in Vivo Structure−Activity Relationship Study Where

Bioconjugate Chem. 2003, 14, 93−102

93

Radiochemical Investigations of 99mTc-N3S-X-BBN[7-14]NH2: An in Vitro/in Vivo Structure-Activity Relationship Study Where X ) 0-, 3-, 5-, 8-, and 11-Carbon Tethering Moieties C. Jeffrey Smith,† Hariprasad Gali,† Gary L. Sieckman,‡ Chris Higginbotham,‡ Wynn A. Volkert,†,‡ and Timothy J. Hoffman*,‡,§ Research Services, Harry S. Truman Memorial Veterans’ Hospital, Columbia, Missouri 65201, and Departments of Internal Medicine and Radiology, University of MissourisColumbia School of Medicine, Columbia, Missouri 65211. Received April 24, 2002; Revised Manuscript Received July 15, 2002

Bombesin (BBN), a 14 amino acid peptide, is an analogue of human gastrin releasing peptide (GRP) that binds to GRP receptors (GRPr) with high affinity and specificity. The GRPr is overexpressed on a variety of human cancer cells, including prostate, breast, lung, and pancreatic cancers. The specific aim of this study was to develop 99mTc-radiolabeled BBN analogues that maintain high specificity for the GRPr in vivo. A preselected synthetic sequence via solid-phase peptide synthesis (SPPS) was designed to produce N3S-BBN (N3S ) dimethylglycyl-L-seryl-L-cysteinylglycinamide) conjugates with the following general structure: DMG-S-C-G-X-Q-W-A-V-G-H-L-M-(NH2), where the spacer group, X ) 0 (no spacer), ω-NH2(CH2)2COOH, ω-NH2(CH2)4COOH, ω-NH2(CH2)7COOH, or ω-NH2-(CH2)10COOH. The new BBN constructs were purified by reversed phase-HPLC (RP-HPLC). Electrospray mass spectrometry (ES-MS) was used to characterize the nonmetalated BBN conjugates. Re(V)-BBN conjugates were prepared by the reaction of Re(V)gluconate with N3S-X-BBN[7-14]NH2 (X ) 0 carbons, β-Ala (β-alanine), 5-Ava (5-aminovaleric acid), 8-Aoc (8-aminooctanoic acid), and 11-Aun (11aminoundecanoic acid)) with gentle heating. Re-N3S-5-Ava-BBN[7-14]NH2 was also prepared by the reaction of [Re(V)dimethylglycyl-L-seryl-L-cysteinylglycinamide] with 5-Ava-BBN[7-14]NH2. ESMS was used to determine the molecular constitution of the new Re(V) conjugates. The 99mTc conjugates were prepared at the tracer level by each the prelabeling, post-conjugation and pre-conjugation, postlabeling approaches from the reaction of Na[99mTcO4] with excess SnCl2, sodium gluconate, and corresponding ligand. The 99mTc and Re(V) conjugates behaved similarly under identical RP-HPLC conditions. In vitro and in vivo models demonstrated biological integrity of the new conjugates.

INTRODUCTION

Detection and treatment of cancers using radiopharmaceuticals that selectively target cancers in human patients has been employed for several decades (1-10). There is accelerated interest in developing new radiolabeled drugs due to the emergence of more sophisticated biomolecular vectors that exhibit high affinity and specificity for in vivo targeting of tumors. Several types of targeting agents are being developed and have been investigated, including monoclonal antibodies and other immuno-derived fragments and assemblies and receptoravid peptides (11-15). The potential utility of using radiolabeled receptor-avid peptides to formulate sitedirected radiopharmaceuticals is readily exemplified by the radiolabeled conjugates of octreotide and analogues that bind to cancer cells that overexpress somatostatin receptors (16-21). 111In-DTPA-octreotide (Octreoscan, Mallinckrodt Medical Inc.) is available for routine use in patients,16 and other derivatized forms are currently * Corresponding author. Address: Harry S. Truman VA Hospital, 800 Hospital Drive, Research F-003, Columbia, MO 65211. E-mail: [email protected]. Phone: (573)814-6000 ext. 2593. Fax: (573)882-1663. † Department of Radiology, University of MissourisColumbia. ‡ Harry S. Truman Memorial Veterans Hospital. § Department of Internal Medicine, University of Missouris Columbia.

being developed as anti-cancer radiolabeled diagnostics and therapeutics (16-21). Bombesin (BBN), a 14 amino acid peptide, is an analogue of human gastrin releasing peptide (GRP) that binds to GRP receptors with high affinity and specificity (22-24). GRP receptors have been shown to be overexpressed on several types of cancer cells including prostate, breast, lung, and pancreatic cancers (22-24). Since BBN agonistically binds to GRP receptors on cancer cells and may function as an autocrine or paracrine growth stimulator, a great deal of work has been devoted to development of BBN or GRP analogues that are antagonists as potential agents for inhibition and control of GRP-expressing tumors (23, 24). These antagonists are designed to competitively inhibit endogenous GRP binding to GRP receptors and reduce the rate of cancer cell proliferation (23, 24). Treatment of cancers using these nonradioactive antagonists requires chronic injection regimens using large quantities of the drug.25 In designing receptor-avid radiopharmaceuticals for use as diagnostic or therapeutic agents, it is important that the drug not only exhibit cancer-specific in vivo targeting and acceptable pharmacokinetic properties, but it must be available as high specific activity products and exhibit long-term residualization in the tumor (26-28). The synthesis and characterization of radiolabeled BBN or GRP analogues has been reported by several groups (26-35). Several of these analogues, labeled with radioiodine and radiometals, have been shown to bind

10.1021/bc020034r CCC: $25.00 © 2003 American Chemical Society Published on Web 12/14/2002

94 Bioconjugate Chem., Vol. 14, No. 1, 2003

Smith et al.

Figure 1. Structures of DMG-S-C-G-X-Q-W-A-V-G-H-L-M-NH2, where the spacer group, X ) 0 (no spacer), ω-NH2(CH2)2COOH, ω-NH2(CH2)4COOH, ω-NH2(CH2)7COOH, or ω-NH2-(CH2)10COOH.

selectively and avidly to GRP receptors on cancer cells, both in vitro and in vivo (26-35). The various BBN analogues studied range from structures in which the radiolabeled moiety is conjugated to the nearly full-length BBN (1-14) sequence, to where it is attached to a smaller truncated amino acid sequence comprising the GRPreceptor binding moiety (e.g., BBN[8-14]NH2). Results of studies to evaluate these conjugates demonstrate that radiolabeled BBN/GRP analogues hold important promise as cancer specific radiopharmaceuticals (26-35). Our laboratory has focused on the design of BBNagonist analogues in which the radiolabeled moiety (e.g., radiometal chelate) is linked either directly to the Nterminal amine group of BBN[7-14]NH2 or via hydrocarbon spacer groups (Figure 1) (33-38). The length and composition of the spacer group, or tether, as well as the physio-chemical properties of the radiolabeled moiety will influence the GRP receptor binding affinity, residualization of radioactivity in cancer cells, and pharmacokinetics of the BBN conjugate (33-38). In this study, we have complexed 99mTc to a triamido-thiol (N3S) bifunctional chelating agent (BFCA). The 99mTc chelate has been conjugated to the N-terminal amine of BBN[7-14]NH2

via a limited series of hydrocarbon spacers of increasing length (i.e., 3, 5, 8, and 11 carbon atoms in length). The purpose of this study is to determine the effects of varying the length of hydrocarbon spacer groups on the in vitro binding affinity with GRP receptors expressed on human prostate PC-3 cells and their in vivo pharmacokinetics in normal CF-1 mice. It would be beneficial to formulate new 99mTc-labeled site-specific tracers since 99mTc is currently the most widely used radionuclide used for diagnostic SPECT imaging studies (39). The N3S-BFCA used to complex 99mTc is N-dimethylglycyl-L-seryl-Lcyteinyl-glycine[(CH3)2-G-S-C-G], which has been shown to form a well-defined and stable complex with TcO3+ (40). EXPERIMENTAL SECTION

