Two Technetium-99m-Labeled Cholecystokinin-8 (CCK8) Peptides for

Technetium-99m with a γ energy of 141 keV has the ideal characteristics for imaging, combined with a relatively short half-life of 6 h. In the presen...
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Bioconjugate Chem. 2004, 15, 561−568

561

Two Technetium-99m-Labeled Cholecystokinin-8 (CCK8) Peptides for Scintigraphic Imaging of CCK Receptors Peter Laverman,†,* Martin Be´he´,‡ Wim J. G. Oyen,† Peter H. G. M. Willems,§ Frans H. M. Corstens,† Thomas M. Behr,‡ and Otto C. Boerman† Department of Nuclear Medicine, University Medical Center Nijmegen, Nijmegen, The Netherlands, Department of Nuclear Medicine, Phillips University, Marburg, Germany, and Department of Biochemistry, Nijmegen Center for Molecular Life Sciences, University Medical Center Nijmegen, Nijmegen, The Netherlands. Received November 10, 2003; Revised Manuscript Received February 2, 2004

A broad spectrum of radiolabeled peptides with high affinity for receptors expressed on tumor cells is currently under preclinical and clinical investigation for scintigraphic imaging and radionuclide therapy. The present paper evaluates two 99mTc-labeled forms of the C-terminal octapeptide of cholecystokinin (CCK8): sulfated (s)CCK8, with high affinity for CCK1 and CCK2 receptors, and nonsulfated (ns)CCK8, with high affinity for CCK2 receptors but low affinity for CCK1 receptors. Peptides were conjugated with the bifunctional chelator N-hydroxysuccinimidyl hydrazino niconitate (s-HYNIC). 99mTc-labeling, performed in the presence of nicotinic acid and tricine, was highly efficient (∼95%) and yielded products with a high specific activity (∼700 Ci/mmol) and good stability (∼5% release of radiolabel during 16 h incubation in phosphate buffered saline at 37 °C). Chinese hamster ovary cells stably expressing the CCK1 receptor (CHO-CCK1 cells) internalized ∼3% of added 99mTcsCCK8 per confluent well during 2 h at 37 °C. Internalization was effectively blocked by excess unlabeled sCCK8. CHO-CCK1 cells did not internalize 99mTc-nsCCK8. Displacement of 99mTc-sCCK8 and -nsCCK8 by unlabeled CCK-8 (performed at 0 °C to prevent internalization) revealed 50% inhibitory concentrations (IC50) of 8 nM and >1 µM, respectively. CHO-CCK2 cells internalized ∼25% and ∼5% of added 99mTc-sCCK8 and -nsCCK8, respectively. In both cases internalization was blocked by excess unlabeled peptide. IC50 values for the displacement of 99mTc-sCCK8 and -nsCCK8 were 3 nM and 10 nM, respectively. CHO-CCK1 cell-derived tumors present in one flank of athymic mice accumulated 2.0% of injected 99mTc-sCCK8 per gram tissue at 1 h postinjection. This value decreased to 0.6% following coinjection with excess unlabeled peptide. Uptake of 99mTc-nsCCK8 was low (0.2%) and not did change by excess unlabeled peptide (0.3%). Accumulation of 99mTc-sCCK8 and -nsCCK8 by CHO-CCK2 cell-derived tumors (present in the other flank) amounted to 4.2% and 0.6%, respectively. In both cases uptake was significantly reduced by excess unlabeled peptide to 1.0% and 0.4% for sCCK8 and nsCCK8, respectively. Accumulation of 99mTc-sCCK8 was also high in pancreas (11.7%), stomach (2.0%), and kidney (2.1%), whereas uptake of 99mTc-nsCCK8 was high in stomach (0.7%) and kidney (1.4%). Both radiolabeled peptides showed a rapid blood clearance. In conclusion, these data show that CCK8 analogues can be efficiently labeled with 99mTc using s-HYNIC as chelator and nicotinic acid/tricine as coligand system without compromising receptor binding. Furthermore, the present study demonstrates that CCK1 tumors hardly accumulate 99mTc-nsCCK8, CCK2 tumors accumulate 2 times more 99mTc-sCCK8 than CCK1 tumors, and CCK2 tumors accumulate 15 times more 99mTc-sCCK8 than 99mTc-nsCCK8. Although accumulation in some nontarget organs was also higher with 99mTcsCCK8, this may not reflect the human situation due to a different receptor expression pattern in humans as compared to mice. Therefore, further studies are warranted to investigate the possible use of 99mTc-sCCK8 for scintigraphic imaging of CCK receptor-positive tumors in humans.

INTRODUCTION

Radiolabeled peptides with high affinity for receptors on tumor cells can be used for scintigraphic imaging and for radionuclide therapy. A broad spectrum of peptides, ranging from somatostatin analogues, RGD1 peptides, neurotensin analogues to gastrin analogues is currently * To whom correspondence should be addressed. Phone: +3124-3615054. Fax: +31-24-3618942. E-mail: p.laverman@ nucmed.umcn.nl. † Department of Nuclear Medicine, University Medical Center Nijmegen. ‡ Phillips University. § Department of Biochemistry, University Medical Center Nijmegen.

under (pre)clinical evaluation (1). The gastrin analogues gained interest since the discovery of the presence of cholecystokinin (CCK)/gastrin receptors on a variety of tumors (2). Several studies described the radiolabeling of gastrin analogues with indium-111 (111In) or iodine131 (131I) for imaging and/or therapy (3-6). However, 1 Abbreviations: CCK8, cholecystokinin-8; sCCK8, sulfated cholecystokinin-8; nsCCK8, nonsulfated cholecystokinin-8; CHO, chinese hamster ovary; DMF, dimethylformamide; DMSO, dimethyl sulfoxide; DTPA, diethylenetriamine pentaacetic acid; EDDA, ethylenediaminediacetic acid; HYNIC, hydrazino nicotinamide; IC50, 50% of the inhibitory concentration; keV, kilo electronvolt; PBS, phosphate buffered saline; RCP, radiochemical purity; RGD, arginine-glycine-aspartic acid; RP-HPLC, reversed phase HPLC; SPE, solid-phase extraction.

