Cholecystokinin-B/Gastrin Receptor Binding Peptides: Preclinical Development and Evaluation of Their Diagnostic and Therapeutic Potential I

The high sensitivity of pentagastr in stimulation in detecting pr imary or metastatic medullary thyroid cancer (MTC) suggests widespread expression of the corresponding receptor type on human MTC. Indeed, autoradiographic studies demonstrated cholecystokinin (CCK)-B/gastrin receptors not only in >90% of MTCs but in a high percentage of small cell lung cancers and potentially a variety of gastrointestinal adenocarcinomas. In a pilot study, we have demonstrated the feasibility of radiolabeled gastrin-I to target CCK-B receptor-expressing tissues in vivo in animals and patients (T. M. Behr et al., Eur. J. Nucl. Med., 25: 424-430, 1998). The aim of the present study was to systematically optimize, in a preclinical model, suitable radioligands for targeting CCK-B receptors in vivo. For this purpose, a variety of CCK/gastrin-related peptides, all having in common the COOH-terminal CCK-receptor binding tetrapeptide sequence Trp-Met-Asp-PheNH 2 or derivatives thereof, were studied. They were radioiodinated by the Iodogen or Bolton-Hunter procedures. The peptides tested were members of the gastrinor cholecystokinin families or possessed characteristics of both, which differ by the intramolecular position of a tyrosyl moiety (occurring in native or sulfated form). Their stability and affinity were studied in vitro and in vivo; their biodistribution and therapeutic efficacy were tested in nude mice bearing s.c. human MTC xenografts. Diethylene-triamine-pentaacetate derivatives of suitable peptides were synthesized, evaluated, and labeled with HlIn. 1 Presented at the "Seventh Conference on Radioimmunodetection and Radioimmunotherapy of Cancer," October 15-17, 1998, Princeton, NJ. Supported by Grant DFG Be 1689/4-1 from the Deutsche Forschungsgemeinschaft and a grant from the Directorate General of the European Commission. 2 To whom requests for reprints should be addressed, at Department of Nuclear Medicine, Georg-August-University, Robert-Koch-Strasse 40, D-37075 G6ttingen, Germany. Phone: 49-551-39-8510; Fax: 49-55139-8526; E-mail: [email protected]. All members of the CCK or gastrin family were stable in serum (with t,/2s of several hours at 37~ nevertheless, the stability of those peptides was highest that bore the NH2-terminal pGlu residues (e.g., big gastrin, gastrin-I, caerulein, and others) or o-amino acids. In accordance to their comparably low affinity, nonsulfated members of the CCK family showed fairly low uptake in the tumor and other CCK-B receptor-expressing tissues (e.g., the stomach). Sulfated CCK derivatives performed significantly better but additionally displayed a high uptake in normal, CCK-A receptor-expressing tissues (such as the liver/gallbladder, pancreas, and bowel). Best tumor uptake and tumor:nontumor ratios were obtained with members of the gastrin family, probably because of their selectivity and affinity for the CCK-B receptor subtype. Pilot therapy experiments in MTC bearing animals showed significant ant i tumor efficacy as compared with untreated controls, l l l In-Labeled diethylene-triamine-pentaacetate derivatives of minigastr in showed excellent targeting of CCK-B receptor-expressing tissues in animals and a normal human volunteer. These data suggest that CCK/gastr in analogues may be a useful new class of receptor binding peptides for diagnosis and therapy of CCK-B receptor-expressing tumors, such as MTC or small cell lung cancer. Nonsulfated gastrin derivatives may be preferable because of their CCK-B receptor selectivity, and hence, lower accretion in normal CCK-A receptor-expressing organs. Fur ther preclinical as well as clinical studies are ongoing. I n t r o d u c t i o n The development of regulatory peptides as tools to visualize and, more recently, also to treat malignant tumors has been an important focus of