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A Preclinical Comparative Study of 68Ga-labeled DOTA, NOTA and HBED-CC Chelated Radiotracers for Targeting PSMA Sangeeta Ray Banerjee, Zhengping Chen, Mrudula Pullambhatla, Ala Lisok, Jian Chen, Ronnie C Mease, and Martin G Pomper Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.5b00679 • Publication Date (Web): 14 Apr 2016 Downloaded from http://pubs.acs.org on April 15, 2016

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Bioconjugate Chemistry

1

A Preclinical Comparative Study of 68Ga-labeled DOTA, NOTA and

2

HBED-CC Chelated Radiotracers for Targeting PSMA

3

Sangeeta Ray Banerjee, Zhengping Chen, Mrudula Pullambhatla, Ala Lisok, Jian Chen, Ronnie

4

C. Mease, Martin G. Pomper

5

Russell H. Morgan Department of Radiology and Radiological Science, Johns Hopkins Medical

6

Institutions, Baltimore, MD 21287

7 8

Corresponding Authors:

Martin G. Pomper, M.D., Ph.D.

9

410-955-2789 (T)

10

443-817-0990 (F)

11

[email protected]

12 13

Sangeeta Ray Banerjee, Ph.D.

14

410-955-8697 (T)

15

410-614-3147 (F)

16

[email protected]

17

Johns Hopkins Medical Institutions

18

1550 Orleans Street, 492 CRB II

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Baltimore, MD 21231

20

KEY WORDS: positron emission tomography, prostate-specific membrane antigen,

21

radiopharmaceutical, urea, chelator.

ACS Paragon Plus Environment


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1

ABSTRACT

2

68

3

antigen (PSMA) are increasingly used clinically to detect prostate and other cancers with

4

positron emission tomography (PET). The goal of this study was to compare the

5

pharmacokinetics of three PSMA-targeted radiotracers: 68Ga-1, using DOTA-monoamide as the

6

chelating agent; 68Ga-2, containing the macrocyclic chelating agent p-SCN-Bn-NOTA, and

7

68

8

HBED-CC. The PSMA-targeting scaffold for all three agents utilized a similar Glu-Lys-urea-

9

linker construct. Each radiotracer enabled visualization of PSMA+ PC3 PIP tumor, kidney, and

10

urinary bladder as early as 15 min post-injection using small animal PET/computed tomography

11

(PET/CT).

12

displayed higher uptake in PSMA+ PC3 PIP tumor compared to 68Ga-1 at 1 h post-injection.

13

There was no significant difference in PSMA+ PC3 PIP tumor uptake for the three agents at 2

14

and 3 h post-injection.

15

normal tissues, including kidney, blood, spleen, salivary glands and PSMA-negative PC3 flu

16

tumors up to 3 h post-injection. In this preclinical evaluation 68Ga-2 had the most advantageous

17

characteristics for PSMA-targeted PET imaging.

Ga-Labeled, low-molecular-weight imaging agents that target the prostate-specific membrane

Ga-DKFZ-PSMA-11, currently in clinical trials, which uses the acyclic chelating agent,

68

Ga-2 demonstrated the fastest rate of clearance from all tissues in this series and

68

Ga-DKFZ-PSMA-11 demonstrated the highest uptake and retention in

18


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1

INTRODUCTION

2

According to the National Cancer Institute approximately 220,800 cases of prostate cancer will

3

be diagnosed in 2015 in the US, with over 27,540 proving lethal (~ 12.5%).1 Existing imaging

4

techniques for detection and therapeutic monitoring of prostate cancer are inadequate for

5

effective management of the disease. The transmembrane glycoprotein prostate-specific

6

membrane antigen (PSMA) is increasingly recognized as an important target for both imaging

7

and therapy of prostate cancer.2, 3 PSMA is found in benign as well as in malignant prostate

8

tissue.4-6 However, expression of PSMA is greatest in prostate adenocarcinoma, particularly in

9

castration-resistant disease.7, 8 PSMA is also present in the neovasculature of solid tumors

10

including kidney, lung,9 stomach, colon, and breast.10-12 Expression of PSMA is associated with

11

the neovascular endothelium in non-prostate tumors.13, 14

12 13

We and others have used PSMA-targeted agents to image patients with prostate cancer using

