Synthesis and Evaluation of a Novel 64Cu-and 67Ga-Labeled

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Article 64

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Synthesis and evaluation of a novel Cu/ Ga-labeled neurokinin 1 receptor antagonist for in vivo targeting of NK1R-positive tumor xenografts Hanwen Zhang, Ananda Kumar Kanduluru, Pooja Desai, Afruja Ahad, Sean D. Carlin, Nidhi Tandon, Wolfgang Weber, and Philip S. Low Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00063 • Publication Date (Web): 21 Feb 2018 Downloaded from http://pubs.acs.org on February 24, 2018

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Bioconjugate Chemistry

Synthesis and Evaluation of a Novel 64Cu/67Ga-labeled Neurokinin 1 Receptor Antagonist for in vivo Targeting of NK1R-positive Tumor Xenografts Hanwen Zhang †,*, Ananda Kumar Kanduluru ‡, Pooja Desai†, Afruja Ahad†, Sean Carlin†, Nidhi Tandon†, Wolfgang A. Weber†,§, and Philip S. Low‡ †

Department of Radiology, Memorial Sloan Kettering Cancer Center, New York, NY, 10065;

‡

Department of Chemistry and Institute for Drug Discovery, Purdue University, West Lafayette,

IN, 47906; §

Molecular Pharmacology & Chemistry Program, Memorial Sloan Kettering Cancer Center, New

York, NY, 10065.

* Corresponding author: [email protected]

ABSTRACT Neurokinin 1 receptor (NK1R) is expressed in gliomas and neuroendocrine malignancies, and represents a promising target for molecular imaging and targeted radionuclide therapy. The goal of this study was to synthesize and evaluate a novel NK1R ligand (NK1R-NOTA) for targeting NK1R-expressing tumors. Using carboxymethyl moiety linked to L-733060 as starting reagent, NK1R-NOTA was synthesized in a 3-step reaction, and then labeled with 64Cu (or 67Ga for in vitro studies) in the presence of CH3COONH4 buffer. The radioligand affinity and cellular uptake was evaluated with NK1R-transduced HEK293 cells (HEK293-NK1R) and NK1R non-transduced HEK293 cells (HEK293-WT) and its xenografts. Radiolabeled NK1R-NOTA was obtained with a radiochemical purity of > 95% and specific activities of >7.0 GBq/µmole for GBq/µmole for

67

Ga. Both

64

Cu and

67

64

Cu and >5.0

Ga-labeled NK1R-NOTA demonstrated high uptake in

HEK293-NK1R cells; whereas, co-incubation with an excess amount of the NK1R ligand L-733060 reduced uptake by 90%. PET imaging showed that

64

Cu-NK1R-NOTA had a rapid high

accumulation in HEK293-NK1R xenografts and 10-fold lower uptake in HEK293-WT xenografts. Radioactivity was cleared by gastrointestinal tract and urinary systems. Biodistribution studies confirmed that the tumor-to-organ ratios were ≥ 5 for all studied organs at 1 h p.i., except kidneys, liver, and intestine, and that the tumor-to-intestine and kidney ratios also were improved at 4 h and 20 h post injection. 64Cu-NK1R-NOTA is a promising ligand for PET imaging of NK1R-expressing

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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

tumor xenografts. Delayed imaging with

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Cu-NK1R-NOTA improves image contrast due to

continuous clearance of radioactivity from normal organs.

Key words: NK1 receptor, NK1 antagonist, Substance P, Tachykinin receptor 1, HEK293-NK1R tumor xenografts, PET imaging, cancer


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INTRODUCTION Neurokinin 1 receptor (NK1R), also known as tachykinin receptor 1 and substance P receptor, induces tumor cell proliferation, angiogenesis, and migration increased NK1R expression, including gliomas

2, 4-6

1-3

, and many human cancers exhibit

and neuroendocrine tumors 4, while normal

organs display significantly lower levels of expression. These observations suggest that a detectable/targetable amount of NK1R on the cell membrane allow for in vivo imaging and targeted radionuclide therapy. However, the natural NK1R receptor ligand, Substance P (SP), shows low stability in the serum and is hydrolyzed within 2 min during circulation 7. Nevertheless, chelator-conjugated SP analogs have been developed which maintained high affinity to NK1R and remained intact in the targeted cancer cells for >72 h 5. Thus, chelator-conjugated SP analogs labeled with therapeutic radioisotopes, such as 177Lu, 90Y, or 213Bi have been tested clinically for local radionuclide-based treatment of gliomas. Examples include

177

Lu,

90

Y, or

213

Bi-DOTAGA-SP

5, 8

, and

213

Bi-DOTA-[Thi8,Met(O2)11]-

substance P 9. However, radiolabeled SP analogs (agonists) showed a limited application for systemic cancer targeting not only because of instability in the circulation, but also due to the pharmacologic effects that can be induced by micromolar amounts of SP. Metabolically stable, radiolabeled nonpeptidic NK1R antagonists are therefore more attractive for the development of radiolabeled NK1R ligands

10

. Several NK1R antagonists have been developed

10, 11

and one of

them, aprepitant (Trade name: Emend) has been FDA approved for treatment of chemotherapyinduced nausea.

