68Ga Small Peptide Imaging: Comparison of NOTA and PCTA

Oct 5, 2012 - Alexander Schmidtke , Tilman Läppchen , Christian Weinmann , Lorenz .... Benjamin P. Burke , Gonçalo S. Clemente , Stephen J. Archibal...
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68

Ga Small Peptide Imaging: Comparison of NOTA and PCTA

Cara L. Ferreira,*,† Donald T. T. Yapp,*,‡ Derek Mandel,† Rajanvir K. Gill,‡ Eszter Boros,§,∥ May Q. Wong,‡ Paul Jurek,⊥ and Garry E. Kiefer⊥ †

Nordion, 4004 Wesbrook Mall, Vancouver, British Columbia, Canada, V6T 2A3 Department of Experimental Therapeutics, BC Cancer Agency, Vancouver, British Columbia, Canada § TRIUMF, Vancouver, British Columbia, Canada ∥ Chemistry Department, University of British Columbia, Vancouver, British Columbia, Canada ⊥ Macrocyclics, Dallas, Texas, United States ‡

ABSTRACT: In this study, a bifunctional version of the chelate PCTA was compared to the analogous NOTA derivative for peptide conjugation, 68Ga radiolabeling, and small peptide imaging. Both p-SCN-Bn-PCTA and p-SCN-BnNOTA were conjugated to cyclo-RGDyK. The resulting conjugates, PCTA-RGD and NOTA-RGD, retained their affinity for the peptide target, the αvβ3 receptor. Both PCTA-RGD and NOTA-RGD could be radiolabeled with 68Ga in >95% radiochemical yield (RCY) at room temperature within 5 min. For PCTA-RGD, higher effective specific activities, up to 55 MBq/nmol, could be achieved in 95% RCY with gentle heating at 40 °C. The 68Ga-radiolabeled conjugates were >90% stable in serum and in the presence of excess apotransferrin over 4 h; 68Ga-PCTA-RGD did have slightly lower stability than 68Ga-NOTA-RGD, 93 ± 2% compared to 98 ± 1%, at the 4 h time point. Finally, the tumor and nontarget organ uptake and clearance of 68Ga-radiolabeled PCTA-RGD and NOTARGD was compared in mice bearing HT-29 colorectal tumor xenografts. Activity cleared quickly from the blood and muscle tissue with >90% and >70% of the initial activity cleared within the first 40 min, respectively. The majority of activity was observed in the kidney, liver, and tumor tissue. The observed tumor uptake was specific with up to 75% of the tumor uptake blocked when the mice were preinjected with 160 nmol (100 μg) of unlabeled peptide. Uptake observed in the blocked tumors was not significantly different than the background activity observed in muscle tissue. The only significant difference between the two 68Ga-radiolabeled bioconjugates in vivo was the kidney uptake. 68Ga-radiolabeled PCTA-RGD had significantly lower (p < 0.05) kidney uptake (1.1 ± 0.5%) at 2 h postinjection compared to 68Ga-radiolabeled NOTA-RGD (2.7 ± 1.3%). Overall, 68Garadiolabeled PCTA-RGD and NOTA-RGD performed similarly, but the lower kidney uptake for 68Ga-radiolabeled PCTA-RGD may be advantageous in some imaging applications.

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INTRODUCTION The high-resolution nuclear imaging technique positron emission tomography (PET) has been growing in clinical use over the past decade. Complementary interest in the generatorproduced positron-emitting radionuclide 68Ga has also been on the rise in recent years. 68Ga can be acquired from a parent/ daughter 68Ge/68Ga generator, much like 99mTc, the mainstay for single photon emission computed tomography (SPECT) imaging, is acquired from a 99Mo/99mTc generator. In contrast, the majority of positron-emitting nuclides used clinically are cyclotron-produced. For example, 18F, a widely used PET nuclide, has a short 110 min half-life and requires local production of the isotope within hours of use. The availability of an appropriate generator-produced radionuclide such as 68Ga could further facilitate the growth of PET centers in areas that are remote from cyclotron facilities.1 68 Ga has a short half-life of only 68 min, which makes it ideal for imaging small molecules and peptides that clear quickly from the background tissue. The short half-life of the isotope necessitates fast radiolabeling chemistry, preferably delivering >95% radiochemical yield in high specific activity to obviate © 2012 American Chemical Society

time-consuming purification processes, in order to minimize significant loss of the imaging agent to decay. The ability to obtain the final radiolabeled compound in high effective specific activity (activity per total moles of both labeled and unlabeled compound) is particularly important for low/medium density receptors where the excess unlabeled compound will compete for binding sites effectively reducing target uptake.2 The ability to radiolabel efficiently at room temperature in buffered aqueous solutions would be ideal because small peptides and molecules can be sensitive to harsh conditions, such as high temperature and low pH. Stability, especially kinetic stability of the radiolabeled agent in the presence of ubiquitous in vivo proteins with an affinity for Ga, such as the iron-transport protein transferrin, is important over a period of several hours. High thermodynamic and long-term stabilities are less relevant, again due to short half-life and early imaging time frame of 68 Ga-based PET agents. Received: June 28, 2012 Revised: September 25, 2012 Published: October 5, 2012 2239

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Figure 1. Structures of bifunctional chelates applicable to Ga radioisotopes.

