Evaluation of 64Cu-Labeled Bifunctional Chelate–Bombesin

TECHNICAL NOTE pubs.acs.org/bc

Evaluation of 64Cu-Labeled Bifunctional Chelate
Bombesin Conjugates Samia Ait-Mohand,† Patrick Fournier,† V
eronique Dumulon-Perreault,† Garry E. Kiefer,‡ Paul Jurek,‡ Cara L. Ferreira,§ Franc-ois B
enard,^ and Brigitte Gu
erin*,† †

Centre d’imagerie mol
eculaire de Sherbrooke (CIMS), Department of Nuclear Medicine and Radiobiology, Universit
e de Sherbrooke, 3001, 12th North Avenue Sherbrooke, Qubec, Canada, J1H 5N4 ‡ Macrocyclics, Dallas, Texas, United States § Nordion, Vancouver, British Columbia, Canada ^ BC Cancer Agency Research Centre, Vancouver, British Columbia, Canada ABSTRACT: Several bifunctional chelates (BFCs) were investigated as carriers of 64Cu for PET imaging. The most widely used chelator for 64 Cu labeling of BFCs is DOTA (1,4,7,10-tetraazacyclododecane-N,N0 , 00 000 N ,N -tretraacetic acid), even though this complex exhibits only moderate in vivo stability. In this study, we prepared a series of alternative chelator
peptide conjugates labeled with 64Cu, measured in vitro receptor binding affinities in human breast cancer T47D cells expressing the gastrin-releasing peptide receptor (GRPR) and compared their in vivo stability in mice. DOTA-, NOTA-(1,4,7-triazacyclononane-1,4,7-triacetic acid), PCTA-(3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-triacetic acid), and Oxo-DO3A-(1-oxa-4,7, 10-triazacyclododecane-4,7,10-triacetic acid) peptide conjugates were prepared using H2N-Aoc-[D-Tyr6,βAla11,Thi13,Nle14]bombesin(6
14) (BBN) as a peptide template. The BBN moiety was selected since it binds with high affinity to the GRPR, which is overexpressed on human breast cancer cells. A convenient synthetic approach for the attachment of aniline-BFC to peptides on solid support is also presented. To facilitate the attachment of the aniline-PCTA and aniline-Oxo-DO3A to the peptide via an amide bond, a succinyl spacer was introduced at the N-terminus of BBN. The partially protected aniline-BFC (p-H2N-Bn-PCTA(Ot-Bu)3 or p-H2N-BnDO3A(Ot-Bu)3) was then coupled to the resulting N-terminal carboxylic acid preactivated with DEPBT/ClHOBt on resin. After cleavage and purification, the peptide-conjugates were labeled with 64Cu using [64Cu]Cu(OAc)2 in 0.1 M ammonium acetate buffer at 100 C for 15 min. Labeling efficacy was >90% for all peptides; Oxo-DO3A-BBN was incubated an additional 150 min at 100 C to achieve this high yield. Specific activities varied from 76 to 101 TBq/mmol. Competition assays on T47D cells showed that all BFCBBN complexes retained high affinity for the GRPR. All BFC-BBN 64Cu-conjugates were stable for over 20 h when incubated at 37 C in mouse plasma samples. However, in vivo, only 37% of the 64Cu/Oxo-DO3A complex remained intact after 20 h while the 64Cu/ DOTA-BBN complex was completely demetalated. In contrast, both 64Cu/NOTA- and 64Cu/PCTA-BBN conjugates remained stable during the 20 h time period. Our results indicate that it is possible to successfully conjugate aniline-BFC with peptide on solid support. Our data also show that 64Cu-labeled NOTA- and PCTA-BBN peptide conjugates are promising radiotracers for PET imaging of many human cancers overexpressing the GRP receptor.

