“Judah is going to cure cancer in two years”, said James Watson, a Nobel laureate “If you have cancer and you are a mouse, we can take good care of you”, replied the eminent oncologist Judah Folkman
Dissatisfaction with something, as a rule, provokes action. So when creating new drugs, the main stimulus is the so-called 'unmet need': when there is no effective treatment against the disease, or the available drugs are not effective enough or have significant side effects. Progress in the study of nosogenic mechanisms and the development of new technologies can facilitate the emergence of new drugs.1,2 An illustrative example which combines all these stimuli is the emergence of such promising fields of study as immunotherapy and targeted therapy (or 'molecular targeting') in oncology.
So where do new drugs come from?
Currently, the search for new drugs usually begins with the disease. In other words, in understanding the pathogenesis – which processes are disturbed in the body by a particular disease, its pathogenesis – the researchers are looking for possible actors: target molecules and substances that can act upon these targets. In this way, study of the virus' interaction with host cells led to the creation of acyclovir, the first highly selective antiviral drug.3 Acyclovir selectively inhibits the reproduction of certain viruses (mainly the herpes simplex virus) and prevents the synthesis of their DNA while not affecting the DNA synthesis of human cells. In 1988, the American scientists Gertrude Elion and Dr. George Hitchings received the Nobel Prize in Physiology or Medicine for their discovery of important principles for molecular target drug treatment, of which acyclovir is an example.4
In the case when known substance has properties that are potentially useful in medicine, the task of scientists and pharmacologists comes down to the search for its application in therapy. Some tetrahydrocannabinol-based drugs are good examples of this approach. The well-known property of cannabis-based drugs to stimulate the appetite has been used to create weight loss drugs and to alleviate the effects of cancer chemotherapy or HIV infection.5
There is also, of course, always the element of chance. Some drugs are discovered unexpectedly. The most famous example here is the discovery of antibiotics, or rather, the first antibiotic – penicillin – by Alexander Fleming in 1928. As story goes, after returning from a holiday Fleming found that certain mould that had grown in dishes of staphylococcus culture killed the bacteria around them. He realised that the fungus produced an antibacterial substance and named it after the fungus penicillin.6
Let us suppose that in one of the aforementioned ways we have obtained a new potential drug against a particular disease. However, it does not matter how promising the drug's intended action is – the drug still has a long way to go through trials to prove its efficacy and safety before pharmaceutical companies start production. The basis of these trials is pre-clinical studies – various studies of the drug before trials on humans, i.e. before clinical trials. It can be said that the scope for 'creativity' is unlimited, and, as they say, any means are good. For example, in our age of information technology, the computer, which used to be merely a medium for analysis, has become a medium for conducting experiments. Thanks to the emergence of huge biological databases and an increase in computing power, the effects of a new drug can now be verified by computer modelling without getting up from one's desk, or, as scientists call it, in silico (a pseudo-Latin word derived from ‘in silicon’ – a reference to the wide use of silicon in production of computer chips). With the help of computer methods, specialists in the field of bioinformatics 'cast' for the roles of potential new drugs, testing the conditions and features of their interaction with their targets. Moreover, the efficacy and toxicity of drugs are tested using virtual biological processes and systems.7
Unfortunately, it is not yet possible to get by completely with just 'dry' methods. Not everything can be modelled using computer simulation (although attempts are being made ) and scientists still have to 'get their hands dirty' with 'wet' chemical and biological experiments. The efficacy of new drugs is tested on a variety of cell cultures, including human ones. Complex model of our skin has been created8. These are the so-called in vitro methods, i.e. ‘in a test tube’ (Latin ‘in the glass’). Sometimes, when attempting to reproduce the most natural conditions in order to achieve greater specificity, pre-clinical experiments are carried out on organs and tissues isolated from a living organism, ex vivo (Latin ‘out of the living’). It is impossible, however, to evaluate the effect of a substance overall organism using these methods, despite their convenience, availability and affordability. The effect of a substance in an organism shown in vitro may simply not manifest itself in the body due to the nature of metabolism, target localisation and the presence of natural barriers. It is also impossible to predict all of a drug's effects, since unintended targets may be found in the body, and affecting them can lead to serious side effects9-10. So far, computer simulation and in vitro studies have been able to reduce the duration and cost of animal in vivo studies (Latin ‘within the living’), but have not completely replaced them. Many experiments cannot be carried out on less complex systems than the whole organism.