interest over the past years (1-3). The successful development of sufficiently stable, radiolabeled somatostatin analogues, such as 123I-Tyr3or 111In-DTPA3-I> PheX-octreotide for diagnostic purposes (1, 2), as well as the introduction of ~61Tb-DTPAor 9~ N,N',N",N'-tetra-acetate-conjugates (3) for therapeutic applications, has opened new horizons in nuclear oncology (3-5). More recently, other regulatory peptides, such as vasoactive intestinal polypeptide (6), substance P (7), or gastrin-releasing peptide/ bombesin derivatives (8), have emerged as potentially useful candidates for in vivo scintigraphy and radiopeptide therapy. Whereas somatostatin receptor scintigraphy has proven as 3 The abbreviations used are: DTPA, diethylene-triamine-pentaacetate; CCK, cholecystokinin; MTC, medullary thyroid cancer; HPLC, highperformance liquid chromatography; MTD, maximum tolerated dose; % ID/g, percentage of injected dose per gram; p.i., post injection. Research. on May 1, 2017. © 1999 American Association for Cancer clincancerres.aacrjournals.org Downloaded from Clinical Cancer Research 3125s valuable tool for the staging of gastroenteropancreatic tumors (e.g., carcinoids), its sensitivity and accuracy in other neoplasms, such as MTC or small cell lung cancer, is limited (9-11). Because of the outstanding diagnostic accuracy of the pentagastrin test in detecting the presence, persistence, or recurrence of malignant C cells, even far below a size detectable by conventional morphological imaging methods, we postulated the expression of the corresponding receptor type in human MTC (12). Indeed, receptor autoradiographic studies by Reubi and Waser (13) had demonstrated the expression and presence of CCK-B/gastrin receptors not only in >90% of MTCs but in a high percentage of other tumor types, such as small cell lung cancer, stromal ovarian cancers, and astrocytomas as well (14). Earlier studies suggested an even more widespread occurrence of gastrin receptors, e.g., in colon, pancreatic, or stomach cancers (15). Therefore, we undertook pilot experiments in nude mice bearing human MTC xenografts and were able to demonstrate the feasibility of radiolabeled gastrin-I to target CCK-B receptor-expressing tumors, as well as CCK receptor-expressing normal organs (e.g., the stomach) in vivo in animals and patients (12). However, a variety of problems remained to be solved, such as the molecular characteristics, which render the peptide as an optimal candidate for in vivo targeting of CCK-B receptorexpressing tumors, considering in vivo stability, affinity to and selectivity for the CCK-B receptor, or the potentially unfavorable accretion in normal organs (such as the liver, bowel, or kidney). The aim of the present study was, therefore, to develop and optimize, in a preclinical model, suitable radioligands for targeting CCK receptors in vivo. Materials and Methods Peptides. The peptides as listed in Table 1 were investigated in the present study. They belong either to the gastrin or cholecystokinin "super" families or possess characteristics of both (16). Both families differ by the location of their tyrosyl moiety. These peptides were commercially obtained from Sigma Chemie (Deisenhofen, Germany), ICN Biomedicals (Eschwege, Germany), or Bachem Biochemicals (Heidelberg, Germany), whereas [Gln56-m]-gastrin-I, [Gln52-6]-minigastrin, D-Leu ~minigastrin, and gastrin-I fragment 11-17 were synthesized by Genosys Biotechnologies (Cambridge, England, United Kingdom). The peptides were stored frozen as lyophilized powder at -20~ Immediately before use, the peptides were dissolved in 0.05 M aqueous NHgOH at concentrations between 1 and 10 mg/ml. Radioiodination of the Peptides. Iodine-131 and iodine125 were purchased as sodium iodide in 0.1 M NaOH from New England Nuclear DuPont (Brussels, Belgium and North Billerica, MA). Radioiodination by the Iodogen method was performed essentially as described earlier (12). Briefly, the