Synthesis and Evaluation of Glycosylated Octreotate Analogues

Dec 23, 2005 - ... substantially lower than that of I-Gluc-TOCA suggesting other factors such as net charge and overall geometry of the peptide may be...
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Bioconjugate Chem. 2006, 17, 195−203

195

Synthesis and Evaluation of Glycosylated Octreotate Analogues Labeled with Radioiodine and 211At via a Tin Precursor G. Vaidyanathan,*,† D. J. Affleck,† M. Schottelius,‡ H. Wester,‡ H. S. Friedman,§ and M. R. Zalutsky† Departments of Radiology and Neurooncology, Duke University Medical Center, Durham, North Carolina 27710 and Technical University of Munich, Munich, Germany. Received August 19, 2005; Revised Manuscript Received October 18, 2005

Carbohydration of N-terminus and substitution of a threonine for the threoninol residue at the C-terminus of Tyr3-octreotide (TOC) has resulted in improved pharmacokinetics and tumor targeting of its radioiodinated derivatives. Yet, these peptides are very susceptible to in vivo deiodination due to the similarity of monoiodotyrosine (MIT) to thyroid hormone. The goal of this work was to develop octreotate analogues containing both a sugar moiety and a nontyrosine prosthetic group on which a radioiodine or 211At can be introduced. Solid-phase synthesis and subsequent modifications delivered an iodo standard of the target peptide NR-(1-deoxy-D-fructosyl)-N-(3iodobenzoyl)-Lys0-octreotate (GIBLO) and the corresponding tin precursor NR-(1-deoxy-D-fructosyl)-N-[(3-trin-butylstannyl)benzoyl]-Lys0-octreotate (GTBLO). GIBLO displaced [125I]TOC from somatostatin receptor subtype 2 (SSTR2)-positive AR42J rat pancreatic tumor cell membranes with an IC50 of 0.46 ( 0.05 nM suggesting that GIBLO retained affinity to SSTR2. GTBLO was radiohalogenated to [131I]GIBLO and NR-(1-deoxy-D-fructosyl)N-(3-[211At]astatobenzoyl)-Lys0-octreotate ([211At]GABLO) in 21.2 ( 4.9% and 46.8 ( 9.5% radiochemical yields, respectively. From a paired-label internalization assay using D341 Med medulloblastoma cells, the maximum specific internalized radioactivity from [131I]GIBLO was 1.78 ( 0.8% of input dose compared to 9.67 ( 0.43% for NR-(1-deoxy-D-fructosyl)-[125I]iodo-Tyr3-octreotate ([125I]I-Gluc-TOCA). Over a 4 h period, the extent of internalization of [131I]GIBLO and [211At]GABLO was similar in this cell line. In D341 Med murine subcutaneous xenografts, the uptake of [125I]I-Gluc-TOCA at 0.5, 1 and 4 h was 21.5 ( 4.0% ID/g, 18.8 ( 7.7% ID/g, and 0.9 ( 0.4% ID/g, respectively. In comparison, these values for [131I]GIBLO were 6.9 ( 1.2% ID/g, 4.7 ( 1.4% ID/g, and 0.8 ( 0.5% ID/g. Both in vitro and in vivo catabolism studies did not suggest the severance of the lys0 along with its appendages from the peptide. Taken together, although GIBLO maintained affinity to SSTR2, its tumor uptake both in vitro and in vivo was substantially lower than that of I-Gluc-TOCA suggesting other factors such as net charge and overall geometry of the peptide may be important.

INTRODUCTION The expression of high levels of somatostatin receptors (SSTR1) on a variety of tumors has provided the impetus for the development of radiolabeled octreotide derivatives, which target SSTR, and can be utilized for imaging and treating these cancers (1-3). Among the radionuclides investigated for these purposes are those of iodine, which have a range of physical characteristics suited for a variety of imaging (SPECT, PET) and therapy (β-particle, Auger electron) approaches. Direct radioiodination of peptides is achieved by substitution on * Correspondence to Ganesan Vaidyanathan, Ph.D., Research Professor, Department of Radiology, Duke University Medical Center, Durham, NC 27710. Phone: (919) 684-7811. Fax: (919) 684-7122. E-mail: [email protected]. † Department of Radiology, Duke University Medical Center. ‡ Technical University of Munich. § Department of Neurooncology, Duke University Medical Center. 1 Abbreviations (uncommon) used: [211At]GABLO, NR-(1-deoxyD-fructosyl)-N-(3-[211At]astatobenzoyl)-Lys0-octreotate; Dde, 1-(4,4dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl; GIBLO, NR-(1-deoxy-Dfructosyl)-N-(3-iodobenzoyl)-Lys0-octreotate; Gluc-Lys0-Lys5(Dde)TOCA, NR-(1-deoxy-D-fructosyl)-Lys0-Lys5(Dde)-TOCA; Gluc-TOCA, NR-(1-deoxy-D-fructosyl)-Tyr3-octreotate; GMIBO, N-(4-guanidinomethyl-3-iodobenzoyl)-Phe1-octreotate; GTBLO, NR-(1-deoxy-D-fructosyl)-N-(3-tri-n-butylstannyl-benzoyl)-Lys0-octreotate; I-Gluc-Lys0TOCA, NR-(1-deoxy-D-fructosyl)-Lys0-TOCA; I-Gluc-TOCA, NR-(1deoxy-D-fructosyl)-iodo-Tyr3-octreotate; MIT, monoiodotyrosine or 3-iodotyrosine; SIB, N-succinimidyl 3-iodobenzoate; SSTR, somatostatin receptor; STB, N-succinimidyl 3-(tri-n-butyl)stannylbenzoate; TOC, Tyr3-octreotide; TOCA, Tyr3-octreotate.