’ INTRODUCTION Positron-emitting 64Cu (T1/2 = 12.7 h, β+ 17.4%, Emax = 0.656 MeV, β
39%, Emax = 0.573 MeV) is a suitable isotope for PET imaging,1 although the availability of appropriate Cu-chelating agents limits its wider application. Several bifunctional chelates (BFCs) have been investigated as carriers of radiometals for positron emission tomography (PET) imaging. Among them, DOTA is the most widely used for metal radioisotopes.2 However, DOTA complexes with some radiometals such as 64Cu show only moderate in vivo stability, resulting in demetalation and subsequent accumulation of the radiometal in nontarget tissues. Previous studies have described the potential of NOTA,3 PCTA,4,5 and Oxo-DO3A4,5 as BFCs for divalent and trivalent r 2011 American Chemical Society

metal ions. These chelators have been radiolabeled efficiently with 64Cu and present higher resistance to transmetalation reactions in vivo as compared to DOTA.3,4,6 We recently reported a practical synthetic approach for the preparation of NOTA-peptides directly on solid support.7 However, no data on coupling of aromatic amine derivatives, such as PCTA and OxoDO3A, to peptides have been reported. The coupling of aromatic amine derivatives of BFC to antibodies or peptides require either large excesses of BFC or long reaction times.8,9 In this Received: May 25, 2011 Revised: July 15, 2011 Published: July 18, 2011 1729

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Bioconjugate Chemistry

TECHNICAL NOTE

Figure 1. Structures of the BFC-BBN conjugate and bifunctional chelates DOTA, NOTA, PCTA, and Oxo-DO3A.

study, we report a convenient synthetic approach for the preparation of PCTA and Oxo-DO3A-peptide conjugates directly on solid support using H2N-Aoc-[D-Tyr6,βAla11, Thi13,Nle14]bombesin(6
14)10 (BBN) as peptide template. The two novel BFCs contain a 12-membered macrocyclic chelate, PCTA-(3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-triacetic acid) or Oxo-DO3A-(1-oxa-4,7,10triazacyclododecane-4,7,10-triacetic acid) and an aniline substituent, which can be attached to the BBN peptide via a succinyl spacer. The BBN derivative acts as an agonist and binds with high affinity to GRPR overexpressed on human prostate,11,12 breast,13
15 and other cancers.16,17 GRPR belongs to a large family of G-protein-coupled receptors that are characterized by high or low affinity for agonist depending on the coupling of a guanine nucleotide (GDP or GTP) on the G-protein.18 Furthermore, the level of GRPR has been shown to correlate positively to the presence of estrogen receptors.14 For PET imaging, Schuhmacher et al. labeled a DOTA-PEG2-[D-Tyr6,β-Ala11,Thi13,Nle14]BBN(6
14) with 68Ga,19 while Chen et al. used DOTA-Lys3bombesin with 64Cu.20 Smith et al. successfully labeled modified BBN(7
14) analogues with 64Cu for potential use in diagnostic imaging using NOTA or NO2A as chelating agents and obtained stable compounds.3,21 Our laboratory has reported the synthesis and the characterization of DOTA- and NOTA-BBN and showed that the NOTA peptide had an inhibition constant (Ki) value slightly lower than that of the analogous DOTA-peptide.7 To determine whether PCTA-BBN and Oxo-DO3A-BBN offered an advantage over the currently used BFCs for Cu-based radiopharmaceuticals, we compared our novel BFCs to the analogous BFC-BBN conjugates containing the DOTA and NOTA moieties. The BFC-BBN conjugates (Figure 1) were examined with respect to 64Culabeling efficacy, GRPR affinity, and stability. The availability of various copper chelators with high in vivo stability would allow fine-tuning of the various biological properties of 64Cu-labeled BFC-BBN conjugates.

