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. 2017 Feb 16;8(3):673–679. doi: 10.1039/c7md00006e

68Ga-Chelation and comparative evaluation of N,N′-bis-[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N′-diacetic acid (HBED-CC) conjugated NGR and RGD peptides as tumor targeted molecular imaging probes†

Drishty Satpati a,✉, Rohit Sharma a, Chandan Kumar a, Haladhar Dev Sarma b, Ashutosh Dash a
PMCID: PMC6071919  PMID: 30108785

graphic file with name c7md00006e-ga.jpgRadiosynthesis and bioevaluation of HBED-CC conjugated RGD and NGR peptides, 68Ga-HBED-CC-c(NGR) and 68Ga-HBED-CC-c(RGD) is described.

Abstract

Peptides containing RGD and NGR motifs display high affinity towards tumor vasculature molecular markers, integrin αvβ3 and CD13 receptors, respectively. In the present study, RGD and NGR peptides were conjugated with the novel acyclic chelator N,N′-bis-[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N′-diacetic acid (HBED-CC) for radiolabeling with 68Ga. The radiotracers [68Ga-HBED-CC-c(NGR)] and [68Ga-HBED-CC-c(RGD)] were quite hydrophilic with respective log P values being –2.8 ± 0.14 and –2.1 ± 0.17. 68Ga-HBED-CC-c(RGD) displayed a significantly higher (p < 0.05) uptake in murine melanoma B16F10 tumors as compared to 68Ga-HBED-CC-c(NGR) indicating its higher specificity towards integrin αvβ3-positive tumors. The two radiotracers showed similar uptake in CD13-positive human fibrosarcoma HT-1080 tumor xenografts (∼1.5 ± 0.2% ID g–1). The tumor uptake of the two radiotracers was significantly reduced (p < 0.05) in both animal models during blocking studies. The tumor-to-blood ratio was observed to be ∼2–2.5 for the two radiotracers, whereas the tumor-to-muscle ratio was significantly higher (p < 0.005) for 68Ga-HBED-CC-c(RGD) in the two animal models. The two radiotracers 68Ga-HBED-CC-c(NGR) and 68Ga-HBED-CC-c(RGD) exhibited renal excretion with rapid clearance from blood and other non-target organs. Thus, 68Ga-chelated HBED-CC conjugated NGR and RGD peptides expressed features conducive towards development as tumor targeted molecular imaging probes. This study further opens avenues for the successful conjugation of different peptides with the acyclic chelator HBED-CC and expansion of 68Ga-based radiopharmaceuticals.

Introduction

The new blood vessels sprouting around the tumor cells feed them oxygen and other nutrients as well as ensure ample blood supply. Angiogenic blood vessels are thus significantly involved in tumor progression and also serve as channels for dissemination of tumor cells leading to distant metastases.1–3 This newly formed tumor vasculature has altered expression of signature molecules [integrins (αvβ3/αvβ5),4 vascular endothelial growth factor receptor (VEGFR),5 platelet-derived growth factor receptor,6 and CD13/aminopeptidase N (APN, referred to as CD13) receptor7,8] distinctly different from normal blood vessels. Therefore, radiotracers targeting these specific markers predominantly expressed on tumor vasculature are attractive candidates for molecular imaging and therapy. Two such markers, integrin αvβ3 and CD13 receptors, show a high level of expression on endothelial cells of angiogenic vessels and also on tumor cells and specifically recognize arginine–glycine–aspartic acid (RGD) and asparagine–glycine–arginine (NGR) motif containing peptides, respectively.9–12 Thus, RGD and NGR peptides are considered to be potential vehicles for delivery of chemotherapeutic drugs, cytotoxic peptides, nanoparticles and radioisotopes allowing tumor-targeted molecular diagnosis and therapeutics with increased efficacy and reduced systemic toxicity.13,14 An NGR peptide conjugated anti-cancer drug, human tumor necrosis factor (hTNF), is already under clinical investigation for malignant pleural mesothelioma, colorectal, lung (small-cell and non-small-cell), liver and ovarian cancers, and soft tissue sarcomas.15