Weirdly enough, quite disparate organisms, from zebrafish to dwarf pigs, are used for in vivo research into new drugs. However, the undisputed leaders here are, of course, mice and rats, averaging 70-85% of all laboratory animals.10-12 The choice of test subject and an adequate model of the disease or syndrome are critical when constructing a pre-clinical study.11 There are currently many approaches to modelling human pathology in laboratory animals. Firstly, it is worth mentioning genetically modified animals, in which human diseases and disorders are replicated by changing the activity of certain genes or by inserting new genes into transgenic animals.11 Thus, rat strains have been created which have high blood pressure, a predisposition to obesity and developing certain cancers, or which suffer from Alzheimer's disease.13-14 However, not all disease causes are found in specific genes, moreover, the 'genes' of the disease may be as yet unknown. Animal models can also be based on following a special diet (diabetes mellitus modelling), being kept in certain conditions (changing the day-night lighting hours, raising the noise level for insomnia modelling)15 or pharmacological effects. The animal can be infected with the necessary virus or bacterium in order to simulate infectious diseases or older specimens can be used to study age-related disorders. A stress model can be obtained by separating the pups from their mother early or by placing a young male in a cage with a more aggressive and dominant older male.16 It is important to understand that absolutely any model can be created, the main requirement is to reproduce the disorders observed in humans as accurately as possible and, ideally, the factors that caused them. We must however remember that a model is an abstraction, which tries to describe the real system. The validity of the conclusions made using a model therefore depends on the quality of the model. E.g., for this purpose, a set of criteria was proposed for assessing models of nervous system disorders in animals: face validity – the manifestation of symptoms similar to the clinical condition; construct validity – the similarity of the underlying mechanisms and causes of the disease; and predictive validity – a similar response to well-known and effective drugs11. Models that accurately reproduce human diseases also exist. A good example is the genetic model of Huntington's disease, as it is caused by the mutation of a single gene.11 However, models mostly recreate only some symptoms of the disease. There are still no accurate models of autoimmune diseases or neurodegenerative diseases such as Alzheimer's.11 It is difficult to create an accurate model when the causes of the disorder are still not fully understood. How is it possible to recreate 'human' behavioural and psychiatric disorders such as depression or anxiety in animals? Here, in addition to the often-unknown causes of diseases, creating a model is also hampered by the subjective, contradictory and often intermittent nature of their symptoms.11 Rats cannot comment on their condition or talk about fears and anxieties.
In addition to choosing a model of the disease, it is also very important to choose the right methods to evaluate the drug's efficacy. They must be adequate and sensitive enough for each specific case. It is important to consider their limitations and specifics. If unsuitable methods are used, there is a chance of not detecting the drug's effect and coming to a false conclusion. For instance, one can overlook the effect of an anti-epileptic drug when evaluating only the behaviour of animals, whereas a long-term video EEG recording will reliably reveal changes in the onset of seizures17. Many different tests are specific enough for particular models of disorders. Spatial memory abnormalities are often assessed by forcing rats to swim in a special pool with opaque water and find a platform to escape the water (Morris water maze), or to look for an escape in a flat arena with holes along the edge (Barnes maze). In elevated plus-maze stress models, the maze has two open and two enclosed arms, and the time the animal spends in the enclosed areas is recorded – it is usually greater for anxious animals.16
One rodent stress test used in pre-clinical experiments is the elevated plus-maze.
