Pharmacology

How Animal Research Aids Pharmacology

Testing on animals is a significant tool in the drug development process, required by law before any new compound can enter a patient. Animal models are set up to not only test the effectiveness of a new drug, but also to observe any potential side effects and to calculate a safe dosage for humans.

Although animal testing is a legal requirement, implemented for our own safety, it must be remembered that it is only a model; a substitute for human physiology, whose results could be completely erroneous if they were derived from a poorly planned experiment. Species differences are a concern when setting up an appropriate animal model, and a lot of time is spent researching 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: physiological differences, differences in metabolism and toxicity, pharmacological differences and behaviour.

Physiology

It is pointless to test a drug on an animal and look for effects that are physically impossible for the animal to manifest. Any tests carried out on one species with implications for another must focus on 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. Rats do not have vomit reflex and therefore a different model, most likely a different species, needs to be used that has a physiology closer to ours.

Metabolism and Toxicity

Drugs metabolise differently in different species, either via other 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 lethal inaccuracies.

For example, dogs should never be given coffee or chocolate. They are poor metabolisers of theobromine, a xanthine alkaloid that occurrs naturally in both. 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 cats, due to their poor glucuronidation ability, while dogs are more susceptible to aspirin’s gastrointestinal side effects.

Pharmacology

Chemical pathways and their associated protein machinery will not necessarily be structurally identical, or indeed act in the same way, in differing species. 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 became obese. Giving leptin to those that could not express it themselves, but still possessed the appropriate receptor, caused them to lose weight – 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 leptin, 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 storm. This highlights the importance of species difference; that it is a real issue and not just a theoretical concern.

Behaviour

Animals are not able to clearly express their feelings to us humans.

During testing 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 pain thresholds, analgesia is associated with an increased latency in moving the tail away from a given 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.

Furthermore, behavioural responses may be specific to the species under investigation. 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.

Strategies

A number of strategies have been devised for combating these issues. In-depth knowledge of the species under investigation is a must. Experience and familiarity with a particular species will lead to a better ability to understand an animal’s behaviour. New animal workers are likely to be more anthropomorphic, drawing from their experience with other people instead of using experience to make a more accurate conclusion.

Another strategy to reduce risks imposed by unknown or overlooked differences, and one eequired by law, is to use at least two differnt species. Doing so reduces the chances that unwanted responses will be seen in different species and therefore they are likely to be exhibited by humans too.

Some proteins remain relatively conserved across a wide range of species. Concentrating on these specific proteins that share a great deal of similarity between their human counterparts are more liekly to lead to more reliable results. For example, the muscarinic receptor family has been well conserved throughout evolution such that the human and rat receptors share a very similar agonist/antagonist profile. Therefore, it is very probable that drugs acting on rat muscarinic receptors will elicit similar responses in humans.

More recently, genetic engineer promises to make animal models even more clinically 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 produced. 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.

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