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One-Step 18F-Labeling of Carbohydrate-Conjugated Octreotate-Derivatives Containing a Silicon-Fluoride-Acceptor (SiFA): In Vitro and in Vivo Evaluation as Tumor Imaging Agents for Positron Emission Tomography (PET) Carmen Wa¨ngler,† Beatrice Waser,‡ Andrea Alke,§ Ljuba Iovkova,| Hans-Georg Buchholz,⊥ Sabrina Niedermoser,† Klaus Jurkschat,| Christian Fottner,# Peter Bartenstein,† Ralf Schirrmacher,∇ Jean-Claude Reubi,‡ Hans-Ju¨rgen Wester,§ and Bjo¨rn Wa¨ngler* Department of Nuclear Medicine, Hospital of the Ludwig-Maximilians-University, Munich, Germany, Institute of Pathology, University of Berne, Berne, Switzerland, Department of Nuclear Medicine, Technical University Munich, Munich, Germany, Department of Inorganic Chemistry II, University of Dortmund, Dortmund, Germany, Department of Nuclear Medicine and I. Medical Clinic, University of Mainz, Germany, and McConnell Brain Imaging Centre, Montreal Neurological Institute, McGill University, Montreal, Canada. Received July 13, 2010; Revised Manuscript Received October 18, 2010
The synthesis, radiolabeling, and initial evaluation of new silicon-fluoride acceptor (SiFA) derivatized octreotate derivatives is reported. So far, the main drawback of the SiFA technology for the synthesis of PET-radiotracers is the high lipophilicity of the resulting radiopharmaceutical. Consequently, we synthesized new SiFA-octreotate analogues derivatized with Fmoc-NH-PEG-COOH, Fmoc-Asn(Ac3AcNH-β-Glc)-OH, and SiFA-aldehyde (SIFAA). The substances could be labeled in high yields (38 ( 4%) and specific activities between 29 and 56 GBq/ µmol in short synthesis times of less than 30 min (e.o.b.). The in vitro evaluation of the synthesized conjugates displayed a sst2 receptor affinity (IC50 ) 3.3 ( 0.3 nM) comparable to that of somatostatin-28. As a measure of lipophilicity of the conjugates, the log Pow was determined and found to be 0.96 for SiFA-Asn(AcNH-β-Glc)PEG-Tyr3-octreotate and 1.23 for SiFA-Asn(AcNH-β-Glc)-Tyr3-octreotate, which is considerably lower than for SiFA-Tyr3-octreotate (log Pow ) 1.59). The initial in vivo evaluation of [18F]SiFA-Asn(AcNH-β-Glc)-PEG-Tyr3octreotate revealed a significant uptake of radiotracer in the tumor tissue of AR42J tumor-bearing nude mice of 7.7% ID/g tissue weight. These results show that the high lipophilicity of the SiFA moiety can be compensated by applying hydrophilic moieties. Using this approach, a tumor-affine SiFA-containing peptide could successfully be used for receptor imaging for the first time in this proof of concept study.
INTRODUCTION Fluorine-18 ( F, t1/2 ) 109.7 min) is the most important radionuclide for imaging by means of positron emission tomography (PET), a noninvasive imaging modality which found its way into human diagnostic imaging more than 30 years ago. The introduction of 2-[18F]FDG, a 18F-labeled sugar derivative, has pushed nuclear medicine forward, and 2-[18F]FDG today accounts for the majority of human PET scans in a variety of human afflictions such as cancer and neurological diseases. Further stimulated by the introduction of PET/CT, it is very likely that the number of PET-scans will continue to rise within the next years particularly as the single-photonemission-tomography (SPECT) technology has recently suffered from radioisotope shortages and thus impaired reliability (1, 2). One apparent drawback of the PET technology is the often complicated synthesis of the respective imaging agents, a circumstance which, despite best efforts, has not yet been 18
* To whom correspondence should be addressed. Bjo¨rn Wa¨ngler: Tel: +49 89 7095 7543, fax: +49 89 7095 4648, bjoern.waengler@ med.uni-muenchen.de. † Hospital of the Ludwig-Maximilians-University. ‡ University of Berne. § Technical University Munich. | University of Dortmund. ⊥ Department of Nuclear Medicine, University of Mainz. # I. Medical Clinic, University of Mainz. ∇ McGill University.
