Webinar Q&A | DMPK Challenges and Strategies for Developing Oligonucleotide Drugs

Oligonucleotides comprise a rapidly growing class of therapeutics. In fact, more than 10 oligo-based drugs have been approved by the U.S. FDA since 2016. However, due to their novelty, complexity and limited regulatory guidance, oligonucleotides have more technical challenges and unknown consequences for drug developers compared to more conventional drugs. As such, oligonucleotides require pharmacokinetic evaluation systems that differ from traditional assays in significant ways. In the preclinical development stage, study strategies should be customized according to the specific characteristics of the oligonucleotide to accelerate its development. For in vitro ADME study, it is necessary to select an appropriate in vitro metabolic model based on the structural characteristics of the drug; for in vivo PK study, different administration methods can be used to investigate the PK properties.

Notably, on March 2, 2023, Dr. Shiyan Chen, Dr. Hong Zhang, and Dr. Nan Zhao in the DMPK Service Department of WuXi AppTec held a webinar on AAPS (American Association of Pharmaceutical Scientists) eLearning, sharing our strategies to tackle these DMPK challenges and accelerate oligonucleotide drug development. Challenges have been divided into three categories: ADME, metabolite identification and profiling, and bioanalysis. This article summarizes some high-frequency questions to provide feedback on the audience's support for this webinar.

Q1. What strategy do you use for LC-MS quantification of uncharged PMOs? What sensitivity can be achieved in plasma and tissue homogenate?

A: An LLOQ at 5 ng/ml could be achieved by using the LC-MS method. The challenges for PMO quantitation in the biological matrix by using LC-MS are nonspecific binding, low extraction recovery, poor chromatography and carryover. The following methods may help：

1. Conduct nonspecific binding experiments as soon as possible.

2. Use μElution SPE extraction plate to increase throughput and reduce the potential loss of the analyte to container surfaces (adsorption, solubility, etc.).

3. Use column switching techniques (back flush, forward flush, etc.) to reduce column carryover and matrix effect.

Q2. Did you detect any impurities other than truncated sequences (which may be metabolites as well)?

A: Yes, in addition to minor truncated sequences, we have occasionally detected other impurities such as low levels of oxidation, desulfurization, and acetylation adducts during oligo parent characterization. These impurities may also be metabolites. To differentiate them from metabolites, we compare the incubation sample with the T0 sample. When the content of impurities is low, they are unlikely to interfere with our MetID experimental results.

Q3. Have you measured siRNA levels in RISC? How is it measured?

A: Yes. We have developed a method to measure siRNA loaded into RISC. siRNA was measured by stem-loop RT-qPCR after Ago2 immunoprecipitation.

Q4: Based on your experience, how strong is the correlation between RISC-bound siRNA and the respective PD effect?

A: Based on the literature references and our experiences so far, the correlation between the observed PD effect and the concentrations of RISC-loaded siRNA is stronger than plasma and tissue concentrations. However, quantification of siRNA levels in RISC has higher demands on the analytical platform and also BA scientists, decision of including this analysis in the PKPD study could be made case by case with "fit for purpose".

Q5. Do you check out urinary excretion? And at what stage?

A: Usually we will have a urinary excretion study in the IND stage but it also depends on the target tissues of the oligonucleotide drugs. For example, for oligonucleotides targeting kidney tissues, we do check out the urinary excretion in the early screening stage.

Q6. Considering in vitro investigation, is there a suggested internal standard to be used for LC-MS/MS analysis?

A: A structurally similar oligonucleotide is often used as an analog internal standard for a target oligonucleotide analyte. This analog internal standard needs to be chromatographically separated from the oligonucleotide analyte to eliminate mass spectral interference.

Q7. Does deamination coelute or is separated from the parent? Can you identify if coeluting?

A: In the case presented, the deamination metabolite was well separated from the parent compound. If the metabolite and parent co-elute completely, it can be difficult to distinguish between them in the MS spectrum due to their molecular weight difference of only 1 Da and multiple charge states. Therefore, it is crucial to optimize the chromatographic conditions to effectively separate the two compounds for accurate metabolite identification.

Q8. What transition did you use in LC-MS/MS?

A: For the target analyte, a Q1 full scan was conducted to obtain the MS scan ion spectra. The most abundant ions were selected as the precursor ions. For oligonucleotides with phosphorothioate backbones, m/z 95 (phosphorothioate ion) is a commonly used production.

Q9. Have you been able to achieve baseline chromatographic separation for A to I metabolic species from the parent molecule? If so, what column chemistry and mobile phases allowed for this?

A: In the case presented, the A to I metabolite was successfully baseline separated from the parent compound using a Waters BEH C4 column. For the mobile phase, we utilized an ion pair reagent consisting of DIPEA, HFIP, and EDTA in both water and methanol.

In the study described in the reference (Bioanalysis (2019) 11(21), 1955–1966), they were also successfully separated as shown in the figure. To achieve this, the researchers utilized an ACQUITY UPLC Oligonucleotide BEH C18 column (1.7 μm, 130 A, 50 ˚ × 2.1 mm; Waters, MA, USA). The LC–MS mobile phases used were as follows: mobile phase A: H2O/HFIP/DIEA (100:1:0.1, v/v/v) with 10 μM EDTA, mobile phase B: H2O/ACN/HFIP/DIEA (35:65:0.75:0.0375, v/v/v/v) with 10 μM EDTA, mobile phase C: H2O/MeOH/ACN (10:45:45, v/v/v). More detailed information can be obtained from the reference mentioned.

For more details, please click here to watch the webinar recording. If you have any other questions, please feel free to talk to a WuXi AppTec expert or send an email to DMPK_Service@wuxiapptec.com.

Committed to accelerating drug discovery and development, we offer a full range of discovery screening, preclinical development, clinical drug metabolism, and pharmacokinetic (DMPK) platforms and services. With research facilities in the United States (New Jersey) and China (Shanghai, Suzhou, Nanjing, and Nantong), 1,000+ scientists, and over fifteen years of experience in Investigational New Drug (IND) application, our DMPK team at WuXi AppTec are serving 1,500+ global clients, and have successfully supported 1,200+ IND applications.