All solvents were either ACS certified or HPLC grade. All solvents were obtained from Fisher Scientific and used as received. Fmoc-amino acids, coupling agents and resins were purchased from Calbiochem-Novabiochem Corp., San Diego, CA. All other reagents were purchased from either Aldrich Chemical Co., Fisher Scientific or ACROS Chemicals. 99mTcO4- was obtained as the sterile

99mTc−N

3S-X-BBN[7−14]NH2

Bioconjugate Chem., Vol. 14, No. 1, 2003 95

Scheme 1

0.9% aqueous NaCl eluant solution from a 99Mo/99mTc generator (Mallinckrodt Medical, Inc.). Electrospray mass spectral (ESMS) Analyses were performed by SynPep Corporation, Dublin, CA. 125I-Tyr4-BBN was obtained from NEN Life Sciences Products, Inc., Boston, MA. Solid-Phase Peptide Synthesis (SPPS). Peptide synthesis was performed on a Perkin-Elmer-Applied Biosystem Model 432 automated peptide synthesizer employing traditional Fmoc Chemistry. The reaction of HBTU activated carboxyl groups on the reactant with the N-terminal amino group on the growing peptide, anchored via the C-terminus to the resin, provided for stepwise amino acid addition. Rink Amide MBHA resin (25 µmole) and Fmoc-protected amino acids, with appropriate side-chain protections, and the Fmoc-protected ω-NH2(CH2)nCOOH compounds used as spacer groups (75 µmol), were used for SPPS of the nonmetalated BBN conjugates. The preselected synthetic sequence was designed to produce the N3S-BBN conjugates with the following general structure DMG-S-C-G-X-Q-W-A-V-GH-L-M-NH2, where the spacer group, X ) 0 (no spacer), ω-NH2(CH2)2COOH, ω-NH2(CH2)4COOH, ω-NH2(CH2)7COOH, or ω-NH2-(CH2)10COOH (See Figure 1 for structures of these N3S-X-BBN[7-14]NH2 conjugates). The final products were cleaved by a standard procedure using a cocktail containing thioanisol, water, ethanedithiol and trifluoracetic acid in a ratio of 2:1:1:36 and precipitated into methyl-tert-butyl ether. The crude peptides were purified by HPLC and the solvents removed on a SpeedVac concentrator. Typical yields of the crude peptides were 80-85%. ES-MS was used in order to determine the molecular constitution of the N3S-X-BBN[7-14]NH2 conjugates. HPLC Purification and Analysis. HPLC analyses and purification of all peptide conjugates and their metal complexes were performed on a Waters 600E instrument equipped with a Varian 2550 variable absorption detector, a Packard Radiometric 150 TR flow scintillation analyzer or a sodium iodide crystal radiometric detector, and an Eppendorf TC-50 column temperature controller and Hewlett-Packard HP3395 integrator. HPLC solvents consisted of H2O containing 0.1% trifluoroacetic acid

Table 1. Mass-Spectral Analyses and IC50 (IC50nM(SD), n ) 3) Determination of the Peptide Series and Re Conjugates peptide/ conjugate Re-0 Re-3 Re-5 Re-8 Re-11

calculated mass

actual mass

IC50(PC-3)

1471.7 1542.8 1570.8 1612.9 1655.0

1472.4 1543.6 1571.4 1612.9 1655.6

3.97(1.07) 0.67(0.21) 1.00(0.20) 0.53(0.25) 1.50(0.26)

(Solvent A) and acetonitrile containing 0.1% trifluoracetic acid (Solvent B). A Phenomenex Jupiter C-18 (5 µm, 4.6 × 250 mm) column was used with a flow rate of 1.5 mL/ min. The HPLC gradient system begins with a solvent composition of 95% A and 5% B and follows a linear gradient of 30% A:70% B from 0 to 25 min, and 30% A:70% B to 5% A:95% B from 25 to 30 min. 99mTc- and Re-N S-X-BBN[7-14]NH Conjugate 3 2 Synthesis. The N3S-X-BBN[7-14]NH2 analogues were labeled with 99mTc and Re using pre-conjugation, posttransmetalation from 99mTc(V)41 or Re(V)-gluconate42 synthons, respectively (Scheme 1). Preparation of Re(V)gluconate was performed as previously described.42 In brief, the preparation of the Re(V) conjugates was performed by addition of excess Re(V)-gluconate solution to a solution of N3S-X-BBN[7-14]NH2. Each mixture was heated for 2h at 60 °C. After incubation of the resulting solutions at room temperature, the Re-N3S-X-BBN[714]NH2 complexes were purified by RP-HPLC. Each of the new complexes were peak-collected and evaporated to dryness. ES-MS analysis was used to determine the molecular constitution of the new Re conjugates (Table 1). For 99mTc, this method involved preparation of a stannous gluconate stock solution by mixing 20 mg SnCl2 in 1 mL ultrapure deoxygenated water. Twenty microliters of this stock solution was mixed with 1.0 mL of a 13 mg/mL sodium gluconate solution. To 100 µL of this solution was added 500 µL of an isotonic saline solution containing approximately 370 MBq of 99mTcO4- and 50 µg of the N3S-X-BBN[7-14]NH2 conjugate.10,30,40 After incubation of the resulting solution at room temperature, the 99mTc-N3S-X-BBN[7-14]NH2 complexes were puri-

96 Bioconjugate Chem., Vol. 14, No. 1, 2003

Smith et al.

Scheme 2

fied by RP-HPLC and the peaks collected into a 1 mL solution of 0.02 M sodium phosphate buffered saline at pH 7.4, containing 0.1 mg of bovine serum albumin (BSA). Prior to use of the purified conjugates for in vitro GRP receptor binding and in vivo biodistribution studies, the residual acetonitrile was evaporated from the solution under a stream of nitrogen. These 99mTc-N3S-X-BBN[7-14]NH2 conjugates were of high specific activity as the N3S-X-BBN[7-14]NH2 analogues eluted approximately 1 min prior to the corresponding metalated 99mTc-N3S-X-BBN[7-14]NH2 conjugates. 99m Tc-N3S-5-Ava-BBN[7-14]NH2 was also synthesized by the prechelate post-conjugation approach, as was Re-N3S-5-Ava-BBN[7-14]NH2 (Scheme 2). The 99mTcN3S bifunctional chelate (BFC) was prepared by transchelation from 99mTc(V)-gluconate similar to the method described earlier. The Re-N3S BFC was prepared by trans-metalation of Re(V) to the N3S-BFCA from the [ReO2(en2)]Cl (en ) ethylenediamine) synthon using a method previously reported (40). ReO-N-dimethyglycylL-seryl-L-cysteinyl-glycine (ReO-N3S-BFCA) was provided by Resolution Pharmaceuticals (Toronto, Canada). The C-terminal carboxy group on both the 99mTc- and Re-N3S-BFCAs were converted to the corresponding tetrafluoro-phenyl (TFP) ester precursors. To produce the Re-N3S-TFP precursor, 0.011 g (0.02 mmol) of Re-N3SBFCA was dissolved in 500 µL of deionized water with gentle stirring. To this solution was added 7 mg of TFP (0.042 mmol) in 500 µL of acetonitrile, followed by addition of 7 mg (0.044 mmol) of N,N′-dicyclohexcylcarbodiimide. The reaction mixture was heated gently for 2 h. Synthesis of Re-N3S-5-Ava-BBN[7-14]NH2 was performed by dissolving 0.011 g (0.11 × 10-5 moles) of N3S5-Ava-BBN[7-14]NH2 in a water/acetonitrile mixture (50:50) with gentle stirring, followed by addition of 1.8 mL of 0.2N NaHCO3. The crude Re-N3S-TFP solution was added to this solution and the reaction mixture was heated at 60 °C for 2 h. The progress of the reaction was incrementally followed by RP-HPLC (Figure 2). The HPLC purified Re-N3S-5-Ava-BBN[7-14]NH2 product was analyzed by ES-MS (m/z for (M+H)+ was found to be 1570.0; calculated 1569.9). The 99mTc-N3S-5-Ava-BBN[7-14]NH2 product was prepared by the same procedure (Scheme 1), however, the HPLC purified 99mTc-N3S-BFCA reagent was present at tracer levels when reacted with excess TFP to produce the 99mTc-N3S-TFP precursor. The progress of the reactions leading to production of the 99mTc-N3S-5-Ava-BBN-

Figure 2. HPLC elution profile of ReO-N3S-5-Ava-BBN[7-14]NH2 and reaction intermediates (prechelate, post-conjugation labeling method).

