physiology and pharmacology of the brushtail possum gastrointestinal tract: relationship to the...

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Advanced Drug Delivery Revie

Physiology and pharmacology of the brushtail possum gastrointestinal tract:Relationship to the human gastrointestinal tract☆

Arlene McDowell a,⁎, Bernie J. McLeod b

a School of Pharmacy, University of Otago, P.O. Box 56, Dunedin, 9054, New Zealandb AgResearch, Invermay Agricultural Centre, Private Bag 50034, Mosgiel, 9024, New Zealand

Received 30 April 2007; accepted 19 June 2007Available online 16 August 2007

Abstract

Oral formulations are typically based on studies from eutherian animal models. This review introduces information relating to oral formulationsfor a marsupial species, the Australian brushtail possum (Trichosurus vulpecula) that has arisen from research into new methods for controllingthis species — a major vertebrate pest in New Zealand. Morphologically, the gastrointestinal tract of the brushtail possum is similar to that ofhindgut fermenting eutherian species, but there are some striking differences in function. Limited data suggests that the pharmacokinetics andbioavailability of administered drugs are similar to that in eutherian species, but there is some evidence that possums may have specificmechanisms for handling the intake of plant toxins and xenobiotics. The development of oral formulations for a free-ranging pest species presentsseveral challenges above those encountered in the development of therapeutic formulations for humans and domestic animals. Use of a marsupialanimal model may lead to new strategies for oral formulations in humans.© 2007 Elsevier B.V. All rights reserved.

Keywords: Oral delivery; Marsupial; Caecum; LHRH; Gastrointestinal pH; Transit time; Trichosurus vulpecula

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11221.1. The common brushtail possum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11221.2. The brushtail possum in New Zealand . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11221.3. Developing oral delivery formulations for possums . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1122

2. Gastrointestinal tract physiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11222.1. Morphology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11222.2. Gastrointestinal transit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11232.3. Gastrointestinal pH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11242.4. Microflora . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1125

3. Barriers to oral delivery of peptides and proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11253.1. Proteolytic activity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11253.2. Permeability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11263.3. Epithelial cell function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1127

Abbreviations: CSM; colonic separating mechanism; GI gastrointestinal; 99mTc; radiolabeled technetium; BSA; bovine serum albumin; LHRH; luteinizinghormone releasing hormone; DGGE; denaturing gradient gel electrophoresis; EDTA; ethylenediamine tetra-acetic acid; SDA; sodium deoxycholic acid; DTT;dithiothreitol; PSM; plant secondary metabolite.☆ This review is part of the Advanced Drug Delivery Reviews theme issue on “Prediction of Therapeutic and Drug Delivery Outcomes Using Animal Models”.⁎ Corresponding author. Tel.: +64 3 479 7145; fax: +64 3 479 7034.E-mail addresses: [email protected] (A. McDowell), [email protected] (B.J. McLeod).

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1122 A. McDowell, B.J. McLeod / Advanced Drug Delivery Reviews 59 (2007) 1121–1132

4. Pharmacology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11274.1. Metabolism of ingested toxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11274.2. Drug pharmacokinetics in possums . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1128

5. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1129References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1129

1. Introduction

1.1. The common brushtail possum

The common brushtail possum (Trichosurus vulpecula Kerr)is a nocturnal, arboreal, marsupial endemic to Australia with anadult body weight of between 1.5 and 3.0 kg. Marsupials are aseparate lineage of mammals that diverged from eutherian1

mammals about 130 million years ago [1]. Marsupials radiatedin Australasia and the group includes many anatomicallydiverse species such as kangaroos (Macropus spp.) and thekoala (Phascolarctos cinereus). The common brushtail possum,one of 28 possum species in Australia (Family Phalangeridae),is distinct from the American opossums (genus Didelphis,Family Didelphidae).

Although the brushtail possum is usually considered to be ageneralist folivore, there is increasing evidence that they obtainadditional energy and/or nutrients from other sources. Brushtailpossums are amongst the smallest folivorous mammals and thusface severe problems in meeting their energy requirements froma leaf diet alone [2]. There is evidence that possums, at leastthose in New Zealand, predate on birds eggs and fledglings [3],invertebrates [4] and molluscs [5]. In our own possum colony,where possums are housed as groups [6], brushtail possumsfrequently kill and eat sparrows [7]. The brushtail possum istherefore, an opportunistic folivore that eats a wide range offoliar and non-foliar foods, including a variety of animals [8].

1.2. The brushtail possum in New Zealand

The brushtail possum was deliberately introduced into NewZealand during the mid-1800s and is now the country's mostsignificant vertebrate pest [9]. It is a major ecological threat toNew Zealand's indigenous biodiversity [10] and an economicthreat as a vector for bovine tuberculosis [11]. Current controlmethods rely largely on poisons, for example sodium mono-fluoroacetate (compound 1080 [12]). However, the use ofpoisons is becoming increasingly unpopular for welfare reasonsand for the impact of poisons on non-target species. In addition,the scale of the possum problem in New Zealand is so large thatpoisons alone have a limited effect on reducing the populationof brushtail possums on a national level. Consequently, anumber of novel biocontrol strategies are currently beinginvestigated [13–15], including contraceptives and sterilants to

1 Eutherians are a sub-class of mammals that includes placental mammals(e.g. humans). Marsupials belong to the sub-class Metatheria. This classifica-tion is based on reproductive features.

interfere with reproduction and lethal toxins that may bespecific to possums or to marsupials in general.

