Design, Synthesis, and Initial Evaluation of High ... - ACS Publications

Potent antagonists of bombesin-like peptides have shown great potential for applications in cancer therapy. A 99mTc-labeled agent capable of identifyi...
14 downloads 0 Views 122KB Size
Download PDF
218

Bioconjugate Chem. 1998, 9, 218−225

Design, Synthesis, and Initial Evaluation of High-Affinity Technetium Bombesin Analogues Kwamena E. Baidoo,* Kuo-Shyan Lin, Yougen Zhan, Paige Finley, Ursula Scheffel, and Henry N. Wagner, Jr. Division of Radiation Health Sciences, Department of Environmental Health Sciences, The Johns Hopkins University, 615 North Wolfe Street, Baltimore, Maryland 21205. Received November 13, 1997; Revised Manuscript Received January 15, 1998

Potent antagonists of bombesin-like peptides have shown great potential for applications in cancer therapy. A 99mTc-labeled agent capable of identifying patients who could benefit from these emerging therapies would have a great impact on patient management. This study involves the synthesis and initial evaluation of technetium diaminedithiolate analogues derived from the potent bombesin analogue Pyr-Gln-Lys-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2 (Lys3-bombesin). We coupled two diaminedithiol (DADT) bifunctional chelating agents (BCAs 1 and 2) to the Lys3 residue at the N-terminal region that is not required for binding to the receptor. 99mTc labeling was performed by ligand exchange on addition of [99mTc]glucoheptonate to a solution of the adduct at room temperature. Two products were obtained from each adduct on analysis by HPLC. The major to minor product ratios of the 99mTc-labeled analogues were 3:1 for products from BCA 1 and 9:1 for the products from BCA 2. Macroscopic amounts of the 99Tc analogues were similarly prepared using [99Tc]glucoheptonate. In this case, the major to minor ratios were 2:1 for the products from both BCAs. For initial evaluation of the binding of the Tc-labeled peptides to bombesin receptors, the 99Tc analogues were used in vitro in competitive binding assays in rat brain cortex membranes against [125I-Tyr4]bombesin. Results of the in vitro assays showed that the inhibition constants (Ki) of the major and minor products were 3.5 ( 0.7 and 3.9 ( 1.5 nM, respectively, for the products from BCA 1; and 7.4 ( 2.0 and 5.2 ( 1.5 nM for the products derived from BCA 2, respectively. The high affinity exhibited by these technetium analogues is an indication of their potential for use in non-invasive in vivo biochemical characterization of cancers that possess receptors for bombesin.

INTRODUCTION

Bombesin is a 14-amino acid peptide initially isolated from frog skin (1). The main mammalian members of the bombesin family of peptides are gastrin-releasing peptide (GRP), a 27-amino acid peptide isolated from porcine stomach (2), and neuromedin B, a 10-amino acid peptide isolated from porcine spinal cord (3). Receptors for bombesin/GRP are expressed in the central nervous system as well as in peripheral tissues such as the intestines and pancreas. Many tumors are also known to express bombesin/GRP receptors. These include glioblastoma, small cell lung cancer, prostate, breast, gastric, colon, and pancreatic cancer (4-17). In many of these cancers, bombesin/GRP analogues function as autocrine growth factors. Activation of bombesin/GRP receptors on cancer cells causes a number of biochemical events such as phosphatidylinositol turnover, activation of protein kinase C, and elevation of cytosolic calcium leading to the expression of early oncogenes such as c-fos and c-jun and ultimately results in stimulated growth and proliferation of the cells (5, 18, 19). It is conceivable that disruption of the biochemical pathways of bombesin action on cancer cells would result in the inhibition of growth. This has been the basis of recent efforts to * Author to whom correspondence should be addressed at Division of Radiation Health Sciences, Department of Environmental Health Sciences, The Johns Hopkins University, 615 N. Wolfe St., Room 2001, Baltimore, MD 21205. Telephone: (410) 955-7706. Fax: (410) 955-6222. E-mail: [email protected].

develop therapeutic regimens in several cancers. For example, an antibody to the receptor binding C terminus of bombesin and GRP was reported to effect clinical remission of small cell lung cancer in a patient and partial remission in others (20). In another approach, several antagonists to the receptor have also been developed. The early antagonists were substance P analogues and were generally of low affinity (21, 22). The second-generation analogues, such as isobutyryl-His-TrpAla-Val-D-Ala-His-Leu-NH-CH3 (23), D-Tpi6,Leu13Ψ(CH2(RC-3095) (24), and NH)-Leu14-bombesin6-14 3-phenylpropanoyl-His20,D-Ala24,Leu26Ψ(CH2NH)-Phe27GRP20-27 (BW2258U89) (25), are highly potent. Among these, D-Tpi6,Leu13Ψ(CH2NH)-Leu14-bombesin6-14 (RC3095) has been shown to inhibit the growth of many cancers (9-14, 16, 17, 26, 27), and 3-phenylpropanoylHis20,D-Ala24,Leu26Ψ(CH2NH)-Phe27-GRP20-27(BW2258U89) has been shown to inhibit the growth of small cell lung cancer cells (28). Furthermore, labeling bombesin analogues with therapeutic radionuclides would result in derivatives that could be used directly in cancer therapy as has been demonstrated using a 105Rh-labeled bombesin analogue (29). A radiolabeled agent capable of identifying the patients that could benefit from these emerging therapies would have a great impact on patient management. This study involves the synthesis and initial evaluation of receptor binding of technetium analogues derived from the bombesin analogue Pyr-Gln-Lys-Leu-Gly-Asn-GlnTrp-Ala-Val-Gly-His-Leu-Met-NH2 (Lys3-bombesin) and

