Gastrin releasing peptide (GRP) receptor targeted radiopharmaceuticals: A concise update

Nuclear Medicine and Biology 30 (2003) 861– 868

www.elsevier.com/locate/nucmedbio

Gastrin releasing peptide (GRP) receptor targeted radiopharmaceuticals: A concise update C.J. Smitha,b,c, W.A. Volkerta,b, T.J. Hoffmana,c,d,* a

Radiopharmaceutical Sciences Institute, University of Missouri, Columbia, MO, 65211 b Department of Radiology, University of Missouri, Columbia, MO 65211 c Department of Internal Medicine, University of Missouri, Columbia, MO 65211 d H.S. Truman Memorial VA Hospital, Columbia, MO 65201

Abstract The gastrin releasing peptide (GRP) receptor is becoming an increasingly attractive target for development of new radiolabeled peptides with diagnostic and therapeutic potential. The attractiveness of the GRP receptor as a target is based upon the functional expression of GRP receptors in several tumors of neuroendocrine origin including prostate, breast, and small cell lung cancer. This concise review outlines some of the efforts currently underway to develop new GRP receptor specific radiopharmaceuticals by employing a variety of radiometal chelation systems. Published by Elsevier Inc.

1. Introduction For over a decade, the field of nuclear medicine has been investigating the potential of radiolabeled peptides to target tumor expression of receptors [1–11]. The most outstanding example of success in this arena has been the results obtained targeting somatostatin receptor expression in the development of both diagnostic and therapeutic radiopharmaceuticals [1,2,12]. This work has paved the way for radiolabeled peptide exploration of other receptor systems including bombesin, alpha-melanocyte stimulating hormone, vasoactive intestinal peptide, cholecystokinin, and neurotensin [5–10]. One recent focus of our laboratory, and others, has been the development of peptide based radiopharmaceuticals which target the mammalian gastrin releasing peptide (GRP) receptor, a subtype of the bombesin receptor family. The bombesin receptor family is currently comprised of four receptor subtypes. These subtypes are classified as the neuromedin B (NMB) subtype (BB1), the GRP subtype (BB2), the orphan receptor subtype (BB3), and the bombesin (BBN) receptor subtype (BB4) [13–18]. High affinity native peptide ligands for the BB1, BB2, and BB4 subtypes have been identified with the structures shown in Table 1. * Corresponding author. Tel.: ⫹1-573-814-6000, ext 2593; fax: ⫹1573-882-1663. E-mail address: [email protected] (T.J. Hoffman). 0969-8051/03/$ – see front matter Published by Elsevier Inc. doi:10.1016/S0969-8051(03)00116-1

Although a high affinity native peptide ligand has not been identified for the BB3 subtype, a high affinity peptide which binds to the BB3 receptor has been synthesized [19]. Of the four known bombesin receptor subtypes, the BB2, or GRP subtype, has been studied the most extensively to date. Investigation of the bombesin receptor system was initiated in 1971 with the isolation of the tetradecapeptide bombesin from the skin of the frog Bombina bombina by Anastasi and co-workers [20]. The mammalian counterpart of bombesin, a 27 amino acid peptide called GRP was isolated from porcine stomach by McDonald and co-workers in 1979 [21]. GRP and bombesin share amidated C terminus sequence homology in the final 7 amino acids, -Trp-Ala-Val-Gly-HisLeu-Met-NH2. The impetus for targeting the bombesin receptor system, and more specifically the GRP subtype, is based on reports demonstrating that a variety of human tumors and tumor cell lines have been shown to overexpress the GRP (BB2) receptor including prostate, breast, and small cell lung cancers [22–26]. With respect to human prostate cancer, high affinity GRP receptor expression has been identified in tissue biopsy samples and immortalized cell lines [27–29]. Markwalder and Reubi demonstrated that GRP receptor expression in primary prostatic invasive carcinoma was present in 100% of the tissues tested (30 of 30 cases). In 83% of these cases, GRP receptor expression was determined to be high or very high (⬎1000 dpm/mg). Of 26 patients studied with high-

