The distinctive advantages of small radiopeptides over other biologically active molecules, such as proteins and antibodies, are summarized in Table 2. Receptors for peptides are often found in higher density on tumor cells than in normal tissues; hence specifically designed receptor-binding radiolabeled peptides could enable efficient visualization of tumors. Because of their small size, peptides usually exhibit rapid pharmacokinetics and good tumor targeting characteristics, with the ability to penetrate into tumors efficiently (Fani et al. 2012a, b; Langer and Beck-Sickinger 2001). As an advantage, the low molecular weight of the conjugate results in a fast diffusion rate. Peptides consist of a small number of amino acids (<50 amino acids) and can easily be prepared by standard solid-phase peptide synthesis (via convenient good manufacturing practices) and the desired pharmacokinetic characteristics can be molecularly engineered (by making appropriate changes in the peptide sequence) during synthesis and/or by adding a biomodifying molecule to the sequence (Antunes et al. 2007; Garcia et al. 2008). Automated peptide synthesizers are readily available for parallel synthesis procedures allowing the synthesis of peptide libraries in short time (Fani et al. 2012a). Peptides can also withstand harsher conditions for modification and labeling than antibodies, they are less likely to produce immunogenic response, and blood clearance and tumor uptake are faster. The faster clearance of non-target tissue leads to a better tumor-to-background ratio (better image contrast) and therefore to less irradiation of non-target organs (Fani et al. 2012b).

The binding affinity and the biodistribution are of major importance in the assessment of radiopharmaceuticals (Reubi et al. 1995). The first reflects the potential to fulfill its objective to target a receptor while the second is elementary for reaching the target and avoiding undesired radiation to non-target organs. Both are related to a major advantage of receptor-mediated peptide radiopharmaceuticals compared to, for example, conventional chemotherapeutics: the selectivity. Most receptors have high affinities for their ligands and are active at low nanomolar concentrations of the ligands. Therefore, radiopeptides with high specific radioactivity are crucial. Even small molar quantities of imaging agents may saturate a receptor, limiting the ability to visualize receptor expression and increasing the background due to non-specific binding. For biodistribution studies, tumor cells for which the bioconjugate is being developed are inoculated to animals (e.g., mice, rats) inducing the expression of a tumor. After administration of the radiopeptide, uptake in different organs, especially the receptor-positive organs and in the critical organs concerning toxicity effects may be determined.

For small size biomolecules, such as peptides the radioisotope label may significantly affect binding to the target receptor and in vivo pharmacokinetics. In this situation, the choices of radionuclide, labeling position, or location of the radiolabel may be important. The attachment of a bulky bifunctional chelator (BFC) to a relatively small peptide may lower also the binding affinity and biological potency. To reduce this negative influence, spacers between the peptide and the chelator may be introduced (Okarvi 2004; Fani et al. 2012a, b).

The biologic role of most receptors important in cancer is derived from their role in the tissue of cancer origin. In general, tumor receptors are expressed in the parent cell lineage and have an established physiologic function. For example, estrogen receptor expression is vital to the function of normal mammary gland epithelial cells. Many tumor receptors also play an important role in promoting carcinogenesis or tumor growth, as is the case for steroid receptors in breast and prostate cancer. The dependency on the receptor signaling pathway for tumor growth makes the receptor a suitable target for therapy, because interruption of the receptor-initiated signaling will result in a cessation of tumor growth and often tumor cell death. Thus, knowledge of the levels of receptor expression, which may vary significantly in different types of tumors and even in different sites in the same tumor, is required to infer the possibility that receptor-directed targeting will be effective (Mankoff et al. 2008).

One major problem associated with unmodified linear peptides is their often short biological half-life due to rapid proteolysis in plasma. As native peptides are degraded rapidly by endogenous peptidases and proteases before reaching the intended target, thus most peptides have to be modified synthetically to minimize rapid enzymatic degradation (Fani et al. 2012a; Behr et al. 2001). Great effort has been focused on developing metabolically stable peptides suitable for clinical use by performing appropriate molecular modifications, such as the use of D-amino acids for L-amino acids, the use of pseudo-peptide bonds, the inclusion of amino alcohols and the insertion of unnatural amino acids or amino acid residues with modified side-chains without compromising the receptor-binding affinity and biological activity of the peptide (Fani et al. 2012a). It is the specific amino acid sequence of a peptide and usually the nature and type of particular amino acid side-chains that determine resistance to enzymatic degradation. For example, native SST, which has a plasma half-life of ~2–3 min, but its molecularly modified synthetic peptide derivative, octreotide has a half-life of 1.5–2 h, making it suitable for clinical application (Mankoff et al. 2008).