Integrin αvβ3 and CD13 receptors are expressed in solid tumors including melanoma, prostate, lung, breast and ovarian tumors and their tumor vasculature.16–19 Radiolabeled RGD and NGR peptides can therefore not only serve as classic tools for detection and staging of several tumor types and malignant sites but also for treatment response monitoring. There are numerous reports on RGD peptides radiolabeled with positron emission tomography (PET) isotopes, 18F, 64Cu, and 68Ga, as well as a single photon emission tomography (SPECT) isotope, 99mTc.20,21 However, there are few reports on 68Ga, 64Cu, and 99mTc-labeled NGR peptides.22–24 The PET isotope 68Ga has established new dimensions in the field of nuclear medicine imaging by virtue of its simpler, economic and cyclotron independent availability through commercial 68Ge/68Ga generators (>1 year shelf-life). In addition, the facile radio-chelation and amenability to kit-type production have brought 68Ga/PET a step closer to the 99mTc/SPECT paradigm. The high sensitivity, higher spatial resolution and accurate quantification offered by PET modality have further enthused the development of 68Ga-based radiotracers.25 The other advantage is the short half life of 68Ga (68 min) which allows it to be a perfect radionuclide for performing imaging studies with rapidly localizing and clearing targeting vectors (peptides and small molecules).

1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) are the most common and thoroughly studied chelators for 68Ga-complexation.26 Recently, the acyclic chelator HBED-CC (N,N′-bis-[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N′-diacetic acid) has gained attention for preparation of 68Ga-radiotracers. The higher thermodynamic stability of 68Ga-HBED-CC (log K = 38.5) complexes as compared to those of 68Ga-DOTA (log K = 21.33) and 68Ga-NOTA (log K = 30.98) complexes explicitly marks HBED-CC as an efficient chelator. Rapid radio-metallation kinetics at low concentration and ambient temperature are other attractive attributes of HBED-CC.27,28 The present study thus aims at the preparation of HBED-CC-conjugated NGR (HBED-CC-cKCNGRC) and RGD peptides (HBED-CC-cRGDfK) for 68Ga-radiolabeling. The two radiotracers, 68Ga-HBED-CC-cKCNGRC [68Ga-HBED-CC-c(NGR)] and 68Ga-HBED-CC-cRGDfK [68Ga-HBED-CC-c(RGD)], were subsequently investigated for their efficacy as tumor targeting molecular imaging probes. The radiochemical yield/purity, partition coefficient and in vitro cell uptake studies of the two radiotracers were carried out. In vivo biodistribution studies of the two radiotracers were performed in mouse xenografts with subcutaneously induced CD13-positive HT-1080 fibrosarcoma as well as in C57BL6 mice bearing melanoma tumors. The tumor targeting specificity was determined by in vivo blocking studies.

Materials and methods

Reagents, cell lines and instruments

Solvents and chemicals were purchased from Aldrich (Milwaukee, WI) unless stated otherwise and used without further purification. 9-Fluorenylmethoxycarbonyl (Fmoc)-protected amino acids, coupling reagents and Novasyn TGR resin were purchased from NovaBiochem, (Germany). The peptide c(RGDfK) and the bifunctional chelator HBED-CC-tris(tBu)ester were purchased from ABX advanced biochemical compounds (Biomedizinische Forschungsreagenzien GmbH, Radeberg, Germany). A 30% solution of Suprapur® hydrochloric acid (HCl) and sodium acetate was purchased from Fluka Analytical (Steinheim, Germany). The B16F10 murine melanoma and HT1080 human fibrosarcoma cell lines were obtained from the National Center for Cell Sciences (NCCS), Pune, India. Female nude mice were purchased from Vivo Bio Tech Ltd. (India). Matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry analysis was carried out at the Mass spectrometry facility of Tata Memorial Centre, Advanced Centre for Treatment, Research and Education in Cancer (ACTREC). High-performance liquid chromatography (HPLC) grade water was obtained from Merck. All other solvents and chemicals were purchased from Sigma Aldrich, USA. All radioactive counting associated with the radiochemical studies were carried out using a well-type NaI(Tl) scintillation gamma counter (Electronic Corporation of India Limited, India). Purification of peptides was carried out using a semi-preparative HPLC system (JASCO, Japan) connected to a JASCO-PU-2086 Plus, an intelligent prep pump, and a JASCO UV-2075 Plus absorption detector and having a Megapak Sil C18-10 column (7.5 × 250 mm). The analytical HPLC measurements were performed using a JASCO PU 2080 Plus dual pump HPLC system, Japan, with a JASCO 2075 Plus tunable absorption detector and a Gina Star radiometric detector system, using a C18 reversed phase HiQ Sil column (5 μm, 4 × 250 mm). The eluting solvents (1 mL min–1) used in HPLC were H2O (solvent A) and acetonitrile (solvent B) with 0.1% trifluoroacetic acid following the gradient: 0–28 min: 90% A–10% A; 28–30 min: 10% A; 30–32 min: 10% A–90% A.

