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

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Advanced Drug Delivery Reviews 59 (2007) 1121 – 1132 www.elsevier.com/locate/addr

Physiology and pharmacology of the brushtail possum gastrointestinal tract: Relationship to the human gastrointestinal tract ☆ Arlene McDowell a,⁎, Bernie J. McLeod b a

b

School of Pharmacy, University of Otago, P.O. Box 56, Dunedin, 9054, New Zealand AgResearch, Invermay Agricultural Centre, Private Bag 50034, Mosgiel, 9024, New Zealand Received 30 April 2007; accepted 19 June 2007 Available online 16 August 2007

Abstract Oral formulations are typically based on studies from eutherian animal models. This review introduces information relating to oral formulations for a marsupial species, the Australian brushtail possum (Trichosurus vulpecula) that has arisen from research into new methods for controlling this species — a major vertebrate pest in New Zealand. Morphologically, the gastrointestinal tract of the brushtail possum is similar to that of hindgut fermenting eutherian species, but there are some striking differences in function. Limited data suggests that the pharmacokinetics and bioavailability of administered drugs are similar to that in eutherian species, but there is some evidence that possums may have specific mechanisms for handling the intake of plant toxins and xenobiotics. The development of oral formulations for a free-ranging pest species presents several challenges above those encountered in the development of therapeutic formulations for humans and domestic animals. Use of a marsupial animal 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.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. The common brushtail possum . . . . . . . . . . . 1.2. The brushtail possum in New Zealand . . . . . . . 1.3. Developing oral delivery formulations for possums . Gastrointestinal tract physiology . . . . . . . . . . . . . . 2.1. Morphology . . . . . . . . . . . . . . . . . . . . . 2.2. Gastrointestinal transit . . . . . . . . . . . . . . . . 2.3. Gastrointestinal pH . . . . . . . . . . . . . . . . . 2.4. Microflora . . . . . . . . . . . . . . . . . . . . . . Barriers to oral delivery of peptides and proteins . . . . . 3.1. Proteolytic activity. . . . . . . . . . . . . . . . . . 3.2. Permeability . . . . . . . . . . . . . . . . . . . . . 3.3. Epithelial cell function . . . . . . . . . . . . . . .

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Abbreviations: CSM; colonic separating mechanism; GI gastrointestinal; 99mTc; radiolabeled technetium; BSA; bovine serum albumin; LHRH; luteinizing hormone 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). 0169-409X/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.addr.2007.06.012

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4.

Pharmacology . . . . . . . . . . . . . . . 4.1. Metabolism of ingested toxins . . . 4.2. Drug pharmacokinetics in possums 5. Conclusions . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . .

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1. Introduction 1.1. The common brushtail possum The common brushtail possum (Trichosurus vulpecula Kerr) is a nocturnal, arboreal, marsupial endemic to Australia with an adult body weight of between 1.5 and 3.0 kg. Marsupials are a separate lineage of mammals that diverged from eutherian1 mammals about 130 million years ago [1]. Marsupials radiated in Australasia and the group includes many anatomically diverse species such as kangaroos (Macropus spp.) and the koala (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 a generalist folivore, there is increasing evidence that they obtain additional energy and/or nutrients from other sources. Brushtail possums are amongst the smallest folivorous mammals and thus face severe problems in meeting their energy requirements from a leaf diet alone [2]. There is evidence that possums, at least those 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 possums frequently kill and eat sparrows [7]. The brushtail possum is therefore, an opportunistic folivore that eats a wide range of foliar 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 New Zealand during the mid-1800s and is now the country's most significant vertebrate pest [9]. It is a major ecological threat to New Zealand's indigenous biodiversity [10] and an economic threat as a vector for bovine tuberculosis [11]. Current control methods rely largely on poisons, for example sodium monofluoroacetate (compound 1080 [12]). However, the use of poisons is becoming increasingly unpopular for welfare reasons and for the impact of poisons on non-target species. In addition, the scale of the possum problem in New Zealand is so large that poisons alone have a limited effect on reducing the population of brushtail possums on a national level. Consequently, a number of novel biocontrol strategies are currently being investigated [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 classification is based on reproductive features.

