**1. Introduction**

Positron emission tomography (PET) is a nuclear medicine imaging technique for the non-invasive quantitative measurement of specific biochemical, physiological, and pharmacological processes in vivo [1]. PET is useful in the diagnosis and staging of neurological, cardiovascular, and various oncology-based diseases [2]. PET imaging is achieved by administering a patient with a PET radiopharmaceutical which will localize into organs and/or tissues that express the desired biological target. Once localized, the distribution of the PET radiopharmaceutical throughout the body can be imaged with a PET scanner and a diagnosis can be made. PET radiopharmaceuticals are biologically active molecules that are labeled with positron-emitting radionuclides such as fluorine-18, gallium-68, or copper-64. A key component of a PET radiopharmaceutical is the targeting entity, which is designed to possess a pharmacophore that has high affinity and specificity towards a desired biological target present in an organ and/or tissue that is associated with a specific disease or malignancy [3]. The targeting entities of radiopharmaceuticals were initially developed as biologically active small molecules, as is the case for the most widely used PET radiopharmaceutical [18F]fluorodeoxyglucose [4]. However, in recent years there has been a rapid development in the use of targeting entities developed from biologics, such as peptides, proteins, antibodies, and antibody fragments for the use as PET radiopharmaceuticals [5–7].

#### *Peptides as Radiopharmaceuticals*

The structure of a peptide-based radiopharmaceutical (Figure 1) typically contains the following components: a peptide to act as the targeting entity, a linker, a radionuclide bearing moiety, and a PET radionuclide. The linker is sometimes an optional component of a radiopharmaceutical that is incorporated to facilitate the conjugation of the targeting peptide and the radionuclide bearing moiety, and/or improve its pharmacokinetics such as by increasing metabolic stability or manipulating biodistribution [8–11]. The linker can also be used as a spacer to distance bulky portions of a radiopharmaceutical, such as chelators from the bioactive portions, to reduce steric interference and maintain high binding affinity [12].

**Figure 1.** The structural components of a peptide-based positron emission tomography (PET) radiopharmaceuticals.

There are many advantages offered by utilizing peptides as the targeting entity in radiopharmaceuticals, especially in the field of oncology. These peptide-based radiopharmaceuticals can take the form of radiopharmaceuticals for the diagnosis of diseases by imaging the biological target associated with the disease in specific tissues, or as radiotherapeutics for the treatment of cancers by subjecting the tissue to localized ionizing radiation. Furthermore, the opportunity to exploit the same targeting agent with either an imaging or therapeutic radionuclide has given rise to the field of 'Theranostics' in nuclear medicine [13].

A key advantage of peptide-receptor targeting peptide-based radiopharmaceuticals is the higher density of peptide receptors expressed on tumor cells than in normal tissues, thus specific receptor-binding radiolabeled peptides can be designed to enable the efficient visualization and treatment of various tumors [14]. Due to the relatively small size of peptides compared to other biologics, such as proteins and antibodies, peptides often exhibit rapid pharmacokinetics, with the ability to efficiently penetrate tumors, fast clearance from the bloodstream and non-target tissues, and are not immunogenic [5,14–16]. Peptides can also be readily synthesized using conventional peptide synthesizers, and any desired modifications to the structure can be easily engineered by making the appropriate changes to the peptide sequence during synthesis and/or by adding other structural modifications after synthesis [9,17,18].

Consequently, the last 20 years have seen an explosive growth in the development of radiolabeled peptides for targeted diagnostic imaging and therapy. While radiolabeled peptides have been applied to various molecular imaging modalities that use nuclear probes, such as scintigraphy and single-photon emission computed tomography (SPECT), the superior image quality and quantitative data available from PET have resulted in a significant amount of research being devoted to the development of PET radiolabeled peptides [19]. Recently, [68Ga]DOTATATE, also known as NETSPOT® (Figure 2), was the first radiolabeled peptide for PET imaging to receive regulatory approval from the Food and Drug Administration (FDA) [20].

**Figure 2.** Structure of [68Ga]DOTATATE (NETSPOT®).

Despite the advantages offered by peptides, there are several challenges inherent to the use of peptides in drug design and development, including for imaging and therapy. The most significant of these is that unmodified peptides often possess prohibitively short half-lives in vivo, primarily due to rapid proteolytic degradation in the blood, liver, and kidneys by endogenous proteases [21]. This liability results in a short duration of in vivo activity, poor bioavailability, and has significantly limited their application in drug development [22].

#### **2. Challenges Faced by Peptide-Based Radiopharmaceuticals In Vivo**

The rate at which a drug is metabolized and removed from the body determines its biological half-life [23]. In the case of radioactive drugs, such as PET radiopharmaceuticals, the physical half-life is determined by the incorporated radionuclide [23]. Thus, to achieve optimal results, the localization process of a radiopharmaceutical has to be fast relative to its biological and physical half-lives, such that the drug will localize to the target with adequate time for imaging or therapy before degrading below an effective activity and/or concentration [24]. In the case of peptide-based radiopharmaceuticals, the two most significant forms of degradation in vivo that impact the efficacy of the drug are loss of the radionuclide and degradation of the conjugated peptide. The premature degradation of peptide-based radiopharmaceuticals in vivo is of pressing concern since these drugs are typically administered in doses that are significantly smaller than conventional drugs and are therefore especially vulnerable to having their efficacy significantly disrupted by any amount of degradation in vivo.

