Advances in technology are paving the way to reduce and potentially replace toxicity and drug safety testing in animals, Katrina Megget reports
Around the world, an estimated 115m animals are used in research each year to test pharmaceuticals, food additives, household cleaners, industrial chemicals and agrochemicals – even cosmetics, despite bans in many countries. But questions are being raised over the efficiency of animal testing, which is costly and time consuming and does not always provide accurate results that are applicable to humans.
According to Carla Owen, Chief Executive of Animal Free Research UK, which funds research into alternative technologies, animals are poor predictors of human reactions to chemicals and drugs and have limited relevance to many human diseases. In many cases, species-specific differences in physiologies and genetic makeup mean animal tests cannot reliably predict efficacy or toxicity of drugs in humans. ‘Around 90% of drugs that proved promising in animal tests fail in clinical trials, mostly due to toxicity or poor efficacy not detected in animals,’ she says.
One of the most high-profile examples of this was at London’s Northwick Park Hospital in 2006, when a Phase 1 clinical study of the drug TGN1412 (theralizumab) caused life-threatening side effects in all six previously healthy human volunteers, even though the drug was previously found safe in animals and trial participants were given a 500-times lower dose. According to Thomas Hartung, a toxicologist and Director of the Center for Alternatives to Animal Testing at Johns Hopkins University, US, animal testing is becoming less of a gold standard. ‘We are not 70kg rats.’
Technology could provide the solution. Several countries, including China, already have research programmes for developing alternative technologies, while many, including the UK, have banned the testing of cosmetics on animals, although the industry had been exploring non-animal alternatives long before this. According to Emma Meredith, Director General of the UK’s Cosmetic, Toiletry and Perfumery Association, the commitment of the industry to embrace non-animal alternatives has led to the development and validation of more than 20 alternative test methods, such as tissue cultures.
While 2D cell culture, where human cells grow as a monolayer on a flat surface, has become one of the most popular alternatives within the chemical industry, its limited organ structure and physiological conditions means more advanced technologies that more accurately mimic the human environment are the potential gamechangers. In recent years, there have been huge advances, with the promise of a dramatic shift in the way chemical toxicity and drug safety testing is done. ‘Advances in non-animal technologies are signalling a new era in medical research and human safety assessment and they offer real hope of an end to animal testing,’ says Owen.
These new technologies include 3D bioprinting (C&I, 2021, 85, 2, 30). Like traditional 3D printing, bioprinting uses a computer-aided design to construct a three-dimensional model made from ‘bioinks’. This generally consists of printing live human cells, such as stem cells, into hydrogels or extracellular matrix-type material, which provides signals for the cells to organise themselves and develop into human tissues or organs.
‘There is currently a big hype around 3D bioprinting and more and more researchers and companies are entering the field,’ says Jens Kurreck, professor of applied biochemistry at the Technische Universität Berlin, Germany. Following the EU animal testing ban on cosmetics in 2013, French firm L’Oréal partnered with US start-up Organovo to use bioprinted human skin tissue to test its cosmetics, and in pharma, Servier uses Poietis’ bioprinting technology for the production of liver tissues to be used in its preclinical research.
Kurreck’s team is focusing on two miniature bioprinted organs – the liver and lung – and is working on a human cancer model utilising technology from the US-based bioink firm CELLINK. The lung model is currently being used to develop an inhibitor for coronavirus, Kurreck says.
Bioprinted mini-organs, or organoids, show promise in shaking up the animal testing space. These clusters of stem cells are just a few micrometres to five millimetres in size but form miniature organs. Liver, kidney, intestines, as well as cancer models, have been created as organoids and experts believe these in vitro models are better suited for drug and chemical testing by studying the behaviour of cells in a more human environment. ‘We strongly believe that these humanised models may overcome the species-specific differences that we experience with results from animal testing,’ Kurreck says.
Bioprinted mini-organs, or organoids, are typically clusters of stem cells that are just a few micrometres to 5mm in size.