Synthesis of the HBED-CC-c(KCNGRC) peptide [HBED-CC-c(NGR)]

The fully protected peptide H-Lys(Boc)-Cys(Trt)-Asn(Trt)-Gly-Arg(Pbf)-Cys(Trt)-NH2 was assembled on Novasyn TGR resin manually by standard Fmoc solid phase peptide synthesis. Coupling of each amino acid was carried out using a standard Fmoc strategy where a 3-fold excess of amino acid as well as O-(7-azabenzotriazol-l-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HATU) was used along with a 6-fold excess of N,N-diisopropylethylamine (DIPEA) in dimethylformamide (DMF) for 90 min. The reaction was monitored by the picrylsulphonic acid test, and the Fmoc groups were removed by treatment with 20% piperidine in DMF for 30 min. Subsequently, on-resin cyclization of cysteine sulphides was carried out with 1.2 eq. of thallium(iii) trifluoroacetate in DMF. The chelator HBED-CC-tris(t-Bu)ester (2 eq.) was then coupled to the N-terminus of the peptide in the presence of HATU and DIPEA. Subsequently, the resin was treated with a cocktail mixture of trifluoroacetic acid (TFA)/tri-isopropylsilane (TIPS) (99 : 1, v/v) for cleavage of the peptide and deprotection of side chain groups. The crude peptide was precipitated and washed three times with diethyl ether. The crude peptide was then purified by semi-preparative HPLC and lyophilized to obtain white fluffy powder. MALDI-TOF MS (1): m/z 1191.98 observed (calcd for C50H74N14O16S2: 1191.3).

Synthesis of HBED-CC-c(RGDfK) [HBED-CC-c(RGD)]

The chelator HBED-CC-tris(t-Bu)ester (5 μmol) and the peptide c(RGDfK) (5.5 μmol) were dissolved in DMF (100 μL), and DIPEA (40 μL) was added to the mixture. The reaction was stirred for 24 h at room temperature followed by purification by semi-preparative HPLC. MALDI-TOF MS (2): m/z 1232.9 [M + CF3COOH] observed (calcd for C53H71N11O16: 1118.2).

68Ga-Labeling of HBED-CC-c(NGR) and HBED-CC-c(RGD) {[68Ga-HBED-CC-c(NGR)] and [68Ga-HBED-CC-c(RGD)]}

For 68Ga-labeling of peptides, 68GaCl3 (222 MBq, 6 mCi) was eluted from an Eckert & Ziegler 68Ge/68Ga generator in 2 mL of 0.1 M HCl. Pre-concentration of 68GaCl3 was performed using a cation-exchange cartridge where the purified 68GaCl3 was eluted in a 500 μL mixture of acetone and HCl (97.6%/0.02 M).

For radiolabeling, purified 68Ga eluate (0.5 mL) was added to sodium acetate buffer (0.5 mL, 1.5 M, pH 4) along with the peptide (20 μg). The reaction was incubated at 90 °C for 5 minutes. Radiochemical yield and purity were determined by paper chromatography and HPLC.