HBED have been reported to be stable to the loss of 68Ga in the presence of serum proteins. Previously, we reported the evaluation of two novel backbone-functionalized chelates, p-NO2-Bn-PCTA and pNO2-Bn-Oxo-DO3A, for use with 68Ga.14 The chelates were directly compared to analogous derivatives of DOTA and NOTA with respect to radiochemistry, in vitro stability, and biodistribution properties. PCTA was found to have superior radiolabeling efficiency compared to DOTA. PCTA required shorter reaction times and lower reaction temperatures to obtain the same radiochemical yields and effective specific activities as DOTA when all other reaction conditions were identical. As well, when challenged with human apo-transferrin, DOTA was shown to lose 68Ga to the protein, in contrast PCTA and NOTA were shown to be impervious to transchelation by the protein. We concluded that PCTA may be a useful BFC for the development of 68 Ga-based radiopharmaceuticals. In this study, we investigated the utility of p-SCN-Bn-PCTA for 68Ga radiolabeling and imaging of small peptides, specifically cyclo-RGDyK. Small peptides containing the RGD motif have been widely investigated for PET imaging of angiogenesis, a hallmark of cancer and potential indicator of metastatic potential. 68 Ga radiolabeling and imaging using RGD containing peptides has been reported with a multitude of chelates,8,15 and the radiolabeling and imaging properties have been shown to be dependent on the choice of chelate. Thus, we chose to utilize conjugates of cyclo-RGDyK to scrutinize the 68 Ga small peptide labeling and imaging properties of PCTA in direct comparison with NOTA.

Bifunctional chelates (BFCs; Figure 1) are used to facilitate the 68Ga radiolabeling of biologically relevant targeting vectors, such as small molecules and peptides. 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), a chelate utilized for coordination of a myriad of d- and f-block metals, was one of the first chelates used for 68Ga radiolabeling. Somatostatintargeted conjugates of DOTA, such as DOTATOC, DOTANOC, and DOTATATE, radiolabeled with 68Ga are now prepared in clinics throughout the world for imaging of neuroendocrine tumors. However, radiolabeling DOTA with 68 Ga requires high temperatures to obtain high radiochemical yields in a reasonable reaction time.3,4 Moreover, DOTA and some DOTA-containing conjugates have also been shown to release 68Ga via transchelation by proteins due to the similar size and charge of Ga3+ and Fe3+;5 iron sequestering and iron transporting proteins are most culpable.6 With the increasing interest in 68Ga, there has been a rising amount of research on novel BFCs for 68Ga. The smaller macrocyclic core of 1,4,7triazacyclononane-1,4,7-triacetic acid (NOTA) has been shown to be more efficiently radiolabeled at lower temperatures with 68 Ga and to yield a radiolabeled agent with superior in vivo stability.7 68Ga is predisposed to form six-coordinate complexes, so while DOTA, which has eight potential coordinating groups, has a “spare” carboxylate available for conjugation, NOTA, which only has six coordinating groups, compromises stability if a carboxylate is used for conjugation. Derivatives of the NOTA chelator such as NOTA-Bn-SCN, NODAGA, and TRAP allow for facile conjugation of biological targeting vectors without compromising one of the six coordinating groups.8−11 The TRAP chelator has also been reported to achieve exceptionally high specific activities, over 700 MBq/nmol with radiochemical yields >95%.11 The acyclic chelates Dedpa and HBED have been reported to be radiolabeled at room temperature with 68 Ga with even higher efficiency, producing higher radiochemical yields in shorter reaction times using lower concentrations of BFC compared to NOTA.12,13 Despite the predicted lower stability of acyclic chelates over macrocyclic chelates, attributed to the increased rigidity and smaller loss of entropy upon coordination of the latter, both Dedpa and

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EXPERIMENTAL SECTION

Materials. All solvents and reagents were used as received unless otherwise noted. The BFCs, p-SCN-Bn-NOTA, and pSCN-Bn-PCTA were acquired from Macrocyclics Inc. (Dallas, TX). 125I-Echistatin was purchased from PerkinElmer (Boston, MA). 67Ga was obtained as a dilute HCl solution (Nordion, Vancouver, Canada). 68Ga was obtained from a generator consisting of 68Ge on a titanium dioxide sorbent and eluted with 1 or 5 mL of 0.1 N HCl. Cyclo-(RGDyK) was purchased 2240