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interfere with reproduction and lethal toxins that may be specific to possums or to marsupials in general. There are no reliable methods of determining a population of brushtail possums, but it has been estimated that there may be as many as 70 million possums in New Zealand, spread across more than 90% of the land mass, including remote and inaccessible bushland. Consequently, the oral route of delivery (in the form of a bait) is the most prudent strategy to administer the biocontrol agent to this widespread, free-ranging, feral animal. For contraceptive and sterility agents at least, it is highly likely that the biocontrol agent will be in the form of a peptide or protein molecule. Therefore, appropriate formulation of the bioactive will be essential, due to the inherently low bioavailability of protein and peptide bioactives, especially if delivered via the oral route. Thus, oral delivery will be a major hurdle to overcome in the development of a new biological control strategy for this species, as it is in eutherians. 1.3. Developing oral delivery formulations for possums Approaches used in the pharmaceutical industry to design efficacious drug delivery systems for humans and domestic animals can be applied to the design of delivery systems for biocontrol agents for use in wildlife management, including the brushtail possum. However, until very recently there has been little information on the function of the brushtail possum gastrointestinal (GI) tract or on the stability or absorption of drugs following oral delivery in this species. This review presents information on the function of the brushtail possum GI tract, on peptide and protein stability within the GI tract and permeability across the epithelial cell layer. Comparisons will also be made with each feature in humans. 2. Gastrointestinal tract physiology 2.1. Morphology The organization of the GI tract of the common brushtail possum is in general, similar to that of other mammals including humans. However, due to differences in digestive strategies, the brushtail possum has some specialised adaptations. The morphology of the GI tract of the common brushtail possum was first described in detail by Lönnberg [16]. This animal is a hindgut fermenter and the morphology of its GI tract is an important adaptation to the nutritionally-poor diet of Eucalyptus leaves that the brushtail possum evolved to utilize in Australia [2,17]. In particular, the caecum and proximal colon (collectively referred to as the hindgut) represents a greater proportion of the GI tract in this species than it does in humans

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(Fig. 1). The enlarged caecum is somewhat simple in structure compared with that in humans, as it has no haustrations. Colonic haustrations have been implicated with extended retention times in the hindgut of poorly indigestible food matter [18], so might also be expected to be present in hindgut fermenting animals such as the brushtail possum. A specialised hindgut process — the colonic separating mechanism (CSM) — that is involved in the separation and selective retention of small, easily digestible plant fragments into the caecum, while at the same time facilitating the passage of indigestible, fibrous material through the colon, has been shown to be present in some marsupial species [19]. However, there is no evidence of a CSM in the brushtail possum [20]. The lack of a CSM in the brushtail possum is considered to be the main reason why brushtail possums are not able to maintain themselves on a diet composed exclusively of Eucalyptus leaves as some other marsupials species do (e.g. koala), and why they are generalist herbivores [20]. We are not aware of the existence of a colonic separating mechanism in humans, however it is reported in rabbits [21]. There is a relationship between diet quality and GI morphometrics [22]. In eutherian hindgut fermenting mammals such as rats, caecal length has been shown to be significantly greater in animals fed diets with a high fibre content (22% bran

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content) than in those fed a low fibre diet (containing 5 or 10.5% cellulose) [23]. A similar correlation between GI tract morphometrics and diet quality has also been observed in rabbits [24]. Brushtail possums in New Zealand have the opportunity to select a higher quality diet than their counterparts in their native habitat in Australia. As the New Zealand flora evolved without the need for protection against browsing herbivores (there are no indigenous land mammals in New Zealand apart from two species of bat), the flora lacks the extensive range of chemical defences found in the leaves of Australian species [8]. Consistent with this, in brushtail possums collected from the Otago region of New Zealand, the tissue mass of the caecum accounted for approximately 12% of the total GI tract mass, compared with 21% in Australian specimens examined by Crowe and Hume [25]. Conversely, the proportion by mass of the small intestine from brushtail possums collected in Otago was 41%, compared with 35% for the Australian specimens [25]. This probably reflects the higher proportion of easily digestible food in the New Zealand diet that does not require microbial digestion in the hindgut. Chiou et al. [21] present data on the weight and length of sections of the GI tract from some hindgut fermenting, eutherian laboratory animals. The proportion of mass that is small intestine is 25.8%, 23.6% and 22.3% for the herbivorous rabbit, guinea pig and hamster, respectively. The rat, an omnivorous hindgut fermenter, has a larger proportion of the GI tract that is small intestine, approximately 41% [21]. Similarly, the length of the small intestine relative to the whole GI tract is greater in the rat compared to the herbivorous species studied (84.2% in the rat compared to 65.5%, 62% and 49% in the rabbit, guinea pig and hamster, respectively) [21]. The digestive strategy of humans is different to the hindgut fermenting species described above and so there are corresponding differences in the anatomy of the GI tract. Humans are omnivores and consume a diet that is more easily digested than that of a herbivore. The most striking difference between the anatomy of the GI tract from a herbivore, such as the brushtail possum, and the human is the length of the small intestine. The small intestine is the longest part of the human GI tract and from measurements commonly cited in the literature, makes up approximately 78% of the GI tract by length. 2.2. Gastrointestinal transit

