Modulation of Pharmacokinetics of Radioiodinated Sugar-Conjugated

Sugar-Conjugated Somatostatin Analogues by Variation of Peptide. Net Charge and ...... F., Rush, B. D., and Ruwart, M. J. (1991) Peptide to glycopepti...
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Bioconjugate Chem. 2005, 16, 429−437

429

Modulation of Pharmacokinetics of Radioiodinated Sugar-Conjugated Somatostatin Analogues by Variation of Peptide Net Charge and Carbohydration Chemistry Margret Schottelius,† Friederike Rau,† Jean Claude Reubi,‡ Markus Schwaiger,† and Hans-Ju¨rgen Wester*,† Nuklearmedizinische Klinik und Poliklinik, Klinikum rechts der Isar, Technische Universita¨t Mu¨nchen, 81675 Mu¨nchen, Germany, and Institute of Pathology, University of Berne, 3010 Berne, Switzerland. Received March 26, 2004; Revised Manuscript Received January 13, 2005

Sugar conjugation of biooactive peptides has been shown to be a powerful tool to modulate peptide pharmacokinetics. In the case of radiolabeled somatostatin analogues developed for in vivo scintigraphy of somatostatin receptor (sst) expressing tumors, it generally led to tracers with predominant renal excretion and low uptake in nontarget organs, and in some cases also with enhanced tumor accumulation. Especially with respect to endoradiotherapeutic applicability of these tracers, however, understanding the structural requirements for minimal kidney accumulation and maximal tumor uptake is important. The aim of this study was therefore the evaluation of the potential of specific glycoside structures in combination with reduced peptide net charge to reduce kidney accumulation without affecting tumor accumulation. Three glyco analogues of radioiodinated Tyr3-octreotate (TOCA) with z ) 0 were evaluated in a comparative study using [125I]Mtr-TOCA (z ) +1), the maltotrioseAmadori analogue of [125I]TOCA, as a reference, [125I]Glucuron-TOCA, the Amadori conjugate with glucuronic acid, and [125I]Gluc-S- and [125I]Gal-S-TOCA, the coupling products with glucosyl- and mannosyl-mercaptopropionate. In cells transfected with sst1-sst5, all three new analogues show sstsubtype binding profiles similar to I-Mtr-TOCA with high, but somewhat reduced, affinity for sst2. In contrast, internalization into sst2-expressing cells (in % of [125I]Tyr3-octreotide ([125I]TOC)) as well as the EC50,R of unlabeled TOC for internalization determined in dual-tracer experiments are substantially enhanced for [123I]Gal-S-TOCA and [123I]Gluc-S-TOCA (internalization, 190% ( 12% and 265% ( 20%, respectively, vs 168% ( 6% of [125I]TOC for [123I]Mtr-TOCA; EC50,R, 2.62 ( 0.07 and 2.96 ( 0.14, respectively, vs 1.81 ( 0.07 for [123I]Mtr-TOCA). The tumor accumulation of [125I]Gal-S-TOCA and [125I]Gluc-S-TOCA in AR42J tumor-bearing nude mice 1 h p.i. is consequently very high (22.6 ( 2.2 and 26.2 ( 5.6%ID/g) and comparable to that of [125I]Mtr-TOCA (25.1 ( 4.4%ID/g). [125I]GlucuronTOCA showed lower uptake in sst-expressing tissues than did [125I]Mtr-TOCA, but considerably enhanced accumulation in nontarget organs such as liver, intestine, and kidney. Due to increased lipophilicity, hepatic and intestinal uptake 1 and 4 h p.i. of [125I]Gal-S-TOCA and [125I]Gluc-S-TOCA was also slightly higher than that of [125I]Mtr-TOCA. Kidney accumulation, however, was reduced by approximately 50% for both compounds (2.6 ( 0.3 and 2.2 ( 0.4, respectively, vs 4.0 ( 0.7%ID/g at 1 h p.i.). Because no sugar-specific effect was detected in the latter case, it is concluded that general ligand pharmacokinetics and especially kidney accumulation of the tracers investigated are mainly determined by physicochemical characteristics such as lipophilicity, net charge, and also charge distribution ([125I]Glucuron-TOCA vs [125I]Gal-S- and [125I]Gluc-S-TOCA). With respect to receptor targeting, however, the structure of the carbohydrate moiety plays an important role, leading to dramatically enhanced ligand internalization, especially in the case of [123I]Gluc-S-TOCA. Taking into account the combined effects of the Gluc-S-moiety both on kidney and on tumor accumulation, this group seems to be a promising synthon for the synthesis of other radiolabeled peptide analogues with improved pharmacokinetics.

INTRODUCTION

That the introduction of carbohydrates into small bioactive peptides may have significant beneficiary impact on peptide pharmacokinetics has been demonstrated in a variety of studies. This approach has been successfully applied to improve drug delivery to the target tissues, either exploiting specific uptake mechanisms or * To whom correspondence should be addressed. Telephone: 49 89 4140 4586. Fax: 49 89 4140 4841. E-mail: h.j.wester@ lrz.tum.de. † Technische Universita ¨ t Mu¨nchen. ‡ University of Berne.

enhanced peptide bioavailability. For example, enkephalin peptides glycosylated near or at the C-terminus were shown to elicit prolonged and profound analgesia in mice due to enhanced peptide delivery to the brain (1-3). Conjugation of Arg8-vasopressin with alkyl-glucosides or -mannosides led to substantially enhanced and specific renal peptide uptake from blood (4). Furthermore, besides a stabilization toward enzymatic degradation in vivo, carbohydration of the tetrapeptide Gly-Gly-Tyr-Arg was shown to entail enhanced intestinal absorption of the peptide via the Na+-dependent glucose transporter SGLT-1 (5). Enhanced bioavailability, both oral and intravenous, was observed for several other glycopeptides

10.1021/bc0499228 CCC: $30.25 © 2005 American Chemical Society Published on Web 02/22/2005

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Schottelius et al.

