8 In Vitro Systems for Oligo Metabolism Study: Pros and Cons and Selection Suggestions

Oligonucleotide (Oligo) drugs have seen a growing trend in recent years, and their metabolic pathways are different from conventional small molecules. The main metabolic enzymes for Oligo drugs are exonucleases and endonucleases, which widely exist in blood and various organs and tissues. There was little difference in nuclease metabolism across species, which led to similar metabolite profiles across the species. The conventional metabolic systems used for small molecule research are not suitable for studying the metabolism of Oligo drugs. Therefore, it is important to select appropriate in vitro metabolism study systems for screening Oligo drugs, particularly in the early stages of development.

Pros and cons of 8 in vitro systems for Oligo metabolism study

The liver is the major organ where most small molecules are metabolized, so hepatocytes, liver microsomes, and the liver S9 system are commonly used for the metabolic study of small molecules. The enzymes involved in the metabolism of small molecule compounds are mainly phase I and phase II metabolizing enzymes. However, Oligo drugs are usually metabolized by nucleases, which are widely distributed in various tissues and organs, so the conventional strategies are not suitable for Oligo drugs. 8 research systems commonly used for the in vitro metabolism of Oligo drugs are shown in Table 1. In the early screening stage, it is difficult to screen compounds or optimize structures in large quantities through in vivo experiments. An appropriate in vitro metabolic system can predict in vivo metabolic, assist in optimizing lead compounds, and save time and cost for researchers.

Table 1. In vitro metabolism study system for Oligo drugs

#1 Nuclease

Nucleases can be classified into endonucleases and exonucleases. Exonucleases act on the end of the strand, leading to the release of mononucleotides, while endonucleases cleave internally, resulting in strands of different lengths. In vitro metabolism studies of pegaptanib using mixed enzyme solutions containing endonucleases, 3' and 5' exonucleases, and ribonucleases showed that the remaining percentage of unchanged was 6.1-17.6% after 5 h of nuclease incubation. Nuclease metabolic stability is only mentioned for Viltolarsen among other marketed Oligo drugs, but no detailed data can be found in publications. According to the literature, the metabolic capacity of nucleases is limited. For example, Kim et al. found that ASO compounds incubated with purified endonuclease (RNase A) and exonuclease (Exo-T) for 2 days did not undergo degradation but did produce degradation in both liver homogenate and in vivo ^[2]. Robin et al. used 3 'exonuclease (Nuclease P1) to study the in vitro metabolism of Oligos and found that single-stranded Oligos were quickly metabolized but double-stranded Oligos were stable ^[1].

#2 Serum/plasma

Of the six approved siRNA drugs, five were using serum for metabolic stability studies and exhibited different results. For approved ASO drugs, only plasma stability data are presented in the application materials of Pegaptanib. After incubation in plasma for 5 h, 12.9% ~ 100% of Pegaptanib remained in human, rabbit, dog, and monkey plasma. Stability data in plasma or serum are not found in other marketed ASO drug application materials. Serum stability was also studied with the marketed aptamer drug Avacincaptad Pegol, a potential metabolite was found which accounted for 9, 12, and 14% of the unchanged drug in monkey serum, and accounted for 0, 6, and 6% of the unchanged drug in rat and human serum after 24, 48, and 72 hours of incubation. The reason that serum is chosen over plasma may be the additional anticoagulant treatment for the preparation of plasma. EDTA, one of the commonly used anticoagulants, is an inhibitor of nuclease, so the plasma anticoagulated with EDTA is not suitable for the in vitro stability study of Oligo drugs.

Table 2. Stability of approved siRNA drugs in serum

M: mouse; R: rat; C: monkey; H: human

#3 Liver S9

Of the six siRNA drugs that have been marketed, five have conducted liver S9 metabolic stability assay and the results showed different levels of metabolism. There is no obvious metabolism pattern from the metabolic trend. In our experience, the GalNAc moieties of the SS strand are quickly cleaved in the liver, with the SS strand losing GalNAc as the main metabolite. However, the SS strand is relatively more stable in the in vitro S9 system than in the in vivo liver, which causes differences between in vitro and in vivo. For approved six ASO drugs, metabolism studies in liver S9 were not presented in their application materials. Avacincaptad Pegol, an aptamer drug, was also studied for stability in liver S9, with 48%, 0%, and 0% remaining in human, monkey, and rat liver S9 after 24 hours of incubation, respectively.

Table 3. Stability of approved Oligo drugs in liver S9

M: mouse; C: monkey; H: human; R: rat

#4 Liver homogenate

The liver homogenate is a recommended system for in vitro metabolism studies. Rosanne et al. performed in vitro metabolic studies of ASO in mouse liver homogenates and found a good correlation with in vivo metabolism ^[3]. Babak et al. studied the in vitro metabolism of GalNAc-conjugated siRNA in rat liver homogenates and compared it with phosphodiesterase, endonuclease/exonuclease, plasma and serum, hepatocytes, and liver lysosomes, and found that the metabolic system of liver homogenates was optimal and presented a good correlation between the metabolic results of liver homogenate systems and in vivo metabolism ^[4]. Babak et al. also suggested using an in vitro liver homogenate stability assay to screen Oligo drugs during lead optimization progress.

#5 Liver microsomes

Although it is generally accepted that common microsomal enzymes such as CYPs and FMO are not involved in the metabolism of Oligo drugs, it has also been reported that Oligo drugs can also be metabolized in human liver microsomes. Kim et al. found that an unmodified ASO compound was completely metabolized after incubation with human liver microsomes for 12 h, and the major metabolites were 3' terminal degradation products, indicating that there exists 3' exonuclease in human liver microsomes. However, the same ASO compound was completely metabolized after incubation with nuclease or liver homogenate for 1 h, indicating that the nuclease activity of liver microsomes was low^[5]. Although the liver microsomal system may metabolize Oligo drugs, it is not related to the contribution of CYP enzymes. So far, there is no report on the involvement of CYP enzymes in Oligo metabolism.