14

positron emission tomography (PET).15-21 Although there are debatable advantages and

15

disadvantages with respect to which isotope to use for detection with PET, namely 18F vs. 68Ga,

16

the radiometal 68Ga can be produced on-site with a generator, followed by simple synthesis of

17

the radiotracer.22 We previously reported 68Ga-1, a radiotracer that employed the DOTA-mono-

18

amide chelator with conjugation to H2N-Lys-(CH2)3-Lys-urea-Glu for targeting to PSMA

19

(Figure 1).23 We chose that chelator to make it possible to complex imaging radiometals, such

20

as 68Ga, 86Y, 203Pb, as well as therapeutic radiometal nuclides, such as 177Lu, 90Y, 212Pb or 225Ac,

21

within the same scaffold. Since then two 68Ga-based agents have demonstrated excellent clinical

22

results for detection of prostate cancer, namely, 68Ga-PSMA-11 (Glu-urea-Lys-(Ahx)-HBED-

23

CC) and EuK-Sub-kff-68Ga-DOTAGA (68Ga-PSMA I&T).24-27 Those compounds both employ



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1 2 3 4 5 6 7 8 9 10 11 12 13

Figure 1. Structure of 68Ga-labeled PSMA inhibitors.

14

the Glu-Lys-urea-based PSMA-targeted moiety, while 68Ga-DOTA-DUPA-Pep, also recently

15

tested clinically, uses DOTA-monoamide as the chelating agent and Glu-Glu-urea as the PSMA-

16

targeting moiety.28 A recent preclinical study also evaluated 68Ga-(CHX-A”-DTPA)-Pep using

17

CHX-A”-DTPA as the chelating agent.29 Among the agents, 68Ga-DKFZ-PSMA-11 has been

18

most widely studied clinically.2, 16-18, 30, 31

19 20

Due to the growing number clinical trials employing 68Ga-based, PSMA-targeted PET, we

21

decided to investigate structural elements that could promote the least off-target uptake of this

22

class of radiotracers. Specific attention was given to decrease activity within renal and salivary

23

gland tissue, commonly seen with these agents. Here we describe a head-to-head, preclinical


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1

comparison of 68Ga-1, 68Ga-2, a new radiotracer containing the macrocyclic chelating agent

2

NOTA, and 68Ga-DKFZ-PSMA-11. Tumor uptake, selectivity and pharmacokinetics were

3

assessed. Although we would prefer head-to-head studies in clinical subjects, we believe that a

4

preclinical study such as this retains value as it is carefully controlled and all of the

5

aforementioned agents were evaluated for pharmacokinetics in preclinical studies23, 24 – with

6

similar comparisons performed – before their successful move to the clinic.24, 25

7 8

RESULTS

9 10 11

Chemical and Radiochemical Syntheses and Characterization

12

Structures of the radioligands used for the study are shown in Figure 1. Lys-Glu urea was used

13

as the PSMA-targeting moiety in all cases. Selected physical properties of 1, 2 and the

14

corresponding natural Ga-complexes are summarized in Table 1. Since NOTA is a hexadentate

15

N3O3 macrocyclic chelator, 68Ga-2 was expected produce a neutral compound.32 DKFZ-PSMA-

16

11 chelated with HBED-CC is reported to provide a uni-negative, hexadentate chelation (N2O4)

17

to Ga(III) at room temperature, with two carboxylates and two phenolates.33-35 All three

18

radiotracers were synthesized in high radiochemical yield (~ 95-99 %) and purity (> 98 %), with

19

specific radioactivity >168 GBq/µmol (4.05 mCi/ µmol).

20 21

We have investigated two radiolabeling methods, one in the presence of HEPES buffer as

22

reported by Eder et al24 and the other by a method reported by us23 following the literature.36 For

23

the latter method, pre-concentrated 68Ga(III)Cl3 could be directly used for radiolabeling, without

24

adjusting pH, and radiolabeling could be done in a total volume of 300-350 µL using as low as 4



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1

µg of any of the three ligands. Based on the HPLC retention time (Table 1), the non-

2

radiolabeled precursor DKFZ-PSMA-11 was the least hydrophilic, although, after radiolabeling,

3

68

4

coefficient (log P) between n-octanol and PBS was also determined. Both 68Ga-2 (−4.04 ± 0.16)

5

and 68Ga-DKFG-PSMA-11 (−3.89 ± 0.16) were found to be one order of magnitude more

6

hydrophilic than 68Ga-1 (−3.0 ± 0.1).

