**Abstract**

Aptamers are single-stranded DNA or RNA that can mimic the functional properties of monoclonal antibodies. Aptamers have high affinity and specificity for their target molecules, which can make them a promising alternative to therapeutic antibodies or peptide ligands. However, many aptamer drug candidates in clinical development have been discontinued due to suboptimal metabolic stabilities and pharmacokinetics. To address these issues, chemical modification can be used to enhance the metabolic stability and prolong the half-life of aptamer candidates. The chapter reviewed published data regarding the metabolic stability and pharmacokinetics of aptamer drug candidates from preclinical and clinical studies. The benefits and possible shortcomings of current modification strategies used in these aptamers were briefly discussed.

**Keywords:** metabolic stability, pharmacokinetics, aptamer, chemical modification, renal clearance

### **1. Introduction**

Aptamers are single-stranded DNA or RNA oligonucleotide-based synthetic molecules that can replicate the functional features of monoclonal antibodies. Nucleic acid aptamers are generally screened from a library of random nucleic acids utilizing systematic evolution of ligands by exponential enrichment (SELEX) technology [1]. To effectively create an aptamer against a given target molecule, the SELEX procedure requires multiple phases. The aptamer and desired target molecule are incubated to initiate the binding affinity process, which is followed by the division of bound and unbound sequences. After that, binding sequences are amplified by PCR, and ssDNA is extracted to start a new cycle [2, 3]. As a result, aptamers have a high affinity and selectivity for a wide range of target molecules, including peptides, proteins, tiny compounds, and even living cells. Because inhibitory aptamers that affect the activity of pathogenic target proteins may be utilized as therapeutic agents directly, they are a viable alternative to therapeutic antibodies or peptide ligands [4]. Aptamers

#### **Figure 1.**

*The common strategies in the chemical modifications of nucleic acid aptamers and their purposes.*

have several benefits over antibodies, including ease of synthesis, reduced time and cost, lesser immunogenicity, greater stability, and superior refold ability. As a result, aptamers are prospective replacements for homologous antibodies. The United States Food and Drug Administration (FDA) granted approval for the initial aptamer drug (Macugen®) to treat wet-form neovascular age-related macular degeneration (AMD) in 2004 [5, 6]. Several therapeutic aptamers are currently undergoing clinical trials in phases II and III.

However, aptamer candidates' druggability can be considerably impacted by their metabolic stability and pharmacokinetics. Indeed, due to inadequate qualities in these domains, certain aptamers under clinical development have been abandoned. The renal filtration cut-off value is 30–50 kDa. Native nucleic acid aptamers face challenges *in vivo* due to their susceptibility to nuclease-mediated cleavage and rapid renal filtration, primarily attributed to the abundance of serum nucleases and their relatively low molecular weight (20 kDa). Consequently, these aptamers are rapidly eliminated from the body, leading to a short biological half-life. These are key issues that are impeding the clinical translation of the nucleic acid aptamer. Chemical modification can be utilized to improve the metabolic stability and half-life of aptamer candidates to solve these difficulties. To begin, changes in metabolic stability may increase nucleic acid aptamer formation while increasing nuclease resistance. Second, long-term alterations might permit nucleic acid aptamer boosting molecular weight substantially over the renal filtration cut-off level (**Figure 1**). The chapter reviewed published data regarding the metabolic stability and pharmacokinetics of aptamer drug candidates from preclinical and clinical studies. The advantages and disadvantages of current modification procedures employed in these aptamers were briefly explored.