S1043-1802(97)00195-X CCC: $15.00 © 1998 American Chemical Society Published on Web 02/18/1998

High-Affinity Technetium Bombesin Analogues

Figure 1. Diaminedithiol bifunctional chelating agents.

the diaminedithiol (DADT) bifunctional chelating agents (BCA) 1 and 2 (Figure 1) (30, 31), which are capable of forming highly stable complexes with technetium. MATERIALS AND METHODS

Solvents and chemicals were reagent grade and used as received unless specified. [99Tc]NH4TcO4 and [125ITyr4]bombesin were purchased from NEN Life Science Products (Boston, MA). [99mTc]NaTcO4 was purchased as a solution in physiologic saline from Syncor (Timonium, MD). The Glucoscan kit was purchased from the Du Pont Radiopharmaceutical Division (Billerica, MA). Bombesin, Lys3-bombesin, and somatostatin were purchased from Advanced ChemTech (Louisville, KY). Male CD-1 mice and Sprague-Dawley rats were purchased from the Charles River Laboratories (Charles River, MA). The DADT bifunctional chelating agents 1 and 2 were synthesized according to our previously published procedures (30, 31). Infrared spectroscopic determinations of neat liquids or KBr pellets were performed on a Nicolet 205 FTIR system. 1H NMR spectra were recorded in a deuteriochloroform or deuteriomethylene chloride solution using a Bruker WM 300 MHz spectrometer. Elemental analyses were performed by Atlantic Microlabs (Norcross, GA). MALDI mass spectral analyses were performed by the University of Minnesota Mass Spectrometry Service Laboratory (Minneapolis, MN). HPLC was performed with a Waters Chromatography Division HPLC System equipped with two model 510EF pumps, a model 680 automated gradient controller, a model 490 UV absorbance detector, an EG&G NaI scintillation detector, and a Hewlett-Packard HP 3392A or Shimadzu CR3A integrating recorder. HPLC solvents consisted of acetonitrile containing 0.1% trifluoroacetic acid (solvent A) and water containing 0.1% trifluoroacetic acid (solvent B). Liquid scintillation counting for quantification of 99Tc activity was performed on an LKB Wallac instrument model 1219 Rackbeta using Ecolume (ICN Pharmaceuticals, Inc., Costa Mesa, CA) as a cocktail. Filtration of in vitro binding assay mixtures was performed on a BRANDEL, 48M Harvester. 125I activity on filters from in vitro assays and 99mTc activity of tissues from biodistribution studies were counted in an automated γ-counter (Pharmacia-Wallac model 1282). Analyses of in vitro binding assay results were performed using the EBDA/LIGAND programs from Biosoft (Milltown, NJ). 99mTc activity for synthetic reactions was measured in a Capintec CRC-7 Dose Calibrator. Coupling of the Diaminedithiol Bifunctional Chelating Agents 1 and 2 to Pyr-Gln-Lys-Leu-GlyAsn-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2 (Lys3Bombesin). BCAs 1 and 2 were coupled to Lys3bombesin according to Scheme 1. For coupling reactions using BCA 1 (30), 1‚2HCl (19.4 mg, 53.4 µmol) and PyrGln-Lys-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-MetNH2 (9.0 mg, 5.3 µmol) were dissolved in borate buffer (0.8 mL, 0.1 M, pH 9) and acetonitrile (0.9 mL) and the pH was readjusted to 9 with triethylamine. The mixture