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Tris, pH 7.4, 150 mM NaCl, 2 mM CaCl2, 1 mM MgCl2, 1 mM MnCl2, 0.1% bovine serum albumin). HT-29 human colon adenocarcinoma cell lines were obtained from the ATCC (Manassas, VA, USA) and were certified Mycoplasma negative. Cells were cultured in McCoy’s 5A (StemCell Technologies; Vancouver, CA), supplemented with 1% L-glutamine, 1% penicillin/streptomycin, and 10% FBS. Plated flasks were kept in a humidified incubator (5% CO2, 37 °C) and passaged at ∼80−90% confluence. Tissue culture flasks, tubes, and plates (BD Falcon) were obtained from BD Sciences (Bedford, MA). Prior to use, cells were detached from the surface of the tissue culture flask by treatment with 0.25% Trypsin/EDTA (Sigma). In Vitro Studies. The method for determining the binding affinity of each of the bioconjugates has been reported previously.8,16 Briefly, filter plates (Multiscreen DV plates, 96 well, 0.65 μm pore size, Millipore, Ireland B.V.) were seeded with αvβ3 positive U87MG glioblastoma cells, 105 cells per well, and incubated with 125I-Echistatin (20 000 cpm) along with increasing concentrations (0 to 2 mM) of one of the bioconjugates. After incubating for two hours, unbound 125IEchistatin was removed with several washes, the filters were collected and the activity measured (γ counter, Cobra-II Auto Gamma, Canberra Packard Canada) to determine the amount of bound 125I-Echistatin. In Vivo Studies. Animal studies were conducted with the full approval of the University of British Columbia’s Animal Care Committee, operating under the auspices of the Canadian Animal Care Committee. HT29 tumors were grown subcutaneously on Rag2M (disrupted recombination activating gene) mice as reported previously;17 briefly, 5 × 106 cells (50 uL) were injected subcutaneously on the lower back of Rag2M mice. Once the tumors reached ∼100−150 mm3, the mice were anesthetized with isoflurane (5% induction, 3% maintenance) and injected intravenously with the 68Ga radiolabeled cyclo(RGDyK) conjugates of NOTA or PCTA (∼7−11 MBq, 18.5 GBq/μmol). Groups (n = 4−5) of mice were injected with one of the three radiolabeled bioconjugates and were imaged for 110 min postinjection, framed as 5 × 2 min, 6 × 5 min, 5 × 10 min, and 2 × 15 min. Imaging was carried out in the Siemens Inveon multimodality CT-PET small animal scanner.18 Images were reconstructed in 3D using OSEM-MAP3D algorithms supplied by Siemens following CT-based attenuation scans to correct for the animal’s body mass. 3D regions of interest (ROIs) were placed on the heart (to approximate the activity present in the blood), kidney, liver, muscle (lower leg), and tumor in reconstructed images of the imaged mice. Only ROIs greater than 75 mm3 were used in the analysis to minimize partial volume effects. The activity present in each ROI was determined in Bq/mL based on a calibration scan done with a phantom of known activity to determine camera efficiency and sensitivity. Two hours after injection, mice were euthanized. The blood, liver, kidney, muscle, and tumor tissue were harvested, weighed, and placed in a scintillation counter to determine the activity present per gram of tissue. Statistical Analysis. Data from the animal studies for each of the radiolabeled conjugates, both the blocked and nonblocked groups, were compared using ANOVA analysis for comparing means. P-values ≤0.05 were considered statistically significant. The %ID/g calculated from the biodistribution at 2 h and determined from the ROI analysis at 110 min were compared using a Pearson correlation coefficient.

