Meeting report: oligonucleotide ADME workshop

Abstract

The current regulatory landscape for the development of oligonucleotide drugs may lead companies to perform a variety of small molecule-focussed absorption, distribution, metabolism and elimination (ADME) studies in support of filing packages. Asking the question, if the current activities are suitable for these modalities and should science-driven decisions on development of such molecules be implemented more in the industry.

Challenges and opportunities within oligonucleotide ADME were presented and discussed at the online oligonucleotide ADME workshop (17th and 18th of November 2021). This article summarises the presentations and discussions from the workshop.

The following topics were covered:

1. Introduction to Delivery of Antisense RNA Therapeutics (DARTER), Nucleic Acid Therapy Accelerator (NATA) and OligoNova initiatives.

2. Presentation of various oligonucleotide ADME strategies.

3. Update on the Oligonucleotide Safety Working Group (OSWG) pharmacokinetics (PK)/ADME subcommittee’s recommendations.

4. Oligonucleotide quantitative whole-body autoradiography (QWBA) hot or not?

5. Multimodal imaging of therapeutic oligonucleotides.

Keywords:

• Oligonucleotides
• ADME
• drug development
• Oligonucleotide initiatives
• ADME strategies

Introduction

This meeting report covers the topics presented and discussed at the oligonucleotide absorption, distribution, metabolism and elimination (ADME) workshop, which was held as an online event 17th and 18th November 2021. There was great interest in the meeting with 118 registered delegates with a balanced representation of pharma and contract research organisation (CRO) representatives assuring good input from both worlds. The oligonucleotide ADME workshop is organised under the umbrella of the Drug Metabolism and Discussion Group (DMDG, www.dmdg.org).

The aspiration of this interactive workshop was for DMDG member company scientists to discuss and exchange ideas and experience of oligonucleotide ADME related challenges and opportunities. And furthermore, strengthen the network of scientists working with oligonucleotide ADME.

Topics for discussion included initiatives within oligonucleotides, ADME and drug–drug interactions (DDI) strategies, study types and designs, and not least regulatory requirements, interactions, and feedback. The workshop was closed after concluding discussions on the topics covered by the presenters. Abstracts of the presentations by the authors follows below:

Introduction to delivery of antisense RNA therapeutics (DARTER) initiative, Virginia Arechavala-Gomeza (Biocruces Bizkaia Health Research Institute, Spain)

European Cooperation in Science and Technology (COST) Actions are EU-funded networks that aim to facilitate networking. Funding helps organising workshops and training schools, funding travel expenses for research visits to other laboratories and for attending congresses, as well as disseminating research to other researchers and general public.

The DARTER (www.antisenserna.eu) network includes over 350 researchers in 31 countries with expertise in specific target tissues/disorders, oligonucleotide chemistry or drug delivery and represents a range of backgrounds, mostly academic and clinical, but also small and large industry, and patient representatives. The main goal is to achieve consensus in models and methods applied to assess the delivery of ASOs. Furthermore, there is a specific interest in sharing ‘negative’ results and training of new researchers.

Since late 2018, many different activities have been organised and there are already several tangible outcomes of the collaboration amongst the stakeholders: several stakeholders have started new research projects together, a comprehensive review of the state of the art has recently been published (Hammond et al. 2021), a book including 28 chapters with detailed protocols of oligonucleotide research methods in vitro and in vivo will be published in the next few months (as part of Springer’s ‘Methods in molecular biology series’), and several white papers on model systems and safety and toxicology are being drafted. Also, a special issue on ‘negative’ results was published in ‘Nucleic Acids Therapeutics’, edited by some of our members (Echevarría et al. 2021; Eilers and Denti 2021) and many dissemination activities, such as videos or a YouTube channel, are now in place.

Despite the problems derived by the COVID-19 pandemic, the action has taken advantage of online tools to continue their activities and will continue to do so at least until late 2022, when the current grant period expires.

Oligonucleotide ADME packages, Ben-Filippo Krippendorff (Roche, Switzerland)

While oligonucleotides from a regulatory perspective are treated as small molecules, there exist important differences regarding the ADME strategy and the packages needed for successful development programs. For many typical small molecule packages, the scientific validity for oligonucleotides is questionable and the additional work to perform these studies has to be weighed against the regulatory risk of not performing them. One example for this is relating plasma concentrations to efficacy and toxicities. While many programs have characterised plasma concentrations and have justified dose setting with similar methods as for small molecules, for oligonucleotides tissue concentrations are driving efficacy and toxicities and are, if possible, to be measured, more informative for relevant exposure.

