Somatostatin receptor (SSTR) ligand analogs are the most well-developed and extensively investigated group of small regulatory peptides. Somatostatin receptors are expressed in neuroendocrine tumors, renal cell carcinoma, small cell lung cancer, breast cancer, prostate cancer, and malignant lymphoma (Reubi 2001). The receptor binding affinity, internalization, and biodistribution of different SST ligands have been shown to be dependent on the peptide constitution, chelator type (1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), desferrioxamine (DFO), diethylenetriaminepentaacetic acid (DTPA), and their derivatives), metal cations (In, Y, Ga, Tc, Lu), linkers, and pharmacokinetic modifiers (Kowalski et al. 2003; Eisenwiener et al. 2002; Maecke 2005; Rivier et al. 2005; Wild et al. 2003; 2005; Ginj et al. 2005; Hofland et al. 1999; De Jong et al. 1998; Reubi et al. 2000; Froidevaux and Eberle 2002; Forrer et al. 2004; Schmitt et al. 2005; Smith-Jones et al. 1994; Froidevaux et al. 2002; Heppeler et al. 1999; Henze et al. 2001; Deshmukh et al. 2005; Hofmann et al. 2001; Pettinato et al. 2008; Antunes et al. 2007; Erchegyi et al. 2008; Grace et al. 2008a, b; Cescato et al. 2008; Fani et al. 2010).

Quantification is one of the prominent advantages of the PET technique, allowing estimation of receptor density in vivo (Zhang et al. 2010). However, the accuracy and interpretation of results depend on a number of parameters, including SRA. The latter was demonstrated to be an essential tool for optimization of imaging quality and correlation of radioactivity uptake and receptor expression (Velikyan et al. 2008; Velikyan et al. 2010). In particular, the quantification accuracy of receptor binding of [⁶⁸Ga]Ga-DOTA-TOC was investigated as a function of SRA in vitro on frozen sections of Rhesus monkey brain expressing SSTR. The dependence was described by a sigmoidal function with the region around the inflection point being the most sensitive to SRA variation (Fig. 3). The values of SRA corresponding to the top plateau provided stable target-to-nontarget ratio and thus high reproducibility of the in vitro biological assay and more accurate quantification (Velikyan et al. 2008). However, it should be mentioned that the correlation of the in vitro and in vivo uptake as a function of SRA might not be linear. Optimization of SRA is required, considering the losses of the tracer in vivo due to nonspecific adsorption, blood protein binding, and binding to physiologically expressed sites consuming tracer, thus demanding higher masses of vector and consequently lower SRA.

The short half-life of ⁶⁸Ga, allowing repetitive sequential examinations, in combination with fast target localization and blood clearance of SST analogs is of special interest for clinical use, providing a means for individual patient treatment design (Maecke et al. 2005; Velikyan et al. 2010). Modulation of [⁶⁸Ga]Ga-DOTA-TOC uptake in tumor and healthy organs was achieved by variation of the total amount of administered peptide. In particular, three sequential examinations of a patient were conducted on the same day, and the amount of octreotide administered immediately prior to [⁶⁸Ga]Ga-DOTA-TOC during the second and third examinations was escalated (Velikyan et al. 2010). The uptake increased in the tumor and metastases and at the same time decreased in the liver and spleen upon injection of 50 μg. Further enhancement to 250 and 500 μg resulted in a blocking effect in all tissues (Fig. 4, row 1) except for one patient (Fig. 4, row 2). The latter observation clearly demonstrates the importance of individualized patient diagnostic and therapeutic management.

[⁶⁸Ga]Ga-DOTA-TOC, [⁶⁸Ga]Ga-DOTA-TATE, and [⁶⁸Ga]Ga-DOTA-NOC have been extensively used in clinical studies, demonstrating fast pharmacokinetics, short scanning time, low radiation dose, and high sensitivity, resolution, detection rate, and image contrast, as well as the possibility for quantification (Kowalski et al. 2003; Baum et al. 2008; Henze et al. 2001; Hofmann et al. 2001; Velikyan et al. 2010; Hofmann et al. 2004; Koukouraki et al. 2006; Gabriel et al. 2007; Henze et al. 2004; Dimitrakopoulou-Strauss et al. 2006; Koukouraki et al. 2006; Kayani et al. 2008; Luboldt et al. 2010; Tan and Goh 2010; Ambrosini et al. 2010; Lincke et al. 2009; Rominger et al. 2010). Personalized diagnosis using these tracers is becoming a standard for selection of patient therapeutic management (Maecke et al. 2005; Henze et al. 2001; Gabriel et al. 2010; Putzer et al. 2010; Modlin et al. 2008; Win et al. 2007; Srirajaskanthan et al. 2010; Putzer et al. 2009; Milker-Zabel et al. 2006; Gehler et al. 2009; Prasad and Baum 2010; Ambrosini et al. 2010; Campana et al. 2010; Frilling et al. 2010; Versari et al. 2010; Van Riet et al. 2009; Ambrosini et al. 2008; Sainz-Esteban et al. 2010; Ambrosini et al. 2011; Traub-Weidinger et al. 2010; Boss et al. 2010; Henze et al. 2005; Conry et al. 2010). The vast experience of the clinical studies has been summarized in the form of guidelines for the assistance of nuclear medicine physicians in examination protocols as well as in interpretation and reporting of results (Virgolini et al. 2010; Janson et al. 2010).