10.1021/bc034208w CCC: $27.50 © 2004 American Chemical Society Published on Web 04/10/2004

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Chart 1. Structural Formulas of HYNIC-Conjugated Nonsulfated CCK8 (upper panel) and Sulfated CCK8 (lower panel)

both radionuclides have less favorable imaging characteristics due to their high γ energies (171 and 245 keV for 111In and 364 keV for 131I). Technetium-99m with a γ energy of 141 keV has the ideal characteristics for imaging, combined with a relatively short half-life of 6 h. In the present paper, we report on the 99mTc labeling of two CCK8 analogues which are known to bind to CCK receptors with high affinity. CCK receptors can be distinguished pharmacologically by their affinity for gastrin, a 33 amino acid peptide hormone involved in gastric motility (7). The CCK1 receptor (formerly known as CCK-A) has a low affinity for gastrin, whereas the CCK2 receptor (formerly known as CCK-B) has a high affinity for gastrin. Both receptors belong to a superfamily of G-protein-coupled receptors. The peptides of the gastrin and CCK family are characterized by an amidated C-terminal receptor binding sequence Trp-Met-Asp-Phe-NH2. Both groups of peptides differ by the location of their tyrosyl residue in relation to the receptor binding sequence. Peptides of the gastrin family have one amino acid (usually Gly), and peptides of the CCK-family have two amino acids (Met-Gly or ThrGly), between this Tyr moiety and the receptor binding sequence. This Tyr residue plays a role in the receptor specificity. When this Tyr residue is sulfated, the peptides display high affinity for both the CCK1 and the CCK2 receptor. In contrast, the nonsulfated peptides show a 1000-fold lower affinity for the CCK1 receptor than for the CCK2 receptor (8). The CCK2 receptor is abundantly expressed in most small cell lung cancers, medullary thyroid carcinomas and in some stromal ovarian cancers and astrocytomas (2). For preclinical evaluation, the rat pancreatic AR42J tumor and the human medullar thyroid carcinoma cell line TT, both expressing the gastrin/CCK2 receptor are generally used (3,4). To investigate CCK1 and CCK2 receptor binding affinity of the peptides separately we used chinese hamster ovary (CHO) cells stably transfected with cDNA encoding for either the CCK1 or CCK2 receptor. These cells are well defined with regard to their

receptor expression and allow a more detailed study of the receptor specificity of the radiolabeled CCK8 peptides (8). Several methods are described to radiolabel peptides with 99mTc, mostly based on the use of a bifunctional chelator. N-Hydroxysuccinimidyl hydrazino nicotinate (sHYNIC) is a well-known bifunctional chelator which has proven to yield 99mTc-labeled conjugates with excellent stability, high specific activity, and high radiochemical purity (9). HYNIC has been used for 99mTc-labeling of proteins, peptides, and lipids (9-11). HYNIC can be conjugated to primary amino groups by using N-hydroxysuccinimidyl-HYNIC. The succinimidyl group facilitates conjugation of HYNIC with primary amino groups, either N-terminal or Lys residues. The 99mTc is bound to the hydrazino moiety by forming a TcdN bond. This binding requires coligands for proper coordination of the Tc(V) species (12). The nature of the coligand determines the labeling efficiency, stability, and specific activity of the 99mTc-labeled compound. Moreover, it has been reported that these coligands can affect the in vivo behavior of the 99mTc-labeled peptide or protein (13-16). In the present study we investigated the HYNIC conjugation of both sulfated and nonsulfated CCK8 (Chart 1) (sCCK8 and nsCCK8, respectively). Furthermore, the effect of different coligand systemsstricine, ethylenediaminediacetic acid (EDDA), and tricine/nicotinic acidson the labeling efficiency, radiochemical stability, and purity was investigated. Receptor binding and internalization studies were performed with both 99mTcnsCCK8 and 99mTc-sCCK8 on the CCK receptor-transfected CHO cells. In addition, the in vivo receptor targeting was studied in athymic mice with CCK1 or CCK2 receptor expressing tumors on each flank. EXPERIMENTAL PROCEDURES

Chemicals. Sulfated and nonsulfated cholecystokinin fragment 26-33 (sCCK8 and nsCCK8, respectively) were obtained from Sigma-Aldrich Chemical Co. (St. Louis,

