTA conjugation, the affinities of both

15

compounds decreased to some extent. However, there were no distinct disparities

16

discovered between NOTA-Aca-BBN7-14 and NOTA-PEG3-RM26 either.

were

assessed

by

competitive

binding

assay

using

I-[Tyr4]BBN as the radioligand. The results of these assays were shown in Figure 3.

17


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1 2

Figure 3. Inhibition of

3

P3-RM26, NOTA-Aca-BBN7-14, and NOTA-PEG3-RM26 (n = 3/group, mean ± SD).

125

I-[Tyr4]BBN binding to GRPR on PC-3 cells by BBN7-14,

4 5

Cell Uptake, Internalization and Efflux

6

Time dependent cellular uptake pattern in GRPR positive PC-3 cells was

7

observed for both

8

uptake of

9

incubation, and that of 68Ga-NOTA-PEG3-RM26 was slightly lower (Figure 4A). The

68

68


68

Ga-NOTA-PEG3-RM26. The

Ga-NOTA-Aca-BBN7-14 increased rapidly to nearly 27% within 1 h of

10

agonist

11

around 74 % of the radioactivity uptake was internalized within 1 h of incubation. By

12

contrast,

13

uptake) (Figure 4A). After washing and medium replacement, both the tracers showed

14

efflux with a similar pattern (Figure 4B). At 60 min, 50% of radioactivity uptake was

15

still retained with the cells.

68

Ga-NOTA-Aca-BBN7-14 showed distinctively high internalization and

68

Ga-NOTA-PEG3-RM26 showed very low internalization (< 15 % of total



Page 12 of 35

1 2

Figure

3

68

4

and internalization assay of 68Ga-NOTA-Aca-BBN7-14 and 68Ga-NOTA-PEG3-RM26 on

5

PC-3 tumor cells (n = 3, mean ± SD). (B) Cell efflux assay of 68Ga-NOTA-Aca-BBN7-14

6

and 68Ga-NOTA-PEG3-RM26 on PC-3 tumor cells (n = 3, mean ± SD).

7

In vivo PET Imaging

4.

In

vitro

cell


uptake, 68

internalization,

and

efflux

studies

of

Ga-NOTA-PEG3-RM26 on PC-3 cells. (A) Cell uptake

8

Representative coronal PET images of PC-3 tumor-bearing mice at different

9

time points are shown in Figure 5. The tumors were clearly visualized with high

10

contrast at all the time points for 68Ga-NOTA-PEG3-RM26 (n = 3), as well at 15 min


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1

and 30 min for 68Ga-NOTA-Aca-BBN7-14 (n = 4). However, at 60 min post injection,

2

the

3

with 68Ga-NOTA-Aca-BBN7-14 (Figure 5A). Meanwhile, both the tracers showed

4

considerable accumulation and retention in the abdominal regions including pancreas

5

and intestines, though less was observed for

6

68

7

radioactivity was observed while the radioactivity in the bladder was constantly high

8

for these two probes, suggesting the tracers were excreted mainly by the renal system.

9

Activity accumulation in the tumor was quantified by measuring the regions of

10

interest (ROIs) on the coronal images (Figure 5B, 5C). The mean tumor uptake was

11

determined to be 4.40 ± 0.29, 3.28 ± 0.47, and 2.04 ± 0.34 %ID/g (percentage of

12

injected dose per gram of tissue) for 68Ga-NOTA-Aca-BBN7-14, and 2.99 ± 0.44, 2.96

13

± 0.45, and 3.01 ± 0.45 %ID/g for

14

with the corresponding P value of

Positron Emission Tomography Imaging of Prostate Cancer with Ga

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