One of the 3Rs stands for ‘Reduce’

“Not responding is a response – we are equally responsible for what we don’t do.”  (Jonathan Safran Foer, 2011).

Life-saving therapeutics and vaccines undergo a sophisticated array of both in-vitro, and later in the drug development process, in-vivo testing.  Different animal models are used, with the aid of establishing drug safety, as well as parameters of use in human beings.

The importance of ADME Tox Testing

DMPK, or Drug Metabolism and Pharmacokinetics, is an important part of studies often referred to as ADME (Absorption, Distribution, Metabolism, and Elimination).

  • Absorption (how much and how fast, often referred to as the absorbed fraction or bioavailability)
  • Distribution (where the drug is distributed, how fast and how extensive)
  • Metabolism (how fast, what mechanism/route, what metabolite is formed, and whether they are
  • active or toxic)
  • Elimination (how fast, which route).

In the drug discovery process, early in vitro ADME screening and in vivo PK profiling provide a basis for choosing new molecular entities (NMEs) and lead compounds that have desirable drug metabolism, PK or safety profiles, necessary for drug candidate selection (CS) and late‐stage preclinical and clinical development. The ADME properties of a drug allow the drug developer to understand the safety and efficacy data required for regulatory approval.

Toxicology tests are often a part of this process, yielding the acronym ADMET.

Today, such studies are performed both in vitro and in vivo, and have led to more standardised procedures across the pharmaceutical industry.

Importance of the 3Rs in Drug Discovery

There has been understandable concern over the ways in which animals have been used and treated as part of this process.  As such, the principles of the 3Rs (Replacement, Reduction and Refinement) were developed over 50 years ago providing a framework for performing more humane animal research.  Since then they have been embedded in national and international legislation and regulations on the use of animals in scientific procedures, as well as in the policies of organisations that fund or conduct animal research.  Opinion polls of public attitudes consistently show that support for animal research is conditional on the 3Rs being put into practice.

Tools that enable cloud-based remote operation, tracking and sharing of critical protocols have become an essential requirement

Increased effort has gone into the development of so called ‘alternative’ methods over the last decade.

Replacement and Refinement of Animal Models

Replacement refers to technologies or approaches which directly replace or avoid the use of animals in experiments where they would otherwise have been used, for example the use of methods employing human embryonic stem cells as alternative ways of conducting ADMET studies.  Refinement refers to methods that minimise the pain, suffering, distress or lasting harm that may be experienced by research animals, and which improve their welfare. Refinement applies to all aspects of animal use, from their housing and husbandry to the scientific procedures performed on them.

By contrast, reduction refers to methods that minimise the number of animals used per experiment or study consistent with the scientific aims. It is essential for reduction that studies with animals are appropriately designed and analysed to ensure robust and reproducible findings.

Funders have taken a stronger position regarding the use of animal testing in founded research.  The European Union’s Horizon 2020 program, for example, forbids use of animal models in its funded research, rather advocating methods validated through organisations such as the European Centre for the Validation of alternative methods (ECVAM), based at the Joint Research Centre in Ispra, Italy.

Reducing the need for animal models by minimising unnecessary waste

Reduction also includes methods which allow the information gathered per animal in an experiment to be maximised in order to reduce the use of additional animals. Examples of this include the micro-sampling of blood, where small volumes enable repeat sampling in the same animal. In these scenarios, it is important to ensure that reducing the number of animals used is balanced against any additional suffering that might be caused by their repeated use.  Sharing data and resources (e.g. animals, tissues and equipment) between research groups and organisations can also contribute to reduction.

It’s still about the ‘reproducibility crisis’!

Regarding ‘reduction’ much emphasis is placed on the importance of minimising the number of animals used in a study.  Over the past several years, it’s become increasingly apparent that many lab studies, especially in the life sciences, are not reproducible. As a result, many putative drug targets or diagnostic biomarkers can’t be validated. Some estimates suggest that more than 50% of all published life sciences research is irreproducible, and some indicate that the figure might be even higher.  The problem flew below the radar for years. A 2012 comment in Nature by C. Glenn Begley, a former vice president at Amgen, and Lee M. Ellis, an oncologist at the University of Texas M. D. Anderson Cancer Center, drew attention to the problem. They described Amgen scientists’ attempts to replicate the key findings in 53 “landmark” fundamental cancer studies that claimed to identify potential new drug targets. They were able to replicate the findings in only 11% of the cases.

This ‘concern’ has not dissipated but has triggered a number of subsequent studies aimed at identifying the reasons for such has high levels of irreproducibility.  There a number of causes, which vary from the deliberate falsification of research, with increased pressure being brought to bear on the peer review process, to the tools we use in the laboratory and the ways in which those can contribute to erroneous data.  These include, but are not limited to, the means by which powders (e.g. precision weighing scale) and liquids (e.g. pipettes) are handled, mixed and transferred; as well as the way in which one research group might interpret and repeat the work of another.  This latter point might seem odd when considered alongside tools but bear in mind that the way in which the tools are used depends upon an accurate description and interpretation of the protocol used, an important area of research and development in its own right.

In order to respect the 3Rs, researchers must constantly strive to ensure that the latest techniques tools are fully used.  Pipetting is one such cause of error, as is the incorrect interpretation and replication of an experiment protocol. 

Supporting the 3Rs with ‘the Connected Lab’

It is important to reduce unnecessary replication of studies due to such errors if the means exist to mitigate the cause.  Andrew Alliance developed its OneLab cloud-native software with the intent of minimizing error in setting up pipettes on their Andrew+ pipetting robot or Pipette+ smart pipetting system (they are programmed remotely), and ensuring full experiment traceability.  Moreover, it also ensures accurate sharing of protocols between different research groups, alongside the even greater benefit of a ‘connected laboratory’ where the implications of one experimental action (e.g. weighing) are able to inform other experimental actions (e.g. pipetting) or vice versa.  Put another way, it is about getting smarter in the way we do things in the laboratory, including minimizing waste.

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