Various ⁶⁸Ga-labeled agents for imaging of the human epidermal growth factor receptor family overexpressed in malignant tumors such as breast, ovary, lung, colorectal, and urothelial have been developed and investigated (Yarden and Sliwkowski 2001; Walker and Dearing 1999; Witton et al. 2003; Hirsch et al. 2003; Neal and Mellon 1992; Vosjan et al. 2011). Thus, 53-amino-acid residue EGFR natural ligand (Velikyan et al. 2005; Sandstrom et al. 2011; Velikyan et al. 2006), 58-amino-acid residue Affibody^® ligands (Tolmachev et al. 2010; Tolmachev and Orlova 2009; Ahlgren and Tolmachev 2010; Tolmachev et al. 2009), antibody fragments (Vosjan et al. 2011; Smith-Jones et al. 2004; Smith-Jones et al. 2006), two-helix Affibody derivative (Ren et al. 2009), as well as tyrosine kinase inhibitor (Theeraladanon et al. 2010) have been thoroughly evaluated in vitro and in vivo in xenograft animal models.

The ligand hEGF was conjugated with DOTA (Velikyan et al. 2005; Velikyan et al. 2006) and NOTA (Sandstrom et al. 2011), and the resulting conjugates were labeled with ⁶⁸Ga and ⁶⁷Ga, respectively, under microwave heating and room temperature. The biological activity maintenance of the tracers was thoroughly studied by performing cell-binding assays, biodistribution studies, and microPET imaging in tumor-bearing mice (Fig. 5, left). [⁶⁸Ga]Ga-DOTA-hEGF demonstrated high affinity and fast pharmacokinetic (Fig. 5, right) compatible with application for in vivo imaging of breast or non-small cell lung cancer. However, imaging of head and neck squamous cell carcinoma would be challenging due to the high physiological expression of EGFRs particularly in salivary glands, thus resulting in high background uptake and masking of the signal from tumor cells. Blocking of uptake in healthy organs was necessary to decrease the background and improve the contrast. This could be achieved by pre-injection of nonlabeled EGF ligand. However, the required high amount of the latter would stimulate growth of cancer cells. To avoid the pharmacological effect, anti-EGFR Affibody^® ligands [Z_EGFR:1907, (Z_EGFR:1907)₂ or (Z_EGFR:955)₂] were used for saturation of the physiologically expressed EGFR prior to administration of the tracer molecule based on hEGF ligand ([⁶⁷Ga]Ga-NOTA-Bn-NCS-hEGF) (Sandstrom et al. 2011). The dimeric Affibody molecule (Z_EGFR:1907)₂ exhibited the best results, and the tumor-to-organ ratio in liver, salivary glands, and colon was improved in nude mice bearing UTSCC-7 cell xenograft.

⁶⁸Ga-labeling of DOTA-conjugated Affibody^® molecules can be accomplished using either conventional or microwave heating without compromising the receptor binding capability (Velikyan 2005; Tolmachev et al. 2010). The influence of radiometal exchange on the ligand in vivo distribution was studied in a dual experiment, allowing direct comparison of ⁶⁸Ga- and ¹¹¹In-labeled DOTA-Z_{HER2:342-pep2} (ABY-002). Co-injection of the tracers provided uniform conditions for the biology validation experiments, eliminated the variation that might originate from individual differences amongst animals, and decreased the use of animals. Both tracers demonstrated specific uptake in vivo in SKOV-3 xenografts in mice (Figs. 6, 7). However, due to the faster fade of the ⁶⁸Ga-based counterpart from blood, lungs, gastrointestinal tract, and muscle, the tumor-to-organ ratio for this tracer was higher, thus resulting in better contrast (Tolmachev et al. 2010). Metastatic breast cancer was successfully visualized in clinical patient examination using [⁶⁸Ga]Ga-ABY-002 (Baum et al. 2010). The agent was well tolerated and rapidly cleared from blood, providing high image contrast. This was found to be valuable specifically for HER2 status of metastases not amenable to biopsy. To accelerate clearance, the size of the Affibody^® molecule was decreased to the two-helix molecule ([⁶⁸Ga]Ga-DOTA-MUT-DS) (Ren et al. 2009). Although the clearance became faster, this modification resulted in a drop of the affinity and tumor uptake in SKOV3 xenografts.