#6 Hepatocytes

As the application materials of Lumasiran mentioned the deaminated metabolite of Lumasiran which was found in monkey liver samples was not found in liver S9 of various species. So human hepatocytes HepatoPac^® were further used to study the deaminated metabolite of Lumasiran in in vitro metabolism. The results showed that the deaminated metabolite was detected at a higher concentration than unchanged Lumasiran after 168 hours of incubation, followed by 3'N-1 (AS), indicating that deaminated metabolite can be produced in human hepatocytes. In the application materials of Nedosiran, metabolic studies were performed using mixed human hepatocytes, but no metabolites were found after 24 hours of incubation, the supposed reason is the short incubation time. Robin et al. used rat, monkey, and human hepatocytes to study the in vitro metabolism of GalNAc-modified siRNAs and found that GalNAc hydrolysis and linker hydrolysis products, as well as 3'N-1 (AS) metabolites were all detected. In terms of metabolic rates, GalNAc and linker were faster hydrolyzed in rat hepatocytes than in monkeys and humans. Robin et al. also compared the results of liver metabolism of four different siRNAs in hepatocytes in vitro and in vivo and found that the major metabolites could be found in both in vitro and in vivo ^[1]. OSWG recommends hepatocytes as an in vitro study system ^[6], because they provide the closest approximation of uptake into specific compartments of hepatocytes in vivo, whereas liver S9 or tissue homogenates can result in exposure to nonrelevant compartments and, thus, overexposure to nucleases relative to in vivo systems. There are not many cases of using hepatocytes as an in vitro study system, possibly because the membrane barriers make it difficult for poorly permeable Oligo drugs to enter cells.

#7 Lysosome

Lysosomes are the immediate environment when Oligo drugs enter cells by endocytosis. Acidic lysosomes (pH = ~ 5) containing abundant enzyme systems, including nucleases and various hydrolases, are an important metabolism site for Oligo drugs, so lysosomes are an efficient experimental system to study the metabolic stability of Oligo drugs. As reference suggested, GalNAc-modified siRNA, after 4 hours of incubation with mouse liver lysosomes at pH 7 and pH 5, the remaining were 95% and 32%, respectively, which suggested that acidic conditions are more conducive to the hydrolysis of GalNAc ^[1]. The application document of Nedosiran also showed that four GalNAc fragments were metabolized in lysosomes as well as four amylamine linker groups.

#8 Target tissue-related matrices

The liver is the main distribution and metabolism organ of Oligo drugs as well as a common target tissue. So, liver-related matrices are often used to study the in vitro metabolism of Oligo drugs. In addition to that, some local delivery Oligo drugs can be distributed to other tissues and organs. Due to the wide distribution of nucleases in the body, extrahepatic tissues also have metabolic potential. Metabolic studies using target tissue-related matrices are of certain significance for understanding the metabolism and efficacy of Oligo drugs. For example, we can study the stability of vitreous fluid for intravitreal injected Oligo drugs; the stability of cerebrospinal fluid (CSF) and brain tissue homogenates for intrathecal injected Oligo drugs; the stability of kidney homogenates for kidney-targeted Oligo drugs; the stability in urine for intravesical instillation administrated Oligo drugs.

General suggestions on selecting in vitro metabolism study systems

Oligo drugs are generally developed based on platforms. The platform refers to the combination of structure, length, backbone chemistry, chemical modifications, conjugate, etc. In vitro metabolism studies are also selected based on the structural characteristics and development stages. For the selection of the in vitro metabolism study system, our suggestions are as follows:

a. For Oligo drugs developed based on known platforms with similar metabolic characteristics, it is not necessary to carry out massive metabolic screening studies in the early stage. After obtaining in vivo PK data, in vitro metabolism studies can be retrospectively carried out, or in vitro metabolism studies can be performed to help explain the data when abnormalities are found in in vivo data. The selection of the metabolic system can refer to the known platforms.

b. For Oligo drugs of the new platform, in vitro metabolism studies are important for understanding the metabolic characteristics of the Oligo drugs on the new platform. In our experience, for Oligo drugs developed by the new platform, it is very difficult to accurately predict in vivo stability or metabolism with only 1 or 2 in vitro metabolic systems in the early stage. Therefore, it is recommended to conduct multi-system metabolic studies to verify each other at an early stage, and even continuously optimize the metabolic systems with better in vitro and in vivo correlations.

c. In addition, for liver-targeted Oligo drugs, it is appropriate to select liver homogenate or liver S9 for in vitro metabolism study; for kidney-targeted Oligo drugs, it is recommended to select kidney homogenate or kidney S9 for in vitro metabolism study. Theoretically, systemic tissues have the potential to metabolize Oligo drugs, and for locally administered or locally active Oligo drugs, we need to pay attention to the metabolism of local matrices (such as CSF, brain homogenate, vitreous fluid, urine, etc.) , as it is crucial for predicting efficacy.

Concluding remarks

Metabolite information obtained from in vitro metabolism studies play a significant role in evaluating the pharmacological activity and predicting the clinical metabolism of Oligo drugs. For Oligo drugs developed by the new platform, in vitro metabolism studies can help verify the success of the development of the platform at an early stage and assess the metabolic characteristics. WuXi AppTec DMPK has a complete in vitro metabolism research platform, which can provide appropriate in vitro metabolism systems for the evaluation of Oligo drugs according to the structural characteristics and experimental purposes, assisting the early research and development of Oligo drugs.

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Authors: Qian Li, Liping Ma, Jing Jin

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