EthicsMedicine

In vivo modelling within pharmacological research, its limitations and alternative methods of research

Abstract

In vivo or animal testing has been relied on for years in drug development for studying drug dosages, therapeutic indices, contraindications, mechanisms of action and adverse reactions of drugs undergoing various levels of testing. This article aims to review the importance and basis of in vivo models in drug development and discuss the ethical constraints that have been raised about the method in the recent years. The novel discovery of alternative methods has sparked debates about whether alternatives have the potential to eliminate the need for in vivo testing. Though alternative drug testing strategies have seen some promising developments, particularly in fulfilling ethical objectives, modern methods still have a long way to go before the use of animals in research can be completely eliminated.

Introduction

In vivo testing (Latin for testing “in the living”), widely known as animal testing, is the use of animals for experiments in universities, medical schools, and pharmaceutical laboratories, for instance. It is a reliable tool for studying the life processes of animals in health and disease as well as to test for safety and success of new drugs. Animal testing therefore plays a central role in paving the way for new treatments and drugs in clinical practice.

Today, a great deal about the physiology and anatomy of the human body is known by testing on animals that are biologically similar to humans. Non-human animal models usually serve as the penultimate testing method for a drug before it progresses to clinical trials in humans [1]. It is common for animal research to involve mice, rats, birds and fish, among others (Fig. 1).

Figure 1. Pie chart showing the animals most commonly used in experimental procedures in the United Kingdom in 2015, with mice making up the largest fraction. Reprinted from [2]

Chimpanzees, which share 99% of their DNA with humans respectively, also serve as reliable models for researching diseases due to their similar genetic makeup [3]. Studying them enables scientists to explore how the disease affects the body and the kind of immune response that is triggered, which then makes it possible for scientists to develop potential therapies [4].

However in vivo testing does not eliminate the need for testing on humans, instead it allows scientists to determine whether a drug is too dangerous to be tested on humans. In a letter to the British Medical Journal, pharmacologist William D H Carey asked his reader to consider a hypothetical study for the profiling of four novel anti-HIV drugs on animals, the results of which would report drugs A, B and C-administered animals being killed while drug D being well-tolerated with no adverse effects [5]. He then explained that it would still be inappropriate for drug D to be given to humans, because its success in animals does not guarantee the same in humans. Instead it would be required to be microdosed in humans as part of phase 0 clinical trials (more on microdosing in later section). His statement reiterated the need for us to appreciate that animal testing is not capable of replacing human testing, but rather as a stepping-stone in the drug development process.

The limitations and ethical constraints of in vivo testing

According to the Office of Technology Assessment approximately 17 to 22 million animals were used in 1986 for research in laboratories across the United States [6]. These figures sparked outrage and garnered criticism from animal welfare activists around the world as well as the general public with regards to the pain and suffering animals were being subject to. In a survey that reported the general public’s views on animal testing, 73% of the questioned individuals agreed to banning animal testing due to it being unethical. Their responses have been recorded in Fig. 2.


Figure 2. Proportion of questioned individuals in agreement / disagreement with the statement: “Experimenting on animals is unethical and should be banned” Reprinted from [7]


There have been several arguments made by activists – that it is morally wrong to let such a huge number of animals suffer and die because animals possess the same inherent living rights as humans and the human benefits from animal testing remains intangible [8]. Even if the ethical limitations are to be disregarded, it has been argued that some animals do not express the human pathologies that are research priorities e.g. HIV, schizophrenia, Parkinson’s disease and major heart diseases. To combat this inconsistency however, transgenic animal models are now used where human genes are inserted into test animals’ DNA to develop certain diseases artificially for the purpose of human benefit [8].

Even so, in vivo testing by inducing diseases belittles the role of genetics, psychological factors and inter-personal experiences which all impact an organism’s response to drugs [8,9]. These experiments are artificially regulated (often in windowless rooms, away from natural habitat), which may produce unreliable results due to behavioural changes [9]. It must be noted that in vivo experiments take place in tightly regulated conditions, and thus do not take into account the differences in size, diet and life expectancy of the animals being tested and these parameters are not standardised to give a reliable equivalent dosage for humans [9,10]. In studies where dose conversion calculations are performed, it is common for researchers to simply extrapolate the dosage/pharmacokinetic parameters from animals to humans based on just body weight (allometric scaling). In order to get more accurate conversions, a complex combination of other parameters such as oxygen utilisation, caloric expenditure, basal metabolism, blood volume and circulating plasma protein also need to be accounted for [11]. These considerations can further lengthen and complicate the process which again raises the dilemma of evaluating whether the benefit and reliability of experimental outcomes outweigh the efforts and cost of these methods.