Figure 3. HPLC elution profile of 99mTcO-N3S-5-Ava-BBN[714]NH2 and reaction intermediates (prechelate, post-conjugation labeling method).

[7-14]NH2 product was also incrementally monitored by HPLC (Figure 3). 99m Tc- and Re-N3S BFCs and their respective TFPactivated esters reveal the presence of two readily identifiable peaks for each chelate reflecting the presence of syn- and anti-isomers40 (Figures 2 and 3). The final 99m Tc- and Re-N3S-5-Ava-BBN[7-14]NH2 conjugates, which eluted at retention times of 14.32 and 14.33 min, respectively, are single Gaussian peaks (Figures 2 and 3) which are expected to include both the syn- and antiN3S-BFCA conjugates which are not resolvable by this HPLC elution method. 99mTc-N3S-5-Ava-BBN[7-14]NH2 and Re-N3S-5-Ava-BBN[7-14]NH2, synthesized by each the preformed chelate method and the post-conjugation transmetalation approach, produce 99mTc/Re species that

99mTc−N

3S-X-BBN[7−14]NH2

have identical HPLC retention times (i.e., approximately 14.3 min). 99mTc-N3S-5-Ava-BBN[7-12] was also synthesized. This analogue has both the C-terminal Leu13 and Met14 deleted and is expected to exhibit neither agonist nor antagonist binding to GRP receptors (4344). In Vitro Cell Binding Studies. In vitro GRP receptor binding affinities and specificities of the BBN conjugates were assessed utilizing displacement cell-binding assays using 125I-Tyr4-BBN as the GRP receptor specific radioligand with PC-3 human prostate cancer cells (ATCC, Manassas, VA) by a method previously described.22 Briefly, the PC-3 cells were grown in HAM’s F-12K media supplemented with 7% fetal calf serum. The 3 × 104 PC-3 cells were suspended in 300µL RPMI 1640 media at pH 7.4 containing 2.4 mg/ml HEPES, 0.1µg/mL Bacitracin, and 2 mg/ml BSA. They were incubated at 37 °C (5% CO2) for 40min in the presence of approximately 20,000 cpm of 125I-Tyr4-BBN and increasing concentrations of the N3S-X-BBN[7-14]NH2 conjugates or the Re-N3S-XBBN[7-14]NH2 conjugate ranging from 10-11M to 10-5M. After incubation, the reaction medium was centrifuged (1 min, 8000 rpm) and aspirated and the cells washed three times with media. The radioactivity bound to the cells was counted in a Packard Riastar gamma counting system. The percent 125I-Tyr4-BBN bound to the cells was plotted versus increasing concentration of the BBN[7-14]NH2 conjugates to determine the specific IC50 values (Table 1, n ) 3). The degree of specific 99mTcN3S-X-BBN analogue binding to the GRP receptors expressed on PC-3 cells was determined by incubating approximately 0.01-0.02 µCi of each 99mTc analogue with 3 × 104 PC-3 cells in the absence of displacing BBN[714]NH2 ligands and in the presence of 10-5 M of the corresponding N3S-X-BBN[7-14]NH2 analogue. Internalization Studies. In vitro studies were performed to determine the efficiency of internalization of 99m Tc-N3S-5-Ava-BBN[7-14]NH2 (Scheme 2) and the degree of residualization of 99mTc in the cells as a function of time. These studies were performed by a method similar to that described by Rogers et al. (28). In short, 3 × 104 PC-3 cells were suspended in RPMI 1640 media at pH 7.4 containing 2.4 mg/mL HEPES, 0.1 µg/mL Bacitracin and 2 mg/mL BSA in the presence of approximately 20 000 cpm 99mTc-N3S-5-Ava-BBN[7-14]NH2 (3) for a period of 40 min at 37 °C (5% CO2). After incubation, the reaction medium was centrifuged (1min, 8000rpm) and aspirated, and cells were washed with the incubation media. The percent of 99mTc cell-associated activity were determined as a function of time (in the incubating medium at 37 °C). The percentage of 99mTc activity trapped in the cells was determined after removing 99mTc activity bound to the surface of the cells by washing with a pH 2.5 (0.2 M acetic acid and 0.5 M NaCl) buffer at 10, 20, 30, 60, and 90 min (n ) 3) following the washing with incubation media (Figure 5). A control study with 125I-Tyr4-BBN was performed using the same method (Figure 6). Biodistribution of 99mTc-N3S-X-BBN[7-14]NH2 Analogues in Normal Mice. Normal CF-1 mice (average weight 20-25 g) were used for the biodistribution studies (Tables 2-5). The pH of the HPLC-purified 99mTc-N3S-X-BBN[7-14]NH2 solutions was adjusted to physiological conditions using a 0.01 M phosphate buffer (pH 7.4). Aliquots (80-100 µL) of the labeled peptide solution (55-75 kBq) were injected into each animal via the tail vein. Tissues and organs were excised from the sacrificed animals 30 min, 1 h, and 4 h post-injection (p.i.). The organs and tissue were weighed and the

Bioconjugate Chem., Vol. 14, No. 1, 2003 97

Figure 4. Typical IC50 cell-binding studies of ReO-N3S-5-AvaBBN[7-14]NH2 in human prostate (PC-3) cancerous cells (IC50 ) 1.0 ( 0.2nM). Each point is an average of three separate values. IC50 values from the best-fit line from four separate studies with each conjugate were used to calculate the means and respective standard deviations reported in Table 1.

Figure 5. Efflux determination of 99mTcO-N3S-5-Ava-BBN[7-14]NH2 in human prostate (PC-3) cancerous cells.

Figure 6. Efflux determination of prostate (PC-3) cancerous cells.

125I-Tyr4-BBN

in human

activity was counted in a NaI counter. The percentinjected dose per organ and the percent-injected dose per gram were calculated. The % ID in whole blood was estimated assuming a blood volume of 6.5% of the total body weight. Receptor blocking studies were also carried out where excess BBN was administered to animals prior to injection of 99mTc-N3S-5-Ava-BBN[7-14]NH2. In these studies, each animal received a subcutaneous injection of 1 mg of BBN dissolved in 100 µL of the solvent mixture of saline/acetonitrile/DMF (90:5:5) 35 min prior to the injection of 99mTc-N3S-5-Ava-BBN[7-14]NH2. Control (unblocked) animals received an injection of 100 µL of the solvent mixture without BBN 35min prior to the injection of 99mTc-N3S-5-Ava-BBN[7-14]NH2. The animals were sacrificed 30 min post-injection and the tissues removed, weighed, and counted as previously described.

98 Bioconjugate Chem., Vol. 14, No. 1, 2003

Smith et al.

Table 2. In Vivo Biodistribution Analyses (%ID/organ (SD), 1 h p.i., n ) 5) of Non-Tumor-Bearing Mice Models (CF-1) tissue/ organ blooda heart lung liver spleen stomach l. intestine s. intestine kidney pancreas muscle urine (%ID) a

99mTc-N

3S-X-BBN[7-14]NH2

in

0 carbon

3 carbon

5 carbon

8 carbon

11 carbon

1.85(0.36) 0.03(0.01) 0.07(0.01) 5.09(0.62) 0.04(0.02) 0.28(0.03) 11.4(17.1) 36.9(18.4) 1.87(0.18) 1.48(0.13) 0.01(0.01) 38.0(2.45)

0.24(0.08) 0.01(0.00) 0.02(0.01) 1.94(0.36) 0.10(0.03) 0.36(0.07) 14.2(8.60) 24.1(10.0) 1.26(0.14) 5.54(0.67) 0.01(0.00) 47.5(2.08)

0.34(0.12) 0.00(0.00) 0.03(0.01) 1.79(0.70) 0.03(0.01) 0.43(0.10) 16.1(9.26) 33.7(8.64) 0.90(0.19) 3.19(0.35) 0.00(0.00) 38.8(1.71)

0.31(0.06) 0.01(0.01) 0.05(0.01) 4.62(1.06) 0.06(0.02) 0.38(0.05) 21.2(18.3) 46.0(17.3) 1.30(0.15) 2.87(0.35) 0.00(0.00) 19.7(0.31)

2.97(2.01) 0.07(0.03) 0.30(0.27) 6.00(2.80) 0.13(0.11) 2.37(0.79) 4.71(6.45) 58.0(18.5) 1.99(2.13) 0.99(0.16) 0.02(0.01) 14.3(10.5)

%ID in blood was estimated assuming the whole blood volume to be 6.5% of the total body weight.

