Today, I thought I’d share another one of my essays I had to do recently. This one looks at animal testing, problems concerning species differences and what we can do to avoid them. This essay is a little more sciency than my other one on living forever, so I’ll include the references this time. Here goes:
The use of non-human animals in the drug development process can attract criticism due to the issue of species differences. How significant is this problem and what strategies can be employed to minimise the impact of species differences?
Animal testing is a major tool in the drug development process, required by law before any new drug can enter the market. Animal models are set up to not only test the efficacy of a compound for its intended effect, but also to observe any potential side effects, to calculate a safe dosage for humans and to check for any addiction potential. Although animal testing is a legal requirement, implemented for our own safety, it is still only a model; a substitute for human physiology, whose results could be completely erroneous if they were derived from a poorly planned experiment. Differences between species are always a concern when setting up an appropriate animal model, and a lot of time is spent agonising over them to ensure any results obtained are both accurate and applicable to humans. When it comes to experimental design, species differences can be broadly classified into the following categories: anatomical/physiological differences, differences in metabolism and subsequent toxicity, pharmacological differences and behaviour.
This is perhaps the most obvious class of species difference. It is no good testing a drug on an animal and looking for effects that are physically impossible for the animal to manifest. Any tests carried out on one species with implications for another must only test parts of the physiology common to both species, or identify an analogous symptom that corresponds to the effect you are looking for.
A prime example of this kind of difference crops up when investigating the emetogenic potential of a drug – unfortunately, evolution has not provided rats with a vomiting reflex, so an different model would have to be devised looking for an alternative behaviour or using another species with a physiology closer to ours.
Metabolism & Toxicity Differences
Different species also metabolise drugs differently – either via different metabolic pathways or with different kinetics. As such, a drug toxic to one species may have little effect on another, which is particularly important when trying to determine the toxicity in humans. A drug’s LD50, the amount required to kill 50% of subjects in a particular sample, is usually given in mg/kg of body mass, scaled up from animal experiments. If a drug’s toxicity or pharmokinetics are only determined from one animal species and extrapolated for the average human, the data would not take into account any differences in metabolism that may be present, resulting in potentially extreme inaccuracies.
For example, dogs should never be given coffee or chocolate, as they are poor metabolisers of theobromine1, a xanthine alkaloid occurring naturally in both, as well as being a metabolite of caffeine. As little as 50g of chocolate can result in theobromine poisoning for small dogs, while humans can metabolise it fast enough without issue.
Similarly, metabolism of NSAIDs shows a huge variation across different species. The plasma half-life of aspirin ranges from 1 hour in ponies up to 37 hours in cats2, due to their poor glucuronidation ability, while dogs are more susceptible to aspirin’s gastrointestinal side effects3.
One final example would be the varying MPTP toxicity between species. MPTP can be formed as an unintended byproduct in the manufacture of MPPP, a synthetic opioid with great potential for abuse. MPTP on its own is not harmful, but MPP+, the natural metabolite of MPTP, is a potent neurotoxin. MPP+ is produced via MonoAmine Oxidase B in neuroglia and the capillary endothelia comprising the blood-brain barrier, and results in rapid-onset Parkinsonian symptoms barely indistinguishable from typical Parkinson’s disease4. These symptoms are also reduced by L-DOPA, a drug commonly used in Parkinson’s disease. Rats, however, are almost entirely immune to MPTP toxicity, most likely due to a different level of expression of MAO B5. Mice, on the other hand, do produce MPP+, but clear it from their brain in a matter of hours, unlike the primate brain, in which clearance can take days.
The chemical pathways and their associated protein machinery will not necessarily be structurally identical, or indeed act in the same way. Pathways may be more or less complex, depending on the species, with more or less scope for modulation by other factors. Receptors too may also differ in structure, ligand affinity and the type of G proteins they may couple with. All of these factors may be of huge importance when designing a drug with a particular molecular target in mind.
A few interesting cases have resulted from these types of differences. For a while, Leptin was theorised to suppress hunger, as knockout mice that did not express leptin or its associated receptor got fat. Giving leptin to those that could not express it themselves, but still possessed the appropriate receptor, caused them to lose weight6 – a potential gold mine if the results were also applicable to humans. Unfortunately, they were not. Leptin showed little effect in humans, as weight problems tended to concern signal transduction rather than a lack of leptin7, in much the same way as insulin-resistant diabetes.
Another, rather more serious example is that of TGN1412, a monoclonal antibody with not only a high affinity for the human CD28 receptor, but a strong agonist ability too. Originally intended to help patients with rheumatoid arthritis and B cell chronic lymphocytic leukaemia, TGN1412 was initially tested on animals and an apparently safe dosage calculated. Of the 6 volunteers hospitalised, each given a dose 500 times smaller than that given to their animal counterparts, 4 developed multiple organ failure as a result of cytokine storm8. Hopefully, this example highlights the importance of species difference; that it is a real issue and not just a theoretical concern.
The final category, and perhaps least obvious, is that concerning animal behaviour. Unfortunately for us, animals are not able to clearly express their feelings, so we are left to try and interpret that behaviour, which can be particularly difficult. Humans seem to have an intrinsic penchant for anthropomorphism – we are always unconsciously trying to attribute characteristics that are uniquely human, such as complex emotions or intention, onto animals and even non-living objects. Children are especially guilty of this, smacking a rock, perhaps, as a punishment because it tripped them up. It is only as we grow older and put in a little more thought that we realise that perhaps the rock was not to blame. With animal models, we must also put in that extra thought when it comes to interpreting an animal’s behaviour, instead of opting for the instinctive, humanised interpretation.