Ga-DKFZ-PSMA-11 became the most hydrophilic compound in the series. The partition

7 8

9

Table 1. Selected physical properties of compounds studied.

(nM)

1

1284.4

0.70

0.42 - 1.16

19.2-19.8

Ga-1

1352.5

0.33

0.17 - 0.66

26.8-31.7

2

1054.2

0.81

0.35 - 1.89

23.9-24.9

Ga-2

1120.9

0.38

2.26 - 6.28

20.8-22.5

DKFZ-PSMA-11

947.0

0.03

0.016 - 0.06

34.5-40.0

Ga-DKFZ-PSMA-11

1013.7

N.A.

N.A.

14.5-20.0 N.A.

Ki

95% CI of Ki

HPLC (RP) retention time (min)

Molar Mass (g/mol)

(nM)

a a b b b b

ZJ43 304.3 0.31 0.20 - 0.48 Compounds 1 and 2 are the unlabeled agents containing DOTAmonoamide and NOTA-Bn-SCN chelating agents, respectively. aIsocratic solution of 80% A and 20% B. bIsocratic solution of 85% water and 15% B. Flow rate was 1 mL/min for both methods.

10

Precursor ligands and the corresponding stable metal-labeled compounds demonstrated high

11

binding affinity to PSMA, with Ki values ranging from 0.03 to 0.81 nM (Table 1). The known,

12

high-affinity PSMA inhibitor ZJ4337 was used as a reference ligand and exhibited a Ki of 0.31


60 10 min

68

Ga-1

68

Ga-2

68

Ga-PSMA-11

30 min

1

nM (Table 1). DKFZ-PSMA-11 displayed the

2

highest PSMA-binding affinity from the compounds

60 min

3

tested in this comparative study.

50

4

40 30

5

Cellular uptake and internalization studies.

6

Comparative cell uptake and internalization

7

experiments using isogenic human prostate cancer

8

PSMA+ PC3 PIP and PSMA− PC3 flu revealed

9

significantly higher uptake and internalization of

20 10

PS M A + A PS P + C M PC A 3 P 3 - P IP PI C3 P + flu bl PS oc M PS k A M + A PS P C + P C MA 3 P 3 - P IP PI C3 P + flu bl PS oc M PS k A + M A PS P + C P C MA 3 P 3 - P IP PI C3 P + flu bl oc k

0

50

10 min

30 min

10

68

Ga-2 and 68Ga-DKFZ-PSMA-11 compared to

11

68

Ga-1 as shown in Figure 2. The HBED-CC

12

conjugate 68Ga-DKFZ-PSMA-11 demonstrated

13

significantly higher cell surface uptake and

14

internalization at 30 and 60 min post-incubation

15

compared to 68Ga-1 (P < 0.001 for both cell lysate

60 min

40 30 20 10

l gl yc ysa te in e w as h

l gl yc ysa te in e w as h

0 l gl yc ysa te in e w as h

% Incubated dose/106 cell

PS M

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


% Incubated dose/106 cell

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Figure 2. (Top) PSMA+ PC3 PIP and PSMA− PC3 flu cell binding16 for 68Ga-1, 68Ga-2 and 68GaPSMA-11 and blocking studies of17 the agents in PSMA+ PC3 PIP cell after 10 min of pre-incubation in 18 the presence ZJ43 (10 µM). 19 (Bottom) Internalization studies of at 37°C in PSMA+ PC3 PIP cells 20 at 10 min, 30 min and 60 min postincubation. Values are calculated 21 as percentage of incubated 6 radioactivity dose bound to 10 cells. Data are expressed as mean22 ± SD (n=3). 23

and glycine wash) and for 68Ga-2 (P < 0.001 glycine wash at 30 and 60 min and cell lysate at 60 min P < 0.05) from the series. Cell uptake of the three agents can be specifically blocked nearly completely with excess ZJ43 (10 µM). For all three agents there was a fast cell internalization rate from 10 min to 30 min and a slow rate of internalization was noted from 30 min to 1 h post-incubation at 37°C.



