99mTc-Labeled Neuropeptide Y Analogues as Potential Tumor

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Bioconjugate Chem. 2001, 12, 1028−1034

99mTc-Labeled

Neuropeptide Y Analogues as Potential Tumor Imaging Agents

Michael Langer,†,‡ Roberto La Bella,§ Elisa Garcia-Garayoa,§ and Annette G. Beck-Sickinger*,† Institute of Biochemistry, University of Leipzig, 04103 Leipzig, Germany, Department of Applied Biosciences, Federal Institute of Technology, 8057 Zu¨rich, Switzerland, and Center for Radiopharmaceutical Science, Paul Scherrer Institute, 5232 Villigen, Switzerland. Received May 16, 2001; Revised Manuscript Received September 11, 2001

The possible use of neuropeptide Y (NPY) as a novel radiopeptide has been investigated. NPY is a 36-amino acid peptide of the pancreatic polypeptide family, which is expressed in the peripheral and central nervous system, and is one of the most abundant neuropeptides in the brain. Its receptors are produced in a number of neuroblastoma and the thereof derived cell lines. As structure-activity relationships of NPY are well-known, we could assume where a radionuclide might be introduced without affecting receptor affinity. We applied the novel [99mTc(OH2)3(CO)3]+ aqua complex and PADA (2-picolylamine-N,N-diacetic acid) as bifunctional chelating agent. The peptides were synthesized by solid-phase peptide synthesis, and PADA was coupled to the side chain of Lys4 of the resin-bound peptide. Upon postlabeling of [K4(PADA)]-NPY, 99mTc(CO)3 did not only bind to the desired PADA, but presumably as well to the His in position 26. Since the replacement of His26 by Ala only slightly decreased binding affinity, [K4(PADA),A26]-NPY was specifically postlabeled, and the 185Re surrogate maintained high binding affinity. Furthermore, the prelabeling approach has been applied for the centrally truncated analogue [Ahx5-24]-NPY, which is highly selective for the Y2 receptor. The resulting Ac-[Ahx5-24,K4(99mTc(CO)3-PADA)]-NPY was produced with a yield of only 16%. Therefore, postlabeling was applied for the short analogue as well, again substituting His26 by Ala. Competitive binding assays using 185Re as a surrogate for 99mTc showed high binding affinity of Ac-[Ahx5-24,K4(185Re(CO)3PADA),A26]-NPY. Internalization studies with the corresponding 99mTc-labeled analogue revealed receptor-mediated internalization. Furthermore, biodistribution studies were performed in mice, and stability was tested in human plasma. Our centrally truncated analogue revealed a 6-fold increased stability compared to the natural peptide NPY. We conclude that Ac-[Ahx5-24,K4(99mTc(CO)3-PADA),A26]NPY has promising characteristics for future applications in nuclear medicine.

INTRODUCTION

Peptides can be considered as ideal agents for diagnostic and therapeutic applications, adopting Paul Ehrlichs concept of the “magic bullet” (1). Tumor tissues expressing receptors for a peptide can be addressed specifically by the peptide or differently labeled analogues (2). To date, radiolabeled receptor-binding peptides have emerged as a new class of radiopharmaceuticals (3, 4). However, there are several prerequisites for peptides used for scintigraphy. The corresponding receptors have to be expressed on the target in suitable amounts, and it is crucial that the radiolabeled analogue retains a high affinity. Specificity is to be considered as well. Whereas the natural ligand usually binds with similar affinities to several receptor subtypes, chemically modified synthetic analogues often display pronounced subtype selectivity for only one or few receptor subtypes (3). Peptides that are taken up by receptor-mediated internalization may be preferred, as this results in accumulation of the radioligand in the target, providing an * Correspondence to Prof. Dr. A. G. Beck-Sickinger, Institute of Biochemistry, University of Leipzig, Talstrasse 33, D-04103 Leipzig, Germany, Phone: ++49-(0)341-97 36 901, Fax: ++49(0)341-97 36 998, E-mail: [email protected]. † University of Leipzig. ‡ Federal Institute of Technology. § Paul Scherrer Institute.

