Oligonucleotide Drugs: Metabolism & Pharmacokinetics Studies

Oligonucleotide therapeutics have made great strides in recent years – with new drugs targeting particular diseases via RNAi and gene regulation. But the efficacy of oligonucleotide compounds depends upon their metabolism and PK. A clear understanding of these mechanisms is essential to the development of drug therapies with a greater bioavailability, specificity, and safety profile.

Oligonucleotide Drugs

Oligonucleotide drugs are drugs whose structure consists of short chains of nucleotides (10 - 30 bases), targeting RNA or DNA in order to control gene expression. They have become a revolutionary new therapeutic approach that can be applied to a variety of disorders including genetic disorders, viral infections, cancers and other rare conditions where traditional small molecules are not as effective. The biological activity of oligonucleotide drugs derives from the fact that they can uniquely bind to nucleic acids and influence gene expression, either transcriptionally or post-transcriptionally. There are multiple ways that oligonucleotide molecules work to have therapeutic effects, depending on the oligonucleotide and its target. With more breakthroughs in RNA therapy, oligonucleotide drugs hold great promise. Continued work on new oligonucleotide platforms, along with better delivery technologies, will help to extend oligonucleotide therapeutic potential.

Schematic representation of the basic mechanism of action of antisense oligonucleotide (ASO) drugs and RNA interference (RNAi). (Di, F.D.; et al, 2019)

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What is Drug Metabolism?

Drug metabolism refers to the biochemical alteration of pharmaceutical substances within the body. This process typically involves enzymes that modify the chemical structure of drugs to facilitate their elimination from the body. Drug metabolism is a crucial component of pharmacokinetics (PK) and influences the drug's absorption, distribution, metabolism, and excretion (ADME) characteristics. Drug metabolism is a critical determinant of drug action and pharmacokinetics. Several factors influence the metabolic fate of drugs, including genetics, age, liver function, and interactions with other drugs. Understanding drug metabolism is essential for drug development, as it affects the drug's:

• Bioavailability: The fraction of an administered dose that reaches the systemic circulation and is available to exert its effects.
• Duration of action: Metabolic processes help determine how long a drug remains active in the body.
• Toxicity: Some metabolites may be harmful or toxic, so understanding metabolism pathways helps in predicting adverse effects.

Drug metabolism can also influence the design of new therapies, particularly when developing drugs with specific metabolic profiles. For example, drugs can be designed to avoid certain metabolic pathways that might lead to toxic metabolites or to create prodrugs that require metabolic activation to become effective. In the context of oligonucleotide drugs, metabolism involves complex interactions between the drug molecules and cellular machinery, including nucleases, transporters, and enzymes. These metabolic processes impact the drug's efficacy, stability, and half-life, which ultimately affects its therapeutic potential.

Oligonucleotides Drug Metabolism

Oligonucleotide drugs undergo distinct metabolic pathways compared to traditional small-molecule drugs. Since these drugs are large, charged molecules, they are poorly absorbed via oral administration and typically require alternative delivery strategies. Once administered, oligonucleotides are subject to degradation by exonucleases and endonucleases, which cleave the phosphodiester bonds between nucleotides. These enzymatic processes can result in the rapid elimination of the therapeutic agent unless protected or modified. To overcome these challenges, chemical modifications are often incorporated into oligonucleotide drugs to enhance their stability and resistance to nucleases. Such modifications include modifications to the sugar backbone (e.g., 2'-O-methyl or 2'-fluoro substitutions), phosphorothioate linkages, and the incorporation of locked nucleic acids (LNAs). These modifications not only improve the stability of oligonucleotides but also enhance their tissue penetration and bioavailability.

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Oligonucleotide Drugs Metabolism Examples

ASO Metabolism

Antisense oligonucleotides (ASOs) are subject to complex metabolic processes following systemic administration. Upon entering the bloodstream, ASOs are rapidly bound by serum proteins, which can influence their distribution and elimination. The metabolism of ASOs involves the enzymatic degradation of the phosphodiester backbone by exonucleases, primarily in the liver and kidneys. As a result, ASOs typically have a short half-life and require frequent dosing for sustained therapeutic efficacy. To mitigate this issue, ASOs are often modified to include phosphorothioate linkages, which replace one of the oxygen atoms in the phosphodiester bond with sulfur. This modification significantly enhances the resistance of ASOs to nuclease-mediated degradation, extending their half-life and improving their pharmacokinetic profile.

siRNA Metabolism

Small interfering RNA (siRNA) molecules are designed to mediate RNA interference and silence specific genes. After siRNA molecules are delivered into cells, they are incorporated into the RNA-induced silencing complex (RISC), where they guide the complex to target mRNA for degradation. siRNA metabolism involves extensive degradation by ribonucleases, such as Dicer, before being processed for incorporation into the RISC complex. To improve the stability of siRNA and extend its therapeutic effect, modifications such as 2'-O-methylation and phosphorothioate incorporation are employed. These modifications prevent the degradation of siRNA molecules, ensuring their efficient delivery and uptake by target cells.

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Metabolic Pathways of Oligonucleotide Drugs

The metabolism of oligonucleotides is primarily influenced by their chemical structure, length, and modifications. Unlike small-molecule drugs, oligonucleotides are large, charged molecules that are not readily absorbed by cells and tissues. As such, their metabolism involves several specialized mechanisms, from initial entry into the bloodstream to degradation in cells. The key processes in the metabolic pathways of oligonucleotides are:

Degradation by Nucleases

The stability of oligonucleotides in the bloodstream and within cells is primarily governed by nucleases. Exonucleases and endonucleases are enzymes that break down nucleic acids by cleaving phosphodiester bonds between nucleotides.