constituent tyrosine residues; the absence of tyrosines in octreotide led to the development of Tyr3-octreotide (TOC) (4). Although TOC can be readily labeled, it is not a useful radiopharmaceutical because it exhibits only marginal tumor targeting, due in part to the fact that it undergoes rapid deiodination. A number of TOC structural modifications have been investigated with the goal of providing a labeled peptide with greater clinical potential. These include N-terminal sugar conjugation and Thr-for-Thr(ol) exchange (octreotide f octreotate), both of which have been shown to improve the intracellular retention of radioiodine in vitro as well as tumorto-normal tissue ratios in mice bearing SSTR-expressing xenografts (5, 6). Synergistic enhancements in tumor targeting were achieved when both tactics were combined, exemplified by the results obtained with NR-(1-deoxy-D-fructosyl)-[131I]ITyr3-octreotate ([131I]I-Gluc-TOCA). For example, 1 h after injection, tumor uptake of [131I]I-Gluc-TOCA was about 5 times higher than that of coadministered [125I]TOC (7). An important consideration in peptide radiohalogenation is whether the procedure utilized for radioiodination can also be adapted for use with 211At. Astatine-211 is a radiohalogen that emits R-particles, a type of radiation with many potential advantages for targeted radiotherapy, notably a higher cytotoxicity compared to β-particles (8). Furthermore, its half-life of 7.2 h is well matched to peptide pharmacokinetics. Even though peptides such as Gluc-TOCA offer many advantages compared with TOC, they still undergo radiohalogenation by direct substitution on Tyr3. With radioiodine, this feature results in

10.1021/bc0502560 CCC: $33.50 © 2006 American Chemical Society Published on Web 12/23/2005

196 Bioconjugate Chem., Vol. 17, No. 1, 2006 Chart 1. Chemical Structures of Octreotide, I-Gluc-TOCA, I-Gluc-Lys0-TOCA, and GIBLO

extensive deiodination in vivo, presumably due to recognition by endogenous deiodinases; with 211At, the known instability of astatotyrosine even under in vitro conditions excludes direct radioastatination as a viable labeling strategy (9, 10). To circumvent these problems, we have developed derivatives of octreotide and octreotate in which the radiohalogen resides in a prosthetic group attached to the N-terminus of the peptide (11, 12). Radioiodinated and 211At-labeled peptides could be synthesized in reasonable yield and were shown to be resistant to dehalogenation. Moreover, the second generation peptide, N-(4-guanidinomethyl-3-iodobenzoyl)-Phe1-octreotate (GMIBO) had a significantly higher intracellular accumulation in vitro compared with [125I]I-Gluc-TOCA (12). Unfortunately, normal organ levels, particularly in the liver and kidney, of [131I]GMIBO were several-fold higher than those of [125I]I-Gluc-TOCA, consistent with previous results demonstrating the ameliorative effects of N-terminal sugar conjugation on normal tissue uptake. The goal of this study was to investigate the strategy of synthesizing an SSTR-avid peptide combining the three structural elements noted above: N-terminal sugar conjugation, Thrfor-Thr(ol) exchange, and a prosthetic group carrying the radiohalogen. Glucose was selected as the sugar, and as the prosthetic group, the halobenzoyl template was utilized (Chart 1). Because two functional groups are needed to attach these two subunits, an extra lysine was introduced at the N-terminus of octreotate. The synthesis, radiohalogenation, and evaluation of the radiohalogenated peptides are described herein.

