By Associate Professor Merran Govendir, Sydney School of Veterianry Science, The University of Sydney

 

Koalas have adapted to detoxify their eucalypt leaf diet, but these adaptations reduce the efficacy of their medicines

 

This was first published in Chemistry in Australia journal September 2019

https://chemaust.raci.org.au/sites/default/files/pdf/2019/CiA_Sep%3AOct%202019.pdf pp 16-44

 

The koala is one of the most iconic of the Australian marsupials. It is immediately recognisable as an Australian symbol and greatly loved by the Australian people. Additionally, it is estimated that koalas add significant value to the Australian economy of between AUD $1.1 – 2.5 billion annually by way of domestic and international tourism (NSW Government 2015 http://www.environment.nsw.gov.au/animals/thekoala.htm).

With the Anthropocene era upon us, survival of our wild koalas is threatened. Urban development, forestry and mining are fragmenting and destroying their habitat, especially that located proximal to the hinterland of cities and towns along the east coast of mainland Australia (Species Profile and Threats Database 2019 http://www.environment.gov.au/sprat.). Increasing frequency of droughts and bushfires also threaten habitat and animal survival. Koalas being struck by cars while crossing freeways and roads, attacks by feral and pet dogs, contribute to the alarming reduction in their numbers. Wild koalas can be infected by many infectious diseases, one of these being the bacterial disease chlamydiosis, which can result in painful inflammation of the membranes around the eye (conjunctivitis) and / or serious painful infections of the urinary-genital tract which invariably results in incontinence and infertility. As a result of disease and trauma, koalas are treated medically at wildlife hospitals in significant numbers, annually.

Koalas had been administered medicines such as antibiotics and analgesics at the same dosages as for dogs and cats, for decades. Application of canine and feline medicine dosages to koalas was probably due to the fact that the koala is comparable in body mass to a small to middle size dog (the average weight range of a mature koala is 5 to 8 kilograms with some of the larger males reaching 10 kg). But surprisingly, it has only been in the last fifteen years that is has been recognised that treating koalas with those medicine dosages for dogs and cats was not appropriate, due to the significant differences in diet and digestive anatomy between these species. This article will outline some of the anatomical and physiological adaptations of koalas to digest and absorb their diet and then provide an example why this is significant when dosing them with medicines.

The koala is classified as a specialised folivore, i.e. it has an almost exclusive eucalypt leaf diet and has anatomical and physiological adaptations for detoxifying this diet. This diet is low in minerals, proteins and carbohydrates (such as sugars and starches) but extremely high in oils. Eucalyptus leaves contain high concentrations of plant secondary metabolites (PSMs). Most plants contain PSMs as a defence to avoid ingestion by herbivores or insects, or attack by microbial disease. However eucalyptus leaves have many PSMs such as lignin which inhibits gastrointestinal (git) microbial digestion of plant cell-wall constituents, tannins which bind to nutrient protein inhibiting its gut absorption, essential oils (or terpenoids), polyphenolic compounds and formylated phloroglucinol compounds (i.e. 1,3,5-benzenetriol), with these latter compounds being toxic to animal cells. Consequently the koala’s diet has one of the highest concentrations of ingested PSMs, and is one of the most toxic diets of any terrestrial animal. Even a small proportion of the koala’s daily dietary intake of PSMs, especially the terpenoid concentration, would induce toxicity in people (Pass et al., 2001, Xenobiotica, 31, 205-221). Therefore koalas have to expend significant energy to minimise PSM absorption and maximise their elimination. This constant and great energy demand to detoxify their diet is a contributory factor to their characteristic sleepy demeanour.

 

Factors that affect nutrient absorption from the git

There are a couple of factors that affect the rate of food absorption from the koala’s git. The eucalypt leaf ingested mass within the stomach and short small intestine is extremely fibrous and although there is some PSM breakdown by normal gut microbes it takes a long time to break down these substances at these locations. It is the duodenum (the first section of the small intestine) where the dietary nutrients and PSMs are absorbed into the blood stream. However the small intestine of the koala is relatively short compared to that in carnivores and even other herbivores. Koalas have the largest hindgut (caecum) relative to the rest of their digestive tract of all marsupials and an expanded proximal colon. This large colon and hindgut acts as a fermentation chamber to facilitate decomposition and eventual elimination of residual git PSMs.

 

Factors that accelerate absorbed PSMs and their elimination

Once those nutrients and PSMs from the eucalyptus leaves have been absorbed from the git into the blood stream, the liver has an important role for further breaking down the toxic PSMs. The liver is the principal organ for metabolism of oily / fat-soluble substances in the blood. The word ‘metabolism’ can have many different meanings depending on context, but pharmacological metabolism which is discussed here, means that the original parent molecule in the blood is catalysed and undergoes chemical conversion into one or more metabolites prior to elimination.