A lung-on-a-chip has been produced, which is lined with lung cells and capillary cells to emulate the airway and includes the mechanical process of breathing.
Organ chip technology can provide new human models for tests where no animal models currently exist.
Roche says the new toxicology methods have allowed the company to reduce the number of animals in experimental use by almost 40% over the past eight years.
However, organoids do have some limitations around physiology and environmental responses, such as vascular flow or immune response, which are significant in understanding chemical effects. A new technology, dubbed organ-on-a-chip, helps address this limitation. These microfluidic devices of clear, flexible polymer are about the size of a USB memory stick and contain hollow channels where different organoids are placed or printed and connected together to form a 3D human-like environment perfect for testing drugs and chemicals.
Harvard University’s Wyss Institute in Massachusetts, US, is one of the frontrunners in this technology. Its lung-on-a-chip is lined with lung cells and capillary cells to emulate the airway, and includes the mechanical process of breathing. Air flows over the lung cells while liquid medium containing blood cells flows through the channel below the capillary cell layer. The Institute has models for intestine, kidney, skin, liver and blood-brain barrier, and has even linked 10 different organ chips together by transferring fluid between their common vascular channels in order to mimic whole-body physiology and essentially create a human-body-on-a-chip.
‘Organ chips are essentially living, three-dimensional cross-sections of major functional units of whole living organs,’ explains Donald Ingber, professor and Founding Director of the Wyss Institute for Biologically Inspired Engineering. He says they have been scientifically shown to ‘faithfully recapitulate’ normal physiology, disease states and responses to drugs, toxins and radiation exhibited by many different human organs, while body-on-chips can predict drug pharmacokinetics in humans. Furthermore, this technology can provide new human models for tests where no animal models currently exist, like testing for human-specific antibodies or rare human genetic diseases, he says.
‘Human organ chips are better alternatives than animals because they better replicate human responses and don’t require killing sentient beings,’ Ingber says. ‘They also offer a window on molecular scale activities inside living human cells growing within a tissue and organ context, which enables insight into human-relevant disease mechanisms, as well as mechanisms of action and toxicity of drugs.’ According to Ingber, most of the world’s pharma companies are using organs-on-a-chip technology and it is now starting to be used in other industries such as cosmetics and agrochemicals.
While in vitro alternatives are fast developing, other scientists are looking at what computers can bring to drug and chemical testing. These in silico models are leveraging artificial intelligence (AI) to screen chemicals and simulate the human body and predict drug dosages and effects. Hartung and colleagues, for instance, have developed an algorithm that can predict the toxicity of a chemical based on its similarity to others.
First, the team generated a database of 10,000 known chemicals that mapped relationships between chemical structures and toxic properties, based in part on 800,000 separate toxicology tests. Then they developed additional software to determine the toxicity of new compounds based on their properties compared with those in the database. The team found that the AI system could predict toxicity more accurately than a single animal test.
We envisage a future where drug development programmes require little to no animal tests prior to entering clinical trials.
Jo Varshney founder and Chief Executive, VeriSIM Life, San Francisco, US
On average, the AI programme was 87% accurate in reproducing animal test results. In contrast, any single animal test only had an 81% chance, on average, of obtaining the same result for toxicity when repeated (Toxicological Sciences, 2018; 165(1):198). The US Food and Drug Administration and the US Environmental Protection Agency are now evaluating the technology to assess whether it could replace a significant proportion of animal tests, while Australian authorities have accepted it for registering chemicals including cosmetic ingredients, says Hartung.
VeriSIM Life in San Francisco has developed BIOiSIM, an artificial intelligence-enabled biosimulation platform that integrates in vivo physiological models across seven species, including humans, to generate a ‘digital’ representation of animals and humans. ‘This enables simulated tests, so we could significantly reduce reliance on animals or forgo animal testing in some scenarios altogether during drug development,’ says Jo Varshney, Founder and Chief Executive of VeriSIM Life. BIOiSIM has been used to test more than 650 compounds in partnership with one top pharma firm and is being used to test drug combinations against Covid-19. Varshney says BIOiSIM is more than 81% accurate in its predictions and could reduce animal testing by more than 50%.
