In 1998, Andrew Fire and Craig Mello discovered the phenomenon of RNA interference (RNAi) in Caenorhabditis elegans, laying the foundation for the development of RNAi technology and earning them the 2006 Nobel Prize in Physiology or Medicine. In the following years, it was successively confirmed that the RNAi phenomenon also exists in mammals and humans. With advances in nucleic acid chemical modification and targeted delivery systems, siRNA drug development has made significant breakthroughs. In 2018, the first siRNA drug, Patisiran, developed by Alnylam Pharmaceuticals, was approved for market. To date, a total of six siRNA drugs have been approved for commercial use, as listed below.
| Brand Name | Generic Name | Approval Year | Developer Company | Indication |
| ONPATTRO | Patisiran | 2018 | Alnylam | Hereditary transthyretin-mediated amyloidosis with polyneuropathy |
| GIVLAARI | Givosiran | 2019 | Alnylam | Acute hepatic porphyria |
| OXLUMO | Lumasiran | 2020 | Alnylam | Primary hyperoxaluria type 1 |
| LEQVIO | Inclisiran | 2021 | Alnylam/Novartis | Hypercholesterolemia |
| AMVUTTRA | Vutrisiran | 2022 | Alnylam | Hereditary transthyretin-mediated amyloidosis with polyneuropathy |
| RIVFLOZA | Nedosiran | 2023 | Novo Nordisk | Primary hyperoxaluria type 1 |
siRNA drugs are typically composed of 20–30 nucleotides and function by forming a RNA-induced silencing complex (RISC) with human Ago2 protein. This complex mediates the cleavage and degradation of target mRNA, thereby blocking its translation and achieving the silencing of specific gene expression. Unlike traditional small molecules and biologics that are primarily metabolized and hydrolyzed, siRNA drugs are mainly degraded by endogenous human endonucleases and exonucleases. In addition to differences in mechanism of action and metabolism, siRNA drugs also exhibit distinct PK/PD characteristics compared to other drug types.
The pharmacodynamic (PD) effects of siRNA drugs are dissociated from systemic exposure both temporally and spatially. After systemic exposure, siRNAs are rapidly taken up by cells with the aid of chemical conjugates or delivery technologies, and are phagocytosed by immune system cells, resulting in a relatively short plasma half-life. However, the efficacy of siRNA drugs is determined by the concentration at the intracellular target site, particularly in the cytoplasm. This explains why siRNA drugs tend to have long-lasting PD effects and require infrequent dosing. Additionally, the off-target effects of siRNA drugs are mainly related to base pairing with endogenous non-target RNA sequences. Therefore, compared to traditional small molecules and biologics, siRNA drugs differ in many aspects, necessitating special considerations in their clinical pharmacology studies.
Based on the six currently approved siRNA drugs, the following summarizes key points in the clinical pharmacology of siRNA therapeutics:
All six marketed siRNA drugs are double-stranded RNAs. Except for patisiran, which lacks chemical conjugation, the other five siRNAs use N-acetylgalactosamine (GalNAc) conjugation for liver-targeted delivery. The GalNAc sugar moiety binds to the asialoglycoprotein receptor (ASGPR) on hepatocyte surfaces, facilitating siRNA delivery into liver cells.
Pharmacokinetics (PK)
Except for patisiran, which mainly distributes in the liver and has low plasma protein binding, the other siRNA drugs exhibit 80%–90% binding to plasma proteins at clinical therapeutic doses. Systemic exposure increases proportionally with dose, and no accumulation is observed with repeated dosing. With the exception of nedosiran, the elimination half-life of the other siRNAs is under 10 hours, significantly shorter than the dosing interval. This is a typical feature of siRNA drugs—dissociation between PK and PD. Therefore, the plasma exposure–effect relationship is not suitable for dose selection. For example, the dosing of givosiran is based on the dose–effect relationship rather than plasma exposure–effect correlation.
Population PK data indicate that among the six FDA-approved siRNA drugs, body weight is the primary intrinsic factor affecting pharmacokinetics.
Analytical Methods
All siRNA drugs are analyzed using liquid-phase techniques, either pure liquid chromatography or liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). The specific RNA components analyzed vary: lumasiran, vutrisiran, and nedosiran measure the antisense strand; givosiran and patisiran measure the double-stranded RNA; and inclisiran measures the ratio of antisense to sense strands.
| Generic Name | Tmax | T1/2 | Vd | Plasma Protein Binding | Bioanalytical Method |
| Patisiran | 70 min | 3 days | 0.26 L/kg | <2.1% | LC |
| Givosiran | Parent: 3 h Active Metabolite: 7 h | 6 h | 10.4 L/kg | 90% | LC-MS/HRAM LC-MS/MS |
| Lumasiran | 4.1 h | 5.2 h | 4.9 L/kg | 77–85% | LC-TOF-MS |
| Inclisiran | 4 h | 7 h | 500 L/kg | 87% | LC-TOF-MS |
| Vutrisiran | 4 h | 5.2 h | 10.1 L/kg | 80% | LC-MS/HRAM |
| Nedosiran | 6 h | 15 h | 126 L | 86% | HPLC |
Drug-Drug Interactions (DDIs)
GalNAc-conjugated siRNAs typically do not interact with drug-metabolizing enzymes or transporters. This is primarily because siRNAs are effectively delivered to target tissues and do not induce changes in cytokine levels. Cytokines are generally known to influence the expression of hepatic CYP enzymes and drug transporters. Many biologics indirectly affect CYP enzymes and transporters by modulating cytokine levels. In addition, siRNAs are mostly administered subcutaneously, making it unlikely for them to affect gastrointestinal P-glycoprotein (P-gp) transporters.