alleviated. Especially for larger molecules, such as peptides and proteins, simple one-step labeling procedures have been evasive despite several advancements in the field (3-7). Although direct labeling via nucleophilic substitution has recently been shown to be a possible route for the 18F-labeling of some selected peptides with nonacidic side chain functionalities (8), this path lacks general applicability to more complex and temperaturesensitive peptides. A further drawback of all the described methods for the radiolabeling of peptides and proteins based on the formation of a carbon-18F bond is the necessity for intricate purification methods (e.g., HPLC) (9). Recently, our group and Ametamey et al. independently described the synthesis of sterically hindered silicon-18F building blocks used as prosthetic groups in the synthesis of radiolabeled peptides and proteins (10-13). Our method was coined SiFA chemistry (SiFA: Silicon-Fluoride-Acceptor), whose concept is based on a simple isotopic exchange reaction, yet yielding compounds of high specific activity. Single-step labeling as well as twostep labeling procedures were described, and the first true 18Fkit labeling procedure for proteins emerged from these efforts (14-16). Despite this success for protein labeling, the initial application of silicon-18F derivatized peptides for tumor imaging unambiguously revealed a pronounced lipophilicity imparted by the Si-F building blocks, leading to a high unspecific liver and low tumor uptake (17, 13). In order to circumvent this problem, we report here the application of our SiFA chemistry together with hydrophilic PEG spacers and carbohydrates in the syntheses of octreotate derivatives in order
10.1021/bc100316c 2010 American Chemical Society Published on Web 11/17/2010
2290 Bioconjugate Chem., Vol. 21, No. 12, 2010
to obtain compounds with improved properties for the in vivo imaging of somatostatin receptor positive tumors (18-20). The peptides Tyr3-octreotate as well as Tyr3-octreotide (an alcohol derivative of Tyr3-octreotate) are analogues of the peptide hormone somatostatin and have a preferentially high affinity to the somatostatin receptor subtype 2, which is highly overexpressed on the cell surface of several neuroendocrine tumors. Thus, these peptides represent ideal compounds for receptormediated tumor targeting and imaging (21-23). As of now, the radiolabeling of somatostatin analogues with 18F is complicated and time-consuming, as it necessitates a two-step procedure for (i) the synthesis of a secondary labeling precursor (which has in most cases to be purified by HPLC) and (ii) the subsequent reaction with the peptidic compound. Thus, a more simple and efficient method for the introduction of 18F into peptides is highly desirable. A promising approach is the use of the SiFA technology, as this would allow the introduction of 18F into peptides in a one-step reaction. Thus, the aim of this work was to demonstrate that (i) the introduction of hydrophilic groups in combination with the SiFA moiety into peptides yields radiofluorinated products retaining their binding affinity to the target receptor while (ii) simultaneously displaying improved in vivo behavior for PET imaging compared to radiofluorinated peptides using silicon-fluoride building blocks alone, and that furthermore (iii) one-step synthesis of these radiotracers is feasible.