But is this technology enough to replace animal testing? There is no denying the new technologies are promising – pharma company Roche, for instance, has embraced the technological alternatives and says the new methods have allowed the company to reduce the number of animals in experimental use by almost 40% over the past eight years. But Ingber says these technologies, as they currently stand, are unlikely to completely replace all animal testing because of human body complexity and the need to review whole-body functions. Organ chips are currently unable to totally recreate this, and for many AI programmes, their development and improved precision will require animal research to continue in order to generate the necessary data to feed into the AI databases.
Even for cosmetics it’s not that simple. Meredith notes the exceptional progress in the industry but says major scientific challenges remain. ‘Science today is unable to predict complex, multi-stage toxicological events such as reproductive toxicity, carcinogenicity, chronic toxicity and some specific-organ effects. Until new technologies and integrated methods for safety assessment covering all endpoints are established and accepted by regulators, the cosmetics industry is limited in its ability to use completely new ingredients.’ She says research is by no means completed and is ongoing to further develop technologies.
But it’s not just about the technology, explains Kristie Sullivan, VP for research policy at US-based non-profit Physicians Committee for Responsible Medicine. ‘Technology certainly helps – we need technological advancements in order to develop better models and there has been quite a bit of progress in finding alternatives; but the replacement of animal testing with those alternatives is another step altogether. Technology is not the only solution – we also need to change the laws and policies.’ These are broader challenges to be addressed, she notes. For instance, there is the current regulatory requirement that drugs be tested in at least two different species of live mammal before they move into human clinical trials, and uptake of new tech in general by regulatory authorities has been slow.
In addition, this ‘traditional’ view of how research is done means broader adoption of new technology could be a challenge, both Sullivan and Owen highlight. Not only is there a ‘serious knowledge gap’ among regulatory inspectors about the available alternative technologies but the editorial policies of some scientific journals that require validating their approaches against animal models are outdated, Owen says. ‘People may be nervous or resistant to move away from their animal testing comfort zone. A change in mindset from animal tests being the gold standard is vital,’ she says. ‘Key is for the government to provide supportive infrastructure, funding, education and training and regulations that enable human-relevant research.’
However, Jennifer Harris, Medicines Development Policy Manager at the Association of the British Pharmaceutical Industry, says there is a commitment for change within the life sciences sector but that it requires a collaborative approach with regulators to turn it into reality. ‘This isn’t about mindset change, it’s more about developing the evidence to demonstrate how new medicines, treatments and vaccines can be developed safely without the use of animals.’
Hartung agrees, saying the system isn’t flexible to change. He points to the vital need for incentives to critically assess and validate the alternative technologies and for new approaches to be funded so that they can be used. ‘Waiting 10-20 years for alternative methods to be used is not attractive for biotech companies… public safety and a lot of money is at stake.’
Yet despite these challenges and the need for technology to improve, scientists are optimistic that change is afoot. ‘I do expect that over the next five years, we will see organ chips being used to replace the first animal model for generation of data in support of moving a drug into clinical trials,’ says Ingber. In the AI space too, Varshney says it’s reasonable to expect significant results could be achieved within five to seven years as computational platforms continue to outperform animal tests. ‘We envisage a future where drug development programmes require little to no animal tests prior to entering clinical trials.’
In many ways, Owen hopes some of that future is already here given the shakeup Covid-19 has had on R&D. ‘This demonstrates a real and exciting opportunity for progress and change and modernisation in medical research. There has never been a more pertinent moment to embrace new approach methodologies and set up investment in advancing technologies that are representative of the whole human system.’
Image credit: WLADIMIR BULGAR/SCIENCE PHOTO LIBRARY