from Peptides International (Louisville, KY). Water was deionized using a Milli-Q biocel A-10 water purification system. Conjugation Chemistry. The bioconjugates were prepared by combining cyclo-(RGDyK) and a slight excess (1.1 equiv) of each of the BFCs in pH 9, 50 mM HEPES buffer. The solutions were left at room temperature overnight. The resulting conjugates were purified by HPLC with a Waters XBridge BEH130 4.6 × 150 mm analytical column using a gradient of 5% water/95% ethanol to 100% ethanol over 30 min at 1 mL/ min. The bioconjugates were characterized by mass spectroscopy and HPLC. Mass spectroscopy was done using positiveion mode electrospray ionization with a Micromass Q-TOF Ultima API Time-of-Flight mass spectrometer by Mr. Andras Szeitz (University of British Columbia, Department of Pharmaceutical Sciences). A calibrated VWR symphony pH meter was used to adjust the pH of all buffer solutions. Radiochemistry. Initial radiolabeling and stability studies were conducted using the longer-lived gallium radioisotope 67 Ga. The radioisotope (37−185 MBq) was added to the conjugate (1−100 nmol) dissolved in 1 mL of 10 mM sodium acetate buffer pH 4.5 and then incubated at room temperature or in a heated water bath or in a Biotage Initiator 2.5 microwave reactor for a predetermined amount of time ranging 5−30 min. 68 Ga radiolabeling was accomplished similarly. The radioisotope (37−740 MBq) was added to the conjugate (1−50 nmol) dissolved in 1 mL of 200 mM sodium acetate buffer pH 4.5. Alternatively, sodium acetate (10.2 mg) was added to the conjugate (1−50 nmol) in a minimal amount of water followed by 1 mL of the generator eluent containing the radioisotope (185−740 MBq) yielding a reaction solution with a pH of ∼4.5. The radiolabeling reactions were then incubated at room temperature or heated in a water bath or in a Biotage Initiator 2.5 microwave reactor for a predetermined period of time. The radiochemical yield and purity were determined by HPLC analysis using a Waters Alliance HT 2795 separation module equipped with a Raytest Gabi star NaI detector and a Waters 996 photodiode array (PDA) detector. The HPLC method used a Waters XBridge BEH130 RPC18 4.6 × 150 mm analytical column eluting with a binary solvent system; solvent A = acetonitrile, solvent B = 0.05% trifluoroacetic acid in water, gradient elution 5% A/95% B to 100% A over 30 min at 1 mL/ min. The guard column was monitored after each run to confirm no colloids or impurities remained on the column. In Vitro Stability. The stability of the radiolabeled conjugates was examined by challenging each radiolabeled conjugate with human apo-transferrin. Each bioconjugate was first radiolabeled with 67Ga in >95% radiochemical purity. Aliquots of the radiolabeled conjugates containing ∼37 MBq of radioisotope were added to a 3 mg/mL solution of apotransferrin in 10 mM NaHCO3 buffer at pH 6.9. The challenge samples were incubated in a hot water bath at 37 °C for different periods of time and then analyzed using the HPLC method described above or using size-exclusion PD-10 columns (GE) eluting with PBS (pH 7.2). Cell Lines. U87MG human glioblastoma cells were maintained in DMEM supplemented with 2 mM L-glutamine (Invitrogen, Vancouver, BC) and 10% fetal bovine serum (FBS) (Invitrogen, United States) under 95% air and 5% CO2 at 37 °C. Prior to use, the cells were washed twice with PBS, detached from the surface of the tissue culture flask by treatment with 0.25% Trypsin/EDTA (Sigma), and then resuspended (1 × 106 cells/mL) in binding buffer (20 mM 2241

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RESULTS Bioconjugate Preparation and Characterization. Both of the BFCs were conjugated to cyclo-(RGDyK) by coupling the lysine amine of the peptide with the isothiocyanate functionality of the BFC, as reported previously.8 Characterization information for the bioconjugates (Figure 2) can be

found in Table 1. The identity of the conjugates was confirmed by mass spectroscopy, in which peaks of both the singly charged parent and the dication were observed. The purity of the bioconjugates was determined by HPLC and was >90% in all cases. Finally, the affinity of the bioconjugates for the αvβ3 integrin receptors was examined by competitive binding assay with 125I-echistatin, a known high-affinity ligand for αvβ3. Both of the bioconjugates produced concentration-dependent inhibition of 125I-echistatin and the determined IC50 values for PCTA-RGD (1.8 ± 0.6 μM) and NOTA-RGD (1.0 ± 0.3 μM) were not statistically different from one another. Radiochemistry. The radiochemistry was initially evaluated using the longer-lived radioisotope 67Ga. Once the optimized reaction parameters were determined using 67Ga, the same parameters were used with 68Ga. Previously, it was determined that the optimal pH conditions for efficient Ga radiolabeling of these BFCs is between pH 4 and pH 5, although PCTA and NOTA were less sensitive to changes in pH than DOTA.14 So, all reactions were done by adding the radioisotope to the bioconjugate dissolved in a pH 4.5 sodium acetate solution at a concentration with an appropriate buffering capacity. Comparison of the reaction conditions and radiochemical yields are given in Table 2. Similar labeling efficiency was observed for both Ga radioisotopes. NOTA-RGD was labeled most efficiently, achieving >95% RCY in 5 min. PCTA-RGD could also achieve >95% RCY in 5 min, but required slightly elevated temperatures. The majority of 68Ga-radiolabeled RGD conjugates, including the NOTA-RGD conjugate used in this study, have been previously reported with an effective specific activity of ∼18 MBq/nmol achievable with optimized reaction conditions.3,8,9,15,19,20 In this study, higher effective specific activities, up to 55 MBq/nmol, were achievable for both the NOTA-RGD and the PCTA-RGD systems radiolabeling at room temperature. Stability. To estimate the potential in vivo stability of each of the bioconjugates, challenge experiments were undertaken where each of the radiolabeled bioconjugates was incubated with apo-transferrin. The amount of the Ga-radiolabeled bioconjugate that remained intact and not associated with the protein at various time points was measured with size exclusion chromatography. There was no difference in the measured stability of the two complexes within the first 2 h of incubation. By the 4 h time point, Ga-radiolabeled PCTA-RGD and NOTA-RGD had only lost 7 ± 2% and 2 ± 1% to transferrin, respectively, which was significantly different (p = 0.03). In Vivo Studies. The in vivo properties of both of the 68Garadiolabeled RGD conjugates were compared in a human colorectal tumor model (HT-29), that has been shown to have a high vascular density and high levels of αvβ3 expression