EXPERIMENTAL SECTION Materials. Commercially available chemical compounds were purchased in highest purity and used without further purification. The resin for peptide synthesis (Fmoc-Thr(tBu)-Wang resin), Fmoc-protected amino acids, Fmoc-8-amino-3,6-dioxaoctanoic acid (Fmoc-NH-PEG-COOH), N-R-Fmoc-N-β-[3,4,6-tri-Oacetyl-2-(acetylamino)-deoxy-2-β-glucopyranosyl]-L-asparagine (Fmoc-Asn(Ac3AcNH-β-Glc)-OH), and bis-Boc-aminooxy acetic acid (Bis-boc-Ao-OH) were purchased from NovaBiochem (Nottingham, UK). 4-(Di-tert-butylfluorosilyl) benzaldehyde (silicon-fluoride-acceptor aldehyde, SiFA-A) (1) as well as [18F]-SiFA-Tyr3-octreotate ([18F]-2) were synthesized according to a published procedure (11). SepPak C-18 cartridges were obtained from Waters Corporation (Milford, MA, USA), Chromafix PS-HCO3 cartridges from GE-Healthcare Europe (Freiburg, Germany). Instruments. The analytical and semipreparative HPLC system used was an Agilent 1200 system equipped with a Raytest Gabi Star radioactivity detector together with an analytical Chromolith Performance column (RP-18e, 100 × 4.6 mm, Merck, Germany) at a flow rate of 4 mL/min and a semipreparative Chromolith column (RP-18e, 100 × 10 mm, Merck, Germany) at a flow rate of 8 mL/min, respectively. ESI mass spectra were obtained by using a Finnigan MAT95Q. The PET scans were performed using a Siemens Inveon 120 small animal PET system (Siemens Preclinical Imaging, Knoxville, TN, USA) or a Philips MOSAIC small animal scanner (Philips Healthcare, Eindhoven, The Netherlands). Gamma counting was performed using a Packard Canberra apparatus. Peptide Synthesis. Synthesis of Aoa-Asn(AcNH-β-Glc)-PEGTyr3-octreotate (3). Tyr3-octreotate was synthesized on solid support applying standard Fmoc solid-phase peptide synthesis as described by Wellings et al. (24) on a Fmoc-Thr(tBu)-Wang resin (standard coupling procedure: 4 equiv amino acid, 3.9 equiv HBTU, 4 equiv DIPEA in DMF; Fmoc-cleavage: 50% piperidine in DMF). After cyclization of the Tyr3-octreotate peptide sequence (Fmoc-D-Phe-Cys(Acm)-Tyr-D-Trp-Lys-ThrCys(Acm)-Thr-Wang) by incubating the resin with Tl(III)trifluoroacetate in DMF for 45 min, the terminal Fmoc protecting group was removed. Subsequently, Fmoc-NH-PEG-COOH,
Wa¨ngler et al.
Fmoc-Asn(Ac3AcNH-β-Glc)-OH, and Bis-boc-Aoa-OH were successively coupled using standard coupling conditions. The peptide was cleaved from the resin by incubation with a mixture of TFA (trifluoroacetic acid)/TIS (triisopropylsilane)/H2O (95: 2.5:2.5, 1.5 mL) for 45 min, precipitated in diethyl ether, washed twice with diethyl ether, and dried. For cleavage of the carbohydrate acetyl protecting groups, the dried crude peptide was dissolved in methanol (1 mL), and a sodium methanolate solution in methanol (0.5M) was added until the pH of the mixture reached 12-13 (approximately 0.6 mL; pH determined on moistened pH indicator strips). After 30 min reaction at room temperature, the mixture was acidified with neat TFA (approximately 50 µL) and purified by semipreparative HPLC using a gradient of 15-40% MeCN + 0.1% TFA over 5 min. The product was isolated as white solid after lyophilization (23.2 mg, 14.6 µmol, 19% yield). HR-ESI-MS (m/z) for [M+H]+ (calculated) 1584.63 (1584.64); (m/z) for [M+2H]2+ (calculated) 792.82 (792.82). Synthesis of SiFA-Asn(AcNH-β-Glc)-Tyr3-octreotate (4). The intermediate Aoa-Asn(AcNH-β-Glc)-Tyr3-octreotate was synthesized by reacting fully protected Tyr3-octreotate on resin with Fmoc-Asn(Ac3AcNH-β-Glc)-OH and Bisboc-Aoa-OH and cleaved from the resin as described before for the synthesis of 3. The deprotection of the O-acetyl protecting groups was also performed analogously to 3 by reacting the peptide for 30 min with sodium methanolate solution in methanol. When the reaction was finished, the