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Animal Testing & Species Differences

By John Clarke

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?

lab ratAnimal 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.

Anatomical/physiological Differences

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.
chicken-anatomy

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.

Pharmacological Differences

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.

Behavioural Differences

hedgehog ballThe 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.

Strategies

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.

References

  1. Kahn CM, editor. The Merck Veter­inary Manual. 9th Ed. New Jersey: Merck & Co., Inc; 2008.
  2. 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
  3. 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://​vet​medi​cine​.about​.com/​c​s​/​a​l​t​v​e​t​m​e​d​g​e​n​e​r​a​l​/​a​/​d​o​g​c​a​t​a​s​p​i​r​i​n​.​htm
  4. 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.
  5. 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
  6. 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
  7. 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
  8. 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
  9. 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
  10. 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
  11. Katoh M, Tateno C, Yosh­izato K, Yokoi T. Chi­meric mouse with human­ized liver. Tox­ic­o­logy. 2008 Apr 3;246(1):9 – 17

One Response to Animal Testing & Species Differences

  1. Hannah says:

    Really helpful with my school essay on IS IT MORALLY DEFENS­IBLE TO TEST ON ANIMALS. Thanks! :D

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