Improvement of Pharmacokinetics of Radioiodinated Tyr3-Octreotide

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Bioconjugate Chem. 2002, 13, 1021−1030

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Improvement of Pharmacokinetics of Radioiodinated Tyr3-Octreotide by Conjugation with Carbohydrates Margret Schottelius,† Hans-Ju¨rgen Wester,*,† Jean Claude Reubi,‡ Reingard Senekowitsch-Schmidtke,† and Markus Schwaiger† 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 January 16, 2002; Revised Manuscript Received May 10, 2002

Among a variety of other factors, the clearance kinetics and routes of excretion of radiopharmaceuticals are of crucial importance for early and high tumor/background ratios and thus signal intensity in diagnostic imaging by single photon emission tomography (SPECT) or positron emission tomography (PET). To overcome the unfavorable pharmacokinetics of radiohalogenated octreotide analogues, we evaluated three carbohydrated conjugates of Tyr3-octreotide (TOC). Glucose ([125I]Gluc-TOC), maltose ([125I]Malt-TOC), and maltotriose ([125I]Mtr-TOC) derivatives of [125I]TOC were synthesized via Maillard reaction and subsequent radioiodination. In cells transfected with sst1-sst5, I-Malt-TOC, and I-MtrTOC show sst-subtype binding profiles similar to I-TOC with high affinity for sst2. Comparative biodistribution studies 10, 30, and 60 min pi in nude mice bearing rat pancreatic tumor xenografts showed fast blood clearance for all glycosylated derivatives. Due to their markedly increased hydrophilicity, [125I]Gluc-TOC and [125I]Malt-TOC were mainly cleared via the kidneys, which led to a significant decrease in activity accumulation in liver and intestine (5.3 and 1.4 versus 10.6%ID/g for [125I]TOC in the liver, 1.7 and 1.0 versus 3.8%ID/g for [125I]TOC in the intestine 60 min pi). For all compounds, hydrophilicity and uptake in liver and intestines correlate. Uptake of the carbohydrate conjugates in the kidney was comparable. Compared to the parent compound, the accumulation of the carbohydrated compounds in sst-rich tissues (pancreas, adrenals) was increased by a factor of 1.5-3.5. While tumor uptake of [125I]TOC (6.7 ( 2.6%ID/g), [125I]Malt-TOC (5.3 ( 1.9%ID/g), and [125I]Mtr-TOC (4.9 ( 2.2%ID/g) at 30 min postinjection was comparable, accumulation of [125I]Gluc-TOC was significantly increased (10.1 ( 2.8%ID/g at 30 min pi). Somatostatin receptor specificity of tumor uptake was confirmed by pretreatment, competition, and displacement experiments in vivo using 0.8 mg TOC/kg and γ-camera imaging. Glycosylation proved to be a powerful tool for the development of high affinity sst ligands with excellent excretion profiles and improved tumor accumulation.

INTRODUCTION

On the basis of the findings that many human tumors overexpress a variety of receptors for regulatory peptides and peptide hormones (1-6), neuropeptide receptor targeted diagnostic imaging and radiotherapy has become a major concept for the detection, localization, and therapeutic intervention of malignant neoplasms in Nuclear Medicine (7-11). For this purpose, a number of radiolabeled analogues of endogenous peptides are presently investigated and under preclinical evaluation: e.g., analogues of somatostatin (SS) (9, 12, 13), substance P (14), gastrin (15), bombesin (BB) (16), R-melanocyte stumulation hormone (R-MSH) (17, 18), neurotensin (NT) (19), or vasoactive intestinal peptide (VIP) (20). The clinical potential of these tracers will rely, among a variety of other factors, such as high receptor affinity and selectivity, high metabolic stability, low nonspecific uptake, and high specific accumulation, upon two main factors with crucial importance for early and high tumor/ * To whom correspondence should be addressed. HansJ.Wester, Ph.D., Nuklearmedizinische Klinik und Poliklinik, Klinikum rechts der Isar, Technische Universita¨t Mu¨nchen, Ismaninger Strasse 22, D - 81675 Mu¨nchen, Germany. Tel: 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.

background ratios, namely favorable blood clearance and excretion kinetics. New radiolabeling strategies mainly focus on improved in vivo stability against dehalogenation (e.g., 21, 22, 23) or transchelation (e.g., 24, 25, 26), fast and high yield radiolabeling, or basic aspects such as radiolabeling without affecting biological integrity (27, 28) of the peptide investigated. However, peptide radiotracers often only suffer from disadvantages of less than optimal blood clearance, excretion pathways and nonspecific uptake (29, 30). Thus, to generate more adequate radioligands, the use and evaluation of suitable chemical tools with broad utility is recommended. What are the demands for such a tool? To increase target-to-nontarget tissue ratio (tumor-to-nontumor ratio), the tracer should be rapidly cleared from the circulation. Furthermore, to reduce the whole body radiation exposure, the modification used should reduce hepatobiliary excretion. The tracer should be cleared via the kidneys. However, to avoid nephrotoxicity after application of therapeutic dosages, renal uptake should be minimized ([90Y]DOTATOC) (31, 32). Receptor affinity and selectivity should remain unchanged, and the modification used has to be compatible with the radiolabeling strategy, thus not affecting radiolabeling yield. To influence receptor binding and enhance peptide delivery to the brain or spinal cord, attachment of carbohydrate groups to enkephalin peptides has been

10.1021/bc0200069 CCC: $22.00 © 2002 American Chemical Society Published on Web 08/31/2002

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demonstrated in vivo by pretreatment, competition, and displacement experiments. MATERIALS AND METHODS

Figure 1. Structures of [125I]Gluc-TOC, [125I]Malt-TOC, and [125I]Mtr-TOC.