#### *2.1. Loss of the Radionuclide*

Many radiopharmaceuticals are labeled with radiometals (e.g., technetium-99m, gallium-68, and copper-64). Ensuring that the radiometal is not lost from the radiopharmaceutical in vivo is of critical concern when developing these drugs as the free radiometal may exhibit high toxicity and cause significant damage to the body [25]. In the case of fluorine-18 labeled radiopharmaceuticals, in vivo radiodefluorination results in the release of free [18F]fluoride ions that can readily accumulate into the calcium-rich fluorophilic bones of the body [26]. Radiodefluorination and in vivo metabolism of [18F]radiopharmaceuticals also present major challenges to imaging studies as non-specific uptake of free [18F]fluoride ions and [18F]metabolites can lead to a degradation of the signal to noise ratio [27]. Further information on the radiodemetallation and radiodefluorination of radiopharmaceuticals, and the strategies that have been applied to mitigate these issues, are beyond the scope of this review but have been thoroughly reviewed elsewhere [28–30].

#### *2.2. Degradation of the Peptide*

The major challenge of using peptides in the active component of a pharmaceutical is that naturally occurring peptides are usually rapidly degraded in vivo [21,22]. Compared to other biologics, such as proteins and antibodies, peptides are generally more susceptible to enzymatic and/or chemical degradation. One of the key reasons a peptide sequence can also be susceptible to proteolytic degradation is due to its backbone containing a recognition motif for an endogenous protease [31]. In addition to proteolytic degradation, peptide bonds can also undergo spontaneous degradation under physiological conditions when particularly labile sequence motifs are present [32].

In peptide-based radiopharmaceuticals, degradation of the conjugated peptide will significantly disrupt its ability to localize to the target tissue, and the subsequent radioactive metabolites will undergo non-specific binding to other tissues and/or be rapidly cleared from the body. In the case of radiopharmaceuticals, this will reduce imaging sensitivity due to increasing background radiation [33]. In radiotherapeutics, this can result in insufficient irradiation of the target tissue while increasing the irradiation of non-target tissues [34,35].

Another point to consider is that amide bonds are often used to conjugate the radionuclide bearing moiety to the biomolecule or linker and these bonds are also susceptible to the same degradation pathways as peptide bonds via proteases and amidases [36]. As a result of these challenges, most peptides for use in radiopharmaceuticals are synthetically modified to minimize metabolic degradation in vivo [15,37].

#### **3. Increasing the In Vivo Stability of Peptide-Based Radiopharmaceuticals**

The peptide amide bond represents the central repeating structural element of peptides and proteins. This bond possesses partial double bond character, which is one of the key attributes that contributes to the rigidity of peptide chains. Its ability to form hydrogen bonds also makes the peptide bond play a crucial role in its recognition by and interactions with other proteins. Normally, the peptide amide bond is stable to hydrolysis, requiring harsh conditions involving concentrated acids or bases at increased temperatures [38,39]. However, the peptide bond can be readily cleaved under mild conditions at or even below room temperature in the presence of an appropriate protease or peptidase [39,40].

Peptidases can either be classified as exopeptidases, which specifically hydrolyze the *C*- or *N*-termini of a peptide, or as endopeptidases which are capable of hydrolyzing amide bonds within a peptide [41,42]. Mechanistically, hydrolysis of an amide bond with a peptidase occurs via a nucleophilic attack at the carbonyl carbon of the amide bond (Figure 3), with its pathway dependent on the amino acids present in the peptidase's active site [43]. Peptidases that have nucleophilic amino acids residues such as cysteine and serine in their active site, can attack the carbonyl carbon of the amide/peptide bond and form an acylated enzyme (as a thioester for cysteine or ester for serine), which is more vulnerable to attack by water than the original peptide bond (Figure 3A). Often these peptidases also have histidine and aspartic acid or glutamic acid within the active site, as a 'catalytic triad' [43,44]. With peptidases that have amino acid residues such as aspartate/aspartic acid or glutamate/glutamic acid (but no serine or cysteine) in their active site, these residues directly assist a water molecule in its nucleophilic attack of the carbonyl carbon of the amide bond, leading to direct hydrolysis of the amide bond (Figure 3B) [43].

**Figure 3.** General mechanisms of hydrolysis via a peptidase (**A**) with nucleophilic amino acids; (**B**) with acidic amino acid residues [43,44].

Several strategies have been developed to synthesize peptide analogues in which vulnerable peptide bonds are either modified or obscured such that they are no longer targeted by proteolytic enzymes. The goal of this is to generate metabolically stable peptide analogues that maintain the biological activity of the original peptide. This review is focused on strategies to enhance metabolic stability of PET peptide-based radiopharmaceuticals. It includes modifications of the *C*- and/or *N*-termini, introduction of d- or other unnatural amino acids, backbone modification, PEGylation and alkyl chain incorporation, cyclization, and peptide bond substitution. While some of the examples discussed in this review have only been applied to SPECT radiopharmaceuticals, radiotherapeutics, or non-radioactive peptide-based pharmaceuticals, the strategies are applicable also to PET peptide-based radiopharmaceuticals.