18

F-labeled aprepitant analogs are able to cross the blood-brain barrier (BBB) for

targeted imaging of NK1R expression in the human brain

12

, but these may not be suitable for

imaging at longer time points such as 24 h post injection or later. We first designed and synthesized a near-infrared fluorescent (NIRF) dye conjugated to the NK1R antagonist, L-733060 for fluorescent imaging of NK1R expression in vivo. The recently published results

13

showed that modification on the piperidine ring of L-733060 with a linker and NIRF dye

did not significantly compromise the antagonist’s low nM affinity for NK1R and allowed visualization of HEK293-NK1R xenografts in vivo. To investigate whether a chelator for radiometal labeling can be similarly integrated in L-733060 without compromising its receptor binding, and to explore whether the radiometal-conjugated NK1R antagonists might be suitable for non-invasive PET imaging of NK1R expressing cancers following intravenous injection, we designed and


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synthesized a hydrophilic chelator conjugated to the L-733060 analog (Figure 1) for potential labeling with Al18F,

68/67

Ga, or 64/67Cu. This work is the first to: 1) evaluate the binding and uptake

of the newly synthesized NK1R-targeted radioimaging agent, NK1R-NOTA, by NK1R-expressing cells and xenografts; and (2) measure its in vivo stability in circulation.

MATERIALS AND METHODS General All chemicals were obtained from commercial sources and used without further purification. 64

CuCl2 and 67GaCl3 solution were obtained from Washington University in St. Louis and Nordion

(Ottawa, ON, Canada), respectively. Radioactivity was quantified with a WIZARD™ 3” 1480 γcounter (PerkinElmer, Waltham, MA) or a dose calibrator (Capintec CRC-30BC, Ramsey, NJ). An HPLC system equipped with Shimadzu LC-20 AB binary pumps, SPD-20A Prominence UV/VIS detector, and a BioScan Flow Count radiodetector was used for analysis of radiolabeled ligands. NK1 receptor-transduced HEK293 (HEK293-NK1R)

13

and non-transduced HEK293

(HEK293-WT) cells were cultured in DME medium supplemented with 10% FBS, 2.25 g/L glucose, 2 mM L-glutamine, 1mM sodium pyruvate, and 0.8 mg G418 per mL. In vivo and ex vivo images were acquired on a small animal PET/CT system (Inveon PET/CT, Siemens, Malvern, PA).

Synthesis and characterization of NK1R-NOTA (Figure 1) Synthesis of 3: To a solution of 1 (0.015 g, 0.032 mmole in 1 mL DMSO) were added HATU (0.015 g, 0.039 mmole), tert-butyl (2-(2-(2-aminoethoxy)-ethoxy)ethyl)carbamate (2, 0.096 g, 0.039 mmole), followed by DIPEA (0.128 mmole) under nitrogen. After the reaction mixture was stirred overnight, the reaction was quenched by adding water (3 mL) dropwise and then extracted with EtOAc (3 × 10 mL). The combined organics were washed with brine (10 mL), dried over anhydrous Na2SO4, then filtered and concentrated. After the crude product was purified by silica-gel column chromatography using DCM-EtOAc, the final product 3 (19 mg in 85 % yield) was confirmed by LC/MS.

Synthesis of 4: To the solution of 3 (0.019 g, 0.027 mmole in 0.5 mL of dry CH2Cl2) was added TFA (0.54 mmole) under nitrogen. The reaction mixture was stirred for 1 h and the progress of the


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reaction was monitored by LC/MS. After evaporation of the reaction solvents, the residue was diluted with water and extracted with CH2Cl2 (3 × 5 mL). The combined organic layers were washed with brine, dried (Na2SO4) and concentrated to generate the final 4 without further purification in a quantitative yield. The purity and mass spectrum of 4 was confirmed by LC/MS.