Figure 1. Structures of [125I]Mtr-TOCA, [125I]Glucuron-TOCA, [125I]Gluc-S-TOCA, and [125I]Gal-S-TOCA.

such as an N-linked glycopeptide renin inhibitor (6), glycosylated vasopressin analogues (7), and several Amadori sugar derivatives of octreotide (8). This effect was attributed to enhanced in vivo stability of the peptides, a shift from hepatobiliary toward renal excretion, leading to higher plasma levels of the drug (6, 8), and to improved absorption from the small intestine (7). These findings also constitute the basis of the recent development of a series of radiolabeled, glycosylated octreotide (9-15) and RGD analogues (16, 17) as receptor ligands for the in vivo scintigraphic detection of somatostatin receptor (sst) and Rvβ3-integrin expressing human malignancies, respectively. Especially the sstreceptors are of high clinical relevance in nuclear oncology, because many tumors, predominantly those of neuroendocrine origin, show overexpression of somatostatin receptors (18), and especially of the sst2-subtype (19, 20). This overexpression allows high-level targeting of sstexpressing tumors both for scintigraphic and for endoradiotherapeutic applications. For these purposes, however, radiolabeled sst analogues need to meet certain prerequisites: fast clearance kinetics, predominant renal excretion, as well as high receptor affinity and selectivity, all of which lead to high target-to-nontarget tissue ratios (tumor-to-nontumor ratios). That these can be substantially improved by glycosylation has been demonstrated in the case of various radiolabeled Tyr3-octreotide (TOC) and Tyr3-octreotate (TOCA) derivatives, which have been modified with different aldoses via Amadori reaction. As compared to their nonglycosylated counterparts, all showed substantially reduced hepatobiliary clearance in favor of fast renal excretion. This effect was found to correlate with increasing carbohydrate size, with the maltose derivatives showing the lowest extent of nonspecific accumulation in nontarget tissues such as liver and intestine (915). Interestingly, the influence of carbohydration on the biodistribution of [125I]TOC and [125I]TOCA was not only limited to the above expected effect. For both glucose analogues, [125I]Gluc-TOC and [125I]Gluc-TOCA, ligand internalization into sst-expressing cells was significantly enhanced as compared to the nonmodified parent peptide (14, 15). This effect was also reflected in the increased tumor accumulation of these radioligands. Despite an internalization efficiency comparable to that of [125I]TOCA, however, the maltotriose analogue [125I]MtrTOCA (Figure 1) showed the highest tumor accumulation in AR42J tumor-bearing nude mice of all glyco derivatives investigated (25.1 ( 4.4%ID/g at 60 min p.i., 11).

Furthermore, although the glucose- and maltose-Amadori derivatives of [125I]TOC showed an increasing preference of renal over hepatobiliary excretion, their renal accumulation remained unaffected as compared to [125I]TOC (10). This finding suggested that, because the peptides investigated all bear approximately the same net charge under physiological conditions (+1.8 to +2 (positive charges on d-Phe1 and Lys5)), charge-dependent endocytosis into renal tubular cells may be the main contributor to kidney accumulation of these peptides, rendering an influence of carbohydrate structure on kidney accumulation improbable. The contrary, however, was observed in the case of [125I]TOCA and its sugar analogues, where both the glucose and the maltose analogues showed a reduction of kidney accumulation by up to 50% as compared to [125I]TOCA and its maltotriose analogue [125I]Mtr-TOCA (15), indicating a certain influence of the sugar moiety on renal tracer accumulation in these cases. It was therefore of interest to investigate in more detail the physicochemical and structural requirements for combined optimization of both receptor targeting and maximal reduction of kidney uptake of radiolabeled TOCA derivatives. As demonstrated in a previous study using different 111In-DTPA-conjugated octreotide analogues, reduction of peptide net charge can lead to reduced renal tracer accumulation (21). This methodology was therefore chosen to attempt a further reduction of kidney uptake of radioiodinated, glycosylated TOCA analogues. Two approaches leading to glycopeptides with a net charge z ) 0 instead of z ) 1 were investigated: introduction of an additional negative charge by Amadori reaction of TOCA with glucuronic acid, leading to Glucuron-TOCA (Figure 1), and “removal” of the N-terminal positive charge via an alternative glycosylation route, that is, N-terminal acylation, leading to Gluc-S- and GalS-TOCA (Figure 1). In the latter case, the glycosylation method was based on results of Suzuki et al., who had demonstrated that kidney accumulation of Arg8-vasopressin could not only be specifically enhanced by conjugation with certain O- and S-alkyl-glycosides, but could also be significantly reduced, depending on the length of the alkyl chain and the structure of the carbohydrate used (4, 22). In this study, the structure of the alkylglycoside was chosen such that renal targeting via the basolateral membrane was highly improbable (22, 23), that is, a very short alkyl chain (mercaptopropionyl). Due to the higher in vivo stability of S- versus O-glycosides, this spacer was used to link two different carbohydrates to [125I]TOCA to study eventual influences of carbohy-

Pharmacokinetics of Somatostatin Analogues

drate structure on the pharmacokinetics of the corresponding radiolabeled peptide. All three new compounds were evaluated in detail in vitro and in vivo, and data on receptor affinity, ligand internalization, and pharmacokinetics, especially with respect to renal uptake and tumor accumulation, were compared to those obtained with [125I]Mtr-TOCA. EXPERIMENTAL PROCEDURES

Peptide Synthesis. General Conditions. TCP-resin was obtained from PepChem (Tu¨bingen, Germany). Fmoc- and other amino acids were supplied by Novabiochem or Bachem (Heidelberg, Germany). All other organic reagents were purchased from VWR (Darmstadt, Germany), Alexis (Gru¨nberg, Germany), Aldrich, or Fluka (Neu-Ulm, Germany). Solvents were used without further purification. Solid-phase peptide synthesis was carried out manually using a flask shaker (St. John Associates Inc., USA). Thin-layer chromatography was performed using Kieselgel 60 F254 TLC plates from Merck (Darmstadt, Germany). UV absorption at 254 nm was detected using a Fluotest UV lamp. Compounds that showed no UV absorption were detected by treating the TLC plate with a solution of 0.5 mL of anisaldehyde and 1 mL of concentrated sulfuric acid in 50 mL of acetic acid and subsequent heating to 150-200 °C. Flash-chromatographic separations were carried out using silica gel 60 (particle size 0.04-0.063 mm) with a mesh of 230-400 from Fluka (Neu-Ulm, Germany). Analytic RP-HPLC was performed on Nucleosil 100 C18 (5 µm, 125 × 4.0 mm) columns (CS GmbH, Langerwehe, Germany) using a Sykam gradient HPLC System (Sykam, Fu¨rstenfeldbruck, Germany). The peptides were eluted applying various gradients of 0.1% TFA (trifluoroacetic acid) in H2O (solvent A) and 0.1% TFA in acetonitrile (solvent B) in 15 min at a constant flow of 1 mL/min. UV-Detection was performed at 220 nm in a 206 PHD UV-vis detector (Linear Instruments Corp., Reno, NV). For radioactivity measurement, the outlet of the UV-photometer was connected to a NaI(Tl) well-type scintillation counter from EG&G Ortec (Mu¨nchen, Germany). Preparative RP-HPLC was performed on the same HPLC system using a Multospher 100 RP 18-5 (250 × 10 mm) column (CS GmbH, Langerwehe, Germany) at a constant flow of 5 mL/min. Mass spectra were recorded on the LC-MS system LCQ from Finnigan (Bremen, Germany) using the HewlettPackard series 1100 HPLC system. Mtr-TOCA. Mtr-TOCA was synthesized as described previously (11). Briefly, the sequence H2N-D-Phe-Cys(Trt)-Tyr(tBu)-D-Trp-Lys(Dde)-Thr(tBu)-Cys(Trt)Thr(tBu) was assembled on TCP-resin using a standard Fmoc-protocol. After cleavage from the solid support using TFA/H2O/TIBS (triisobutylsilane) (95/2.5/2.5 (v/v/ v)), Lys5(Dde)-TOCA (TOCA(Dde)) was cyclized using H2O2 in an aqueous THF (tetrahydrofurane) solution at pH 7. Derivatization with maltotriose was performed via Maillard reaction and subsequent Amadori rearrangement by treating the Lys5(Dde)-protected peptide with 5 equiv of maltotriose in methanol/acetic acid (95/5) at 60 °C. Subsequent removal of the Lys5(Dde) protecting group was carried out using 2% of hydrazine hydrate in DMF (dimethylformamide). After product isolation via preparative HPLC, Mtr-TOCA was obtained in 23% yield (based on Lys5(Dde)-TOCA).