increased signal for scintigraphy (5). A main concern of radiolabeled peptides is their stability under physiological conditions, i.e., peptide fragmentation by peptidases and the stability of the radiometal-chelator complex (6). As the natural peptides usually exhibit a short half-life in plasma, metabolically more stable analogues had to be developed. Somatostatin (SST) receptor scintigraphy using different radiolabeled SST analogues has been extensively clinically applied for tumor diagnosis (7, 8). Furthermore, bombesin and VIP have been evaluated as radiopharmaceuticals (9-11), and many further peptides are promising candidates, for instance neurotensin, R-MSH, substance P, and CCK (12). Here, we investigate the possibility to use neuropeptide Y (NPY) as a novel radiopeptide, while taking into account the above-mentioned prerequisites. NPY is a 36amino acid peptide of the pancreatic polypeptide family. It is expressed in the peripheral and central nervous system and is one of the most abundant neuropeptides in the brain (13). Several physiological activities such as induction of food intake, inhibition of anxiety, increase in memory retention, presynaptic retention of neurotransmitter release, and vasoconstriction have been attributed to NPY (14). Its receptors are produced in a number of neuroblastoma and the thereof derived cell lines, making them optimal targets for tumor scintigraphy. Five distinct NPY receptors have been cloned, which have been named Y1, Y2, Y4, Y5, and y6 receptor subtype

10.1021/bc015514h CCC: $20.00 © 2001 American Chemical Society Published on Web 10/24/2001

99mTc-Labeled

NPY Analogues

Figure 1. Structures of 99mTc-labeled NPY analogues. (A) Full length NPY analogue [K4(99mTc(CO)3-PADA),A26]-NPY and (B) Centrally truncated analogue Ac-[Ahx5-24,K4(99mTc(CO)3PADA),A26]-NPY. Ahx stands for 6-aminohexanoic acid, and PADA for 2-picolylamine-N,N-diacetic acid.

(15). Because structure-activity relationships of NPY are well-known (16, 17), we could assume where the radionuclide 99mTc might be introduced without affecting receptor affinity. The nuclear properties of 99mTc (monoenergetic γ-radiation of 140 keV, t1/2 of 6 h) are virtually ideal for diagnostic imaging (18). We applied the method of Alberto (19) by using the [99mTc(OH2)3(CO)3]+ aqua complex and PADA (2-picolylamine-N,N-diacetic acid) as bifunctional chelating agent (BFCA). Pre- and postlabeling approaches with attachment of PADA on the Lys4 side chain of NPY were investigated, and the full length analogue [K4(99mTc(CO)3-PADA),A26]-NPY as well as centrally truncated ones such as Ac-[Ahx5-24,K4(99mTc(CO)3PADA),A26]-NPY (Ahx ) 6-aminohexanoic acid) were produced (Figure 1). Synthesis, labeling, stability, binding affinities, internalization, and biodistribution studies are presented. MATERIALS AND METHODS

Materials. Fmoc-protected amino acids, 4-(2’,4’-dimethoxyphenyl-Fmoc-aminomethyl)phenoxy resin, and 1H-benzotriazolium,1-[bis(dimethylamino)methylene]-, tetrafuoroborate(1-),3-oxide (TBTU) were obtained from NovaBiochem (La¨ufelfingen, Switzerland), diisopropylcarbodiimide (DIC) and thiocresol from Aldrich, 1-hydroxybenzotriazole (HOBt), N-ethyldiisopropylamine (DIPEA), trifluoroacetic acid, thioanisole, acetic anhydride, hydrazine, and piperidine from Fluka, and tert-butyl alcohol from Merck. Dimethylformamide and diethyl ether were purchased from Scharlau (La Jota, Barcelona, Spain). Acetonitrile was obtained from Romil (Cambridge, England). Bovine serum albumin and bacitracin were purchased from Sigma, Minimum Essential Medium with Earle’s salts (MEM), Dulbeccos MEM/NutMix F12 medium, fetal calf serum, trypsine-EDTA, Dulbecco’s phosphate-buffered saline (PBS) without calcium and magnesium, glutamine, sodium pyruvate, and nonessential amino acids from Gibco. EDTA, Pefabloc SC, and all other chemicals were from Fluka. 3H-propionylNPY (specific activity of 3.63 TBq/mmol) was purchased from Amersham. Na[99mTcO4] was eluted from a Mallinckrodt 99Mo/99mTc generator (Petten, The Netherlands) using 0.9% saline. Peptide Synthesis. The peptides were synthesized by automated multiple solid-phase peptide synthesis using a robot system (Syro, MultiSynTech, Bochum, Germany). To obtain the peptide amide, 4-(2’,4’-dimethoxyphenyl-

