99mTc(CO)3-DTMA bombesin conjugates having high affinity for the GRP receptor
Introduction
Bombesin peptide (BBN) is a 14-amino acid analog of human gastrin releasing peptide (GRP) originally isolated from the skin of the frog Bombina bombina in 1970 [1]. There are four known receptor subtypes of BBN, including the neuromedin B receptor (subtype 1), the GRP receptor (GRPr, subtype 2), the orphan receptor (subtype 3) and the BBN receptor (subtype 4) [2]. In recent years, our group and others have focused upon development of site-directed molecular imaging agents targeting human cancers expressing the GRPr subtype [3], [4], [5], [6], [7], [8], [9]. These studies have been based primarily upon reports that specific human tumors tend to express the GRPr in very high numbers, therefore providing an approach to selectively target GRP receptor-expressing neoplasms with minimal accumulation in nontarget collateral tissue [1], [2], [10], [11], [12].
The use of radiolabeled BBN targeting vectors to selectively target receptor-expressing tumors offers innovative diagnostic and treatment strategies for patients suffering from specific human cancers such as breast or prostate [1], [2], [10], [11]. The utility to effectively image prostate and breast cancer tumor xenografts via site-directed single photon emission computed tomography (SPECT) or positron emission tomography (PET) using 111In- or 64Cu-radiolabeled BBN molecular imaging agents provides impetus toward further development of new molecular imaging strategies in order to overcome the limitations of current procedures for diagnosis, staging and restaging of the disease. 99mTc is a versatile radiometal for use in molecular imaging of human tumors due to its ideal physical properties (t½=6 h and 140 keV gamma emission), onsite availability from a 99Mo/99mTc generator and diverse labeling chemistry [13], [14], [15]. 99mTc has proven its utility in nuclear medicine by its use in ∼85% of all diagnostic procedures in clinical nuclear medicine [16].
Organometallic, tricarbonyl, technetium chemistry has become a focus for future 99mTc radiopharmaceuticals due to the ease of reducing generator pertechnetate eluent from a 7+ oxidation state to a kinetically-inert, d6, 1+ oxidation state via an Isolink radiolabeling kit [14], [17], [18]. The development of the aqua ion complex, [99mTc(H2O)3(CO)3]+, has generated interest for new chelating ligand frameworks that can stabilize the cationic Tc(I) metal center under in vivo conditions [13], [17], [19]. Studies have shown bi- and tridentate ligands that are composed of primary, secondary and aromatic amines to be effective chelators for the low-valent metal center, providing the stability necessary for in vivo molecular imaging of human tumor tissue [3], [18], [20], [21], [22], [23], [24]. Amines are relatively soft donor atoms and are known to have high affinity for soft acceptors [25], hence the strong binding and kinetic inertness of 99mTc-conjugates based upon these ligand frameworks. The effectiveness of using bidentate ligands for imaging of GRP receptor-expressing prostate tumors has been demonstrated by the high-quality microSPECT images obtained by Prasanphanich et al. [25], [26]. Tridentate ligand frameworks, however, occupy all three binding sites on the fac-[99mTc(CO)3]+ metal fragment, alleviating the possibility for trans-metallation reactions in the presence of serum proteins and nontarget accumulation of tracer in tissue, thus offering the possibility of higher-contrast, higher-quality SPECT images.
Herein, we report the synthesis of the tridentate 2-(N,N′-Bis(tert-butoxycarbonyl)diethylenetriamine) acetic acid (DTMA) ligand and its conjugation to the N-terminal primary amine of H2N-(X)-BBN(7-14)NH2 to produce DTMA-(X)-BBN(7-14)NH2. The BBN analog BBN(7-14)NH2 was used in this study due to its compact size and similar homology to mammalian GRP in the amidated C-terminal region of the peptide. GRP and BBN share an amidated C-terminus with an identical sequence homology of the seven terminal amino acids, W-A-V-G-H-L-M-NH2. All of the conjugates were synthesized by solid phase peptide synthesis (SPPS) and fully characterized by negative ion electrospray-ionization mass spectrometry (ESI-MS). The conjugates were radiolabeled with [99mTc(H2O)3(CO)3]+, synthesized via an Isolink radiolabeling kit, to produce [99mTc(CO)3-DTMA-(X)-BBN(7-14)NH2] conjugates in high yield. Internalization and externalization studies and competitive displacement binding assays [inhibitory concentration at 50% (IC50)] were performed in PC-3 human prostate cancer cells, a cell line that expresses the GRPr in very high numbers (2.7±0.6×105 receptors per cell) [26]. The pharmacokinetics of the series of [99mTc(CO)3-DTMA-(X)-BBN(7-14)NH2] conjugates were determined in CF-1 normal mice. The [99mTc(CO)3-DTMA-(β-Ala)-BBN(7-14)NH2]+ conjugate, which showed favorable uptake and retention of tracer in vitro and in normal mouse models, was evaluated in SCID mice bearing PC-3 xenografted tumors.