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HPLC (15%f45% in 15 min): tR ) 7.88 min; K′ ) 3.52. Calculated monoisotopic mass for Mtr-TOCA (C67H94N10O27S2) ) 1534.6; found: m/z ) 1535.2 [M + H]+, m/z ) 1557.2 [M + Na]+. Glucuron-TOCA. The synthesis of Glucuron-TOCA was performed according to the procedure described for MtrTOCA. For the conjugation of Lys5(Dde)-TOCA with glucuronic acid, 10 equiv of the carbohydrate was used. Yield after preparative HPLC: 24%. HPLC (10%f60% in 15 min): tR ) 10.7 min; K′ ) 4.79. Calculated monoisotopic mass for Glucuron-TOCA (C55H72N10O18S2) ) 1224.4; found: m/z ) 1225.3 [M + H]+, 1247.3 [M + Na]+, 1263.1 [M + K]+. Gluc-S-TOCA and Gal-S-TOCA. Both 1-S-(2,3,4,6tetraacetyl-glucopyranosyl)-3-mercaptopropionate (Gluc(4Ac)-S-OH) and 1-S-(2,3,4,6-tetraacetyl-galactopyranosyl)3-mercaptopropionate (Gal(4Ac)-S-OH) were prepared according to the literature (24). Synthesis of the corresponding pentafluorophenyl (Pfp) active esters Gluc(4Ac)S-OPfp and Gal(4Ac)-S-OPfp was carried out as described previously using N,N′-diisopropyl carbodiimide (1.1 equiv) and pentafluorophenol (1.1 equiv) in THF (13). For sugar conjugation of TOCA(Dde), the peptide (50-100 mg, 1 equiv) and Gluc/Gal(4Ac)-S-OPfp (1.1 equiv) were dissolved in 1-2 mL of DMF and stirred at room temperature for 2 h in the presence of 1 equiv of Hu¨nigs Base. After product precipitation using Et2O and washing of the precipitate with Et2O, the respective glycosylated peptides were obtained in 46-67% yield. Gluc(4Ac)-STOCA(Dde): HPLC (30%f80% B in 15 min): tR ) 12.1 min; K′ ) 6.31. Gal(4Ac)-S-TOCA(Dde): HPLC (30%f80% B in 15 min): tR ) 12.0 min; K′ ) 6.42. After Dde-deprotection for 10 min at room temperature, the peptides were precipitated using Et2O (diethyl ether). For subsequent sugar deacylation, peptides were redissolved in 1 mL of methanol containing 0.5 equiv of KCN (25). Deacylation was complete after stirring at room temperature for 4-48 h. The product was then precipitated using Et2O and purified via preparative HPLC, yielding Gluc-S-TOCA and Gal-S-TOCA in yields of 12-15% (based on TOCA(Dde)). Gluc-S-TOCA: HPLC (20%f70% B in 15 min): tR ) 8.2 min; K′ ) 3.99. Gal-S-TOCA: HPLC (20%f70% B in 15 min): tR ) 8.3 min; K′ ) 3.96. Calculated monoisotopic mass for Gluc-S-TOCA and Gal-S-TOCA (C58H78N10O18S3) found: m/z ) 1299.3 [M + H]+, 1321.3 [M + Na]+, 1337.2 [M + K]+. 3-Iodo-Tyr3-Reference Compounds. For the synthesis of the 3-iodo-Tyr3-reference compounds, Fmoc-Tyr(tBu)OH was replaced by Fmoc-3-iodo-Tyr(tBu)-OH during SPPS. All other reaction steps were performed analogously to the syntheses presented in the previous section. I-Mtr-TOCA. HPLC (10%f60% B in 15 min): tR ) 11.2 min; K′ ) 6.33. Calculated monoisotopic mass for I-Mtr-TOCA (C67H93N10O27S2I) ) 1660.5; found: m/z ) 1661.2 [M + H]+. I-Glucuron-TOCA. HPLC (10%f60% B in 15 min): tR ) 11.1 min; K′ ) 6.41. Calculated monoisotopic mass for I-Glucuron-TOCA (C55H71N10O18S2I) ) 1350.3; found: m/z ) 1351.3 [M + H]+, 1373.3 [M + Na]+, 1389.2 [M + K]+. I-Gluc-S-TOCA. HPLC (20%f70% B in 15 min): tR ) 9.2 min; K′ ) 4.72.