There are no reliable methods of determining a population ofbrushtail possums, but it has been estimated that there may be asmany as 70 million possums in New Zealand, spread acrossmore than 90% of the land mass, including remote andinaccessible bushland. Consequently, the oral route of delivery(in the form of a bait) is the most prudent strategy to administerthe biocontrol agent to this widespread, free-ranging, feralanimal. For contraceptive and sterility agents at least, it is highlylikely that the biocontrol agent will be in the form of a peptide orprotein molecule. Therefore, appropriate formulation of thebioactive will be essential, due to the inherently lowbioavailability of protein and peptide bioactives, especially ifdelivered via the oral route. Thus, oral delivery will be a majorhurdle to overcome in the development of a new biologicalcontrol strategy for this species, as it is in eutherians.

1.3. Developing oral delivery formulations for possums

Approaches used in the pharmaceutical industry to designefficacious drug delivery systems for humans and domesticanimals can be applied to the design of delivery systems forbiocontrol agents for use in wildlife management, including thebrushtail possum. However, until very recently there has beenlittle information on the function of the brushtail possumgastrointestinal (GI) tract or on the stability or absorption ofdrugs following oral delivery in this species. This reviewpresents information on the function of the brushtail possum GItract, on peptide and protein stability within the GI tract andpermeability across the epithelial cell layer. Comparisons willalso be made with each feature in humans.

2. Gastrointestinal tract physiology

2.1. Morphology

The organization of the GI tract of the common brushtailpossum is in general, similar to that of other mammals includinghumans. However, due to differences in digestive strategies, thebrushtail possum has some specialised adaptations. Themorphology of the GI tract of the common brushtail possumwas first described in detail by Lönnberg [16]. This animal is ahindgut fermenter and the morphology of its GI tract is animportant adaptation to the nutritionally-poor diet of Eucalyp-tus leaves that the brushtail possum evolved to utilize inAustralia [2,17]. In particular, the caecum and proximal colon(collectively referred to as the hindgut) represents a greaterproportion of the GI tract in this species than it does in humans

1123A. McDowell, B.J. McLeod / Advanced Drug Delivery Reviews 59 (2007) 1121–1132

(Fig. 1). The enlarged caecum is somewhat simple in structurecompared with that in humans, as it has no haustrations. Colonichaustrations have been implicated with extended retention timesin the hindgut of poorly indigestible food matter [18], so mightalso be expected to be present in hindgut fermenting animalssuch as the brushtail possum.

A specialised hindgut process — the colonic separatingmechanism (CSM) — that is involved in the separation andselective retention of small, easily digestible plant fragmentsinto the caecum, while at the same time facilitating the passageof indigestible, fibrous material through the colon, has beenshown to be present in some marsupial species [19]. However,there is no evidence of a CSM in the brushtail possum [20]. Thelack of a CSM in the brushtail possum is considered to be themain reason why brushtail possums are not able to maintainthemselves on a diet composed exclusively of Eucalyptusleaves as some other marsupials species do (e.g. koala), andwhy they are generalist herbivores [20]. We are not aware of theexistence of a colonic separating mechanism in humans, how-ever it is reported in rabbits [21].

There is a relationship between diet quality and GImorphometrics [22]. In eutherian hindgut fermenting mammalssuch as rats, caecal length has been shown to be significantlygreater in animals fed diets with a high fibre content (22% bran

Fig. 1. Line drawing of the gastrointestinal tract from the common brushtailpossum (Trichosurus vulpecula). Note the expanded caecum. The dashed linesindicate the points of distinction between adjacent gut sections (stomach, smallintestine, caecum, proximal colon and distal colon). The proximal colon wasdefined as the section of gut extending from the ileo-cecal junction to the firstappearance of the faecal pellets. Scale bar=5 cm. Reprinted from McDowellet al. [22] with permission from Australian Mammalogy.

content) than in those fed a low fibre diet (containing 5 or 10.5%cellulose) [23]. A similar correlation between GI tractmorphometrics and diet quality has also been observed inrabbits [24]. Brushtail possums in New Zealand have theopportunity to select a higher quality diet than their counterpartsin their native habitat in Australia. As the New Zealand floraevolved without the need for protection against browsingherbivores (there are no indigenous land mammals in NewZealand apart from two species of bat), the flora lacks theextensive range of chemical defences found in the leaves ofAustralian species [8]. Consistent with this, in brushtailpossums collected from the Otago region of New Zealand, thetissue mass of the caecum accounted for approximately 12% ofthe total GI tract mass, compared with 21% in Australianspecimens examined by Crowe and Hume [25]. Conversely, theproportion by mass of the small intestine from brushtailpossums collected in Otago was 41%, compared with 35% forthe Australian specimens [25]. This probably reflects the higherproportion of easily digestible food in the New Zealand diet thatdoes not require microbial digestion in the hindgut.