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Biodistribution

2

Tables 2 show the pharmacokinetics in selected organs for 68Ga-1, 68Ga-2 and 68Ga-DKFZ-

3

PSMA-11, respectively. All compounds exhibited clear PSMA-dependent binding in PSMA+

4

PC3 PIP tumor xenografts. The tumor uptake for 68Ga-1 was 19.5 ± 1.8 % ID/g at 1

5

h, highest at 2 h (24.8 ± 1.1 % ID/g) and remained high at 3h post-injection (19.5 ± 5.1 % ID/g)

6 7

Table 2. Tissue biodistribution of 68Ga-1, 68Ga-2 and 68Ga-DKFZ-PSMA-11 in mice bearing

8

PSMA+ PC3 PIP and PSMA− PC3 flu tumors (n = 4). Values are expressed as mean ± SD. 68

68

Ga-2 3h

1h

Ga-3

Tissue

1h

2h

3h

1h

2h

blood

0.5 ± 0.1

0.3 ± 0.0

0.2 ± 0.0

0.4 ± 0.2

0.1 ± 0.0

0.1 ± 0.0

0.8 ± 0.2

0.4 ± 0.1

0.3 ± 0.1

heart

0.2 ± 0.0

0.1 ± 0.0

0.1 ± 0.0

0.2 ± 0.1

0.0 ± 0.0

0.0 ± 0.0

0.4 ± 0.2

0.3 ± 0.1

0.2 ± 0.0

lung

0.4 ± 0.0

0.2 ± 0.0

0.1 ± 0.0

1.0 ± 0.2

0.3 ± 0.0

0.2 ± 0.1

2.2 ± 0.5

2.1 ± 0.7

1.3 ± 0.2

liver

0.2 ± 0.0

0.2 ± 0.0

0.1 ± 0.0

0.5 ± 0.2

0.2 ± 0.0

0.2 ± 0.0

0.8 ± 0.2

0.3 ± 0.1

0.4 ± 0.2

spleen

1.0 ± 0.3

0.4 ± 0.0

0.4 ± 0.2

4.9 ± 0.7

0.8 ± 0.2

0.7 ± 0.1

12.4 ± 3.8

8.9 ± 1.5

7.2 ± 2.3

kidney

26.5 ± 6.9

11.9 ± 1.0

12.1 ± 5.6

106 ± 23

34.7 ± 5.7

12.7 ± 4.9

133 ± 21

89 ± 20

120 ± 16

muscle

0.1 ± 0.1

0.0 ± 0.0

0.0 ± 0.0

0.1 ± 0.0

0.0 ± 0.0

0.0 ± 0.0

0.3 ± 0.1

0.2 ± 0.1

0.1 ± 0.0

small intestine

0.2 ± 0.0

0.1 ± 0.0

0.1 ± 0.0

0.2 ± 0.1

0.0 ± 0.0

0.1 ± 0.0

0.4 ± 0.1

0.2 ± 0.1

0.2 ± 0.1

2h

3h

salivary gland

0.3 ± 0.0

0.2 ± 0.0

0.1 ± 0.0

0.2 ± 0.1

0.2 ± 0.1

0.1 ± 0.0

1.4 ± 0.3

1.0 ± 0.2

0.9 ± 0.1

PSMA+ PC3 PIP

19.5 ± 1.8

24.8 ± 1.1

19.5 ± 5.1

42.2 ± 6.7

21.7 ± 3.7

17.4 ± 5.6

26.0 ± 9.7

21.1 ± 1.2

26.9 ± 5.6

PSMA− PC3 flu

0.2 ± 0.0

0.2 ± 0.0

0.2 ± 0.0

0.4 ± 0.1

0.10 ± 0.0

0.1 ± 0.0

0.6 ± 0.1

0.4 ± 0.2

0.3 ± 0.1

84 ± 4

149 ± 16

122 ± 31

110 ± 22

232 ± 26

182 ± 15

47 ± 18

58 ± 27

111 ± 21

PIP:flu

9

68

Ga-1

PIP:kidney

0.8 ± 0.3

2.1 ± 0.1

1.8 ± 0.6

0.4 ± 0.1

0.7 ± 0.1

1.6 ± 0.1

0.2 ± 0.1

0.2 ± 0.0

0.2 ± 0.0

PIP:blood

59 ± 25

101 ± 10

236 ± 24

120 ± 29

321 ± 79

207 ± 33

36 ± 13

52 ± 11

80 ± 10

PIP:salivary gland

77 ± 3

157 ± 3

173 ± 27

188 ± 35

212 ± 3

222 ± 35

18.6 ± 9.0

21 ± 3

31 ± 5

10 11

(Table 2). PSMA+ PC3 PIP-to-PSMA− PC3 flu tumor uptake ratios were 84 ± 4 at 1 h and 149

12

± 16 at 2 h. The distribution within normal organs and tissues was also favorable, with low

13

blood and normal tissue uptake and rapid clearance. The highest accumulation of radioactivity

14

was observed in the kidneys, where uptake was expectedly high and peaked at 26.5 ± 6.9 %ID/g