Lipophilicity studies

Radiotracers (50 μL, ∼1.8 MBq) were mixed with distilled water (950 μL) and n-octanol (1 mL) in a vortex mixer for about 1 min and then centrifuged (3500g) for 5 min to allow clear separation of the two layers. The radioactivity in 100 μL samples of both the aqueous and the organic phases was measured using a γ-counter. The experiment was repeated thrice to calculate the log Po/w of radiotracers.

Cell uptake studies

B16F10 murine melanoma cells and HT-1080 human fibrosarcoma cells were maintained in Minimum Essential Medium (MEM) supplemented with 10% fetal calf serum (Invitrogen, Carlsbad, CA) and 1% antibiotic/antimycotic formulation in a humidified atmosphere of 5% CO2 at 37 °C. For cell uptake studies, B16F10 cells or HT-1080 cells (1 × 105) were seeded in 24-well tissue culture plates and incubated at 37 °C overnight. The cells were subsequently incubated with 68Ga-HBED-CC-c(NGR) or 68Ga-HBED-CC-c(RGD) (37 kBq per well), at 37 °C for 1 h. After incubation, cells were washed twice with ice-cold phosphate buffer saline (PBS) and the cells were harvested by trypsinization. At the end of trypsinization, wells were examined using a light microscope to ensure complete detachment of cells. Cell suspensions were collected and the radioactivity associated with the cells was measured using a NaI (Tl) gamma counter. The activity in these cell suspensions as percentage of total input radioactivity was calculated to determine the cell uptake data. The experiment was carried out in triplicate. Specific uptake was determined by pre-incubation of cells with a 100-fold excess of the peptide [c(KCNGRC)/c(RGDfK)]. Percentage inhibition values were calculated as [{difference between the cell binding of the radiotracer in the absence and in the presence of the cold peptide/cell binding in the absence of the peptide} × 100].

Biodistribution studies

Biodistribution studies of 68Ga-HBED-CC-c(NGR) and 68Ga-HBED-CC-c(RGD) were carried out in C57BL6 mice bearing melanoma tumors and in athymic nude mice bearing HT-1080 tumors. The radiotracers (150 μL, 3.7 MBq) were intravenously injected into the tail vein of each mice (n = 4). Studies were carried out at 60 min post injection (p.i.). The radioactivity associated with each tissue was counted using a NaI(Tl) flat geometry detector. The results are expressed as percentage of injected dose per gram [% ID g–1, mean ± standard deviation (SD), n = 4/radiotracer]. All the animal experiments were carried out in compliance with the relevant national laws and institutional guidelines, as approved by the Institutional Animal Ethics Committee (IAEC) on the conduct and ethics of animal experimentation.

Statistical analysis using the paired two-tailed student's t test was performed to compare values between 68Ga-HBED-CC-c(NGR) and 68Ga-HBED-CC-c(RGD) animal groups at 1 h p.i.; values of p < 0.05 were considered statistically significant.

Results and discussion

Chemistry, radiochemistry and in vitro stability

The peptide HBED-CC-cKCNGRC was synthesized in ∼15% yield and >98% purity. The peptide HBED-CC-cRGDfK could be prepared in 70% yield, and the purity of the conjugated peptide was >98%.

68Ga-Labeling of HBED-CC-c(NGR) and HBED-CC-c(RGD) could be achieved within 5 minutes of incubation at 90 °C. The structures of 68Ga-HBED-CC-c(NGR) and 68Ga-HBED-CC-c(RGD) are presented in Fig. 1. The two radiotracers were prepared in >98% radiochemical yield and purity and with a specific radioactivity of >10 GBq μmol–1 (270 mCi μmol–1). Radiochemical yield was determined by paper chromatography carried out using acetonitrile/water (1 : 1, v/v) as the mobile phase. The Rf value of the 68Ga-radiotracers was 0.9, whereas an Rf value of 0 was observed for 68GaCl3. The radiochemical purity of the radiotracers was analyzed by radio-HPLC chromatograms. The retention times of 68Ga-HBED-CC, 68Ga-HBED-CC-c(NGR) and 68Ga-HBED-CC-c(RGD) were 9.8, 10.5 and 12.2 min, respectively (Fig. 2A and B). The radiotracers were used for in vitro and in vivo studies without any further purification.