Bioconjugate Chem., Vol. 12, No. 6, 2001 1029

Fmoc-aminomethyl)phenoxy resin was used. The polymer matrix was polystyrene-1%-divinylbenzene (30 mg; 15 µmol). The side-chain protection was chosen as follows: Tyr(tert-butyl), Glu(tert-butyl), Arg(2,2,3,5,5-pentamethylchromansulfonyl), His(trityl), Gln(trityl), Asn(trityl), Thr(tert-butyl), Lys(tert-butyloxy-carbonyl), or Lys(4,4-dimethyl-2,6-dioxocyclohex-1-ylideneethyl) (Lys(Dde)). Double coupling procedures were performed with diisopropylcarbodiimide/1-hydroxybenzotriazole (DIC/HOBT) activation, 10-fold excess and a coupling time of 40 min (20). Upon completion of the peptide sequence, the coupling of Re(CO)3-PADA was performed on the resin (after hydrazine-based deprotection of Lys4(Dde)) (21) by DIC/HOBT activation, 10-fold excess, and a coupling time of 24 h. For the PADA-peptides, PADA was activated by diisopropylcarbodiimide for 45 min and subsequently coupled with a 10-fold excess to the selectively deprotected Lys4 side chain. The N-termini were either Bocprotected or had been acetylated. The peptide amides were cleaved with 1 mL of trifluoroacetic acid/thioanisole/thiocresole (90:5:5) within 3 h. The peptide was precipitated from cold diethyl ether, collected by centrifugation, and lyophilized from water/ tert-butyl alcohol (1:1). Analysis of the product was performed by analytical reversed-phase HPLC at 220 nm on nucleosil C-18 columns from Merck-Hitachi (5 µm, 3 × 125 mm). Acetonitrile (25% to 75%) with 0.1% trifluoroacetic acid within 30 min was used as an eluent for the full length analogues, whereas for the centrally truncated ones 5% to 60% within 30 min was applied. Correct mass was identified by ion-spray mass spectrometry (SSQ 710, Finnigan MAT, Bremen, Germany). Radiolabeling. The preparation of [99mTc(OH2)3(CO)3]+ was done according to earlier description (19). A mixture of 4 mg disodium carbonate (0.04 mmol), 5.5 mg sodium borohydride (0.15 mmol), and 20 mg potassium sodium tartrate tetrahydrate (0.07 mmol), inclosed in a Wheaton sample vial, were flushed with 1 atm CO for 15 min. One milliliter of a [99mTcO4]- generator eluate in saline (about 1 GBq) was added, and the solution was heated for 30 min at 75°C. The solution was adjusted with approximately 100 µL of a mixture of 1 M phosphate buffer pH 7.4 and 1 N HCl (1:2) to pH 7. For postlabeling, 10 µL of 0.001 M solutions of the different PADA-NPY analogues were added to 90 µL of the neutralized 99mTc(I)-tricarbonyl solution and heated for other 60 min at 75°C. Prelabeling was done as described in (10). The labeling procedures and conjugates have been analyzed by RP-HPLC on a Macherey-Nagel CC Nucleosil 100-5 C18 reversed-phase column (10 mm, 250 × 4.6 mm). A gradient system from 5% to 60% acetonitrile with 0.1% trifluoroacetic acid in 30 min and a flow rate of 1.5 mL/ min were applied. Stability. To analyze the chemical stability of the radiolabeled NPY conjugates, challenging assays were done according to the method of Stalteri et al. (22) with some modifications. A 10 µL amount of the 99mTc labeled peptide was added to 90 µL of 0.1 M solution of histidine or cysteine (1000-fold molar excess of challenging agents). The resulting solutions were incubated at 37 °C, and progress of the reaction was followed by RP-HPLC with radiometric detection. The proteolytic degradation in plasma of the NPY analogues was determined in vitro in freshly prepared human plasma. Plasma from healthy donors was incubated with the 99mTc-labeled NPY analogues at a concentration of 0.2 pmol/mL for different time periods at 37 °C. After incubation, samples were precipitated with acetonitrile/ethanol and centrifuged at