Bioconjugate Chem., Vol. 9, No. 2, 1998 219

was incubated at room temperature for 4.5 h and then extracted with ether (3 × 2 mL). The aqueous fraction was chromatographed by semipreparative reversed phase HPLC [linear gradient from solvent B (100%) to solvent A (80%)/solvent B (20%) over the course of 40 min at a flow rate of 6 mL/min using a C-18 Novapak cartridge (25 × 100 mm, 6 µm, Waters Chromatography Division, Milford, MA) monitored on-line for UV absorption at 220 nm]. The product with a retention time of 25.6 min was isolated from the HPLC eluate by solid phase extraction using a C-18 Sep-Pak cartridge (Waters Chromatography Division), followed by lyophilization. The isolated yield of the adduct named Pyr-Gln-Lys(Pm-DADT)-Leu-GlyAsn-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2 [Lys3(PmDADT)-bombesin] was 5.2 mg (57%). High-resolution MALDI MS: calcd MW for Lys3(Pm-DADT)-bombesin 1880.96, found [M + Na]+ 1904.05. Preparation of the BCA 2 (31) adduct was similarly performed by incubating a mixture of 2 (20.0 mg, 66 µmol) and Pyr-Gln-Lys-Leu-Gly-Asn-Gln-Trp-Ala-ValGly-His-Leu-Met-NH2 (10.0 mg, 6.3 µmol) in a borate buffer (0.7 mL, 0.1 M, pH 9) containing acetonitrile (0.9 mL) for 2.5 h at room temperature. Using the same isolation procedure as above gave the product Pyr-GlnLys(Hx-DADT)-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-HisLeu-Met-NH2 [Lys3(Hx-DADT)-bombesin] with a 26.7 min retention time and a 52% (6.2 mg) isolated yield. High-resolution MALDI MS: calcd MW for Lys3(HxDADT)-bombesin 1894.97, found [M + Na]+ 1917.95. 99mTc Labeling of the Lys3(DADT)-Bombesin Adducts. 99mTc labeling of Lys3(Pm-DADT)-bombesin and Lys3(Hx-DADT)-bombesin was performed as depicted in Scheme 1. A glucoheptonate kit (Glucoscan) was reconstituted with water (1.0 mL). From this solution, [99mTc]glucoheptonate was prepared by addition of an aliquot (200 µL) to a [99mTc]pertechnetate solution (400 µL, 8 mCi) from the generator and vortexed for 1 min. The [99mTc]glucoheptonate solution (300 µL) was then added to a solution of the Lys3(DADT)-bombesin (0.5 µmol) in water (500 µL) and the mixture vortexed for 1 min and then incubated at room temperature for 10 min. The reactions were followed by reversed phase HPLC. Two products were obtained from either adduct. The total yield of products in either case was >90%. The HPLC conditions for the isolation of the products from Lys3(Pm-DADT)-bombesin were a linear gradient from solvent A (15%)/solvent B (85%) to solvent A (50%)/ solvent B (50%) over the course of 70 min at a flow rate of 4 mL/min using a C-18 Novapak cartridge (8 × 100 mm, 4 µm, Waters Chromatography Division) monitored on-line for UV absorption at 220 nm and scintillation for radioactivity. These products were designated Lys3(99mTc-Pm-DADT)-bombesin-1 and Lys3(99mTc-Pm-DADT)bombesin-2 with retention times of 41.3 and 44.5 min, respectively, and a ratio of Lys3(99mTc-Pm-DADT)bombesin-1:Lys3(99mTc-Pm-DADT)-bombesin-2 of 3:1. To isolate the products obtained from Lys3(Hx-DADT)bombesin, the HPLC conditions were solvent A (34%)/ solvent B (66%) using the same column as above at a flow rate of 4 mL/min. The products were designated Lys3(99mTc-Hx-DADT)-bombesin-1 and Lys3(99mTc-Hx-DADT)bombesin-2 with retention times of 11.3 and 13.5 min, respectively, and a ratio of Lys3(99mTc-Hx-DADT)bombesin-1:Lys3(99mTc-Hx-DADT)-bombesin-2 of 1:9. Preparation of the 99Tc Analogues of the Lys3(DADT)-Bombesin Adducts. The general method for the preparation of the 99Tc analogues was as follows. To a solution of Lys3(Pm-DADT)-bombesin or Lys3(Hx-

220 Bioconjugate Chem., Vol. 9, No. 2, 1998

Baidoo et al.

Scheme 1. Synthesis of Technetium Diaminedithiolate Analogues of Bombesin

DADT)-bombesin (0.9 µmol) in methanol (850 µL) and water (200 µL) was added a solution of [99Tc]glucoheptonate (32) in water (45 µL, 20 mM), and the mixture was stirred at room temperature for 1.0 h. Similar products were obtained as described above for the 99mTc analogues. The overall yield of products was 48-69%. The products were isolated by reversed phase HPLC

using the same conditions above for the separation of the Tc analogues. The product ratios were as follows: 2.2:1 Lys3(99mTc-Pm-DADT)-bombesin-1:Lys3(99Tc-PmDADT)-bombesin-2 and 1:2.3 Lys3(99Tc-Hx-DADT)bombesin-1:Lys3(99Tc-Hx-DADT)-bombesin-2. Highresolution MALDI MS: calcd MW for Lys3(99Tc-PmDADT)-bombesin-1 1993.94, found [M]+ 1993.86; calcd 99m