Synthesis of NK1R-NOTA (6): To the solution of 4 (0.01 g, 0.017 mmole in 0.5 mL of dry DMSO) was added NOTA-NHS ester (5, 0.011 g, 0.017 mmole), followed by DIPEA (0.034 mmole) under argon. The reaction mixture was stirred for 12 h and the reaction progress was monitored by LC/MS. The final product was purified by RP-HPLC (mobile phase A = 20 mM ammonium acetate buffer, pH 7, B = MeCN, gradient 10−100% B in 30 min, 13 mL/min, λ = 254 nm). Pure fractions were combined, concentrated under vacuum, and lyophilized to yield the desired product 6 in 40 % yield. NK1R-NOTA (6) was confirmed by LC/MS. Preparation of 64/natCu and 67/natGa-labeled NK1R-NOTA All radiolabeled NK1R-NOTA was prepared by dissolving 5-10 nmole of NK1R-NOTA in 5-10 µL H2O, adding 80 µL 0.5 M ammonium-acetate buffer (pH 5.4), 37-74 MBq of

67

GaCl3 or

64

CuCl2

solution (10-20 µL), and incubating for 10 min at 95 °C. After incubation, purification was performed with an HLB cartridge (Waters Corp., Milford, MA). The cartridge was pre-condition with ethanol (5 mL) and H2O (5 mL) first; the radioactive solution together with 0.5 mL of EDTA buffer (pH 5.5, 0.25 M) was loaded on the cartridge. After elution with H2O (2 × 1 mL), the pure product was obtained from the cartridge with ethanol elution (0.2 mL), and the solvent was evaporated with nitrogen flow. The final product was reformulated in PBS with 1.0% bovine serum albumin (BSA) solution and analyzed with analytical HPLC (Column: Luna C8(2), 100 Å, 2 × 100 mm; flow rate: 0.6mL/min; mobile phase: 0.1% TFA in water and MeCN; gradient: 0–1 min, 10%– 20% MeCN; 1–17 min, 20%–80% MeCN; 18 min, 95% MeCN; 23–25 min, 95%–10% MeCN). For the purpose of in vitro saturation assay, three equivalents of Ga(NO3)35H2O or Cu(SO4)25H2O was added, and the final solution was incubated for another 10 min to generate structurally homogeneous ligands.

nat

Ga/natCu-NK1R-NOTA was synthesized with a similar procedure, and the

final product in H2O was lyophilized and characterized with UPLC-MS and high-resolution mass spectrum.


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Binding affinity assays Saturation binding studies for determining Kd and Bmax values were performed on HEK293-NK1R cells with varied concentrations of

67/nat

Ga or

64/nat

Cu-chelated NK1 receptor antagonist

6

triplicate samples containing 0.5×10 cells and various amounts of [

67/nat

14

. Briefly,

64/nat

Ga] or [

Cu]-NK1R-

NOTA (0.1 to 300 nM at final incubation volume) were incubated with HEK293-NK1R cells. After 1 h incubation at 37 °C, the cells were isolated by rapid filtration through glass microfiber filters (Cat. No.: FP-100, Brandel, Gaithersburg, MD) and washed with 3 × 3 mL of ice-cold tris-buffered saline (pH 7.4). The radioactivity in the cells was measured with a γ-counter. To determine the nonspecific binding, 30 µM of the NK1 receptor antagonist L-733060 (without conjugation of spacer and NOTA to NK1R-NOTA) was used for co-incubation. The Kd (Bmax) values were estimated using a least-squares fitting routine (GraphPad Prism 6, San Diego, CA, USA).

In vitro cell uptake The uptake studies were performed with HEK293-NK1R and HEK293-WT cells to investigate the pharmacological binding of radiolabeled NK1R-NOTA

15

. Briefly, 0.5×106 cells were seeded into

6-well plates one day before the experiment; triplicate samples containing ~1.85 kBq of

67

Ga or

64

Cu-labeled NK1R-NOTA (2.5 pmole in 1 mL, 2.5 nM) were added and incubated at 37 °C for 2.0

h. L-733060 (1.0 nmole) was used as a blocking agent to determine the non-specific binding.

In vitro and in vivo stability Three male athymic NCr-nu/nu mice (7 to 9 weeks old, Taconic, Albany, NY) were used for in vivo stability studies of

64

Cu-NK1R-NOTA. After administration of 0.7 nmole (5.5 MBq) of

64

Cu-

NK1R-NOTA (n = 3), the animals were sacrificed at 0.5 h post-injection, and the serums from all three mice were collected and combined. After precipitation with three-fold volume of acetonitrile at room temperature for 10 min, the mixture was then centrifuged at 15,000 g for 5 min, and the supernatant passed through a 0.45-µm filter to eliminate any precipitated proteins. The filtered supernatant was evaporated with Biotage® V-10 evaporator (Charlotte, NC) at room temperature and reformulated into 0.4 - 0.6 mL deionized H2O/MeCN (3/1) for injection into the (radio)HPLC system. 64Cu-NK1R-NOTA stored at room temperature for 3 h was used as positive control.