1030 Bioconjugate Chem., Vol. 12, No. 6, 2001

4 °C. The supernatant was analyzed by reverse-phase HPLC using a Superdex peptide HR 10/30 (300 mm, 1315 µm). Cell Culture. SK-N-MC cells were grown in Minimum Essential Medium (MEM) with Earl’s salts containing 10% fetal calf serum, 4 mM glutamine, 1 mM sodium pyruvate, and 1% nonessential amino acids. SMS-KAN cells were grown under the same conditions using Dulbecco’s MEM/Nutrient mix F12 1:1 with 15% fetal calf serum. Cells were grown to confluency at 37°C and 5% CO2. The growth medium was removed, and the cells were washed with Dulbecco’s PBS. After incubation at room temperature for 3 min with PBS containing 0.02% EDTA, the cells were detached by mechanical agitation and suspended in new medium or incubation buffer. Receptor Binding Studies. Binding assays on SKN-MC and SMS-KAN cells were performed as previously described (23). In brief, cells were washed with PBS and resuspended in incubation buffer (minimum essential medium with Earl’s salts containing 0.1% bacitracin, 50 µM Pefabloc SC, and 1% bovine serum albumin). A 200 µL volume of the suspension containing 400000 cells was incubated with 25 µL of 10 nM 3H-propionyl-NPY and 25 µL 10 pM to 10 µM of NPY-analogues. Nonspecific binding was defined in the presence of 1 µM cold NPY. After 1.5 h at room temperature, the incubation was terminated by centrifugation at 2000g and 4 °C for 5 min. The pellets were then washed once with PBS by centrifugation, resuspended in PBS, transferred to scintillation vials, and mixed with scintillation cocktail. Radioactivity was determined using a β-counter. Internalization Studies. Studies of internalization of receptor-bound 99mTc labeled NPY analogues were performed as previously described (24, 25) with some modifications. Briefly, SK-N-MC or SMS-KAN cells were incubated at 37 °C in 6-well plates with about 300000 cpm [K4(99mTc(CO)3-PADA),A26]-NPY or Ac-[Ahx5-24, K4(99mTc(CO)3-PADA),A26]-NPY, respectively. To determine nonspecific binding and internalization, the incubation was also performed in the presence of 1 mM NPY or [Ahx5-24]-NPY. Incubation was terminated after different time periods (5, 30, 60, 120, and 180 min), and the cells were washed with ice-cold medium containing 1% bovine serum albumin. Cell-surface bound radioligand was removed by two steps of acid wash (50 mM glycine‚HCl/ 100 mM NaCl, pH 2.8) at room temperature for 5 min. The pH were neutralized with PBS, and subsequently the cells were solubilized by incubation with 1 N NaOH at 37°C to determine internalized radioligand. Biodistribution. Biodistribution of the radioconjugate Ac-[Ahx5-24,K4(99mTc(CO)3-PADA),A26]-NPY was estimated in BALB/c mice. The analogue was injected intravenously into the tail vein (3.7 MBq/mouse). For blocking studies, coinjections with [Ahx5-24]-NPY (0.3 mg/ mouse) and NPY (0.25 mg/mouse) were performed. Organs and tissues (blood, heart, lung, spleen, kidneys, stomach, pancreas, duodenum, ileum, colon, liver, muscle, bone, brain) were excised from the sacrificed animals 1.5 and 24 h postinjection. The tissues were weighed, and the radioactivity was determined by γ-counting. Results were expressed as percentage of injected dose per gram of tissue (% ID/g). RESULTS

Peptide Synthesis. Upon completion of the peptide sequence on the robot, the coupling of PADA or Re(CO)3PADA was performed manually to the resin-bound peptide. For the conjugation of Re(CO)3-PADA, activation by

Langer et al.