Tc-99m Labeling of CCK8 Peptides

MO). Amino acid sequences were as follows. Sulfated CCK8: Asp-Tyr(SO3H)-Met-Gly-Trp-Met-Asp-Phe-NH2. Nonsulfated CCK8: Asp-Tyr-Met-Gly-Trp-Met-Asp-PheNH2. Tricine (N-[tris(hydroxymethyl)methyl]glycine) was purchased from Fluka (Buchs, Switzerland). Ethylenediaminediacetic acid (EDDA) and nicotinic acid were from Sigma-Aldrich. The propylaldehyde hydrazone of succinimidyl hydrazinonicotinate (s-HYNIC) was synthesized essentially as described by Abrams et al. (17) and Schwartz et al. (18). Na99mTcO4 was eluted from a commercial 99Mo/99mTcO4- generator (Tyco Mallinckrodt, Inc., Petten, The Netherlands). 111InCl3 was also obtained from Tyco Mallinckrodt. Cell Lines. Chinese hamster ovary (CHO) cells stably transfected with cDNA encoding for either the CCK1 or CCK2 receptor were used. Generation and characterization of the cell lines is described elsewhere (8). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco Life Technologies, Gaithersburg, MD), supplemented with 2 mmol/L glutamine, 10% (v/v) fetal calf serum, penicillin (100 U/mL), streptomycin (100 µg/mL), and gentamicin (50 µg/mL). The cells were cultured in humidified atmosphere with 5% CO2 at 37 °C. RP-HPLC. An Agilent 1100 series LC system (Agilent Technologies, Palo Alto, CA) equipped with an in-line Radiomatic A-500 series radiodetector (Canberra-Packard, Meriden, CT) was used for analytical and preparative reversed phase HPLC. A Zorbax Rx C18 column (5 µm, 4.6 × 250 mm) was used at a flow rate of 1 mL/min and the following solvent system. Gradient: 0-10 min 100% TFA in water (0.1% v/v), 10-20 min 0% to 100% acetonitrile. Retention times for the nonsulafted peptide were as follows: nsCCK8: 17.5 min, HYNIC-nsCCK8: 17.7 min and 99mTc-HYNIC-nsCCK8 (nicotinic acid/tricine as coligands): 18.1 min. Retention times of the sulfated peptide were: sCCK8: 17.4 min, HYNIC-sCCK8: 17.6 min, and 99mTc-HYNIC-sCCK8 (nicotinic acid/tricine as coligands): 17.9 min. Unbound pertechnetate eluted with the void volume (3.4 min). SPE Purification. Solid-phase extraction (SPE) was used for purification of the radiolabeled peptides in the stability studies. A C18 SepPak-cartridge (Waters, Inc., Milford, MA) was activated with ethanol and rinsed with 5 mL of water. The radiolabeled peptide was loaded onto the cartridge. The cartridge was washed with 5 mL of water, the radiolabeled peptide was eluted with 1 mL of acetonitrile and subsequently the solvent was evaporated under vacuum. The eluted fraction was reconstituted in phosphate buffered saline (PBS), pH 7.4. HYNIC Conjugation of CCK8. The CCK8 peptides were conjugated with s-HYNIC essentially as described previously (10). Briefly, 10 µL of NaHCO3 (1.0 M, pH 8.2) was added to 200 µL of peptide (0.1 µmol). Subsequently, a 10-fold molar excess of s-HYNIC in 10 µL of dry DMSO was added dropwise to the mixture. The conjugation mixture was incubated for 30 min at room temperature. To remove unreacted s-HYNIC, the mixture was passed through an activated C18 SepPak cartridge as described above. The dried fraction was reconstituted in water, and the HYNIC-conjugated peptide was separated from underivatized peptide by preparative RP-HPLC. The fraction containing the HYNIC-CCK8 was collected and the solvent was evaporated under a stream of nitrogen. HYNIC-nsCCK8: MALDI-TOF-MS: analysis (C58H71N13O14S2) calculated 1238.4 (M + H)+, found 1238.7 (M + H)+. HYNIC-sCCK8: MALDI-TOF-MS: analysis (C58H71N13O17S3) calculated 1318.5 (M + H)+, found 1318.6 (M + H)+.