EXPERIMENTAL PROCEDURES General. All chemicals were purchased from Sigma-Aldrich unless otherwise noted. Sodium [125I]iodide and sodium [131I]iodide with specific activities of 2200 Ci/mmol and 1200 Ci/ mmol, respectively, were obtained from Perkin-Elmer Life and Analytical Sciences (Boston, MA). The 211At activity was

Vaidyanathan et al.

produced on the Duke University Medical Center CS-30 cyclotron via the 209Bi(R, 2n)211At reaction by bombarding natural bismuth metal targets with 28 MeV R-particles (13), and the radioactivity was isolated from the target by dry distillation and extracted into chloroform. Unlabeled N-succinimidyl 3-iodobenzoate (SIB) as well as N-succinimidyl 3-(trin-butyl)stannylbenzoate (STB) were synthesized as reported (14). Octreotide (Sandostatin) was purchased as an aqueous solution (0.5 mg/mL) from the Duke University Medical Center Pharmacy (Durham, NC). TCP (trityl chloride polystyrene) resin was purchased from PepChem (Tubingen, Germany). Appropriately protected amino acids were obtained from Novabiochem (Bad Soden, Germany). Solid-phase peptide syntheses were carried out manually using a flask shaker (St. Johns Associates, Inc., Beltsville, MD). Three HPLC systems were used. For radiochemistry, either of two Beckman systems (System Gold) was used. System 1 was equipped with a Model 126 programmable solvent module, a Model 168 diode array detector, and a Model 170 radioisotope detector, which was interfaced using a Model 406 analog interface module. System 2 was equipped with a Model 126 programmable solvent module, a Model 166 NM variable wavelength detector, a Model 170 radioisotope detector, and a Beckman System Gold remote interface module SS420X; data were acquired using 32 Karat software. For nonradioactive and preparative work, a Waters Model Delta 600 semi-prep system with a Model 600 controller and a Model 2487 dual wavelength absorbance detector were used; data were acquired using Millenium software. Waters XTerra C18 columns of dimensions 4.6 × 250 mm (5 µm) and 19 × 150 mm (7 µm) were used for analytical and semipreparative chromatography, respectively. Mass spectra were obtained on either a JEOL SX-102 highresolution mass spectrometer (FAB), an Applied Biosystems Voyager DE Pro (MALDI), or an Agilent 1100 LC/MSD Trap SL (electrospray). Amino acid analysis was performed at the Protein Chemistry Lab, University of North Carolina (Chapel Hill, NC). Peptide Syntheses. Gluc-TOCA. As reported previously (7), Tyr3-octreotate, with its Lys5, -amino function protected with a Dde group, was prepared by solid-phase synthesis. The peptide was cleaved from the resin and cyclized. The N-terminal amino group was glycosylated via a Maillard reaction and the Dde group was subsequently removed. NR-(1-Deoxy-D-fructosyl)-N-(3-iodobenzoyl)-Lys0-octreotate (GIBLO). The peptide sequence Boc-Lys(Fmoc)-DPhe-Cys(Trt)-Phe-DTrp(Boc)-Lys(Dde)-Thr(tBu)-Cys(Trt) (Scheme 1) was assembled on resin-bound Thr(tBu) by manual solid-phase synthesis using the standard Fmoc protocol. The peptide was cleaved from the resin, cyclized, and glycosylated as reported previously for similar peptides (2, 5, 6). Then, the Fmoc protecting group on the Lys0 side chain was removed using 20% piperidine in DMF and purified using preparative reversed-phase HPLC. To the above peptide (3.5-4 mg) in a 1 mL Reacti-vial was added a solution of SIB (5 mg) in 0.2 mL of 1% TEA in DMF. The homogeneous reaction mixture was left at room temperature under argon for 18 h. The progress of reaction was followed by analytical HPLC using the following gradient: 0.1% (w/v) TFA in water (solvent A) and 0.1% (w/v) TFA in acetonitrile (solvent B) at a flow rate of 1 mL/min, with %B increased from 30 to 60 over 30 min and then to 90 over another 10 min and then held at 90 for 5 min. The retention times of the starting peptide, SIB, and the SIB-modified peptide were 17.0, 26.8, and 24.9 min, respectively. The SIB modified peptide was precipitated by the addition of a large excess of ether, isolated by centrifugation, and washed several times with ether. MS (MALDI) m/z: Calcd for C78H101IN12O20S2: 1717.7. Found: 1717.6 (M+), 817.8, 556.5. The above peptide was taken

Radiohalogenated Glycosylated Octreotate Derivatives Scheme 1. Synthesis of GIBLO and Its Tin Precursora

a Reagents and conditions: (a) i. 95:2.5:2.5 (v/v/v/) TFA:water: triisopropylsilane, ii. H2O2, THF, 0.05 M NH4OAc; (b) i. D-glucose, HOAc/MeOH, ii. 20% piperidine in DMF; (c) i. N-succinimidyl 3-iodobenzoate or N-succinimidyl 3-(tri-n-butylstannyl)benzoate, DIEA/ NMP, ii. 1:1 ethanolamine:EtOH.