• Exonucleases: These enzymes remove nucleotides from the ends of the oligonucleotide chain. For example, 3'-exonucleases and 5'-exonucleases cleave from the ends of RNA or DNA molecules, leading to progressive degradation.
• Endonucleases: These enzymes cleave internal bonds within the oligonucleotide chain, producing shorter fragments. Endonucleolytic cleavage is particularly important for degrading long oligonucleotide chains that are no longer needed or active.

The susceptibility of oligonucleotides to nuclease degradation is influenced by the presence of certain chemical modifications that protect the nucleotides from enzymatic cleavage.

Tissue Distribution

Oligonucleotides are absorbed into the bloodstream, though their poor oral bioavailability requires alternative delivery methods, such as intravenous or subcutaneous injections. In circulation, serum proteins like albumin can protect oligonucleotides from degradation and affect cellular uptake. The liver is a key site for metabolism, where oligonucleotides undergo modifications, potentially leading to degradation or the formation of active fragments that may cause off-target effects.

Cellular Uptake and Endosomal Processing

Oligonucleotides enter cells via endocytosis, a process aided by surface receptors and transporters. Once internalized, they are transported to endosomes, from where they must escape to reach the cytoplasm or nucleus. For siRNA, this escape is crucial for RNA interference. However, endosomal release is often inefficient, limiting the therapeutic potential of many oligonucleotide drugs. Lipid nanoparticles (LNPs) have been developed to enhance endosomal release.

Pharmacokinetics - Oligonucleotide Drugs

The pharmacokinetics (PK) of oligonucleotide drugs is a critical aspect of their therapeutic efficacy. These molecules, due to their large size, negative charge, and hydrophilicity, exhibit distinct PK properties compared to traditional small molecules. The PK of oligonucleotides encompasses their absorption, distribution, metabolism, and elimination (ADME), which collectively determine the drug's overall bioavailability, therapeutic duration, and tissue targeting.

• Absorption and Bioavailability: Oligonucleotides are typically administered parenterally due to poor oral bioavailability. Their large size and negative charge limit passive diffusion across cell membranes. Lipid-based formulations, such as lipid nanoparticles (LNPs), protect these drugs from degradation and enhance their cellular uptake.
• Distribution: Oligonucleotides are distributed to tissues like the liver, kidneys, and spleen. Targeted delivery systems, such as LNPs or nanoparticles, improve drug accumulation in specific organs, like the liver or CNS, based on the drug's size, charge, and transporters involved.
• Metabolism: Oligonucleotides are metabolized mainly by exonucleases and endonucleases, leading to degradation in the liver and kidneys. Chemical modifications, like phosphorothioate or 2'-O-methyl substitutions, can increase stability and prolong the drug's half-life in circulation.
• Elimination: The elimination of oligonucleotides is primarily renal, with degraded fragments cleared through the kidneys. Modifications like PEGylation or lipid encapsulation can slow elimination, extending the therapeutic window.
• Half-life and Clearance: Oligonucleotides typically have shorter half-lives due to rapid degradation, but modifications such as PEGylation and lipid formulations can extend circulation time, reducing clearance and improving PK profiles.
• Dose and Frequency: Due to rapid clearance, oligonucleotides often require frequent dosing. However, strategies like sustained-release formulations can reduce dosing frequency, improving patient compliance.

Optimizing the PK of oligonucleotide drugs through formulation strategies and chemical modifications is crucial to enhancing their therapeutic efficacy and minimizing off-target effects.

Pharmacokinetic Enhancement Strategies for Oligonucleotide Drugs

Pharmacokinetic enhancements for oligonucleotide drugs focus on improving bioavailability, stability, and tissue targeting. Several strategies have been employed to achieve these objectives:

Lipid-based Formulations

Lipid-based formulations such as lipid nanoparticles (LNPs) and lipid-core micelles are widely used to improve the delivery of oligonucleotides. LNPs protect oligonucleotides from degradation, facilitate cellular uptake, and enhance tissue targeting. These formulations are particularly effective for delivering RNA-based therapeutics, such as mRNA vaccines and siRNA therapies, to target cells.

Chemical Modifications

Chemical modifications, including PEGylation and sugar modifications, are frequently employed to enhance the pharmacokinetic properties of oligonucleotides. PEGylation involves the attachment of polyethylene glycol (PEG) to the oligonucleotide, which increases its stability and reduces immune recognition. Sugar modifications, such as the addition of 2'-O-methyl or 2'-fluoro groups, improve stability against nucleases and extend the drug's half-life.

Targeted Delivery Systems

Targeted delivery systems, such as nanoparticle-based drug delivery and ligand-based targeting, are essential for ensuring that oligonucleotide drugs reach their intended site of action. Nanoparticle-based systems can encapsulate oligonucleotides and release them in a controlled manner at the target site. Ligand-based targeting uses specific molecules, such as antibodies or aptamers, to guide the drug to specific tissues or cells, enhancing therapeutic precision.

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Reference

1. Di, F.D.; et al. Antisense Oligonucleotide: Basic Concepts and Therapeutic Application in Inflammatory Bowel Disease. Front. Pharmacol. 2019, 10: 305.

Oligonucleotide Drugs: Metabolism & Pharmacokinetics Studies

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