The biodistribution of 99mTc-N3S-5-Ava-BBN[7-12] analogue, which does not bind with high affinity to GRP receptors, was also determined at 1 h p.i. RESULTS

The N3S-X-BBN[7-14]NH2 (Figure 1) conjugates were conveniently synthesized by SPPS. The yields of the HPLC purified conjugates were approximately 50%. ESMS analyses were consistent with the molecular weights calculated for each conjugate. Figure 4 shows an example of the displacement binding of 125I-Tyr4-BBN from GRP receptors expressed on PC-3 cells as a function of increasing concentrations of the metalated N3S-X-BBN[7-14]NH2 conjugate, Re-N3S-5Ava-BBN[7-14]NH2. In the absence of competitors, approximately 5% of the 125I-Tyr4-BBN was bound to the cells after 40 min incubation. In the presence of 10-5 M cold Tyr4-BBN, 90% of the 125I-Tyr4-BBN associated with the cells corresponds to specific GRP receptor binding. The IC50 values obtained for the series of ReO-N3SX-BBN[7-14]NH2 conjugates are summarized in Table 1. These results show that the metalated N3S-X-BBN[7-14]NH2 derivatives with 3-, 5-, and 8-carbon spacers (i.e., X ) ω-NH2(CH2)2COOH, ω-NH2(CH2)4COOH and ω-NH2(CH2)7COOH) exhibit IC50 values in the single direct nanomolar range which are lower than derivatives containing the 0- and 11-carbon spacers. The IC50 for N3S5-Ava-BBN[7-12], the analogue with deletion of Leu13 and Met14, is >10-6M. The IC50 for ReO-N3S-5-Ava-BBN[7-14]NH2 is 1.0 nM. Radiolabeling of the N3S-X-BBN[7-14]NH2 series of chelates with 99mTc was performed by the pre-conjugation post-labeling approach using 99mTc(V) gluconate as the synthon (40). The yields of the 99mTc- N3S-X-BBN[714]NH2 conjugates ranged from 90% to 95%. 99mTc-N3SX-BBN[7-14]NH2 products were purified by RP-HPLC to produce the respective 99mTc conjugates in high radiochemical purity (RCP). The 99mTc(metalated) conjugates eluted 1 min later than the corresponding nonmetalated N3S-X-BBN[7-14]NH2 reactants, making it possible to collect the 99mTc N3S-X-BBN[7-14]NH2 conjugates as high specific activity products. The HPLC chromatograms in Figures 2 and 3 show the elution profiles of the prechelated 99mTc and Re reactants and the final 99mTcand Re-N3S-5-Ava-BBN[7-14]NH2 products. These figures also show that both the Re- and 99mTc-N3S-5-AvaBBN[7-14]NH2 species coelute at 14.3 min retention times.

Specific binding of all of the 99mTc- N3S-X-BBN[7-14]NH2 conjugates to GRP receptors expressed on PC-3 cells was demonstrated following incubation (40min) of 3 × 104 PC-3 cells with high specific activity 99mTc analogues. In the absence of the corresponding nonmetalated analogue, approximately 3-6% of the 99mTc activity was associated with the PC-3 cells. In contrast, if 10-5M of the corresponding unlabeled N3S-X-BBN[7-14]NH2 conjugate or BBN(1-14) is present during the 30 min incubation, less than 0.5% of the 99mTc activity is cell associated. Figure 5 summarizes the results of studies to assess the degree of trapping (or internalization) of the 99mTcN3S-5-Ava-BBN[7-14]NH2 in PC-3 cells. The total 99mTc activity associated with the cells after the 40 min incubation was measured following washing the cells with the pH 7.4 incubation media. After washing these cells with the pH 2.5 buffer to remove surface bound 99mTc activity, approximately 84% remained trapped by the cells (Figure 5). Results of measurements at 10, 20, 30, 60, and 90 min show that the majority of activity remains trapped by the PC-3 cells, with approximately 70% of the 99m Tc activity associated with the cells at t ) 0 remaining residualized at 90 min. Thus, at 90 min, approximately 83% of the activity remains residualized when normalized to the 84% trapped in the cells at t ) 0. The same studies, when performed with 125I-Tyr4-BBN, show that after a 40 min incubation of PC-3 cells with 125I-Tyr4BBN, nearly 100% of the cell-associated 125I activity is internalized (Figure 6). Furthermore, efflux of radioactivity is comparable to that of the 99mTc conjugate. Therefore, incorporation of the 99mTc-N3S chelate onto BBN[714]NH2 has little or no effect on the internalization properties of the 99mTc conjugate in GRP receptor-specific PC-3 cells. The binding of these radioligands to PC-3 cells is receptor-specific since the addition of 10-5 M of the corresponding unlabeled BBN analogues essentially eliminated the uptake of radioactivity by these cells. Furthermore, incubation of 99mTc-N3S-5-Ava-BBN[7-14]NH2 and 125I-Tyr4-BBN with MDA-MB-438 human breast cancer cells, a cell line that exhibits no measurable GRPr expression, showed no significant uptake or internalization of the radioligand. Biodistribution of 99mTc-N3S-BBN[7-14]NH2 Conjugates. Tables 2 and 3 summarize the results of the biodistribution studies in normal CF-1 mice at 1 h postintravenous injection for the series of 99mTc-N3S-X-BBN[7-14]NH2 conjugates. As the hydrocarbon chain length of the spacer group, X, increases from 0 to 11, the % ID cleared by the renal-urinary pathway decreases. There is no significant uptake or retention in the stomach

99mTc−N

3S-X-BBN[7−14]NH2

Bioconjugate Chem., Vol. 14, No. 1, 2003 99

Table 3. In Vivo Biodistribution Analyses (%ID/g (SD), 1h p.i., n ) 5) of Non-Tumor-Bearing Mice Models (CF-1) tissue/ organ blooda heart lung liver spleen stomach l. intestine s. intestine kidney pancreas muscle urine (%ID)

in

0 carbon

3 carbon

5 carbon

8 carbon

11 carbon

0.14(0.04) 0.10(0.07) 0.14(0.10) 1.18(0.18) 1.04(0.45) 1.13(0.29) 16.5(10.7) 17.4(7.59) 3.15(0.30) 19.5(2.24) 0.10(0.05) 47.5(2.08)

0.23(0.09) 0.02(0.04) 0.25(0.10) 1.22(0.58) 0.59(0.25) 1.05(0.31) 22.9(11.8) 25.0(7.90) 2.89(0.67) 13.8(1.62) 0.10(0.06) 38.8(1.71)

0.20(0.04) 0.09(0.08) 0.31(0.06) 2.85(0.61) 0.70(0.26) 1.05(0.36) 28.2(25.7) 31.3(11.5) 3.47(0.60) 10.5(1.07) 0.06(0.05) 19.7(0.31)

1.66(1.21) 0.64(0.29) 1.48(1.39) 3.74(1.68) 0.97(0.67) 5.51(1.83) 4.54(5.76) 35.3(11.2) 5.04(5.40) 3.43(0.31) 0.12(0.09) 14.3(10.5)

tissue/ organ

0.5 h

1h

4h

bloodb heart lung liver spleen stomach l. intestinea s. intestinea kidneya pancreas muscle urinea

0.55(0.07) 0.01(0.01) 0.04(0.02) 3.96(1.40) 0.08(0.03) 0.46(0.08) 2.89(0.73) 45.8(3.52) 1.45(0.24) 4.34(1.60) 0.01(0.00) 34.2(6.05)

0.34(0.12) 0.00(0.00) 0.03(0.01) 1.79(0.70) 0.03(0.01) 0.43(0.10) 16.1(9.26) 33.7(8.64) 0.90(0.19) 3.19(0.35) 0.00(0.00) 38.8(1.71)