In vitro studies have shown that the six FDA-approved siRNAs are neither inducers, inhibitors, nor substrates of hepatic CYP450 enzymes. Similarly, these siRNAs are not inhibitors or substrates of drug transporters. Among them, only givosiran has been investigated in clinical DDI studies; the others have not undergone clinical DDI evaluations.
However, this does not mean siRNAs are completely free of DDI risks. For example, siRNAs may bind off-target to the mRNA of P450 enzymes or transporters, potentially modulating their activity. Liver-targeted siRNAs may influence hepatic biological functions, thereby posing DDI risks. This may explain why givosiran underwent DDI studies. Givosiran affects the heme biosynthesis pathway in hepatocytes, which could reduce P450 enzyme activity. Indeed, it was found that givosiran increases the exposure of substrates of CYP1A2, 2C9, 2C19, and 3A4. Additionally, some small molecules may bind to chemically modified siRNAs, potentially enhancing cellular uptake and increasing DDI risks. That said, no such PK alterations have been observed with the approved siRNAs due to small molecule interactions.
Immunogenicity
siRNAs can stimulate the innate immune system, and both the siRNA molecules and their delivery systems may carry some immunogenicity risk. Therefore, it is necessary to evaluate the generation of anti-drug antibodies (ADAs). However, the ADA risk for the currently approved siRNAs is low. Clinical ADA incidence rates are as follows: patisiran (3.6%), givosiran (0.9%), lumasiran (6%), inclisiran (1.7%), vutrisiran (2.5%), and nedosiran (0%). Furthermore, ADA formation has not affected the pharmacokinetics (PK), pharmacodynamics (PD), efficacy, or safety of these drugs.
Impact of Hepatic and Renal Impairment
For patisiran, givosiran, lumasiran, and vutrisiran, population PK modeling has been used to assess the impact of hepatic and renal impairment. Inclisiran and nedosiran have undergone dedicated clinical studies for this purpose.
No clinically significant impact on PK was observed for patisiran, givosiran, lumasiran, or vutrisiran in patients with mild to moderate hepatic or renal impairment. Clinical trials of nedosiran in patients with mild or moderate renal impairment and mild hepatic impairment showed no significant PK changes. However, no studies have been conducted in patients with severe renal impairment or moderate to severe hepatic impairment.
Inclisiran studies showed increased Cmax and AUC in patients with mild to moderate hepatic impairment or mild to severe renal impairment, but there was no observed impact on PD compared to healthy individuals. Therefore, dose adjustments are not necessary.
Given that siRNAs are primarily metabolized by nucleases rather than CYP450 enzymes and have short plasma half-lives, hepatic impairment is generally not considered a major concern for their PK. Some concerns may arise regarding whether hepatic impairment could affect ASGPR expression, which is crucial for GalNAc-siRNA delivery to hepatocytes. However, even with a 50% reduction in ASGPR expression, therapeutic doses of GalNAc-siRNA do not saturate the receptor, unless in extreme cases.
QTc Interval
Among the six approved siRNAs, only inclisiran has undergone a dedicated clinical QT study. The results indicated no QT interval prolongation at therapeutic doses. While no dedicated QT studies were performed for the other siRNAs, ECG monitoring was included in other clinical trials, and no QT prolongation was observed.
Furthermore, preclinical in vitro hERG assays and in vivo safety pharmacology studies in monkeys also suggest no risk of QT interval prolongation at clinical exposure levels. For example, nedosiran showed no QT or QTc prolongation in repeat-dose (30 and 300 mg/kg) subcutaneous studies in monkeys. The in vitro IC50 for hERG inhibition by nedosiran is over 390 times higher than its clinical Cmax. Overall, the risk of QT prolongation by siRNA drugs is considered very low.
Conclusion
In summary, siRNA drugs present relatively low risks in terms of DDI, ADA formation, the impact of hepatic/renal impairment, and QTc prolongation. They can be considered a relatively low-maintenance class of drugs. However, one important consideration is the typical dissociation between plasma PK and PD observed with siRNAs. Thus, exposure–response relationships are usually not based on plasma concentrations. Preclinical target tissue siRNA concentration/PD modeling and physiologically based PK (PBPK) modeling can be useful for dose selection.
For clinical pharmacology study design, in addition to referencing existing approved siRNA drugs, it is recommended to consult the FDA’s guidance on clinical pharmacology studies for oligonucleotide therapeutics.
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