Section snippets
Synthesis of N,N′-Bis(tert-butoxycarbonyl)diethylenetriamine
All chemicals were purchased from Aldrich Chemical (St. Louis, MO, USA) and used without further purification. American Chemical Society-certified solvents were purchased from Fisher Scientific (Pittsburgh, PA, USA) and used without further purification. 2-(tert-butoxycarbonyloxyimino)-2-(phenlacetonitrile) (BOC-ON) (4.46 g, 18.1 mmol) dissolved in tetrahydrofuran (THF) (250 ml) was slowly added dropwise to diethylenetriamine (0.933 g, 9.04 mmol) in THF (10 ml) at <0°C [ice, NaCl and (CH3)2CO].
Results
DTMA was synthesized in a three-step procedure according to Scheme 1. Briefly, BOC protection of the primary amines of diethylenetriamine, alkylation of the secondary amine and subsequent ester hydrolysis afforded the product in good yield. The final product and product reaction intermediates were purified and fully characterized by 1H and 13C NMR and HR-FAB MS. DTMA was stored in a vial under nitrogen atmosphere. No decomposition was observed over a period of several months. DTMA peptide
Discussion
Small peptides continue to be effective delivery vehicles for diagnostic and therapeutic radionuclides due to their ability to bind receptors expressed on specific human cancers [26], [27]. The model of success in this arena has been Octreoscan ([111In-DTPA-Octreotide]), with its targeting and diagnostic imaging of somatostatin receptor-positive neuroendocrine tumors [28]. As a result of this success, there is a basis for investigating in diagnostic and therapeutic radiopharmaceuticals based
Conclusion
DTMA provided a versatile synthetic route via traditional SPPS to produce new peptide conjugates that were easily radiolabeled with the fac-[99mTc(CO)3]+ metal center using the Isolink radiolabeling kit. DTMA-(X)-BBN(7-14)NH2 formed well-defined conjugates in good yield upon radiolabeling with the [99mTc(H2O)3(CO)3]+ precursor. [99mTc(CO)3-DTMA-(X)-BBN(7-14)NH2]+ conjugates presented some variability in excretion properties depending upon the pharmacokinetic modifier that was chosen. All of the
Acknowledgments
This material was 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, Columbia, MO 65211, USA. This work was funded in part by The United States Department of Veterans' Affairs VA Merit Award. Salary support for T.L.R. and G.L.S. also acknowledges the National Institutes of Health (1 P50 CA103130-01).
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2012, International Journal of PharmaceuticsCitation Excerpt :This is mainly accomplished by the covalent attachment of a bifuctional chelator, for the complexation of the [Tc(V) = O]3+ core (Wong et al., 1997; Lin et al., 2004, 2005; Blok et al., 2004; Van de Wiele et al., 2000, 2001a,b, 2008; Smith et al., 2003a,b, Trejtnar et al., 2000; Gourni et al., 2009; Fragogeorgi et al., 2009), or the tricarbonyl [Tc(I)(CO)3]+ core (La Bella et al., 2002a,b; Smith et al., 2003a,b; Alves et al., 2006; Veerendra et al., 2006) to the targeting biomolecule with or without the presence of an intermediate linker/spacer chain. Among the BN analogues studied, two basic structure types can be recognised: the first includes analogues, in which only a small portion of natural peptide sequence is retained, usually BN(7–14), with the addition of a chelator group and a spacer chain (Decristoforo and Mather, 2002; Rogers et al., 2003; Smith et al., 2003a,b; Giblin et al., 2005; Veerendra et al., 2006; Alves et al., 2006; Yang et al., 2006; Kunstler et al., 2007; Parry et al., 2007; Prasanphanich et al., 2007; Garayoa et al., 2007, 2008; Ananias et al., 2008; Schweinsberg et al., 2008; Lane et al., 2008; Gourni et al., 2009), while the second type includes analogues availing the full length of the BN peptide chain, with one or more amino acid residues of the N-terminal region being selectively replaced by a chelator group and/or a spacer chain (Baidoo et al., 1998; Breeman et al., 1999a,b; Chen et al., 2004; Lin et al., 2004, 2005; Yang et al., 2006; Fragogeorgi et al., 2009). In general, the spacer technology seems to be one of the most promising proposals concerning the design of new BN analogues (Garayoa et al., 2008; Schweinsberg et al., 2008; Fragogeorgi et al., 2009; Lane et al., 2010) as well as of other bioactive peptides targeting G-protein coupled receptors (Kolenc-Peitl et al., 2011) that may be applied as radiodiagnostic/radiotherapeutic agents.