## **2. Strategies for enhancing metabolic stability of aptamers**

Modification tactics for improving metabolic stability of aptamer drug candidates may be carried out in two ways: pre-SELEX chemical modifications and

#### *The Modification Strategies for Enhancing the Metabolic Stabilities and Pharmacokinetics… DOI: http://dx.doi.org/10.5772/intechopen.112756*

post-SELEX chemical modifications. Pre-SELEX alterations mostly entail chemical changes that are critical for the aptamer's functions [6]. Nucleobase modifications and genetic alphabet extension are typical examples. The goal of adding these new chemical moieties or bases to aptamers is to improve their functionality and allow them to engage with additional targets. These changes immediately affect the threedimensional structure of aptamers [7]. Aptamer activity may be fully eliminated by adding these chemical groups following SELEX. As a result, pre-SELEX alteration is the most effective strategy to minimize activity loss. The fundamental disadvantage of this method is that the alteration might impair the nucleotide's capacity to act as a substrate for DNA or RNA polymerase [8]. During solid-phase chemical synthesis, changes at multiple sites are added to preselected aptamers in post-SELEX techniques for the best performances such as high affinity, high stability, and high specificity. Because aptamer affinity/specificity and function are structure-dependent, post-SELEX alteration may modify the intrinsic characteristics and folding structures of the original aptamers, compromising binding affinity [9]. As a result, alterations must be properly tailored to the intended functions [10]. Unfortunately, general guidelines do not exist for all aptamers, and tedious evaluation/optimization is frequently required [11]. The following are some of the most prevalent nucleic acid aptamer modifications:

### **2.1 Nucleobases modifications**

The research on SELEX using unnatural nucleobases has seen rapid growth in recent years, with two main categories of efforts: (1) creating unnatural base pair systems that are independent of Watson-Crick base pairs and (2) integrating peptidelike functional groups into native nucleobases [12]. However, a significant challenge in using unnatural nucleobases in SELEX is the need for encoding and decoding these nucleotides throughout the selection process, which often requires compatibility with polymerases. Ensuring high accuracy in base pairing selection with or between modified bases during the encoding and decoding stages is another obstacle in nucleobase modifications. To overcome these challenges and expand the range of nucleobase changes available for in vitro aptamer selection, researchers have developed several innovative strategies.

#### **2.2 Ribose modifications**

The creation of medicinal antisense oligonucleotides spurred early advances in chemical modifications of aptamers. These changes were made largely to improve resistance to nuclease-mediated degradation [13]. Initially, changes were introduced at the 2′-position of the ribose sugar unit. Modifications at all nucleotides, on the other hand, are seldom tolerated since a sugar alteration reduces aptamer activity [14]. Point-by-point changes and activity testing take time and money. As a result, including changed nucleotides in the SELEX procedure should be investigated. The presence of a polymerase that can be modified is the most significant aspect. Nowadays, replacing fluorine (2′-F) (**Figure 2a**), methoxy (2′-OMe) (**Figure 2b**), or amino (2′-NH2) (**Figure 2c**) groups for the 2′-hydroxy residue of RNA greatly improves aptamer stability against nuclease degradation [15].

2′-Aminopyrimidines were utilized in the first ribose-modified SELEX experiment [16]. Then, a change was made to the T7 RNA polymerase to improve substrate compatibility. It has been demonstrated that 2′-fluoro and 2′-deoxypyrimidine are

**Figure 2.**

*Chemical structures of modifications for the improvement of nuclease resistance of aptamers: (a) 2*′*-fluoro-RNA (2*′*-F); (b) 2*′*-methoxy-RNA (2*′*-OMe); (c) 2*′*-amino-RNA (2*′*-NH2); (d) Thiophosphorus-DNA/RNA; (e) Phosphorothioate-DNA/RNA.*

incorporated by T7 RNA polymerase with the Y639F mutation [17–19]. Pyrimidine alterations have frequently been used as a starting point for aptamer synthesis because they prevent RNase A-mediated degradation [5]. However, in contrast to 2′-fluoropyrimidine, 2′-aminopyrimidine is seldom used in the present SELEX technique due to reduced coupling efficiencies in chemical synthesis, a predilection for the C2′-endo ribose conformation, and a detrimental influence on base pairing stability [20].