A more anxious animal will try to spend more time in the labyrinth's enclosed arms.
Scientists have even created a virtual reality 'room' for rodents: the rat has the illusion of staying in and moving between different environments thanks a spherical treadmill and the screens surrounding it.18
Virtual reality room for rodents
A rat running on a spinning ball has the illusion of moving in different directions in different conditions created by the screens located around the treadmill. Various parameters of the animal can be registered online, right up to the activity of individual brain cells.
In order for pre-clinical studies to be reproducible and to make it easier to use the results in subsequent clinical trials, there are various recommendations for their preparation, implementation, and analysis. There are special documents for conducting such studies – standards. Thus, the global system of standards aimed at ensuring the consistency and reliability of laboratory results is the so-called Good Laboratory Practice, (GLP) 19. This is something of a mark of quality. GLP standards are quite stringent and are mainly used in safety testing substances and materials. However, regardless of national laws and regulations, it is important in any research to adhere to such key principles as the blind method – by which the researcher does not know which animal is given the drug and which is not, randomisation – the random distribution of animals into experimental groups, adequate control groups, proper analysis and statistical manipulation of results. As for the ethics of biomedical research on animals, they are enshrined in national legislation as well as a number of international agreements and guidelines.10 In the European Union, experiments on laboratory animals conducted for research purposes are regulated by special Directive 2010/63/EU and local ethics committees, which are usually present in every scientific research institute.10 The directive establishes the so-called 'three Rs principle' – Replacement, Reduction and Refinement – which primarily involves reducing the number of experimental animals where they can be adequately replaced by alternative methods.8-10
In conclusion, I would like to mention perhaps the most urgent and painful problem of pre-clinical studies – the problem of translating data obtained from models into clinical trials. Why do a large number of drugs that are successful in pre-clinical studies fail clinical trials? A striking example is the drug Endostatin, which, having shown extreme efficacy in the fight against cancer in mice without side effects, was only partially effective in humans12. There are many factors20-21 that make it difficult to interpret data obtained from pre-clinical studies. Some of these factors are down to the scientists – flaws in methodological approaches such as non-observance of randomisation or blinding, lack of control groups, and incorrect statistical analysis20-21, while others can be attributed to the established rules in the scientific community. Scientific journals practically never publish the results when a drug does not have the desired effect.20-21. Another example is standardisation: all around the world, it is usually customary to use only one kind of animal in experiments. Moreover, they are young males from a pure strain living in practically sterile and unnatural conditions. The sample is extremely homogeneous, in contrast to the humanity11. However, one of the main factors is the limitations of animal models, in particular in rodents11-12. Although we are genetically very close to rats and mice, and many physiological processes are similar, we are not mice, we diverged from a common ancestor 85 million years ago and took different evolutionary paths.12 First and foremost, of course, we differ in size – an average human is 2500 times larger than a mouse! This difference forms the basis of many others such as metabolism (a mouse's heart beats at about 600 bpm) and life expectancy, which, in turn, affect the structure of internal organs, tissues and cells, and the biochemistry of processes.12 Unlike humans, mice produce vitamin C, their haemoglobin has a lower oxygen-binding capacity, mice have more metabolically active tissues (liver, kidneys), no appendix, and are more resistant to infections and increased cholesterol.12 Mice and rats are more prone to neoplasms (cancer), and less prone to cardiovascular diseases.12 In addition, rats and mice do not vomit11 and they have completely different bacteria in the intestine and immune system.12 Therefore, it is impractical to test antiemetic drugs on these animals or study immune diseases. Given the many differences in rodent and human biology, it is not surprising that the disease patterns of these species can be different, and therefore the results of studies conducted on rodents may differ greatly from the results that would be observed in humans. Therefore, the model approach in biology and medicine cannot be taken as the ultimate truth, as Nobel laureate Jacques Monod sarcastically put it: "Anything found to be true of E. coli must also be true of elephants.” The problem of translating data from test subjects to humans is significant, but research on rodents and other species is important and yet has no alternative. A more careful approach towards their planning and interpretation is needed, which takes into account both the differences and similarities between the chosen model type and a human one.