studied (33-36). It has been shown, that glycosyl groups, if properly placed, will preserve previously determined structure-activity relations (SAR) of the native peptides (34, 35, 37, 38). Enkephalin analogues glycosylated in the near or at the C-terminus were shown to elicit prolonged and profound analgesia in mice (34, 35). Furthermore it has been demonstrated, that the in vivo stability of an (N-linked) glycopeptide renin inhibitor is enhanced (39). Glucosyl, mannosyl, galactosyl, and 2-deoxy-glucosyl derivatives of Arg8-vasopressin exhibited a sugar dependent renal uptake and intrarenal distribution (40). Glycosylated vasopressin analogues displayed an increased bioavailability resulting from absorption from the small intestine and most likely from an increased stability toward enzymatic degradation (41). Studies on the uptake of a glycosylated derivative of the tetrapeptide GlyGly-Tyr-Arg into brush border membrane vesicles and transport though the intestinal membrane has indicated increased resistance to aminopeptidases. Furthermore, the Na+-dependent glucose transporter SGLT-1 played an important role in the intestinal absorption of both the R- and β-glucopyranoside derivative (42). Recently, sugar-conjugated integrin antagonists have been investigated in our laboratory (27, 30, 43). It was shown that coupling of sugar amino acids significantly improves the pharmacokinetics of RVβ3-integrin antagonists (27, 43). Furthermore we introduced radiolabeled carbohydrated octreotide analogues as a new series of somatostatin receptor (sst) binding radiotracers with excellent physicochemical characteristics (44-48). The goal of the current study was to directly compare three carbohydrated conjugates of Tyr3-octreotide (TOC), an agent previously evaluated for targeted imaging and radiotherapy of somatostatin receptor (sst) expressing tumors. The glucose ([125I]Gluc-TOC), maltose ([125I]MaltTOC), and maltotriose ([125I]Mtr-TOC) derivatives of [125I]TOC were synthesized via formation of an N-terminal NR-glycosylamine with the respective aldose followed by Amadori rearrangement (48) and subsequent radioiodination (Figure 1). The affinity profiles of I-Malt-TOC, I-Mtr-TOC and I-TOC were assessed in cells stably expressing sst1-sst5. We compared the effects of carbohydration on biodistribution in rat pancreatic tumor (AR42J) xenografted mice, particularly on hepatic uptake, biliary excretion, renal uptake, and tumor accumulation and demonstrate the suitablity of carbohydration to modify the physicochemical tracer characteristics in vivo by γ-camera imaging of tumor bearing mice. Specificity of tumor uptake was

Instrumentation. Analytic reversed phase high performance liquid chromatography (RP-HPLC) was performed using a Sykam gradient HPLC System (Sykam GmbH, Gilching, Germany) and a Nucleosil 100 C18 (5 mm, 250 × 4.0 mm) column. The peptides were eluted (if not stated otherwise in the text) applying a gradient of 10-60% B (solvent A: 0.1% TFA (trifluoro acetic acid) in H2O, solvent B: 0.1% TFA in acetonitrile) in 30 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 Corporation, Reno, NV). Preparative RP-HPLC of all peptides was performed on the Sykam HPLC system using a Multospher 100 RP 18-5 (250 × 10 mm) column 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. Sample preparation for peptide sequence analysis was performed according to the following protocol: For reduction of the disulfide bridge, 1 nmol of the synthetic peptide was dissolved in 5 µL of 10 mM NH4HCO3 containing 10 mM dithiothreitol and incubated at 56 °C for 30 min. The reduced peptide (10 pmol) was desalted over a POROS R2 microcolumn (Perseptive, Framingham, MA), packed in a GELoader Tip (Eppendorf, Hamburg, Germany), and eluted with 1 µL of 5% formic acid/60% methanol directly into the nanoelectrospray needle (MDS Proteomics, Odense, Denmark). All experiments were performed on a QSTAR Pulsar quadrupole time-of-flight tandem mass spectrometer (AB/ MDS Sciex, Toronto, Canada) equipped with a nanoelectrospray ion source (MDS Proteomics). The MS experiments were performed in the positive ion mode. Nitrogen was used as the collision gas at a pressure of 5.3 × 10-5 Torr. Peptide fragmentation data are listed according to the Roepstorff-Fohlmann-Biemann nomenclature (Figure 2). Purification of all radioiodinated peptides was carried out on a J’Sphere R&D ODS-H80 (4 mm, 150 × 4.6 mm, 80A) column using a Sykam HPLC system connected to a UVIS 200 Photometer (Linear Instruments Corporation, Reno, NV) using a gradient of 10-60% B in 15 min. For radioactivity measurement, the outlet of the UVphotometer was connected to a NaI(Tl) well-type scintillation counter from EG&G (Mu¨nchen, Germany). Radioactivity measurements for the determination of lipophilicity and biodistribution assays were performed on a 1480 Wizard 3 gamma counter from Wallac (Turku, Finland). Reagents. Fmoc-(9-fluorenylmethoxycarbonyl) amino acids as well as the DHP-HM (3,4-Dihydro-2H-pyran2-ylmethoxymethylpolystyrene) resin were purchased from novabiochem (Bad Soden, Germany). IodoGen (1,3,4,6-tetrachloro-3R,6R-diphenylglycoluril) came from Pierce (Rockford, IL). N.c.a. [125I]NaI and [123I]NaI were supplied by Amersham (Buckinghamshire, UK). All other organic reagents were purchased from Merck Eurolab (Darmstadt, Germany), Alexis (Gru¨nberg, Germany), Aldrich, or Fluka (Neu-Ulm, Germany). Synthesis of the Glycosylated Derivatives of Tyr3Octreotide. Reduction of Fmoc-Thr(tBu)-OH to the Corresponding Amino Alcohol (49). A solution of FmocThr(tBu)-OH (1.391 g, 3.5 mmol) in 30 mL of THF

Pharmacokinetics of Radioiodinated Tyr3-Octreotide

Bioconjugate Chem., Vol. 13, No. 5, 2002 1023

Figure 2. Fragmentation scheme and fragment nomenclature for Tyr3-octreotide and Gluc-Tyr3-octreotide in MS/MS-sequence analysis. Data are given in Tables 1 and 2.

