**3.1 ASO's pharmacokinetics and toxicity**

The pharmacokinetics of ASOs has been extensively studied; they rapidly distribute to all tissues *in vivo* [55]. First and second-generation PTO ODs are broadly distributed to all peripheral tissues. The highest concentrations of ODs are found in the liver, kidney, spleen, lymph nodes and bone marrow with no measurable distribution to the brain [52]. Moreover, they not only distribute to tissues but also accumulate within cells in tissues [56]. The mechanism(s) by which ODs accumulate within cells following parenteral administration is currently unknown [57]. However, ODs that do not contain a PTO linkage appear to be rapidly excreted in the urine [58]. This difference in tissue distribution appears to be due largely to interactions with plasma proteins.

**21**

*Antisense Oligonucleotides, A Novel Developing Targeting Therapy*

(ALT), thrombocytopenia, and hyperglycemia [52].

delivery via the use of delivery vehicles.

*3.2.1 Mechanism of cellular uptake*

Although the clinical relevance of ASOs has been demonstrated, inefficient cellular uptake, both *in vitro* and *in vivo*, limit the efficacy of ASOs and has been a barrier to therapeutic development. Cellular uptake can be <2% of the dose resulting in too low ASO concentration at the active site for an effective and sustained outcome [22]. Indeed, many researches are conducted on the improvement of ASO

Cellular uptake refers to the combination of both OD membrane-binding and internalization. It is challenged by numerous barriers, most importantly, the lipophilic nature of the cell membrane, which makes the passage of these large anionic molecules complicated. Several barriers to cellular uptake exist such as the lipophilic cell membrane, through which these large, anionic molecules must pass to reach the

**3.2 ASO's delivery**

In summary, pharmacokinetic studies of PTO ASO demonstrate that they are well absorbed from parenteral sites, distribute broadly to all peripheral tissues, do not cross the blood-brain barrier (BBB), and are eliminated primarily by slow metabolism. Altogether, their pharmacokinetic properties depend on chemistry

Concerning ASO's toxicity, there are two broad categories of potential toxicities for ASOs; toxicities due to exaggerated pharmacology and toxicities due to nonantisense effects of the ASO. The former category of side effects results from the ASO binding to off-target RNA, producing an undesirable effect. The potential for such toxicities can be further minimized by vigilant selection of the drug target and homology searches against human genomic databases. The second category of potential toxicities (non-antisense effects) has been documented at higher doses of ASOs. In clinical trials PTO ASOs have proven to be safer than originally anticipated [31, 34, 35]. ASOs drugs principally induce mild-to-moderate toxicities related to the administered dose. *In vivo,* the two mains mechanism that induce acute toxicity through ASOs administration are for the first one the activation of the transient complement cascade and for the second one the inhibition of the clotting cascade. The increase in the clotting times is defined by a transient concentration-dependent escalation of activated partial thromboplastin times (aPTT) [59]. Another frequently occurring sub chronic toxicity is immune stimulation, manifested as splenomegaly, lymphoid hyperplasia and diffused multi-organ mixed mononuclear cell infiltrates [60]. This is due to an unmethylated cytosine-phosphorous-guanine (CpG) motif in the ASO sequence that can be recognized by Toll-like receptor (TLR)-9 in immune cells. This results in the release of cytokines, interleukin (IL)-6, IL-12, interferon (INF)-γ), B cell proliferation, antibody production and activation of T lymphocyte and natural killer (NK) cells [61]. In order to avoid the immune response, the cytosine of the sequences including CpG motif are substituted by methylated cytosine. Also, for preventing this side-effect, the sequences of ASOs drug can be designed without CpG motif. These both stimulatory immune responses are related to the sequence and the chemical modification of the ASOs [6]. Indeed, second-generation ASOs have been shown to have smaller immune stimulation that ASOs PTO. In addition, introduction of LNA into the PTO-ASO has been shown to reduce, and even eliminate, CpG dinucleotide-mediated immunostimulation [62, 63]. When ASO's plasma concentration is high, some toxicities have been observed, such as elevated liver enzyme such as aspartate aminotransferase (AST) and alanine amino-transferase

*DOI: http://dx.doi.org/10.5772/intechopen.82105*

rather than sequence.