Fig. 1. Line drawing of the gastrointestinal tract from the common brushtail possum (Trichosurus vulpecula). Note the expanded caecum. The dashed lines indicate the points of distinction between adjacent gut sections (stomach, small intestine, caecum, proximal colon and distal colon). The proximal colon was defined as the section of gut extending from the ileo-cecal junction to the first appearance of the faecal pellets. Scale bar = 5 cm. Reprinted from McDowell et al. [22] with permission from Australian Mammalogy.

Designing an oral delivery system requires knowledge of the time taken for the dosage form to reach different regions of the GI tract and of the factors that affect transit and residence times within each region. The GI transit of pharmaceutical dosage forms has been studied extensively in humans [26–30]. Among other factors, gastric transit time for pharmaceutical dosage forms is dependent on the rate of gastric emptying, which in turn is determined by whether the stomach is in the fed or fasted state [31]. Gastric emptying is slower in the fed state compared to the fasted state and is dependent on the calorific value of the meal [31]. The process of gastric sieving in the fed state also means that the size of the dosage form can influence the gastric residence time, with larger particles being retained longer than

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smaller particles and fluid [31]. The rate of transit through the small intestine, reported as being approximately 3–4 ± 1 h, is independent of the size of the dosage form [32], is more consistent than gastric transit and is not affected by fed or fasted state [29]. Colonic transit in humans, is reported to be in the order of 35–60 h, is less well understood and is thought to be strongly influenced by the control of movement of material through the ileo-caecal junction [33]. The majority of human studies on GI transit also report both intra- and inter-subject variability. Stevens and Hume [34] present data on the GI transit time for different sized particulates in a range of animal species including pig, horse and dog. Previous studies have investigated the GI transit time of fluid and particulates in brushtail possums fed different types of diets (semi-purified pellets or eucalypt leaf particles) (Table 1). A limitation with these studies is that typically mouth to anus transit time was calculated and the rate of passage through different sections of the GI tract was not determined. In addition, there is evidence that the heavy metals frequently used as a marker, may not remain bound to any one particle throughout transit or that marker may be preferentially bound to small particles [20], which would confound interpretation of the data. Using gamma scintigraphy, we investigated the distribution of radioactivity following oral administration of 99m Tc labeled solution (diethylenetriamine pentaacetic acid) and 99m Tc labeled indigestible (anion exchange resin) particles of two different size ranges (75–125 or 500–700 μm diameter). Transit time was variable between individual animals, but was independent 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 of transit through each section of the GI tract in the common brushtail possum were found to be similar for all three Table 1 Summary of studies on gastrointestinal transit in the common brushtail possum using different markers Marker size

Marker

Mean retention Transit time (h) time (h)

Fluid

51

51

–

64

3

36

5.8

49

–

71

3

40

5.8

33

5.8

Cr–EDTA (Eucalypt diet) 51 Fluid Cr–EDTA (semi-purified diet) Fluid Co–EDTA (semi-purified diet) b75 μm 103Ru–P (Eucalypt diet) b75 μm 103Ru–P (semi-purified diet) b75 μm Yb CWC (semi-purified diet) N300 μm Cr CWC (semi-purified diet) 51

Reference Foley and Hume [20] Wellard and Hume [97] Sakaguchi and Hume [98] Foley and Hume [20] Wellard and Hume [97] Sakaguchi and Hume [98] Sakaguchi and Hume [98]