Chiou et al. [21] present data on the weight and length ofsections of the GI tract from some hindgut fermenting, eutherianlaboratory animals. The proportion of mass that is smallintestine is 25.8%, 23.6% and 22.3% for the herbivorous rabbit,guinea pig and hamster, respectively. The rat, an omnivoroushindgut fermenter, has a larger proportion of the GI tract that issmall intestine, approximately 41% [21]. Similarly, the length ofthe small intestine relative to the whole GI tract is greater in therat compared to the herbivorous species studied (84.2% in therat compared to 65.5%, 62% and 49% in the rabbit, guinea pigand hamster, respectively) [21].

The digestive strategy of humans is different to the hindgutfermenting species described above and so there arecorresponding differences in the anatomy of the GI tract.Humans are omnivores and consume a diet that is more easilydigested than that of a herbivore. The most striking differencebetween the anatomy of the GI tract from a herbivore, such asthe brushtail possum, and the human is the length of the smallintestine. The small intestine is the longest part of the human GItract and from measurements commonly cited in the literature,makes up approximately 78% of the GI tract by length.

2.2. Gastrointestinal transit

Designing an oral delivery system requires knowledge of thetime taken for the dosage form to reach different regions of theGI tract and of the factors that affect transit and residence timeswithin each region. The GI transit of pharmaceutical dosageforms has been studied extensively in humans [26–30]. Amongother factors, gastric transit time for pharmaceutical dosageforms is dependent on the rate of gastric emptying, which inturn is determined by whether the stomach is in the fed or fastedstate [31]. Gastric emptying is slower in the fed state comparedto the fasted state and is dependent on the calorific value of themeal [31]. The process of gastric sieving in the fed state alsomeans that the size of the dosage form can influence the gastricresidence time, with larger particles being retained longer than


smaller particles and fluid [31]. The rate of transit through thesmall intestine, reported as being approximately 3–4±1 h, isindependent of the size of the dosage form [32], is moreconsistent than gastric transit and is not affected by fed or fastedstate [29]. Colonic transit in humans, is reported to be in theorder of 35–60 h, is less well understood and is thought to bestrongly influenced by the control of movement of materialthrough the ileo-caecal junction [33]. The majority of humanstudies on GI transit also report both intra- and inter-subjectvariability.

Stevens and Hume [34] present data on the GI transit time fordifferent sized particulates in a range of animal speciesincluding pig, horse and dog. Previous studies have investigatedthe GI transit time of fluid and particulates in brushtail possumsfed different types of diets (semi-purified pellets or eucalypt leafparticles) (Table 1). A limitation with these studies is thattypically mouth to anus transit time was calculated and the rateof passage through different sections of the GI tract was notdetermined. In addition, there is evidence that the heavy metalsfrequently used as a marker, may not remain bound to any oneparticle throughout transit or that marker may be preferentiallybound to small particles [20], which would confoundinterpretation of the data.

Using gamma scintigraphy, we investigated the distributionof radioactivity following oral administration of 99mTc labeledsolution (diethylenetriamine pentaacetic acid) and 99mTclabeled indigestible (anion exchange resin) particles of twodifferent size ranges (75–125 or 500–700 μm diameter). Transittime was variable between individual animals, but wasindependent of gender (P=0.184) and body mass (P=0.640),at least over the liveweight range 1.4 to 3.7 kg. The profiles oftransit through each section of the GI tract in the commonbrushtail possum were found to be similar for all three

Table 1Summary of studies on gastrointestinal transit in the common brushtail possumusing different markers

Markersize

Marker Mean retentiontime (h)

Transittime (h)

Reference

Fluid 51Cr–EDTA(Eucalypt diet)

51 – Foley and Hume[20]

Fluid 51Cr–EDTA(semi-purified diet)

64 3 Wellard andHume [97]

Fluid Co–EDTA(semi-purified diet)

36 5.8 Sakaguchi andHume [98]

b75 μm 103Ru–P(Eucalypt diet)

49 – Foley and Hume[20]

b75 μm 103Ru–P(semi-purified diet)

71 3 Wellard andHume [97]

b75 μm Yb CWC(semi-purified diet)


N300 μm Cr CWC(semi-purified diet)


51Cr–EDTA (chromium–ethylene diamine tetra-acetic acid complex), 103Ru–P(ruthenium-labelled Tris (1, 10-phenanthroline)-ruthenium (II) chloride com-plexed with phenanthroline), Co–EDTA (cobalt–EDTA), Yb CWC (Ytterbiummordanted cell wall components of hay), Cr CWC (chromium mordanted cellwall components of hay). Transit time is the duration between administrationand first appearance in the faeces. Mean retention time is the duration taken forthe marker to move through the digestive tract [34].

formulations [35]. Maximum concentration of the radioactivelabel present in the caecum, was recorded between 12 and 24 hafter dosing [35].