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1

at 1 h and decreased to 11.9 ± 1.0 %ID/g by 2 h and remained roughly the same at 3 h post-

2

injection.

3 4

68

5

injection. Tumor uptake remained high, with faster clearance from 1 h to 2 h. The PSMA+ PC3

6

PIP-to-PSMA− PC3 flu tumor ratios were 110 ± 22 at 1 h, 232 ± 26 at 2 h and 182 ± 15 at 3 h.

7

Renal uptake for 68Ga-2 was highest at 1 h, 106 ± 23 %ID/g, much higher than that seen for

8

68

9

injection. In addition, non-target organs, such as blood, heart, liver, spleen, stomach, pancreas,

10

showed lower uptake (≤ 1 %ID/g at 1 h, except for spleen) and faster clearance than for 68Ga-1.

Ga-2 showed the highest PSMA-dependent tumor uptake with 42.2 ± 6.7 %ID/g at 1 h post-

Ga-1 and showed faster renal clearance, which decreased to 34.7 ± 5.7 %ID/g by 2 h post-

11 12

Unlike 68Ga-1, 68Ga-DKFZ-PSMA-11 showed the highest PSMA-dependent tumor uptake with

13

26.9 ± 5.6 % ID/g at 3 h post-injection. Tumor uptake was nearly comparable from 1 to 3 h

14

post-injection. The PSMA+ PC3 PIP-to-PSMA− PC3 flu ratios were 46.6 ± 7.6 % ID/g at 1 h,

15

57.7 ± 27.1 % ID/g at 2 h and 110.6 ± 21.3 % ID/g at 3 h post-injection.

16 17

Figure 2 summarizes several comparative tissue uptake properties of the three agents. PSMA+

18

PC3 PIP tumor uptake of 68Ga-2 was significantly higher than 68Ga-1 at 1 h post-injection (P
0.05) at

Ga-2 and 68Ga-DKFZ-PSMA-11 (P < 0.001) at 1 h, although there was no significant



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1

At 2 h post-injection renal uptake of both 68Ga-1 and

68Ga-1 68Ga-2

Ga-2 were significantly lower than for 68Ga-DKFZ-

2

68

3

PSMA-11 (P < 0.001) and renal uptake of 68Ga-1 was still

4

significantly lower than 68Ga-2 (P < 0.01). At 3 h post-

5

injection renal uptake of both 68Ga-1 and 68Ga-2 were

6

significantly lower than for 68Ga-DKFZ-PSMA-11 (P
95%.

5 6

HPLC purification of stable compounds was performed using a Phenomenex C18 Luna 10 × 250

7

mm2 column and elution with water (0.1% TFA) (A) and CH3CN (0.1% TFA) (B) on a Waters

8

600E Delta LC system with a Waters 486 variable wavelength UV/Vis detector, both controlled

9

by Empower software (Waters Corporation, Milford, MA). HPLC purifications of 68Ga-1, 68Ga-

10

2 and 68Ga-DKFZ-PSMA-11 were performed on a Varian Prostar System (Palo Alto, CA),

11

equipped with a Varian ProStar 325 UV-Vis variable wavelength detector and a Bioscan Flow-

12

count in-line Radioactivity detector (Washington DC), all controlled by Galaxie software (Varian

13

Inc., Walnut Creek, CA).