(tetrahydrofuran) was cooled to -10 °C. N-Methylmorpholine (386 µL, 3.5 mmol) and ethyl chloroformate (333 µL, 3,5 mmol) were added successively. The reaction mixture was stirred at -10 °C for 30 min. Then NaBH4 (396 mg,10.5 mmol) was added. Over a period of 30 min, 60 mL of MeOH (methanol) were slowly added to the reaction mixture, which was then stirred at 0 °C for 3 h. After adding 50-70 mL of 1 N HCl (the cloudy reaction mixture has to become transparent), the organic solvents were evaporated and the remaining aequous phase was extracted twice with DCM (dichloromethane). The combined organic layers were dried over MgSO4 and evaporated to dryness. The crude product, a yellowish oil, was purified by flash chromatography (ethyl acetate). Yield: 94%. Calculated monoisotopic mass for C23H29NO4 ) 383.21; found: m/z ) 406.1 [M + Na]+ 1H NMR (CDCl3): δ(ppm) 1.16 (3H, d, J ) 6.2 Hz, CHCH3), 1.20 (9H, s, tBu), 2.88 (1H, broad, OH), 3.61 (1H, broad, CHCH2OH), 3.66 (2H, broad, CHCH2OH), 3.94 (1H, m, CHCH3), 4.22 (1H, t, J ) 6.8 Hz, CHCH2CO), 4.40 (2H, m, CHCH2CO), 5.28 (1H, d, J ) 7.5 Hz, NH), 7.30 (2H, d, J ) 7.4 Hz, aromatics), 7.38 (2H, t, J ) 7.2 Hz, aromatics), 7.59 (2H, d, J ) 7.4 Hz, aromatics), 7.74 (2H, d, J ) 7.4 Hz, aromatics). SPPS of Tyr3-Octreotide (TOC) (50). DHP-HM-resin (1.000 g, load: 0.94 mmol linker/g) was allowed to preswell in 10 mL of dry DCE (1,2-dichloroethane) for 1 h. Then a solution of Fmoc-Thr(tBu)-ol (1.266 g, 3.3 mmol) and of pyridinium-p-toluenesulfonate (414 mg, 1.75 mmol) in 7 mL of abs. DCE was added, and the reaction mixture was stirred overnight at 80 °C under argon. To cap free functional groups on the resin, 5 mL of pyridine was added, and the suspension was stirred for another 15 min at RT. The loaded resin was then filtered off, washed twice with DMF (N,N-dimethylformamide) and DCM, respectively, and dried in vacuo. Synthesis of the peptide sequence Cys(Trt)-Thr(tBu)Lys(Boc)-DTrp-Tyr-Cys(Trt)-DPhe-NH2 on the resinbound amino alcohol was performed according to Standard Fmoc-protocol using 1.5 equiv of HOBt (1hydroxybenzotriazole) and TBTU (O-(1H-benzotriazol-1yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate) as coupling reagents. After removal of the N-terminal Fmoc protection-group with 20% piperidine in DMF, the peptide was cleaved from the resin using a mixture of 95% TFA/2.5% TIBS (triisobutylsilane)/2.5% H2O and DCM (1:1). After 90 min, the resin was filtered and washed twice with DCM. The combined filtrates were concentrated in vacuo, and the crude product was precipitated with Et2O (diethyl ether). Cyclization of TOC. The crude peptide was resuspended in 50 mL of THF (per 300 mg of peptide), and 5 mM NH4OAc was added, until a clear solution was obtained. The pH was adjusted to 7 by dropwise addition

Table 1. Fragmentation of Tyr3-Octreotide in MS/ MS-sequence Analysis. Fragment Nomenclature and Fragmentation Schemes Are Given in Figure 2 fragment

sum formula

calculated mass [m/z]

found [m/z]

y1 y2 y3 y4 y5 y6 y7 b2 b3 b4 b5 b6 b7 [M + 2H]2+

[C4H12NO2]+ [C7H17N2O3S]+ [C11H24N3O5S]+ [C17H36N5O6S]+ [C28H46N7O7S]+ [C37H55N8O9S]+ [C40H60N9O10S2]+ [C12H15N2O2S]+ [C21H24N3O4S]+ [C32H34N5O5S]+ [C38H46N7O6S]+ [C42H53N8O8S]+ [C45H58N9O9S2]+ [C49H70N10O11S2]+

106.09 209.10 310.14 438.24 624.32 787.38 890.39 251.09 414.15 600.23 728.32 829.37 932.38 519.23