Pharmacological metabolism may occur by one or two ‘phases’ to catabolize the oily parent molecule into one or more, water soluble metabolites which can be excreted in urine and / or via bile and into the faeces. Cytochrome (CYP) P450 monooxygenases, are a multi-gene family of enzymes that have a significant role as catalysts for many metabolic phase I reactions whereby the absorbed molecule undergoes either hydroxylation, dealkylation or oxidation, and ring-opening and reduction can also occur. Although CYPs’ gene sequence and function is not greatly dissimilar between species; species differences of CYP activity with respect to substrate specificity and rate of metabolic activity occurs. This enzyme superfamily is subdivided into various families e.g. CYP1, CYP2, CYP3 etc. This division into CYP families is based on the similarity of gene sequences (for a CYP gene to be assigned to a family there must be > 40% amino sequence similarity with the rest of the members of that family), and these families are further divided into sub-families e.g. CYP2A, CYP2B, and CYP2C etc (> 55% amino acid sequence similarity). However the difference between CYP families and subfamilies along divisions of amino acid sequence divergence results in the different CYP families catabolising different phase I chemical reactions as well as differences in enzymic activity.

Investigations into the metabolism of various substrates by marsupial CYPs has occurred over the last few decades concluding that some CYP metabolism pathways, but not all, are recognized to have superior activity in the koala (Liapis et al. 2000 Comp. Biochem. Physiol. C. 127, 351-357; El-Merhibi et al. 2007 Aus. J Ecotox. 13, 53-64). In people, CYP2C is subdivided into four isoforms (CYP2C8, CYP2C9, CYP2C18, and CYP2C19) and is reportedly responsible for the clearance of approximately 15% of those medicines that undergo phase I reactions including non-steroidal anti-inflammatory drugs  (NSAIDs), however koala CYP2C activity is ten times that of rats (Liapis et al. 2000  Comp. Biochem. Physiol. C. 127, 351-357). Recently the koala genome has been sequenced and it was surprising that the greatest number of genes identified was those for the CYP2C enzymes (Johnson et al. 2018 Nat. Gen. 50, 1102-1111). This high number of genes for CYP2C has not been observed in any other species’ genome identified to date, probably reflecting that a great variety of genes are required for the diverse range of enzymes required to detoxify a eucalypt leaf diet.

Phase II metabolism is characterized by conjugation of a glucuronyl, sulfate, methyl, acetyl or glycyl moiety to either the parent molecule, or the phase I metabolite, via transaminases such as uridine diphosphate (UDP)-glucuronosyltransferases (UGTs) and sulfotransferases. Glucuronidation is an important pathway for the excretion of phenolic PSMs in koalas (McLean et al. 2003 J. Chem. Ecol. 29, 1465-1477). Thus in those plant eating marsupials that have been studied, they use both phase I (oxidative) and phase II (conjugative) metabolic reactions, which target different dietary PSMs (McLean et al. 2003  J. Chem. Ecol. 29, 1465-1477) and these different biotransformation pathways can likewise, target specific medicines.

 

Why is this information important with respect to giving koalas medicines?

Understanding the above information has resulted in recent changes in recommendations for treating koalas with medicines. One of the best examples is that NSAID meloxicam has been demonstrated to have little therapeutic effect when administered to koalas.

Meloxicam is one of the most frequently prescribed NSAIDs for people and animals in Australia and globally. It is used for its anti-inflammatory and analgesic properties for animals.  One of the trade-names of this NSAID for people is ‘Mobic’, or ‘Metacam’ for pets and now there are many generic meloxicam trade names for both people and pets. Meloxicam was the most popular analgesic administered orally to injured koalas for analgesia. Our group found that when it was administered by mouth to koalas, meloxicam had very poor absorption in contrast to the excellent absorption in people, dogs and cats. We think that the poor git absorption seen in the koala is due to the drug binding to the fibrous ingesta in the gut and not being free to be absorbed by the duodenal wall. In contrast, the relatively low fibrous diet of people and carnivorous pets, does not inhibit meloxicam’s oral absorption. So the first lesson learnt there was little point giving this drug (and many others) orally to koalas.  But there was a further very important lesson to learn with administering meloxicam.  We can also administer the meloxicam to koalas by injection straight into their blood (i.e. an intravenous injection) or inject into their muscles (intramuscular injection) or tissues (subcutaneous injection), where the local capillaries pick up medicine in the blood. Going back to the fact that koalas are known to have very efficient CYP oxidative pathways to detoxify terpenoids, this very efficient oxidative metabolism pathway is the same pathway that the koala uses to catabolise meloxicam. Due to this active pathway, meloxicam is eliminated in less than 2 hours in koalas (Kimble et al, 2012 J. Vet. Pharmacol. Ther. 36, 486-493) compared with other species that have much slower oxidative pathways such as in the dog where a single dose of meloxicam is eliminated relatively slowly i.e. over 24 hours, and 15 to 20 hours in people. So the second lesson is that due to the koala’s innate superior oxidative metabolism pathway, current formulations of meloxicam don’t last long enough in the koala to provide analgesia. So when possible, veterinarians that treat koalas realise that when giving medicine to koalas it is advisable to avoid oral administration, preferably administer the drug by injection; and additionally there is a need to check the rate of elimination of the drug to ensure it is in the body long enough to be efficacious.

CiA

The koala / meloxicam story summarises a few of the issues we have observed when medications are administered to koalas. We are the only group researching this area. We have conducted many studies, and continue to conduct studies to improve the efficacy of many antibiotics and analgesics administered to koalas. Our biggest current challenges are to improve antibiotic treatment for chlamydiosis and to find analgesics that only need to be administered once daily to koalas, but provides significant pain relief over that 24 hour period.