TBTU/HOBT/DIPEA was tested, but resulted in an incomplete coupling, even after several repetitive coupling steps. Therefore, the activation with DIC/HOBT was used, where a 10-fold excess of DIC/HOBT and Re(CO)3-PADA and a coupling time of 24 h was applied. For the PADA-peptides, a preactivation of PADA with DIC for 45 min and subsequently coupling with a 10fold excess to the peptide was suitable. As the BFCA PADA was coupled to the selectively deprotected Lys4 side chain, the N-termini had to be protected, which was achieved by either Boc-protection or acetylation. The PADA-peptides and Re(CO)3-PADA conjugates yielded in high purity (>95%), as determined by analytical HPLC. A synthesis scheme is shown in Figure 2, and the analytical data are summarized in Table 1. Radiolabeling. Upon postlabeling of [K4(PADA)]NPY, the aquaion 99mTc(CO)3 did not only bind to the desired PADA, but as well to a secondary labeling site, as indicated by two peaks in the HPLC chromatogram. As His is known to be a strong binding chelator for the aquaion as well (26), we assumed that the His in position 26 of NPY is the second labeling site. Therefore, His26 was substituted by Ala, and this [K4(PADA),A26]-NPY was selected for postlabeling. Specific labeling could be achieved, as shown by a single peak in the HPLC chromatogram. By the comigration of the resulting [K4(99mTc(CO)3-PADA),A26]-NPY with its cold 185Re surrogate on HPLC (retention times 23.5 min and 23.7, respectively), identity was confirmed. The prelabeling approach has been applied for labeling of the centrally truncated analogue [Ahx5-24]-NPY. The resulting Ac-[Ahx5-24,K4(99mTc(CO)3-PADA)]-NPY was produced with a yield of about 16%. As subsequent purification steps make the prelabeling approach very time-consuming, we applied postlabeling for the short analogue as well, again substituting His26 by Ala. The resulting Ac-[Ahx5-24,K4(99mTc(CO)3-PADA),A26]-NPY nicely comigrated with its cold analogue on RP-HPLC (retention times 20.5 min and 20.8, respectively). Stability. Neither histidine nor cysteine led to significant ligand exchange and/or decomposition after 24 h incubation at 37 °C. The results of these challenge experiments are shown in Table 2. In addition, the degradation of [K4(99mTc(CO)3-PADA),A26]-NPY and Ac[Ahx5-24,K4(99mTc(CO)3-PADA),A26]-NPY was determined in human plasma. The former full length NPY analogue showed almost no degradation after 1 h. In contrast, the centrally truncated analogue Ac-[Ahx5-24,K4(99mTc(CO)3PADA),A26]-NPY was metabolized as shown in Figure 3, and a half-life time of 24.7 ( 2.0 min was calculated. Receptor Binding Studies. To determine the affinity of the NPY analogues, competition binding assays at two neuroblastoma cell lines were performed (n ) 3). The SKN-MC cell line has been shown to express the NPY Y1 receptor, whereas the SMS-KAN line is known to express the Y2 receptor (27, 28). 185Re was used as a cold surrogate for 99mTc. As shown in Table 3, the peptides were still able to bind to the corresponding receptors. The natural ligand pNPY has a high affinity to the Y1 and Y2 receptor subtype (IC50 1 and 0.5 nM, respectively). By replacing His26 by Ala, this affinity decreased to 8.5 and 5.3 nM, respectively. The introduction of the BFCA PADA on the Lys4 side chain resulted only in a slight affinity reduction, and finally the cold radiopeptide analogue [K4(185Re(CO)3-PADA),A26]-NPY exhibited an IC50 value of 16.0 nM for the Y1 and 8.5 nM for the Y2 receptor. A similar stepwise reduction of receptor binding upon acetylation, attachment of PADA and Re(CO)3-PADA was also seen for the short NPY analogue on the Y2 receptor.

99mTc-Labeled

NPY Analogues

Bioconjugate Chem., Vol. 12, No. 6, 2001 1031

Figure 2. Synthesis scheme for 99mTc-labeled NPY analogues. (A) Synthesis and postlabeling of the full length NPY analogue. (B) Synthesis and pre- or postlabeling of the centrally truncated analogue. Table 1. Analytical Data of the Prepared NPY Analogs molecular weight compound

calcd

exptl

retention time

[K4(PADA),A26]-NPY [K4(185Re(CO)3-PADA),A26]-NPY Ac-[Ahx5-24,K4(PADA), A26]-NPY Ac-[Ahx5-24,K4(185Re(CO)3-PADA)]-NPY Ac-[Ahx5-24,K4(185Re(CO)3-PADA),A26]-NPY

4393.7 4662.2 2402.6 2737.8 2670.5

4394.3 4663.5 2403.0 2739.2 2672.1

21.1a 21.7a 26.2b 27.2b 28.5b

a 25% to 75% acetonitrile with 0.1% trifluoroacetic acid within 30 min. b 5% to 60% acetonitrile with 0.1% trifluoroacetic acid within 30 min.