1. 1. Step 1: discovery and development. 2018. FDA.
2. Making a medicine. Step 1: pre-discovery. (2015). EUPATI.
3. Bryan-Marrugo, O.L., Ramos-Jiménez, J., Barrera-Saldaña, H., Rojas-Martinez, A., Vidaltamayo, R., & Rivas-Estilla, A.M. (2015). History and progress of antiviral drugs: From acyclovir to direct-acting antiviral agents (DAAs) for Hepatitis C. http://www.elsevier.es/en-revista-medicina-universitaria-304-articulo-history-progress-antiviral-drugs-from-S166557961500037X
4. The Nobel Prize in Physiology or Medicine 1988.
5. Robson, P.J. Therapeutic potential of cannabinoid medicines. Drug Test Anal. 2014. 6 (1-2): 24-30.
6. Diggins, F.W. The true history of the discovery of penicillin, with refutation of the misinformation in the literature. Br J Biomed Sci. 1999. 56 (2): 83-93.
7. Ekins S, Mestres J, Testa B. In silico pharmacology for drug discovery: methods for virtual ligand screening and profiling. Br J Pharmacol. 2007 Sep;152(1):9-20. doi: 10.1038/sj.bjp.0707305. Epub 2007 Jun 4. PMID: 17549047; PMCID: PMC1978274.E.
8. Sutterby, P. Thurgood,S. Baratchi, K. Khoshmanesh, E. Pirogova,VIEW.2022,3, 20210012.https://doi.org/10.1002/VIW.20210012
9. In Vivo vs. In Vitro: What Are the Differences? https://www.verywellhealth.com/what-does-in-vivo-and-in-vitro-mean-2249118
10. The Ethics of Animal Models in Preclinical Testing https://www.news-medical.net/life-sciences/The-Ethics-of-Animal-Models-in-Preclinical-Testing.aspx
11. McGonigle, P., Ruggeri, B. (2014). Animal models of human disease: Challenges in enabling translation. Biochemical Pharmacology, 87 (1), 162-171.
12. Perlman, R.L. (2016). Mouse Models of Human Disease: An Evolutionary Perspective. Evolution, Medicine, and Public Health, eow014.
13. Curated disease models. European Mouse Mutant Archive.
14. MICE & SERVICES. The Jackson Laboratories (Bar Harbor, USA).
15. Revel, F. G., Gottowik, J., Gatti, S., Wettstein, J. G., Moreau, J.-L. (2009). Rodent models of insomnia: A review of experimental procedures that induce sleep disturbances. Neuroscience & Biobehavioral Reviews, 33 (6), 874–899.
16. Wang Q., Timberlake M.A., Prall K., Dwivedi Y. The recent progress in animal models of depression. Prog Neuropsychopharmacol Biol Psychiatry. 2017. 77: 99–109. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5605906/
17. Cambiaghi M., Magri L., Cursi M. Importance of EEG in validating the chronic effects of drugs: suggestions from animal models of epilepsy treated with rapamycin. Seizure. 2015. 27: 30-9.
18. Harvey C.D., Collman F., Dombeck D.A., Tank D.W. Intracellular dynamics of hippocampal place cells during virtual navigation. Nature. 2009 Oct 15; 461(7266):941-6. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2771429/
20. Caestecker M., Humphreys B.D., Liu K.D., Fissell W.H., Cerda J., Nolin T.D., Askenazi D., Mour G., Harrell F.E. Jr., Pullen N., Okusa M.D., Faubel S. Bridging Translation by Improving Preclinical Study Design in AKI. J Am Soc Nephrol. 2015. 26 (12): 2905-16.
21. Begley C.G., Ellis L.M.: Drug development: Raise standards for preclinical cancer research Nature. 483: 531–533, 2012.