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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

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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! 😀

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