0.17(0.16) 0.00(0.00) 0.01(0.01) 1.20(0.53) 0.02(0.01) 0.29(0.04) 36.6(11.9) 5.64(3.32) 0.49(0.09) 1.36(0.27) 0.00(0.00) 52.0(9.35)

99mTc

At 4 h, feces containing had been excreted from each animal, and the % in the urine was estimated to be approximately 35% of the ID. b%ID in blood was estimated assuming the whole blood volume to be 6.5% of the total body weight. Table 5. In Vivo Biodistribution Analyses (%ID/g (SD), n ) 5) of 99mTc-N3S-5-Ava-BBN[7-14]NH2 in Normal Mice Models (CF-1) tissue/organ

3S-X-BBN[7-14]NH2

0.48(0.10) 0.15(0.02) 0.24(0.03) 2.30(0.42) 0.21(0.02) 0.44(0.05) 8.65(13.0) 19.2(10.4) 3.29(0.45) 2.79(0.33) 0.04(0.03) 38.0(2.45)

Table 4. In Vivo Biodistribution Analyses (%ID/organ (SD), n ) 5) of 99mTc-N3S-5-Ava-BBN[7-14]NH2 in Normal Mice Models (CF-1)

a

99mTc-N

0.5 h

1h

4h

blood heart lung liver spleen stomach l. intestine s. intestine kidney pancreas muscle

0.38(0.05) 0.16(0.13) 0.30(0.13) 2.53(0.91) 1.10(0.47) 1.04(0.28) 3.88(1.03) 29.8(3.43) 4.22(0.81) 20.3(6.15) 0.18(0.13)

0.23(0.09) 0.02(0.04) 0.25(0.10) 1.22(0.58) 0.59(0.25) 1.05(0.31) 22.9(11.8)) 25.0(7.90) 2.89(0.67) 13.8(1.62) 0.10(0.06)

0.12(0.12) 0.06(0.06) 0.10(0.07) 0.82(0.28) 0.29(0.22) 0.82(0.11) 50.6(13.8) 4.04(2.38) 1.53(0.31) 6.28(1.29) 0.01(0.01)

pancreas/blood pancreas/muscle

53.4(23.0) 113(88.3)

60.0(24.5) 138(84.4)

52.3(53.4) 628(641)

indicating that there is minimal, if any, in vivo dissociation of 99mTc from these ligands to produce 99mTcO4-. Pancreatic uptake of this series of conjugates at 1 h p.i. shows considerable variability, with the highest value associated with the three-carbon conjugate (Tables 2 and 3). As the tethering moiety is lengthened beyond three carbons, the uptake in normal pancreas decreases. There is retention of the 99mTc activity localized in the pancreas; however, there is some efflux. For example, for the 99mTc-BBN analogue where X ) NH2(CH2)4COOH (5-Ava), the percent 99mTc activity retained in the pancreas at 1 and 4 h, relative to 99mTc activity localized in that organ at 30 min is approximately 75% and 35%, respectively. Tables 4 and 5 summarize the results of the biodistribution studies in normal CF-1 mice at 30 min, 1 h, and

4 h post-intravenous injection for the 99mTc-N3S-5-AvaBBN[7-14]NH2 conjugate. There is 4.22 ( 0.81% ID/gm retained in the kidneys at 30 min p.i., but in all cases, the 99mTc activity in the kidneys decreases to a level at 4 h p.i., which is approximately 35% of that observed at 30 min p.i. The 99mTc-N3S-5-Ava-BBN[7-12] analogue with the Leu13 and Met14 deletion does not bind to GRP receptors and shows no significant accumulation in pancreatic tissue. Blocking studies in which high levels of cold BBN[1-14] was administered 35 min prior to the 99m Tc ligands, reduced the % ID/gm uptake/retention in the pancreas at 30 min p.i. by a factor of 8-10, demonstrating the in vivo specificity of these analogues for GRPreceptor expressing cells. DISCUSSION

Considerable efforts have been made by several research groups to identify structural features of BBNanalogues that produce binding and stability properties necessary for in vivo targeting of GRP receptor expressing cancers (43-48). Insights gained from these research efforts can provide the basis for designing radiolabeled conjugates of BBN derivatives that maintain high GRP receptor-binding affinities. The W-A-V-G-H-L sequence BBN[8-13] has been shown to be a sequence that is directly involved in a specific high-affinity binding interaction with GRP receptors (44, 49). Modifications or a deletion at position 14 (i.e., the position normally occupied by methionine Met14) is an important factor in determining whether or not the derivative will possess antagonistic or agonistic properties (23, 45, 47, 48). Minor structural variations (e.g., reduction of the Leu13-Met14 peptide bond or cyclization of Met14 on the BBN analogue structure) can confer agonist or antagonist properties (44, 47, 50). While the vast majority of research with BBN analogues has been devoted to the development of antagonists, BBN[8-14]NH2 is an example of a truncated BBN[1-14] sequence that will form conjugates that frequently bind GRP receptors as agonists (34, 43, 44). The strategy used in designing the limited series of 99mTc-N3S-X-BBN[7-14]NH2 derivatives was to covalently link the 99mTc-N3S chelate via a spacer group to the BBN[8-14]NH2 binding sequence (32). Previous studies demonstrated that the inclusion of Gln7 in linking radiometal chelates to BBN[8-14]NH2 was beneficial in reducing renal retention of 105Rh-S4-BBN analogues and is compatible with maintaining high GRP receptor binding affinity (15, 35). For these reasons, the various hydrocarbon spacer groups used in this study were linked to the BBN[8-14]NH2 receptor binding sequence via the Gln7 residue (Figure 1). Since the Re-N3S-5-Ava-BBN[7-14]NH2 and 99mTcN3S-5-Ava-BBN[7-14]NH2 conjugates each have a 14.3

100 Bioconjugate Chem., Vol. 14, No. 1, 2003

min retention time by RP-HPLC (Figures 2 and 3), it can be deduced that both the Re and 99mTc conjugates have the same structure. These results provide evidence that 99m Tc is present as the 99mTcO-N3S chelate, attached via the spacer groups to the BBN[7-14]NH2 GRP receptor binding sequence. These results are consistent with previous studies which show that 99mTc(V) precursors react with N3S ligand systems to form stable chelates that contain the mono-oxo-Tc(V) (i.e., TcO3+) core, as do the corresponding Re(V) chelates (40). Complexation of ReO3+ and TcO3+ with these types of N3S ligands results in deprotonation of two amide groups to produce metal chelate structures that have an overall neutral charge (40). The observation that 99mTcO-N3S-5-Ava-BBN[714]NH2 is formed in high yields by transchelation from 99m Tc-gluconate (Scheme 1), and, coelutes with 99mTcOand ReO-N3S-5-Ava-BBN[7-14]NH2 conjugates synthesized by the preformed chelate route demonstrates the feasibility of producing well defined 99mTc-N3S-BBN[7-14]NH2 chemical entities via the post conjugation labeling method. These results are consistent with previous studies that utilized other N3S bioconjugates to formulate 99mTc(V)O-labeled radiopharmaceuticals in high specific activities using similar labeling approaches (40, 41). In vitro binding studies with the N3S-X-BBN[7-14]NH2 analogues demonstrate that derivatives where the spacer group, X, comprises a 3-, 5-, and 8-carbon tether (i.e., β-Ala, 5-Ava and 8-Aoc), produce derivatives that have high GRP-receptor binding affinities (i.e., IC50 < 10 nM) (Table 1). In the absence of a spacer group and with an 11-carbon (11-Aun) tether, the IC50 values increase (Table 1). This is similar to the results obtained with analysis of 105Rh-S4-X-BBN[7-14]NH2 constructs (35). These data, therefore, indicate that the most promising analogues of this design, for in vivo targeting of cells expressing GRP receptors, are those with X ) 3-8 carbon atoms. Maximizing residualization of radioactivity in tumors after binding of the activity is an important factor in optimizing the diagnostic and/or therapeutic efficacy of radiotracers (1, 36, 51). Results of studies with 99mTcN3S-5-Ava-BBN[7-14]NH2 show that most of the 99mTc activity associated with the PC-3 cells is not surface bound and is not lost from the cells by incubation in pH 2.5 buffer (Figure 5). Furthermore, there is only minimal efflux of 99mTc activity from the cells for at least 90 min. These results indicate that residualization of 99mTc activity results from GRP receptor-mediated endocytosis of 99m Tc-N3S-5-Ava-BBN[7-14]NH2 with subsequent trapping of activity. Similar studies with 125I-Tyr4-BBN demonstrate that GRP receptor mediated trapping and efflux of 125I radioactivity is comparable to that of the 99m Tc conjugate (Figure 6). Internalization of both the 125I and 99mTc-BBN analogues can be attributed to their agonistic binding to GRP receptors (26, 28, 36, 44). GRP receptors are 7-TMS-Gprotein coupled receptors which are capable of internalizing the agonist-receptor complex (48, 49, 52, 53). Since the BBN[7-14]NH2 receptor binding sequence is consistent with agonistic binding, GRP receptor mediated endocytosis of the 99mTc-N3S-X-BBN[7-14]NH2 constructs used in this study is not unexpected. The specific intracellular trapping mechanism of the 99mTc activity in these PC-3 cells is not understood. It is likely that following internalization of 99mTc-N3S-X-BBN[7-14]NH2, that lysosomal proteases degrade the 99mTc-BBN conjugate to peptide fragments.54,55 The fragments to which 99mTc remain attached would be the chemical species that are residualized in the cell for extended