Other topics that were discussed included the characterisation of metabolites preclinically and in the clinic, measurements of protein binding, DDI and transporter studies, antidrug antibodies (ADA) strategy, QWBA, and mass balance studies.

From these topics, especially protein binding studies and transporter studies are considered in Roche projects to be likely uninformative due to different binding, distribution and elimination pathways being used by oligonucleotides. It was discussed that the characterisation of metabolites preclinically and in humans is feasible with established methods, however most Roche programs would focus on the metabolites retaining activity (n-1, n-2). Often, the characterisation of metabolites related to potential toxicity is not performed due to the metabolites in safety testing (MIST) guidance focussing on systemic exposure thresholds. DDI and ADA measurements are usually included in oligonucleotide programs at Roche due to the perceived regulatory risk although ongoing discussions on their relevance. The requirement and rationale for conducting QWBA studies were discussed in detail in a separate talk during the meeting.

Introduction to nucleic acid therapy accelerator (NATA) initiative, Martin Kerr (Rutherford Appleton Laboratory, UK)

The NATA (www.natahub.org) is a major UK government initiative that will unlock the potential of precision genetic medicines by enabling state-of-the-art interdisciplinary and international research partnerships to solve critical issues in drug delivery. By providing a dedicated discovery research and development platform into disruptive drug delivery technologies, the NATA will address a crucial barrier to progressing this new generation of genomic medicines towards clinical studies and ultimately patients. This internationally unique resource has dedicated infrastructure located on the Harwell Campus near Oxford, UK, and will bring together world-leading academic expertise with innovative businesses in public–private partnerships, anchoring the nucleic acid therapy industry in the UK for many years to come.

It is widely accepted that for nucleic acid medicines to truly effect a paradigm shift in healthcare the major scientific and industrial challenge of effective nucleic acid drug delivery must be overcome. While there have been a number of recent high-profile nucleic acid therapy drug approvals (e.g. Eteplirsen FDA approval 2016 (Aartsma-Rus and Krieg 2017), Nusinersen FDA approval 2016 (Wood et al. 2017), Patisiran FDA approval 2018 (Ledford 2018), Givosiran FDA approval in 2019 (US Food and Drug Administration (FDA) 2019) and Inclisiran FDA approval 2021 (US Food and Drug Administration (FDA) 2021), success has been modest and confined mainly to liver and local delivery indications. Further development of nucleic acid delivery systems will allow more accurate dosing and targeting of these therapeutics to a broader range of specific cells and tissues, reducing or eliminating toxicity. In recognition of this, NATA has launched a £6 M research funding call ([UKRI] UK Research and Innovation 2022) with the aim of funding an international academic-industry consortium to tackle the challenges faced with safe and efficient delivery of nucleic acid therapies. The successful consortium will be announced in summer 2022.

Oligonucleotide quantitative whole-body autoradiography (QWBA) hot or not? David Kenworthy (GSK, UK)

Synthetic oligonucleotides are an increasing part of pharma portfolios and they provide many atypical development challenges for chemistry and biology disciplines alike. Neither small nor large biopharm molecule, these synthetic, intermediate size molecules share characteristics of both. A forward looking review of the challenges of interpretation of quantitative tissue distribution and metabolism derived from radiolabelled oligonucleotides was shared. This was demonstrated and discussed with reference to literature and a review of GSK’s own experience of ADME development with drisapersen (GSK2402968, PRO051), a fully phosphorothioated, 2’O-methyl modified, exon skipping antisense oligonucleotide. The presenter questioned the reliability and the value of a radiolabelled approach for quantification of oligonucleotide (and metabolites), recognising the specific activities of metabolites formed following nuclease mediated cleavage will often be lower than that of the original radiolabelled oligonucleotide. Literature and experience have shown tissue distribution and metabolism of oligonucleotides is largely predictable. Radiolabelled ADME has traditionally often been used as a tool to quantify small molecule ADME, but the presenter questioned whether such an approach was quantitatively informative or even required at all for oligonucleotide ADME. Whilst several delegates agreed that previously conducted QWBA studies could be used to inform the tissue distribution of subsequent oligonucleotides in the same class, it was suggested that additional investigations, possibly using non-radiolabelled techniques, may be required to satisfy regulatory expectations for novel oligonucleotide modalities.