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In vivo and ex vivo imaging of HEK293-NK1R and HEK293-WT xenografts Male athymic NCr-nu/nu mice (7 to 9 weeks old, Taconic, Albany, NY) were used for subcutaneous implantation. HEK293-NK1R and HEK293-WT cells (5 × 106 cells suspended in 200 µL of cell culture medium/matrigel (BD Bioscience, Franklin Lakes, NJ) (v/v = 1/1) were implanted on the shoulder area. At 20-30 days post-inoculation, imaging and tissue sampling were performed when the tumor sizes were between 100-350 mm3.

Nuclear imaging with PET/CT Mice implanted with dual xenografts were administrated 5.5 MBq (0.7 nmole) of

64

Cu-NK1R-

NOTA (n = 4) via tail vein injection. At 1, 4, and 20 h post-injection, PET/CT imaging was performed on Inveon PET/CT system (Siemens, Malvern, PA) with the tumors centered in the field of view, and the animal under 2% isoflurane anesthesia. After 10 min of data acquisition on the PET scanner, the animal was moved to the CT scanner. CT acquisition was performed for 4 min at 60 kVp and 0.8 mA with 2 mm aluminum filtration. PET images were reconstructed by 2D-ordered subsets expectation maximization (2D-OSEM) on the Inveon PET/CT system. The reconstructed data of PET and CT images were rendered in 3D or 2D using an Inveon Research Workstation (Siemens, Malvern, PA). The calibration factor of the Inveon PET/CT scanner was measured with a mouse-sized phantom composed of a cylinder uniformly filled with an aqueous solution of 18F with a known activity concentration. Region-of-interest (ROI) analysis of the acquired images was performed using ASIPro software (Siemens, Malvern, PA), and the observed maximum pixel value (%ID/mL) was utilized to diminish partial volume effects of tumor.

Autoradiography imaging and H&E staining Following PET imaging, tumors were excised, embedded in optimal-cutting-temperature mounting medium (OCT, Sakura Finetek), and frozen on dry ice prior to cutting a series of 10 µm frozen sections. Digital autoradiography was performed on a phosphor imaging plate (Fujifilm BASMS2325; Fuji Photo Film) at −20°C. Phosphor imaging plates were read at a pixel resolution of 25 µm with a Typhoon 7000 IP plate reader (GE Healthcare). Following autoradiography imaging, the same section was stained with H&E and whole-mount bright field images acquired in a similar manner.


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Biodistribution of 64Cu-labeled NK1R-NOTA in dual xenografts The dual xenografts (n = 12) were randomly assigned into three cohorts and administrated 5.5 MBq (0.7 nmole) of

64

Cu-NK1R-NOTA via tail vein injection. The cohort of 20 h post-injection was

imaged first with PET/CT. At 1, 4, and 20 h post-injection, the animals were sacrificed for tissue dissection. The organs of interest were collected, rinsed, blotted, weighed, and counted with the γcounter. The total injected radioactivity per animal was determined from the measured radioactivity in an aliquot of the injectate. Data were expressed as percent of injected dose per gram of tissue (%ID/g).

Statistical analysis Data calculated using Microsoft Excel are expressed as mean ± SD. The Student’s unpaired t-test (GraphPad Prism 6, San Diego, CA) was used to determine statistical significance at the 95% confidence level. Differences with P values 95% at specific activities of >5.0 GBq/µmole for

67

Ga, and >7.0 GBq/µmole for

64

Cu at the end of

radiosynthesis. Ga- or Cu-labeled NK1R-NOTA was analyzed by UPLC-MS and high-resolution mass spectrometry (Table 1).


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Figure 1. Synthetic scheme of NK1R-NOTA antagonist (6) Table 1. Analytic and affinity data for Ga/Cu-labeled NK1R antagonists. 67/natGa or 64/natCu-labeled NK1R-NOTA was used for saturation binding assays in HEK293-NK1R cells and lipophilicity measurement. The values (mean ± SD) were from three independent studies, with triplicates in each independent experiment. NM, not measured.

Mass ([M+H]+)

Compound

Purity

Kd

Bmax

LogP 6

calculated measured

(%)

(nmole/L) (mol./cell) x 10 (1-Octanal/PBS)

Ga-NK1R-NOTA 943.2956 943.3004

> 98

12.0 ± 1.4

1.24 ± 0.40

NM

Cu-NK1R-NOTA 937.2996 937.3044

> 98

15.2 ± 1.1

1.49 ± 0.20

0.60 ± 0.01

Binding affinity assays Table 1 summarizes the binding affinities of

67/nat

Ga/64/natCu-chelated NK1R antagonists to NK1

receptors on HEK293-NK1R cells. Kd and Bmax assays confirmed that both

67/nat

Ga and

64/nat

Cu-

labeled NK1R-NOTA had a similar nM affinity (12.0 ± 1.4 nM vs. 15.2 ± 1.1 nM) to NK1 receptor, and binding capability (Bmax: (1.24 ± 0.40 vs. 1.49 ± 0.20)×106 molecules per cell). Representative saturation binding profiles of 64/natCu- and 67/natGa-labeled NK1R-NOTA are shown in Supplemental Figure 2.