14 15

All radiotracers were purified using a Varian microsob-MV 100-5 C8 25×4.6 mm column with a

16

flow rate 1 mL/min with water (0.1% TFA) (A) and CH3CN (0.1% TFA) (B) as the eluting

17

solvents. In order to ensure uniform purity of the compounds undergoing comparison, various

18

HPLC methods were applied to separate excess ligand from the radiolabeled compound. For

19

68

20

PSMA-11, an isocratic solution of 85% water and 15% B was employed.

Ga-1, an isocratic solution of 80% A and 20% B was used. For 68Ga-2 and 68Ga-DKFZ-

21 22

Retention times of the radiolabeled compound and unlabeled free ligands are listed in Table 1.

23

The radiochemical yield and purity of the radiotracers were further checked by withdrawing 1 µL


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1

aliquots of the radiolabeled solution and were analyzed by radio-TLC on RP-18 thin layer plates

2

using 5/1 saline/methanol as the mobile phase. The specific radioactivity was calculated as the

3

radioactivity eluting at the retention time of product during the preparative HPLC purification

4

divided by the mass corresponding to the area under the curve of the UV absorption.

5 6

Radiolabeling Methods

7

68

8

and following other literature procedures.24, 36 Briefly, 488 MBq (13 mCi) of 68GaCl3 in 7 mL of

9

0.1 N HCl were obtained from an 18-month-old 1,850 MBq (50 mCi) 68Ge/68Ga generator,

Ga-Labeling of target ligands was performed according to our previously reported method23

10

Eckert-Ziegler (Berlin, DE). Pre-concentration was performed on a cation-exchange cartridge.

11

The purified 68Ga(III)Cl3 was obtained in a total volume of 400 µL, eluted in 2.4/97.6 0.05 N

12

HCl/acetone. The 68Ga(III) in HCl/acetone was used immediately for the radiolabeling of 1, 2 or

13

DKFZ-PSMA-11.

14 15

We investigated two radiolabeling techniques. The first was undertaken in water (without any

16

added buffer solution), as we reported earlier,23 and the second used 2.1 M HEPES buffer at pH

17

~ 4, as reported by Eder et al.24 Using HEPES buffer, each ligand (12.5 µg), was radiolabeled in

18

> 95% yield in a total volume of ~ 120 µL, however the yield was dependent on the total volume

19

of the radiolabeling solution. In water the pre-concentrated 68Ga(III)Cl3 solution could be

20

directly used for radiolabeling at pH ~ 3-4.23 The total volume of the radiolabeling solution was

21

~ 300-350 µL to produce > 93% yield using ~ 4-6 µg of each precursor ligand.

22



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1

In a typical reaction 50 µL of the concentrated radioactivity was added to 250 µL of deionized

2

H2O in a 1.5 mL polypropylene vial, followed by addition of 3-5 µL of a solution of precursor

3

ligand (2 µg/µL in water, pH ~ 3.5-4). The reaction vial was heated at 95ºC for 10 min for

4

ligand 1, ~ 3 min for ligand 2 and the complex was allowed to form at room temperature for 10

5

min for both 2 and DKFZ-PSMA-11. Complex formation was monitored by iTLC as above,

6

using 5/1 saline/methanol.

7 8

For the comparison studies, all three radiotracers were purified by HPLC to remove excess

9

precursor ligand so that all three radioligands could be obtained in > 98% purity. The acidic

10

eluate was neutralized with 50 µL 1 M Na2CO3 and the volume of the eluate was reduced under

11

vacuum to dryness. The solid residue was diluted with saline to the desired radioactivity

12

concentration for biodistribution and imaging studies.

13 14

PSMA Inhibition Assay

15

The PSMA inhibitory activity of 1, 2 and DKFZ-PSMA-11 and the corresponding natural Ga-

16

labeled analogs Ga-1 and Ga-2 were determined using a fluorescence-based assay according to a

17

previously reported procedure (Table 1).51 Briefly, lysates of LNCaP cell extracts (25 µL) were

18

incubated with the inhibitor (12.5 µL) in the presence of 4 µM N-acetylaspartylglutamate

19

(NAAG) (12.5 µL) for 2 h. The amount of the glutamate released by NAAG hydrolysis was

20

measured by incubating with a working solution (50 µL) of the Amplex Red Glutamic Acid Kit

21

(Life Technologies, Grand Island, NY) for 1 h.