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

formulations [35]. Maximum concentration of the radioactive label present in the caecum, was recorded between 12 and 24 h after dosing [35]. Circadian patterns in the bioavailability of orally-administered drugs have been documented for a number of compounds in human patients [36]. In a study by Coupe et al. [37], patients dosed in the evening with pressure-sensitive radiotelemetry capsules, had longer gastric residence times and longer small intestine transit times compared to those dosed in the morning. However, total transit time (mouth to anus) was not significantly different between morning and evening dosing (approximately 25 h and 32 h for morning and evening dosing, respectively) [37]. Similarly, feeding and activity level of a study animal may affect the GI transit of oral dosage forms. Brushtail possums are nocturnal and in the wild they emerge from their nesting site about 2–3 h after sunset to feed and return to their nest several hours before sunrise. Feeding behavior involves 2–3 sessions for feeding of 1–2 h in duration throughout the night. Thus, in our transit studies in which animals were maintained under natural daylight, the animals that were dosed in the morning would have received the radiolabeled formulation when the stomach was the fullest. After morning dosing, possums were inactive and did not eat. Conversely, animals that were dosed in the evening would have had an emptier stomach, than those in the morning dose group and they would have been active and consuming food within a few hours after receiving the radiolabeled dose. Therefore, to investigate diurnal differences in GI transit in this species, we compared the distribution of radioactivity throughout the GI tract 12 h after dosing, in groups of animals that were dosed at either 06:00 or 18:00 h. Observations made on the fullness of the stomach at the time of dissection concur with those described above. Although the stomachs of animals dosed at 18:00 contained less food, transit through the GI tract of the common brushtail possum was not different between animals dosed in the evening or the morning [35]. This indicates that by 12 h after administration of the oral dose, GI transit of formulations is relatively constant, at least in this captive population where food resources are unlimited. It remains to be determined whether a longer period of fasting prior to the dose would influence transit times. 2.3. Gastrointestinal pH The progressive change in pH along the human GI tract has been well characterised [38,39]. Comparing pH values found in the human GI tract and those in the brushtail possum, the pH of digesta from the small intestine of the brushtail possum are higher than those recorded for human subjects (Table 2). The pH recorded for digesta from the GI tract of brushtail possums from the Otago region of New Zealand are generally in agreement with previous reports [40–42]. Foley et al. [40] found that there was little change in the pH between the small intestine, caecum and proximal colon. However, in our study the pH of digesta from the small intestine (average 7.5 ± 0.34) was higher than that measured by Foley et al. using Australian specimens [40], and there was a significant difference in pH between the small intestine and the hindgut [22]. There was,

A. McDowell, B.J. McLeod / Advanced Drug Delivery Reviews 59 (2007) 1121–1132 Table 2 Comparison of pH of digesta between humans and the brushtail possum in the fed state Section of GIT Stomach Fundus Pyloric region Small intestine Duodenum Jejunum Ileum Caecum Proximal Median Distal Colon Proximal Distal

Brushtail possum

Human

3.3 ± 0.19 2.4 ± 0.04

1.0–2.5 [39]

7.5 ± 0.25 7.8 ± 0.27 8.3 ± 0.25

5.1–6.6 [39,99] 5.2–6.2 [99] 6.8–7.8 [39,99]

6.7 ± 0.21 6.8 ± 0.30 6.9 ± 0.21

5.7–6.4 [39] – –

6.5 ± 0.28 6.4 ± 0.17

6.5 [39,99] 7.0 [39]

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

however, no difference in pH of the digesta from the caecum compared to the proximal colon. The characteristic change in pH across the ileo-caecal junction that is recorded in humans [43] is also evident in the brushtail possum (Table 2). When the pH of the mucosal surface of GI tract was compared between fed and fasted brushtail possums, the pH in the caecum of fasted animals was higher than that in fed animals, but was not significantly different in all other sections of GI tract [22]. 2.4. Microflora The indigenous microorganisms that inhabit the GI tract are particularly important for herbivores. The hindgut contains microorganisms which, through the process of fermentation, convert the indigestible cellulose in plant cell walls into nutrients (short chain fatty acids) that can be absorbed and utilised by the animal [34]. There are very few records in the literature of the microflora associated with the GI tract of marsupials and the accounts are usually quite general with the bacteria identified classified into broad morphological, rather than taxonomic, groups [44–46]. There has been one report of Helicobacter spp. being identified in colonic tissue from a range of marsupial species including the brushtail possum [47]. Specialised Gram-negative enterobacteria that degrade tannin– protein complexes have been identified in the caecal wall from the GI tract of the koala [44]. The microflora of the GI tract of the brushtail possum remains largely unexplored with one report that the hindgut has a high density of both rod and cocci bacteria [45]. Recently, the number of bacteria colonising different regions of the GI tract of brushtail possum pouch young has been investigated using the molecular technique of denaturing gradient gel electrophoresis (DGGE) of 16S ribosomal DNA. The greatest amount of bacterial diversity was present in the caecum [48] with a maximum of 11 different bacterial species found in the caecum. This is lower than the number of species reported for human infants [49]. With age, the number of bacterial species present in the gut is reported to increase [48],