Figure 2. Bioconjugate structures.

Table 1. Characterization of the Bioconjugates (MS, HPLC, binding affinity) mass spectroscopy

bioconjugate

calculated

observed

PCTA-RGD

1147.2

NOTA-RGD

1070.2

1147.0 [M+], 574.7 [M+2]2+ 1070.0 [M+], 535.7 [M+2]2+

HPLCa retention time

IC50 μmol/L

12.8 min

1.8 ± 0.6

13.4 min

1.0 ± 0.3

Waters XBridge BEH130 RPC18 4.6 × 150 mm analytical column gradient elution 5% A/95% B to 100% A over 30 min at 1 mL/min; solvent A = acetonitrile, solvent B = 0.05% trifluoroacetic acid in water. a

Table 2. Optimized Reaction Conditions (Temperature and Time), Product HPLC Retention Times, and Radiochemical Yields (%RCY) for 67Ga and 68Ga Labeling of PCTA and NOTA Conjugates of cyclo-RGDyK bioconjugate

temp.

reaction time

PCTA-RGD PCTA-RGD PCTA-RGD NOTA-RGD NOTA-RGD

Ambient Ambient 40 °C Ambient Ambient

30 min 5 min 5 min 5 min 5 min

% RCY with 67Ga 95.9 94.9 95.8 99 99.8

± ± ± ± ±

0.8 0.8 0.5 1 0.5

% RCY with 68Ga 96.2 95.2 97.1 99 98.9

± ± ± ± ±

0.5 0.8 0.4 1 0.3

specific activity MBq/nmol 55 17.5 55 18 17.5

± ± ± ± ±

1 0.4 0.4 2 0.4

Waters XBridge BEH130 RPC18 4.6 × 150 mm analytical column gradient elution 5% A/95% B to 100% A over 30 min at 1 mL/min; solvent A = acetonitrile, solvent B = 0.05% trifluoroacetic acid in water. a

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filtration rates for animals under isoflurane anesthesia combined with the larger amount of peptide injected.

attributed to tumor neovasculature.21 Mice were inoculated with HT-29 human colon cancer cells, and the tumors were allowed to grow to ∼150−200 mm3. Immunohistochemical staining of the cells and excised tumors confirmed the absence of αvβ3 expression on the HT-29 cells, but the presence of αvβ3 expression on the activated endothelial cells present in tumor. Groups of 3−5 mice with HT-29 xenografts were injected with one of the 68Ga BFC-RGD conjugates and imaged over 110 min. At 120 min postinjection, the animals were sacrificed and the %ID/g present in select tissues was determined. Results from the imaging and biodistribution studies are summarized in Table 3 and Figures 3 and 4. The imaging results at 112 min