Multimodal imaging of therapeutic oligonucleotides, Prabuddha Mukherjee and Aneesh Alex (University of Illinois Urbana-Champaign (UIUC), US and GSK, US

For more efficient and selective uptake, antisense oligonucleotides (ASOs) are often conjugated with molecules with high binding affinity for transmembrane receptors. Triantennary N-acetyl-galactosamine conjugated phosphorothioate ASOs (GalNAc-PS-ASO) were developed to enhance targeted ASO delivery into liver via the hepatocyte-specific asialoglycoprotein receptor (ASGR). We assessed the kinetics of uptake and subsequent intracellular distribution of AlexaFluor 488 (AF488)-labelled PS-ASOs and GalNAc-PS-ASOs in mouse macrophages and primary mouse or rat hepatocytes monocultures as well as liver complex in vitro models (CN Bio Platform, www.cn-bio.com) using simultaneous Coherent Anti-Stokes Raman Scattering (CARS) and two-photon fluorescence (2PF) imaging (Mukherjee et al. 2022). The CARS modality captured the dynamic lipid distributions and overall morphology of the cells, 2PF measured the time- and dose-dependent intracellular localisation of ASOs. We found that both regular PS-ASOs and GalNAc-PS-ASOs were taken up at a similar rate in mouse macrophages. However, GalNAc-PS-ASOs exhibited a peripheral uptake distribution compared to a polar distribution by the regular untargeted PS-ASOs in hepatocytes. GalNAc-PS-ASOs showed improved uptake compared to PS-ASOs at earlier timepoints in CN Bio samples as well. This work demonstrates the relevance of multimodal optical imaging for elucidating the uptake mechanism, accumulation, and fate of different ASOs in liver cells that can be used in monocultures, complex in vitro models, and liver tissues to evaluate ASO distribution and activity.

Introduction to OligoNova initiative, Shalini Andersson (AstraZeneca, Sweden)

OligoNova was initiated in 2020 by the University of Gothenburg and AstraZeneca through support from SweLife and the Swedish Government Innovation Agency, Vinnova.

The aim of OligoNova is to gather national experts on therapeutic oligonucleotides representing academia, national regions, research and healthcare institutes, industries as well as small and medium sized enterprises (SMEs). The expectation is to also collaborate with international researchers and biotechs as the centre matures.

OligoNova is comprised of four cornerstones, the OligoNova Hub, the OligoNova Accelarate, the OligoNova Ventures and the OligoNova scientific network. https://oligonova.org/

The OligoNova Hub is a national research platform that will provide academic researchers in Sweden with the support and capabilities essential to transform ideas and discoveries into new therapeutic oligonucleotides that will benefit patients. The Hub is building expert capabilities for the design, synthesis, testing and evaluation of oligonucleotides to progress early drug projects from idea to in vitro proof of concept (PoC) and is currently placed within the AstraZeneca Bioventure Hub in Gothenburg, Sweden. The OligoNova Hub has received substantial funding from Knut and Alice Wallenberg Foundation, SciLifeLab and the University of Gothenburg.

The second cornerstone, the OligoNova Accelerate, will help drive the most promising projects from in vitro PoC to early clinical evaluation including access to expert advice and external capital. The investments required to progress these programmes into novel therapies for patients with unmet medical need will be funded through OligoNova Ventures consisting of industrial and institutional investors.

Oligonucleotides represent a powerful therapeutic modality capable of selectively impacting the production or splicing of RNA that might otherwise be difficult to target with classical small molecule drugs or biologics. This has been clearly demonstrated by the large number of oligonucleotides in clinical trials and successfully launched therapies on the market. However, specifically targeting mRNA in extra-hepatic tissues and cell types after systemic administration continues to be a challenge. The OligoNova scientific network aims to build national and international collaborations to tackle scientific challenges within the area of therapeutic oligonucleotides such as targeted delivery, enhanced cellular uptake and intracellular trafficking. All together OligoNova will help catalyse and translate novel ideas into oligonucleotide therapeutics that will benefit patients and strengthen research capabilities in the field in Sweden.

Update on the oligonucleotide safety working group (OSWG) PK/ADME subcommittee's recommendations, Cindy Berman (Independent Consultant, US)

This presentation summarised the recommendations of the ADME Subcommittee of the OSWG for the characterisation of absorption, distribution, metabolism and excretion of oligonucleotide therapeutics in nonclinical studies. In general, the recommended approach is similar to that for small molecule drugs. However, some differences in timing and/or scope may be warranted due to the greater consistency of results across oligonucleotide classes as compared with the diversity among small molecule classes. For some types of studies, a platform-based approach may be appropriate; once sufficient data are available for the platform, presentation of these data should be sufficient to support development of additional oligonucleotides of the same platform. These recommendations can serve as a starting point for nonclinical study design and foundation for discussions with regulatory agencies.