up in 2.5 mL of ethanol, and 0.5 mL of ethanolamine was added. The reaction was determined to be complete in 1 h by HPLC (tR ) 16.9 min for the final product). The product was isolated by semipreparative HPLC using the same solvents and gradient described above but at a flow rate of 10 mL/min and using the semipreparative column. Solvents were evaporated from the HPLC fractions, and the peptide was redissolved in ethanol. The concentration of this solution was determined by amino acid analysis. MS (MALDI) m/z: Calcd for C68H89IN12O18S2: 1553.5. Found: 1553.6 (M+). NR-(1-Deoxy-D-fructosyl)-N-(3-tri-n-butylstannyl-benzoyl)Lys0-octreotate (GTBLO). This was prepared essentially as above but with STB as the acylation agent. The intermediate peptide with the Dde group had a retention time of 41.5 min (vs 49 min for STB) under the HPLC conditions used for GIBLO. MS (FAB+) m/z: Calcd for C90H128N12O20S2120Sn: 1882.1. Found: cluster peaks around 1883.6 (M + H)+. For the final step (removal Dde) a different gradient was used for both analytical and semipreparative chromatography. The solvents used were 0.2% HOAc in 30% methanol in water (A) and 0.2% HOAc in 90% MeOH in water (B). The %B was increased from 0 to 100 over 30 min and then run for another 10 min. The retention time of the product peptide under the analytical conditions was 29.5 min. MS (MALDI) m/z: Calcd for C80H116N12O18S2120Sn: 1717.9. Found: 1740 (M + Na)+, 1717.5 (M+). NR-(1-Deoxy-D-fructosyl)-Lys0(Dde)-TOCA (Gluc-Lys0-TOCADde). This was synthesized as reported before (15, 16). NR-(1-Deoxy-D-fructosyl)-N-(3-iodobenzoyl)-lysine. This compound was synthesized for use as an HPLC standard. To a suspension of NR-Boc-N-(3-iodobenzoyl)lysine (17) (24 mg; 0.05 mmol) in 0.2 mL of methylene chloride in a Reacti-vial was added 0.2 mL of TFA, and the mixture was stirred at room temperature for 30 min. The solvents were evaporated using a stream of argon, and the residue was taken up in 0.5 mL of 5% acetic acid (v/v) in methanol. To the resultant solution was added 90 mg of R-D-glucose, and the capped vial was heated overnight

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at 60 °C. The reaction was followed by reversed-phase HPLC. For this, the analytical column was eluted at 1 mL/min with a gradient consisting of 0.1% TFA in each 90:10 water:acetonitrile (solvent A) and 10:90 water:acetonitrile (solvent B). The %B was held at 0 for 10 min, increased to 100 over the next 30 min, and held at 100 for another 5 min. Under these conditions, the starting material had a tR of 25.1 min and the product, 24.3 min. Because HPLC analysis did not indicate much progress in the reaction, 107 mg of R-D-glucose in 1.3 mL of solvent was added and the heating continued for 2 days, after which only moderate progress was seen. Semipreparative HPLC using the same gradient as above at a flow rate of 10 mL/min yielded 0.6 mg of product: MS (ESI) m/z: 539.72 (MH+); 561.52 (M + Na)+ (100%); 583.02 (M + 2Na - H)+. Radiohalogenation. [125I]I-TOC and [125I]I-Gluc-TOCA. These were prepared as reported (7). Briefly, to a solution of sodium [125I]iodide (1-2 mCi in 3 µL of 0.1 N NaOH) in a Reacti-vial was added 20 µL of 0.05 M phosphate buffer (pH 7.5) and a solution of 14 µg of Gluc-TOCA in 20 µL 0.05 M acetic acid. After vortexing this mixture, 0.16 µg of Chloramine-T in 20 µL of the above phosphate buffer was added. After a 1-min reaction at room temperature, the labeled peptide was isolated from the reaction mixture by reversed-phase HPLC. [131I]I-Gluc-Lys0-TOCA. Gluc-Lys0-Lys5(Dde)-TOCA was labeled with 131I essentially as described above for [125I]I-GlucTOCA. The reaction mixture was passed through an activated C-18 cartridge, which was further washed with 2 × 5 mL water, and the product was eluted with 0.25 mL portions of ethanol. The fractions containing the product were pooled and ethanol was evaporated. To the residual activity, 10 µL of a solution of hydrazine in DMF (2% v/v) was added. Ten minutes later, the reaction mixture was diluted with 25 µL of ethanol and injected onto a reversed-phase HPLC column. It was eluted with a linear gradient (10-40% B over a period of 30 min) consisting of solvents 0.1% TFA in water (A) and 0.1% TFA in acetonitrile (B) at a flow rate of 1 mL/min. The retention times of [131I]IGluc-Lys0-TOCA and its Dde-protected precursor under these conditions were 26.1 and 36.2 min, respectively. The fractions containing the final labeled product were isolated and concentrated by solid-phase extraction as described above for the intermediate peptide. [131I]GIBLO and [211At]GABLO. A solution of GTBLO (∼100 µg) in ethanol (50 µL) was transferred to a 1 mL Reactivial and the ethanol was evaporated with a gentle stream of argon. To this were added 131I (0.5-2 mCi in 1-3 µl) and N-chlorosuccinimide (5 µg in 10 µL of HOAc), and the mixture was sonicated at room temperature for 2 min. The entire reaction mixture was injected into an analytical reversed-phase column. The column was eluted using the gradient described for the synthesis of unlabeled GIBLO (tR ) 17-18 min). The labeled peptide was isolated in 21.2 ( 4.9% radiochemical yields. For astatination, the 211At activity in chloroform was first extracted into 1-2 µL of 0.1 N NaOH in a Reacti-vial, and the procedure described above for iodination was followed. [211At]GABLO was obtained in about 46.8 ( 9.5% radiochemical yields. The HPLC fractions containing the labeled peptide were concentrated using a C18 solid-phase cartridge. A Waters tC18ENV Seppak cartridge was activated by elution with 5 mL each of water and ethanol. HPLC fractions were purged with an argon stream to remove most of the acetonitrile, diluted with water, and passed through the solid-phase cartridge. The cartridge was washed twice with 5 mL of water and then eluted with 0.25 mL portions of ethanol. The labeled peptide generally eluted in fractions 4-6. Ethanol was evaporated from the combined fractions, and the product was reconstituted in PBS containing 1% BSA. Cells and Culture Conditions. The SSTR-expressing AR42J rat pancreatic tumor cell line (18) was obtained from American