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Calculated monoisotopic mass for I-Gluc-S-TOCA (C58H77N10O18S3I) ) 1424.4; found: m/z ) 1425.6 [M + H]+, 1447.6 [M + Na]+, 1463.5 [M + K]+. I-Gal-S-TOCA. HPLC (20%f70% B in 15 min): tR ) 9.1 min; K′ ) 4.79. Calculated monoisotopic mass for I-Gal-S-TOCA (C58H77N10O18S3I) ) 1424.4; found: m/z ) 1425.6 [M + H]+, 1447.6 [M + Na]+, 1463.5 [M + K]+. Peptide Radioiodination. Radioiodination was generally performed using the Iodogen method. A solution of 100-200 µg of peptide in 200 µL of PBS (phosphate buffered saline, 0.1 M, pH 7.4) was transferred to an Eppendorf cap coated with 30 µg of Iodogen (Pierce, Rockford, USA). After the addition of 5-20 µL of solution of radioiodide (Amersham, Buckinghamshire, UK) in 0.05 M NaOH ([125I]NaI (n.c.a.), 18-74 MBq; [123I]NaI (c.a.), 37-185 MBq), the cap was vortexed and the labeling reaction was allowed to proceed for 20 min at room temperature. The peptide solution was then removed from the insoluble oxidizing agent. The radioiodinated peptides were purified via RPHPLC using an isocratic solvent mixture of 25% ([123I]Mtr-TOCA), 28% ([123I]Glucuron-TOCA), or 33% ([123I]Gluc-S-TOCA and [123I]Gal-S-TOCA) EtOH (0.5% AcOH) in water (0.5% AcOH). For the biodistribution experiments, an excess of absolute ethanol was added to the collected fraction, and the solvents were evaporated to dryness. The radioiodinated product was reconstituted in PBS to yield a solution of radiolabeled peptide with an activity concentration of approximately 370 kBq/100 µL. For the paired-label internalization experiments, [125I]TOC and the respective 123I-labeled radioligand were each separately redissolved in assay medium (containing 5% BSA) and diluted to an activity concentration of approximately 200 000 cpm/10 µL. A 1:1 (v/v) mixture of both solutions containing approximately 100 000 cpm/ 10 µL of each radioligand was then used for the internalization experiment. In Vitro Studies. sst-Receptor Binding Affinities. Cells stably expressing human sst1, sst2, sst3, sst4, and sst5 were grown as described previously (26). Cell membrane pellets were prepared, and receptor autoradiography was performed on pellet sections (mounted on microscope slides), as described in detail previously (26). For each of the tested compounds, complete displacement experiments were performed with the universal somatostatin radioligand 125I-[Leu8, D-Trp22, Tyr25]-somatostatin 28 using increasing concentrations of the unlabeled peptide ranging from 0.1 to 1000 nM. Somatostatin 28 was run in parallel as control using the same increasing concentrations. IC50 values were calculated after quantification of the data using a computer-assisted image processing system. Tissue standards (autoradiographic [125I]microscales Amersham), containing known amounts of isotopes, cross-calibrated to tissue equivalent ligand concentrations, were used for quantification (26). Internalization and Determination of EC50,R. CHO cells stably transfected with human sst2 (epitope tagged at the N-terminal end) were kindly provided by Dr. Jenny Koenig, University of Cambridge, UK. Cells were grown in DMEM/Nutrition Mix F-12 with Glutamax-I (1:1) (Gibco BRL) supplemented with 10% FCS and 500 mg/L Geneticin (Gibco BRL). For the preparation of the solutions used for the internalization studies containing various concentrations of unlabeled TOC, FCS was replaced by 5% BSA (Sigma, St. Louis, MO).

Schottelius et al.

For cell counting, a CASY1-TT cell counter and analyzer system (Scha¨rfe System GmbH, Reutlingen, Germany) was used. Internalization experiments were carried out as previously described in detail (13). Briefly, after preconditioning of the cells (approximately 100 000 cells/well) with 190 µL of unsupplemented medium for a minimum of 15 min, 50 µL (per well) of DMEM (5% BSA) containing increasing concentrations of unlabeled TOC (Tyr3-octreotide) was added, followed by the addition of approximately 100 000 cpm of both the respective 123I-labeled glycosylated peptide and the reference [125I]TOC in 10 µL of DMEM (5% BSA). Final TOC concentrations in the incubation medium used for the determination of the EC50,R were 0.1, 0.2, 0.5, 0.8, 1, 2, 4, 6, 8, 10, 12, 15, 20, 50, 100, 500, and 1000 nM. In a control experiment (n.c.a. conditions), TOC-free DMEM (5% BSA) was added. Nonspecific internalization was determined by including 5 µM unlabeled TOC. Experiments were carried out in triplicate for each concentration. After incubation at 37 °C (10 min), the incubation medium was removed, and cells were rinsed with 250 µL of fresh medium. The combined medium fractions represent the amount of free radioligand. Receptor bound (acid releasable) radioactivity was then removed using 2 × 250 µL of ice-cold acid wash buffer (0.02 M NaOAc buffered with AcOH to pH ) 5). The internalized activity was released by incubation with 250 µL of 1 N NaOH, tranferred to vials, and combined with 250 µL of PBS used for rinsing the wells. Quantification of the amount of free, acid-releasable, and internalized activity was performed in a γ-counter. For numerical analysis of the EC50 of TOC for internalization, data for both the 123I-labeled compound of interest and the reference [125I]TOC in the same experiment were first corrected by the amount of nonspecific internalization, respectively, and then each normalized to the amount of internalized ligand in the absence of unlabeled competitor (100%). Data were fitted with a weighted two-parameter logistic function using Sigma Plot. To eliminate the influence of cell count and cell viability on the absolute EC50-values, data are expressed as the ratio (EC50,R) of the EC50 observed for the compound of interest (COI) and the EC50 found for [125I]TOC in the same experiment (EC50,R ) EC50(COI)/EC50([125I]TOC)). Biodistribution Experiments. Tumor Model. The AR42J cell line as a transplantable rat pancreatic tumor model with high sst2 somatostatin receptor expression (27) was used. AR42J cells were obtained from ECACC (European Collection of Cell Cultures, Salisbury, UK). Cells were maintained in RPMI 1640 (Seromed, Berlin, Germany) supplemented with 10% FCS (Seromed) and 2 mM L-glutamine (Gibco BRL Life Technologies, Karlsruhe, Germany). To establish tumor growth, cells were detached from the surface of the culture flasks using 1 mM EDTA in PBS, centrifuged, and resuspended in serum-free culture medium. Concentration of the cell suspension was (2.5-5) × 106 cells/100 µL serum. Into nude mice (Swiss nu/nu, female, 6-8 weeks, from Charles River WIGA GmbH (Sulzfeld, Germany)) was injected 100 µL of the cell suspension subcutaneously into the flank. Ten days after tumor transplantation, all mice showed solid palpable tumor masses (tumor weight 20200 mg) and were used for the experiments. Biodistribution Studies. The radiolabeled peptides, 370 kBq (10 µCi) in 100 µL of PBS (pH 7.4), were injected i.v. into a tail vein of nude mice bearing an AR42J tumor. For competition studies, 10 µg TOC (0.4 mg/kg) was co-

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Pharmacokinetics of Somatostatin Analogues