Fig. 1. Schematic representation of the structures of 68Ga-HBED-CC-cNGR and 68Ga-HBED-CC-cRGD.

Fig. 1

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Fig. 2. HPLC radiochromatograms of (A) 68Ga-HBED-CC and 68Ga-HBED-CC-cNGR and (B) 68Ga-HBED-CC and 68Ga-HBED-CC-cRGD.

Fig. 2

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The partition coefficients (log P) of radiotracers 68Ga-HBED-CC-c(NGR) and 68Ga-HBED-CC-c(RGD) were determined to be –2.8 ± 0.14 and –2.1 ± 0.17, respectively, suggesting that the two radiotracers have a nearly similar hydrophilic character. The in vitro stability of the radiotracers was determined 3 h after incubation at 37 °C by analytical radio-HPLC. No change in the radio-HPLC chromatogram was observed for the two radiotracers and the radiochemical purity was found to be >98%. These results suggest sufficient in vitro stability of the radiotracers for carrying out any further studies.

In vitro cell uptake studies

Cell uptake studies were performed in HT-1080 and B16F10 cells for both radiotracers. The studies revealed a significantly higher cell uptake of 68Ga-HBED-CC-c(RGD) (5.02 ± 0.73%; p = 0.022) in comparison to that of 68Ga-HBED-CC-c(NGR) (3.32 ± 0.6%) in B16F10 murine melanoma cells. On pre-incubation of cells with the cKCNGRC peptide, the cell uptake of 68Ga-HBED-CC-c(NGR) was reduced to 2.64 ± 0.17% (p = 0.10), whereas that of 68Ga-HBED-CC-c(RGD) was significantly reduced (1.49 ± 0.24%; p = 0.001) on incubation with the cRGDfK peptide. The cell uptake of 68Ga-HBED-CC-c(RGD) and 68Ga-HBED-CC-c(NGR) in HT-1080 cells was observed to be 1.34 ± 0.3% and 1.23 ± 0.6%, respectively, with the corresponding inhibition values being 0.77 ± 0.4% and 0.96 ± 0.3%.

Biodistribution studies

The results of the biodistribution studies of 68Ga-HBED-CC-c(NGR) and 68Ga-HBED-CC-c(RGD) in C57BL6 mice bearing melanoma tumors and in nude mice bearing HT-1080 tumors are summarized in Fig. 3.

Fig. 3. Biodistribution studies of 68Ga-HBED-CC-cNGR and 68Ga-HBED-CC-cRGD in (A) C57BL6 mice bearing B16F10 murine melanoma tumors and (B) nude mice bearing human fibrosarcoma HT-1080 tumors at 1 h p.i. The error bars represent standard deviation.

Fig. 3

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The uptake value of 68Ga-HBED-CC-c(RGD) in B16F10 tumors (1.6 ± 0.1% ID g–1) was significantly higher (p = 0.01) than that of 68Ga-HBED-CC-c(NGR) (1.1 ± 0.2% ID g–1). However, no significant difference (p > 0.05) in the HT-1080 tumor uptake of 68Ga-HBED-CC-c(NGR) (1.5 ± 0.1% ID g–1) and 68Ga-HBED-CC-c(RGD) (1.4 ± 0.2% ID g–1) was observed. The tumor-to-blood (T/B) ratio of the two radiotracers was nearly similar and remained unchanged in both animal models (Fig. 4A). However, the tumor-to-muscle (T/M) ratio was observed to be significantly higher for 68Ga-HBED-CC-c(RGD) in comparison to that of 68Ga-HBED-CC-c(NGR) in mice bearing B16F10 tumors (5.3 : 1 vs. 2.7 : 1, p < 0.005) as well in mice bearing HT-1080 tumors (7 : 1 vs. 5 : 1, p < 0.005) (Fig. 4B).