Circadian patterns in the bioavailability of orally-adminis-tered drugs have been documented for a number of compoundsin human patients [36]. In a study by Coupe et al. [37], patientsdosed in the evening with pressure-sensitive radiotelemetrycapsules, had longer gastric residence times and longer smallintestine transit times compared to those dosed in the morning.However, total transit time (mouth to anus) was not significantlydifferent between morning and evening dosing (approximately25 h and 32 h for morning and evening dosing, respectively)[37]. Similarly, feeding and activity level of a study animal mayaffect the GI transit of oral dosage forms. Brushtail possums arenocturnal and in the wild they emerge from their nesting siteabout 2–3 h after sunset to feed and return to their nest severalhours before sunrise. Feeding behavior involves 2–3 sessionsfor feeding of 1–2 h in duration throughout the night. Thus, inour transit studies in which animals were maintained undernatural daylight, the animals that were dosed in the morningwould have received the radiolabeled formulation when thestomach was the fullest. After morning dosing, possums wereinactive and did not eat. Conversely, animals that were dosed inthe evening would have had an emptier stomach, than those inthe morning dose group and they would have been active andconsuming food within a few hours after receiving theradiolabeled dose. Therefore, to investigate diurnal differencesin GI transit in this species, we compared the distribution ofradioactivity throughout the GI tract 12 h after dosing, in groupsof animals that were dosed at either 06:00 or 18:00 h.Observations made on the fullness of the stomach at the timeof dissection concur with those described above. Although thestomachs of animals dosed at 18:00 contained less food, transitthrough the GI tract of the common brushtail possum was notdifferent between animals dosed in the evening or the morning[35]. This indicates that by 12 h after administration of the oraldose, GI transit of formulations is relatively constant, at least inthis captive population where food resources are unlimited. Itremains to be determined whether a longer period of fastingprior to the dose would influence transit times.

2.3. Gastrointestinal pH

The progressive change in pH along the human GI tract hasbeen well characterised [38,39]. Comparing pH values found inthe human GI tract and those in the brushtail possum, the pH ofdigesta from the small intestine of the brushtail possum arehigher than those recorded for human subjects (Table 2).

The pH recorded for digesta from the GI tract of brushtailpossums from the Otago region of New Zealand are generally inagreement with previous reports [40–42]. Foley et al. [40]found that there was little change in the pH between the smallintestine, caecum and proximal colon. However, in our studythe pH of digesta from the small intestine (average 7.5±0.34)was higher than that measured by Foley et al. using Australianspecimens [40], and there was a significant difference in pHbetween the small intestine and the hindgut [22]. There was,

Table 2Comparison of pH of digesta between humans and the brushtail possum in thefed state

Section of GIT Brushtail possum Human

StomachFundus 3.3±0.19 1.0–2.5 [39]Pyloric region 2.4±0.04

Small intestineDuodenum 7.5±0.25 5.1–6.6 [39,99]Jejunum 7.8±0.27 5.2–6.2 [99]Ileum 8.3±0.25 6.8–7.8 [39,99]

CaecumProximal 6.7±0.21 5.7–6.4 [39]Median 6.8±0.30 –Distal 6.9±0.21 –

ColonProximal 6.5±0.28 6.5 [39,99]Distal 6.4±0.17 7.0 [39]

Values for brushtail possum are mean (±SD) of digesta from successive sectionsof the gastrointestinal tract (GIT) (n=4) (Chiu and McDowell, unpubl. data).


however, no difference in pH of the digesta from the caecumcompared to the proximal colon. The characteristic change inpH across the ileo-caecal junction that is recorded in humans[43] is also evident in the brushtail possum (Table 2). When thepH of the mucosal surface of GI tract was compared betweenfed and fasted brushtail possums, the pH in the caecum of fastedanimals was higher than that in fed animals, but was notsignificantly different in all other sections of GI tract [22].

2.4. Microflora

The indigenous microorganisms that inhabit the GI tract areparticularly important for herbivores. The hindgut containsmicroorganisms which, through the process of fermentation,convert the indigestible cellulose in plant cell walls intonutrients (short chain fatty acids) that can be absorbed andutilised by the animal [34]. There are very few records in theliterature of the microflora associated with the GI tract ofmarsupials and the accounts are usually quite general with thebacteria identified classified into broad morphological, ratherthan taxonomic, groups [44–46]. There has been one report ofHelicobacter spp. being identified in colonic tissue from a rangeof marsupial species including the brushtail possum [47].Specialised Gram-negative enterobacteria that degrade tannin–protein complexes have been identified in the caecal wall fromthe GI tract of the koala [44]. The microflora of the GI tract ofthe brushtail possum remains largely unexplored with onereport that the hindgut has a high density of both rod and coccibacteria [45].

Recently, the number of bacteria colonising different regionsof the GI tract of brushtail possum pouch young has beeninvestigated using the molecular technique of denaturinggradient gel electrophoresis (DGGE) of 16S ribosomal DNA.The greatest amount of bacterial diversity was present in thecaecum [48] with a maximum of 11 different bacterial speciesfound in the caecum. This is lower than the number of speciesreported for human infants [49]. With age, the number ofbacterial species present in the gut is reported to increase [48],

presumably with the associated changes in diet composition asthe animal grows. Identification of the species of bacteriapresent in the GI tract was not done in this study, althoughEscherichia coli was identified in the samples of digesta [48].The authors conclude that this was due to the fact that youngmarsupials are maintained in the mother's pouch as E. coli hasbeen cultured from the pouch female brushtail possums [50].

A better knowledge of the microflora present in the GI tractof the brushtail possum is required and in particular, theenzymes that are secreted by those bacteria. There may bepossum-specific bacteria, such as the tannin–protein degradingbacteria in the koala, present in the hindgut that could betargeted for selective degradation of different polymer coatingsused to protect bioactive compounds from degradation in theupper sections of the GI tract.