volatile components of the mixture were evaporated, the obtained solid was dissolved in phosphate buffer (0.5 M, pH ) 4.0, 400 µL) and reacted with SiFA-A (1) dissolved in acetonitrile by adding SiFA-A in small amounts until an excess of SiFA-A could be detected by analytical HPLC (gradient of 0-100% MeCN + 0.1% TFA over 5 min) after reaction times of 5 min. The crude product was purified by semipreparative HPLC using a gradient of 10-50% MeCN + 0.1% TFA over 5 min. The product was isolated as white solid after lyophilization (5.5 mg, 3.3 µmol, 13.3%). HR-ESI-MS (m/z) for [M+H]+ (calculated): 1686.70 (1686.69). Synthesis of SiFA-Asn(AcNH-β-Glc)-PEG-Tyr3-octreotate (5). For the oxime formation, a modified protocol from that described by Poethko et al. was used (25). A solution of AoaAsn(AcNH-β-Glc)-PEG-Tyr3-octreotate (3, 10 mg, 6.3 µmol) in phosphate buffer (0.25 M, pH ) 4.0, 400 µL) was added to a solution of SiFA-A (1, 2.1 mg, 7.9 µmol, 1.25 equiv) in acetonitrile (500 µL). The pH was adjusted to 4.0 by adding phosphate buffer (0.5 M, pH ) 4.0, ∼50 µL). The mixture was incubated for 5 min at ambient temperature, and subsequently, the product was purified by semipreparative HPLC using a gradient of 40-80% MeCN + 0.1% TFA over 4 min. The product was isolated as a white solid after lyophilization (9.3 mg, 5.1 µmol, 80%). HR-ESI-MS (m/z) for [M+H]+ (calculated) 1832.02 (1832.77); (m/z) for [M+2H]2+ (calculated) 917.39 (917.88). Radiolabeling. Preparation of a Stock Solution Containing [18F]F-/Kryptofix 2.2.2/K+ Complex for the Labeling of SiFAAsn(AcNH-β-Glc)-Tyr3-octreotate (4) and SiFA-Asn(AcNH-β-Glc)PEG-Tyr3-octreotate (5). Aqueous [18F]fluoride (4000-7500 MBq) that had been produced by the 18O(p,n)18F nuclear reaction on an enriched [18O]water (95%) target was loaded onto a Chromafix PS-HCO3 cartridge and eluted with a mixture of acetonitrile (800 µL), water (200 µL), potassium oxalate solution (1 M, 10 µL), and Kryptofix 2.2.2 (12.5 mg). The solvents were removed by coevaporation to dryness under reduced pressure (650 mbar) using a stream of helium at 87 °C for 4 min. The drying step was repeated twice with CH3CN (0.8 mL) (3 min) and full vacuum (∼10 mbar) was applied in the final drying step (4 min). The dried [18F]F-/Kryptofix 2.2.2/K+ complex was
18
F-Labeling of Peptides by SIFA
dissolved in dry DMSO (500-1000 µL) and used as stock solution for labeling. Radiosynthesis of [18F]SiFA-Asn(AcNH-β-Glc)-Tyr3-octreotate ([18F]-4) and [18F]SiFA-Asn(AcNH-β-Glc)-PEG-Tyr3-octreotate ([18F]-5). The SiFA containing peptides 4 and 5 (10-25 nmol, 10-25 µL of a 1 mmol/L stock solution in dry DMSO) were added to 450-500 µL of the stock solution containing [18F]F-/ Kryptofix 2.2.2/K+ complex (2-3 GBq) in dry DMSO and reacted at ambient temperature for 5 min without stirring. Subsequently, the reaction mixture was added to HEPES buffer (0.1 M, pH 4, 9 mL) and loaded on a Waters SepPak C-18 light cartridge, previously conditioned by subsequent rinsing with ethanol (5 mL) and water for injection (10 mL). The trapped [18F]SiFA-peptides were washed with water for injection (5 mL), eluted from the cartridge with ethanol (200 µL), and diluted with isotonic saline solution (2 mL). The solution was filtered sterile for further use. The overall synthesis time was 15 min. Reverse-phase HPLC revealed radiochemical purities ranging from 92% to 96%. 18F-fluoride impurities were in all cases 1000b >10 000c >1000 (2) >1000 (2)
a
sst2 b
sst3
2.1 ( 0.3 3.1 ( 1.8b 1.3 ( 0.3c 4.4 ( 0.2 (2) 3.3 ( 0.3 (2) b
sst4
3.2 ( 0.2 330 ( 146b 128 ( 22c 756 ( 176 (2) 184 ( 66 (2) b
sst5
3.0 ( 0.3 346 ( 114b 867 ( 33c 444 ( 96 (2) 93 ( 26 (2) b
2.5 ( 0.2b 10.8 ( 1.8b 50 ( 12c 302 ( 25 (2) 126 ( 10 (2)
Values are given as mean ( SD. b Data from ref 34. c Data from ref 18.