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presumably with the associated changes in diet composition as the animal grows. Identification of the species of bacteria present in the GI tract was not done in this study, although Escherichia coli was identified in the samples of digesta [48]. The authors conclude that this was due to the fact that young marsupials are maintained in the mother's pouch as E. coli has been cultured from the pouch female brushtail possums [50]. A better knowledge of the microflora present in the GI tract of the brushtail possum is required and in particular, the enzymes that are secreted by those bacteria. There may be possum-specific bacteria, such as the tannin–protein degrading bacteria in the koala, present in the hindgut that could be targeted for selective degradation of different polymer coatings used to protect bioactive compounds from degradation in the upper sections of the GI tract. The lack of information about the microflora that inhabits the GI tract of the brushtail possum is in contrast to the well characterised intestinal bacteria in humans. The composition of the microflora that inhabits the human GI tract changes with the region of the gut and, as has been found in the marsupial GI tract, the colonic region contains the greatest diversity of microflora. There is a sharp increase in the concentration of bacteria between the ileum and the colon where there are approximately 1012 cfu/ml [51]. It has been estimated that there are 400 species of bacteria that inhabit the human colon and most are obligate anaerobes including species in the genera Bacteroides, Bifidobacterium, Fusobacterium and Clostridium [52]. The proliferation of bacterial diversity in the human colon is thought to be facilitated by the near neutral pH and the reduced rate of digesta transit [53]. The enzymes produced by the colonic microflora have been utilized as a site-specific drug delivery strategy to target the colon. For example the azoreductase enzymes produced by Clostridium spp. cleave the azo bond of the prodrug sulfasalazine to release the active drug 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 absorption of proteins and peptides, including their large molecular size, enzymatic degradation, short plasma half-life, ion permeability and tendencies for aggregation, adsorption and denaturation [55]. In this review only the biochemical (enzymatic degradation) and physiological (physical barriers) factors that determine the extent to absorption of proteins and peptides following oral delivery will be discussed, with emphasis on the brushtail possum. 3.1. Proteolytic activity The GI tract is rich in proteolytic enzymes and will digest therapeutic or pest biocontrol proteins as readily as it digests dietary proteins. Peptides and proteins are subject to proteolysis at several sites within the GI tract; within the luminal contents, at the brush-border membrane of mucosal cells and within the epithelial cells. Enzymatic degradation of peptides and proteins

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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 pig and pig [56–61]. The proteolysis of these model peptide and protein molecules was assessed in a series of experiments in the brushtail possum. Prior to this, there was little if any information available for proteolysis in the GI tract for any marsupial species. The rate of degradation of model peptide and protein molecules was determined in luminal and mucosal extracts isolated from the brushtail possum duodenum, jejunum, ileum, caecum, proximal colon and distal colon. The degradation of LHRH by luminal contents was also compared with that of known concentrations of the pancreatic enzymes, chymotrypsin, trypsin and elastase. Proteolytic extracts of luminal extracts were significantly greater, and up to 1000 times higher, in the small intestine than in the hindgut (Table 3, [62]). All pancreatic enzymes hydrolysed LHRH, but chymotrypsin had the greatest activity [62,63]. Therefore, in the brushtail possum, proteolysis occurs primarily in the small intestine through luminal enzymes, with chymotrypsin playing a major role. It appears that the brushtail possum hindgut contributes little to the metabolism of peptides and proteins, identifying it as a potential site to target for the delivery of peptides and proteins [64]. To reduce the metabolic barrier presented to peptides and proteins in the GI tract, their co-administration with protease inhibitors to counter the proteolytic enzymes is required. Again, there is no background literature concerning the protection of peptides and proteins in the marsupial GI tract. However, as earlier studies demonstrated the dominant proteolytic role of chymotrypsin in the brushtail possum GI tract [64], a range of inhibitors of chymotrypsin were tested for their efficacy to protect LHRH and BSA from degradation in extracts from the possum small intestine. These inhibitors included soybean trypsin inhibitor, sodium deoxycholate, carbopol 934P, bacitracin, and bestatin. All compounds investigated significantly inhibited degradation of both the model peptide and model protein tested (Fig. 2, [65]). Thus there is potential for Table 3 Mean (±S.E.M) specific activity of luminal and mucosal extracts from the intestinal tract of the brushtail possum against bovine serum albumin (BSA) and luteinizing hormone releasing hormone (LHRH) Substrate

BSA

LHRH

Gut region

Duodenum Jejunum Ileum Caecum Proximal colon Distal colon Jejunum Ileum Caecum Proximal colon