The lack of information about the microflora that inhabits theGI tract of the brushtail possum is in contrast to the wellcharacterised intestinal bacteria in humans. The composition ofthe microflora that inhabits the human GI tract changes with theregion of the gut and, as has been found in the marsupial GItract, the colonic region contains the greatest diversity ofmicroflora. There is a sharp increase in the concentration ofbacteria between the ileum and the colon where there areapproximately 1012 cfu/ml [51]. It has been estimated that thereare 400 species of bacteria that inhabit the human colon andmost are obligate anaerobes including species in the generaBacteroides, Bifidobacterium, Fusobacterium and Clostridium[52]. The proliferation of bacterial diversity in the human colonis thought to be facilitated by the near neutral pH and thereduced rate of digesta transit [53]. The enzymes produced bythe colonic microflora have been utilized as a site-specific drugdelivery strategy to target the colon. For example theazoreductase enzymes produced by Clostridium spp. cleavethe azo bond of the prodrug sulfasalazine to release the activedrug moiety and is used in the treatment of Crohn's disease[54].

3. Barriers to oral delivery of peptides and proteins

There are physicochemical factors that limit the absorptionof proteins and peptides, including their large molecular size,enzymatic degradation, short plasma half-life, ion permeabilityand tendencies for aggregation, adsorption and denaturation[55]. In this review only the biochemical (enzymatic degrada-tion) and physiological (physical barriers) factors that determinethe extent to absorption of proteins and peptides following oraldelivery will be discussed, with emphasis on the brushtailpossum.

3.1. Proteolytic activity

The GI tract is rich in proteolytic enzymes and will digesttherapeutic or pest biocontrol proteins as readily as it digestsdietary proteins. Peptides and proteins are subject to proteolysisat several sites within the GI tract; within the luminal contents,at the brush-border membrane of mucosal cells and within theepithelial cells. Enzymatic degradation of peptides and proteins

Fig. 2. Specific chymotrypsin activity (μg/min/mg protein) present in the variousregions of the brushtail possum intestinal tract. All values are means±SD, n=3.Reproduced from Wen et al. [65] with permission from Journal of ComparativePhysiology.


has been reported for several eutherian species. For example,the degradation of the protein, bovine serum albumin (BSA)and the peptide, luteinizing hormone releasing hormone(LHRH) has been investigated in the rat, rabbit, guinea pigand pig [56–61]. The proteolysis of these model peptide andprotein molecules was assessed in a series of experiments in thebrushtail possum. Prior to this, there was little if any infor-mation available for proteolysis in the GI tract for any marsupialspecies.

The rate of degradation of model peptide and proteinmolecules was determined in luminal and mucosal extractsisolated from the brushtail possum duodenum, jejunum, ileum,caecum, proximal colon and distal colon. The degradation ofLHRH by luminal contents was also compared with that ofknown concentrations of the pancreatic enzymes, chymotryp-sin, trypsin and elastase. Proteolytic extracts of luminal extractswere significantly greater, and up to 1000 times higher, in thesmall intestine than in the hindgut (Table 3, [62]). All pancreaticenzymes hydrolysed LHRH, but chymotrypsin had the greatestactivity [62,63]. Therefore, in the brushtail possum, proteolysisoccurs primarily in the small intestine through luminal enzymes,with chymotrypsin playing a major role. It appears that thebrushtail possum hindgut contributes little to the metabolism ofpeptides and proteins, identifying it as a potential site to targetfor the delivery of peptides and proteins [64].

To reduce the metabolic barrier presented to peptides andproteins in the GI tract, their co-administration with proteaseinhibitors to counter the proteolytic enzymes is required. Again,there is no background literature concerning the protection ofpeptides and proteins in the marsupial GI tract. However, asearlier studies demonstrated the dominant proteolytic role ofchymotrypsin in the brushtail possum GI tract [64], a range ofinhibitors of chymotrypsin were tested for their efficacy toprotect LHRH and BSA from degradation in extracts from thepossum small intestine. These inhibitors included soybeantrypsin inhibitor, sodium deoxycholate, carbopol 934P, bacitra-cin, and bestatin. All compounds investigated significantlyinhibited degradation of both the model peptide and modelprotein tested (Fig. 2, [65]). Thus there is potential for

Table 3Mean (±S.E.M) specific activity of luminal and mucosal extracts from theintestinal tract of the brushtail possum against bovine serum albumin (BSA) andluteinizing hormone releasing hormone (LHRH)

Substrate Gut region Specific activity (μg h−1 mg−1)

Luminal extracts Mucosal homogenates

BSA Duodenum 419±220 NDJejunum 6491±647⁎⁎ 47±37Ileum 4767±413⁎⁎ 21±25Caecum 133±67 NDProximal colon 91±33 NDDistal colon ND ND

LHRH Jejunum 546.7±39.8⁎⁎ 41.46±5.15⁎⁎

Ileum 286.5±30.33⁎⁎ 30.00±3.01⁎⁎

Caecum 60.86±14.6 16.73±0.55Proximal colon 56.78±12.73 11.39±1.24

(ND) not detectable, ⁎⁎ significantly different from other segments (Pb0.01)(adapted from [71]).

formulation strategies that incorporate protease inhibitors toprotect biocontrol peptides and proteins after oral dosing.

3.2. Permeability

The epithelial lining of the GI tract also presents a majorphysical barrier to the absorption of peptides and proteins. Thisphysical barrier includes the unstirred water layer that may bindpeptides and proteins, the tight intercellular junctions that limitpermeation via the paracellular pathway, and the lipid matrix ofthe cell membrane that limits permeation of such hydrophiliccompounds via the transcellular pathway.