Bioconjugate Chem., Vol. 15, No. 3, 2004 563 99m

Tc-Labeling of HYNIC-CCK8. (i) Tricine as Coligand. Tricine (150 µL, 100 mg/mL in PBS) was added to 5 µL of HYNIC-CCK8 (1 mg/mL). Fifteen microliters of a freshly prepared solution of SnSO4 (1 mg/mL in nitrogen purged 0.1 N HCl) and 100-370 MBq of Na99mTcO4 were added. The solution was incubated at room temperature for 30 min. (ii) EDDA as Coligand. EDDA (500 µL, 10 mg/mL in PBS) was added to 5 µL of HYNIC-CCK8 (1 mg/mL). Five microliters of a freshly prepared solution of SnSO4 (1 mg/ mL in nitrogen purged 0.1 N HCl) and 100-370 MBq of Na99mTcO4 were added. The mixture was incubated at 75 °C for 30 min. (iii) Nicotinic Acid/Tricine as Coligands. Tricine (400 µL, 100 mg/mL in PBS) and nicotinic acid (100 µL, 20 mg/mL in 25 mM benzoate buffer, pH 5.0) were added to 5 µL of HYNIC-CCK8 (1 mg/mL). Twenty five microliters of a freshly prepared solution of SnSO4 (1 mg/mL in nitrogen purged 0.1 N HCl) and 100-370 MBq of Na99mTcO4 were added. The mixture was incubated at 75 °C for 30 min. (iv) Stability Tests. The in vitro stability of both the sulfated and the nonsulfated 99mTc-labeled CCK8 peptides was investigated. Peptides were radiolabeled and purified by SPE as described above. After evaporation of the solvent, the dried 99mTc-labeled peptide was reconstituted in 200 µL of PBS. The mixture was incubated at 37 °C for 16 h. Samples of the incubation mixture were taken at 5 min and 16 h and radiochemical purity (RCP) was assessed by RP-HPLC analysis according to the method described above. 111 In-Labeling of sCCK8. As a control, sCCK8 was conjugated with dicyclic anhydride of diethylenetriamine pentaacetic acid (DTPA) to allow labeling with 111In (6). Conjugation was performed at a 1:10 molar ratio, and the DTPA-conjugated peptide was purified by SPE and RP-HPLC as described above. DTPA-sCCK8 was radiolabeled with 111In by incubation with 111InCl3 in 0.15 M ammonium acetate buffer, pH 5.0, for 30 min at room temperature. In Vitro Characterization of 99mTc-Labeled HYNICCCK8. IC50 Determination. The 50% inhibitory concentration (IC50) for binding the CCK1 and CCK2 receptor of both peptides was determined on CCK-receptor expressing CHO cells. CHO-CCK1 and CHO-CCK2 cells were grown to confluency in 24-well plates. Cells were washed twice with binding buffer (HEPES-buffered DMEM with 1% (w/v) bovine serum albumine). After 10 min incubation at 37 °C with binding buffer, unlabeled peptide was added in a range from 0.1 to 1000 nM in combination with a trace amount of radiolabeled peptide. To prevent internalization, cells were incubated for 2 h at 0 °C. Medium was removed, and cells were washed twice with ice-cold binding buffer. Cells were scraped, and cell-associated radioactivity was counted in 3” well-type γ counter (Perkin-Elmer, Boston, MA). Internalization Assay. Internalization of the radiolabeled peptide was assessed as follows. Cells were cultured and pretreated as described above. Radiolabeled peptides were added at a concentration of 0.1 nM (∼10 000 cpm/ well). Cells were incubated at 37 °C for 2 h, medium was removed, and cells were washed with ice-cold binding buffer. To remove receptor bound radiolabeled peptide, cells were incubated with acid buffer (0.1 M acetic acid, 154 mM NaCl, pH 2.0) for 10 min at 4 °C. After washing the cells twice with ice-cold binding buffer, the internalized fraction was determined by counting the cells in a γ counter.

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Table 1. Labeling Efficiency (%) and Specific Activity (Ci/mmol) of Sulfated and Nonsulfated HYNIC-CCK8 Peptides, Labeled with 99mTc in the Presence of Different Coligands (n ) 3/coligand)a tricine

99mTc-HYNIC-nsCCK8 99mTc-HYNIC-sCCK8

EDDA

NA/tricine

labeling efficiency (%)

S.A. (Ci/mmol)

labeling efficiency (%)

S.A. (Ci/mmol)

labeling efficiency (%)

S.A. (Ci/mmol)

93 ( 2 98 ( 1

400 445

9(1 19 ( 1

88 200

93 ( 3 96 ( 3

678 764

a Labeling conditions are described in the Methods section. Tricine: N-[tris(hydroxymethyl)methyl]glycine; EDDA: ethylenediaminediacetic Acid; NA: nicotinic acid. S.A.: specific activity.

Release of radiolabel from the cells was studied in a separate experiment; 99mTc-sCCK8 or 111In-sCCK8 (each 0.1 nM) were added to CHO-CCK-2 cells and incubated at 37 °C for 2 h. Cells were washed with acid-buffer as described above and subsequently incubated for 2 h at 37 °C with fresh medium (without peptide). Subsequently, the medium was removed and eluted over an activated C18 SepPak-cartridge to determine whether the radioactivity released by the cells was pertechnetate or another metabolite. Receptor Targeting of 99mTc-Labeled HYNICCCK8 in Vivo. Tumor-targeting of the radiolabeled peptides was studied in female athymic BALB/c mice bearing subcutaneous tumors of CCK-receptor transfected CHO-cells. Mice were inoculated with 4 × 106 CHO-CCK1 cells in the right flank and with 4 × 106 CHO-CCK2 cells in the left flank. When tumors had reached a weight of approximately 0.2 g, mice were randomly divided into four groups of six mice each. Mice were injected intravenously with 100 µCi (17 ng) 99mTclabeled sulfated CCK8 or nonsulfated CCK8 (tricine/ nicotinic acid as coligands) with or without a 1000 molar excess of unlabeled sulfated or nonsulfated CCK8, respectively. Mice were killed by CO2 asphyxiation 1 h after injection. Organs of interest, including the tumors, were dissected, weighed, and counted in a gamma-counter. The animal experiments were approved by the local animal welfare committee and performed according to national regulations. RESULTS

HYNIC Conjugation and 99mTc Labeling. The efficiency of the conjugation reaction was 28 ( 3% (n ) 3) for the sulfated and 34 ( 2% (n ) 3) for the nonsulfated CCK8 peptide, as based on the HPLC chromatograms of the conjugation mixtures. Performing the reaction with a 50:1 or 2:1 molar excess of s-HYNIC instead of 10:1 or when using dimethylformamide (DMF) rather than PBS as the solvent, the conjugation efficiency did not improve (data not shown). The conjugated peptides were obtained carrier-free, i.e., without nonderivatized CCK8. After 6 months of storage in PBS at -20 °C, the peptides could still be labeled with high labeling efficiency, comparable to the freshly conjugated peptides (data not shown). Technetium-99m labeling of both peptides was studied using three coligand systems: (i) tricine, (ii) EDDA and (iii) a combination of nicotinic acid and tricine. The labeling efficiency was determined by RP-HPLC. Results of the labeling efficiency are summarized in Table 1. Labeling efficiency after labeling with tricine or the combination of tricine and nicotinic acid was always higher than 90%, whereas labeling efficiency with EDDA as coligand was lower than 20%. Varying the amount of tin (from 5 to 50 µg), used for the reduction during the 99m Tc-labeling, did not improve the labeling efficiency (data not shown). Since serum proteins are known to stabilize 99mTcHYNIC labeled peptides and proteins (19), the stability