The 3 R’s of animal testing

Scientists are now much more aware about finding ways to overcome the ethical constraints associated with animal testing and acknowledge that it should be carried out not only in the most humane and responsible way, but the number and duration of animals used in drug screening is also reduced, in addition to the need for non-animal alternatives to animal research [12]. To achieve this goal, modern science has been developing and implementing various types of computerised modelling, biokinetic-modelling, and in vitro systems involving tissue cultures as testing strategies [13]. This technological progress is greatly a result of the “The Principles of Humane Experimental Technique” published by William Russell and Rex Burch in 1959 in which the three R’s, reduce, replace and refine, serve as a framework for a more ethically justified animal research [14]. Russell and Burch proposed that more priority should be given to replace animal-oriented experiments with non-animal alternatives, and if animals must absolutely be used, that the number of animals are reduced, and experiments are refined in such a manner that animals are subject to minimum pain and distress. Following this, the UK government went further than other countries by making it a requirement by law for animal-using researchers to submit a proposal for the critical evaluation of the suitability and cost-benefit analysis of using animals [15]. If approved, researchers are wary of unnecessary suffering of animals and as a result take certain actions to maximise reduction and refinement [15]. For example, pain-relieving drugs can be given to prevent any post-surgical pain in animals, as well as taking other actions such as improving laboratory living environments may aid with their behavioural and emotional well-being during experiments [14,15].

Computerised models

Computerised (In silico) models can be used to stimulate diseases and accurately predict how different drugs behave in the human body based on existing databases [12]. The drugs that clear the primary screening process as harmless are then used for in vivo experimentation thus lowering the overall number of animals used and fulfilling the “reduction” objective of the 3R’s.

Quantitative structure-activity relationships (QSARs) computationally determine a substance’s biological activity or likeliness of toxicity based on its physicochemical properties using statistical approaches and mathematical equations. QSARs have garnered broad scientific attention, particularly in the pharmaceutical industry for toxicity studies in drug discovery projects [16,17]. QSARs allow for relatively fast and reproducible data for the prediction of activity of previously untested molecules, and it has certainly been successful in markedly saving time, cost and human resources [17]

However, these computerised models too have their own shortcomings- though 2D QSARs are ideal for efficiently supporting high-speed technological methods, the results they generate can be less realistic as they tend to ignore the 3D conformation of the ligand being studied (in drug trials, the ligand can be a drug molecule interacting with a receptor). This can result in less-than-ideal accuracy for a biological endpoint which is dependent on small molecule-receptor interactions [17]. The updated form of QSAR is known as 3D-QSAR, which considers the surrounding biological environment of molecules during visualisation, taking into account the heterogeneity and bioactive conformations of each molecule. Nonetheless 3D-QSARs require expensive 3D conformers which are known to be very sensitive to alignment procedures; these time-consuming processes are not present with 2D-QSARs, which makes it a point of comparison when choosing which method is suitable for a problem [17,18]. Considering everything, while QSARs serve as excellent models for initial structure-activity correlations to be made for a molecule, the findings from these methods still need to be confirmed with whole animals due to their inconsistencies [10].

Microdosing and fMRI

Functional magnetic resonance imaging (fMRI) and microdosing are modern advancements that have ensured that human volunteers are studied without having any detrimental effects on them. fMRI is an imaging technique that measures brain activity by detecting changes associated with blood flow; its main principle is that when a cerebral region is upregulated due to neuronal activity (may be triggered by drugs), the increased neural firing results in increased energy requirement and thus increased oxygen saturation due to blood inflow [19]. fMRI allows the human brain to be studied and visualised as specifically as possible whilst also allowing researchers to reversibly induce brain disorders, especially in transgenics [16]. Though fMRI is included in preclinical animal studies, it also has immense potential in target identification, clinical trials and post-approval studies. The potential applications of fMRI in the stepwise development of central nervous system (CNS) drugs have been outlined in Fig. 3.