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Table 1 Bombesin Receptor Subtypes Subtype

Native Peptide

BB1 BB2

Neuromedin B (NMB) Gastrin Releasing Peptide (GRP)

BB3 BB4

BRS-3 Bombesin (BBN):

Origin Gly-Asn-Leu-Trp-Ala-Thr-Gly-His-Phe-Met-NH2 Val-Pro-Leu-Pro-Ala-Gly-Gly-Gly-Thr-Val-Leu-ThrLys-Met-Tyr-Pro-Arg-Gly-Asn-His-Trp-Ala-Val-GlyHis-Leu-Met-NH2 Not identified Pyr-Gln-Arg-Leu-Gly-Asn-Gln-Trp-Val-Gly-His-Leu-Met-NH2 Pyr-Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Phe-Met-NH2

Mammalian Mammalian

Mammalian Amphibian

The sequences for the native peptides which bind to the mammalian bombesin receptor subtypes BB1 and BB2 and the amphibian bombesin receptor subtype BB4 are represented. Two distinct forms of the peptide bombesin have been identified for the BB4 receptor subtype differing by either ⫺Leu13- or ⫺Phe13-. A high affinity native peptide has not been identified for the mammalian bombesin receptor subtype BB3.

grade prostatic intraepithelial neoplasia, all but one demonstrated high to very high densities of GRP receptors. This study also demonstrated that more than 50% of androgen independent prostate cancer bone metastases were GRP receptor positive (4 of 7 cases). GRP receptor density analysis in non-neoplastic prostatic tissue, and in particular, benign prostatic hyperplasia was either completely absent or of low incidence. The study by Sun and co-workers further confirmed the preferential expression of GRP receptors in prostatic carcinoma with 91% of the samples tested (20 of 22) expressing mRNA for the GRP receptor [29]. Also of note in this study was the detection of mRNA for two other bombesin receptor subtypes, the NMB receptor and the orphan receptor BB3. Prior to human tissue analysis, both androgen-dependent and androgen-independent human prostate cancer cell lines were shown to express high affinity GRP receptors [30]. A large body of evidence is available demonstrating the expression of the GRP receptor subtype in estrogen receptor positive (ER⫹) and estrogen receptor negative (ER-) immortalized breast cancer cell lines [31–36]. Two studies using primary human breast carcinoma and axillary metastastic tissue have confirmed the presence of the GRP receptor subtype, as well the expression of the GRP receptor gene in a large percentage of the tissues sampled [24,33,37]. Halmos and co-workers examined the binding of radioiodinated bombesin to a membrane preparation from 100 individual human breast carcinomas and found significant GRP receptor expression in 33% of these samples [33]. The study of Gugger and Reubi demonstrated that primary breast carcinomas had a 62% incidence (32 of 52 cases) of GRP receptor expression [24]. They further demonstrated 100% GRP receptor incidence in all lymph node metastases (n ⫽ 33) sampled from GRP receptor positive primary breast tumors. These results combined with those obtained in prostatic carcinoma tissue clearly make the GRP receptor an attractive candidate to target for diagnostic and therapeutic purposes. This review highlights some of the more recent studies aimed at developing synthetic peptide analogues that exhibit specificity for the GRP preferring subtype of the bombesin receptor system. The results obtained with these systems

demonstrate several rationales for formulation of new diagnostic and therapeutic radiopharmaceuticals targeting GRP receptor expression in neoplastic tissues.