Table 2. Stability of 99mTc-Labeled HYNIC-nsCCK8 and HYNIC-sCCK8 after SPE, during Overnight Incubation in PBS at 37 °Ca

99mTc-HYNIC-nsCCK8 99mTc-HYNIC-sCCK8

5 min 16 h 5 min 16 h

tricine (%)

EDDA (%)

NA/tricine (%)

81 ( 3 27 ( 4 88 ( 2 42 ( 3

97 ( 1 92 ( 3 99 ( 1 92 ( 2

91 ( 2 87 ( 4 83 ( 3 77 ( 2

a Aliquots of the incubation mixture were taken at times indicated, and RCP was assessed by RP-HPLC. Values are expressed as percentage RCP ( SD (n ) 3).

of the 99mTc-labeled HYNIC-peptides was studied in PBS. Moreover, the excess of coligand was removed from the labeling mixture by SPE, to prevent possible stabilization by excess unbound coligand. As summarized in Table 2, the 99mTc-peptides labeled in the presence of tricine are highly unstable. Immediately after the SPE purification, the RCP dropped to 81 ( 3% and 88 ( 2%, for 99mTcHYNIC-nsCCK8 and 99mTc-HYNIC-sCCK8, respectively (n ) 3) and were as low as 27 ( 4% and 42 ( 3% (n ) 3) after overnight incubation at 37 °C. Although the labeling efficiency was low when EDDA was used as coligand, after SPE purification the peptides released less than 5% radiolabel during 16 h in PBS. Although less stable than the EDDA-labeled peptides, the combination of nicotinic acid and tricine also showed good stability, with only 4-6% of radiolabel released from the peptides during 16 h at 37 °C in PBS. Liu et al. (20) recently suggested that acetonitrile could also act as coligand and could lead to the formation of mixed coligand systems. Therefore, in additional experiments we used methanol instead of acetonitrile to elute peptides during SPE and found similar results, indicating no exchange of coligands with acetonitrile (data not shown). In Vitro Characterization of 99mTc-Labeled HYNICCCK8. The 50% inhibitory concentration (IC50) of the peptides was determined on the CCK-receptor expressing CHO cells. On the basis of the results obtained in the stability test, peptides were labeled with 99mTc using nicotinic acid and tricine as coligands. The IC50 value of nsCCK8 was 10 nM for the CCK2 receptor, and >1 µM for the CCK1 receptor. IC50 values for sCCK8 were 8 nM and 3 nM, for the CCK1 and CCK2 receptor, respectively. Both 99mTc-labeled CCK8-analogues showed rapid binding to the respective receptors and a time-dependent internalization (Figure 1) in both CCK receptor-positive cell lines. Since the nsCCK8 has virtually no affinity for the CCK1 receptor no binding and internalization was observed. After 2 h incubation at 37 °C, 2.7% of the added 99m Tc-sCCK8 was internalized by the CHO-CCK1 cells and 27% in the CHO-CCK2 cells. After 2 h, 4.6% of nsCCK8 was internalized in CHO-CCK2 cells, while no binding and internalization was observed in the CHOCCK1 cells. Addition of excess unlabeled peptide blocked the binding and internalization of the radiolabeled peptides, indicating their receptor-specific binding and internalization (data not shown).

Tc-99m Labeling of CCK8 Peptides

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Table 3. Biodistribution of 99mTc-Labeled SCCK8 and NsCCK8 in Athymic Mice Bearing CHO-CCK1 and CHO-CCK2 Receptor Expressing Tumors in Each Flanka 99mTc-sCCK8

blood muscle CCK1 tumor CCK2 tumor lung pancreas spleen kidney stomach liver intestine tumor 1-to-blood tumor 2-to-blood tumor 2/tumor 1

0.31 ( 0.13 0.08 ( 0.04 2.04 ( 0.65 4.15 ( 0.28 0.34 ( 0.25 11.7 ( 0.93 0.09 ( 0.03 2.14 ( 0.23 2.01 ( 0.46 0.29 ( 0.06 0.38 ( 0.08 7.1 ( 2.6 14.7 ( 4.3 2.2 ( 0.8

99mTc-sCCK8

excess unlabeled

0.38 ( 0.08 0.09 ( 0.02 0.57 ( 0.13 1.02 ( 0.12 0.37 ( 0.09 0.56 ( 0.09 0.12 ( 0.03 2.87 ( 0.57 0.86 ( 0.13 0.39 ( 0.10 0.46 ( 0.25 1.5 ( 0.2 2.8 ( 0.6 1.8 ( 0.4