Table 3. Tissue Distribution [ID/g] of 18F-SiFA-Asn(AcNH-β-Glc)PEG-Tyr3-Octreotate ([18F]-5) in AR42J Tumor-Bearing Mice tissue
10 min p.i.a
60 min p.i.b
blood lung liver pancreas spleen stomach large intestine small intestine kidney adrenals muscle femur tumor
13.43 ( 2.58 8.30 ( 2.96 29.55 ( 7.04 5.64 ( 1.22 3.94 ( 0.87 4.36 ( 0.85 1.59 ( 1.16 2.93 ( 0.45 6.67 ( 1.11 6.14 ( 2.46 0.89 ( 0.19 1.88 ( 1.15 3.25 ( 1.06
5.92 ( 1.52 5.82 ( 1.59 16.11 ( 3.80 11.99 ( 2.89 2.72 ( 0.48 14.46 ( 2.14 2.14 ( 1.15 6.90 ( 1.10 6.35 ( 0.98 3.92 ( 1.13 0.59 ( 0.21 3.79 ( 1.24 7.73 ( 1.90
a Group of 4 mice. b Group of 5 mice. Values are mean ( SD injected dose per gram tissue (% ID/g).
As 18F-SiFA-Asn(AcNH-β-Glc)-PEG-Tyr3-Octreotate ([18F]5) showed the most promising in vitro evaluation profile and the most favorable properties with regard to hydrophilicity, we evaluated the compound in vivo in AR42J tumor-bearing nude mice. The tissue distribution after 10 and 60 min postinjection is shown in Table 3. As expected, and despite the hydrophilic derivatization, the lipophilicity of [18F]-5 leads to a high accumulation of 30% and 16% ID/g tissue in the liver after 10 and 60 min, respectively. The radioactivity concentration in the blood decreases from ∼13.4% (10 min p.i.) to 6% (60 min p.i.), displaying a relatively high plasma half-life of the derivative.
The accumulation in pancreas, stomach, and intestines increases by the factor 2-3 over time. In Vivo Animal PET Data for a Silicon-18F Derivatized [18F]SiFA-Octreotate of High Lipophilicity Lacking PEG and/or Glucose Moieties. Initial laboratory results for the in vivo evaluation of the SiFA-derivatized octreotate derivative [18F]SiFA-Tyr3-octreotate ([18F]-2), the radiosynthesis of which was published by our group in 2007 (11) as well as the results published by Ho¨hne et al. (13) for the in vivo evaluation of an 18 F-Si derivatized bombesin derivative revealed only a negligible uptake in the tumor tissue of the corresponding mouse model (Figure 2A). These findings can most probably be attributed to the high lipophilicity of the evaluated compounds which lead to an extraordinarily high uptake of the radiolabeled peptides in the liver and gallbladder. This high lipophilicity is a result of the tert-butyl substituents of the SiFA-moiety, which are necessary to stabilize the silicon-based fluoride acceptor systems against hydrolysis. In contrast to these preliminary data found for the small animal PET study of 18F-SiFA-Tyr3-octreotate [18F]-2, we found an uptake of 7.7% ID/g of 18F-SiFA-Asn(AcNH-β-Glc)-PEGTyr3-octreotate ([18F]-5) in the tumor after 60 min p.i.. Compared to the uptake of radioiodinated octreotide and octreotate derivatives in tumor tissue, these findings are comparable to the uptake of a radioiodinated octreotide derivative already used for in vivo imaging, but display significantly lower uptake than the respective radioiodinated octreotate derivative (18, 35).