2. Tc-99m/Re-188 bombesin conjugates Technetium-99m continues to be at the forefront of nuclear medicinal applications due to its wide range availability (99Mo/99mTc generator system), ideal nuclear characteristics (t1/2 ⫽ 6.04h, E␥ ⫽ 140KeV (89%)), and wellestablished labeling chemistries. For example, 99mTc accounts for more than 85% of all diagnostic applications performed in medical facilities each year [38]. Rhenium188 holds potential as an isotope for therapeutic nuclear medicinal applications primarily because of its widespread availability (188W/188Re Generator) and attractive physical characteristics (t1/2 ⫽ 16.94h, ␤⫺max ⫽ 2.12MeV, and E␥ ⫽ 155KeV) [39]. Rhenium-188 is the therapeutic surrogate of Tc-99m. Therefore, production of 188Re-labeled radiopharmaceuticals often parallel the chemistries used to develop technetium-based radiopharmaceuticals [38 –39]. Significant progress towards the design and development of clinically useful 99mTc/188Re-labeled conjugates which target the GRP, or BB2, receptor subtype has been reported [40 – 43]. Baidoo and co-workers have described the radiosyntheses and initial evaluation of receptor binding of 99mTcDADT (Diaminodithol) conjugates (Fig. 1, Structure 1) of Pyr-Gln-Lys-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-LeuMet-NH2 (Lys3-BBN) [40]. Their studies showed that these conjugates had very high affinity for the GRP receptor in rat brain cortex. Competitive binding displacement assays where 125I-Tyr4-BBN was used as the radiolabel, for example, showed Ki values of 3.5–7.4 nM for a series of 99mTcDADT-conjugates (Ki of native BBN ⫽ 4.3 ⫾ 1.0 nM). In vivo biodistribution analyses of the series of conjugates demonstrated little uptake in non-target tissue, with excretion being primarily the hepatobiliary pathway [40], which is not uncommon for conjugates of this type. Gali et al., have demonstrated the usefulness of the P2S2 ligating framework for in vivo stabilization of 99mTc/188Re(V)-conjugates of BBN(7–14)NH2 (Fig. 1, Structure 2) [41]. The

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Fig. 1. Structural drawing of twelve representative examples of radiolabeled peptide constructs which target the GRP, or BB2, subtype of the bombesin receptor. Details regarding specific receptor binding and tumor uptake of these compounds are outlined in Table 2.

competitive binding displacement assay of ReO2-P2S2-5Ava-BBN(7–14)NH2 versus the native radiolabel 125I-Tyr4BBN on Swiss 3T3 fibroblasts showed that the new conju-

gate exhibited an IC50 value of 0.8 ⫾ 0.4 nM. Furthermore, preliminary biodistribution assays in PC-3 tumor-bearing, SCID mice (SCID ⫽ Severely Compromised Immunodefi-

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cient) showed tumor uptake of 4.7 ⫾ 0.8% ID/g [41– 42]. Clearly, further work need be performed to ascertain the usefulness of this therapeutic motif for clinical applications. Constructs of the type 99mTc-N3S-X-BBN (7–14)NH2 ((Fig. 1, Structure 3) X ⫽ 0-Carbons, ␤-Ala (␤-alanine), 5-Ava (5-aminovaleric acid), 8-Aoc (8-aminooctanoic acid), and 11-Aun (11-aminoundecanoic acid)) provide flexibility for designing 99mTc-labeled conjugates that retain high in vitro and in vivo specificity targeting of GRP receptor expressing cells have been studied in our laboratory [43]. In these studies, it was shown that the length of the hydrocarbon spacer group could be varied from at least 3 to 8 carbon atoms in length without compromising agonistic binding affinity to GRP receptors [43]. Receptor-mediated tumor accumulation of 99mTc-N3S-5-Ava-BBN (7–14)NH2 in PC-3, human cancer, xenografted, SCID mouse models demonstrated uptake of 2.1 ⫾ 0.5% ID/g (unpublished results, Table 1). The potential clinical utility of these 99mTcN3S-X-BBN (7–14)NH2 constructs as cancer specific imaging agents was recently demonstrated by Van de Weile, et al., in human patients with either prostate or breast cancer [44 – 45]. Their studies showed that 99mTc-N3S-5-AvaBBN(7–14)NH2 localized in tumors with high specificity producing good tumor-to-normal tissue uptake ratios and high quality SPECT images [44 – 45]. Most recently, Nock et al., have reported some remarkable results for the potent bombesin antagonist 99mTc-Demobesin (Fig. 1, Structure 4) [46]. Receptor-mediated tumor uptake of 16.2 ⫾ 3.1% ID/g in Swiss nu/nu mice bearing human PC-3 xenografts was reported. These values are well above all radiolabeled BBN conjugates that have been previously reported [46]. This uptake is receptor-specific as demonstrated by the 79.5 ⫾ 0.8% ID/g value obtained in the normal pancreas at 1h p.i. The normal pancreas is typically used as an indicator of GRP receptor specificity for BBN conjugates of this type. Co-administration of Tyr4-BBN effectively blocked pancreatic uptake and further reflects the high specificity this conjugate has for the GRP receptor [46]. Aside from traditional approaches [i.e., 99mTc(V) or 188 Re(V) labeling via N or S chelating donors] for radiolabeling small molecules and biologically active targets with technetium, a more recently developed “Organometallic” labeling strategy has been developed [47–52]. Recent investigations by La Bella, Alberto, and Schibli have led to the development of some ‘state-of-the-art’ Tc(I) and Re(I) chemistry [50,51,53]. They reported the organometallic chemistry of Tc(I) and Re(I) tricarbonyl complexes containing the fac-M(CO)3 moiety and demonstrated the lability of the three water molecules coordinated to the fac-M(CO)3 moiety to account for excellent labeling efficiencies with a number of donor groups including amines, thioethers, phosphines, and thiols [50,51,53]. Initial investigations during the development of a clinically useful 99mTc/188Re tricarbonyl radiosynthon for the labeling of even the simplest biomolecules proved futile due to multistep, high-pressure synthetic protocols. They have shown that the direct car-