’ EXPERIMENTAL PROCEDURES Materials. All chemicals and solvents (reagent grade) were used as supplied by the vendors without further purification, unless otherwise stated. NovaSyn TGR resin was obtained from NovaBiochem. Fmoc-protected amino acids were obtained from EMD NovaBiochem (Gibbstown, NJ, USA) or Chem-Impex International Inc. (Wood Dale, IL, USA). 1,4,7,

10-Tetraazacyclododecane-1,4,7-tris-tert-butyl acetate-10-acetic acid (DOTA(Ot-Bu)3OH), 3,6,9,15-tetraazabicyclo[9.3.1]pentadeca1(15),11,13-triene-3,6,9-tris-tert-butyl acetate (p-H2N-Bn-PCTA(Ot-Bu)3), and 1-oxa-4,7,10-triazacyclododecane-4,7,10tris-tert-butyl acetate (p-H2N-Bn-Oxo-DO3A-(Ot-Bu)3) were obtained from Macrocyclics (Dallas, TX, USA). 1.4.7-Triazacyclononane was obtained from TCI America (Portland, OR, USA). 2-(1H-7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU) was purchased from Chem-Impex International Inc. 3-(Diethoxy-phosphoryloxy)-3H-benzo[d][1,2,3] triazin-4-one (DEPBT) and 6-chloro-1-hydroxy-1H-benzotriazole (ClHOBt) were purchased from ChemPep (Wellington, FL, USA) and Matrix Innovation (Quebec, QC, CA), respectively. HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), amphothericin B, RPMI-1640, PBS (phosphatebuffered saline), trypsin, penicillin, streptomycin, and fetal bovine serum (FBS) were purchased from Wisent (StBruno, QC, CA). N,N-Diisopropylethylamine (DIEA), thioanisole, and triisopropylsilane (TIPS) were obtained from Aldrich Chemical Co. Bovine serum albumin (BSA) and bombesin were purchased from Sigma-Aldrich (Saint-Louis, MO, USA). Acetonitrile, dichloromethane (DCM), N,N-dimetylformamide (DMF), and isopropyl alcohol (i-Pr-OH) were obtained from Fisher Scientific (Ottawa, ON, CA). 125I-Bombesin was purchased from Perkin-Elmer Life Science Products (Boston, MA, USA). Finally, T47D human breast cancer cell line was obtained from ATCC (Manassas, VA, USA). DMF was dried over 4 Å molecular sieves for at least one week to remove trace amounts of amines and filtered before its use. Peptide Synthesis. DOTA-BBN (1) and NOTA-BBN (2). We recently reported the synthesis, characterization, and biological activity of these peptides.7 General Procedure for the Coupling of Aniline-BFC to Peptide on Solid Support. The BBN derivative was synthesized by a continuous flow method on a Pioneer Peptide Synthesis System (PerSeptive Biosystems) using the Fmoc strategy. A 2-fold excess of Fmoc-protected amino acid over available resin substitution sites was used for coupling in amine free DMF. Fmoc-protected amino acids were activated for coupling with an equimolar amount of HATU and 2 equiv of DIEA. Fmoc deprotection was performed in 20% piperidine in DMF and monitored through absorbance at 364 nm. The resin was washed with trice with DMF, MeOH, DMF, MeOH, and DCM, respectively. After completion of the synthesis on the automated system, Fmoc-Aoc-OH and the succinyl spacer, as well as the aniline-BFC, 1730