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Type Culture Collection (Manassas, VA) and grown in DMEM containing 2 mM glutamine, 10% FCS, and 5 g/L glucose. D341 Med is a human medulloblastoma cell line derived from a patient tumor biopsy (19) and grown in 10% FCS and zinc option medium. Cells were maintained in a humidified atmosphere (37 °C, 5% CO2). Receptor Binding Assay. The SSTR receptor binding affinity of GIBLO was determined by a competitive binding assay using AR42J membranes according to a literature protocol (20). About 50 µg of membranes was incubated in triplicate at 37 °C for 1 h with ∼25 nCi of [125I]TOC in the absence or presence of various concentrations of unlabeled GIBLO or octreotide. Radioactivity bound to the membranes in the presence of 50 nM unlabeled peptide was considered to be nonspecific binding. The IC50 values were calculated from the specific binding versus peptide concentration plots. Internalization Assay. Three paired-label assays were performed following a previously reported protocol (12). D341 Med cells (5 × 105 per tube per mL) were incubated at 37 °C with (a) [125I]I-Gluc-TOCA (5.1 fmol) and [131I]GIBLO (31.0 fmol), (b) [131I]GIBLO (19.4 fmol) and [211At]GABLO (0.02 fmol), or (c) [125I]I-Gluc-TOCA (13.8 fmol) and [131I]I-GIu-Lys0TOCA (2.6 fmol) for 0.5, 1, 2, 3, and 4 h. At each time point, radioactivity in the cell culture supernatant, that bound to the cell surface, and that trapped inside the cells were determined. The percent of input dose that was internalized was plotted versus the time. Tissue Distribution Experiments. Animal studies were performed under the guidelines established by the Duke University Institutional Animal Care and Use Committee. Athymic BALB/c-nu (SPF) mice weighing 20-25 g with subcutaneous D341 Med human medulloblastoma xenografts were utilized (19). The biodistribution studies were initiated when tumors were about 200-300 mm3. Mice received 4 µCi of [125I]I-Gluc-TOCA and 3 µCi of [131I]GIBLO via the tail vein and groups of five animals were killed 0.5,1, 4, 16, and 24 h after injection. Mice were killed with an overdose of halothane, dissected and tissues of interest were isolated, weighed, and counted in a dual channel gamma counter. A paired Student’s t test was used to determine the statistical significance of differences between the two peptides. Paired-Label Catabolism [125I]I-Gluc-TOCA and [131I]GIBLO. In Vitro. The internalization assay described above was repeated at 0.5 and 4 h with 10 µCi of each tracer. After washing off cell surface-bound radioactivity, the cells were lysed in 100 µL of 0.5% NP-40 in PBS. Reversed-phase HPLC of cell culture supernatant, cell lysate, and acid washes were performed along with intact peptide standards, 3-iodotyrosine, and NR-(1-deoxy-D-fructosyl)-N-(3-iodobenzoyl)-lysine. For this, a C-18 analytical column was eluted at 1 mL/min with a gradient consisting of solvents A, 0.1% TFA in water, and B, 0.1% TFA in acetonitrile. The percent B was held at 10 for 10 min and then increased to 90 over 30 min. Under these conditions, the retention times of I-Gluc-TOCA, GIBLO, 3-iodotyrosine, and NR-(1-deoxy-D-fructosyl)-N-(3-iodobenzoyl)-lysine were 25-26 min, 29-30 min, 18-19 min, and 2526 min, respectively. In ViVo. Two groups of three mice bearing the D-341 Med xenografts were injected with 10 µCi of each tracer. Necropsies were performed 0.5 and 1 h postinjection, and blood, urine, liver, kidney, and tumor were obtained. Serum was isolated from blood by centrifugation and urine was filtered through a Spin-X filter (Corning, New York, NY). Tissues were homogenized in 2 mL of 80:20 EtOH:HEPES (1.0 M, pH 7.0) with a hand-held homogenizer. The homogenates were centrifuged, and the pellets were extracted once more with one volume of the solvent.