Table 2. Chemical Stability [%] of the 99mTc-labeled NPY Derivatives against Ligand Exchange in 10 mM Histidine and Cysteine at 37 °C histidine [K4(99mTc(CO) 1h 4h 24 h 1h 4h 24 h

3

cysteine

-PADA),A26]-NPY

98 99 98 98 96 97 Ac-[Ahx5-24,K4(99mTc(CO)3-PADA),A26]-NPY 98 99 97 98 95 96

[Ahx5-24]-NPY has an IC50 value of 0.2 nM, whereas the cold surrogate Ac-[Ahx5-24,K4(185Re(CO)3-PADA),A26]NPY binds with 2.5 nM affinity. As the replacement of the amino acid sequence 5-24 of NPY by the spacer Ahx leads to subtype-specific high affinity analogue for the Y2 receptor and a complete loss of affinity at all other receptors (29), these short analogues were not tested at the Y1 receptor. Internalization Studies. Internalization of the 99mTclabeled peptides was investigated in vitro by incubating

Figure 3. Proteolytic degradation of Ac-[Ahx5-24,K4(99mTc(CO)3PADA),A26]-NPY after incubation in human plasma at 37 °C.

[K4(99mTc(CO)3-PADA),A26]-NPY on SK-N-MC and Ac[Ahx5-24,K4(99mTc(CO)3-PADA),A26]-NPY on SMS-KAN cells at 37 °C. Almost no specific binding and internalization was observed for [K4(99mTc(CO)3-PADA),A26]-NPY. In contrast, Ac-[Ahx5-24,K4(99mTc(CO)3-PADA),A26]-NPY was specifically internalized in SMS-KAN cells. The uptake was time-dependent, as shown in Figure 4. After 120 min, 25%, and after 180 min, 33% of the radiolabeled ligand

1032 Bioconjugate Chem., Vol. 12, No. 6, 2001

Langer et al.

Table 3. Binding Affinities of PNPY Analogs at SK-N-MC (Y1 Receptor) and SMS-kan (Y2 Receptor) Cells

a

compound

IC50 [nM] at Y1

IC50 [nM] at Y2

NPY [A26]-NPY [K4(PADA),A26]-NPY [K4(185Re(CO)3-PADA),A26]-NPY [Ahx5-24]-NPY Ac-[Ahx5-24]-NPY Ac-[Ahx5-24,K4(PADA),A26]-NPY Ac-[Ahx5-24,K4(185Re(CO)3-PADA)]-NPY Ac-[Ahx5-24,K4(185Re(CO)3-PADA),A26]-NPY

1.0 ( 0.3 8.5 ( 0.2 9.6 ( 0.3 16.0 ( 0.1 >4000 n.da n.d n.d n.d

0.5 ( 0.1 5.3 ( 0.1 6.2 ( 0.1 8.5 ( 0.3 0.2 ( 0.1 0.7 ( 0.3 1.3 ( 0.8 1.4 ( 0.5 2.5 ( 0.1

n.d. ) not determined.

in the uptake with or without coinjection. Differences were observed after coinjection with cold NPY, especially at the gastrointestinal tract, where the highest uptake was found in the duodenum. However, specific blocking by a cold analogue could usually not be observerd, and the interindividual variations in the mice were considerably high. DISCUSSION

Figure 4. Internalization studies. Specific internalization of Ac-[Ahx5-24,K4(99mTc(CO)3-PADA),A26]-NPY on SMS-KAN cells at 37 °C over the time. Surface bound and internalized fractions of the radiolabeled ligand are shown. Table 4. Biodistribution of Ac-[Ahx5-24,K4(99mTc(CO)3-PADA),A26]-NPY in BALB/c Mice after iv Injection. Data Are Expressed as % ID/g of Tissue ( SD 1.5 h tissue blood heart lung spleen kidney pancreas stomach duodenum ileum colon liver muscle bone brain