Table 1. Affinity Profiles of Somatostatin 28 and the Compounds Investigated for Human sst1-sst5 Receptors Using 125I-[Leu8, D-Trp22, Tyr25]-Somatostatin 28 as the Radioligand (Values Are IC 50 ( SD [nM], Number of Experiments in Parentheses) hsst1

hsst2

hsst4

hsst5

SS-28 I-TOC I-TOCA

peptide

3.6 ( 0.3 (5) >10 000 (5) >10 000 (3)

2.1 ( 0.3 (5) 1.3 ( 0.3 (5) 0.47 ( 0.2 (3)

3.2 ( 0.2 (5) 128 ( 22 (5) 187 ( 38 (3)

3 ( 0.3 (5) 867 ( 33 (5) 337 ( 57 (3)

2.5 ( 0.2 (5) 50 ( 12 (5) 50 ( 5.8 (3)

I-Mtr-TOCA I-Glucuron-TOCA I-Gluc-S-TOCA I-Gal-S-TOCA

>10 000 (3) >1 000 (2) >1 000 (3) >1 000 (3)

0.95 ( 0.3 (3) 2.9 ( 1.3 (2) 2.0 ( 0.7 (3) 2.0 ( 0.8 (3)

823 ( 67 (3) >1000 (2) 398 ( 19 (3) 491 ( 63 (3)

823 ( 87 (3) 220 ( 23 (2) 356 ( 60 (3) 482 ( 134 (3)

327 ( 93 (3) 414 ( 160 (2) 310 ( 156 (3) 413 ( 167 (3)

injected with the radioligands. The animals (groups of 3-4) were sacrificed 1 and 4 h postinjection, and the organs of interest were dissected. The radioactivity was measured in weighted tissue samples using a γ-Counter.

hsst3

Table 2. Internalization (in % of the Reference [125I]TOC) and Relative EC50 Values (EC50,R) of Unlabeled TOC for Inhibition of the Internalization of [123I]TOCA and the Sugar Analogues Investigated (CHO cells (hsst2), 10 min Incubation, n ) 3 in Three Separate Determinations, Mean ( SD)

RESULTS

Peptide Synthesis. Solid-phase peptide synthesis using a standard Fmoc-protocol and HOBt/TBTU as coupling reagents yielded TOCA(Dde) and its 3-iodo-Tyr3 analogue, I-TOCA(Dde), in 90-97% yield based on resinbound Fmoc-Thr(tBu)-OH. Quantitative disulfide bridge formation was achieved within less than 30 min at room temperature using H2O2 in an aqueous THF-solution buffered to pH 7. While UV purity (220 nm) of crude cyclized TOCA(Dde) usually exceeded 93%, more side products were observed for the 3-iodo-Tyr3-containing peptide (up to 20%). However, both peptides were used for the following reaction steps without further purification. N-terminal glycosylation of the parent peptides with maltotriose (Mtr-) and glucuronic acid (Glucuron-) via Amadori reaction required reaction times of 16-20 h at 60 °C, and glycosylation yields usually did not exceed 85% (value based on the integration of product and precursor peaks in the reaction control RP-HPLC chromatograms (220 nm)). In contrast, N-terminal conjugation with the respective pentafluorophenyl active esters of (1-S-2,3,4,6tetraacetyl-gluco/galactopyranosyl)-3-mercaptopropionate was quantitative within 30-45 min at room temperature. Subsequent Dde-deprotection (and deacetylation in the case of Gluc-S- and Gal-S-TOCA), followed by preparative RP-HPLC purification, afforded all glycosylated TOCA derivatives as well as their 3-iodo-Tyr3-counterparts in overall yields of 12-25% (based on nonglycosylated precursor) and in purities g98% (220 nm). Radiolabeling. Due to the formation of radioiodinated side products, overall radiochemical yields of the 125I- and 123 I-labeled peptides after RP-HPLC isolation were limited to 45-73%. Radiochemical purities of the isolated radioiodinated peptides, however, usually exceeded 98%. The HPLC conditions applied allowed very efficient separation of the radioiodinated product from the unlabeled precursor (∆tR g 5 min (13)) for all radioligands investigated. Thus, and because no coeluting carrier peak was observed in the labeled peptide peak in the quality control UV-chromatograms, the specific activity of the labeled peptides was assumed to be that of the radioiodide used for their preparation (g2000 Ci/mmol for 125I, ∼5000 Ci/mmol for 123I). In Vitro Studies. The affinities of I-Mtr-TOCA, IGlucuron-TOCA, I-Gluc-S-TOCA, and I-Gal-S-TOCA to the five human sst-subtypes sst1-sst5 are summarized in Table 1 and compared to those of the reference I-TOC and of nonglycosylated I-TOCA. All sugar analogues show very high and comparable affinity to hsst2, no affinity to hsst1, and low to moderate affinity to hsst3-5. As com-

internalized [% of [125I]TOC]

EC50,R

[125I]TOC [123I]TOCA

100 178.8 ( 6.9

1 1.70 ( 0.11

[123I]Mtr-TOCA [123I]Gluc-S-TOCA [123I]Gal-S-TOCA

168.0 ( 5.8 264.7 ( 20.3 189.8 ( 11.7

1.81 ( 0.07 2.96 ( 0.14 2.62 ( 0.07

pared to I-TOCA, all glycosylated derivatives show significantly reduced affinities to hsst3 and hsst5. This effect, however, is more pronounced in the case of the Amadori analogues than for the two thioglycoside-modified peptides. Of the carbohydrated peptides investigated, I-Mtr-TOCA shows the highest hsst2-specificity. It has been shown in a previous study that the sst2affinity of radioiodinated sugar analogues of TOC and TOCA does not correlate with in vivo tumor accumulation (13). In contrast, both the internalization of a ligand in % of the reference [125I]TOC, and the EC50,R of unlabeled TOC for ligand internalization (EC50,R ) EC50(compound of interest):EC50([125I]TOC)) have been shown to be good predictive parameters for tumor uptake in vivo (13). Therefore, these two quantities have also been determined for the four compounds in this study (Table 2). Internalization of all sugar analogues into hsst2expressing CHO cells was substantially increased as compared to the reference [125I]TOC. For both [123I]MtrTOCA and [123I]Gal-S-TOCA, however, it remained almost unchanged as compared to nonmodified [123I]TOCA (Table 2). Interestingly, the EC50,R of [123I]Mtr-TOCA was also comparable to that of [123I]TOCA, while it was significantly higher for [123I]Gal-S-TOCA (2.6 vs 1.7). Only in the case of [123I]Gluc-S-TOCA, however, was the very high EC50,R (3.0) also accompanied by dramatically enhanced ligand internalization (260% vs 170-190% of [125I]TOC for all other TOCA analogues). Biodistribution Studies. The biodistribution of [125I]Mtr-TOCA, [125I]Glucuron-TOCA, [125I]Gluc-S-TOCA, and [125I]Gal-S-TOCA in AR42J tumor-bearing nude mice 1 and 4 h postinjection is shown in Table 3. Clearance of all four glycopeptides from the circulation is very rapid, resulting in low blood activity concentrations (0.470.75%ID/g) already 1 h p.i. Of the four compounds investigated, [125I]Glucuron-TOCA showed the highest blood activity level, and also high nonspecific accumulation in kidney, liver, and intestine, the latter being nearly twice as high as that observed for [125I]Mtr-TOCA. For both [125I]Gluc-S-TOCA and [125I]Gal-S-TOCA, almost identical biodistribution in nontarget organs was observed at all time points. As compared to [125I]Mtr-TOCA, these two compounds show slightly increased hepatic and intestinal uptake, but also a reduction in renal activity