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DISCUSSION Radiochemistry. It has been widely reported that DOTA conjugated peptides require higher temperatures and sometimes longer reaction times to obtain high (>95%) radiochemical yields at reasonable effective specific activities.3,9,19 Although this limitation has not stopped 68Ga-based agents such as 68Ga-DOTATOC from moving into the clinic, more efficient Ga radiochemistry would better facilitate the translation of 68Ga-based radiopharmaceuticals into the clinic. Room temperature radiolabeling in an aqueous buffer appropriate for injection would be ideal, as it would be applicable to most biological targeting vectors, including temperature sensitive peptides. It would also facilitate the preparation of simple kit formulations analogous to the 99mTc-based radiopharmaceutical kits that are widely used in hospital radiopharmacy settings today. The RGD bioconjugate of PCTA was shown to have favorable radiolabeling efficiency. In comparison to Ga radiolabeling of reported RGD bioconjugates of DOTA, which require high temperatures and longer reaction times,3,9,19 PCTA-RGD could be radiolabeled at room temperature in 30 min or in shorter reaction times and/or higher effective SA at 40 °C. Previous studies using NOTARGD or other RGD conjugates of bifunctional NOTA derivatives have reported elevated temperatures and longer reaction times for radiolabeling.8,15 More recent studies have reported high radiochemical yields with short reaction times and room temperature conditions,9,10,20 in agreement with our results for optimized NOTA-RGD 68Ga radiolabeling. Both PCTA-RGD and NOTA-RGD are applicable to room temperature kit type radiolabeling in high RCY at acceptable SA and both were found to be stable under physiological conditions. In Vivo Studies. The objective of the studies was to compare the in vivo properties of the Ga-radiolabeled RGD conjugates prepared from either p-SCN-Bn-PCTA or p-SCNBn-NOTA. Both systems were effective at specifically imaging αvβ3 in HT-29 tumors, which is expressed primarily on the neovasculature in this tumor model. Overall, Ga-radiolabeled PCTA-RGD and NOTA-RGD were similar in their tissue uptake and clearance patterns in tumor bearing mice, with the exception of minor differences in kidney uptake and clearance. Previously, comparative biodistribution studies of Ga-radiolabeled analogous nonconjugated bifunctional chelates p-NO2Bn-PCTA and p-NO2-Bn-NOTA in healthy mice showed substantially higher kidney uptake for Ga-radiolabeled p-NO2Bn-NOTA. The same trend was observed in this study with Garadiolabeled NOTA-RGD having significantly higher kidney uptake than the Ga-radiolabeled PCTA-RGD. The lower kidney uptake and the higher tumor to kidney ratio for Garadiolabeled PCTA-RGD may be advantageous, especially for delineating tumors in the abdominal area. Numerous other publications have investigated different chelates for peptide imaging with 68Ga, several using the RGD motif containing peptides. Previous reports of the mouse biodistribution in nontarget organs for Ga-radiolabeled NOTARGD were consistent with the results reported here.15,20 Our results and previously published results with Ga-radiolabeled NOTA-RGD are also consistent with the biodistribution measurements of Ga-radiolabeled NODAGA-RGD, another system containing the NOTA chelate.9,10 In contrast, other

Table 3. %ID/g and Tumor to Tissue Ratios for Selected Organs 2 h Post-Injection (7−11 MBq) in Rag2M Mice with Subcutaneous HT29 Tumors organ blood tumor kidney liver muscle tumor to blood tumor to kidneys tumor to liver tumor to muscle a

Ga-NOTARGD %ID/g 0.23 1.5 2.7 1.1 0.11

± ± ± ± ±

0.07 1.1 1.3* 0.5 0.09

Ga-NOTARGD blockeda %ID/g 0.5 0.5 5.3 1.5 0.5

± 0.1 ± 0.2 ± 1.5 ± 0.6 ± 0.2 ratios

Ga-PCTARGD %ID/g 0.29 1.3 1.1 0.8 0.15

± ± ± ± ±

0.09 0.7 0.5* 0.5 0.10

6.1 ± 2.2

4.1 ± 1.1

0.7 ± 0.4*

1.5 ± 0.4*

1.9 ± 0.7

1.7 ± 0.8

9.9 ± 5.4

11 ± 7.5

Ga-PCTARGD blockeda %ID/g 0.5 0.3 8.3 0.9 0.4

± ± ± ± ±

0.1 0.2 3.1 0.5 0.2

Blocked injected with 160 nmol (100 μg) of cyclo-(RGDyK). Statistically different p < 0.05.

*

and the biodistribution results at 120 min were highly correlated; Pearson correlation coefficients of 0.97 and 0.90 were determined for Ga-PCTA-RGD and Ga-NOTA-RGD, respectively. Time activity curves in Figure 3 show that both of the radiolabeled bioconjugates cleared from nontarget tissues. Higher uptake was observed in the kidney and liver, but cleared with time. Uptake in the tumor cleared slower than in other tissues, resulting in increasing tumor-to-background ratios over the course of the imaging experiment. Tumor uptake was significantly lower (p < 0.05), reduced by approximately 67− 75%, when αvβ3 receptor sites were blocked with excess (160 nmol) of cyclo-RGDyK peptide. Statistical analysis was done to determine if any of the differences between the radiolabeled bioconjugates observed in the biodistribution study were significant based on a 95% confidence level. The only tissue that showed a statistically different uptake was the kidneys. The %ID/g at 2 h in the kidneys for Ga-PCTA-RGD was 1.1 ± 0.5% which was significantly (p < 0.05) lower compared to Ga-NOTA-RGD at 2.7 ± 1.3%. Tumor-to-background ratios were not found to be significantly different for the two bioconjugates, with the exception of the tumor-to-kidney ratio, which was double at 1.5 ± 0.5% for Ga-PCTA-RGD compared to 0.7 ± 0.4% for GaNOTA-RGD. Interestingly, αvβ3 blocked animals that were administered excess cyclo-RGDyK, showed much higher kidney uptake after 2 h, but this is likely attributable to slower renal 2243

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Figure 3. Time activity (%ID/g) curves from regions of interest analysis of PET images over 112 min for NOTA-RGD (n = 4−5). Note the different %ID/g axis ranges.