 

Figure 3. Role of fMRI in CNS drug discovery development, and the likely locus of imaging at each developmental stage. Flowchart collated using images and information from Borsook et al. [16]

 

However 25 years after fMRI being practised and yielding interesting images of the brain during feelings of love, drug addiction, video games and disease states, Eklund et al.’s study revealed there were significant inconsistencies between 3 well-known and widely-used fMRI software packages. While it was theoretically expected that there would be around 5% of false positives, the experiment showed false-positive rates of upto 70% [20]. These findings indicated that some results were so off-the-mark that they could be showing brain activity in areas where there was none, putting into risk the reliability of over 40,000 fMRI studies [20]. This major scientific revelation may have had a part to play in the lack of research/slowdown of research into fMRI. However Eklund et al. believe that this is not a setback; focusing more on the validation of existing methods and identifying limitations is the key. It can be positively said that fMRI, if further developed, can play a significant role in drug development for disorders associated with the CNS in the near future, including the common neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease.

Often fMRI is used in conjunction with microdosing; microdosing gives extremely small harmless dosages of drugs to an individual after which fMRI, PET scans and the like are used to monitor the effect of the drug on a microscopic level [21]. One of the biggest advantages of microdosing is that the administered concentrations are always under 0.1 mg, making it one of the safest and most risk-free ways of drug trialling. Two principles form the basis of microdosing (a) that there is no better pharmacological indicator of a drug’s pharmacokinetics in humans than humans and (b) that upto a certain concentration, clearance, volume of distribution, and bioavailability are not affected by the dosage. However the biggest disadvantage of microdosing is that the pharmacokinetic effect and solubility of a drug in the blood is only studied at a microscopic level, and since it does not demonstrate preclinical or phase I-level clinical/phenotypic activity, it is known as a Phase 0 study [22]. Microdosing is still not a standard testing paradigm and it is underreported in research papers for this reason. However it holds an impressive potential as a tool for determining initial pharmacokinetic profile of medications in humans, which ultimately may reduce drug attrition at later stages of clinical trials and allow for early availability of drugs, benefitting both patients and the pharmaceutical industry [23].

In Vitro modelling

In Vitro modelling, using human tissues and body fluids isolated from their biological environments can also help achieve the replacement aspect from the 3R’s. At the current level of technology, in vitro is primarily used for measuring distribution, biotransformation (alteration in body) and absorption as well as for the detection of toxic properties of therapeutic drugs [12]. While the advantage of directly using human cells over animals comes into play, in vitro testing focuses on the reactions at cellular level, not taking into account the interactions between all the organ systems of the body, particularly factors that can affect the absorption, distribution, metabolism, and excretion (ADME) of drugs [24]. These include endocrine hormones, leukocytes of the immune system and most importantly metabolising enzymes, such as cytochrome P450, which are responsible for metabolising the majority of small molecule drugs. There is also a remarkable inter-individual variation observed in drug response, particularly attributed to genetic differences in these enzymes [25]. The homogeneity of in vitro assays does not allow for these variabilities to be considered during drug development and as a result cell-based assays fail to demonstrate ADME. This makes it a challenge to predict how long the drug will be part of a system; whether it will be broken down before it becomes toxic or whether it will circulate in the bloodstream long enough to have an effect.

Concluding remarks

The benefits of in vivo research have been enormous, especially in drug discovery and development, and it is because of advances resulting from animal testing that people all over the world benefit by having a prolonged and improved quality of life. It would have dire consequences for public health and pharmaceutical research if in vivo research were to be completely abandoned. Although animal use cannot yet be completely replaced, scientists must prioritise animal well-being through reduction and refinement. With this sentiment, the modern alternatives and simulations that have been discussed so far have undoubtedly reduced the number of animals used in research. However, despite having a pivotal role in early preclinical testing, a successful in vitro outcome is only the first criterion for progression into in vivo testing. Therefore in vitro testing does not eliminate the need for animal testing, but minimises animal use due to the early detection of poor drug candidates. Alternatively, computerised models and fMRI imaging are increasingly being implemented as adjunctive testing strategies, though they are unable to replace animal testing within their current scope of action.

A final statement from the author is that the above-mentioned alternatives can at most complement, not eliminate the need for animal testing. Reinforcing Russell and Burch’s 3R’s for ethical testing, every care should be taken to replace animals whenever possible and if used, experiments should be designed responsibly and more compassionately towards all biological beings.

References

1. “Experimenting on animals.” BBC. 2014. http://www.bbc.co.uk/ethics/animals/using/experiments_1.shtml last accessed [25 May, 2020]

2. “Annual Statistics of Scientific Procedures on Living Animals Great Britain” UK Home Office. 2015. https://assets.publishing.service.gov.uk/government/uploads/system/uploads/attachment_data/file/537708/scientific-procedures-living-animals-2015.pdf last accessed [28 May, 2020]

3. “Why Are Animals Necessary in Biomedical Research?” California Biomedical Research Association. 2014. https://ca-biomed.org/CSBR/pdf/fs-whynecessary.pdf last accessed [1 February, 2019]

4. “Why Animals are Used.” Animal Research Info. 2014. http://www.animalresearch.info/en/designing-research/why-animals-are-used/ last accessed [1 February, 2019]

5. Greek, R. 2002. “Animal studies and HIV research.” BMJ 324: 236a-236.

6. Swindler, D. 1987. “Alternatives To Animal Use In Research, Testing, And Education.” American Journal Of Physical Anthropology 74 (2): 276-277.