Chemical agents have been employed to increase the poresize of the tight junctions between adjacent epithelial cells topromote uptake via the paracellular pathway. For example,formulation approaches for improving paracellular uptake ofproteins and peptides include the use of surfactants, bile salts,chelating agents, fatty acids, sulfhydryl compounds, mucolyticagents and mucoadhesive polymers and their efficacy has beenassessed in rat, rabbit, pig, dog, sheep, salmon and humantissues [66–68].

There is a dearth of information on permeability across theepithelia for any marsupial species. In a recent series of in vitroexperiments, the permeability of the possum hindgut tohydrophilic compounds was investigated, using both achemically-stable compound that is resistant to degradation byluminal or mucosal enzymes (sodium fluorescein) and ahydrophilic molecule that is readily hydrolysed by the luminaland mucosal enzymes (LHRH). Two regions of the brushtailpossum large intestine (caecum and proximal colon) wereselected, with permeability being assessed by measuring themucosal-to-serosal flux of these molecules in the Ussingchamber under short-circuit conditions [69].

The permeability of possum colonic and caecal tissues tofluorescein and LHRH was assessed in untreated tissues and theeffectiveness of a range of permeation enhancers to increaseabsorption was evaluated. The enhancers tested included:


ethylenediamine tetra-acetic acid (EDTA), sodium deoxycholicacid (SDA), dithiothreitol (DTT) and polyacrylic acids ofdifferent molecular weights. In the absence of any treatment, theproximal colon and caecum had comparable, but lowpermeabilities for both compounds. The apparent permeabilityof LHRH (mw 1128) across brushtail possum colon recorded inthis study (1.37×10− 7cm s− 1) was even lower than thatreported in a hindgut fermenting eutherian species (rabbit,2.83×10− 7cm s− 1) to the LHRH agonist nonopeptide(leuprolide, mw 1209) [70].

Polyacrylic acid (50, 90 and 150 kDa, all at 10 mg/ml),EDTA (5 and 10 mM), SDA (2, 5 and 10 mM) and DTT (2 and5 mM) all significantly increased permeability to fluorescein,with a 2–6 fold increase in apparent permeability [69].Similarly, exposure of hindgut tissues to SDA (5 mM) andEDTA (5 and 10 mM) resulted in a 3-fold increase inpermeability to LHRH. There were no significant differencesin permeability to fluorescein and LHRH between colonic andcaecal tissues [69].

Corresponding in vivo studies using an in situ single passperfusion model, determined the absorption of fluorescein andLHRH across measured sections of small intestine (jejunum) orhindgut (caecum and colon) in brushtail possums [71]. Meanplasma concentrations of fluorescein and LHRH over the periodof perfusion in the absence of the permeation enhancer were0.25±0.01 μg/ml and 0.20±0.01 ng/ml, respectively [71].When perfused in the presence of enhancer mean concentrations

Fig. 3. Mean (± S.E.M) plasma concentrations of (a) LHRH and (b) fluoresceinin brushtail possums during perfusion through the proximal colon, without(filled circles) or with (open circles) co-administration of the permeationenhancer sodium deoxycholate. Reproduced from McLeod et al. [71] withpermission from New Zealand Veterinary Journal.

were 7.8±1.64 μg/ml and 8.7±3.14 ng/ml for fluorescein andLHRH, respectively (Fig. 3).

Given the assumptions made in determining the parametersused in the estimation of apparent permeabilities (e.g. surfacearea of perfused gut segment, clearance from i.v. pharmacoki-netic studies) [71], there was reasonable agreement between thevalues obtained in the in situ preparations [71] and those ob-tained from the in vitro study [69]. The increase in permeabilityfollowing addition of the permeation enhancer SDA, was 33-fold and 63-fold for fluorescein and LHRH for the in situ studyand 3-fold and 4-fold for fluorescein and LHRH in the in vitroexperiments. This indicates that Ussing chamber experiments,while useful for assessing permeation and the effect of perme-ation enhancers for promoting uptake across possum intestinaltissues, may underestimate the effectiveness of permeationenhancers.

3.3. Epithelial cell function

A number of unexpected differences have been identified intransport properties of intestinal epithelia in the brushtailpossum compared to those in eutherian mammals. Thesedifferences include: (i) electrogenic ion transport in the intestineof the brushtail possum differs significantly from that ineutherian species [72]; (ii) amiloride-sensitive sodium channels(those that classically play a major role sodium balance) arefound throughout the entire length of the hindgut in brushtailpossums, whereas they are restricted to the distal colon ineutherians [73] and in the brushtail possum these sodiumchannels are not regulated by the same mechanism that controlstheir activity in eutherian species [64]. Furthermore, (iii) thecolon of the possum does not respond to secretagogues aseutherian mammals do [72], while (iv) in the ileum, cAMP-dependent secretagogues such as forskolin and prostaglandinstimulate electrogenic bicarbonate ion secretion [74] rather thanelectrogenic Cl− secretion as is seen in eutherians [75].