In vitro cell uptake The in vitro uptake of

67

Ga or

64

Cu-labeled NK1R-NOTA is depicted in Figure 2. Both


67

Ga- and 8


64

Cu-labeled NK1R-NOTA showed a similar high accumulation in HEK293-NK1R cells (6.00 ±

0.60 and 6.37 ± 0.96 % of added activity per 0.5 × 106 cells, respectively), but low uptake in HEK293-WT cells (0.25 ± 0.07 % for

64

Cu-NK1R-NOTA). When an excess amount of L-733060

was co-incubated with the radiotracers to block radiotracer binding, the uptake of both

67

Ga- and

64

Cu-labeled NK1R-NOTA in HEK293-NK1R cells was reduced to 0.21 ± 0.09 and 0.34 ± 0.15 %,

respectively, which is similar to the uptake of

64

Cu-NK1R-NOTA in HEK293-WT cells (0.29 ±

0.09 %). 64Cu-NK1R-NOTA

8

67Ga-NK1R-NOTA

6

4

Figure 2. In vitro uptake of

67

HEK293-NK1R, Blocked

HEK293-NK1R

HEK293-WT, Blocked

HEK293-WT

HEK293-NK1R, Blocked

HEK293-NK1R

0

HEK293-WT, Blocked

2

HEK293-WT

% of total added activity per 0.5x106 cells

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

Page 10 of 23

Ga/64Cu-labeled NK1R-NOTA in NK1 receptor-expressing cells.

Both 67Ga- and 64Cu-labeled NK1R-NOTA (2.5 nM) showed significantly higher specific uptake in NK1R-positive cells. Values (mean ± SD) were from three independent studies with triplicates in each experiment.

In vitro and in vivo stability After 0.5 h of administration of

64

Cu-NK1R-NOTA, the remaining radioactivity in the circulation

was collected for monitoring the potential decomposition of

64

Cu-NK1R-NOTA. The results

(Figure 3) showed that no metabolite derived from 64Cu-NK1R-NOTA could be detected by HPLC. The retention time of

64

Cu-NK1R-NOTA was identical to that of

64

Cu-NK1R-NOTA in 1%

BSA/PBS at room temperature for 3 h.


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Page 11 of 23

400

Counts (CPM)

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


PBS

Plasma, in vivo

300 200 100 0 0

5

10

15

20

25

Time (min)

Figure 3. In vitro and in vivo stability of 64Cu-NK1R-NOTA. After 0.5 h injection of 64Cu-NK1RNOTA, the blood from three mice was combined for monitoring the potential metabolism. No detectable metabolites were observed in the serum (red solid), which was identical with that of 64

Cu-NK1R-NOTA in PBS solution (blue dash).

In vivo and ex vivo imaging of HEK293-NK1R and HEK293-WT xenografts Nuclear imaging with PET/CT Dual xenografts (HEK293-NK1R and HEK293-WT) were imaged with PET/CT at 1, 4, and 20 h post-injection.

64

64

Cu-NK1R-NOTA

Cu-labeled NK1R-NOTA was able to clearly delineate

HEK293-NK1R xenografts from the HEK293-WT xenografts and the adjacent background radioactivity in the images (Figure 4) at all three-time points, indicating that

64

Cu-NK1R-NOTA

rapidly accumulated in the target tissue and was rapidly excreted from other background organs. However,

64

Cu-NK1R-NOTA showed excretion to the intestine area, which indicated that it was

excreted predominantly through the gastrointestinal system (Figure 4A and 4B). A high contrast ratio between HEK293-NK1R xenografts and whole body was observed at 20 h post-injection (Figure 4C). Region-of-interest (ROI) values (%ID/mL) obtained from the PET images showed that 64

Cu-NK1R-NOTA had significantly higher accumulation in NK1R-positive xenografts than in

NK1R-negative xenografts (1 h: 3.56 ± 0.79 vs. 0.38 ± 0.10 %ID/mL, P = 0.016; 4 h: 3.09 ± 0.86 vs. 0.26 ± 0.08 %ID/mL, P = 0.006; 20 h: 1.92 ± 0.55 vs. 0.26 ± 0.06 %ID/mL, P < 0.0001).


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Figure 4. In vivo PET/CT imaging of dual xenografts (HEK293-NK1R and HEK293-WT) with 64

Cu-NK1R-NOTA. Dual xenografts-bearing mice were imaged at 1, 4, and 20 h post-injection of

64

Cu-NK1R-NOTA (n = 4). A color threshold was optimized to visualize the tumor clearly on the

PET/CT fusion image. An accurate color-intensity scale bar (%ID/mL) is precluded in these meanintensity projection (MIP) images (ROI measurements are provided).