High-Affinity Technetium Bombesin Analogues

MW for Lys3(99Tc-Pm-DADT)-bombesin-2 1993.94, found [M]+ 1993.82; calcd MW for Lys3(99Tc-Hx-DADT)-bombesin-1 2006.94, found [M + Na]+ 2029.85; calcd MW for Lys3(99Tc-Hx-DADT)-bombesin-2 2006.94, found [M + Na]+ 2029.85. In Vitro Binding Assay of the Bombesin Analogues. In vitro binding assays were performed using an adaptation of the method of Moody et al. (33). Briefly, adult male Sprague-Dawley rats were decapitated, the brains dissected on ice, and the medulla/pons removed. The rest of the brain was weighed and homogenized in 50 volumes of Tris-HCl buffer (50 mM, pH 7.4) at 4 °C with a Brinkmann Polytron instrument (setting 5, 15 s). The homogenate was centrifuged at 30000g for 15 min and the resulting pellet resuspended in 10 volumes of Tris-HCl buffer (50 mM, pH 7.4, 4 °C) containing NaCl (100 mM) and incubated at 4 °C for 60 min. The homogenate was centrifuged again at 30000g for 15 min. The resulting pellet was resuspended in 10 volumes of the Tris-HCl buffer (50 mM, pH 7.4). The protein concentration of the membrane suspension was determined using the Pierce (Rockford, IL) BCA Protein Reagent Kit and aliquoted for storage at -70 °C at a concentration of 5 mg protein/mL. For the receptor binding assay, 400 µg of the membrane protein was incubated for 25 min with 0.25 nM [125I-Tyr4]bombesin at 4 °C in a total volume of 125 µL of assay buffer in the presence or absence of 0.01-1000 nM bombesin, Lys3-bombesin, Lys3(Pm-DADT)-bombesin, Lys3(Hx-DADT)-bombesin, or the 99Tc-labeled bombesin analogues or 1-106 nM somatostatin. The assay buffer (50 mM Tris-HCl at pH 7.4) contained 1 mg/mL BSA and 2 µg/mL bacitracin. The incubation was terminated by addition of 5 mL of ice cold Tris-HCl (50 mM, pH 7.4) containing 1 mg/mL BSA followed by rapid filtration through Whatman GF/B glass fiber filters presoaked for 20 min in Tris-HCl buffer (50 mM, pH 7.4) at 4 °C. The filters were washed twice more with 5 mL aliquots of Tris-HCl buffer (50 mM, pH 7.4) at 4 °C and then counted in an automated scintillation γ-counter. Nonspecific binding was determined in the presence of 1 µM bombesin. The inhibition constants (Ki) were calculated using the EBDA/LIGAND computer programs (35). Biodistribution of the Lys3(99mTc-DADT)-Bombesin Analogues in Normal Mice. The HPLC-purified Lys3(99mTc-DADT)-bombesin analogues were isolated from the respective HPLC eluates by solid phase extraction using a C-18 Sep-Pak cartridge. The Sep-Pak cartridge was first washed with ethanol (5 mL) followed by a solution of ammonium acetate (0.05 M, 5 mL). The HPLC eluate containing the Lys3(99mTc-DADT)-bombesin analogue was diluted five times with ammonium acetate (0.05 M) and then passed through the Sep-Pak cartridge. The Sep-Pak cartridge was then washed with an ammonium acetate solution (0.05 M, 5 mL). The Lys3(99mTc-DADT)-bombesin analogues were then eluted with ethanol. The ethanolic solution was diluted with physiologic saline. The final injectate contained 2.5% ethanol. CD-1 mice were injected via the tail vein with the HPLC-purified Lys3(99mTc-DADT)-bombesin analogues (2 µCi, 0.2 mL per mouse). At 15 min and 3 h postinjection, four mice for each time point per tracer were euthanized by cervical dislocation. Major organs were dissected, weighed, and counted in an automated scintillation γ-counter with aliquots of the injectate (as standards) counted at the same time. RESULTS AND DISCUSSION

The development of biomarkers for detection of cancer is important in patient management. Because they are

Bioconjugate Chem., Vol. 9, No. 2, 1998 221

rapidly metabolized in blood, autocrine growth peptides such as bombesin are not suitable as biomarkers for detection. The receptors, on the other hand, could be used as biomarkers for detection if suitable tracers could be developed for identifying them in vivo. Toward this end, bombesin/GRP receptors on cancer cells may be important for early diagnosis of certain cancers. For example, in a study by Preston et al. (36), only 1 of 23 stomach mucosa samples obtained from areas uninvolved with cancer in gastric cancer patients showed highaffinity binding sites for bombesin/GRP. This patient was the only one who had Menetrier’s disease, a mucosa disorder known to be premalignant. In gastric (36) as well as other cancers such as prostate (34) and small cell lung cancers (37, 38), moderate to very high receptor densities are present on the cells. This would make the system amenable to good target to nontarget differentiation that can be taken advantage of in both scintigraphic imaging and therapy. For in vivo scintigraphic studies by planar imaging or single photon emission computed tomography (SPECT), 99m Tc is the best radionuclide by virtue of its short 6.02 h half-life, 140 keV photons, a high photon yield per disintegration unaccompanied by significant particulate radiation, a relatively low dose to patients, ready availability in generator form, and low cost. In order to derivatize small biologically active peptides with the metal, a chelating ligand (39) or a specific sequence of amino acids usually including one or more cysteine residues (40) has to be introduced into the peptide to carry the metal. Care has to be taken so that the Tc chelate does not interfere with the pharmacophore of the substrate so as to preserve its biological activity. Of chelating systems available for Tc, the diaminedithiol ligand forms one of the most stable complexes (41-44). To facilitate the incorporation of Tc into biologically active compounds using the DADT ligand system, we developed the two thiolactone derivatives 1 and 2 (Figure 1) (30, 31). We have evaluated the feasibility of applying the DADT ligand in the form of the two bifunctional chelates as the carrier of the Tc in the derivatization of Lys3bombesin (Figure 2), a potent bombesin agonist. This analogue of bombesin contained a Lys residue at amino acid position 3 in place of Arg in natural bombesin (Figure 2). In bombesin analogues, the binding site resides in the C-terminal octapeptide sequence (23, 45). Because the Lys residue is further from the binding site, we hypothesized that a Tc chelate attached to the -NH2 group of Lys3 would lead to an analogue that still maintained high-affinity binding to the receptor. Coupling of the BCAs to Lys3-bombesin was performed at pH 9 using a BCA:Lys3-bombesin ratio of 10:1 (Scheme 1). After incubation at room temperature, the excess BCA was removed by extraction with ether and the Lys3(DADT)-bombesin adduct purified by semipreparative reversed phase HPLC. No side chain protection was necessary since the indole and imidazole groups of Trp and His, respectively, are weak nucleophiles under the coupling conditions. The purified adducts were characterized by mass spectroscopy. We have prepared both no-carrier-added (99mTc) and milligram quantities (99Tc) of the Tc-derivatized bombesin analogues [Lys3(Tc-DADT)-bombesin]. The compounds were prepared in ligand exchange reactions by incubation of a mixture of the Lys3(DADT)-bombesin adducts with [99mTc]- or [99Tc]glucoheptonate (prepared from glucoheptonate, [99mTc]- or [99Tc[pertechnetate, and stannous ion), respectively, at room temperature (Scheme 1).