Another issue related to radiolabeled peptides is often their high uptake and retention by the kidneys, which is of a concern, particularly for radiotherapy because of the potential nephrotoxicity (Teunissen et al. 2005; Virgolini et al. 2001; Valkema et al. 2005; Imhof et al. 2011). Though procedures have been developed and applied successfully in the clinics to manage nephrotoxicity that includes the infusion of a mixture of amino acids, such as lysine and arginine (Rolleman et al. 2003). There is a need to reduce kidney uptake and/or enhance renal clearance of the radiopeptide. Recently, it has been shown that the use of the cytoprotective drug amifostine (Rolleman et al. 2007a, b), and low doses of the plasma expander succinylated gelatin and gelatin-based gelofusine can inhibit the renal uptake of radiolabeled octreotide analogs (Vegt et al. 2006, van Eerd et al. 2006).

The main steps involved in developing a radiolabeled peptide for successful receptor targeting are as follows: (i) identification of the molecular target (receptor), with relevance to human disease (unmet medical need), and search for a lead peptide, which may be a natural or a synthetic peptide, (ii) receptor density, homogeneity, and incidence are important factors in predicting successful in vivo tumor receptor targeting, (iii) solid-phase peptide synthesis (SPPS) of a tumor targeting peptide or its derivatives. In general, the design of a peptide is based on the structural composition of the endogenous ligand (natural peptide ligand), which exhibits high affinity for the corresponding receptor. The natural peptide molecule is often structurally modified to produce a metabolically stabilized analog, which preserves most of the biological activity and receptor affinity of the original peptide, (iv) covalent attachment of a chelating agent or a prosthetic group to the peptide either directly or through a linker/spacer group. The attachment of the chelating moiety to the peptide sequence may be performed at the non-active end of the main chain, mostly the N-terminus of the molecule, (v) radiolabeling that allows high labeling efficiency and high specific radioactivity, (vi) in vitro characterization, such as the binding of a radiopeptide with tumor cells, determination of receptor affinity, internalization into the tumor cells, and dissociation from the tumor cells, (vii) in vivo evaluation to assess the biological behavior, biokinetics, and tumor targeting capacity of the radiopeptide in animal models. Many aspects should be considered for further development, such as the accumulation of radiopeptide in target and non-target tissues, the rate and extent of the clearance of radioactivity from the body, the excretory pathway, and in vivo stability of the radiopeptide. (viii) Finally, the radiolabeled peptides, which successfully passed all the preclinical tests, after toxicological studies and established radiopharmaceutical preparation, may enter clinical studies in humans (Ambrosini et al. 2011). Tumor receptor targeting poses unique challenges for the design and development of a peptide-based agent. From the design of a new peptide molecule until the use in the clinical settings is a long way and from a large number of developed radiopeptides only few meet the criteria of a radiopharmaceutical for clinical application. The peptide-based radiopharmaceuticals in the field of targeted radiotherapy with impact in patient care are the SST receptor targeting analogs.

