SiRNA drugs exert their pharmacological activities by regulating mRNA levels and modulating the production of disease-related proteins. In order to evaluate changes in mRNA and protein levels, it is crucial to establish appropriate in vitro and in vivo models, which rely not only on the LNP delivery system and GalNac coupling technology, but also have significant implications for extrahepatic targeted delivery, the exploration of novel mechanisms of action of siRNA drugs, pharmacodynamic research, and pharmacokinetics/pharmacodynamics (PK/PD) studies
In contrast to small molecule drugs and protein drugs, siRNA drugs exhibit unique pharmacokinetic (PK) and pharmacodynamic (PD) relationships:
1. Short exposure time in plasma, long exposure time in target organs (e.g., liver), and sustained drug efficacy.
Fig. 1. Givoiran, Inclisiran and Vutrisiran: Relationship between the concentration of siRNA in plasma and the change of PD markers in serum.
2. The key PD material basis lies in the complex formed by siRNA and RISC in the cytoplasm, rather than the total concentration of siRNA in plasma or target organs.
Fig. 2. Relationship between total concentration of siRNA, concentration of siRNA-RISC and PD markers in liver.
3. The in vivo escape efficiency of siRNA and the binding kinetics between siRNA and RISC may vary with different platforms or delivery systems.
Fig. 3. The process and key steps of GalNac SiRNA entering cells, intracellular distribution and pharmacological activity.
Fig. 4. Two schemes can improve the escape efficiency of siRNA endosomes.
These characteristics pose significant challenges for the pharmacodynamic evaluation and clinical translation of siRNA drugs. Moving forward, there is a need for more quantitative research on the mechanism of action and PK processes of siRNA drugs, including intracellular PK, to obtain accurate non-clinical pharmacodynamic data that can enhance clinical effectiveness and guide clinical trial design. Additionally, integrating existing non-clinical and clinical data is vital for model improvement and optimization.
Evaluation of in vitro activity and off-target effects
In vitro testing is a crucial component in the research and development of siRNA drugs, aiming to identify siRNA sequences with high targeting specificity and efficacy. Typically, primary cell lines or transfected cell lines derived from humans or animals are utilized as experimental systems to investigate the effects of siRNA on cell activity, function-related proteins, and target mRNA levels. These in vitro experiments allow for the evaluation of the effects on human targets as well as the assessment of mRNA effects in different species, providing a basis for the selection of relevant species in toxicology studies. According to FDA review reports, six siRNA drugs on the market can regulate target mRNA and modulate protein expression levels in a concentration-dependent manner, demonstrating pharmacological activity in both monkeys and in vitro settings. Moreover, in silico comparisons of target mRNA homology among different species or predictions of pairing with candidate siRNA drugs offer insights for selecting relevant species. Preliminary screening of non-target sequences (off-target sequences) in human transcripts that may hybridize with candidate siRNA drugs, followed by subsequent in vitro testing and comprehensive risk assessment, adds to the overall evaluation.
In vivo pharmacodynamics
The pairing of siRNA drugs with target mRNA relies on a high degree of specificity, as successful pairing only occurs with sequences that exhibit high homology to the target mRNA. Generally, in vivo pharmacodynamic studies are performed in related species or animal models after selecting the appropriate related species and genera. Non-human primates serve as crucial models for evaluating the in vivo pharmacodynamics of siRNA drugs, enabling the determination of effective dose ranges and assessment of dosage regimens. In cases where no related species display pharmacological activity, transgenic animal models can be utilized or corresponding in vitro pharmacodynamic tests may be conducted.
Fig. 5. Correlation analysis of dose-urine ALA/PBG level (top), dose-urine ALAS1 mRNA/ALA/RBG level (middle) and dose-urine ALA/PBG level (bottom) of Givoiran.
Pharmacodynamic experiments conducted in vivo allow for the investigation of dose-response relationships, time-course effects, and efficacy-related biomarkers. Additionally, within the realm of quantitative pharmacology, PK/PD models, dose-effect-time models, and PK/PB models play pivotal roles in the development of siRNA drugs, enabling the simulation, extrapolation, and prediction of data from preclinical to clinical stages. Comprehensive understanding and utilization of the unique PK/PD relationship of siRNA drugs are crucial for guiding their development. The scientifically and rationally designed preclinical pharmacodynamic trials, alongside the study of transformation perspectives, can better support the design of clinical trials, human efficacy prediction of siRNA products, and assist in determining the first-in-human (FIH) clinical trial and dosage regimens in different clinical stages. They also provide support for clinical dosage selection in New Drug Application (NDA). Listed siRNA drugs, such as patisiran and givosiran, have employed the aforementioned pharmacodynamic models.
Conclusion
With strict adherence to ethical standards for animal experiments and regulatory requirements governing drug development in different countries, Prisys Biotechnology is committed to contributing to siRNA drug translational research through the utilization of extensive animal model resources for non-human primate diseases, professional expertise in pharmacodynamics, DMPK, and safety evaluation of small nucleic acid drugs.
Prisys Biotechnology has accumulated abundant experience in pharmacodynamic research utilizing animal models of non-human primate diseases. Leveraging our resources and technological advantages, we have established various animal models for induced non-human primate diseases, which boast high homology with humans and demonstrate closer genotypic and phenotypic characteristics to the disease populations.
In the field of siRNA, Prisys Biotechnology maintains close collaborations with numerous leading nucleic acid drug companies throughout the year, having completed concept verification tests and preclinical pharmacodynamic studies of siRNA drugs, encompassing multiple prominent indications, including hyperlipidemia, NASH, and hypertension. At the same time, Prisys Biotechnology closely monitors industry R&D trends and clinical needs, continuously strengthening forward-looking layouts and technical capacity building in new technology domains. We are dedicated to exploring novel models that meet the R&D requirements of diverse customers in innovative siRNA drugs, encompassing new indications, delivery systems, and combination therapies.