Fig. 4. Comparison of the (A) tumor/blood ratio and (B) tumor/muscle ratio of 68Ga-HBED-CC-cNGR and 68Ga-HBED-CC-cRGD in mice bearing B16F10 tumors and HT-1080 tumor bearing xenografts. The error bars represent standard deviation. *p < 0.05 and **p < 0.005.

Fig. 4

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The renal pathway was the preferred route of excretion for the two radiotracers. The blocking studies carried out by co-injection of either 200 μg of cKCNGRC or cRGDfK significantly reduced the uptake of 68Ga-HBED-CC-c(NGR) (p < 0.05) and 68Ga-HBED-CC-c(RGD) (p < 0.005) in B16F10 tumors. A significantly reduced (p < 0.005) accumulation of the two radiotracers was also observed in HT-1080 tumors during blocking studies (Fig. 5).

Fig. 5. Uptake of 68Ga-HBED-CC-cNGR and 68Ga-HBED-CC-cRGD in B16F10 and HT-1080 tumors with and without blocking. The error bars represent standard deviation. **p < 0.005.

Fig. 5

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The fact that angiogenesis plays a key role in tumor growth and metastasis has stimulated the design and development of tumor vasculature targeted radiotracers for molecular imaging.29,30 The angiogenic markers, integrin αvβ3 and aminopeptidase N (APN/CD13) receptors, are highly expressed on tumor vascular endothelial cells in several solid tumors and mediate tumor cell migration by interacting with signature RGD and NGR peptide motifs, respectively, in extracellular matrix proteins.31 NGR and RGD peptide based PET/SPECT radiotracers are thus under investigation for CD13/αvβ3-targeted molecular imaging. These molecular imaging probes offer an elegant approach towards rapid lesion detection, selection of patients for targeted therapy, monitoring the disease progression and therapeutic response and thus are precious tools for cancer management. 68Ga-labeled DOTA/NOTA conjugated NGR/RGD peptides have been reported for in vivo imaging of CD13/integrin αvβ3 receptor-positive tumors.22,32–34 However, the high kinetic stability of Ga-HBED-CC complexes and rapid reaction kinetics owing to the acyclic structure of HBED-CC prompted us to evaluate 68Ga-HBED-CC-c(NGR) and 68Ga-HBED-CC-(cRGD) as molecular probes for tumor targeted imaging.

In this direction, the cKCNGRC peptide was synthesized manually using the Fmoc strategy and the disulphide bond between two cysteines was formed by on-resin cyclization. The C-terminal amide was synthesized, and the N-terminal amine group was conjugated with the chelator HBED-CC; the resulting peptide-conjugate HBED-CC-c(NGR) was then radiolabeled with 68Ga. The corresponding RGD conjugate, HBED-CC-c(RGD), was synthesized in solution phase using a commercially available cRGDfK peptide.

The in vitro studies were carried out in integrin αvβ3-positive murine melanoma B16F10 tumor cells and CD13-positive human fibrosarcoma HT-1080 tumor cells. The cell uptake of 68Ga-HBED-CC-c(NGR) in HT-1080 cells (1.23 ± 0.6%) was similar to that reported for 68Ga-NOTA-G3-NGR (1.10% ± 0.10% at 1 h incubation)32 and slightly better than 64Cu-DOTA-NGR (0.78 ± 0.04% at 1 h incubation).23 The radiotracer 68Ga-HBED-CC-c(NGR) demonstrated ∼20% reduction in cell uptake in B16F10/HT-1080 cells on blocking with the cKCNGRC peptide.