Pegaptanib (Macugen®), the sole FDA-approved oligonucleotide-based medicine on the market, exemplifies a sugar-modified aptamer. It was developed *in vitro* using a combination of unmodified purine nucleotides and 2′-F-pyrimidine nucleotides [9]. The remaining unmodified purine nucleotides were converted to 2′-OMe units through solid-phase synthesis. Additionally, two 2'-F-modified thrombin-binding aptamers (PG13 and PG14) showed a fourfold increase in thrombin-binding affinity and up to a sevenfold improvement in nuclease resistance. In 10% FBS, the improved aptamer's G-quadruplex stability was enhanced up to 48-fold [21]. Lin et al. created a human neutrophil elastase (HNE) aptamer with a 2'-NH2 group, demonstrating high binding affinity and 10-fold increased stability in human serum and urine compared to the original aptamer [22]. It has also been found that basic fibroblast growth factor (bFGF) can be effectively inhibited by RNA ligands modified with 2′-amino-2′ deoxypyrimidine. When incubated in human serum, 2′-aminopyrimidine-modified RNA aptamers are much more stable than naturally unmodified RNA aptamers (>1000-folds) [23]. In another study by Green et al., Vascular permeability factor/ vascular endothelial growth factor (VPF/VEGF)-binding RNA and DNA aptamers demonstrated a good binding affinity and nuclease resistance [24].

#### **2.3 Phosphate modifications**

Modifications to the phosphate portion of aptamer are thought to be a significant technique for aiding aptamer's resistance to nuclease *in vivo*. Sulfur can replace two nonbridging oxygens to generate thiophosphorus (PS) (**Figure 2d**) and phosphorodithioate (PS2) (**Figure 2e**). Solid-phase synthesis had developed PS-modified DNA. The vulcanization phase replaces the oxidation step in the PS DNA synthesis. Beaucage reagent is used for vulcanizing a recently generated phosphite triester ester. Two isomers would be produced as a result of this reaction [25]. Through high-performance liquid chromatography (HPLC), a single PS modification can be isolated, and synthetic methods can produce several pure diastereomers of PS modifications [26]. The PS modification's ability to enhance the affinity between aptamers and targets can be illustrated with the following examples.

*The Modification Strategies for Enhancing the Metabolic Stabilities and Pharmacokinetics… DOI: http://dx.doi.org/10.5772/intechopen.112756*

Wu et al. created two DNA aptamers, XQ-2 and a shortened variant termed XQ-2d, to target Pancreatic Ductal Adenocarcinoma (PDAC), the most frequent pancreatic adenocarcinoma [27]. They made phosphorothioate and 2'-OMe derivatives of XQ-2d to boost serum stability; however, they discovered that the thioaptamer form of XQ-2d had decreased binding to PL45 cells, while the 2'-OMe version did not [28]. Chen et al. investigated a 50-polyethylene glycol (PEG)-modified form of Adipo8 with phosphonothioate linkages placed right after the first base and just before the last base [29]. When examined in tissue culture and *in vivo*, the addition of these two thioate connections boosted serum stability by a tiny but statistically significant amount. Because this aptamer can identify white adipocytes from brown adipocytes and preadipocytes, it might be used to provide adipocyte-specific treatment [30]. PS2 alterations, which replace both nonbridging oxygen atoms with sulfur, have been incorporated into RNA aptamers chosen for binding to VEGF165. PS2 alterations increase nuclease resistance and aptamer stability in human serum [31].

#### **2.4 Isomerized nucleoside modifications**

#### *2.4.1 Spiegelmers*

A method for altering aptamers is the creation of "mirror-image" aptamers, also known as spiegelmers (**Figure 3a**). Spiegelmers counteract nucleases' stereoselectivity by inverting the chirality centers inside sugar molecules, resulting in a mirror image of wild-type DNA or RNA. This structural change makes spiegelmers more stable *in vivo*, as they evade recognition by nucleases and the immune system [32]. Traditionally, spiegelmer selection involved a two-step process, where researchers first created a mirror-imaged target and selected D-aptamers using standard SELEX. The chosen D-aptamer was then converted into the corresponding spiegelmer [33]. However, recent advancements in molecular systems capable of replicating and transcribing L-nucleotides have simplified and reduced the cost of the selection process [34].