4. Pharmacology

4.1. Metabolism of ingested toxins

Brushtail possums are generalist feeders that have evolvedmetabolic pathways that are able to process the wide range ofplant toxins ingested as part of their diet. In particular, Euca-lyptus leaves contain an array of plant secondary metabolites(PSMs) such as phenols, tannins and cyanogenic compoundsthat retard digestion and are energetically expensive for smallmammals to detoxify [2,17,76]. Herbivores must be able toremove ingested PSMs to avoid their harmful effects. In contrast,ingestion of Eucalyptus oil by humans results in poisoning [77].It is well established that in humans, the majority of drugs areextensively metabolised in the liver and that the cytochromeP450 (CYP) family of enzymes are responsible for the Phase Ibiotransformation of the majority of ingested drugs [78].

McLean and Duncan [79] provide an excellent review onhow PSMs can be viewed as chemical defences that contributeto diet choice of herbivores just as drugs, many of which are


PSMs or their derivatives (morphine and codeine, respectively),are studied in relation to their therapeutic effects in humans. Theimpetus for recent studies of the pharmacology of the brushtailpossum has been to understand how this animal utilises a diet ofEucalyptus leaves that contain an array of PSMs (e.g. phenolicsand tannins) and what factors determine diet choice. Thedeterminants of diet choice and intake for marsupial herbivoresare complex and continue to be an area of active research usingbehavioural feeding studies of captive animals [80].

The mechanism for removal of ingested toxins via detoxi-fication appears to vary within the group of mammalianherbivores. Specialist herbivores, such as the koala (P. cinereus),use oxidation reactions to produce water-soluble metabolitesthat are easily excreted in urine (Phase 1 detoxification) [81].The latter has been illustrated by Boyle et al. who identified anovel, extensively oxidized metabolite, 4-(1,2-dihydroxy-1-methylethyl)-benzoic acid, in the urine of koalas fed a dietcontaining the dietary terpene, p-cymene [82]. Generalistfeeders, such as the brushtail possum, rely on conjugationreactions to remove toxins (Phase 2 detoxification) [81], e.g.glucuronic acid [83].

The detoxification pathways utilised by mammalian herbi-vores such as the brushtail possum can be modified to deal withhigher toxin loads encountered in their diet. An example of this,is the capacity of endemic herbivores in Western Australia totolerate higher levels of the compound fluoroacetate than theirconspecifics in the east of Australia [84]. Some native plants inWestern Australia (e.g. the legumes Oxylobium species andGastrolobium species) contain high concentrations of sodiummonofluoroacetate (Compound 1080) and so in order to utilizethese plants for food, common brushtail possums (and otherindigenous marsupials) have developed a tolerance to this toxin[85]. Similarly, the brushtail possum is able to induce metabolicenzymes when exposed to a xenobiotic in their diet, for example1,8-cineole [86]. As in humans, xenobiotics are detoxified bythe cytochrome P450 enzymes and the CYP450 enzyme has

Table 4Comparison of pharmacokinetic variables for compounds investigated in the brusht

Compound Route Possums Reference

Paracetamol Oral tmax=1–2 h Eason et al. [92t1/2=5.2–12.9 h

Antipyrine i.v. t1/2=1.3 h Eason et al. [92Warfarin Oral tmax=6 min Eason et al. [92

t1/2=11.9 hInsulin i.v. t1/2=14.2 min A. McDowell (Fluorescein Perfused GI tract t1/2=13.5 min McLeod et al.

Cl=4.3 ml/min/kgVd=82 ml/kg


LHRH Perfused GI tract t1/2=18 min McLeod et al.Cl=42 ml/min/kgVd=344 ml/kg


Iophenoxic acid Oral tmax=7 h Eason et al. [10t1/2=0.8 d

been shown to be induced rapidly when brushtail possums areexposed to terpenes as part of the diet [87]. Tissue distributionof CYP3A has been investigated in brushtail possums fed dietswith a mixture of terpenes. It was found that there is adifferential distribution of CYP3A-like isoforms in a variety oftissues (brain, testes, adrenal gland and gut tissue). The jejunumonly produces CYP3A P1; ileum, kidney, testes and adrenaltissues produce CYP3A P2 and all 3 isoforms were identified inthe liver and the duodenum. Such data support the importanceof these enzymes to the ability of brushtail possums to tolerate awide range of PSMs [88].

Marsh et al. [89] have provided evidence that the dietarystrategy of the brushtail possum to eat a variety of plant foods,with a range of PSMs, is to enable the animal to utilize a numberof different detoxification pathways. Brushtail possums use acombination of oxidation, hydrolysis and conjugation todetoxify PSMs [89]. Furthermore, it is thought that thesaturation of the detoxification pathways is the mechanism bywhich PSMs limit foliage intake by marsupial herbivores [90].However, an alternative theory has been proposed by Sorensenand Dearing [91] that efflux transporters in the GI tract (P-glycoproteins) determine the absorption of PSMs that in turnregulate the intake and diet choice of herbivores.