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Bioconjugate Chemistry were coupled manually to the peptide. The Fmoc-Aoc-OH (2.5 equiv) was dissolved in 2 mL of DMF at 0 C, DIEA (2.5 equiv) and PyBOP (2.5 equiv) were added to the cold solution. After 15 min of stirring, the mixture was added to the partially protected peptide on resin preswelled with DCM and mixed with ClHOBt (2.5 equiv) and DIEA (2.5 equiv). For this coupling, a mechanical agitation was maintained for 2 h at room temperature. The resin was washed thrice with DMF, MeOH, DMF, MeOH, and DCM, respectively. Fmoc deprotection was performed in 20% piperidine in DMF during 15 min, and the resin was washed as described above. The coupling and the Fmoc deprotection steps were followed by a Kaiser’s test on resin; the reaction between resin and ninhydrin was followed colorimetrically whereby free primary amines after Fmoc deprotection were detected as blue beads and their absence as yellow beads. Reaction conditions were optimized by monitoring each step by analytical reversed-phase HPLC after cleavage of a small amount (10 mg) of the resin-bound peptide with 95% aqueous TFA. The partially protected peptide on resin was swelled in a minimal volume of DMF (2 mL), succinic anhydride (10 equiv), and DIEA (10 equiv) dissolved in DMF (3 mL) were added. This coupling procedure was performed twice (30 min and 1 h). The resin was washed as described above. The resulting N-terminal carboxylic acid group was activated with ClHOBt (1 equiv) and DIEA (2 equiv) in DMF. The preactivation mixture was stirred for 30 min, then DEPBT (1 equiv) and the aniline-BFC (p-H2N-BnPCTA-(OtBu)3 or p-H2N-Bn-Oxo-DO3A-(OtBu)3, 2 equiv) were added and the reaction was allowed to proceed overnight at room temperature, under mechanical agitation. The coupling was performed twice with 2 equiv of the amine. The resin was washed thrice as described above, and the desired peptide was deprotected and cleaved from the support by treatment with a cocktail of TFA/H2O/thioanisole (92:2:6, v/v) for 4 h at room temperature under mechanical agitation. The resin was removed by filtration and washed with TFA. Combined filtrates were added dropwise to ethyl ether (1 mL of TFA/10 mL of ether). The precipitated peptides were centrifuged at 1200 rpm for 15 min. The ether solution was decanted and the white solid was dissolved in water, frozen, and lyophilized. The crude peptide was purified by flash chromatography on a Biotage SP4 system equipped with a C18 column. Purity of the peptides was verified by HPLC, and their identity was confirmed by API 3000 LC/MS/MS. Analytical high performance liquid chromatography (HPLC) was performed on an Agilent 1200 system equipped with a Zorbax Eclipse XDB C18 reversed-phase column (4.6  250 mm, 5 μ) and Agilent 1200 series diode array UV
vis detector, using a linear gradient of 0% to 100% acetonitrile in water with 0.1% TFA over 30 min at a flow rate of 1 mL/min. PCTA-BBN (3). After purification, fractions containing the desired product were combined and lyophilized to yield peptide 3 as a white solid (10 mg, 23% yield based on resin substitution rate). Product purity was confirmed to be 95% by analytical reversed-phase HPLC: average retention time 16.2 min. MS Calc: 1846.9; Found: 924.6 (M/2 + 1), 1848.3 (M+1). Oxo-DO3A-BBN (4). After purification on Biotage, fractions containing the desired product were combined and lyophilized to provide peptide 4 (8.3 mg, 20% yield based on resin substitution rate). Product purity was confirmed to be 99% by analytical reversed-phase HPLC: average retention time 16.2 min. MS Calc: 1813.9; Found: 908.5 (M/2 + 1), 1815.4 (M+1). Cell Culture. The human breast cancer T47D cells were used in their 25th to 35th passages after receipt and cultured in RPMI