Other problems are encountered when we assume a particular behaviour is a result of a particular effect. For example, in the tail flick assay, designed to measure effects on nociception, analgesia is associated with an increased latency in moving the tail away from a heat source. Approving a new drug as an analgesic based on only this interpretation could be disastrous if the increased tail flick latency was instead due to a loss of muscle control or paralysis.
One final thought concerning animal behaviour, is that some behavioural responses may be unique to the species in question. For example, a hedgehog might curl up into a ball as a typical fear response. While this may be easy to interpret, other idiosyncratic responses may not.
A number of strategies have been devised for combating the issues species difference brings up, ranging from simple common sense to the rather more complex. An in-depth knowledge of the species under investigation is a good start. Experience and familiarity with a particular species will naturally lead to a better ability to read an animal’s behaviour, just as we become better at reading the people around us the longer we spend in their company. Someone new to animal work will be more likely to anthropomorphise, drawing instead from their experience with other people, whereas someone with ample experience could make a more accurate judgement. Another benefit from experience is that any of the more subtle differences between that species and us is more likely to spring to mind, reducing the risk of something important being overlooked. For example, rat models are a useful tool when studying the intestinal bioavailability of drugs, but are a poor choice when it comes to intestinal metabolism9.
Another strategy to reduce the risks imposed by any unknown or overlooked differences, and one that is required by law, is to test on more than one species. Doing so greatly reduces the chances that any observed response is unique to one species in particular, and is therefore likely to be exhibited by humans too.
Although there are an incredible number of individual species, some proteins remain relatively conserved. Working with these specific proteins that share a great deal of similarity between their human counterparts will likely lead to more reliable results. For example, the muscarinic receptor family has remained much the same throughout evolution such that the human and rat receptors share a very similar agonist/antagonist profile10. It is very likely that something acting on rat muscarinic receptors will elicit the same response in humans, making this an accurate model.
More recently, the latest tools and techniques of the genetic engineer promise to make animal models even more relevant. Genetic manipulation has already delivered knockout animals, not expressing particular genes, and transgenic animals, expressing genes belonging to another species, but in 2008 a chimeric mouse with 90% human hepatocytes (liver cells) was produced11. Until now, the best tool for studying the effects of drugs on the liver would be to use actual human liver (another strategy for overcoming species differences is to use human cells if possible), but the chimeric mouse has already shown great potential. The liver is mainly responsible for the pharmacokinetics of a drug, as it is the primary place that drugs are metabolised, which has subsequent effects on the toxicity and efficacy of that drug. The chimeric mouse has shown a similar pharmacokinetic profile to the human donor, as well as human-specific metabolites not ordinarily found in mice, making this an excellent model with which to study pharmacokinetics and toxicity. This advancement brings with it all the benefits of testing drugs on an actual human target, without any of the ethical considerations raised with human testing.
We humans are an animal species like any other, and we may have our own species-specific responses that are impossible to capture or anticipate with any animal model. It is important to remember that an animal model is just that – a model. Species differences will always be an issue; there are even idiosyncratic reactions to drugs within the same species, such as some humans being allergic to penicillin, so we can never eliminate these differences completely. Increasing research, awareness, criticisms from the animal rights campaigners and new genetic techniques will continue to help us reduce the severity of these issues until they can be reduced no further.
- Kahn CM, editor. The Merck Veterinary Manual. 9th Ed. New Jersey: Merck & Co., Inc; 2008.
- Boothe DM. The Analgesic, Antipyretic and Anti-inflammatory Drugs. In: Adams HR, editor. Veterinary Pharmacology and Therapeutics. 8th Ed. Iowa: Iwoa University Press; 2001. p. 433-454
- Crosby JT. Veterinary Questions and Answers – Can you give a dog or cat aspirin? [cited: 2008 Sept 02] About.com: Veterinary Medicine. Available from: http://vetmedicine.about.com/cs/altvetmedgeneral/a/dogcataspirin.htm
- Langston JW, Ballard P. Parkinsonism induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP): implications for treatment and the pathogenesis of Parkinson’s disease. Can J Neurol Sci. 1984 Feb;11(1 Suppl):160-165.
- William Langston JW. The Impact of MPTP on Parkinson’s Disease Research: Past, Present, and Future. In: Factor SA, Weiner WJ, editors. Parkinson’s Disease: Diagnosis and Clinical Management, New York: Demos Medical Publishing, 2002. p. 407-436
- Pelleymounter MA, Cullen MJ, Baker MB, et al. Effects of the obese gene product on body weight regulation in ob/ob mice. Science. 1995 Jul 28;269(5223):540-543
- Considine RV, Sinha MK, Heiman ML, et al. Serum immunoreactive-leptin concentrations in normal-weight and obese humans. N Engl J Med. 1996 Feb 1;334(5):292-295
- Suntharalingam G, Perry MR, Ward S, et al. Cytokine storm in a phase 1 trial of the anti-CD28 monoclonal antibody TGN1412. N Engl J Med. 2006 Sep 7;355(10):1018-1028
- Hurst S, Loi CM, Brodfuehrer J, El-Kattan A. Impact of physiological, physicochemical and biopharmaceutical factors in absorption and metabolism mechanisms on the drug oralbioavailability of rats and humans. Expert Opin Drug Metab Toxicol. 2007 Aug;3(4):469-489
- Venter JC, Eddy B, Hall LM, Fraser CM. Monoclonal antibodies detect the conservation of muscarinic cholinergic receptor structure from Drosophila to human brain and detect possible structural homology with alpha 1-adrenergic receptors. Proc Natl Acad Sci USA. 1984 Jan;81(1):272-276
- Katoh M, Tateno C, Yoshizato K, Yokoi T. Chimeric mouse with humanized liver. Toxicology. 2008 Apr 3;246(1):9-17