68

Ga-radiolabeled PCTA-RGD and

Figure 4. Images of HT-29 xenograft Rag2M Mice anesthetized with isoflurane 120 min after injection with 68Ga-radiolabeled NOTA-RGD and 68 Ga-radiolabeled PCTA-RGD. From left to right: transverse, coronal, and sagittal views for each radiolabeled bioconjugate, respectively. Yellow arrows indicate the location of the tumors.

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chelate systems that have been used to radiolabel RGD peptides with 68Ga, including RGD conjugates of DOTA,3,9,10 SarAr,22 and dedpa,23 have been reported to have higher background uptake than reported for NOTA or NODAGA and compared to what is reported here for PCTA.

CONCLUSION

The bifunctional chelate p-SCN-Bn-PCTA was compared to the analogous NOTA BFC for small peptide imaging. Both pSCN-Bn-PCTA and p-SCN-Bn-NOTA were conjugated to 2244

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cyclo-RGDyK for targeting the αvβ3 integrin. The resulting conjugates, PCTA-RGD and NOTA-RGD, allowed for facile room temperature radiolabeling generating the 68Ga-radiolabeled products in high RCY and effective specific activity. The radiolabeling conditions are amenable to simple kit-type radiopharmaceutical preparation, optimal for translation to a radiopharmacy setting. Although lower stability was observed after 4 h in the presence of apo-transferrin for 68Ga-PCTARGD compared to 68Ga-NOTA-RGD, no difference in stability was observed during the 2 h window that was optimal for imaging. With the exception of kidney uptake, the in vivo uptake and clearance of 68Ga-radiolabeled PCTA-RGD and NOTA-RGD were not statistically different; both demonstrated specific uptake in tumors and cleared quickly from background tissue such as blood and muscle. 68Ga-radiolabeled PCTA-RGD did have slightly lower average kidney uptake and consequentially somewhat better tumor to kidney ratio than 68Garadiolabeled NOTA-RGD, which may be useful for imaging applications in the vicinity of the kidneys. The efficient radiochemistry, kinetic stability, and positive in vivo properties of the bifunctional chelate p-SCN-PCTA make it a valuable addition to the BFCs available for radiopharmaceutical development with 68Ga. The potential of p-SCN-PCTA could be further improved by modifying the conjugation strategy, as has been investigated with the NOTA chelate.9,10