In vivo studies revealed that the renal pathway is the main route of excretion for 68Ga-HBED-CC-c(NGR) as was also expected from the high hydrophilicity determined by the low partition coefficient value. The uptake in other organs (blood, heart, lungs, spleen, intestines, liver) was similar to that reported for 68Ga-NOTA-G3-NGR.32 However, 68Ga-NOTA-G3-NGR had shown higher retention in HT-1080 tumors (4.96% ± 3.18% ID g–1 at 1 h p.i.). A better tumor uptake was also reported for 64Cu-DOTA-NGR (2.35 ± 0.17% ID g–1 at 24 h p.i.) in comparison to that observed for 68Ga-HBED-CC-c(NGR) (1.5 ± 0.1% ID g–1 at 1 h p.i.). The use of a different chelator and the absence of a spacer (three glycine units) in the 68Ga-HBED-CC-c(NGR) radiotracer may be the plausible reason for the difference in the tumor retention. 68Ga-HBED-CC-c(NGR) had lower kidney retention which can be attributed to higher hydrophilicity as compared to 68Ga-NOTA-G3-NGR (log P = –2.8 ± 0.14 and –2.25 ± 0.17, respectively) and/or the uni-negative charge of 68Ga-HBED-CC-c(NGR) as a result of chelation with two carboxylates and two phenolates in comparison to the neutral three carboxylate chelated 68Ga-NOTA-G3-NGR complex. The drawback of 64Cu-DOTA-NGR was high liver uptake (4.14 ± 0.26% ID g–1 at 24 h p.i.) due to the lower kinetic stability of the 64Cu-DOTA chelate.

The significantly higher (p < 0.05) uptake of 68Ga-HBED-CC-c(RGD) in B16F10 tumors demonstrates its higher selectivity for integrin αvβ3-positive tumors as compared to 68Ga-HBED-CC-c(NGR). Knetsch et al. investigated 68Ga-NODAGA-RGD and 68Ga-DOTA-RGD in human melanoma M21 tumor xenografts.33 The tumor uptake of 68Ga-DOTA-RGD was nearly twice that of 68Ga-HBED-CC-c(RGD)/68Ga-NODAGA-RGD, but it was accompanied by higher uptake in all the other non-target organs. Retention in kidneys (4.2 ± 0.1% ID g–1) and intestine (1.7 ± 0.6% ID g–1) was observed to be higher for 68Ga-HBED-CC-c(RGD) in comparison to 68Ga-NODAGA-RGD (<1.5% ID g–1 and <1% ID g–1, respectively) which may be the result of slower excretion. The tumor uptake of 68Ga-NODAGA-RGD (∼1.4% ID g–1) was similar to that of 68Ga-HBED-CC-c(RGD), but a higher accumulation in the spleen (>1% ID g–1) and liver (>1.5% ID g–1) was observed for 68Ga-NODAGA-RGD.

The two radiotracers exhibited a promising pharmacokinetic profile with rapid clearance from the non-target organs. Although the tumor models were different, 68Ga-HBED-CC-c(RGD) exhibited a biodistribution pattern quite comparable to that of 68Ga-NODAGA-RGD.

Conclusions

In this study, HBED-CC conjugated NGR and RGD peptides were synthesized and radiolabeled with 68Ga in high radiochemical purity. The efficacy of the two radiotracers was studied in murine melanoma and human fibrosarcoma tumor models. 68Ga-HBED-CC-c(RGD) displayed a higher specificity in integrin αvβ3-positive B16F10 cells and a higher uptake in B16F10 tumors in comparison to 68Ga-HBED-CC-c(NGR). The two radiotracers exhibited a similar uptake in HT-1080 tumor xenografts. 68Ga-HBED-CC-c(NGR) can be further modified and optimized by introduction of spacer units. 68Ga-HBED-CC-c(RGD) compared favorably well with the reported radiotracer. The favorable pharmacokinetics demonstrated by the two radiotracers asserts their potential for development as tumor targeted molecular imaging probes. This work further highlights the tremendous potential of the acyclic chelator HBED-CC for conjugation with different peptides and their development as tumor targeting radiotracers.

Acknowledgments

The authors are thankful to Dr. B. S. Tomar, Director, Radiochemistry and Isotope Group, Bhabha Atomic Research Centre (BARC) for his support.

Footnotes

†The authors declare no competing interests.

References

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