bonylation of the permetallate salt (i.e., MO⫺ 4 ) by the action of borohydride under atmospheric carbon monoxide pressure produces the new organometallic aquaions [99mTc(H2O)3(CO)3]⫹ and [188Re(H2O)3(CO)3]⫹ [54]. Clearly, a new avenue for the successful radiolabeling of bioactive molecules with low-valent 99mTc/188Re has been identified. The feasibility of using the [99mTc(H2O)3(CO)3]⫹ aquaion as a radiosynthon for the successful labeling of bioactive molecules has been reported [52–53]. By simply functionalizing the N-terminus of Neurotensin (NT) with histidine or (N␣-histidinyl)acetic acid, Egli et al., were able to successfully radiolabel NT, achieving relatively high specific activity radiocomplexes that maintained high biological activity [52]. The potential utility of a [99mTc(CO)3N␣-histidinyl acetate]-bombesin(7–14)NH2, (Fig. 1, Structure 5), construct as a cancer specific imaging agent was recently demonstrated by La Bella et al., in PC-3 tumorbearing mice [53]. They were able to obtain high specificactivity 99mTc(I)-conjugates that demonstrated high affinity for the GRP receptor in vitro and in vivo. Furthermore, they demonstrated the ability of these new conjugates to maintain inherent biological integrity, presumably due to the tridentate ligating fashion of the chelate. However, their studies showed that the [99mTc(CO)3-N␣-histidinyl acetate]-bombesin(7–14) analogue exhibited minimal localization in PC-3 tumors (0.6 ⫾ 0.1% ID/g, 1.5h p.i, Table 2), possibly due to weak vascularization of the tumor model [53]. We have reported the synthesis of [99mTc(X)(CO)3Dpr-SSS-BBN(7–14)NH2] (Fig. 1, Structure 6) (Dpr⫽ 2,3Diaminopropionic Acid, X ⫽ H2O or P(CH2OH)3) conjugates and their in vitro/in vivo evaluation in GRP receptorspecific tissue [55–56]. The new 99mTc(I)-labeled conjugates retain high in vitro and in vivo stability and specifically target GRP receptor-expressing cells. For example, specific targeting of the GRPr in human, PC-3 xenografts showed uptake of 3.7 ⫾ 0.9%ID/g at 1h p.i. (Table 2). Furthermore, it was shown that the ancillary third ligand (H2O or P(CH2OH)3) or tethering moiety (i.e., SSS) could be varied with little or no compromise of agonistic binding to GRP receptors in vivo [55]. Tumor uptake and retention in human prostate (PC-3) cells for the new conjugate [99mTc(X)(CO)3-Dpr-SSS-BBN (7–14)NH2] (Fig. 1, Structure 6), is superior to the 99mTc-N3S-conjugate, (Fig. 1, Structure 3), in the same animal model [56]. Research investigations toward the design and development of an in vivo stable Re-188 surrogate based upon N3S-5-Ava-BBN(7–14)NH2 proved unsuccessful in our laboratory. Schibli and co-workers recently reported the development of the [188Re(H2O)3(CO)3]⫹ synthon, and therefore considerable interest has again been sparked toward the development of an in vivo stable Re-188 conjugate with high affinity for the GRP receptor [54]. The design and development of [188Re(H2O)(CO)3-Dpr-SSS-BBN [7–14]NH2] has been described [39]. In vitro assessment of the new conjugate shows a well defined 188Re(I)-labeled