Drug metabolism and pharmacokinetics (DMPK)/ADME considerations of oligonucleotides in discovery and development, Rasmus Jansson-Löfmark (AstraZeneca, Sweden)

Oligonucleotides are an emerging drug modality with DMPK/ADME challenges both in terms of knowledge gaps and regulatory needs. The purpose of this talk was to provide a DMPK/ADME perspective on discovery and development of oligonucleotides ( antisense oligonucleotides and small interfering RNAs (siRNAs)). In both drug discovery and development phases, one should first consider what type of oligonucleotide chemistry is being used as this choice will steer activities and determine how much of platform chemistry can be applied. In discovery, for established chemistries – pharmacokinetic–pharmacodynamic modelling is a powerful tool to drive projects forward, both in terms of directing the project throughout the discovery phase and later guiding the design of early clinical studies. When generating DMPK data for oligonucleotides with established chemistries, effort should be directed towards addressing pharmacokinetics and pharmacodynamics questions, and tissue targeting. For novel chemistries/oligonucleotide-conjugates, a more extensive DMPK evaluation may be needed, e.g. plasma protein binding, in vitro metabolic stability and in vivo pharmacokinetics as part of the screening, and later for prediction of human pharmacokinetics. In development, the ICH (M3) guideline (2009) that applies for small molecules is generally used. Strategies or justifications for not conducting or back-loading studies can be worked up and confirmed with Health Authorities when there is an opportunity for an interaction. In general, in vitro drug–drug interaction (DDI) studies, such as transporter and cytochrome P450 (CYP) inhibition studies, and CYP induction studies are required but can be back-loaded until New Drug Application (NDA) submission. ADME studies using radiolabelled drug substance can be avoided if the key questions can be addressed via other reliable technologies, such as (semi) quantitative mass spectrometry.

Discussion

In the true spirit of the DMDG, the Oligonucleotide ADME meeting held on the 17th and 18th November provided an opportunity for DMPK scientists from across EU Pharma to share their experiences. While the event was virtual, the delegates had ample opportunity to discuss the pros and cons of various strategies and provide insights into the subsequent responses from the regulators.

The meeting was also enriched by the presence of representative of publicly funded initiatives (DARTER, NATA, OligoNova), demonstrating that many of the challenges faced in developing therapeutic oligonucleotides are common to all parties and the need for pre-competitive initiatives remain core to progression in this field. We were also able to gain insight into the academic contributions in the field of sub-organ and sub-cellular oligonucleotide distribution that will become key to our future understanding of oligonucleotide ADME.

In addition to the formal presentations described above, the meeting devoted time to a more general discussion driven by ‘hot topics’ raised during the previous sessions. These were divided between ‘how have you addressed this with your molecules’ and ‘do we really need to…’ type conversations. While the former question was covered well from the industrial presentations, the latter point was discussed in depth.

The scientific rational for running classical small molecule in vitro DDI assays (CYP inhibition and induction, transporters) was reviewed and the position of ‘because we can, we should’ was challenged. While the majority of the virtual room agreed that a paper argument could be written to exclude these studies from regulatory submissions, some agencies are noting emerging data (Kazmi et al. 2018) and are expecting this to be addressed at file. A similar regulatory position was identified in the need to conduct QWBA studies and while the distribution of subcutaneous dosed ASOs and siRNAs are well described in the literature, novel chemistries and dose routes may still need to be characterised in more detail.

Further discussions on the most appropriate bioanalytical methodologies for plasma and tissue pharmacokinetics as well as ADAs we touched on, but the group concluded that these should be addressed in a future meeting.

Concluding remarks

This oligonucleotide ADME workshop stands out from other fora by focussing on the discussion of oligonucleotide ADME related topics in relation to drug development of this class of molecules. The feedback from the delegates attending the virtual workshop was positive and the presentations stimulated fruitful discussions around the different ADME topics addressed. Diversity of opinion still remains, encouraging a scientific rationale-based approach to oligonucleotide ADME strategy.

Acknowledgements

The organisers would like to thank all speakers for their contributions, and to all participants for fruitful discussions. The authors would also like to acknowledge the DMDG secretariat (AssociAction Enterprises) for support with organizing the workshop.

Disclosure statement

No potential conflict of interest was reported by the author(s).