Figure 2. Small animal PET evaluation of nonmodified [18F]-2 (A) and modified [18F]-5 (B) in AR42J tumor-bearing nude mice (CD1 nu/nu). Both images show coronal slices (sum from 50 to 90 min p.i.). (A) In vivo small animal PET evaluation of 18F-SiFA-Tyr3-Octreotate ([18F]-2) in a tumor bearing nude mouse model was carried out using a Philips Mosaic small animal scanner. The site of the tumor is indicated by an arrow. (B) In vivo evaluation of 18F-SiFA-Asn(AcNH-β-Glc)-PEG-Tyr3-octreotate ([18F]-5) was carried out using a Siemens Inveon small animal PET-scanner in the same tumor model showing the uptake of the radiotracer in the tumor tissue (arrow).
18
F-Labeling of Peptides by SIFA
Though giving no optimal results, this is the first approach demonstrating the applicability of silicon-based building blocks (SiFAs) for the one-step 18F-radiolabeling of a receptor-affine peptide for PET and a successful in vivo imaging of a receptorpositive tumor (Figure 2 B). According to these encouraging findings, one part of further developments will now focus on the remaining challenge of the SiFA-approach, namely, the still intrinsically high lipophilicity deriving from the SiFA-building blocks developed so far. Even the introduction of a small PEGspacer and a carbohydrate as presented in this work could not fully compensate the high lipophilicity of the SiFA building block. In order to further reduce the lipophilicity of SiFAderivatized peptides, we intend to introduce larger sugar moieties, such as maltotriose or cellobiose (19), or charged chemical groups, such as phosphates and quarternary ammonia groups, into the peptides or to use more hydrophilic silicon building blocks stable against hydrolysis.
CONCLUSION This study demonstrates the feasibility of silicon-fluoride building blocks for the labeling of glucose and glucose/PEG derivatized octreotate derivatives. The SiFA moiety can easily be introduced via chemoselective oxime formation between the aminooxy-derivatized octreotate derivatives and the aldehyde group of SiFA-A (1). The conjugates can be radiolabeled with 18 F in a simple one-step isotopic exchange reaction at ambient temperature within 5 min, yielding radiofluorinated octreotate derivatives with reduced lipophilicity suitable for in vivo PETimaging of sst-positive tumors in mice. We further demonstrated that the PEG- and carbohydratederivatized SiFA-octreotate derivatives can be radiolabeled in high specific activities of 29-56 GBq/µmol. In contrast to other studies using Si-F building blocks, no HPLC purification was required for the isolation of 18F-SiFA-Asn(AcNH-β-Glc)-Tyr3octreotate ([18F]-4) and 18F-SiFA-Asn(AcNH-β-Glc)-Tyr3- PEGoctreotate ([18F]-5) from the reaction mixture. Although substantial chemical modifications have been made to the original octreotate, both derivatized octreotate conjugates display preserved binding affinities to the sst2 subtype. Compound [18F]5 could be used for a successful visualization of a sst2-positive tumor in vivo in an AR42J tumor mouse model with a high tumor uptake of 7% after 60 min. In conclusion, we progressed significantly toward a feasible application of the SiFA labeling technique for PET tracer development demonstrating that a reduction of the inherent high lipophilicity of the SiFA building block is the key to successful in vivo imaging.
ACKNOWLEDGMENT Parts of this work were funded from the Deutsche Forschungsgemeinschaft (DFG grant number WA 2132/3-1) to B.W. C.W. would like to thank the Fonds der Chemischen Industrie for financial support.
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