64

Cu-NK1R-NOTA was able

to visualize HEK293-NK1R xenografts (left) on PET imaging with high tumor-to-background contrast, except high uptake was observed in the intestine at early time points.

Autoradiography imaging and H&E staining The dissected dual xenografts were used for in vitro autoradiography and H&E staining (Figure 5). Ex vivo images confirmed in vivo PET imaging findings that 64Cu-NK1R-NOTA had higher uptake in HEK293-NK1R (Figure 5A and 5E) tissue than in HEK293-WT (Figure 5C and 5F), and also demonstrated the lack of uptake in tumor regions that were necrotic by H&E (Figure 5B, 5D and 5G and 5H).


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Figure 5. Ex vivo H&E and autoradiographic imaging of HEK293-NK1R (panel A, B, E, G) and HEK293-WT (panel C, D, F, H) xenografts with 64Cu-NK1R-NOTA. 64Cu-NK1R-NOTA showed a significantly higher uptake in NK1R-transduced tissue (panel A and E) than the non-transduced tissue (panel C and F). Necrotic regions showed by H&E staining (panel B, D, G and H) demonstrated only very low binding of 64Cu-NK1R-NOTA (panel A, C, E and F). Biodistribution of 64Cu-labeled NK1R-NOTA in dual xenografts The results confirmed the observation of the previous PET/CT studies (Fig. 4), summarized in Table 2.

64

Cu-NK1R-NOTA showed a specific high accumulation in NK1R-positive organs,

including HEK293-NK1R xenografts (1 h: 3.23 ± 0.35 %ID/g; 4 h: 2.98 ± 0.60 %ID/g; and 20 h: 1.98 ± 0.47 %ID/g.), whereas low uptake in other normal organs, such as kidneys (1 h: 1.22 ± 0.25 %ID/g; 4 h: 0.93 ± 0.01 %ID/g; and 20 h: 0.99 ± 0.18 %ID/g). However, the high accumulation of 64

Cu-NK1R-NOTA in the liver and intestines suggested that

64

Cu-NK1R-NOTA had a dominant

clearance in the hepatobiliary system. The time-dependent clearance of the radiotracer in the small intestine (1 h: 25.9 ± 14.1 %ID/g; 4 h: 0.82 ± 0.14 %ID/g; and 20 h: 0.51 ± 0.06 %ID/g) and large intestine (1 h: 14.3 ± 16.0 %ID/g; 4 h: 17.5 ± 1.2 %ID/g; and 20 h: 0.73 ± 0.09 %ID/g) further illustrated the observed excretion. The radioactivity in the small and large intestine reached to 60.1 ± 3.6, 24.5 ± 7.3 and 1.6 ± 0.1 % of the total injected dose at these three-time points. 64Cu-NK1RNOTA also showed improved tumor-to-organ ratios at 20 h post-injection than that of the earlier time point.


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Table 2. Biodistribution of 64Cu-NK1R-NOTA (5.5 MBq/0.7 nmole/ in dual xenografts (HEK293NK1R and HEK293-WT). The values were presented as mean ± SD %ID/g (percent of injected dose per gram of mass). * the values listed in the bracket is %ID for small and large intestine. 1h N=4

4h N=4

20 h N=4

HEK293-NK1R

3.23 ± 0.35

2.98 ± 0.60

1.98 ± 0.47

HEK293-WT

0.50 ± 0.14

0.38 ± 0.05

0.45 ± 0.12

Blood

0.44 ± 0.05

0.33 ± 0.03

0.27 ± 0.06

Muscle

0.11 ± 0.09

0.09 ± 0.01

0.11 ± 0.03

Heart

0.44 ± 0.12

0.37 ± 0.06

0.44 ± 0.07

Lung

1.78 ± 1.36

1.15 ± 0.07

0.95 ± 0.12

Liver

4.07 ± 0.92

2.74 ± 0.68

1.82 ± 0.13

Pancreas

0.36 ± 0.10

0.24 ± 0.01

0.25 ± 0.05

Spleen

0.61 ± 0.28

0.45 ± 0.07

0.56 ± 0.10

Kidney

1.22 ± 0.25

0.93 ± 0.01

0.99 ± 0.18

Stomach

0.57 ± 0.20

0.35 ± 0.13

0.27 ± 0.09

Small intestine *(%ID)

25.9 ± 14.1 (43.8 ± 20.5)

0.82 ± 0.14 (9.1 ± 15.3)

0.51 ± 0.06 (0.70 ± 0.11)

Large intestine *(%ID)