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Table 2 Receptor binding affinities and respective tumor uptake data is represented for each of the compounds described in the text and shown in Figure 1. Conjugate 1 2 3 4 5 6 7 8 9 10 11 12

IC50nM (SD) in GRPExpressing Tissue

%ID/g (SD) in GRPExpressing Tumor Tissue

References

Ki ⫽ 3.5(0.7)-7.4(2.0), rat brain cortex 0.8(0.4), Swiss 3T3 1.0(0.2), PC-3 0.7(0.1), PC-3 2.2(2.0), PC-3 0.9(0.2), PC-3 ******* Free ligand, 8.0, rat pituitary 2.4(0.0), PC-3 0.5(1.0), PC-3 4.76(0.79), Swiss 3T3 Kd ⫽ 10.5 nM, PC-3

*******

Baidoo, et al.40

4.7(0.8), PC-3, 1h p.i. 2.1(0.5), PC-3, 1h p.i. 16(3.0), PC-3, 1h p.i. 0.6(0.1), PC-3, 1.5h p.i. 3.7(0.9), PC-3, 1h p.i. ******* 0.048(0.008), 24h p.i. rat pituitary tumor 3.6(1.1), PC-3, 1h p.i. 4.2(1.1), PC-3, 1h p.i. ******* 5.5(0.6), PC-3, 2h p.i.

Gali, et al.41,42 Smith, et al.43 Nock, et al.46 La Bella, et al.53 Smith, et al.55,56 Rogers, et al.64 Breeman, et al.65

conjugate that is chemically similar to the 99mTc surrogate, [99mTc(H2O)(CO)3-Dpr-SSS-BBN [7–14]NH2], based upon RP-HPLC. The [188Re(H2O)(CO)3-Dpr-SSS-BBN (7–14)NH2] conjugate retains high in vitro and in vivo stability and specifically targets GRP receptor expressing cells in PC-3 tumor xenografts (i.e., 3.0 ⫾ 0.7% ID/g at 1h p.i.) [39]. To our knowledge, this is the first site-directed [188Re(I)(CO)3]-conjugate of this type successfully proven to effectively target receptor-specific cancerous tissue.

3. Trivalent radiometallated bombesin conjugates Trivalent metallic radioisotopes, such as the radiolanthanides, are particularly attractive for use in diagnostic and/or therapeutic procedures in nuclear medicine. For example, the radiolanthanide and lanthanide-like (i.e., 90Y3⫹) elements possess similar chemistries in aqueous solution and can be produced as high specific activity reagents [57]. The radiolanthanides exist primarily in an oxidation state of 3⫹ and can be stabilized by hard donor atoms such as nitrogen or oxygen. The mechanism of ligand coordination is more often ionic, rather than covalent, for these trivalent isotopes, and therefore, multidentate, macrocyclic, polyamino-carboxylate ligand frameworks such as DTPA or DOTA (DTPA ⫽ Diethylenetriaminepentaacetic acid; DOTA ⫽ 1,4,7,10-tetraazacyclododecane-N,N⬘,N“,N”’-tetraacetic acid) are often utilized to produce kinetically inert, in vivo stable conjugates [57]. Radiolabeling strategies of radiolanthanides often parallel, and include heating for short periods of time at a pH of 5.5– 6.0 in order to obtain radiochemical yields of ⱖ95%. Adding to the attractive chemical characteristics of the lanthanides is the wide range of nuclear properties exhibited among the series (i.e., t1/2, ␤⫺max, E␥), providing opportunities to chemically tune a particular application by changing only the radioisotope (Table 3)[57].