Aa

Bb

24 h Cc

1.02 ( 0.30 1.17 ( 0.33 1.52 ( 0.28 0.65 ( 0.13 0.82 ( 0.28 1.06 ( 0.19 1.44 (0.29 3.61 ( 1.61 3.74 ( 0.94 0.83 ( 0.20 1.06 ( 0.25 1.15 ( 0.06 8.21 ( 1.69 11.05 ( 2.19 8.74 ( 1.92 1.01 ( 0.34 2.79 ( 3.97 4.50 ( 2.27 3.11 ( 1.34 4.19 ( 1.78 8.27 ( 0.20 4.10 ( 2.53 6.67 ( 3.34 28.29 ( 10.43 8.37 ( 7.06 11.83 ( 9.79 2.59 ( 1.05 1.89 ( 1.67 1.36 ( 0.67 3.54 ( 0.47 8.24 ( 1.64 11.79 ( 2.73 15.40 ( 1.60 0.32 ( 0.08 0.35 ( 0.12 0.53 ( 0.12 0.41 ( 0.103 0.50 ( 0.16 0.60 ( 0.07 0.05 ( 0.01 0.08 ( 0.04 0.11 ( 0.01

Dd 0.17 ( 0.03 0.25 ( 0.05 1.02 ( 0.68 0.36 ( 0.08 4.08 ( 0.59 0.32 ( 0.05 0.36 ( 0.07 0.24 ( 0.06 0.22 ( 0.06 0.24 ( 0.07 4.19 ( 0.91 0.10 ( 0.01 0.10 ( 0.02 0.01 ( 0.00

a Group A (n ) 7) received 3.7 MBq of Ac-[Ahx5-24,K4(99mTc(CO) 3 PADA),A26]-NPY; b Group B (n ) 7) received 3.7 MBq of Ac5-24 4 99m 26 [Ahx ,K ( Tc(CO)3-PADA),A ]-NPY coinjected with cold [Ahx5-24]-NPY (0.3 mg/mouse); c Group C (n ) 3) received 3.7 MBq of Ac-[Ahx5-24,K4(99mTc(CO)3-PADA),A26]-NPY coinjected with cold NPY (0.25 mg/mouse); d Group D (n ) 4) received 3.7 MBq of Ac-[Ahx5-24,K4(99mTc(CO)3-PADA),A26]-NPY.

were internalized, whereas the surface-bound fraction accordingly decreased. Biodistribution. Table 4 shows the tissue distribution of Ac-[Ahx5-24,K4(99mTc(CO)3-PADA),A26]-NPY in BALB/c mice mice after intravenous injection. Time points at 1.5 (row A) and 24 h p.i. (row D) were chosen in order to follow the time course of disappearance from the blood, kidney, and liver. The peptide exhibited a rapid clearance from the blood pool. The levels after 24 h in both kidneys and liver might indicate urinary and hepatobiliary excretion. Blocking has been performed with cold [Ahx5-24]NPY and full length NPY. Blood, heart, spleen, brain, muscle, and bone had small activities and no difference

In the postlabeling approach, a BFCA is first attached to the peptide, and the radionuclide is subsequently coupled to the free chelating group of the BFCA (30). This method can be combined with the advantages of solidphase peptide sythesis (SPPS). Applying a molar excess of BFCA, complete coupling to the resin-bound peptide can be achieved, and unreacted BFCA is easily washed away. Subsequent cleavage from the resin yields the highly pure BFCA-peptide conjugate. Using the organometallic aquaion [99mTc(OH2)3(CO)3]+ has been shown to be very suitable for labeling (31). Efficient postlabeling of histidine derivatives of neurotensin has been performed (32, 33), and a bombesin analogue was successfully prelabeled applying PADA as BFCA (10). Accordingly, we synthesized NPY derivatives on a rinkamide resin, selectively deprotected Lys4 by hydrazine and coupled the BFCA PADA to this side chain. Upon postlabeling of [K4(PADA)]-NPY, the 99mTc did not only bind to the desired PADA, but presumably as well to the His in position 26, as His is a preferred binding chelator (26). Therefore, we tried to replace the disturbing amino acid, which is not an essential position for binding (16, 34). The replacement of His26 by Ala resulted in a specific 99mTc-labeling of PADA. As assumed, high binding affinity at the Y1 and Y2 receptor was maintained, as tested by the cold 185Re surrogate. Another way how to circumvent the attachment of 99mTc to His26 is the prelabeling approach, where the radionuclide-BFCA complex is formed prior to its attachment to the peptide (30). To avoid polymerization due to the activation in solution, we applied the shortened analogue [Ahx5-24]-NPY, in which all the reactive aspartate amino acids are omitted. By replacing the sequence 5-24 in NPY by Ahx, receptor subtype selectivity for the Y2 receptor is achieved (29). The resulting Ac-[Ahx5-24,K4(99mTc(CO)3-PADA)]-NPY was produced with a yield of only 16%. Therefore, we again substituted His26 by Ala and attached PADA on the Lys4 side chain. Competitive binding assays showed surprisingly high affinity of the so produced Ac-[Ahx5-24,K4(185Re(CO)3-PADA,A26)]-NPY, considering the bulky chelator on the short peptide sequence. Thus, although substitution of a single amino acid, introduction of spacer, N-terminal protection and introduction of an organometallic moiety was performed, the NPY analogues maintained high binding to their corresponding receptor.