14.3 ± 16.0 (16.3 ± 18.4)

17.5 ± 1.20 (15.4 ± 9.3)

0.73 ± 0.09 (0.87 ± 0.07)

Adrenals

0.53 ± 0.13

0.51 ± 0.11

0.46 ± 0.05

Bone

0.28 ± 0.05

0.27 ± 0.04

0.28 ± 0.05

Brain

0.05 ± 0.01

0.05 ± 0.01

0.07 ± 0.01

Organs

HEK293-NK1R/tissue ratio HEK293-WT

6.8 ± 1.8

8.1 ± 2.1

4.6 ± 1.3

Blood

7.7 ± 1.6

9.2 ± 2.3

7.7 ± 2.4

Muscle

49 ± 42

36 ± 10

20 ± 6

Liver

0.8 ± 0.2

1.2 ± 0.6

1.1 ± 0.3

Kidney

2.8 ± 0.8

3.2 ± 0.7

2.0 ± 0.5

Small intestine

0.2 ± 0.1

3.7 ± 1.2

3.9 ± 1.0

Large intestine

2.8 ± 3.1

0.2 ± 0.0

2.8 ± 1.0


13

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DISCUSSION Our in vitro and in vivo data indicate that 64Cu-NK1R-NOTA has promising properties for imaging and potentially radionuclide therapy of NK1R expressing tumors. The ligand showed nanomolar affinity for NK1R and high metabolic stability in vivo. Tumor uptake in vivo was high and specific whereas radioactivity was cleared quickly from the circulation by the liver and kidneys. This resulted in very high contrast images of NK1R positive xenografts at 20 h post injection.

Considering that the modification of L-733060 on its piperidine ring with bulky functional group, including NOTA and NIRF dye, did not significantly reduce its affinity to NK1R

13

, other

hydrophilic chelators, such as DOTA, can be integrated into the molecule using the same strategy. DOTA-integrated NK1R antagonists may still maintain their affinity to NK1R, which allows for radiolabeling with other therapeutic radioisotopes, including 177Lu, 90Y, and 225Ac. In addition, both 67/nat

Ga- and 64/natCu-labeled NK1R-NOTA showed similar affinities and accumulation in HEK293-

NK1R cells, which may imply that the different radioisotope-chelated NK1R-DOTA analogs will show similar in vitro and in vivo behaviors. To increase the hydrophilicity of NK1R-NOTA and facilitate its excretion via the urinary pathway, a more hydrophilic linker could be added for further optimization.

HEK293-NK1R cells displayed a fairly high NK1R density in the saturation binding assay of both 67/nat

Ga- and

64/nat

Cu-labeled NK1R-NOTA, which might indicate that the antagonist detected more

binding sites than the agonist, i.e. similar to the behavior observed by other G-protein coupled receptors 16. Notably, however, is that a direct comparison between the radiolabeled SP analog and NK1R-NOTA saturation assays will further verify this assumption, especially on the NK1Rpositive human cancer cell line or tissues. The radiolabeled NK1R agonist [Indium-111-DTPA-Arg1] Substance P displayed rapid enzymatic degradation with a half-life of 3 min during circulation 7. Although the methionine at the 11th position of SP was subsequently replaced with the synthetic amino acid, Met(O2), to improve its in vivo stability 9, the impact of this substitution on DOTA-[Thi8,Met(O2)11]-substance P has not yet been published. In contrast, the non-peptidic NK1R antagonist

64

Cu-NK1R-NOTA proved to be

metabolically stable in vitro and in vivo, which allows for imaging NK1R-expressing cancer by


14


Page 16 of 23

administering the dose intravenously. Therefore, the metabolically stable

64

Cu-NK1R-NOTA may

be more feasible for targeting NK1R-postive cancers outside of the brain, especially neuroendocrine cancers of the pancreas 17, stomach, colon, and breast 18, 19.

Despite the fact that radiolabeled SP analogs (agonists) show high affinity for NK1R and high cell uptake, very few reports have been published on their in vivo accumulation in NK1R-positive xenografts because of their rapid degradation in the circulation 7; e.g., [Indium-111-DTPA-Arg1]SP had a sub-nanomolar affinity in vitro, but a relatively low accumulation in transplantable pancreatic tumor, CA20948. In contrast, the in vivo validation of

64

Cu-NK1R-NOTA with dual xenografts

(HEK293-NK1R and HEK293-WT) showed that this antagonist had high accumulation in HEK293NK1R xenografts, but low uptake in the non-transduced xenografts (HEK293-WT) at all three-time points. Ex vivo imaging and staining confirmed that HEK293-NK1R tissue than in HEK293-WT. Since

64

64

Cu-NK1R-NOTA had higher uptake in

Cu-NK1R-NOTA was rapidly excreted from

all background organs through the gastrointestinal tract system, a high contrast ratio between HEK293-NK1R xenografts and whole body was observed, especially at 20 h post-injection, consistent with the results obtained from the subsequent biodistribution study.