222 Bioconjugate Chem., Vol. 9, No. 2, 1998

Baidoo et al.

Figure 2. Bombesin analogues.

The yield of 99mTc products was >90% after incubation for 10 min as determined by HPLC. Two main products were obtained from each adduct. The products were named Lys3(99mTc-Pm-DADT)-bombesin-1 and Lys3(99mTc-Pm-DADT)-bombesin-2 for the analogues derived from BCA 1. Lys3(99mTc-Pm-DADT)-bombesin-1 was the major component of the complexes derived from Lys3(Pm-DADT)-bombesin. The ratio of the major to minor component in this series was 3:1. The products derived from BCA 2 were named Lys3(99mTc-Hx-DADT)-bombesin-1 and Lys3(99mTc-Hx-DADT)-bombesin-2. Lys3(99mTc-Hx-DADT)-bombesin-2 was the major component of the complexes derived from Lys3(Hx-DADT)-bombesin. The ratio of major to minor component in this case was 9:1. For the 99Tc analogues, the ratio of major to minor components was 2:1 for both series. It should be noted that the chromatographic profiles of the major and minor components were reversed for the two cases; i.e., the major component was more hydrophilic than the minor component in the case of the complexes derived from BCA 1, whereas for the complexes derived from BCA 2, the minor component was more hydrophilic than the major component. The mass spectral analysis of the 99Tc

complexes showed that the major and minor components had the same molecular ion species, indicating that they were isomeric. This is in agreement with the syn-anti geometric isomerism associated with carbon- and nitrogensubstituted Tc-DADT complexes (46-48). As a first step in the evaluation of the biological activity of the bombesin analogues, we have investigated the ability of the Lys3(99Tc-DADT)-bombesin analogues to bind to the bombesin receptor in rat brain membrane preparations according to the method of Moody et al. (33). These studies involved competitive binding experiments using the authentic bombesin receptor ligand [125ITyr4]bombesin. As shown in Figure 3, all the Lys3(99TcDADT)-bombesin analogues were highly effective in displacing [125I-Tyr4]bombesin from the receptor. Results of analysis with the EBDA/LIGAND computer programs (Biosoft) are shown in Table 1. Table 1 shows that coupling of both DADT bifunctional chelating agents to Lys3-bombesin resulted in adducts with binding affinities similar to that of Lys3-bombesin, the original substrate (Ki ) 17.9-24.2 nM). Upon subsequent labeling with Tc, the resulting analogues, however, had an even better affinity (Ki ) 3.5-7.4 nM) for the receptor than either

High-Affinity Technetium Bombesin Analogues

Bioconjugate Chem., Vol. 9, No. 2, 1998 223

Figure 3. Inhibition of [125I-Tyr4]bombesin binding to the bombesin receptor in rat brain membranes by bombesin analogues. Table 1. Results of in Vitro Binding Studies of the Bombesin Analogues to Rat Brain Membranes

99Tc

compound

Kia (nM)

bombesin Lys3-bombesin Lys3(Pm-DADT)-bombesin Lys3(Hx-DADT)-bombesin Lys3(99Tc-Pm-DADT)-bombesin-1 Lys3(99Tc-Pm-DADT)-bombesin-2 Lys3(99Tc-Hx-DADT)-bombesin-1 Lys3(99Tc-Hx-DADT)-bombesin-2 somatostatin

4.3 ( 1.0 21.4 ( 5.9 24.2 ( 12.3 17.9 ( 8.1 3.5 ( 0.7 3.9 ( 1.5 5.2 ( 1.5 7.4 ( 2.0 69500 ( 11200

a

Average Ki ( SEM (n ) 3).

Lys3-bombesin or the adducts Lys3(Pm-DADT)-bombesin and Lys3(Hx-DADT)-bombesin. The affinities of the Lys3(99Tc-DADT)-bombesin analogues were comparable to that of bombesin (Ki ) 4.3 ( 1.0 nM) itself. BCAs 1 and 2 were designed to affect the biodistribution of the Tc complexes of the resulting adducts as a result of the charge carried by the Tc complex. TcDADT complexes contain a [Tc(V)-oxo]3+ core. Complexes in which at least one of the coordinating nitrogens of the DADT ligand is a secondary amine such as adducts from BCA 2 have neutral complex cores because complexation of Tc results in deprotonation from the two thiols as well as a secondary amine nitrogen (30, 41-44, 46-48). Therefore, Lys3(Tc-Hx-DADT)-bombesin-1 and Lys3(Tc-Hx-DADT)-bombesin-2 which are derived from BCA 2 both have neutral Tc complex cores. When both coordinating nitrogens of the ligand are tertiary amines, as is the case for adducts from BCA 1, deprotonation from nitrogen is not possible. Therefore, complexes derived from BCA 1 adducts have a positively charged chelate core (31, 49). These differences in the charge of the Tc complex core are reflected in the biodistribution of the 99mTc analogues (Figure 4A,B). Thus, Lys3(99mTc-Hx-

Figure 4. Biodistribution of the 99mTc-labeled bombesin analogues in normal mice 15 min postinjection (A) and 3 h postinjection (B).