Imaging of integrin and vascular endothelial growth factor (VEGF) receptors overexpressed in tumors and ischemic injuries with extensive formation of new capillaries is an important means not only for diagnosis and monitoring response to therapy but also for antiangiogenic drug development. A ligand for VEGFR, single-chain VEGF (scVEGF), which is a functionally active single-chain version of VEGF (Backer et al. 2007, 2008), was conjugated to PEG linkers in order to adjust the pharmacokinetics and to couple to DOTA- or NOTA-based bifunctional chelators for efficient labeling with ⁶⁸Ga (Blom et al. 2011). The chelators were conjugated to Cys-tag in scVEGF via bifunctional PEG linkers of various lengths (2.0, 3.4, and 5.0 kDa), and the impact of these modifications on the radiolabeling efficiency and functional activity of the ligand was assessed. The labeling efficiency did not depend on the PEG linker length and was comparable for both DOTA and NOTA chelating moiety at elevated temperature provided by either conventional or microwave heating. However, NOTA offered the additional advantage of labeling at ambient temperature, providing mild labeling conditions for the relatively large sc-VEGF (~28 kDa) and the possibility for cold kit-type tracer production. An in vitro binding assay using multicellular spheroids of 293/KDR cell line confirmed the biological functionality of the scVEGF-based tracers. Accumulation of [⁶⁸Ga]Ga-NOTA-PEG-scVEGF could be detected in vivo in mouse 293/KDR xenografts. High kidney uptake was characteristic for all analogs.

An extensive number of peptide ligands with an exposed arginine-glycine-aspartic acid (RGD) sequence for binding to α_vβ₃ integrin receptors have been developed during the last two decades with the objective of visualizing angiogenesis and improving related drug development, diagnosis, and therapy monitoring (Dijkgraaf and Boerman 2009; Bergstrom et al. 2003; Garner and Lappin 2006; Chen et al. 2004; Haubner et al. 2004; Janssen et al. 2002; Liu 2006; Decristoforo et al. 2008; Indrevoll et al. 2006; Haukkala et al. 2009; Liu et al. 2009; Jeong et al. 2008; Li et al. 2008; Dijkgraaf et al. 2011). Specific radioactivity of a DOTA-containing analog could be increased considerably using microwave heating (Velikyan et al. 2004; Långström et al. 2008). Two bioconjugates with the same constitution but dislocation of a few amino acids with the aim of creating RGD sequence in one counterpart and DGF sequence in the other (negative control) were designed with the aim of demonstrating the necessity of the RGD motif for specific binding to α_vβ₃ integrin receptors. The distribution of these analogs was studied in primates using PET/CT imaging (unpublished data). Increased accumulation of the RGD counterpart was detected in the wall of the uterus, probably indicating neovascularization, and most importantly it could partly be blocked by co-injection of excess cold analog, indicating binding specificity. Uptake of the analogs in other tissues was low.

Another class of biological macromolecules for targeted imaging is antisense oligonucleotides, which can be considered for in vivo imaging of gene expression and tumors that express ras oncogene point mutations (Tavitian 2005; Lendvai et al. 2009). An antisense oligonucleotide is a short, synthetic nucleic acid that may selectively hybridize with its complementary sequence in messenger RNA (mRNA). A number of various analogs with modifications in backbone, furanose ring moiety, as well as 3′- and 5′-end nucleotides for subsequent conjugation with bifunctional chelators were designed (Velikyan 2005; Lendvai et al. 2005, 2008; Velikyan et al. 2004). Labeling of oligonucleotides up to 9.8 kDa with ⁶⁸Ga was performed under microwave heating. Tracers based on phosphodiester, phosphorothioate, 2′-O-methyl phosphodiester, locked nucleic acid (LNA), LNA-DNA mixmer (LNA and DNA nucleotides in alternation along the sequence), and peptide nucleic acid were thoroughly assessed in vitro and in vivo in rat (Lendvai et al. 2005, 2008, 2009; Roivainen et al. 2004; Velikyan et al. 2004). The hybridization capability of the analogs was controlled in situ, where the concentration of the ⁶⁸Ga-labeled antisense oligonucleotide was kept constant while the concentration of sense was gradually increased, resulting in a gradual increase of hybrid formation (Fig. 8). The radioactivity organ distribution pattern varied considerably depending on the oligonucleotide modification. Uptake in nonhybridization specific tissue was most probably mediated by scavenger receptors (Lendvai et al. 2009). In vivo tumor accumulation of ⁶⁸Ga-labeled 17-mer antisense phosphorothioate oligonucleotide was observed in rat model bearing xenografts with activated K-ras point mutation mRNA (Fig. 9) (Roivainen et al. 2004). Radioactivity accumulation was not detected in the negative control.