*Antisense Therapy*

**Table 2.**

*Chemical modification characteristics.*

of the ribose [46]. LNA modifications improve significantly the ASO hybridization affinity towards mRNA target, due to the important increase in the thermal stability of the DNA/RNA heteroduplexes [47]. In addition, LNAs avoid nuclease degradation. As their ribose 2'-O position are modified, LNAs are not recruiting RNase H [48]. Nevertheless LNA nucleotides can be freely incorporated at the ends of RNA and DNA sequences to form chimeric oligonucleotides resulting in restoration of RNase H-mediated cleavage of mRNA. It has been shown that the chimeric LNA/ DNA/LNA gapmer with seven to 10 phosphorothioate-modified DNA central gaps flanked by three to four LNA nucleotides on both 5′-end and 3'ends induces efficient mRNA cleavage, because of their high target affinity and nuclease resistance [29]. In the first *in vivo* study reported, the LNA ASOs appeared to be nontoxic in the optimal dosage. Therefore, full LNA and gapmers LNA·DNA·LNA ASOs seem to offer an attractive set of properties, such as potent biological activity and apparent lack of acute toxicity, making it a promising antisense agents [49–51]. **Table 2**

Despite the large number of molecules being evaluated in the clinical trials, the clinical progress of ASOs had to face many challenges; indeed, poor pharmacokinetics [52], poor cell membrane permeation [53], and off-target effects [27]. Last decade, the development of oligonucleotide delivery through lipid or polymer systems has improved the cellular uptake and the pharmacokinetic behavior [54].

The pharmacokinetics of ASOs has been extensively studied; they rapidly distribute to all tissues *in vivo* [55]. First and second-generation PTO ODs are broadly distributed to all peripheral tissues. The highest concentrations of ODs are found in the liver, kidney, spleen, lymph nodes and bone marrow with no measurable distribution to the brain [52]. Moreover, they not only distribute to tissues but also accumulate within cells in tissues [56]. The mechanism(s) by which ODs accumulate within cells following parenteral administration is currently unknown [57]. However, ODs that do not contain a PTO linkage appear to be rapidly excreted in the urine [58]. This difference in tissue distribution appears to be due largely to

recapitulates the chemical modifications characteristics.

**3. ASO's pharmacokinetics, toxicity and delivery**

**3.1 ASO's pharmacokinetics and toxicity**

interactions with plasma proteins.

**20**

In summary, pharmacokinetic studies of PTO ASO demonstrate that they are well absorbed from parenteral sites, distribute broadly to all peripheral tissues, do not cross the blood-brain barrier (BBB), and are eliminated primarily by slow metabolism. Altogether, their pharmacokinetic properties depend on chemistry rather than sequence.

Concerning ASO's toxicity, there are two broad categories of potential toxicities for ASOs; toxicities due to exaggerated pharmacology and toxicities due to nonantisense effects of the ASO. The former category of side effects results from the ASO binding to off-target RNA, producing an undesirable effect. The potential for such toxicities can be further minimized by vigilant selection of the drug target and homology searches against human genomic databases. The second category of potential toxicities (non-antisense effects) has been documented at higher doses of ASOs. In clinical trials PTO ASOs have proven to be safer than originally anticipated [31, 34, 35]. ASOs drugs principally induce mild-to-moderate toxicities related to the administered dose. *In vivo,* the two mains mechanism that induce acute toxicity through ASOs administration are for the first one the activation of the transient complement cascade and for the second one the inhibition of the clotting cascade. The increase in the clotting times is defined by a transient concentration-dependent escalation of activated partial thromboplastin times (aPTT) [59]. Another frequently occurring sub chronic toxicity is immune stimulation, manifested as splenomegaly, lymphoid hyperplasia and diffused multi-organ mixed mononuclear cell infiltrates [60]. This is due to an unmethylated cytosine-phosphorous-guanine (CpG) motif in the ASO sequence that can be recognized by Toll-like receptor (TLR)-9 in immune cells. This results in the release of cytokines, interleukin (IL)-6, IL-12, interferon (INF)-γ), B cell proliferation, antibody production and activation of T lymphocyte and natural killer (NK) cells [61]. In order to avoid the immune response, the cytosine of the sequences including CpG motif are substituted by methylated cytosine. Also, for preventing this side-effect, the sequences of ASOs drug can be designed without CpG motif. These both stimulatory immune responses are related to the sequence and the chemical modification of the ASOs [6]. Indeed, second-generation ASOs have been shown to have smaller immune stimulation that ASOs PTO. In addition, introduction of LNA into the PTO-ASO has been shown to reduce, and even eliminate, CpG dinucleotide-mediated immunostimulation [62, 63]. When ASO's plasma concentration is high, some toxicities have been observed, such as elevated liver enzyme such as aspartate aminotransferase (AST) and alanine amino-transferase (ALT), thrombocytopenia, and hyperglycemia [52].