TECHNICAL NOTE

1640 medium containing phenol red supplemented with 2.5 mM glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, 100 ng/mL amphothericin B, and 10% FBS. Cells were grown in 5% CO2 in air at 37 C; the medium was changed 3 times per week. In Vitro Receptor Binding. Competition assays were performed on T47D human breast cancer cells expressing GRPR. In 24-well plates, the cells were cultured until near confluence and the medium was replaced by 400 μL of reaction medium (RPMI complemented with 2 mg/mL BSA, 4.8 mg/mL HEPES, 1 U/mL penicillin G, and 1 μg/mL streptomycin). For the assay, equal volumes of radioactive and nonradioactive ligands were added. The concentration of the radioactive ligand [125I]I-Tyr4Bombesin (74 TBq/mmol) was 10
12 M. Increased concentrations (10
6 to 10
14 M) of the GRP ligand of interest were added to consecutive wells. The plates were incubated for 40 min at 37 C with agitation. After the incubation, the reaction medium was removed and the cells were washed three times with PBS at room temperature. The cells were harvested and the bound [125I]I-Tyr4-Bombesin was counted in a gamma counter (Cobra II autogamma counter, Packard, MN). Finally, data were analyzed with GraphPad Prism 5 software to determine the IC50 value. The Ki was calculated from the IC50 using the equation of Cheng and Prusoff.22 The Kd = 1.5  10
10 M value for the [125I]I-Tyr4-Bombesin was determined by experiments done under similar conditions.23 Radiolabeling of BFC-Peptide Conjugate with 64Cu. Our cyclotron facility provides 64Cu isotope on a routine basis for research purposes, using a target system developed in collaboration with ACSI (Richmond, BC, CA). 64Cu was prepared on a TR-19 cyclotron (ACSI) by the reaction 64Ni(p,n)64Cu using an enriched 64Ni target electroplated on a rhodium disk.24 64CuCl2 was recovered from the target following the procedure of McCarthy et al.25 and converted to 64Cu-acetate by dissolving the 64CuCl2 in ammonium acetate (0.1 M; pH 5.5), followed by evaporation to dryness. BFC-peptide conjugates were labeled with 64Cu following conditions optimized in our laboratory. Briefly, BFC-peptide (5 μg) was dissolved in a 0.1 M ammonium acetate buffer pH 5.5 with [64Cu]Cu(OAc)2 (296
370 MBq or 8
10 mCi) in a total volume of 250
300 μL, and then the resulting solution was incubated at 100 C for 15 min, except for the Oxo-DO3A derivative which was incubated for 2.5 h at the same temperature. The labeled product was purified by HPLC equipped with a radiodetector. The peptide fraction was collected, evaporated, and 64Cu counted in a Capintec Radioisotope Calibrator to calculate the specific activity of the product. Plasma Stability Studies. 64Cu-labeled DOTA-, NOTA-, PCTA-, and Oxo-DO3A-peptides were poorly soluble in a physiological solution; 10% DMF in PBS was used to solubilize the peptides. 64Cu-labeled peptides (111 MBq (3 mCi); 30
50 μL) were incubated at 37 C in 500 μL of fresh mouse plasma (n = 3) for different time periods, ice-cold acetonitrile was added, and the samples were vortexed and centrifuged. The supernatant was analyzed by reversed-phase HPLC to detect 64Cu metabolites. For radioTLC analysis, plasma stability studies were achieved by incubating 1.85 MBq (50 μCi, 5
10 μL) of 64Culabeled peptides at 37 C in 100 μL of fresh mouse plasma (n = 3) for different time points. Free 64Cu and 64Cu/BFC-BBN conjugate were co-spotted with plasma samples (without treatment) and used as references. TLC was performed either on silica gel coated plastic sheets with methanol/0.1 M ammonium acetate pH 5.5 (1:1) as eluent or on C-18 bonded reversed-phase TLC 1731

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Bioconjugate Chemistry

TECHNICAL NOTE

Table 1. Analytical Data for BFC-BBN Conjugates [M]+ entry

peptide

calcd

founda

yield (%)

purityb (%)

Kic (nM)

labeling yieldd (%)

0.76 ( 0.54

1

Bombesin

2

DOTA-Aoc-BBN(6
14)

1667

1668

11

93

10.61 ( 4.55

>90

3

NOTA-Aoc-BBN(6
14)

1566

1567

16

98

6.13 ( 2.72

>95

4

64

5

Oxo-DO3A-Suc-Aoc-BBN(6
14)

1814

1815

20

99

2.51 ( 1.50

>90e

6

PCTA-Suc-Aoc-BBN(6
14)

1847

1848

23

95

0.36 ( 0.05

>95

3.08 ( 0.74

Cu/NOTA-Aoc-BBN(6
14)

a

Mass values were obtained by MALDI TOF mass spectroscopy or LC/MS/MS. b Purity was determined by HPLC analysis. c Affinities for GRPR were determined with [125I-Tyr4]bombesin in T47D human breast cancer cell line. d Labeling yield was determined by radio-HPLC analysis based on 64Cu starting activity. e This peptide was incubated an extra 150 min at 100 C.