106.08 209.09 310.14 438.24 624.31 787.38 890.39 251.08 414.14 600.21 728.30 829.36 932.36 519.22

of saturated NaHCO3 solution. Then 100 µL (per 300 mg of peptide) of H2O2 (30%) were added. After 30 min of stirring at room temperature cyclization was complete (gradient HPLC control: 30f80% B in 30 min). The solvents were evaporated, and the cyclized product was lyophilized. Purification was performed using preparative gradient RP-HPLC. HPLC (10f60% in 30 min): tR (linear peptide) ) 14.1 min; K′ ) 4.96; tR (cyclic peptide) ) 12.9 min; K′ ) 4.48; Calculated monoisotopic mass for TOC (C49H66N10O11S2) ) 1034.49; found: m/z ) 1035.2 [M + H]+; the fragmentation of Tyr3-octreotide (linear) in MS/MS-sequence analysis is shown in Table 1. Boc Protection of the Lys5-Side Chain. The peptide was redissolved in DMF (1 mL/100 mg). After the addition of 1.1 equiv (with respect to free Lys-N-amino groups in the peptide) of Boc2O (di-tert-butyl dicarbonate) in DMF, the solution was stirred at RT for 30 min. The protected peptide was precipitated by the addition of Et2O, washed with Et2O, and dried in vacuo. HPLC (30f80% in 30 min): tR ) 10.94 min; K′ ) 3.63; calculated monoisotopic mass for C54H74N10O13S2 ) 1134.49; found: m/z ) 1135.3 [M + H]+. Conjugation with Carbohydrates. On the basis of a method reported previously (51), 1 equiv of Lys5(Boc)TOC and 10 equiv of the respective aldose (glucose, maltose, maltotriose) were dissolved in methanol/acetic acid (95/5 (v/v)), and the reaction mixture was stirred at 60°C for 24-48 h. The solvents were then evaporated, and the crude product was precipitated by the addition of Et2O. Boc Deprotection. A solution of protected peptide in TFA/H2O/TIBS (95:2,5:2,5 (v/v/v); 1 mL/100 mg) was stirred at RT for 15-60 min. The deprotected peptide was precipitated by the addition of Et2O, washed with Et2O,

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Table 2. Fragmentation of Gluc-Tyr3-octreotide in MS/MS Sequence Analysis. Fragment Nomenclature and Fragmentation Schemes Are Given in Figure 2 fragment

sum formula

calculated mass [m/z]

found [m/z]

y1 y2 y3 y4 y5 y6 y7 a1 a1 - H2O b1 b1 - H2O b2 - H2O b4 - 3 H2O b5 - 3 H2O b6 - 3 H2O b7 - 3 H2O [M + 2H]2+

[C4H12NO2]+ [C7H17N2O3S]+ [C11H24N3O5S]+ [C17H36N5O6S]+ [C28H46N7O7S]+ [C37H55N8O9S]+ [C40H60N9O10S2]+ [C14H20NO5]+ [C14H18NO4]+ [C15H20NO6]+ [C15H18NO5]+ [C18H23N2O6S]+ [C38H38N5O7S]+ [C44H50N7O8S]+ [C48H57N8O19S]+ [C51H62N9O10S2]+ [C55H80N10O16S2]+

106.09 209.10 310.14 438.24 624.32 787.38 890.39 282.13 264.12 310.13 292.12 395.13 708.25 836.34 937.39 1040.40 600.26

106.08 209.09 310.14 438.24 624.32 787.38 890.37 282.14 264.12 310.14 292.13 395.12 708.28 836.34 937.39 1040.41 600.26

and dried in vacuo. Purification was carried out using preparative gradient RP-HPLC. Gluc-TOC: HPLC (10f60% in 30 min): tR ) 13.1 min; K′ ) 4.13; calculated monoisotopic mass for C55H76N10O16S2 ) 1196.5, found: m/z ) 1197.1 [M + H]+, 1219.7 [M + Na]+. Fragmentation of Gluc-TOC (linear) in MS/MS-sequence analysis is shown in Table 2. Malt-TOC: HPLC (10f60% in 30 min): tR ) 12.56 min; K′ ) 3.85; calculated monoisotopic mass for C61H86N10O21S2 ) 1358.54, found: m/z ) 1359.1 [M + H]+. Mtr-TOC: HPLC (10f60% in 30 min): tR ) 13.07 min; K′ ) 3.78; calculated monoisotopic mass for C67H96N10O26S2 ) 1520.59, found: m/z ) 1521.1 [M + H]+. Radioiodination. TOC, Gluc-TOC, Malt-TOC, and Mtr-TOC were labeled with 125I (or 123I for γ-camera imaging) using the IodoGen method. A solution of 100500 µg of peptide in 200 µL of PBS (phosphate-buffered saline, pH 7.4) was transferred to an Eppendorf cap coated with 150 µg of IodoGen. A 5-10 µL (18-37 MBq) amount of n.c.a. [125I]NaI (specific activity > 2000 Ci/ mmol) or c.a. [123I]NaI (specific activity ∼5000 Ci/mmol) was added. The labeling reaction was allowed to proceed for 20 min at RT. The peptide solution was then removed from the insoluble oxidizing agent. Separation of the labeled products was achieved using gradient RP-HPLC. For biodistribution experiments, the collected fraction was evaporated to dryness, and the residue was redissolved in PBS to yield a solution of radiolabeled peptide with an activity concentration of about 370 kBq/100 µL. Determination of Lipophilicity. To a solution of approximately 2 kBq of radiolabeled peptide in 500 µL of PBS (pH 7.4) was added 500 µL of octanol (n ) 6). Vials were vortexed vigorously for 3 min. To achieve quantitative phase separation, the vials were centrifuged at 14600g for 6 min in a Biofuge 15 (Heraeus Sepatech, Osterode, Germany). The activity concentrations in 100 µL samples of both the aequous and the organic phase were measured in a γ-counter. Both the partition coefficient Pow, which is defined as the molar concentration ratio of a single species A between octanol and water at equilibrium, and log Pow, which is an important parameter used to characterize lipophilicity of a compound (52), were calculated. Determination of the Affinity Profiles to hsst. Cells stably expressing human sst1, sst2, sst3, sst4, and sst5 were grown as described previously (53). All culture reagents were supplied by GIBCO/BRL and Life Tech-