It is worth mentioning here that common to all receptors is the interaction of a ligand and the receptor, in which specific binding of the ligand to the receptor results in downstream biochemical or physiologic changes. Ligands that cause physiological functional changes upon receptor binding, typically the naturally occurring ligands, are called “agonists”. Ligands that bind to the receptor and block the binding of agonists but that do not activate changes are known as “antagonists” (Mankoff et al. 2008). It is generally believed that receptor agonist radioligands are more suited for tumor targeting as they exhibit good receptor-mediated internalization into the tumor cell upon binding to the respective cell-surface receptors, thus promoting active accumulation in the target results in optimal visualization (Langer and Beck-Sickinger 2001). Interestingly, however, a recent study performed with two potent SST receptor-selective antagonists demonstrated that the high affinity SST receptor antagonists that poorly internalized into tumor cells can also be effective in terms of in vivo tumor uptake characteristics in animal models as compared to the corresponding agonists, which highly internalized into tumor cells. This observation which was made both for sst₂ and sst₃-selective SST analogs, demonstrates that the SST receptor antagonists are preferable to SST agonists for in vivo tumor targeting (Ginj et al. 2006). In another study, ⁶⁴Cu-CB-TE2A-sst₂-ANT, a SST antagonist was evaluated for in vivo PET imaging of sst₂-positive tumors and compared to ⁶⁴Cu-CB-TE2A-TATE. The radioantagonists showed somewhat better pharmacokinetics over receptor agonists (Wadas et al. 2008).

The most recent study on the newly developed sst₂-antagonist, namely LM3, demonstrated high and persistent tumor uptake of radiolabeled SST antagonists. Also, profound influence of the chelator and the radiometal was observed on the receptor-binding affinity of the radioconjugates (Fani et al. 2011). The first clinical study of SST antagonists confirmed the preclinical data as it showed higher tumor uptake of the antagonist ¹¹¹In-DOTA-sst₂-ANT compared to the agonist ¹¹¹In-DTPA⁰-octreotide and improved tumor-to-background contrast, in particular tumor-to-kidney ratio (Wild et al. 2011a, b).

The introduction of ¹¹¹In-DTPA⁰-octreotide (Fig. 1) for the diagnosis of SST receptor-positive tumors has opened the way to search for new receptor-based target-specific diagnostic and therapeutic peptides. The continued efforts have led to the research and development of several new peptide-based radiopharmaceuticals for the diagnosis and therapy of various human cancers as well as the discovery of several new chelating agents for peptide labeling with different therapeutic radionuclides (Fani et al. 2012a, b; Reubi et al. 2005).

Therapeutic radiopharmaceuticals are radiolabeled compounds specifically designed to deliver high radiation doses to receptor-specific disease sites (i.e., tumor lesions). The systemic noninvasive administration of radiopharmaceuticals that are designed for specific localization at tumor sites hold the great potential to treat disseminated disease. Most systemically administered therapeutic radiopharmaceuticals are small organic or inorganic compounds with definite structural composition. They can also be macromolecules, such as monoclonal antibodies or antibody fragments labeled with a therapeutic radionuclide. Ideally, the therapeutic radiopharmaceutical should localize at the tumor sites (even when their actual locations are unknown) in sufficient concentration to deliver a cytotoxic radiation dose to the tumor cells and clear efficiently from the blood and other normal tissues in order to minimize radiation damage to healthy tissues. In general, it was observed that radionuclide therapies have lower toxicities and longer term antitumor effects than conventional chemotherapy or external beam radiotherapy (Liu and Edwards 2001; Lee and Jeong 2012).

The utilization of radiometals in research and clinical practice has increased over the past 10 years and their radiochemistry has received renewed interest. One of the reasons is the development of medical radionuclide generators which produce positron emitting radiometals along with the tremendous advancement in medical imaging technology, such as the invention of PET/CT. But the main reason is the fast development of clinical targeted radiotherapy in nuclear oncology. In principle this technique can deliver high linear energy transfer (LET) radiation specifically to the tumor site. The search for an ideal therapeutic radionuclide for clinical use has been the driving force to produce new radionuclides with different half-lives and energy or even emitting different particles. Furthermore, efforts to produce some of the interesting lanthanides in carrier-free form appear to be successful and may lead to new therapeutic radionuclides. Of particular interest in this regard is the class of lanthanides including ⁹⁰Y³⁺; several of them can be produced in high-flux reactors. As they have similar chemical properties, their production and use in therapeutic radiopharmaceuticals would allow studying the influence of dose rate and β⁻ energy on tumor response as it can be assumed that the same radiolabeling chemistry can be applied to all lanthanide-based radiopharmaceuticals. This will most likely result in identical or at least very similar pharmacokinetic characteristics. (Fani et al. 2011b; Lee and Jeong 2012).