Table 2. Peptide Coupling Optimization of p-H2N-Bn-DO3A(OtBu)3 in Solution and Solid Phase entry

carboxylic acid segment

coupling conditions

reaction time (h)

conversiona (%)

1

HO-Suc-Aoc-BBN(6
14)-resin

HATU (2.5 equiv), DMF

72

0

2

HO-Suc-Aoc-BBN(6
14)-resin

PyBOP and ClHOBt (2 equiv), DIEA

72

trace

3

BOC-Gly-OH (2 equiv)

72

>99

24

>99

(4 equiv), N-methylpyrrolidone:DMF (1:1)

4

HO-Suc-Aoc-BBN(6
14)-resin

PyBOP and ClHOBt (2 equiv), DIEA (4 equiv), N-methylpyrrolidone:DMF (1:1) ClHOBt (1 equiv), DIEA (2 equiv), DMF, then DEPBT (1 equiv), (2)

a

Conversion refers to fraction of the carboxylic acid fragment consumed in the reaction and was determined by TLC for coupling in solution or by HPLC and LC/MS/MS analysis after peptide cleavage.

plates with 0.1 M sodium citrate at pH 5, and 64Cu was detected an Instant Imager system. In Vivo Stability Studies. 64Cu-labeled peptides (15
25 MBq) were intravenously injected in female balb/c mice (n = 3). For the PCTA-BBN conjugate, this corresponded to about (6.47 ( 0.08)  10
10 mol and in the case of the Oxo-DO3A-BBN derivative to about (1.06 ( 0.2)  10
9 mol. Blood was taken from the mice’s back paw at different times and directly analyzed by TLC either on silica gel with methanol/0.1 M ammonium acetate at pH 5.5 (1:1) or on C-18 coated plates with 0.1 M sodium citrate at pH 5.

’ RESULTS Preparation and Characterization of BFC-BBN Conjugates. We previously reported the synthesis and characteriza-

tion of DOTA- and NOTA-BBN peptides (Table 1).7 These peptides were prepared with overall yields of 11
16% based on the substitution rate of the resin, determined photometrically from the amount of Fmoc chromophore released upon treatment of the resin with piperidine/DMF. According to analytical HPLC, the purity was 93% for DOTA-BBN and 98% for NOTA-BBN. For the preparation of the Oxo-DO3A-BBN, different coupling reagents were tested to activate the succinylated peptide on resin (Table 2). Our first attempt using HATU in DMF followed by the addition of p-H2N-BnDO3A(Ot-Bu)3 failed to afford BFC-BBN conjugate, even after 72 h of mechanical agitation (Table 2, entry 1). A similar product pattern along with a trace amount of the desired OxoDO3A-BBN conjugate (LC/MS/MS analysis after peptide cleavage) was observed when the succinylated-BBN on resin was treated with PyBOP and ClHOBt in the presence of DIEA and a 1:1 ratio of N-methylpyrrolidone/DMF (Table 2, entry 2).

Under similar conditions, coupling of p-H2N-Bn-DO3A(OtBu)3 to BOC-Gly-OH, a model compound, led to the desired BFC-BBN product when the reaction was performed in solution (Table 2, entry 3). We then found an effective coupling reagent, DEPBT, that was used for the activation of the carboxylic acid on resin and the subsequent coupling of the aniline-Oxo-DO3A chelate. As presented in Table 2, DEPBT was highly effective for the coupling of aniline-BFC to peptide on resin with complete conversion of the starting material (Table 2, entry 4). Using these conditions, the OxoDO3A-Suc-Aoc-BBN(6
14) and PCTA-Suc-Aoc-BBN(6
14) were prepared with overall yields of 20% and 26%, respectively, in >95% purity (Table 1). BFC-BBN conjugates were also tested for their receptor binding affinity on T47D cells. DOTA-BBN peptide displayed good binding affinities to GRPR, while NOTA- and Oxo-DO3ABBN peptides gave inhibition constant (Ki) values slightly lower than that of DOTA-peptide (Table 1). The lowest Ki value (0.36 ( 0.05 nM) was obtained with the PCTA-BBN derivative. The Ki value of the Cu/NOTA-BBN derivative is in the low nanomolar range and similar to that of the nonlabeled-peptide (Table 1, entries 3 and 4). Preparation and Characterization (Radiolabeling, Purification, Stability) of 64Cu-BFC-BBN Conjugates. We produce 64 CuCl2 with a yield of 70 mCi/μA hr/μg using 99% enriched Ni-64, with typical production batches on the order of 3.70
9.25 GBq (100
250 mCi) with specific activity of 296
444 TBq/ mmol (8000
12 000 Ci/mmol) of Cu determined by TETA titrations.25 The BFC-BBN conjugate 64Cu-labeling results are summarized in Table 1. An incubation time of 15 min at 100 C was found to produce the highest labeling yields. Using this condition, labeling yields were greater than 90% for DOTA-, 1732