7. “Experimenting on Animals is Unethical and Should be Banned” Animal Friends Croatia. n.d. https://www.prijatelji-zivotinja.hr/index.en.php?id=903 last accessed [28 May, 2020]

8. “Arguments against animal testing.” Cruelty Free International. 2015. https://www.crueltyfreeinternational.org/why-we-do-it/arguments-against-animal-testing last accessed [1 February, 2019]

9. Akhtar, A. 2015. “The Flaws And Human Harms Of Animal Experimentation”. Cambridge Quarterly Of Healthcare Ethics 24(4): 407-419.

10. “In Vivo Studies”. 2019. Belgium Bioelectromagnetics Group. https://www.bbemg.ulg.ac.be/en/main-research/research-methods/info-invivo-studies.html last accessed [4 February, 2019]

11. Shin, J., Seol, I. and Son, C. 2019. “Interpretation Of Animal Dose And Human Equivalent Dose For Drug Development”. The Journal Of Korean Oriental Medicine 31(3): 1-7.

12. Sharma, K., Arora, T., Joshi, V., Rathor, N., Mehta, A., Mehta, K. and Mediratta, P. 2011. “Substitute Of Animals In Drug Research: An Approach Towards Fulfillment Of 4R′S”. Indian Journal Of Pharmaceutical Sciences 73(1): 1-6.

13. Hester, R., and Harrison, R. 2006. Alternatives To Animal Testing. Cambridge, U.K.: Royal Society of Chemistry.

14. Russell, W. M. S, and R. L Burch. 1959 (as reprinted 1992). The Principles Of Humane Experimental Technique. Wheathampstead (UK): Universities Federation for Animal Welfare.

15. Festing, S. and Wilkinson, R, 2007. “The ethics of animal research.” EMBO reports 8(6): 526-530.

16. Borsook, D., Becerra, L., and Hargreaves, R. 2006. “A Role For Fmri In Optimizing CNS Drug Development”. Nature Reviews Drug Discovery 5 (5): 411-425.

17.Perkins, R., Fang, H., Tong, W. and Welsh, W., 2003. “Quantitative structure–activity relationship methods: perspectives on drug discovery and toxicology.” Environmental Toxicology and Chemistry 22(8): 1666.

18. Roncaglioni, A. and Benfenati, E. 2008. “In Silico-Aided Prediction Of Biological Properties Of Chemicals: Oestrogen Receptor-Mediated Effects”. Chemical Society Reviews 37(3): 441-50.

19. Glover, G., 2011. “Overview of Functional Magnetic Resonance Imaging” Neurosurgery Clinics of North America 22(2): 133-139.

20. Eklund, A., Nichols, T. and Knutsson, H. 2016. “Cluster Failure: Why Fmri Inferences For Spatial Extent Have Inflated False-Positive Rates”. Proceedings Of The National Academy Of Sciences 113 (28): 7900-7905.

21. “Alternatives to Animal Testing.” People for the Ethical Treatment of Animals (PETA). 2016. http://www.peta.org/issues/animals-used-for-experimentation/alternatives-animal-testing/ last accessed [2 February, 2019]

22. Atkinson, A. 2013. Principles Of Clinical Pharmacology. 3rd ed. Elsevier.

23. Usha Rani, P. and Naidu, M. 2008. “Phase 0 – Microdosing strategy in clinical trials.” Indian Journal of Pharmacology 40(6):240-242.

24. Vela, J. 2014. In Vivo Models For Drug Discovery. Weinheim: Wiley-VCH.

25. Thummel, K. and Lin, Y. 2014. “Sources of Interindividual Variability.” Methods in Molecular Biology 1113:363-415.

About the Author

Salonee is currently pursuing a master’s degree in Drug Discovery and Pharmaceutical Sciences at the University of Nottingham. An undergraduate assignment sparked her interest in the subject and drove her to dig deeper into the role of ethics in animal testing. Her research interests include immunopharmacology and of late the therapeutic potential of plant-derived compounds in humans. 

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