peptide in 0.05 M NH4OH was transferred into an Iodogencoated glass vial (100 p,g of Iodogen coating the inner surface of a 2-ml vial) with a magnetic stirbar placed inside. Five hundred Ixl of 0.5 M sodium phosphate buffer (pH 7.4) were added. The vial was placed on a magnetic stirrer, and the activity was added in 500 ~xl of 0.05 M sodium phosphate (pH 7.4; up to 200-250 mCi/mg of peptide). After a stirring time of 5-10 min, the radioiodinated peptide was purified from unreacted iodine by gel filtration chromatography on a PD-10 column (Sephadex G-25 preequilibrated in a 15 • 50-mm polypropylene column, which was purchased from Sigma Chemie). The column was eluted with 0.05 M PBS buffer (pH 7.4), collecting l0 drops/ fraction. Fractions containing the labeled peptide were combined and were subsequently filtered through a sterile Millex-G filter (pore size, 0.22 ]xm; Millipore, Molsheim, France). Radiochemical purity was shown by HPLC (Nucleosil 120-3 C t s, 250 • 4 mm, Marchery-Nagel; solvent A: 0.1% trifluoroacetic acid in water; solvent B: AcCN; flow rate 0.5 ml/min; gradient: 0-5 rain 100% solvent A, 0% solvent B; 5-15 min 20% solvent A, 80% solvent B). Radioiodination by the BoRon-Hunter procedure was performed essentially as described previously (17). Briefly, 2.5 mg of crystalline succinimidyl-3-(4-hydroxyphenyl-)propionate (Sigma Chemie) were dissolved in 10 ml of 0.25 M PBS (pH 7.5). One hundred pul of this solution were mixed with 10 mCi of radioiodine (Nam31I or Na125I) in 400 pA of 0.25 M PBS (pH 7.5) and 2 mg of chloramine-T (Sigma Chemie). The reaction was stopped after 10 s by the addition of 1.2 mg of sodium metabisulfite in 600 wl of 0.05 M PBS (pH 7.5). After the addition of 200 txl of dimethylformamide, the radioiodinated BoRon-Hunter reagent was extracted with two 500-~1 portions of benzene and recovered by evaporating the solvent under a gentle stream of dry nitrogen. The peptide (100 txg) was added in 200 txl of 0.1 M sodium borate (pH 8.5) in an ice bath. After vortexing the mixture for 15 min in the ice bath, the reaction mixture was separated on a Sephadex G-25 Superfine column (1 • 55 cm). The radiochemical purity was shown by HPLC as described above. Synthesis of DTPA-Derivatives and Labeling with l l t In . Five mg of minigastrin (obtained from Bachem Biochemicals, Heidelberg, Germany), its [o-Leul]-derivative (Genosys, Cambridge, United Kingdom), or sulfated CCK-8 (Bachem Biochemicals) were dissolved in 1 ml of 0.05 M aqueous NH4OH. Two hundred lxl of 2 M NaHCO 3 were added, and the pH was adjusted to 8 with 1 M HC1. Cyclic DTPA anhydride (Sigma Chemie) was added as a dry powder (18) in 10-fold molar excess over the peptide (i.e., 12 rag). After incubating the reaction mixture for 60 rain at room temperature, it was diluted to 10 ml with water. This solution was applied to a DEAE Sephadex A-50 weakly basic anion exchange resin column (1 • 20 cm) at 4~ preequilibrated with 10 bed volumes each of saturated, 2 M, and 0.05 M NH4HCO 3. The column was eluted at 4~ with 30 ml of 0.05 M NH4HCO 3, followed by a linear gradient of 0.05 M versus 2 M NH4HCO 3 (150 ml each; Ref. 19). The elution of the peptide was monitored by determination of the absorbance at 280 nm and by a modified bicinchoninic acid procedure (Micro BCA assay; Pierce, Rockford, IL). Fractions containing the DTPA-derivatized peptide were combined and lyophilized. The peptide was stored frozen at 2 0 ~ in 0.5 M NH4OAc/0.05 M ammonium ascorbate at pH 5.5 in a concentration of 5 mg/ml. Immediately before use, the peptide was thawed. 11 tinC13 (Mallinckrodt, Petten, Netherlands) was added in 100 pxl of 0.5 M NH4OAc (pH 5.5) to yield a specific activity of up to 400 txCi/p~g of peptide. The radiochemical purity was shown by HPLC as described above. Research. on May 1, 2017. © 1999 American Association for Cancer clincancerres.aacrjournals.org Downloaded from 3126s CCK-B/Gastrin Receptor Binding Peptides