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TECHNICAL NOTE

NOTA-, PCTA-BBN, and the Oxo-DO3A-peptide; the latter was incubated for an additional 150 min to reach this yield (Table 1). The purification by HPLC using a C-18 column to remove unreacted 64Cu yielded the radiolabeled BFC-BBN conjugate with >99% radiochemical purity and specific activity between 76 and 101 TBq/mmol (2050
2740 Ci/mmol) for all labeled peptides. The stability of the 64Cu-radiolabeled BFC-BBN, in terms of percentage of intact conjugate was determined in mouse plasma and female balb/c mice (Table 3). We monitored 64Cu by both HPLC and radio-TLC on silica gel or C18 to determine any 64Cu metabolites and free 64Cu. Similar results were obtained using these two different approaches. Since radio-TLC can be performed directly on plasma and blood samples without precipitating plasma proteins, this approach was used to determine the stability of the other 64Cu/BFC-BBN conjugates. As illustrated in Table 3, 64Cu/DOTA-BBN was stable for 1 h in plasma, but for only 30 min after injection in mice. The radio-TLC analysis showed no degradation of the 64Cu-labeled NOTA-and PCTABBN peptides after 20 h incubation in mouse plasma or in mice. The 64Cu-labeled Oxo-DO3A derivative was stable in vitro, but only 37% intact conjugate remained 20 h post-injection in mice. Table 3. 64Cu/chelator Complex Stability of BBN(6-14) Derivatives Cu/BFC stabilitya (%)

64

stability plasma in vivo (blood)

time (min) 60 1200 30

DOTA

NOTA

PCTA

Oxo-DO3A

>99

>99

>99

>99

n.d. >99

>99

>99

>99

60

67

90

41

>99

>99

>99

1200

0

>99

>99

37
40

a Stability is presented as % intact 64Cu-labeled BFC-peptide as determined by HPLC analysis or using TLC on silica gel with methanol/ 0.1 M ammonium acetate at pH 5.5 (1:1).

’ DISCUSSION Previously, we reported the synthesis and characterization of two BFC-conjugated peptides: DOTA-BBN and NOTA-BBN.7 It was found that NOTA-chelates are readily synthesized in a two-step process starting with bromo-acetylated peptides and that all NOTA peptides had inhibition constant (Ki) values slightly lower than the analogous DOTA-peptides. The synthesis of PCTA- and Oxo-DO3A-BBN conjugates on solid phase is summarized in Scheme 1. Although both isothiocyanate and aniline derivatives of PCTA and Oxo-DO3A are commercially available, we selected aniline derivatives for conjugation to our BBN peptide since they generally display greater long-term stability than their isothiocyanate analogues. To facilitate the coupling of the aniline-PCTA and the aniline-Oxo-DO3A to the peptide, a succinyl spacer was introduced at the N-terminus of BBN linked to the resin. Even if the activation of carboxylic moieties on resin is known to present challenges, a complete conversion of the succinylated-BBN was observed using DEPBT/ClHOBt as coupling reagents (Table 2, entry 4). Indeed, DEPBT has already been employed with success in difficult couplings, such as primary and secondary anilines.26,27 Using their respective partially protected aniline chelates, milligram amounts of PCTA and Oxo-DO3A conjugates are rapidly and efficiently made and purified. An attractive aspect of this procedure is that a very stable amide bond between the BFC and the succinylated peptide is obtained. Also, increasing the distance between the chelate and the peptide backbone by using a succinyl spacer enhanced the BBN binding affinity. Indeed, the Ki values of Oxo-DO3A-BBN and PCTA-BBN conjugates were more than 5-fold and 30-fold lower, respectively, as compared to that found for DOTA-BBN when tested in T47D human breast cancer cells (Table 1). PCTA-Suc-Aoc-BBN(6
14) displayed the highest binding affinity of all BFC-BBN conjugates tested. The higher affinity of the NOTA conjugate for GRPR as compared to DOTA-BBN suggests that the introduction of a smaller chelating unit at the N-terminus of the peptide allowed the BBN peptide to interact more efficiently with its receptor. No significant