nologies (Grand Island, NY). Cell membrane pellets were prepared, and receptor autoradiography was performed on pellet sections (mounted on microscope slides), as described in detail previously (53). 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, crosscalibrated to tissue equivalent ligand concentrations, were used for quantification (53). Tumor Xenografts. AR42J is a rat pancreatic acinar tumor cell line with high somatostatin receptor (sst2) expression (54) and was obtained from ECACC (European Collection of Cell Cultures, Salisbury, UK). Cells were maintained in RPMI 1640 medium supplemented with 10% FCS (Biochrom Seromed (Berlin, Germany)) and 2 mM L-glutamine (Gibco BRL Life Technologies (Karlsruhe, Germany)) at 37 °C and 5% CO2. Animal Model. For all in vivo experiments, nude mice (CD1 nu/nu, male and female, 6-8 weeks) were used. To establish tumor growth, subconfluent monolayer cells were treated with 1 mM EDTA in PBS, suspended, centrifuged, and resuspended in serum free RPMI 1640. Mice were inoculated subcutaneously in the flank with 2.5-3 × 10E6 cells. Ten days postinoculation all mice showed solid palpable tumor masses (tumor weight 150500 mg) and were used for the experiments. Biodistribution Studies. About 370 kBq (10 µCi) of the 125I-labeled peptides in 100 µL of PBS (pH 7.4) were injected intravenously (iv) into the tail vein of nude mice bearing an AR42J tumor. The animals were sacrificed 10, 30, and 60 min postinjection (pi) (n ) 5), and the organs of interest were dissected. The radioactivity was measured in weighted tissue samples using a γ-counter. Data are expressed in %ID/g tissue (mean ( SD). Pretreatment, Coinjection, and Displacement Studies. For pretreatment studies, 0.8 mg/kg Tyr3octreotide (20 µg/mouse) was injected iv into the tail vein as a 100 µL bolus 10 min prior to the injection of 370 kBq (10 µCi) of radioligand in 100 µL of PBS. Coinjection was performed injecting a solution of radioligand and 0.8 mg/kg Tyr3-octreotide (20 µg/mouse) in 100 mL of PBS. For displacement studies, 0.8 mg/kg Tyr3-octreotide (20 µg/mouse) were administered 10 min after the injection of the radioiodinated tracer. All animals were sacrificed 30 min pi of the radioligand. Subsequent determination of the activity accumulation in all organs of interest was performed as described above (biodistribution studies). γ-Camera Imaging. The 123I-labeled peptides (3.7 MBq (100 µCi) in 100 mL of PBS (pH 7.4)) were injected iv into the tail vein of nude mice bearing an AR42J tumor. Mice were euthanized at 120 min pi of the radioligand. Acquisition of γ-camera images was performed on a Siemens Diacam γ-camera (20 min, medium energy general purpose collimator) by positioning the mice directly on the collimator. Region-of-interest (ROI) analysis was performed using the IconTM software, version 5.2.1 (1994), from Siemens Medical Systems, applying the RegionRatio protocol. RESULTS

Peptide Synthesis. Solid-phase peptide synthesis yielded TOC in 82-86% yield, based on the amount of

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Bioconjugate Chem., Vol. 13, No. 5, 2002 1025

Table 3. Lipophilicity of [125I]TOC and Its Glycosylated Derivatives (n ) 6, values are mean ( SD)) [125I]TOC POW 1.50 ( 0.09 log POW 0.18 ( 0.03

[125I]Gluc-TOC [125I]Malt-TOC [125I]Mtr-TOC 0.26 ( 0.01 -0.59 ( 0.02

0.06 ( 0.01 -1.21 ( 0.03

0.10 ( 0.01 -0.99 ( 0.03

Fmoc-Thr(tBu)ol initially coupled to the resin, and in UVpurities > 90%. Subsequent synthesis steps were performed without further purification of the peptide. Bocprotection of the Lys5-side chain usually led to quantitative transformation of the precursor. Final yields of the glycosylated octreotide derivatives generally ranged from 14 to 33% with respect to Boc-protected starting peptide. The glycosylation reaction itself usually proceeded smoothly within 16 h and yielded ratios of glycosylated product to precursor of 60/40 up to 90/10 (values are based on the results of the integration of RP-HPLC chromatograms (UV absorption at 220 nm); impurities were excluded from integration). Product-to-precursor ratios were higher for the maltose and maltotriose derivatives than for the glucose derivative. The moderate overall yields of the sugar-octreotide conjugates can mainly be attributed to the preparative HPLC conditions. On the RP-18 column generally used, baseline-separation of the carbohydrate conjugate and the nonglycosylated precursor could hardly be achieved, even when a very slow gradient was used. The structure of radioiodinated Gluc-TOC is shown in Figure 1. For the sugar moiety, only one of four possible conformations is shown. Beside the β-pyranoid structure (Figure 1), the R- and β-furanoid and the open-chain conformations of the deoxy-ketoses are possible and may be present in equilibrium mixtures (55).