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Table 3. Biodistribution of [123I]Mtr-TOCA, [123I]Glucuron-TOCA, [125I]Gluc-S-TOCA, and [125I]Gal-S-TOCA in AR42J Tumor-Bearing Nude Mice 1 and 4 h p.i. (Data Are %ID/g (mean ( SD, Groups of 3-4)) [123I]Mtr-TOCA blood liver intestine kidney lung spleen muscle stomach adrenals pancreas tumor

[123I]Glucuron-TOCA

[125I]Gluc-S-TOCA

[125I]Gal-S-TOCA

1 h p.i.

4 h p.i.

1 h p.i.

4 h p.i.

1 h p.i.

4 h p.i.

1 h p.i.

4 h p.i.

0.47 ( 0.10 0.43 ( 0.07 4.29 ( 1.54 3.98 ( 0.71 1.93 ( 1.04 0.57 ( 0.10 0.08 ( 0.02 17.47 ( 2.78 5.30 ( 1.78 14.38 ( 4.39 25.11 ( 4.40

0.26 ( 0.03 0.20 ( 0.03 5.24 ( 0.56 1.69 ( 0.21 0.90 ( 0.31 0.31 ( 0.07 0.08 ( 0.03 5.92 ( 0.74 2.71 ( 0.07 3.93 ( 0.39 12.80 ( 2.97

0.75 ( 0.08 0.95 ( 0.16 8.05 ( 0.73 4.34 ( 0.57 1.57 ( 0.50 0.70 ( 0.21 0.17 ( 0.02 9.47 ( 0.98 3.65 ( 0.83 6.44 ( 2.58 19.78 ( 3.12

0.34 ( 0.11 0.27 ( 0.06 7.44 ( 2.91 1.36 ( 0.29 0.68 ( 0.27 0.34 ( 0.05 0.09 ( 0.04 3.30 ( 1.06 2.11 ( 0.60 1.37 ( 0.14 5.87 ( 0.37

0.54 ( 0.13 0.61 ( 0.13 5.23 ( 1.46 2.24 ( 0.41 2.21 ( 1.32 0.61 ( 0.12 0.10 ( 0.02 18.82 ( 3.39 4.98 ( 1.76 14.18 ( 4.57 26.15 ( 5.56

0.28 ( 0.06 0.18 ( 0.05 6.97 ( 1.30 0.69 ( 0.13 0.77 ( 0.24 0.28 ( 0.07 0.09 ( 0.05 4.24 ( 0.79 2.37 ( 0.15 3.35 ( 0.42 7.54 ( 2.22

0.64 ( 0.09 0.80 ( 0.12 5.53 ( 1.08 2.58 ( 0.31 2.81 ( 1.26 0.94 ( 0.46 0.14 ( 0.03 21.96 ( 1.61 6.29 ( 1.14 21.17 ( 6.57 22.55 ( 2.16

0.30 ( 0.06 0.23 ( 0.04 8.73 ( 3.73 0.70 ( 0.07 0.79 ( 0.41 0.49 ( 0.01 0.07 ( 0.02 3.93 ( 0.94 3.95 ( 0.88 2.87 ( 0.38 6.35 ( 0.86

Receptor-specificity of ligand accumulation in sstpositive organs was demonstrated in a competition study (Figure 3). Co-injection of 10 µg unlabeled TOC per mouse led to a dramatic and comparable reduction of ligand accumulation in stomach, adrenals, pancreas, and tumor to 7-13%, 22-30%, 8-14%, and 18-31% of control (1 h p.i.) for all compounds investigated. DISCUSSION

Figure 2. Tumor-to-organ ratios of [125I]Mtr-TOCA, [125I]Glucuron-TOCA, [125I]Gluc-S-TOCA, and [125I]Gal-S-TOCA in AR42J tumor-bearing nude mice 60 min p.i. (mean ( SD, n ) 3-4).

accumulation by up to 45% and 60% at 1 and 4 h p.i., respectively. For all four peptides, the observed decrease in hepatic activity within the observation period was accompanied by an increase in intestinal activity accumulation, indicating a certain contribution of hepatobiliary excretion to overall tracer clearance. The substantial drop in kidney activity levels between 1 and 4 h p.i. detected for all four carbohydrated TOCA analogues indicates that, despite predominant renal excretion of the radiopeptides, no real tracer accumulation, that is, uptake and retention in renal tissue, occurs. Of the glycopeptides investigated, [125I]Glucuron-TOCA showed the lowest, albeit still very high, accumulation in sst-expressing tissues, that is, stomach, adrenals, pancreas, and tumor. Interestingly, tracer uptake in stomach and pancreas was highest for [125I]Gal-S-TOCA, whereas tumor accumulation of this peptide was slightly lower than that of [125I]Mtr-TOCA and [125I]Gluc-S-TOCA (22.6 vs 25.2 and 26.2%ID/g at 1 h p.i., respectively). In most cases, tumor-to-organ ratios are therefore highest for these compounds, both 1 (Figure 2) and 4 h p.i. Due to their particularly low renal accumulation, however, tumor/kidney ratios are highest for [125I]Gluc-S-TOCA and [125I]Gal-S-TOCA (11.7 and 8.7 vs 6.3 and 4.6 for [125I]Mtr-TOCA and [125I]Glucuron-TOCA, respectively, at 1 h p.i.). As expected for radioiodinated tracers, washout of the four glycopeptides investigated from sst-expressing tissues between 1 and 4 h p.i. was considerable. Interestingly, tumor retention of [125I]Mtr-TOCA was significantly higher than that of the other three peptides; that is, 50% instead of 28-30% of the tumor-localized activity at 1 h p.i. were still found inside the tumor at 4 h p.i. in the case of [125I]Mtr-TOCA.