Scheme 1. Synthesis of PCTA- and Oxo-DO3A-BBN Conjugates on Solid Phase

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Bioconjugate Chemistry difference in affinity is observed between Cu-labeled and nonlabeled peptides (Table 1). After 64Cu-labeling, the radiochemistry of the PCTA-BBN and Oxo-DO3A-BBN was compared to that of DOTA- and NOTA-BBN conjugates. All four 64Cu complexing systems can be used to prepare peptide radioligands with high radiochemical purity and high specific activity. The radiolabeling yields for 64Cu were found to depend on the temperature of the reaction mixture and the time with optimal results being obtained at 100 C in 15 min. Even though previous publications reported that NOTA and DOTA are able to incorporate Cu-64 at much lower temperatures,28 we used identical labeling conditions for all BFC-BBN conjugates in order to facilitate a proper comparison of the different peptides. Higher labeling efficacies are obtained when DOTA-, NOTA-, and PCTA-BBN conjugates are labeled using 0.1 M ammonium acetate buffer pH 5.5 and 64Cu(OAc)2 than with Oxo-DO3A-BBN. No radiation damage to the BBN peptide was observed even after heating at 100 C for 150 min. In vitro and in vivo stability assays confirm that 64Cu-labeled NOTA- and PCTA-BBN conjugates remain stable over the 20 h time period. In contrast, both 64Cu/DOTA- and 64Cu/OxoDO3A-BBN have poor in vivo stability. Because the liver is one of the primary transchelation sites for Cu-64 complexes,29 we also studied the stability of the 64Cu/PCTA-BBN peptide in the liver 2 h post injection and detected 25% of free 64Cu and 75% of the intact radiolabeled peptide. It appears that the free Cu-64 remains sequestered in liver (gallbladder) and does not go back in circulation, since only the 64Cu/PCTA-BBN peptide is detected in blood after 20 h. In conclusion, our data show that 64Cu-labeled NOTA- and PCTA-BBN peptides are promising radiotracers for PET imaging. Their high binding affinity for GRP receptors and good stability in mice render them suitable as model compounds for the fine-tuning of biological properties of 64Cu-labeled BFCBBN conjugates. Further in vitro/in vivo evaluation of these tracers as potential cancer PET imaging agents is warranted.

’ AUTHOR INFORMATION Corresponding Author

*Prof. Brigitte Gu
erin, Centre d’imagerie mol
eculaire de Sherbrooke (CIMS), Department of Nuclear Medicine and Radiobiology, Universit
e de Sherbrooke, 3001, 12th North Avenue Sherbrooke, Qc, Canada, J1H 5N4. Phone: (819) 820-6868 ext. 15285; fax: (819) 829-3238. E-mail: [email protected].

’ ACKNOWLEDGMENT B.G. is a member of the FRSQ-funded Centre de recherche
tienne-Le Bel. The work was financially supported by clinique E the Natural Sciences and Engineering Research Council of Canada (NSERC), the Canadian Institute of Health Research (CIHR grant MOP-89875), and the BC Leadership Chair in Functional Cancer Imaging. Conflicts of interest: Cara L. Ferreira is employed by Nordion. Paul Jurek and Garry Kiefer are employed by Macrocyclics Inc. The other authors declare that they have no conflicts of interest. ’ REFERENCES (1) Williams, H. A., Robinson, S., Julyan, P., Zweit, J., and Hastings, D. (2005) A comparison of PET imaging characteristics of various copper radioisotopes. Eur. J. Nucl. Med. Biol. 32, 1473–1480.

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