DADT)-bombesin-1 and Lys3(99mTc-Hx-DADT)-bombesin-2 which are more lipophilic in character showed about 4-fold greater accumulation in the intestines than the Lys3(99mTc-Pm-DADT)-bombesin-1 and Lys3(99mTcPm-DADT)-bombesin-2 analogues which are derived from BCA 1 and therefore contain positively charged chelate cores. The 99mTc-labeled analogues derived from BCA 1 would therefore be more suitable for imaging in the abdominal area. The 99mTc-labeled bombesin analogues described above have a potential for use in the in vivo biochemical characterization of bombesin/GRP receptors by scintigraphy. Their application in cancer may allow noninvasive identification of patients that would benefit from therapies based on the disruption of the bombesin/GRP growth promotion pathway as well as early cancer

224 Bioconjugate Chem., Vol. 9, No. 2, 1998

detection. Use of the agents in this manner would greatly impact patient management. DADT ligands are also capable of forming highly stable complexes with rhenium, the congener of Tc. Therefore, the 186Re and 188Re analogues of the 99mTc-labeled bombesin compounds above would be directly applicable for the therapy of many cancers. ACKNOWLEDGMENT

This work was supported by U.S. Public Health Service Grants CA-32845 and CA-019199. LITERATURE CITED (1) Anastasi, A., Erspamer, V., and Bucci, M. (1973) Isolation and amino acid sequences of slytesin and bombesin: Two analogous active tetradecapeptides from skin of European discoglossid frogs. Arch. Biochem. Biophys. 148, 443-446. (2) McDonald, T. J., Jornvall, J., Nilsson, G., Vagne, M., Ghatei, M., Bloom, S. R., and Mutt, V. (1979) Characterization of a gastrin releasing peptide from porcine non-antral gastric tissue. Biochem. Biophys. Res. Commun. 90, 227-233. (3) Minamino, N., Kangawa, K., and Matsuo, H. (1983) Neuromedin, B: A novel bombesin-like peptide identified in porcine spinal cord. Biochem. Biophys. Res. Commun. 114, 541-548. (4) Alexander, R. W., Upp, J. R., Jr., Poston, G. J., Gupta, V., Townsend, C. M., Jr., and Thompson, J. C. (1988) Effects of bombesin on growth of human small cell lung carcinoma in vivo. Cancer Res. 48, 1439-1441. (5) Moody, T. W., Carney, D. N., Cuttita, F., Quattrocchi, K., and Minna, J. D. (1985) High affinity receptors for bombesin/ GRP-like peptides on human small cell lung cancer. Life Sci. 37, 105-113. (6) Moody, T. W., Mahmoud, S., Staley, J. S., Naldini, L., Cirillo, D., South, V., Felder, S., and Kris, R. (1989) Human glioblastoma cell lines have neuropeptide receptors for bombesin/ GRP. J. Mol. Neurosci. 1, 235-242. (7) Thomas, F., Arvelo, F., Antoine, E., Jacrot, M., and Poupon, M. F. (1992) Antitumoral activity of bombesin analogues on small cell lung cancer xenografts: Relationship with bombesin receptor expression. Cancer Res. 52, 4872-4877. (8) Farre, A., Ishizuka, J., Gomez, G., Parekh, D., Koo, J. Y., Townsend, C. M., and Thompson, J. C. (1993) Bombesin stimulates growth of colon cancer in mice and decreases their survival. Surg. Oncol. 2, 169-173. (9) Qin, Y., Ertl, T., Cai, R. Z., Halmos, G., and Schally, A. V. (1994) Inhibitory effect of bombesin receptor antagonist RC3095 on the growth of human pancreatic cancer cells in vivo and in vitro. Cancer Res. 54, 1035-1041. (10) Qin, Y., Halmos, G., Cai, R. Z., Szoke, B., Ertl, T., and Schally, A. V. (1994) Bombesin antagonists inhibit in vitro and in vivo growth of human gastric cancer and binding of bombesin to its receptors. J. Cancer Res. Clin. Oncol. 120, 519-528. (11) Pinski, J., Halmos, G., Yano, T., Szepeshazi, K., Qin, Y., Ertl, J., and Schally, A. V. (1994) Inhibition of growth of MKN human gastric adenocarcinoma xenografts in nude mice by treatment with gastrin releasing peptide antagonist (RC3095) and somatostatin analogue RC-160. Int. J. Cancer 57, 54-58. (12) Pinski, J., Schally, A. V., Halmos, G., Yano, T., Szepeshazi, K., and Groot, K. (1994) Somatostatin analogues and bombesin/gastrin releasing peptide antagonist RC-3095 inhibit the growth of glioblastomas in vitro and in vivo. Cancer Res. 54, 5895-5901. (13) Yano, T., Pinski, J., Szepeshazi, K., Groot, K., and Schally, A. V. (1992) Stimulation by bombesin and inhibition by bombesin/gastrin releasing peptide antagonist RC-3095 of growth of human breast cancer cell lines. Cancer Res. 52, 4545-4547. (14) Yano, T., Pinski, J., Szepeshazi, K., Halmos, G., Radulovic, S., Groot, K., and Schally, A. V. (1994) Inhibitory effect of bombesin/gastrin releasing peptide antagonist RC-3095 and lutenizing hormone-releasing hormone antagonist SB-75 on