Maximizing tumor targeting and minimizing nonreceptor-mediated accumulation in excretion organs generally are the most important goals in the development of new receptor-targeted peptide tracers for application in nuclear oncology. The first can only be achieved by strutural modifications of the receptor ligand itself; for example, in the case of various radiometalated and radiohalogenated analogues of the somatostatin receptor ligand Tyr3-octreotide (TOC), the desired improvement of receptor-mediated ligand accumulation was achieved by substitution of Thr(ol)8 by Thr8, leading to Tyr3octreotate (TOCA) (13-15, 28, 29). Furthermore, conjugation of [125I]TOC and [125I]TOCA with, for example, glucose via Amadori reaction (10, 14, 15) or the substitution of 111In by 68Ga in chelator-conjugated TOC and TOCA (30) also led to enhanced internalization in vitro and tumor uptake in vivo. Of course, nonspecific radioligand uptake in the excretion organs is also mainly determined by the physicochemical properties of the molecule itself such as lipophilicity and net charge, which are dependent on the labeling method and/or the nature of further functionalities introduced into the peptide. In contrast to receptor targeting, however, radioligand accumulation in nontarget organs can also be influenced “externally”. For example, the high kidney uptake of radiometalated TOC analogues, which leads to high nephrotoxicity and thus limits the applicable dose, has been efficiently reduced by coinfusion of basic amino acids (Lys/Arg) (31). This methodology relies on the assumption that the positively charged amino acids compete for charge-dependent endocytosis into the renal tubular cells. Recent studies indicated that significantly reduced kidney accumulation may also be achieved by a reduction of peptide net charge by amino acid substitution in radiometalated octreotide (21) as well as by certain carbohydrates N-terminally linked to [125I]TOCA (15). To assess the relative impact of both of the above modifications in the same molecule, three new glycosylated [125I]TOCA analogues, [125I]Glucuron-TOCA, [125I]Gluc-S-, and [125I]Gal-S-TOCA (Figure 1), were evaluated in vitro and in vivo with particular focus on the single and combined effects of carbohydrate structure, reduced net charge, and different charge distribution on receptor

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Bioconjugate Chem., Vol. 16, No. 2, 2005 435

Figure 3. Effect of co-injection of 10 µg of unlabeled TOC per mouse (0.4 mg/kg) on the uptake of [125I]Mtr-TOCA, [125I]GlucuronTOCA, [125I]Gluc-S-TOCA, and [125I]Gal-S-TOCA in stomach, adrenals, pancreas, and tumor of AR42J tumor-bearing nude mice 60 min p.i. (mean ( SD, n ) 3).

affinity, ligand internalization, and pharmacokinetics, and compared to the reference [125I]Mtr-TOCA. In this context, the potential identification of carbohydrate structures that might be generally applicable for a reduction of peptidic radiotracer accumulation in the kidney, ideally without affecting its receptor affinity and tumor accumulation, was of major interest. So far, the differences in the sst-affinity profiles of I-Glucuron-TOCA, I-Gluc-S-, and I-Gal-S-TOCA as compared to I-Mtr-TOCA cannot be attributed unambiguously to the influence of either carbohydrate structure or a charge effect. However, one indicator for a chargedependent effect is the slightly reduced sst2-affinity of I-Glucuron-TOCA, I-Gluc-S-TOCA, and I-Gluc-S-TOCA (z ) 0) as compared to I-Mtr-TOCA (z ) +1). A detailed study including four octreotide analogues with net charges z ) +1 to z ) +3 demonstrated a strong correlation between peptide net charge and ligand receptor affinity. The more electropositive charges are on the peptide, the higher is the effective concentration of the peptide at the negatively charged membrane, and thus the higher are the receptor affinity and also functional activity observed for the respective peptide analogue (32). A second indicator for the (indirect) influence of peptide net charge on the sst-affinity profile is the reduced sst2-specificity of the three new glyco-peptides with z ) 0. In the case of radiometalated TOC analogues, a lower hydrophilicity (induced by reduced net charge) leads to increased affinity to sst-subtypes 3-5 (26). The same seems to be the case especially for I-Gluc-S-TOCA and I-Gluc-STOCA. Both peptides show a lower hydrophilicity than

I-Mtr-TOCA (log Pow ) -1.67 ( 0.02 vs -1.73 ( 0.04), and both show substantially increased hsst3 and hsst4 affinities. It has already been demonstrated in several studies (13, 28, 29, 33) that the sst2-affinity of an octreotide analogue is of limited predictive value for the efficiency of ligand internalization into sst2-expressing cells. The same was observed in this study. While both [123I]GlucS- and [123I]Gal-S-TOCA showed reduced sst2-affinity as compared to [123I]Mtr-TOCA (Table 1), they exhibited an increase in internalization by a factor of 1.57 and 1.13, respectively (Table 2). Furthermore, the effective concentration of unlabeled TOC needed to inhibit ligand internalization to 50% of maximum (normalized to the value found for the internal standard [125I]TOC in the same experiment), the EC50,R, of [123I]Gluc-S- and [123I]Gal-S-TOCA was also substantially increased (Table 2). Thus, both compounds have an enhanced ability to compete with unlabeled competitor for receptor binding and subsequent endocytosis into the cell and thus a higher internalization potency than [123I]Mtr-TOCA. So far, it remains unclear as to what extent either the reduced net charge or the change in carbohydrate structure or both are responsible for the divergence between data for [123I]Mtr-TOCA and the two thioglycoside analogues. In contrast to the sst-affinity profiles, however, which were very similar for both [123I]Gluc-S- and [123I]Gal-S-TOCA, both the internalization and the EC50,R determined for these two compounds with identical charge distribution differ significantly. This finding indicates that, although the structure of the carbohydrate (Gluc vs Gal) has no significant influence on the binding