Hoffman, et al.66 Smith, et al.67,68 Ning and Hoffman70,71 Rogers, et al.72

The potential utility of using radiolabeled receptor-avid peptides to produce clinically-useful site-directed radiopharmaceuticals is readily exemplified by the current use of 111 In-DTPA-octreotide (Octreoscan®, Mallinckrodt Medical Inc.) and other radiolabeled octreotide analogues that bind to cancer cells over-expressing somatostatin receptors [57– 63]. Investigations into the synthesis and characterization of *M3⫹-radiolabeled BBN or GRP analogues have also been reported by several groups [64 – 68]. Rogers and Brechbiel have recently described the synthesis and subsequent 111In-radiolabeling of CHX-B-DTPA-8-Aoc-BBN(7– 14) (Fig. 1, Structure 7). In their study, they reported high binding of 111In-CHX-B-DTPA-Aoc-BBN [7–14] to adenoviral vector AdCMVGRPr infected SKOV3.ipl ovarian cancer cells (⬃42.7%) and a high degree of internalization and retention of the conjugate (60% at 15min, 58% at 2h) [64]. Breeman et al., have described the design and development of radiolabeled BBN conjugates based upon the general structure shown in Fig. 1, Structure 8 [65]. In this study, two conjugates of the general type [111In-DTPA-Pro1,Tyr4]BN (agonistic) and [111In-DTPA-Tyr5, D-Phe6]BN(5–13)NHEt (antagonistic) were described. Each of the two conjugates expressed high affinity for the GRP receptor in 7315b rat pituitary tumor cell membranes. [111In-DTPA-Pro1, Tyr4]BBN, which demonstrated in vitro internalization inherent to agonistic binding, also showed higher uptake in rat pituitary tumors [65]. The in vitro and in vivo evaluation of a series of 111In-DOTA-X-BBN(7–14)NH2 analogs, where (X) is a 0 Carbon, ␤-Ala, 5-Ava, 8-Aoc, or 11-Aun tether between the active site of the biomolecule and the chelating ligand (Fig. 1, Structure 9) was recently reported [66]. Conjugates exhibiting high binding affinities for GRP receptors in human PC-3 androgen independent prostate cancer cells in vitro (i.e., IC50 values ranging from 0.6 –2.4 nM) included those with 3, 5, or 8-carbon spacer moieties [66]. In vivo biodistribution studies demonstrated that analogs where X ⫽ 5-Ava and 8-Aoc exhibit high specific localiza-

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Table 3 Nuclear properties for the trivalent metallic radioisotopes currently being employed to radiolabel peptides for targeting the GRP, or BB2, subtype of the bombesin receptor. Isotope