Baidoo et al. the growth of MCF-7 MIII human breast xenografts in athymic nude mice. Cancer 73, 1229-1238. (15) Bold, R. J., Lowry, P. S., Ishizuka, J., Battey, J. F., Townsend, C. M., Jr., and Thompson, J. C. (1994) Bombesin stimulates the in vitro growth of a human gastric cancer cell line. J. Cell. Physiol. 161, 519-525. (16) Radulovic, S., Miller, G., and Schally, A. V. (1991) Inhibition of growth of HT-29 human colon cancer xenografts in nude mice by treatment with bombesin/gastrin releasing peptide antagonist (RC-3095). Cancer Res. 51, 6006-6009. (17) Radulovic, S., Schally, A. V., Reile, H., Halmos, G., Szepeshazi, K., Groot, K., Milovanovic, S., Miller, G., and Yano, T. (1994) Inhibitory effects of antagonists of bombesin/gastrin releasing peptide (GRP) and somatostatin analogue (RC-160) on growth of HT-29 human colon cancers in nude mice. Acta Oncol. 33, 693-701. (18) Moody, T. W., Murphy, S., Mahmoud, S., and Fiskum, G. (1987) Bombesin like peptides elevate cytosolic calcium levels in small cell lung cancer cells. Biochem. Biophys. Res. Commun. 147, 189-195. (19) Draoui, M., Chung, P., Park, M., Birrer, M., Jakowlew, S., and Moody, T. W. (1995) Bombesin stimulates c-fos and c-jun mRNA in small cell lung cancer cells. Peptides 16, 289-292. (20) Kelley, M. J., Linnoila, R. I., Avis, I. L., Georgiadis, M. S., Cuttitta, F., Mulshine, J. L., and Johnson, B. E. (1997) Antitumor activity of a monoclonal antibody directed against gastrin-releasing peptide in patients with small cell lung cancer. Chest 112, 256-261. (21) Jensen, R. T., Jones, S. W., Folkers, K., and Gardner, J. D. (1984) A synthetic peptide that is a bombesin receptor antagonist. Nature 309, 61-63. (22) Woll, P. J., and Rozengurt, E. (1988) [D-Arg1,D-Phe5,DTrp7,9,-Leu11]Substance P, a potent bombesin antagonist in murine Swiss 3T3 cells, inhibits the growth of human small cell lung cancer in vitro. Proc. Natl. Acad. Sci. U.S.A. 85, 1859-1863. (23) Camble, R., Cotton, R., Dutta, A. S., Garner, A., Hayward, C. F., Moore, V. E., and Scholes, P. B. (1989) iso-ButyrylHis-Trp-Ala-Val-D-Ala-His-Leu-NH-CH3 (ICI-216140). A potent in vivo antagonist of bombesin/gastrin releasing peptide (BN/GRP) derived from the C-terminal sequence lacking the final methionine residue. Life Sci. 45, 1521-1527. (24) Radulovic, S., Cai, R. Z., Serfozo, P., Groot, K., Redding, T. W., Pinski, J., and Schally, A. V. (1991) Biological effects and receptor binding affinities of new pseudononapeptide bombesin/GRP receptor antagonists with N-terminal D-Trp or Tpi. Int. J. Pept. Res. 38, 593-600. (25) Singh, P., Gao, Y. S., Kull, F. C., Jr., and Leban, J. J. (1992) A novel bombesin receptor antagonist, potently inhibits bombesin evoked release of gastrointestinal hormones from rats and dogs in vitro and in vivo. Regul. Pept. 40, 75-86. (26) Szepeshazi, K., Schally, A. V., Cai, R. Z., Radulovic, S., Milovanovic, S., and Szoke, B. (1991) Inhibitory effect of bombesin/gastrin releasing peptide antagonist RC-3095 and high dose somatostatin analogue RC-160 on nitrosamineinduced pancreatic cancers in hamsters. Cancer Res. 51, 5980-5986. (27) Szepeshazi, K., Schally, A. V., Halmo, G., Groot, K., and Radulovic, S. (1992) Growth inhibition of estrogen dependent and estrogen-independent MTX mammary cancers in mice by the bombesin and gastrin releasing peptide antagonist RC3095. J. Natl. Cancer Inst. 84, 1915-1921. (28) Moody, T. W., Venugopal, R., Zia, R., Patierno, S., Leban, J. J., and McDermed, J. (1995) BW2258U89: A GRP antagonist which inhibits small cell lung cancer. Life Sci. 56, 521529. (29) Li, N.,. Hoffman, T. J., Sieckman, G. L., Ochrymowycz, L. A., Higginbotham, C., Strutman, M., Volkert, W. A., and Ketring, A. R. (1996) In vitro and in vivo characterization of a Rh-105-Tetrathiamacrocycle conjugate of a bombesin analogue. J. Nucl. Med. 37 (Suppl.), 61P. (30) Baidoo, K. E., and Lever, S. Z. (1990) Synthesis of a diaminedithiol bifunctional chelating agent for the incorporation of technetium-99m into biomolecules. Bioconjugate Chem. 1, 132-137.