Smith et al.

periods. Further work is needed to identify the structure of the 99mTc fragments to help elucidate the trapping mechanisms. Efflux of 125I activity (Figure 6), on the other hand, is expected, as lysosomal breakdown of the 125ITyr4-containing peptides results in the production of free 125 I tyrosine, which is exported from the lysosome and the cell by specific transport pathways (55-57). The degree of efflux for the 125I-Tyr4-BBN species does occur at a slightly more rapid rate than that of the corresponding 99mTc conjugate (Figures 5 and 6). Results from the biodistribution studies, where X ) 0-11 carbons, are summarized in Tables 2 and 3. These data, along with the data acquired from in vitro cell binding assays, suggest that those 99mTc-N3S-X-BBN[7-14]NH2 conjugates, where X ) 3-8, are the most promising candidates for further in vivo evaluation. It is interesting to note that with this limited series of 99mTc-BBN conjugates, the 3-carbon spacer (β-Alanine) analogue exhibits the highest localization in the pancreas at 1 h p.i. As the length of the spacer increases, there is a decline in pancreatic uptake (Tables 2 and 3). The reasons for this observation are not clear. However, this relationship may result from the higher degree of plasma protein binding of the 99mTc-BBN analogues as their hydrophobicity increases. As plasma protein binding increases, the availability of the respective 99mTc analogue for binding to the GRPr expressing cells in the pancreas possibly decreases. Results from biodistribution studies with 99mTc-N3S5-Ava-BBN[7-14]NH2 in normal CF-1 mice at 30 min, 1 h, and 4 h p.i. are summarized in Tables 4 and 5. This 99m Tc conjugate clears efficiently from the blood with only 0.34 ( 0.12 ID remaining in the blood at 1 h p.i. (Table 3). 99m Tc-N3S-5-Ava-BBN[7-14]NH2 clears by each the hepatobiliary and renal/urinary pathways. Retention in the kidneys is significant (i.e., 2.89% ( 0.67% and 1.53% ( 0.31% ID/g at 1 and 4 h, respectively, Table 4) but is less than the level of retention in that organ reported for several other radiolabeled peptides and proteins.12 Uptake and retention of 99mTc-N3S-5-AvaBBN[7-14]NH2 in the pancreas is high (i.e., 13.8% ( 1.62% and 6.05% ( 0.28% ID/g at 1 and 4 h p.i., respectively, Table 4). The observation that the uptake of this 99mTc conjugate in the pancreas is reduced to 2.10% ( 0.80% ID/g at 0.5h p.i., when the animals were administered 1 mg of BBN 35 min prior to injection of the radiotracer, demonstrates the high specificity of 99mTc-N3S-5-Ava-BBN[7-14]NH2 for in vivo targeting of the GRP receptor expressing cells in this organ. The high pancreas-to-blood and pancreas-to-muscle ratios that are maintained at g4 h p.i. (Table 4) is a reflection of the high GRP binding affinity of this 99mTc-BBN analogue and its receptor-mediated transport to promote intracellular trapping. While retention of 99mTc-N3S-5-AvaBBN[7-14]NH2 is extended, washout of 99mTc activity from the organ occurs over time with 1.36% ( 0.27% ID remaining at 4h p.i. in comparison to 3.19% ( 0.35%ID at 1h p.i. (Table 2). For this reason, the pancreas to blood and pancreas-to-muscle ratios at 4h are not significantly different than those at 1 h p.i. CONCLUSION

The results of this study demonstrate that the 99mTcN3S-X-BBN[7-14]NH2 construct provides flexibility for designing 99mTc-labeled conjugates that retain high in vitro and in vivo specificity targeting of GRP receptor expressing cells. In this particular instance, it was shown that the length of the hydrocarbon spacer group can be

99mTc−N

3S-X-BBN[7−14]NH2

varied from at least 3-8 carbon atoms in length without comprising agonistic binding affinity to GRP receptors. These findings, coupled with previous reports, show that there is a significant degree of bulk tolerance when attaching radiometals via the N-terminal end of the BBN[8-14]NH2 sequence (31-35). The potential clinical utility of the 99mTc-N3S-X-BBN[7-14]NH2 constructs as cancer specific imaging agents was recently demonstrated by Van de Weile et al. (10, 30) in human patients with either prostate or breast cancer. Their studies showed that 99mTc-N3S-5-Ava-BBN[7-14]NH2 localizes in tumors with high specificity producing good tumor-to-normal tissue uptake ratios and high-quality SPECT images (10, 30). To design improved radiolabeled synthetic BBN analogues for diagnostic or therapeutic applications, it is essential to devote more effort to better understand the structurally sensitive mechanisms involved in the binding of these derivatives to GRP receptors and subsequent residualization of the radiotracer in cancer cells. ACKNOWLEDGMENT

This material is the result of work supported with resources and the use of facilities at the Harry S. Truman Memorial Veterans’ Hospital, Columbia, MO 65201, and the University of Missouri-Columbia School of Medicine Departments of Radiology and Internal Medicine, Columbia, MO 65211. This work was also funded in part by grants from the American Cancer Society (RPG-99331-01-CDD), the National Cancer Institute (DHHSRO1-CA72942), the National Institute of Health (DHHS1P20-CA86290), and Resolution Pharmaceuticals Inc., Mississauga, Ontario, Canada. The University of Missouri holds a patent on the bombesin agonists cited in this paper. LITERATURE CITED (1) Anderson, C. J., and Welch, M. J. (1999) Radiometal-labeled agents (non-technetium) for diagnostic imaging. Chem. Rev. 99, 2219-2234. (2) Behr, T. M., and Goldenberg, D. M. (1996) Improved prospects for cancer therapy with radiolabeled antibody fragments and peptides? J. Nucl. Med. 37, 834-836. (3) Bakker, W. H., Albert, R., Bruns, C., Breeman, W. A., Hofland, L. J., Marbach, P., Pless, J., Pralet, D., Stolz, B., and Koper, J. W. (1991) [Indium-111-DTPA-D-Phe-1]octreotide, a potential radiopharmaceutical for imaging somatostatin receptor positive tumors: Synthesis, radiolabeling, and in vivo validation. Life Sci. 49, 1583-1591. (4) deJong, M., Bakker, W. H., Krenning, E. P., Breeman, W. A. P., van der Pluijm, M. E., Bernard, B. F., Visser, T. J., Jermann, E., Behe, M., Powell, P., and Macke, H. (1997) Yttrium-90 and Indium-111 labeling, receptor binding, and biodistribution of [DOTAo-dPhe1,Tyr3]octreotide, a promising somatostatin analogue for radionuclide therapy.” Eur. J. Nucl. Med. 24, 368-371. (5) Eckelman, W. C., and Gibson, R. E. (1993) The design of site-directed radiopharmaceuticals for use in drug discovery. In Nuclear Imaging in Drug Discovery, Development, and Approval (H. D. Burns, R. E. Gibson, R. Dannals, and P. Siegl, Eds.) Birkhauser, Boston. (6) Hom, R. K., and Katzenellenbogen, J. A. (1997) Technetium99m-labeled receptor-specific small-molecule radiopharmaceuticals: Recent developments and encouraging results. Nucl. Med. Biol. 24, 485-498. (7) Kvols, L. K. (1999) Somatostatin receptor imaging of neuroendocrine carcinomas: Implications for patient management and radiotherapy. In Technetium, Rhenium and Other Metals in Chemistry and Nuclear Medicine (M. Nicolini and U. Mazzi, Eds.) pp 153-756, SGEditoriali, Padova, Italy. (8) Press, O. W., Appelbaum, F. R., Early, J. F., and Bernstein, I. D. (1994) Radiolabeled antibody therapy of lymphomas. Biol. Ther. Cancer Update 4, 1-13.