99mTc-nsCCK8

0.21 ( 0.05 0.05 ( 0.01 0.20 ( 0.08 0.59 ( 0.06 0.23 ( 0.05 0.13 ( 0.02 0.10 ( 0.02 1.36 ( 0.25 0.68 ( 0.13 0.39 ( 0.07 0.24 ( 0.04 0.9 ( 0.2 4.0 ( 2.6 4.3 ( 2.9

99mTc-nsCCK8

excess unlabeled

0.23 ( 0.08 0.09 ( 0.03 0.33 ( 0.15 0.40 ( 0.07 0.38 ( 0.16 0.24 ( 0.11 0.18 ( 0.04 1.68 ( 0.14 0.58 ( 0.14 0.68 ( 0.12 0.31 ( 0.09 1.5 ( 0.8 1.9 ( 0.7 1.4 ( 0.5

a Mice were injected with 17 ng of Tc-99m-labeled peptide (100 µCi) and were killed at 1 h postinjection. Values are expressed as percentage of the injected dose per gram tissue (% ID/g) ( SD (n ) 5 mice/group).

Figure 1. Internalization curve of 99mTc-nsCCK8 (dotted lines) and 99mTc-sCCK8 incubated with CHO-CCK1 (square symbols) or CHO-CCK2 (triangle symbols) cells at 37 °C. Values are expresssed as percentage of the added radioactivity present in the cells after acid wash.

In a separate in vitro experiment it was shown that the internalized radioactivity (i.e., after adding 99mTcsCCK8) was rapidly excreted by the CHO-CCK-2 cells. After 2 h incubation in fresh medium without peptide, 36% of the internalized radiolabel was excreted into the medium, indicating that the 99mTc was not residualized in the cell. As a control, the internalization and residualization of sCCK8 labeled with the residualizing radionuclide111In was investigated. In this case, no release of radioactivity from the cell was measured over a 2-h period, indicating residualization of the 111In label. An additional experiment using SPE showed that 14 ( 4% (n ) 3) of the excreted 99mTc activity was pertechnetate. Receptor Targeting of 99mTc-Labeled HYNICCCK8 in Vivo. Both 99mTc-labeled CCK8 analogues showed a rapid blood clearance. At 1 h pi, the blood level of 99mTc-nsCCK8 was 0.21 ( 0.05% ID/g and that of 99mTcsCCK8 was 0.31 ( 0.13% ID/g. Uptake of 99mTc-sCCK8 in CCK1 receptor-positive pancreas was 90-fold higher than that of 99mTc-nsCCK8. The biodistribution data are summarized in Table 3 and Figure 2. Marked differences between the sulfated and nonsulfated peptides were observed in the tumor uptake in CCK2 receptor-positive tumors. Uptake of 99mTc-sCCK8 was 4.15 ( 0.28% ID/g at 1 h pi, whereas the tumor uptake of 99mTc-nsCCK8 was 7-fold lower (0.59 ( 0.06% ID/g). Blocking with an excess of unlabeled peptide resulted in a significantly lower uptake in the tumor. Uptake of 99mTc-sCCK8 dropped to only 1.02 ( 0.12% ID/g and uptake of 99mTc-nsCCK8 was reduced to 0.40 ( 0.07% ID/g, indicating CCK receptor-mediated binding of the radiolabeled peptides in the tumor. Blood levels of

Figure 2. Biodistribution of 99mTc-nsCCK8 and 99mTc-sCCK8 in athymic mice bearing CCK1 and CCK2 receptor expressing tumors in each flank. Values are expressed as percentage of the injected dose per gram tissue (n ) 5 mice/group). Blocking was performed by coinjection of a 20 000 molar excess of unlabeled minigastrin. Mice were dissected at 1 h postinjection.

the radiolabeled peptides were unaffected by the excess of unlabeled peptide. Therefore, the tumor-to-blood ratios decreased from 14.7 ( 4.3 to 2.8 ( 0.6 and from 4.0 ( 2.6 to 1.9 ( 0.7 for 99mTc-sCCK8 and 99mTc-nsCCK8, respectively. Uptake of the 99mTc-sCCK8 in the CHO-CCK1 tumor was 2.04 ( 0.65% ID/g, whereas uptake of the 99mTcnsCCK8 was only 0.20 ( 0.08% ID/g. Tumor-to-blood ratios were 7.1 ( 2.9 and 0.9 ( 0.2 for 99mTc-sCCK8 and 99m Tc-nsCCK8, respectively. 99mTc-sCCK8 uptake in the CCK1 receptor positive tumor could be specifically blocked by an excess of unlabeled sCCK8 (0.57 ( 0.13% ID/g). An excess of unlabeled nsCCK8 did not affect the tumor uptake of 99mTc-nsCCK8, indicating that this nonsulafted peptide did not localize specifically in the CCK1 receptor positive tumor. The tumor uptake remained in the same low range at 0.33 ( 0.15% ID/g. DISCUSSION

The present study describes the characterization of the CCK8 peptide both in its sulfated and nonsulfated form and labeled with 99mTc via the bifunctional chelator hydrazino nicotinamide. Both peptides were conjugated with an efficiency of approximately 30%. This efficiency is in the same range as has been reported previously for the HYNIC conjugation of other peptides (10). The HYNIC-conjugated peptides showed a good shelf life