Radiolabeling. Radioiodination efficiency generally exceeded 99%. Radiochemical yields ranged from 45 to 85% and were limited both by formation of radioiodinated sideproducts and by restrictions during [125I]glycopeptide product-fractioning caused by tailing of the unreacted precursor. After RP-HPLC isolation, all radioiodinated compounds were obtained in high radiochemical purity (>99.8%). Specific activities of all 123I- and 125I-labeled compounds were >1800 Ci/mmol. Lipophilicity. The lipophilicity of the radioiodinated compounds is shown in Table 3. While log Pow for the parent compound [125I]TOC is > 0, all glycosylated derivatives exhibited log Pow values below zero in the following order: log Pow [125I]Gluc-TOC > log Pow [125I]Mtr-TOC > log Pow [125I]Malt-TOC. Affinity Profiles to hsst. In vitro binding properties of the three compounds were evaluated in cells stably expressing sst1-sst5 in displacement experiments using [125I]-]Leu8,D-Trp22,Trp25]-somatostatin 28 as radioligand. Table 4 shows the IC50 of somatostatin-28, I-TOC, I-MaltTOC, and I-Mtr-TOC to the five somatostatin receptor subtypes sst1-5. Natural somatostatin 28, which binds with high affinity to all five subtypes, was used as control peptide. Both sugar conjugates of octreotide showed a binding profile to sst similar to I-TOC: no affinity to sst1, high affinity to sst2, and moderate or low affinity to sst3-5. In Vivo Biodistribution. The tissue distributions of all radioiodonated tracers in AR42J tumor bearing nude mice 10, 30, and 60 min postinjection are shown in Table 5.

Table 4. Affinity Profiles (IC50) of Somatostatin 28 and the Compounds Investigated for Human sst1-sst5 Receptors (values are IC50 ( SD [nM], Number of Experiments in Parentheses) peptide

sst1

sst2

sst3

sst4

sst5

SS-28 I-TOCa I-Malt-TOCb I-Mtr-TOCc values are mean ( SD

3.6 ( 0.3 (5) >10000 (3) >10000 (3) >10000 (3)

2.1 ( 0.3 (5) 1.3 ( 0.3 (3) 1.2 ( 0.2 (3) 1.1 ( 0.2 (3)

3.2 ( 0.2 (5) 128 ( 22 (3) 243 ( 47 (3) 210 ( 15 (3)

3 ( 0.3 (5) 867 ( 33 (3) >1,000 (2) >1,000 (3)

2.5 ( 0.2 (5) 50 ( 12 (3) 75 ( 19 (3) 110 ( 35 (3)

a (3-iodo-Tyr3)-octreotide. b NR-(R-D-glucopyranosyl-(1-4)-1-deoxy-D-fructosyl)-(3-iodo-Tyr3)-octreotide. c NR-(O-R-D-glucopyranosyl-(14)-(O-R-D-glucopyranosyl-(1-4)-1-deoxy-D-fructosyl)-(3-iodo-Tyr)3-octreotide.

Table 5. Tissue Biodistribution [%ID/G] of [125I]TOC and Its Glycosylated Derivativesa organ blood liver intestine kidney tumor pancreas adrenals muscle values are mean ( SD a

time pi [min]

[125I]TOC

[125I]Gluc-TOC

[125I]Malt-TOC

10 30 60 10 30 60 10 30 60 10 30 60 10 30 60 10 30 60 10 30 60 10 30 60

1.69 ( 0.54 1.06 ( 0.24 1.23 ( 0.30 7.36 ( 0.92 5.95 ( 1.19 3.76 ( 1.28 3.58 ( 1.20 7.02 ( 1.32 10.60 ( 2.81 5.55 ( 0.71 6.70 ( 1.59 6.95 ( 1.44 4.95 ( 1.53 6.72 ( 2.60 5.53 ( 1.06 1.65 ( 0.55 1.22 ( 0.37 1.17 ( 0.32 1.63 ( 0.76 1.63 ( 0.74 1.25 ( 0.21 0.39 ( 0.14 0.18 ( 0.03 0.26 ( 0.08

2.60 ( 0.45 1.58 ( 0.48 0.88 ( 0.38 3.51 ( 0.77 2.44 ( 0.16 1.73 ( 0.59 2.80 ( 0.72 3.99 ( 1.19 5.28 ( 1.27 7.44 ( 1.77 6.35 ( 0.66 6.25 ( 1.94 4.42 ( 1.15 10.12 ( 2.81 8.73 ( 1.45 4.10 ( 0.82 4.48 ( 0.67 3.36 ( 1.20 2.08 ( 0.40 2.40 ( 0.73 2.05 ( 0.97 0.60 ( 0.13 0.39 ( 0.37 0.17 ( 0.05

3.42 ( 1.09 1.38 ( 0.20 0.74 ( 0.13 2.43 ( 0.78 1.33 ( 0.32 0.96 ( 0.17 1.46 ( 0.47 1.25 ( 0.55 1.48 ( 0.18 9.63 ( 3.24 7.23 ( 1.39 6.78 ( 1.52 6.79 ( 2.58 5.30 ( 1.87 5.24 ( 0.61 3.61 ( 1.71 2.21 ( 0.59 1.92 ( 0.26 3.08 ( 0.92 2.07 ( 0.21 1.93 ( 0.68 0.94 ( 0.79 0.33 ( 0.14 0.20 ( 0.08

[125I]Mtr-TOC 2.66 ( 0.19 2.66 ( 0.17 3.31 ( 0.58 9.61 ( 1.69 4.87 ( 2.15 2.12 ( 0.44 2.98 ( 0.82 0.51 ( 0.08

Groups of five nude mice bearing AR42J tumors. Values are mean ( SD. %ID/g ) percentage of injected dose per gram tissue

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Figure 3. Activity accumulation of [125I]TOC (A), [125I]GlucTOC (B), [125I]Malt-TOC (C) and [125I]Mtr-TOC (D) in liver and intestine of AR42J tumor bearing nude mice 10, 30, and 60 min pi (n ) 5, mean ( SD).