99mTc-Labeled

NPY Analogues

Internalization studies with the two 99mTc labeled analogues revealed receptor-mediated internalization only for the short analogue Ac-[Ahx5-24,K4(99mTc(CO)3PADA,A26)]-NPY. We have previously shown that fluorescent and 3H-marked NPY internalizes in a time- and temperature-dependent manner (25), determined to be 20-30%, which corresponds to our current measurements. As internalization may lead to higher uptake and retention in the target tissue, the short analogue seems to be more appropriate for future application as a tumor imaging agent. Moreover, it revealed to be stable to enzymatic degradation. Most peptides undergo rapid proteolysis in plasma by endogenous peptidases (35) and have to be molecularly engineered to prevent their enzymatic destruction (12). The N-terminal acetylation and introduction of an unnatural spacer Ahx within the sequence made our analogue a stable candidate, as the natural ligand NPY exhibits a half-life time of only 4 min in humans (36), whereas we were able to increase the stability to a half-life time of about 24 min. This is a crucial step in the development of a novel peptide radiopharmaceutical, since the normally rapid enzymatic degradation is one of the major disadvantages of peptides. As an example, only the discovery of the stable somatostatin analogue octreotide (t1/2 90 min vs the t1/2 3 min of somatostatin) enabled the use and success of OctreoScan, the most commonly used radiopharmaceutical to date. The full length NPY analogue [K4(99mTc(CO)3-PADA), 26]-NPY seems to have lost its biological activity upon A postlabeling, as no specific binding to the Y1 receptor or receptor-mediated internalization could be detected. This might explain as well its too-good stability in plasma: the enzymes involved in the metabolism of NPY might be not capable of degrading this biologically inactive form. In the biodistribution studies, the short peptide exhibited a rapid clearance from the blood pool. As seen by the low activity in the brain, the peptide is not crossing the intact blood-brain barrier. Blocking studies have been performed with cold [Ahx5-24]-NPY (Y2 selective) and full length NPY (binds to all NPY subtype receptors). Blood, heart, spleen, muscle, and bone had small activities and no difference in the uptake with or without coinjection, indicating either no receptors or at least no Y2 receptors in these tissues. Differences were observed after coinjection with cold NPY, especially at the gastrointestinal tract, an area which expresses NPY receptors. An explanation for this fact could be the inhibitory effect of the cold NPY on contractility and secretion in the intestine (37). Upon injection of the labeled short analogue alone, the highest activity was found at the ileal level, whereas upon coinjection with cold NPY, the highest activity corresponded to the duodenal level. This might be provoked by the high NPY dosage inducing slower transit and inhibition of intestinal fluid secretion. However, this high accumulation at intestinal level could hide a possible specific binding or blocking of the labeled analogue to its receptor. Generally, there is little known about the Y2 receptor distribution in mice, and distribution varies from one species to another, as shown in autoradiographic studies performed in rabbit and rat (38, 39). In conclusion, we here demonstrate for the first time the novel application of modified NPY analogues as radiopharmaceuticals. Using our knowledge of structureactivity relationships of NPY, and combining the advantages of SPPS and a stable, well-defined BFCA such as PADA for complexing [99mTc(OH2)3(CO)3]+, we could produce a chemically and metabolically stable candidate,

Bioconjugate Chem., Vol. 12, No. 6, 2001 1033

which maintains high binding affinity to its receptor and even exhibits receptor-mediated internalization. Therefore, Ac-[Ahx5-24,K4(99mTc(CO)3-PADA,A26)]-NPY has promising characteristics for future applications in tumor diagnosis or even therapy, when using a suitable radionuclide such as 188Re. ACKNOWLEDGMENT

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