High-Affinity Technetium Bombesin Analogues (31) Baidoo, K. E., Lever, S., Z., and Scheffel, U. (1994) A bifunctional chelator for facile preparation of neutral technetium complexes. Bioconjugate Chem. 5, 114-118. (32) de Kieviet, W. (1981) Technetium Radiopharmaceuticals: Chemical characterization and tissue distribution of Tcglucoheptonate using Tc-99m and Tc-99. J. Nucl. Med. 22, 703-709. (33) Moody, T. W., Pert, C. B., Rivier, J., and Brown, M. R. (1978) Bombesin: Specific binding to rat brain membranes. Proc. Natl. Acad. Sci. U.S.A. 75, 5372-5376. (34) Reile, H., Armatis, P. E., and Schally, A. V. (1994) Characterization of high-affinity receptors for bombesin/ gastrin releasing peptide on the human prostate cancer cell lines PC-3 and DU-145: Internalization of receptor bound 125I-(Tyr4)-Bombesin by tumor cells. Prostate 25, 29-38. (35) McPherson, G. A. (1985) Analysis of radioligand binding experiments. A collection of computer programs for the IBM PC. J. Pharmacol. Methods 14, 213-228. (36) Preston, S. R., Woodhouse, L. F., Jones-Blackett, S., Wyatt, J. I., and Primose, J. N. (1993) High affinity binding sites for gastrin releasing peptides on human gastric cancer and Menetrier’s disease. Cancer Res. 53, 5090-5092. (37) Sunday, M. E., Kaplan, L. M., Motoyama, E., Chin, W. W., and Spindel, E. R. (1988) Biology of disease: Gastrinreleasing peptide (mammalian bombesin) gene expression in health and disease. Lab. Invest. 59, 5-24. (38) Moody, T. W., and Cuttitta, F. (1993) Growth factor and peptide receptors in small cell lung cancer. Life Sci. 52, 11611173. (39) Sundberg, M. W., Meares, C. F., Goodwin, D. A., and Diamanti, C. I. (1974) Chelating agents for the binding of metal ions to macromolecules. Nature 250, 587-588. (40) Pearson, D. A., Lister-James, J., McBride, W. J., Wilson, D. M., Martel, L. J., Civitello, E. R., Taylor, J. E., Moyer, B. R., and Dean, R. T. (1996) Somatostatin receptor-binding peptides labeled with technetium-99m: Chemistry and initial biological studies. J. Med. Chem. 39, 1361-1371. (41) Epps, L. A. (1984) The chemistry of neutral lipid soluble technetium (V) complexes of aminoalcohols and aminothiols. Ph.D. Thesis, The Johns Hopkins University, Baltimore, MD.

Bioconjugate Chem., Vol. 9, No. 2, 1998 225 (42) Lever, S. Z., Burns, H. D., Kervitsky, T. M., Goldfarb, H. W., Woo, D. V., Wong, D. F., Epps, L. A., Kramer, A. V., and Wagner, H. N., Jr. (1985) Design, preparation, and biodistribution of a technetium-99m triaminedithiol complex to assess regional cerebral blood flow. J. Nucl. Med. 26, 12871294. (43) Kung, H. F., Molnar, M., Billings, J., Wicks, R., and Blau, M. (1984) Synthesis and biodistribution of neutral, lipid soluble Tc-99m complexes that cross the blood-brain barrier. J. Nucl. Med. 25, 326-332. (44) Kung, H. F., Liu, B.-L., Wei, Y., and Pan, S. (1990) A study of the structure-stability relationship of technetium compounds. Appl. Radiat. Isot. 41, 773-781. (45) Knight, M., Pineda, J. D., Burke, T. R., Jr., Cohen, S. N., Mahmoud, S., and Moody, T. W. (1990) Design of cyclic bombesin analogue. In Peptides: Chemistry, Structure and Biology (J. Rivier and G. Marshal, Eds.) pp 185-187, ESCOM, Leiden, The Netherlands. (46) Epps, L. A., Burns, H. D., Lever, S. Z., Goldfarb, H., and Wagner, H. N., Jr. (1987) Brain imaging agents: Synthesis and characterization of (N-piperidinylethyl)-hexamethyl diamino-dithiolate oxo technetium(V) Complexes. Appl. Radiat. Isot. 38, 361-364. (47) Kung, H. F., Guo, Y.-Z., Yu, C.-C., Billings, J., Subramanyam, V., and Calabresse, J. (1989) New brain perfusion agents based on the Tc-99m bis-aminoethanethiol (BAT) complexes: stereoisomers and biodistribution. J. Med. Chem. 32, 433-437. (48) Lever, S. Z., Baidoo, K. E., and Mahmood, A. (1990) Structure Proof of Syn/Anti Isomerism in N-Alkylated Diaminedithiol (DADT) Complexes of Technetium. Inorg. Chem. Acta 176, 183-184. (49) Faggiani, R., Lock, C. J. L., Epps, L. A., Kramer, A. V., Brune, D., and Burns, H. D. (1988) (3,6-Dimethyl-3,6-diazaoctan-1,8-dithiolato-S1, N3, N6, S8) oxotechnetium(V) pertechnetate. Acta Crystallogr. C 44, 777-779.

BC9701959