64

Cu-NK1R-NOTA

showed relatively higher accumulation in the liver and lung than that of the NIRF-conjugated NK1R antagonist NK1RL-peptide-LS288 conjugate the nervous system and peripheral tissues

20

13

. Since NK1 receptor is widely expressed in

, a relatively high lung uptake of

64

Cu-NK1R-NOTA

might indicate that the expression level of NK1R in the lung is worthy to be investigated further in the future. Potential explanations for high liver uptake are: 1) 64Cu-NK1R-NOTA may release 64Cu from its 64Cu-NOTA complexation; or 2) the optical signals from NK1RL-peptide-LS288 conjugate may be attenuated in these two organs. To investigate whether the accumulation of 64Cu in the liver is resulted from the dissociation of 64Cu-NK1R-NOTA 21, other metabolically stable chelators, such as CB-TE2A 22, NODAGA 23 or AmBaSar 24 can be used to replace NOTA.

In conclusion, we first synthesized and evaluated a novel radiolabeled NK1R antagonist,

64

Cu-

NK1R-NOTA, which is suitable for non-invasive PET imaging of NK1R-expressing cancers following intravenous administration. Our results showed that

64

Cu-NK1R-NOTA was

metabolically stable, had high nM affinity to NK1R, and displayed in vivo and ex vivo image contrast between NK1R-expressing xenografts and healthy tissues. Therefore,


64

Cu-NK1R-NOTA

15

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PET imaging may be a potential alternative approach to image NK1R-positive cancers if translated into patients. Compared to previously described aprepitant analogs

12

18

F-labeled NK1R antagonists such as

18

F-labeled

the advantages of NK1R-NOTA include the ability for delayed imaging to

allow for clearance of intestinal activity and the labeling with therapeutic isotopes for theranostic applications.

AUTHOR INFORMATION

Corresponding author * Email: [email protected] ORCID Philip S. Low: 0000-0001-9042-5528; Ananda Kumar Kanduluru: 0000-0003-2669-8665. Author contributions HZ and AKK contributed equally in this project. The experiments were performed through the contributions of all authors, and all authors have approved to the final version of the manuscript.

NOTE All authors have nothing to disclose. All animal experiments were performed with the approved protocol of 13-10-015, which is in accordance with the ethical standards of the Institutional Animal Care and Utilization Committee of MSKCC. The current Address of AKK: On Target Laboratories, LLC. West Lafayette, IN 47906.

ACKNOWLEDGEMENTS This project was funded in part by the MSKCC IMRAS grants and NIH grant, P50-CA84638 (H. Zhang, W.A. Weber), Technical services from the MSK Core Facilities were supported by NIH Small-Animal Imaging Research Program grant R24 CA83084, NIH/NCI Cancer Center Support Grant (P30 CA008748-48), and NIH shared instrumentation grants (1 S10 RR020892-01 and 1 S10 OD016207-01).

Supporting Information Supporting Information contains additional figures showing LC-MS analysis of NK1R-NOTA and


16


Page 18 of 23

its intermediates, and a representative saturation-binding assay. Supporting Information is available free of charge on the ACS Publications Website. ABBREVIATION NK: neurokinin; NK1R: neurokinin 1 receptor; HEK293-NK1R: NK1R-transduced HEK293 cell; HEK293-WT: NK1R non-transduced HEK293 cell; PET: positron emission tomography; SP: Substance P; NIRF: near-infrared fluorescent; DMSO: dimethyl sulfoxide; DIPEA: N,Ndiisopropylethylamine;

EtOAc:

ethyl

acetate;

DCM:

dichloromethane;

LC/MS:

liquid

chromatography/mass spectrometry; NOTA: 1,4,7-triazacyclononane-1,4,7-trisacetic acid; RPHPLC: reverse phase-high performance liquid chromatography; HLB: hydrophilic–lipophilic balance; %ID/g: percent of injected dose per gram of tissue; ROI: region-of-interest; DOTA: 1,4,7,10-tetrazacyclononane-1,4,7,10-tetraacetic Tetraazabicyclo[6.6.2]hexadecane-4,11-diacetic acid;

acid;

CB-TE2A:

1,4,8,11-

NODAGA: 1,4,7-triazacyclononane,1-

glutaric acid-4,7-acetic acid; AmBaSar: 4-((8-amino-3,6,10,13,16,19-hexaazabicyclo [6.6.6] icosane-1-ylamino) methyl) benzoic acid.

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Synthesis and Evaluation of a Novel 64Cu-and 67Ga-Labeled

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