Vaidyanathan et al. Scheme 2. Synthesis of Gluc-Lys0-Lys5(Dde)-TOCAa

a Reagents and conditions: (a) i. 95:2.5:2.5 TFA:water:triisopropylsilane, ii. H2O2, THF, 0.05 M NH4OAc; (b) i. D-glucose, HOAc/MeOH. ii. piperidine.

Aliquots of the combined supernatants were analyzed by HPLC as described above.

RESULTS AND DISCUSSION The goal of this study was to synthesize an octreotateglucose conjugate that also possessed a pendant group on which radioiodine or 211At could be introduced in a stable fashion via a halodestannylation reaction in a single radiochemical step. To accomplish this, it was necessary to append an extra lysine at the N-terminus of octreotate. Because a tyrosine residue might compete with the tin precursor as a radioiodination site, octreotate was chosen as the peptide instead of TOCA. Unfortunately, a disadvantage of this approach is that the higher lipophilicity of octreotate compared with TOCA has been shown to yield less favorable pharmacokinetics (21, 22). Solid-phase peptide synthesis without the removal of the Fmoc group in the last step and subsequent cleavage from the resin delivered the N-Fmoc-Lys0-N-Dde-Lys5-octreotate (Scheme 1). A fructose unit was appended to the R amino group of Lys0 of this peptide via a Maillard reaction with glucose and subsequent Amadori rearrangement (23). The Fmoc group on the -amino group of Lys0 was removed, and the resultant peptide was treated with either N-succinimidyl 3-iodobenzoate (SIB) or N-succinimidyl 3-(tri-n-butylstannyl)benzoate (STB). Removal of the Dde group from the -amino group of Lys5 of these peptides delivered the iodo standard of the target peptide (GIBLO; Chart 1) and its tin precursor, respectively. To investigate the effect of an extra lysine (Lys0), Gluc-Lys0Lys5(Dde)-TOCA was also synthesized (Scheme 2). The peptide was assembled on the resin as for the GIBLO synthesis except a Tyr3 instead of a Phe3 was used. The target peptide was obtained by glycosylation and subsequent removal of the Fmoc group (15, 16). Radioiodination of the tin precursor with N-chlorosuccinimide as the oxidant and acetic acid as the solvent gave [131I]GIBLO in about 21.2 ( 4.9% radiochemical yield (n ) 10) (Scheme 3). The radiochemical yield of [211At]GABLO was 46.8 ( 9.5% (n ) 5); the difference between radioiodination and astatination yields is statistically significant. These results are enigmatic as astatination of the same tin precursor generally gives somewhat lower yields than radioiodination, presumably due to steric hindrance caused by the larger atomic size of astatine. It should be noted, however, that a radioactive peak of similar size as the product but with slightly higher retention time was seen in the HPLC trace of the radioiodination reaction mixture; the

Radiohalogenated Glycosylated Octreotate Derivatives

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Scheme 3. Synthesis of [*I]GIBLO and [211At]GABLO

a

Reagents and conditions: (a)

Scheme 4. Radioiodination of

a

131I

or

211At,

NCS, HOAc.