Bioconjugate Chem., Vol. 14, No. 1, 2003 101 (9) Seregni, E., Chiti, A., and Bombardieri, E. (1998) Radionuclide imaging of neuroendocrine tumors: Biological basis and diagnostic results. Eur. J. Nucl. Med. 25, 639-658. (10) Van de Wiele, C., Broecke, R. V., Cocquyt, V., Dumont, F., Oosterlinck, W., Thornback, J., and Peers, S. H. (2000) 99mTc-RP-527, a gastrin releasing peptide(GRP) analogue for visualization of GRP receptor expressing malignancies: A feasibility study. Nucl. Med. Commun. 21, 21. (11) Boerman, O. C., Oyen, W. J. G., and Corstens, F. H. M. (2000) Radio-labeled receptor-binding peptides: A new class of radiopharmaceuticals. Sem. Nucl. Med. 30, 195-208. (12) Giblin, M. F., Wang, N., Hoffman, T. J., Jurisson, S. S., and Quinn, T. P. (1998) Design and characterization of alphamelanotropin peptide analogues cyclized through rhenium and technetium metal coordination. Proc. Natl. Acad. Sci. U.S.A. 95, 12814-12818. (13) Ugur, O., Kostakoglu, L., Hui, E. T., Fisher, D. R., Garmestani, K., Gansow, O. A., Cheung, N. V., and Larson, S. M. (1996) Comparison of the targeting characteristics of various radioimmunoconjugates for radioimmunotherapy of neuroblastoma: Dosimetry calculations incorporating crossorgan beta doses. Nucl. Med. Biol. 23, 1-8. (14) Brechbiel, M. W., Gansow, O. A., Atcher, R. W., Schlom, J., Esteban, J., Simpson, D. E., and Colcher, D. (1986) Synthesis of 1-(p-Isothiocyanatobenzyl) derivatives of DTPA and EDTA. Antibody labeling and tumor-imaging studies. Inorg. Chem. 25, 2772-2781. (15) Ning, L., Ochrymowcycz, L. A., Higginbotham, C., Struttmann, M., Abrams, M. J., Vollano, J. F., Skerlj, R. T., Ketring, A. R., and Volkert, W. A. (1995) Pharmacokinetic studies of 105Rh(III) Complexes with macrocycles. J. Labeled Compd. Radiopharm. 37, 426-428. (16) Krenning, E. P., Kweekkeboom, D. J., Bakker, W. H., Breeman, W. A., Kooij, P. P., Oei, H. Y., van Hagen, M., Postema, P. T., de Jong, M., and Reubi, J. C. (1993) Somatostatin receptor scintigraphy with [111In-DTPA-D-Phe1]- and [123I-Tyr3]-Octreotide: The Rotterdam experience with more than 1000 patients. Eur. J. Nucl. Med. 20, 716-731. (17) Lewis, J. S., Lewis, M. R., Srinivasan, A., Schmidt, M. A., Wang, J., and Anderson, C. J. (1999) Comparison of four 64Cu-labeled somatostatin analogues in vitro and in a tumorbearing rat model: Evaluation of new derivatives for positron emission tomography imaging and targeted radiotherapy. J. Med. Chem. 42, 1341-1347. (18) Smith-Jones, P. M., Stolz, B., Albert, R., Ruser, G., Maecke, H., Briner, U., Tolcfai, L., Weckbecker, G., and Bruns, C. (1995) Synthesis, radiolabeling, and evaluation of DTPA/ Octreotide conjugates for radiotherapy. J. Labeled Compd. Radiopharm. 37, 499-501. (19) Stolz, B., Smith-Jones, P. M., Weckbecker, G., Albert, R., Knecht, H., Haller, R., Tolcsvai, L., Hofman, G., Pollehn, K., and Bruns, C. (1997) Radiotherapy with Yttrium-90 labeled DOTA-Tyr3-Octreotide in tumor bearing rodents. J. Nucl. Med. 38, 18p. (20) Otte, A., Mueller-Brand, J., Goetze, M., Hermann, R., Knecht, H. R., and Maecke, H. R. (1998) Yttrium-90-DOTAOctreotide treatment of somatostatin receptor positive tumors. J. Nucl. Med. 39, 70P. (21) Anderson, C. J., Jones, L. A., Bass, L. A., Sherman, E. L. C., McCarthy, D. W., Cutler, P. D., Lanahan, M. V., Cristel, M. E., Lewis, J. S., and Schwarz, S. W. (1998) Radiotherapy, toxicity, and dosimetry of Copper-64-TETA-Octreotide in tumor-bearing Rats. J. Nucl. Med. 39, 1944-1951. (22) Mahmoud, S., Staley, J., Taylor, J., Bogden, A., Moreau, J.-P., Coy, D., Avis, I., Cuttitta, F., Mulshine, J. L., and Moody, T. W. (1985) [Psi13, 14] Bombesin analogues inhibit growth of small cell lung cancer in vitro and in vivo. Life Sci. 37, 105-113. (23) 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. (24) Qin, Y., Ertl, T., Cai, R.-Z., Horvath, J. E., Groot, K., and Schally, A. V. (1995) Antagonists of bombesin/gastrin-releasing peptide inhibit growth of SW-1990 human pancreatic