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(7) Velikyan, I., Maecke, H., and Langstrom, B. (2008) Convenient preparation of Ga-68 based PET-radiopharmaceuticals at room temperature. Bioconjugate Chem. 19, 569−573. (8) Jeong, J. M., Hong, M. K., Chang, Y. S., Lee, Y.-S., Kim, Y.-J., Cheon, G. J., Lee, D. S., Chung, J.-K., and Lee, M. C. (2008) Preparation of a Promising Angiogenesis PET Imaging Agent: 68GaLabeled c(RGDyK)-Isothiocyanatobenzyl- 1,4,7-triazacyclononane1,4,7-Triacetic Acid and Feasibility Studies in Mice. J. Nucl. Med. 49, 830−836. (9) Dumont, R. A., Deininger, F., Haubner, R., Maecke, H., Weber, W. A., and Fani, M. (2011) Novel 64Cu- and 68Ga-labeled RGD conjugates show improved PET imaging of αvβ3 integrin expression and facile radiosynthesis. J. Nucl. Med. 52, 1276−1284. (10) Knetsch, P. A., Petrik, M., Griessinger, C. M., Rangger, C., Fani, M., Kesenhemer, C., von Guggenberg, E., Pichler, B. J., Virgolini, I., Decristoforo, C., and Haubner, R. (2011) [68Ga]NODAGA-RGD for imaging αvβ3 integrin expression. Eur. J. Nucl. Med. Mol. Imaging 38, 1303−1312. (11) Notni, J., Simecek, J., Hermann, P., and Wester, H.-J. (2011) TRAP, a powerful and verstaile framework for Gallium-68 radiopharmaceuticals. Chem.Eur. J. 17, 14718−14722. (12) Eder, M., Krivoshein, A. V., Backer, M., Backer, J. M., Haberkorn, U., and Eisenhut, M. (2010) ScVEGF-PEG-HBED-CC and scVEGF-PET-NOTA conjugates: comparison of easy-to-label recombinant proteins for [68Ga]PET imaging of VEGF receptors in angiogenic vasculature. Nucl. Med. Biol. 37, 405−412. (13) Boros, E., Ferreira, C. L., Cawthray, J. F., Price, E. W., Patrick, B. O., Wester, D. W., Adam, M. J., and Orvig, C. (2010) Acyclic chelate with ideal properties of 68Ga PET imaging agent elaboration. J. Am. Chem. Soc. 132, 15726−15733. (14) Ferreira, C. L., Lamsa, E., Woods, M., Duan, Y., Fernando, P., Bensimon, C., Kordos, M., Guenther, K., Jurek, P., and Kiefer, G. E. (2010) Evaluation of bifunctional chelates for development of gallium based radiopharmaceuticals. Bioconjugate Chem. 21, 531−536. (15) Li, Z.-B., Chen, K., and Chen, X. (2008) 68Ga-labled multimeric RGD peptides for microPET imaging of integrin αvβ3 expression. Eur. J. Nucl. Med. Mol. Imaging 35, 1100−1108. (16) Shi, J., Kim, Y.-S., Zhai, S., Liu, Z., Chen, X., and Liu, S. (2009) Improving tumor uptake and pharmcokinetics of 64Cu-labeled cyclic RGD peptide dimers with Gly3 and PEG4 linkers. Bioconjugate Chem. 20, 750−759. (17) Baker, J. H. E., Lam, J., Kyle, A. H., Sy, J. T., Oliver, T., Co, S., Dragowska, W. H., Ramsay, E., Anatha, M., Ruth, T., Adam, M., Yung, A., Kozloxski, P., Minchinton, A. I., Ng, S. S. W., Bally, M. B., and Yapp, D. T. T. (2008) Irinophore CTM, a novel nano-formulation of irinotecan, alters tumor vascular function and enhances the distribution 5-FU and doxorubicin. Clin. Cancer Res. 14, 7260−7271. (18) Wieholt, C., Hsiao, I., Lin, K., Chung, Y., Chen, C., and Yen, T. (2008) Performance evaluation of small animal PET system using NEMA standards. J. Nucl. Med. 49, 119P. (19) Haukkala, J., Laitinen, I., Luoto, P., Iveson, P., Wilson, I., Karlsen, H., Cuthbertson, A., Laine, J., Leppänen, P., Ylä-Herttula, S., Knuuti, J., and Roivainen, A. (2009) 68Ga-DOTA-RGD peptide: biodistribution and binding into atherosclerotic plaques in mice. Eur. J. Nucl. Med. Mol. Imaging 36, 2058−2067. (20) Singh, A. N., Liu, W., Hao, G., Kumar, A., Gupta, A., Oz, O. K., Hsieh, J.-T., and Sun, X. (2011) Multivalent bifunctional chelator scaffolds for Gallium-68 based positron emission tomography imaging probe design: signal amplication via multivalency. Bioconjugate Chem. 22, 1650−1662. (21) Zhou, Y., Kim, Y.-S., Chakraborty, S., Shi, J., Gao, H., and Liu, S. (2011) 99mTc-Labeled cyclic RGD peptides for noninvasive monitoring of tumor integrin αvβ3 expression. Mol. Imaging 10, 386−397. (22) Ma, M. T., Neels, O. C., Denoyer, D., Roselt, P., Karas, J. A., Scanlon, D. B., White, J. M., Hicks, R. J., and Donnelly, P. S. (2011) Gallium-68 complex of a macrobicyclic cage amine chelator tethered to two integrin-targeting peptides for diagnostic tumor imaging. Bioconjugate Chem. 22, 2093−2103.

AUTHOR INFORMATION

Corresponding Author

*E-mail [email protected], Tel +1 604 228 8952, Fax +1 604 228 5990 (C.F.); E-mail [email protected], Tel +1 604 675 8023, Fax +1 604 675 8183 (D.Y.). Notes

The authors declare the following competing financial interest(s): Cara Ferreira is currently employed by Nordion. Paul Jurek and Garry Kiefer are currently employed by Macrocyclics. No other authors have any competing interests to disclose.

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ACKNOWLEDGMENTS The authors wish to thank the Natural Sciences and Engineering Research Council (NSERC) for an industrial undergraduate research award for Derek Mandel.

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REFERENCES

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dx.doi.org/10.1021/bc300348d | Bioconjugate Chem. 2012, 23, 2239−2246

Bioconjugate Chemistry

Article

(23) Boros, E., Ferreira, C. L., Yapp, D. T., Gill, R. K., Price, E. W., Adam, M. J., and Orvig, C. (2012) RGD conjugates of the H2dedpa scaffold: synthesis, labeling and imaging with 68Ga. Nucl. Med. Biol. 39, 785−794.

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dx.doi.org/10.1021/bc300348d | Bioconjugate Chem. 2012, 23, 2239−2246