#### **Figure 3.**

*Chemical structures of second-generation modifications for the improvement of nuclease resistance of aptamers: (a) Spiegelmers; (b) d−/l-isonucleoside; (c) inverted thymidine; (d) circular Aptamers.*

Spiegelmers have found applications in cancer research and therapy. For example, Roccaro et al. developed a spiegelmer targeting SDF-1 to inhibit bone marrow metastasis of multiple myeloma cells [35]. In cell and animal tests, the spiegelmer effectively neutralized SDF-1, demonstrating its potential to inhibit metastasis. NOX-A12, an SDF-1 binding spiegelmer, is currently undergoing phase II clinical trials, showing an 86% response rate against relapsed/refractory chronic lymphocytic leukemia. However, further research is needed before NOX-A12 can be approved as a therapeutically viable drug [33].

## *2.4.2 d−/l-Isonucleoside modifications*

Isonucleosides are nucleoside analogs where the bases are repositioned to the nucleoside's 2′ or 3′ position (**Figure 3b**). Oligonucleotides incorporating isonucleosides exhibit increased resistance to nuclease-mediated hydrolysis [36]. Yang et al. explored the potential of d−/l-isonucleosides as modifiers for three aptamers (TBA, GBI-10, and AS1411). The strategic incorporation of d−/l-isonucleosides, particularly in the loop regions, led to notable enhancements in spatial conformation stability and chemical robustness within the modified aptamers [37, 38]. Consequently, the modified aptamers exhibited significantly heightened resistance against biodegradation. Interestingly, modifications with L-isonucleosides had a more profound impact on enhancing the biological activity of the aptamers compared to changes with D-isonucleosides. These findings underscore the significance of isonucleoside chirality in influencing the functional characteristics of modified aptamers.

### *2.4.3 Inverted nucleoside modifications*

Oligodeoxynucleotides were commonly modified with inverted thymidine (5′-,3′-inverted T) (**Figure 3c**) to render them resistant to nucleases. Pegaptanib also has a 40 kDa poly (ethylene glycol) moiety at the 5′ end to help with renal clearance, as well as a 3′-3′-linked deoxythymidine residue to help with nuclease destruction. Despite these changes, pegaptanib retained an exceedingly high affinity for its VEGF165 target and demonstrated sustained in vitro [39] stability. Moreover, the antifactor IXa RNA aptamer RB06 is composed of unmodified purine nucleosides, 2′-F-pyrimidine nucleosides, 5′-terminal 40 kDa-PEG moiety, and 3′-terminal 3′-inverted deoxythymidine preserved excellent affinity to the target, high in vivo stability and robust anticoagulant efficacy [40].

#### **2.5 Nuclease-resistant circular Aptamers**

The creation of circular aptamers (**Figure 3d**), which solve the difficulty of metabolic instability, is a recent accomplishment in aptamer modification. Aptamers can avoid exonuclease degradation by connecting the 5′ and 3′ termini of nucleic acids to create a closed circular shape, resulting in increased resistance to nucleases [40]. King et al. created multivalent circular aptamers with anticoagulant activity, illustrating the power of cyclization in the creation of functional aptamers [41]. Cyclization enhances aptamer resistance to nucleases, increasing heat stability and guaranteeing structural homogeneity. Tan et al. reported the development of bivalent circular aptamers employing three aptamers that target live cancer cells (Sgc8, TD05, and XQ-2d) [42–44]. The cyclization technique provides an economical and practical approach to improving the stability and binding ability of aptamers, allowing them to *The Modification Strategies for Enhancing the Metabolic Stabilities and Pharmacokinetics… DOI: http://dx.doi.org/10.5772/intechopen.112756*

be used in diagnostic and therapy. The usage of circular aptamers is a viable option for increasing aptamer stability and functional qualities, bringing up new possibilities for their use in a variety of biological applications [45].