566 Bioconjugate Chem., Vol. 15, No. 3, 2004

stability over a 6-month period. Although it is wellrecognized that HYNIC has good stability in lyophilized products, its stability in aqueous solutions is reduced (21). Therefore, in the present study we used the propylaldehyde hydrazone protected HYNIC, which previously has shown improved stability in aqueous solutions (15). The propylaldehyde protecting group can be easily removed under mild conditions (12). To obtain 99mTc-labeled peptides with maximal stability and specific activity, three different coligand systems were tested. Although tricine is the most commonly used coligand, mainly due to the high labeling efficiency obtained at room temperature, we showed that the combination of tricine and nicotinic acid yielded 99mTclabeled peptides with the highest specific activity and improved stability in PBS. The labeling efficiency with the tricine coligand system was high, while the stability of these 99mTc-labeled peptides was poor. After 16 h incubation in PBS at 37 °C, only 27% of the 99mTc activity was still peptide associated. This is in line with other studies in which it was found that the 99mTc-tricineHYNIC complex is stable in serum, but unstable when diluted in PBS (22). Most likely, the 99mTc-tricine-HYNIC complex is stabilized by the serum proteins. Ono et al. hypothesized that one of the two tricine molecules in the complex is replaced by plasma proteins which might greatly affect the circulatory half-life, especially of smaller molecules such as peptides (19). The EDDA coligand system showed excellent in vitro stability. However, the specific activity which could be obtained with EDDA was only 12%-25% of that obtained with the other coligand systems. Since the sulfated form of CCK8 is extremely biologically active, its possible clinical application will markedly improve when the peptide can be labeled with high specific activity preventing pharmacological side effects. Therefore, EDDA is less favorable as coligand for the CCK8 peptides. Receptor binding and internalization studies were performed on CHO cells stably transfected with cDNA encoding for either the CCK1 or CCK2 receptor. This enabled us to investigate the receptor specifity and binding to each receptor in more detail than when using tumor cells. We found a high affinity of 99mTc-sCCK8 for both the CCK1 and CCK2 receptor and high affinity of 99m Tc-nsCCK8 for the CCK2 receptor. These results are in line with those of Smeets et al., using the same cell lines but underivatized peptides (8). Moreover, these low IC50 values are in the same range as those reported for indium-111-labeled DTPA-nsCCK8 (5). Both CCK receptors belong to the superfamily of G-protein-coupled receptors (GPCR) and are known to be internalized when gastrin or CCK8 binds to the receptor. Once internalized, the receptor can be recycled and re-expressed on the cell surface or be metabolized. Recycling of the receptor takes approximately 1 h (23). It has also been demonstrated that after internalization the peptides can end up in lysosomes and are rapidly degraded by proteolytic enzymes (23). In our studies we found a steady release of the 99mTc label after internalization. This released radioactivity could not be bound by the cells again, confirming processing of the radiolabeled peptide. Studies with 111In-labeled nsCCK8 described internalization and subsequent retention of the radiolabel (4). Duncan et al. showed that 111In-DTPAlabeled peptides are degraded in the lysosomes to yield 111 In-DTPA-amino acids, which are retained by the lysosomes (24). Shankar and colleagues stated that trapping in lysozomes requires the complex to be protonated at lysozomal pH ∼ 5 (25). Ono et al. confirmed

Laverman et al.

that 99mTc-HYNIC-labeled peptides are degraded in a similar way as 111In-DTPA-labeled peptides (26). Here we show that the 99mTc-coligand-HYNIC-amino acid complex is apparently not positively charged at lysozomal pH and is excreted by the lysosomes, because radioactivity was released by the tumor cells. Although the residualizing property of 111In is beneficial for therapy purposes, residualization may not be required for receptor-mediated peptide scintigraphy, due to the shorter time frame (1-3 h postinjection) necessary for imaging with 99mTc. The in vivo characteristics of both 99mTc-labeled peptides were investigated in athymic mice bearing subcutaneous tumors expressing either the CCK1 or CCK2 receptor. This allowed us to investigate the specificity of receptor binding in vivo. Because 99mTc-nsCCK8 had no affinity for the CCK1 receptor it did not localize specifically in the CHO-CCK1 tumor. The same holds for the pancreas, in which the CCK1 receptor is abundantly expressed in mice (27). The biodistribution of 99mTcsCCK8 was markedly different from that of the 99mTclabeled nonsulfated peptide. In general, uptake of 99mTcsCCK8 was higher in all CCK-receptor (either CCK1 or CCK2) expressing tissues, including both tumors. Uptake in the CCK2 tumor could be blocked by an excess of unlabeled (non)sulfated CCK8, indicating receptor-mediated uptake of the 99mTc-labeled peptide in this tumor. The 99mTc-nsCCK8 uptake in the CCK1 tumor could not be blocked, indicating that the low uptake of the 99mTcnsCCK8 was not receptor-mediated. We showed that both CCK1 and CCK2 tumor uptake of 99mTc-sCCK8 was approximately 15-fold higher than that of 99mTc-nsCCK8. Several studies reported on the use of radiolabeled nonsulfated CCK8 for the scintigraphic imaging and/or therapy of CCK2/gastrin receptor positive tumors. The rationale for the use of nsCCK8 is its CCK2 receptor specificity, resulting in low background accumulation in CCK1 receptor-positive tissues (3, 4). However, the results of the present study suggest that 99m Tc-labeled sulfated CCK8 might be much more favorable for imaging and/or therapy, based on its significantly higher uptake in both the CCK1 and the CCK2 receptor positive tumors. This higher uptake may be due to the higher affinity for the CCK2 receptor (2). We showed in mice that uptake of the 99mTc-labeled sulfated peptide in nontarget tissue such as pancreas and stomach is also higher as compared to the accumulation of the 99mTclabeled nonsulfated peptide, but this may be different in humans. Weinberg et al. (28) analyzed the CCK-receptor expression in a wide range of human tissues and in several human tumors. They showed that the CCK1 receptor is expressed mainly in gall bladder, small intestine, colon, and spleen. The CCK2 receptor is expressed in a much broader range of tissues, including pancreas, small intestine, liver, colon, stomach, spleen, and lung. Interestingly, the CCK1 receptor is expressed in pancreatic adenocarcinomas but not in normal pancreas (28). In contrast, in mice and rats the CCK1 receptor is abundantly expressed in the pancreas (27, 29). Therefore, it is tempting to speculate that the biodistribution of both peptides in humans will be different from that observed in mice and that 99mTc-labeled sulfated CCK8 might be a good candidate for clinical testing. CONCLUSION

