Preclinical Safety Evaluation of Oligonucleotide Drugs
What are oligonucleotide drugs?
Oligonucleotide drugs (ONs) refer to short-chain nucleotides containing about 20 bases that act on transcription and translation by binding to the complementary strand of mRNA based on the Watson-Crick base complementary pairing principle. According to their different structures and mechanisms of action, they are divided into antisense oligonucleotides (ASO), small interfering RNA (siRNA), small activating RNA (saRNA), microRNA (miRNA), aptamers, etc.
Mechanism of action of oligonucleotide drugs
Oligonucleotides act on mRNA or its precursor pre-mRNA and block its expression through the Watson-Crick base-pairing principle, thereby inhibiting protein production. For example, ASO complements the target mRNA to form an RNA-DNA hybrid double strand. This double-stranded structure is then recognized by RNase H and degrades mRNA, preventing protein synthesis. siRNA reduces gene expression by guiding the RNA-induced silencing complex (RISC) to degrade target mRNA. In addition to blocking mRNA function, oligonucleotides can also regulate RNA splicing and translation processes through a stereotyped mechanism of blocking the ribosome.
Safety challenges of oligonucleotide drugs
Unlike small molecule drugs, ONs enter the body, they mainly bind to the target mRNA to achieve their effects; therefore, there are two main sources of toxicity here: one is sequence-dependent hybridization and the other is sequence-independent hybridization; Sequence-dependent hybridization has two toxicities, one is targeted toxicity and the other is off-target toxicity; sequence-independent impurities can produce the following reactions: immunogenicity, high tissue concentration, thrombocytopenia, coagulation abnormalities, complement activation, etc.; Higher concentrations in organs and tissues are affected by off-target toxicity and immune response; and thrombocytopenia is also affected by immune response. Among them, targeted toxicity, off-target toxicity, and thrombocytopenia are mainly affected by the sequence of the drug; while immune response and high tissue concentration are mainly affected by the specific body and drug sequence.
Targeted toxicity of oligonucleotide
Targeted toxicity mainly means that the drug binds to the target mRNA, and the efficacy is too strong and exceeds the expected efficacy (such as hypoglycemia in the treatment of diabetes) or is not the expected target tissue, resulting in adverse reactions in other tissues. Although there are no clear examples at present, due to the action time of small nucleic acid drugs, the action time is longer than that of small molecule drugs, which leads to a longer elution time. Therefore, PMDA recommends that evaluation be conducted with reference to ICH S6. For targeted toxicity, non-clinical safety evaluation can be carried out in 1 or 2 animals with pharmacological activity. When there is no pharmacologically relevant animal species, targeted toxicity studies can be conducted using alternatives that are pharmacologically active in animals. Toxicity studies using alternatives are usually conducted in only one species. For dose selection, similar to biosimilars, reference ICHS6 recommends that the high dose group should produce the greatest pharmacological effect.
Off-target toxicity of oligonucleotide
Off-target toxicity refers to the off-target effect caused by hybridization of sequences similar to the target sequence. In 2009, bevasiranib, an siRNA drug developed by Miami pharmaceutical company OPKO for wet macular degeneration, was terminated due to poor effect in a phase III clinical trial. Off-target toxicity is usually serious, such as acute liver toxicity. For off-target toxicity, OSWG recommends following some simple steps first, such as: ① Using computer models (in silico) to screen out potential complementary sequences (off-target sequences);② Remove some sequences, such as those that are rarely expressed in tissues;③ Conduct in vitro screening in major target and off-target cells to explore the concentration of maximum reactivity in target and off-target cells;④ The in vitro concentration should be above the expected dose level so that potential toxicity to off-target effects can be reflected. For hybridization-dependent off-target toxicity, PMDA recommends that, considering the differences between human and animal genomes, it is recommended to use bioinformatics methods and methods for in vitro investigation of gene expression in human-derived cells for evaluation.
Immunotoxicity of oligonucleotide
General oligonucleotides have some of the same molecular characteristics as oligonucleotides in nature and may cause immunotoxicity. However, these compounds are often chemically modified during development to improve stability, safety, cellular absorption and efficacy. Common modification sites include heterocyclic bases, nucleic acid chains bonded to different groups, sugar or dilipid bonds, and these modifications may increase immune responses. Therefore, OSWG recommends conducting necessary studies on immunogenic reactions, while PMDA recommends that immunotoxicity tests can be evaluated based on the results of repeated dose toxicity tests, and independent immunotoxicity tests may not be conducted.
Organ toxicity of oligonucleotide
The highest concentration of oligonucleotide drugs after systemic administration is in the liver and kidney. Generally, we think that liver toxicity and kidney toxicity are mainly caused by accumulation in the liver and kidney. However, according to literature reports, the cause of liver toxicity and kidney toxicity has nothing to do with accumulation. Minor modifications to the base and sequence backbone of the ASO will have a huge impact on affinity and toxicity; In many cases, liver damage or necrosis was observed microscopically in subrodent studies, and the dose exposure of the drug in the liver was much smaller than the dose accepted for hepatotoxicity.
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Preclinical safety evaluation strategy for oligonucleotide drugs
Guidelines for safety evaluation: Safety assessments of ON generally follow guidelines developed for biotechnology-derived drugs, such as ICH S6 (International Conference on Harmonization). These guidelines emphasize the importance of selecting appropriate animal models for preclinical studies. Typically, one or two related species are used to assess safety, but other models can be used if necessary. Research must ensure that the drug's effects in different systems are fully characterized, including the cardiovascular, respiratory and central nervous systems.
Study design: Repeat-dose toxicity studies are critical to assess the long-term effects of ON, as these drugs may accumulate over time or cause delayed toxicity. Extended study time can help identify potential risks that will not manifest during short-term testing periods in assessing delayed toxicity and recovery of affected tissues.
Safety pharmacology: It can be evaluated together in repeat-dose toxicity experiments. The items evaluated include cardiovascular system, respiratory and central nervous system functions, and part of the in vitro hERG (human Ether-a-go-go Related Gene) experiments will be carried out; in addition, supplement liver and kidney safety pharmacology experiments will be considered based on the needs of liver and kidney metabolism.
General toxicity: ① Single dose: if acute toxicity information can be obtained from other experiments, it may not be carried out alone; It is also mentioned in ICHS6(R1) that a single dose toxicity test can be used as part of the pharmacological test. ② Repeated dosing: mainly refer to the ICHM3(R2) design, but pay attention to the drug concentration in liver and kidney tissues; Considering the long half-life of ONs, it is necessary to extend the recovery period to investigate the delayed toxicity and toxic recovery.
Special toxicity: ① According to the needs of the population to be used, toxicity experiments on young animals need to be carried out. ② Considering the route of administration, an in vitro hemolysis experiment needs to be carried out. ③ Since ONs have some biopharmaceutical properties, it is necessary to increase immunity toxicity and immunogenicity as needed, as well as genotoxicity, reproductive toxicity and carcinogenicity.