Gluc-Lys0-TOCA

Reagents and conditions: (a) 131I, Chloramine-T, phosphate buffer.

intensity of this peak was considerably lower in the case of astatination. The radiochemical purity of the labeled peptides was >90% with no detectable coeluting UV peaks present. [131I]I-Gluc-Lys0-TOCA also was synthesized to investigate the effect of an extra lysine at the N-terminus on peptide behavior. Although it should be possible to remove the Dde protecting group from Gluc-Lys0-Lys5(Dde)-TOCA and generate [131I]I-Gluc-Lys0-TOCA from the resultant peptide in a single radiochemical step, we opted to first label Gluc-Lys0-Lys5(Dde)TOCA and then remove the Dde group (Scheme 4) simply because of the limited availability of Gluc-Lys0-Lys5(Dde)TOCA. Only a few reactions could be attempted, and [131I]IGluc-Lys0-TOCA was obtained after two steps in 25% radiochemical yield and >95% radiochemical purity. The affinity of GIBLO was measured indirectly by a competitive binding assay using AR42J rat pancreatic tumor membranes. The “gold standard” approach in the literature used to determine the binding affinity of octreotide derivatives to SSTR involves the use of SSTR2-expressing AR42J membranes. The ability of GIBLO to displace [125I]TOC bound to AR42J membranes was determined. For comparison, the ability of octreotide to displace this tracer was also measured in a parallel assay. As shown in Figure 1, both GIBLO and octreotide displaced AR42J membrane-bound [125I]TOC in a concentrationdependent manner. The calculated IC50 values for GIBLO and octreotide were 0.46 ( 0.05 nM and 0.99 ( 0.13 nM, respectively, indicating that the novel peptide bound with high affinity to SSTR. An IC50 of 2.8 ( 0.4 nM was reported for

Figure 1. Competitive displacement of [125I]TOC from sst2-expressing AR42J rat pancreatic cell membranes by GIBLO (circles) and by octreotide (triangles). AR42J membranes were incubated with [125I]TOC and none or increasing concentrations of either octreotide or GIBLO. Specific binding, determined by subtracting the value at 50 nM concentration of the ligand from total binding, was plotted as a function of logarithm of ligand concentration.

Gluc-Lys0-fluoropropionyl-TOCA, another peptide with a Lys0 modification, to membranes from CHO cells transfected to express SSTR2 (15, 16). The IC50 values reported for other octreotide derivatives in displacing labeled analogues bound to AR42J rat pancreatic tumor membranes have been in the low nanomolar and sub-nanomolar range (see for example ref 21). Taken together, our results suggest that conjugation of an N-terminal sugar and a pendant prosthetic group for radiohalogenation to octreotate can be accomplished without compromising SSTR2 binding affinity. Receptor binding affinity assay with D341 membranes was not performed, as we were not successful in the past in obtaining reproducible results using these membranes with other octreotate derivatives. Because of our clinical interest in utilizing radiolabeled octreotide conjugates for the targeted radiotherapy of medulloblastoma, the internalization assays and the biodistribution study were performed with the D341 Med human medulloblastoma line. Previously we reported that, of the four radioiodinated octreotide analogues investigated, [125I]I-Gluc-TOCA has the highest level of intracellularly trapped radioactivity, reaching 10-15% of added radioactivity after a 4 h incubation with D341 Med cells (7, 12). Therefore, this peptide was selected as a positive control to evaluate the internalization of the carbohydrated peptides [131I]GIBLO and [211At]GABLO. As shown in Figure 2A, consistent with earlier results, about 10% of the input dose of [125I]I-Gluc-TOCA was internalized at 4 h. Disappointingly however, the specific internalized radioactivity for [131I]GIBLO was considerably lower, 0.05).

Figure 4. Paired-label in vitro catabolism: HPLC profiles of cell extracts (internalized) (A) and cell culture supernatants (B) at 4 h. A paired-label internalization assay was performed by incubating D341 Med cells with 10 µCi each of [125I]Gluc-TOCA (filled circle) and [131I]GIBLO (open circle). Cell lysates (internalized), cell culture supernatants, and surface-bound fractions at 30 min and 4 h were analyzed by reversed-phase HPLC. Data shown are for cell lysates and cell culture supernatants at 4 h.

not present as this elutes at 21-22 min under the HPLC conditions used for the catabolism experiments. We next evaluated the biodistribution of [131I]GIBLO in comparison with [125I]I-Gluc-TOCA in a paired-label format in athymic mice bearing subcutaneous D341 Med xenografts; data for selected time points are shown in Table 1. The tumor localization of [125I]I-Gluc-TOCA was 2- to 3-fold higher than that of [131I]GIBLO at early time points. For example, the tumor uptake of [125I]I-Gluc-TOCA at 0.5 and 1 h was 21.5 ( 4.0% ID/g and 18.8 ( 7.7% ID/g compared to 6.9 ( 1.2% ID/g and 4.7 ( 1.4% ID/g, respectively for [131I]GIBLO. The differences in uptake between the two tracers were statistically significant (p < 0.05). The tumor uptake of [125I]I-Gluc-TOCA at these time points was consistent with that seen earlier in this xenograft model (7). Tumor levels of radioiodine for [131I]GIBLO were