CCK8 peptides can be rapidly and efficiently labeled with 99mTc using HYNIC as chelator and tricine/nicotinic acid as coligand system, without compromising the receptor binding affinity of the peptides. Both 99mTc-

Tc-99m Labeling of CCK8 Peptides

labeled sulfated and nonsulfated CCK8 bind with high affinity to the CCK2 receptor, whereas 99mTc-sCCK8 also showed high affinity toward the CCK1 receptor. Studies in athymic mice revealed that uptake of 99mTc-sCCK8 in CCK1 or CCK2 receptor-positive tumors was fifteen-fold higher than that of 99mTc-nsCCK8. Although accumulation in some nontarget organs was also higher with 99mTcsCCK8, this may not reflect the human situation, due to a different receptor expression pattern in human tisssues as compared to that in mice. Therefore, further studies are warranted to investigate the possible use of 99mTcsCCK8 for the scintigraphic imaging of CCK receptorpositive tumors in humans. ACKNOWLEDGMENT

We thank Mr. Gerrie Grutters and Mrs. Bianca Lemmers-de Weem (Central Animal Laboratory, University of Nijmegen, Nijmegen, The Netherlands) for skilled assistance in the animal experiments. We thank Mr. Matthias Broekema (Department of Medicinal Chemistry, Utrecht University, Utrecht, The Netherlands) for performing the MALDI-TOF-MS analysis. Supporting Information Available: RP-HPLC, MALDITOF-MS data. This material is available free of charge via the Internet at http://pubs.acs.org/BC. LITERATURE CITED (1) Boerman, O. C., Oyen, W. J., and Corstens, F. H. (2000) Radio-labeled receptor-binding peptides: a new class of radiopharmaceuticals. Semin. Nucl. Med. 30, 195-208. (2) Reubi, J. C., Schaer, J. C., and Waser, B. (1997) Cholecystokinin(CCK)-A and CCK-B/gastrin receptors in human tumors. Cancer Res. 57, 1377-1386. (3) Behr, T. M., Jenner, N., Behe, M., Angerstein, C., Gratz, S., Raue, F., and Becker, W. (1999) Radiolabeled peptides for targeting cholecystokinin-B/gastrin receptor-expressing tumors. J. Nucl. Med. 40, 1029-1044. (4) de Jong, M., Bakker, W. H., Bernard, B. F., Valkema, R., Kwekkeboom, D. J., Reubi, J. C., Srinivasan, A., Schmidt, M., and Krenning, E. P. (1999) Preclinical and initial clinical evaluation of 111In-labeled nonsulfated CCK8 analog: a peptide for CCK-B receptor-targeted scintigraphy and radionuclide therapy. J. Nucl. Med. 40, 2081-2087. (5) Reubi, J. C., Waser, B., Schaer, J. C., Laederach, U., Erion, J., Srinivasan, A., Schmidt, M. A., and Bugaj, J. E. (1998) Unsulfated DTPA- and DOTA-CCK analogues as specific high-affinity ligands for CCK-B receptor-expressing human and rat tissues in vitro and in vivo. Eur. J. Nucl. Med. 25, 481-490. (6) Behe, M., Becker, W., Gotthardt, M., Angerstein, C., and Behr, T. M. (2003) Improved kinetic stability of DTPA-dGlu as compared with conventional monofunctional DTPA in chelating indium and yttrium: preclinical and initial clinical evaluation of radiometal labeled minigastrin derivatives. Eur. J. Nucl. Med. Mol. Imag. 30, 1140-1146. (7) Wank, S. A., Pisegna, J. R., and de Weerth, A. (1994) Cholecystokinin receptor family. Molecular cloning, structure, and functional expression in rat, guinea pig, and human. Ann. N. Y. Acad. Sci. 713, 49-66. (8) Smeets, R. L., Fouraux, M. A., van Emst-de Vries S. E., De Pont, J. J., and Willems, P. H. (1998) Protein kinase Cmediated inhibition of transmembrane signaling through CCK(A) and CCK(B) receptors. Br. J. Pharmacol. 123, 11891197. (9) Liu, S., and Edwards, D. S. (1999) 99mTc-Labeled Small Peptides as Diagnostic Radiopharmaceuticals. Chem. Rev. 99, 2235-2268. (10) Rennen, H. J., Boerman, O. C., Koenders, E. B., Oyen, W. J., and Corstens, F. H. (2000) Labeling proteins with Tc-99m via hydrazinonicotinamide (HYNIC): optimization of the conjugation reaction. Nucl. Med. Biol. 27, 599-604.

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