While [125I]TOC was rapidly cleared from the blood via hepatobiliary excretion, resulting in high initial liver uptake (7.36 ( 0.92%ID/g 10 min pi), the glycosylated derivatives exhibited delayed initial blood clearance. Activity accumulation in the liver was decreased for all glycosylated analogues, in particular for [125I]Malt-TOC. The maltose derivative exhibited only 25% of the liver uptake of the parent compound 60 min pi, while for [125I]TOC activity accumulation in the intestine reached 10.6 ( 2.8%ID/g at 60 min pi, intestinal uptake was reduced to 5.28 ( 1.27%ID/g for [125I]Gluc-TOC and to 1.48 ( 0.18%ID/g for [125I]Malt-TOC (Figure 3). Renal activity accumulation for [125I]TOC, [125I]Gluc-TOC, and [125I]Malt-TOC remained almost unchanged (6.95 ( 1.44%ID/g for [125I]TOC, 6.25 ( 1.94 for [125I]Gluc-TOC and 6.78 ( 1.52%ID/g for [125I]Malt-TOC 60 min pi). While for sst-rich tissues such as pancreas and adrenals activity accumulation was increased by a factor of 1.53.5 for all glycosylated derivatives compared to [125I]TOC, tumor uptakes of [125I]TOC and [125I]Malt-TOC were comparable at all time points. In contrast, [125I]Gluc-TOC exhibited an increase in tumor uptake of up to 160% compared to [125I]TOC. For [125I]Mtr-TOC, highest activity concentrations 30 min pi could be detected in the kidneys (9.61 ( 1.69%ID/ g), the tumor (4.87 ( 2.15%ID/g) and in the intestine (3.31 ( 0.58%ID/g). Pretreatment, Competition, and Displacement Experiments. Both for [125I]Gluc-TOC and for [125I]MaltTOC binding to sst-rich tissues in vivo was demonstrated to be specific. While tumor accumulation was significantly reduced in the pretreatment and co-injection experiments compared to the control experiment, resulting in tumor/sst-negative tissue ratios < 1 (Figure 4), displacement of the radioiodinated tracers by injection of 20 µg of Tyr3-octreotide/mouse 10 min pi of the labeled compound was less effective, demonstrating fast internalization of the radiotracer.

Figure 4. Tumor/organ ratios for [125I]Malt-TOC and [125I]GlucTOC under different experimental conditions in AR42J tumor bearing nude mice 30 min pi (n ) 3, mean ( SD) pretreatment: injection of 0.8 mg/kg TOC (20 µg/mouse) 10 min prior to injection of the respective radioiodinated tracer, competition: injection of 0.8 mg/kg TOC (20 µg/mouse) with the respective radioiodinated tracer, displacement: injection of 0.8 mg/kg TOC (20 mg/mouse) 10 min after injection of the respective radioiodinated tracer.

γ-Camera Imaging. Images acquired 120 min pi of [123I]TOC, [123I]Gluc-TOC, and [123I]Malt-TOC in AR42J tumor-bearing nude mice are shown in Figure 5. In the case of [123I]TOC, the tumor is barely discernible from the high intestinal background. ROI analysis revealed a low tumor/intestine ratio of 0.62 (0.52 ( 0.17 in the biodistribution study 60 min pi). As expected from the biodistribution data, [123I]Gluc-TOC still shows considerable nonspecific accumulation in the intestine, but also an increased tumor/intestine ratio due to its higher tumor accumulation (ROI analysis: 1.48; biodistribution study (60 min pi): 1.65 ( 0.48). This results in an improved contrast of the scintigraphic image. When [123I]Malt-TOC was administered, only the kidneys and the tumor showed significant uptake of the radioligand, while abdominal activity accumulation was negligible. The tumor/kidney ratio determined via ROI analysis (0.81) is in accordance with the value found in the biodistribution study (0.77 ( 0.19 at 60 min pi). In all three scintigraphic images, the stomach is also visible. The intensity of activity accumulation in stomach shows some correlation with the tumor uptake of the respective radioligand. This observation is in accordance with the detection of somatostatin receptors in stomach in rats (56).

Pharmacokinetics of Radioiodinated Tyr3-Octreotide

Bioconjugate Chem., Vol. 13, No. 5, 2002 1027

Figure 5. γ-Camera images of AR42J tumor-bearing nude mice 120 min pi of 100 µCi of [125I]TOC (A), [125I]Gluc-TOC (B), and [125I]Malt-TOC (C). Large arrows indicate the tumor, small arrows indicate the kidneys, dashed arrows indicate the stomach.

Figure 6. Correlation between the activity accumulation of [125I]Malt-TOC (A), [125I]Mtr-TOC (B), [125I]Gluc-TOC (C), and [125I]TOC (D) in liver and intestine of AR42J tumor bearing nude mice (n ) 5, mean ( SD) 10 (0) min, 30 (b) min, and 60 (4) min pi and the lipophilicity (log POW) of the respective compound. DISCUSSION

[123I]Tyr3-octreotide (TOC) (57) was the first radiolabeled SRIF analogue that was applied for scintigraphic in vivo localization of sst2-expressing primary tumors and metastases. However, already in the first studies it was shown, that two major drawbacks restricted the applicability of this new tracer: its predominant hepatobiliary excretion and low tumor retention (58, 59). Due to these disadvantages interpretation of planar as well as SPET images of the abdominal region are complicated considerably. In a pharmacokinetic study with patients with neuroendocrine tumors 55% of the administered activity was found in the feces 40 h pi (29). While average tumor doses were approximately 0.9-5 rad/mCi ([131I]TOC), doses calculated for the gallbladder wall and the lower colon were 2.37 rad/mCi and 16.4 rad/mCi, respectively. Therefore [131I]TOC is not suited for PRRT, and there are certainly restrictions with respect to its suitability for tumor scintigraphy. To make radioiodinated derivatives of TOC accessible for these applications, we evaluated the suitability of radiolabeled, carbohydrate-conjugated octreotide analogues and optimized the biokinetics in vivo (44-47). In previous studies we successfully applied sugar amino acid conjugation to improve the biokinetics of Rvβ3-antagonists (27, 43). Furthermore, carbohydration was applied to increase peptide solubility (60), to enhance in vivo stability (51), to improve blood-brain barrier penetration (33, 34) or intestinal absorption (42). In previous studies