higher than those for [125I]I-Gluc-TOCA at 16 and 24 h; however, the difference was statistically significant only at 24 h (0.90 ( 0.20% ID/g versus 0.40 ( 0.07% ID/g). Normal tissue radioiodine levels observed after administration of [131I]GIBLO and [125I]I-Gluc-TOCA are summarized in Table 1. As expected, the strategy of avoiding radioiodination of Tyr3 resulted in significantly lower levels of radioiodine in the thyroid, consistent with a lower degree of deiodination. For example, 16 h after injection, the % injected dose in the thyroid for [131I]GIBLO was more than 4 times lower than that for [125I]I-Gluc-TOCA. The lower tumor uptake of [131I]GIBLO at early time points might be due to its concomitantly higher uptake in liver, kidney and particularly, small intestine (Table 1). In contrast, uptake of [125I]I-Gluc-TOCA was consistently higher in lungs and stomach, which are some of the tissues known to express somatostatin receptors (7). Alternatively, because of the proclivity of free iodide for the stomach, the higher accumulation of [125I]I-Gluc-TOCA in this organ could reflect the greater degree of dehalogenation of this peptide. HPLC analyses of tumor, kidney, liver, blood, and urine samples obtained 30 and 60 min after [125I]I-Gluc-TOCA and [131I]GIBLO injection were performed to determine the nature of the labeled species present in these tissues. In blood and urine, the majority of the radioactivity present at 30 and 60 min for both peptides eluted with a retention time corresponding to the intact peptide. In blood, 15 and 38% of the total 125I activity eluted in the solvent front peak at 30 and 60 min, respectively; for 131I, these values were 9 and 13%. In urine, about 3% of total 125I activity was in the solvent front peak at both time points and for 131I, 19% and 6% of total activity was in this peak at 30 and 60 min, respectively. In kidney and liver, very low levels of intact [125I]I-Gluc-TOCA were seen at both time points; most of the radioactivity eluted at the solvent front. The HPLC profiles for [131I]GIBLO catabolites indicated a distribution of 131I in ratios of 3:1 and 2:1 for kidney and liver, respectively, between solvent front peak and intact peak. It is possible that the solvent front peak may reflect free iodide, and this is consistent with the fact that it accounts for a smaller fraction of the eluted activity for [131I]GIBLO compared with [125I]I-Gluc-TOCA. On the other hand, the radioactivity present in tumor for both peptides was predominantly in the solvent front peak (Figure 5) (∼65-70% at both time points). Consistent with the results obtained in vitro with D341 Med medulloblastoma cells, peaks corresponding to [125I]MIT and NR-(1-deoxyD-fructosyl)-N-(3-[131I]iodobenzoyl)-lysine were not seen in the HPLC profiles of any of the in vivo samples. Unfortunately, these catabolism results fail to offer a cogent explanation for the inferior tumor targeting properties of [131I]GIBLO. Phe for Tyr substitution, addition of Lys0, and the presence of bulky aromatic prosthetic group could have had a combined effect in

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Figure 5. Paired-label in vivo catabolism: HPLC profile of tumor extracts in vivo at 60 min. Mice were injected with 10 µCi each of [125I]Gluc-TOCA (filled circle) and [131I]GIBLO (open circle). Necropsies were performed at 30 and 60 min postinjection. Tumor (and other tissues) was isolated and homogenized. The supernatant, after removal of cell debris, was analyzed by reversed-phase HPLC.

decreasing the extent of internalization and consequent decrease in tumor uptake. In conclusion, we have developed methods for synthesizing octreotate derivatives with two prosthetic groups, one containing a carbohydrate unit and the other, a tin group-containing aryl moiety suitable for radiohalogenation. The new peptide conjugate, GIBLO, maintained high affinity for SSTR and exhibited lower levels of deiodination than I-Gluc-TOCA. Its internalization properties in vitro and tumor targeting in vivo, while inferior to that of I-Gluc-TOCA, were similar to those of I-TOC. Although the factors responsible for the less than ideal internalization and tumor targeting of GIBLO are not known at this time, it is clear that alternate approaches for maximizing the intracellular trapping of radiohalogen and concomitant improvement in pharmacokinetics are needed. Toward that goal, we are currently working on next-generation octreotate analogues that include hydrophilic, charged aryl tin precursors that can be labeled with either radioiodine nuclides or 211At.

ACKNOWLEDGMENT The authors want to thank Philip C. Welsh, Holly Legrand, and Kevin Alston for their excellent technical assistance. The work was supported by Grants CA91927, CA78417, CA42324, and CA93371 from the National Institutes of Health, as well as by a grant from the Pediatric Brain Tumor Foundation.

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