Production/ Availability

t1/2

E␤⫺max

E␥

In-111

Cyclotron

2.80d

*******

Sm-153 Dy-166 Ho-166 Lu-177 Pm-149 Y-90

Reactor Reactor Reactor Reactor Reactor Generator

1.93d 3.40d 1.12d 6.71d 2.21d 2.67d

0.69MeV 0.40MeV 1.80MeV 0.50MeV 1.10MeV 2.28MeV

171.3 and 245.4 KeV 103KeV 82.5KeV 80.6KeV 208KeV 286KeV ******

tion in the pancreas, a normal GRP receptor expressing tissue, and efficient clearance from the blood primarily via the renal/urinary pathway. These results suggested that the 111 In-DOTA-X-BBN(7–14)NH2 construct, where X is a tether of either 5 or 8 carbons in length, might form the basis of development of radiometallated diagnostic or therapeutic radiopharmaceuticals for selective in vivo targeting of GRPreceptor expressing cancers. In vivo uptake of 111In-DOTA8-Aoc-BBN(7–14)NH2 in human prostate PC-3 xenografted flank tumors, demonstrated the efficacy of tumor targeting for agonistic conjugates of this type (i.e., 3.63 ⫾ 1.11% ID/g at 1h p.i.) [66]. Pre-clinical evaluation of the radiolanthanide surrogate, 177Lu-DOTA-8-Aoc-BBN(7–14)NH2 (Fig. 1, Structure 10), has shown that this conjugate exhibits an IC50 of 0.5 ⫾ 0.1nM in GRP receptor-expressing PC-3 tumors cells [67– 68]. Receptor-mediated, tumor targeting of the PC-3 xenografted SCID mice resulted in tumor uptake and retention values of 4.22 ⫾ 1.09%ID/g, 3.03 ⫾ 0.91%ID/g, and 1.54 ⫾ 1.14%ID/g at 1, 4, and 24h, respectively [67– 68].

4. Other metallated bombesin conjugates Rhodium-105 continues to draw interest as a therapeutic radionuclide due to its availability (reactor-produced in high specific activity, [104Ru (n, ␥) 105Ru(␤⫺) ➩ 105Rh] and desirable physical characteristics (t1/2 ⫽ 1.4d, ␤⫺max ⫽ 0.57MeV). Ning and co-workers have reported the pharmacokinetic studies of numerous thioether-containing macrocycles with 105Rh [69]. The 105Rh-S4-BBN conjugate, (Fig. 1, Structure 11), has also been reported. The competitive binding displacement assay of Rh-S4-5-Ava-BBN(7– 14)NH2, (Fig. 1, Structure 11), versus the native radiolabel 125 I-Tyr4-BBN on Swiss 3T3 fibroblasts showed that the conjugate exhibited an IC50 value of 4.76 ⫾ 0.79 nM. For this conjugate, GRP receptor-specific uptake in normal pancreas was found to be 2.25 ⫾ 1.02% ID/organ [70 –71]. Receptor-mediated tumor uptake in rodent models was not reported. Copper-64 (t1/2 ⫽ 12.7h, ␤⫺ ⫽ 0.655MeV (19.3%), ␤⫺

⫽ 0.573 MeV (39.6%)) can be produced in high specific activity using a biomedical cyclotron [59]. Cu-64 is an ideal isotope for PET imaging studies and targeted radiotherapeutic applications. Rogers and co-workers have prepared and studied the in vitro/in vivo investigations of 64Cu-radiolabeled BBN analogues [72]. Recently, they reported the design and development of 64Cu-BBN conjugates based upon the general structure shown in Fig. 1, Structure 12. This conjugate was produced in high specific activity by incubation of DOTA-8-Aoc-BBN(7–14)NH2 with the radiolabel (pH ⫽ 5.5, 30 min, 37oC). Binding of this conjugate to human prostate, PC-3 cells showed that the conjugate had a Kd of 10.5nM (␤max of 2.9 ⫻ 105 receptors/cell). Receptor-mediated, tumor targeting of the PC-3 xenografted athymic mice resulted in tumor uptake and retention values of 5.5 ⫾ 0.6%ID/g, 4.2 ⫾ 1.1%ID/g, and 2.5 ⫾ 0.5%ID/g at 2, 4, and 24h, respectively [72].

5. Conclusion Radiolabeled, small, receptor-avid peptides continue to receive much interest toward the development of site-directed radiopharmaceuticals. The radiolabeled BBN conjugates discussed in this review have shown promising diagnostic/therapeutic efficacy toward the design of site-specific radiopharmaceuticals targeting cancer cells over-expressing the GRP receptor subtype. These studies provide important insight for identifying structural characteristics that are optimal for radiolabeled constructs that can be used for diagnostic or therapeutic applications in patients. Further progress in developing GRP receptor targeted radiopharmaceuticals will require additional efforts in understanding the structurally sensitive mechanisms involved in the binding of these derivatives to GRP/BBN receptors, the subsequent residualization of the radiotracer in GRP receptor expressing cancer cells, and finally, efficient clearance of nonresidualizing radiolabeled peptide from non-target tissues.

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