4.2. Drug pharmacokinetics in possums

The pharmacokinetics of drugs used for humans has beenextensively characterised both in vivo and in vitro. In contrast,there is a paucity of information about the metabolism of drugcompounds in the brushtail possum. Recent and ongoing workin our laboratory is the first time that the pharmacokinetics ofinsulin has been investigated in this species (A. McDowell,unpubl. data) (Table 4). However, some pharmacokineticinformation for paracetamol is known for this species.Following intravenous administration, paracetamol was rapidlyabsorbed in the brushtail possum (Tmax 1–2 h) and the plasma

ail possum and eutherian mammals

Route Humans Reference

] Oral tmax=0.3–1 h Forrest et al. [100]t1/2=2 h

] i.v. t1/2=12 h Branch et al. [101]] Oral t1/2=37 h Chan et al. [102]

unpubl. data) i.v. t1/2=2–4 min Duckworth et al. [103][71] – – –

Route Rats Reference

[71] i.v. t1/2=2.6 min Berger et al. [104]Cl=54.4 ml/min/kgVd=954.2 ml/kg

Route Cats Reference

5] Oral tmax=12 h Eason et al. [105]t1/2=107 d


elimination half-lives were similar to those reported foreutherian species (Table 4) [92]. However, in contrast toeutherians, the highest paracetamol dose tested (2000 mg kg− 1)did not saturate plasma clearance in the brushtail possum andthere were no signs of hepatic damage from the formation oftoxic metabolites due to excess paracetamol in the liver [92].The in vivo pharmacokinetics of antipyrine and warfarin wasalso investigated in the same study by Eason et al. [92]. Thehalf-life for antipyrine was 1.2 h and warfarin 11.9 h was withinthe range recorded for eutherian mammals (t1/2=1–1.4 h and10.9–12.9 h for antipyrine and warfarin, respectively, Table 4).After intravenous administration of LHRH to possums (n = 24),mean distribution and elimination half-lives were 1 min 50 s and11 min, respectively [93], similar to that reported in rats [104](Table 4).

Generally, the pharmacokinetics of the compounds tested todate has been similar in the brushtail possum to that in eutheriananimals. However, there are instances (e.g. paracetamol) inwhich possums appear to be able to tolerate very high doses of adrug, which may be indicative of the presence of specialisedefflux transporters in this species of marsupial.

5. Conclusions

This review gives an overview of information on thebrushtail possum that is pertinent to the development of oraldelivery formulations. In this instance, the brushtail possum isnot being investigated as a potential animal model, but is thespecies being targeted. Consequently, information obtained onthe delivery strategies and therapeutic outcomes using thisspecies is directly applicable [94]. For any new biologicalcontrol program for the brushtail possum to be successful, itmust be underpinned by the ability to effectively administer thecontrol agent(s) to the target population. Given that the oralroute is the preferred route of administration, it is essential thatthe features and processes of the GI tract of the brushtail possumare well characterised and understood. In addition, it is essentialto have information on the uptake and fate of the bioactive andof the delivery system in vivo. In the development of oraldelivery systems for brushtail possums, an understanding of themechanisms by which these animals metabolise and/or excretethe plant toxins that they ingest, may be of relevance.

How can the development of oral delivery strategies for thebrushtail possum be of significance to the development oftherapeutic outcomes for humans? The information presented inthis review encompasses a major proportion of all the dataavailable for any marsupial species. As marsupials divergedfrom placental mammals some 130 million years ago [1] andhave developed some different strategies for vital physiologicalmechanisms [74], such studies may lead to novel methods fortherapeutic oral delivery systems that would not have becomeevident from studies using standard eutherian animal models.Another important difference in using wild-caught brushtailpossums as an animal model is that this is an outbred populationthat will have considerable genetic variation in those mechan-isms that influence the efficacy of an oral formulation dosebetween individuals. This contrasts with the use of standard

laboratory animal models, which are usually highly inbredpopulations. In this case, there can be gross underestimation ofbetween-animal variation.

There are a number of challenges to be met when attemptingto develop oral delivery formulations for possums. For example,the formulation itself must be extremely robust and stable undera wide range of environmental conditions. Firstly, the formu-lation must withstand processing conditions when it is in-corporated into an oral bait. Secondly, it must withstand therigours that it will be exposed to in the field where it may bestored for long periods of time under uncontrolled environ-mental conditions. At this time and again when distributed tothe possum population, the formulation will be exposed to awide range of temperatures, to changes in humidity, to ultra-violet light and to degradation by microorganisms.

The microorganisms that inhabit the GI tract play animportant role in the metabolism of ingested substances,including therapeutic compounds. To date records of themicroflora from the GI tract of the brushtail possum are generaland very few species have been identified.While there have beenadvances in the characterisation of microflora from humansusing molecular techniques [95], identifying the bacteria in thebrushtail possum gut may be challenging because of thelimited database of reference sequences for herbivores. Further-more, to characterise the enzymes produced by the gut flora, itis necessary to use traditional culture techniques to grow thebacteria, which is particularly difficult for anaerobic species.

An additional challenge for oral formulations that aredistributed in the field for the control of free-ranging animalsis that there is no control over the dose taken by individualanimals or the frequency of dosing. Ideally, the bioactive agentwould be extremely potent such that consumption a single baitpellet, would achieve the desired effect. Exposure to a sub-lethaldose of a toxin that causes nausea for example has disastrouseffects on possum control operations because possums have ahighly-developed bait aversion response referred to as “bait-shyness” [12].

A major purpose for reducing possum numbers in NewZealand is to reduce their impact on native fauna. Therefore, ifthe action of the bioactive is not possum-specific, then theformulation needs to be such that the bioactive is unavailable tonon-target species, or at least unattractive to them [96].

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