22


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1

Fluorescence was measured with a VICTOR3V multilabel plate reader (Perkin Elmer Inc.,

2

Waltham, MA) with excitation at 490 nm and emission at 642 nm. Inhibition curves were

3

determined using semi-log plots and IC50 values were determined at the concentration at which

4

enzyme activity was inhibited by 50 %. Enzyme inhibitory constants (Ki values) were generated

5

using the Cheng-Prusoff conversion.56 Assays were performed in triplicate. Data analysis was

6

performed using GraphPad Prism version 4.00 for Windows (GraphPad Software, San Diego,

7

California).

8 9

Determination of lipophilicity

10

To a solution of 0.5 to 1 MBq of each 68Ga-1, 68Ga-2 and 68Ga-DKFG-PSMA-11 in 3mL of

11

phosphate-buffered saline (PBS, pH 7.4), was added 3 mL of n-octanol (n = 3) in 15 mL

12

Eppendorf tubes. At ambient temperature tubes were vortexed vigorously for 3 min and were

13

centrifuged at 3,000 g for 5 min. The activity concentrations in 100 µL samples of both the

14

aqueous and the organic phases were measured in a γ-spectrometer (1282 Compugamma CS;

15

Pharmacia/LKB Nuclear, Inc., Gaithersberg, MD). The partition coefficient was calculated as a

16

ratio between counts in the n-octanol phase to counts in the water phase.

17 18

Cell Lines

19

Sublines of the androgen-independent PC3 human prostate cancer cell line, originally derived

20

from an advanced androgen independent bone metastasis, were used. Those sublines have been

21

modified to express high levels of PSMA [PSMA-positive (+) PC3 PIP] or are devoid of target

22

[PSMA-negative (−) PC3 flu]. They were generously provided by Dr. Warren Heston

23

(Cleveland Clinic). Cells were grown in RPMI 1640 medium (Corning Cellgro, Manassas, VA)



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

containing 10 % fetal bovine serum (FBS) (Sigma-Aldrich, St.Louis, MO) and 1 % penicillin-

2

streptomycin (Corning Cellgro, Manassas, VA). PSMA+ PC3 PIP cells were grown in the

3

presence of 20 µg/mL of puromycin to maintain PSMA expression. All cell cultures were

4

maintained in an atmosphere containing 5 % carbon dioxide (CO2), at 37.0°C in a humidified

5

incubator.

6

Cell uptake and Internalization

7

Cell uptake studies were performed as previously described57. Cells (1 million) were incubated

8

with 37 kBq/mL (1 µCi/mL) of each radiolabeled agent in the growth medium in 6-well plates.

9

To determine specific uptake, cells were pre-blocked with ZJ43 to a final concentration of 10

10

µM. Cellular uptake was terminated by washing with 1 mL of ice-cold PBS. After incubation at

11

37°C for 10 min, 30 min and 60 min, cells were washed with binding buffer, trypsinized using

12

non-enzymatic buffer, and cell-associated activity was determined in a γ-spectrometer.

13 14

For internalization assays, cells were detached using non-enzymatic buffer, and aliquots of 1

15

million cells per tube were incubated with 37 kBq (1 µCi) of each radiolabeled agent per

16

milliliter of solution for 10 min, 30 min and 1 h at 37°C. Assuming minimal receptor

17

endocytosis at 4°C, the internalization assay was performed only with cells incubated at 37°C.

18

At 10-, 30-, 60-min intervals the medium was removed and cells were washed once with binding

19

buffer followed by a mild acidic buffer (50 mM glycine, 150 mM NaCl [pH 3.0]) at 4°C for 5

20

min. The acidic buffer was then collected and cells were washed twice with binding buffer.

21

Pooled washes (containing cell surface–bound 68Ga-labeled agent) and cell pellets (containing

22

internalized 68Ga-labeled) were counted in an automated γ-spectrometer along with the

23

standards. All radioactivity values were converted into percentage of incubated dose (%ID) per


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1

million cells. Experiments were performed in triplicate and repeated 3 times. Data were fitted

2

according to linear regression analysis.