102 Bioconjugate Chem., Vol. 14, No. 1, 2003 adenocarcinoma and production of cyclic AMP. Int. J. Cancer 63, 257-262. (25) Plonowski, A., Nagy, A., Schally, A. V., Sun, B., Groot, K., and Halmos, G. (2000) In vivo inhibition of PC-3 human androgen-independent prostate cancer by a targeted cytotoxic bombesin analogue, AN-215. Int. J. Cancer 88, 652-657. (26) Breeman, W. A. P., Hofland, L. J., De Jong, M., Bernard, B. F., Srinivasan, A., Kwekkeboom, D. J., Visser, T. J., and Krenning, E. P. (1999) Evaluation of radio-labeled bombesin analogues for receptor-targeted scintigraphy and radiotherapy. Int. J. Cancer 81, 658-665. (27) Breeman, W. A. P., De Jong, M., Bernard, B. F., Kwekkebom, D. J., Srinivasan, A., van der Pluijm, M.E., Hofland, L. J., Visser, T. J., and Krenning, E. P. (1999) Pre-clinical evaluation of [111In-DTPA-Pro1, Tyr4]Bombesin, a new radioligand for bombesin-receptor scintigraphy. Int. J. Cancer 83, 657-663. (28) Rogers, B. E., Brechbiel, M. W., Kirkman, R. L., Clarkson, M., and Buchsbaum, D. J. (1999) In Vitro binding and internalization of an Indium-111 labeled bombesin derivative to cells expressing the gastrin releasing peptide receptor. In Technetium, Rhenium and Other Metals in Chemistry and Nuclear Medicine (M. Nicolini and U. Mazzi, Eds.) pp 519525, SGE Editoriali, Padova, Italy. (29) Rogers, B. E., Curiel, D. T., Mayo, M. S., Laffoon, K. K., Bright, S. J., and Buchsbaum, D. J. (1997) Tumor localization of a radiolabeled bombesin analogue in mice bearing human ovarian tumors induced to express the gastrin-releasing peptide receptor by an adenoviral vector. Cancer Suppl. 80, 2419-2424. (30) Van de Wiele, C., Dumont, F., Broecke, R. V., Oosterlinck, W., Cocquyt, V., Serreyn, R., Peers, S., Thornback, J., Slegers, G., and Dierckx, R. A. (2000) Technetium-99m RP525, a GRP analogue for visualization of GRP receptor-expressing malignancies: A feasible study. Eur. J. Nucl. Med. 27, 16941699. (31) Breeman, W. A. P., Hofland, L. J., DeJong, M., Bernard, B. F., Srinivasan, A., Kweekeboom, D. J., Visser, T. J., and Krenning, E. P. (1999) Evaluation of radio-labeled bombesin analogues for receptor-targeted scintigraphy and radiotherapy. Int. J. Cancer 81, 658-665. (32) Baidoo, K. E., Lin, K.-S., Zhan, Y., Finley, P., Scheffel, U., and Wagner, Jr. H. N. (1998) Design, synthesis, and initial evaluation of high-affinity technetium bombesin analogues. Bioconjugate Chem. 9, 218-225. (33) Karra, S. R., Schibli, R., Gali, H., Katti, K. V., Hoffman, T. J., Higginbotham, C., Sieckman, G. L., and Volkert, W. A. (1999) 99mTc-labeling and in vivo studies of a bombesin analogue with a novel water-soluble dithiadiphosphine-based bifunctional chelating agent. Bioconjugate Chem. 10, 254-260. (34) Hoffman, T.J., Quinn, T. P., and Volkert, W. A. (2001) Radio-metalated receptor-avid peptide conjugates for specific in vivo targeting of cancer cells. Nucl. Med. Biol. 28, 527-539. (35) Hoffman, T. J., Li, N., Volkert, W. A., Sieckman, G. L., Higginbotham, C., and Ochrymowcycz, L. A. (1997) Synthesis and characterization of 105Rh labeled bombesin analogues: Enhancement of GRP receptor binding affinity utilizing aliphatic carbon chain linkers. J. Labeled Compd. Radiopharm. 40, 490-493. (36) Volkert, W. A., and Hoffman, T. J. (1999) Therapeutic radiopharmaceuticals. Chem. Rev. 99, 2269-2292. (37) Gali, H., Hoffman, T. J., Owen, N. K., Sieckman, G. L., and Volkert, W. A. (2000) In vitro and in vivo evaluation of 111In-labeled DOTA-8-Aoc-BBN[7-14]NH conjugate for spe2 cific targeting of tumors expressing gastrin releasing peptide receptors (GRP-R). J. Nucl. Med. (Supplement), 41(5), 119P. (38) Smith, C. J., Hoffman, T. J., Hayes, D. L., Owen, N. K., Sieckman, G. L., and Volkert, W. A. (2001) Radiochemical investigations of 177Lu-DOTA-8-Aoc-BBN[7-14]NH2: A new gastrin releasing peptide receptor (GRPr) targeting radiopharmaceutical. J. Labeled Compd. Radiopharm. 44, 706-708. (39) Jurisson, S., Berning, D., Jia, W., and Ma, D. (1993) Coordination compounds in nuclear medicine. Chem. Rev. 93, 1137-1156. (40) Wong, E., Fauconnier, T., Bennett, S., Valliant, J., Nguyen, T., Lau, F., Lu, L. F. L., Pollak, A., Bell, R. A., and Thornback,

Smith et al. J. R. (1997) Rhenium(V) and technetium(V) oxo complexes of an N2N′S peptidic chelator: Evidence of interconversion between the syn and anti conformations. Inorg. Chem. 36, 5799-5808. (41) Guhlke, S., Schaffland, A., Zamora, A., P. O., Sartor, J., Diekmann, D., Bender, H., Knapp, F. F., and Biersack, H.-J. (1998) 188Re- and 99mTc-MAG3 as prosthetic groups for labeling amines and peptides: Approaches with pre- and postconjugation labeling. Nucl. Med. Biol. 25, 621-631. (42) Noll, B., Kniess, T., Friebe, M., Spies, H., and Johannsen, B. (1996) Rhenium (V) gluconate, a suitable precursor for the preparation of rhenium (V) complexes. Isotopes Environ. Health Stud. 32, 21-29. (43) Jensen, R. T., Mrozinski, J. E. Jr., and Coy, D. H. (1993) Bombesin receptor antagonists: different classes and cellular basis of action. Recent Results Cancer Res. 129, 87-113. (44) Moody, T. W. (1996) Peptides and growth factors in nonsmall cell lung cancer. Peptides 17, 545-555. (45) Leban, J. J., Landavazo, A., McDermed, J. D., Diliberto, E. J. Jr., Jansen, M., Stockstill, B., and Kull, F. C. Jr. (1994) Potent gastrin-releasing peptide (GRP) antagonists derived from GRP (19-27) with a C-terminal DPro psi [CH2NH]PheNH2 and N-terminal aromatic residues. J. Med. Chem. 37, 439-445. (46) Kahan, Z., Sun, B., Schally, A. V., Arencibia, J. M., Cai, R. Z., Groot, K., and Halmos G. (2000) Inhibition of growth of MDA-MB-468 estrogen-independent human breast carcinoma by bombesin/gastrin-releasing peptide antagonists RC3095 and RC-3940-II. Cancer 88, 1384-1392. (47) Cai, R. Z., Reile, H., Armatis, P., and Schally, A. V. (1994) Potent bombesin antagonists with C-terminal Leu-psi(CH2N)-Tac-NH2 or its derivatives. Proc. Natl. Acad. Sci. U.S.A. 91, 12664-12668. (48) Coy, D. H., Heinz-Erian, P., Jiang, N. Y., Taylor, J., Moreau, J. P., Gardner, J. D., and Jensen, R. T. (1988) Progress in the development of competitive bombesin antagonists. Ann. N.Y. Acad. Sci. 547, 150-157. (49) Coy, D. H., Jensen, R. T., Jiang, N. Y., Lin, J. T., Bogden, A. E., and Moreau, J. P. (1992) Systematic development of bombesin/gastrin-releasing peptide antagonists. J. Natl. Cancer Inst. Monogr. 13, 133-139. (50) Davis, T. P., Crowell, S., Taylor, J., Clark, D. L., Coy, D., Staley, J., and Moody, T. W. (1992) Metabolic stability and tumor inhibition of bombesin/GRP receptor antagonists. Peptides 13, 401-407. (51) Schubiger, P. A., Alberto, R., and Smith, A. (1996) Vehicles, chelators, and radionuclides: choosing the “building blocks” of an effective therapeutic radioimmunoconjugate. Bioconjugate Chem. 7, 165-179. (52) Bijsterbosch, M. K. (1995) Selective drug delivery by means of receptor-mediated endocytosis. Q. J. Nucl. Med. 39, 4-9. (53) Beck-Sickinger, A. G. (1996) Structural characterization and binding sites of G-protein-coupled receptors. Drug Discovery Today 1, 502-513. (54) Duncan, J. R., Stephenson, M. T., Wu, H. P., and Anderson, C. J. (1997) Indium-111-diethylenetriaminepentaacetic acidoctreotide is delivered in vivo to pancreatic, tumor cell, renal, and hepatocyte lysosomes. Cancer Res. 57, 659-671. (55) Press, O. W., Shan, D., Howell-Clark, J., Eary, J., Appelbaum, F. R., Matthews, D., King, D. J., Haines, A. M., Hamann, P., Hinman, L., Shochat, D., and Bernstein, I. D. (1996) Comparative metabolism and retention of iodine-125, yttrium-90, and indium-111 radioimmunoconjugates by cancer cells. Cancer Res. 56, 2123-2129. (56) Naruki, Y., Carrasquillo, J. A., Reynolds, J. C., Maloney, P. J., Frincke, J. M., Neumann, R. D., and Larson, S. M. (1990) Differential cellular catabolism of 111In, 90Y and 125I radiolabeled T101 anti-CD5 monoclonal antibody. Int. J. Radiat. Appl. Instrum. - Part B, Nucl. Med. Biol. 17, 201207. (57) Mattes, M. J., Griffiths, G. L., Diril, H., Goldenberg, D. M., Ong, G.L., and Shih, L. B. (1994) Processing of antibodyradioisotope conjugates after binding to the surface of tumor cells. Cancer 73 (Suppl. 3), 787-793.

BC020034R