436 Bioconjugate Chem., Vol. 16, No. 2, 2005

affinity to immobilized receptors (Table 1), it does have substantial impact on ligand internalization into live cells. This is supported by data from a previous study in which the glucose-Amadori analogues of [123I]TOC and [123I]TOCA, [123I]Gluc-TOC and [123I]Gluc-TOCA, exhibited a considerable enhancement of ligand internalization and EC50,R as compared to their corresponding maltoseand maltotriose-counterparts (13), although the glycopeptides from each series (TOC and TOCA) had identical net charge (z ) +2 and z ) +1, respectively). In the same study, it has been demonstrated that ligand internalization and also the EC50,R represent more reliable predictive parameters concerning the in vivo tumor accumulation of radiolabeled octreotide analogues in AR42J tumor-bearing mice than their sst2-affinity. Also, in the case of [125I]Mtr-TOCA, [125I]Gluc-S-TOCA, and [125I]Gal-S-TOCA, tumor accumulation (Table 3) rather reflects the tendencies found for ligand internalization than those for sst2-affinity, but differences in tumor accumulation are by far not as pronounced as the observed variation in ligand internalization efficiency. This is not surprising, however, taking into account that tumor accumulation of the peptides investigated is very rapid and takes place early after ligand administration. The relatively fast blood clearance of the peptides, however, leads to rapidly decreasing tracer levels in blood and thus prevents optimal exploitation of the enhanced internalization efficiency of, for example, [125I]Gluc-STOCA as compared to [125I]Mtr-TOCA. Altogether, both in vitro uptake of [*I]Mtr-TOCA, [*I]Glucuron-TOCA, [*I]Gluc-S-TOCA, and [*I]Gal-S-TOCA into sst-expressing cells as well as their in vivo accumulation in sst-positive tissues seem to be mainly determined by the structure of the carbohydrate-modification introduced into the molecule, and not by the net charge of the respective peptide. This is not true, however, for the biodistribution of these tracers in nontarget organs. In the case of both [125I]Gluc-S-TOCA and [125I]Gal-STOCA, the reduced net charge leads to a loss in hydrophilicity as compared to [125I]Mtr-TOCA (log Pow ) -1.67 ( 0.02 vs -1.73 ( 0.04), which is reflected in the slightly increased accumulation of these tracers in liver and intestine. These data are in accordance with results from a previous study, which demonstrated a correlation between the lipophilicity (log Pow) of radioiodinated, glycosylated TOC analogues with peptide accumulation in these organs (10). Interestingly, the data obtained for [125I]Glucuron-TOCA are in contradiction to this correlation. Due to its higher charge density, the peptide has a nearly identical log Pow as compared to [125I]Mtr-TOCA (log Pow ) -1.74 ( 0.04 vs -1.73 ( 0.04). Both hepatic and intestinal uptake of [125I]Glucuron-TOCA, however, are increased by a factor of g2 as compared to the maltotriose analogue. Furthermore, instead of the expected reduction of kidney uptake due to reduced positive net charge, [125I]Glucuron-TOCA showed an increase in renal accumulation. In contrast, both [125I]Gluc-S-TOCA and [125I]Gal-S-TOCA having the same peptide net charge as [125I]Glucuron-TOCA (z ) 0), albeit a significantly different charge distribution within the peptide, showed the anticipated reduction in renal activity accumulation by up to 50% as compared to the reference peptide [125I]Mtr-TOCA. The same had been observed for the L-Asp1 analogue of [111In]DTPA-L-Phe1-octreotide, which showed an activity accumulation in the kidney of only approximately 7%ID/g at 1 h p.i. in normal mice as opposed to approximately 16%ID/g found for the L-Phe1parent peptide (21).

Schottelius et al.

Thus, based on these findings and on the results obtained with the two thioglycoside analogues investigated in this study, modulating peptide net charge can be efficiently used to modify ligand accumulation in the kidney. The unexpected biodistribution pattern of [125I]Glucuron-TOCA as compared to the other two peptides with z ) 0, however, indicates that not only peptide net charge, but also charge distribution may have a substantial impact on peptide uptake not only in the kidney, but also in the other excretion organs. Interestingly, and in contrast to the results obtained with respect to receptor targeting both in vitro and in vivo, the exact carbohydrate structure only seems to have secondary influence on general ligand biodistribution and excretion of radiolabeled sst-ligands. This is illustrated by the nearly identical pharmacokinetics of [125I]Gluc-S-TOCA and [125I]GalS-TOCA found in this study and is also supported by data from previous investigations comparing the pharmacokinetics of different sugar analogues of [125I]TOC and [125I]TOCA (10, 15). Especially in the case of the [125I]Gluc-S-TOCA/[125I]Gal-S-TOCA-pair, however, this finding was unexpected. A large variety of approaches for high-level (pre)targeting of bioactive molecules to the liver rely on the specific internalization of galactosylmodified compounds into parenchymal liver cells via a high-affinity receptor for asialoglycoproteins (34). Surprisingly, because liver accumulation of [125I]Gal-S-TOCA is very low at all time points investigated and nearly identical to that of [125I]Gluc-S-TOCA, this specific transport system does not seem to be targeted by [125I]Gal-STOCA. In conclusion, both the reduction of peptide net charge as well as the methodology employed for N-terminal carbohydrate conjugation of radioiodinated TOCA have a significant impact on peptide pharmacokinetics. The excretion profile and especially kidney accumulation of the four analogues investigated, [125I]Mtr-TOCA, [125I]Glucuron-TOCA, [125I]Gluc-S-TOCA, and [125I]Gal-STOCA, were found to be dominated by physicochemical characteristics such as lipophilicity, peptide net charge, and charge distribution within the peptide. A substantial influence of these factors on accumulation in sst-expressing cells and tissues, however, was not detectable, whereas it was greatly dependent on the structure of the N-terminal carbohydrate moiety. In this context, conjugation of TOCA with the Gluc-S-moiety allowed the most efficient optimization of peptide pharmacokinetics as compared to the reference [125I]Mtr-TOCA, a reduction of kidney accumulation by approximately 50%, and a substantial increase of ligand internalization in vitro and also of tumor uptake in vivo. The Gluc-S-group therefore seems to be a promising synthon for the synthesis of a variety of other neuropeptide analogues with improved pharmacokinetics. LITERATURE CITED (1) Polt, R., Porreca, F., Szabo, L. Z., Bilsky, E. J., Davis, P., Abbruscato, T. J., Davis, T. P., Horcath, R., Yamamura, H. I., and Hruby, V. (1994) Glycopeptide enkephalin analogueues produce analgesia in mice: Evidence for penetration of the blood-brain barrier. Proc. Natl. Acad. Sci. U.S.A. 91, 71147118. (2) Mitchell, S. A., Pratt, M. R., Hruby, V. J., and Polt, R. (2001) Solid-Phase Synthesis of O-Linked Glycopeptide Analogueues of Enkephalin. J. Org. Chem. 66, 2327-2342. (3) Bilsky, E. J., Egleton, R. D., Mitchell, S. A., Palian, M. M., Davis, P., Huber, J. D., Jones, H., Yamamura, H. I., Janders, J., Davis, T. P., Porreca, F., Hruby, V. J., and Polt, R. (2000) Enkephalin glycopeptide analogueues produce analgesia with reduced dependence liability. J. Med. Chem. 43, 2586-2590.

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