3 4

Small-animal PET Imaging and Analysis

5

Whole-body PET and CT images were acquired on a SuperArgus PET-CT preclinical imaging

6

system (SEDECAL SA4R PET-CT, Madrid, Spain). For imaging studies mice were

7

anesthetized with 3% and maintained under 1.5% isoflurane (v/v). PET-CT Imaging studies

8

were performed on NOD/SCID mice bearing PSMA+ PC3 PIP and PSMA− PC3 flu tumors.

9

After intravenous injection of ~ 150 µCi (5.55 MBq) of 68Ga-1, 68Ga-2 or 68Ga-DKFZ-PSMA-

10

11, whole-body PET emission images (two bed positions, 15 min per position) were acquired at

11

the indicated (30 min, 1 h, 2 h and 3 h) time points after injection of radiotracer.

12 13

For binding specificity studies, a mouse was subcutaneously administered a blocking dose of the

14

known PSMA inhibitor ZJ4337 (50 mg/kg) at 30 min before the injection of 68Ga-2, and another

15

mouse was injected with 68Ga-2 alone. A CT scan was acquired after each PET scan in 512

16

projections using a 50 keV beam for anatomic co-registration. PET emission data were corrected

17

for decay and dead time and were reconstructed using the 3-dimensional ordered-subsets

18

expectation maximization algorithm. Data were displayed and analyzed using AMIDE software

19

(http://sourceforge.net/amide).

20 21

Biodistribution

22

Mice bearing PSMA+ PC3 PIP and PSMA− PC3 flu xenografts were injected via the tail vein

23

with 740 kBq (20 µCi) of 68Ga-1, 68Ga-2 and 68Ga-DKFZ-PSMA-11 in 150 µL of saline (n = 4).



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1

At 1 h, 2 h, and 3 h post-injection, mice were sacrificed by cervical dislocation and the blood

2

was immediately collected by cardiac puncture. The heart, lungs, liver, stomach, pancreas,

3

spleen, fat, kidney, muscle, small and large intestines, urinary bladder, PSMA+ PC3 PIP and

4

PSMA− PC3 flu tumors were collected. Each organ was weighed, and the tissue radioactivity

5

was measured with an automated gamma counter (1282 Compugamma CS, Pharmacia/

6

LKBNuclear, Inc., Mt. Waverly, Vic. Australia). The percentage of injected dose per gram of

7

tissue (% ID/g) was calculated by comparison with samples of a standard dilution of the initial

8

dose. All measurements were corrected for decay.

9 10

Data Analysis

11

Data are expressed as mean ± standard deviation (SD). Prism software (GraphPAD, San Diego,

12

California) was used to determine statistical significance. Statistical significance was calculated

13

using a two-tailed Student’s t test. A P-value < 0.05 was considered significant.

14 15

LIST OF ABBREVIATIONS

16

PSMA: prostate-specific membrane antigen; PET: positron emission tomography; NOTA: 1,4,7-

17

triazacyclononane-1,4,7-triacetic acid; HBED-CC: N,N'-bis-[2-hydroxy-5-

18

(carboxyethyl)benzyl]ethylenediamine-N,N'-diacetic acid); DOTA-mono-amide: 1,4,7,10-

19

tetraazacyclododecane-N,N′,N″,N′″-triaacetic acid mono-amide; CHX-A”-DTPA: cyclohexyl-

20

diethylenetriaminepentaacetic acid; 2-PMPA: 2-phosphonomethyl pentanedioic acid.

21


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1

ACKNOWLEDGMENTS

2

We are grateful to NIH K25 CA148901, U54 CA151838, R01 CA134675, R01 CA184228, U01

3

CA183031, and the Patrick Walsh Foundation for Prostate Cancer Research. We thank the JHU

4

PET Center for access to the 68Ga generator. We would like to thanks Gilbert Green and

5

Desmond Jacob for their assistant with imaging studies.

6 7

ASSOCIATED CONTENT

8

Supporting Information. Comparison of PSMA+ PC3 PIP tumor to PSMA-negative flu tumor

9

and selected tissues for the agents and 1H NMR, preparative HPLC chromatograms for 68Ga-1, Ga-2 and 68Ga-PSMA-11. The Supporting Information is available free of charge on the

10

68

11

ACS Publications website.

12 13



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