it has been demonstrated that derivatization of SMS 201-995 at D-Phe1 with different aldoses via Maillard reaction yielded potent sst agonists with high receptor affinity (51). These compounds failed to misuse the transport system in hepatocyte sinusoidal plasma membranes (61) and thus showed significantly reduced hepatobiliary clearance and intestinal excretion. In a rat model, the maltose derivative of Phe3-octreotide ((N-R(R- D -Glucosyl(1-4)-1-deoxy- D -fructosyl))Phe 3 -octreotide), SDZ CO-611, showed a cumulated biliary excretion of only 5% of the injected activity over a period of 120 min, while 65% were found for SMS 201-995. The biodistribution studies in rat pancreatic tumor bearing mice demonstrate that carbohydrate conjugation of [125I]TOC leads to significant changes in the pharmacokinetics of this tracer, too. The most lipophilic compound in our series, the reference [125I]TOC, is mainly excreted via the liver and the intestine. The glucose derivative [125I]Gluc-TOC already shows a 50% decrease in liver and intestinal uptake, while for the most hydrophilic derivative, [125I]Malt-TOC, the activity accumulation in the liver and intestine is only 25% and 14%, respectively, of that of [125I]TOC. These data indicate that the hydrophilicity of the tracers in this series has a major influence on the favored excretion pathway. For the mono- and disaccharide derivatives log POW decreased gradually with the size of the carbohydrate moiety, which correlated with decreasing hepatobiliary excretion (Figure 6).

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when 8 mg unlabeled TOC per kg was given prior to the tracer administration or when the cold compound was coadministered. Less effective displacement already 10 min postinjection of the tracer indicates fast internalization of the radioligand, a prerequisite for activity retention in the tumor. CONCLUSION

Figure 7. Tumor/organ ratios found for [125I]TOC, [125I]GlucTOC, [125I]Malt-TOC, and [125I]Mtr-TOC in AR42J tumor bearing nude mice 30 min pi (n ) 5, mean ( SD).

Conjugation of [125I]TOC with maltotriose (a trisaccharide) yielded a tracer with a log POW similar to that of [125I]Gluc-TOC and therefore with a comparable degree of accumulation in liver and intestine. As previously suggested for non-carbohydrated radiometal labeled octreotide derivatives (62) and demonstrated in Figure 6 for the carbohydrated compounds, the correlation between the log POW’s of [125I]TOC and its glycosylated derivatives and their accumulation in liver and intestine may be applied for predictions concerning the degree of hepatobiliary clearance of radiolabeled TOC-derivatives with N-terminal modifications. Almost identical renal uptake was found for all compounds investigated. Whether these peptides are subject to renal tubular peptide-reabsorption by transport-mediated processes or charge dependent endocytosis (31) needs further investigations. In some studies, coadministration of lysine was reported to reduced renal accumulation of radiometalated octreotide derivatives (63, 64). This effect was explained by competition of lysine at the charge dependent endocytosis of octreotide in the kidneys. Since the net charge of the four derivatives investigated are assumed to be nearly identical under physiological conditions (+1.7 to +2.0) no major differences in renal uptake were expected. Concerning activity accumulation in tumor and other SSTR-positive tissues (pancreas, adrenals), we observed significant differences between [125I]Gluc-TOC and the other derivatives. Tumor uptake of [125I]Gluc-TOC . [125I]TOC, [125I]Malt-TOC, and [125I]Mtr-TOC. However, due to the improved excretion profile of the carbohydrated compounds, improved tumor/nontumor ratios were found for [125I]Gluc-TOC, [125I]Malt-TOC, and [125I]Mtr-TOC (Figure 7), especially for liver, kidney and intestine, wellknown to be critical organs in [123I]TOC-scintigraphy with tumor/nontumor ratios of about 1 or less. Interestingly, although the carbohydrated compounds are mainly cleared via the kidneys, tumor/kidney ratios of [125I]TOC and its glycosylated analogues were comparable. In cells transfected with sst1-sst5, I-Malt-TOC, and I-Mtr-TOC show high affinity for sst2- and sst-subtype binding profiles similar to I-TOC. Knowing that human tumors preferably express sst2 (1, 4), a radiotracer with high selectivity and high binding affinity for sst2 such as the carbohydrated octreotide derivatives may be extremely valuable for clinical use. As demonstrated by pretreatment, competition, and displacement experiments in tumor-bearing mice, activity uptake in AR42J tumors was sst2 receptor specific. As expected, tumor/nontumor ratios decreased to 1 or less

Carbohydration fulfils the demands for a chemical tool suitable to modify the physicochemical behavior of TOC. Carbohydration leads to significantly improved general pharmacokinetics. The carbohydrated tracers were rapidly cleared from the circulation via the kidneys without increased renal uptake. Additionally, the reduced hepatic uptake and biliary excretion, the improved tumor uptake, and thus increased tumor-to-nontumor ratios minimize the effective whole body dose. Especially the glucose and the maltose analogue of Tyr3-octreotide will be the basis for a new series of carbohydrated peptide receptor ligands with high potential for targeted imaging and radiotherapy of somatostatin receptor postive tumors. We assume that the concept of carbohydration is also a promising strategy for the development of tracers for other peptide receptor systems. ACKNOWLEDGMENT

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