Specific activity (μg h− 1 mg− 1) Luminal extracts

Mucosal homogenates

419 ± 220 6491 ± 647⁎⁎ 4767 ± 413⁎⁎ 133 ± 67 91 ± 33 ND 546.7 ± 39.8⁎⁎ 286.5 ± 30.33⁎⁎ 60.86 ± 14.6 56.78 ± 12.73

ND 47 ± 37 21 ± 25 ND ND ND 41.46 ± 5.15⁎⁎ 30.00 ± 3.01⁎⁎ 16.73 ± 0.55 11.39 ± 1.24

(ND) not detectable, ⁎⁎ significantly different from other segments (P b 0.01) (adapted from [71]).

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

formulation strategies that incorporate protease inhibitors to protect biocontrol peptides and proteins after oral dosing. 3.2. Permeability The epithelial lining of the GI tract also presents a major physical barrier to the absorption of peptides and proteins. This physical barrier includes the unstirred water layer that may bind peptides and proteins, the tight intercellular junctions that limit permeation via the paracellular pathway, and the lipid matrix of the cell membrane that limits permeation of such hydrophilic compounds via the transcellular pathway. Chemical agents have been employed to increase the pore size of the tight junctions between adjacent epithelial cells to promote uptake via the paracellular pathway. For example, formulation approaches for improving paracellular uptake of proteins and peptides include the use of surfactants, bile salts, chelating agents, fatty acids, sulfhydryl compounds, mucolytic agents and mucoadhesive polymers and their efficacy has been assessed in rat, rabbit, pig, dog, sheep, salmon and human tissues [66–68]. There is a dearth of information on permeability across the epithelia for any marsupial species. In a recent series of in vitro experiments, the permeability of the possum hindgut to hydrophilic compounds was investigated, using both a chemically-stable compound that is resistant to degradation by luminal or mucosal enzymes (sodium fluorescein) and a hydrophilic molecule that is readily hydrolysed by the luminal and mucosal enzymes (LHRH). Two regions of the brushtail possum large intestine (caecum and proximal colon) were selected, with permeability being assessed by measuring the mucosal-to-serosal flux of these molecules in the Ussing chamber under short-circuit conditions [69]. The permeability of possum colonic and caecal tissues to fluorescein and LHRH was assessed in untreated tissues and the effectiveness of a range of permeation enhancers to increase absorption was evaluated. The enhancers tested included:

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ethylenediamine tetra-acetic acid (EDTA), sodium deoxycholic acid (SDA), dithiothreitol (DTT) and polyacrylic acids of different molecular weights. In the absence of any treatment, the proximal colon and caecum had comparable, but low permeabilities for both compounds. The apparent permeability of LHRH (mw 1128) across brushtail possum colon recorded in this study (1.37 × 10− 7cm s− 1) was even lower than that reported in a hindgut fermenting eutherian species (rabbit, 2.83 × 10− 7 cm 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 and 5 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) and EDTA (5 and 10 mM) resulted in a 3-fold increase in permeability to LHRH. There were no significant differences in permeability to fluorescein and LHRH between colonic and caecal tissues [69]. Corresponding in vivo studies using an in situ single pass perfusion model, determined the absorption of fluorescein and LHRH across measured sections of small intestine (jejunum) or hindgut (caecum and colon) in brushtail possums [71]. Mean plasma concentrations of fluorescein and LHRH over the period of perfusion in the absence of the permeation enhancer were 0.25 ± 0.01 μg/ml and 0.20 ± 0.01 ng/ml, respectively [71]. When perfused in the presence of enhancer mean concentrations

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were 7.8 ± 1.64 μg/ml and 8.7 ± 3.14 ng/ml for fluorescein and LHRH, respectively (Fig. 3). Given the assumptions made in determining the parameters used in the estimation of apparent permeabilities (e.g. surface area of perfused gut segment, clearance from i.v. pharmacokinetic studies) [71], there was reasonable agreement between the values obtained in the in situ preparations [71] and those obtained from the in vitro study [69]. The increase in permeability following addition of the permeation enhancer SDA, was 33fold and 63-fold for fluorescein and LHRH for the in situ study and 3-fold and 4-fold for fluorescein and LHRH in the in vitro experiments. This indicates that Ussing chamber experiments, while useful for assessing permeation and the effect of permeation enhancers for promoting uptake across possum intestinal tissues, may underestimate the effectiveness of permeation enhancers. 3.3. Epithelial cell function A number of unexpected differences have been identified in transport properties of intestinal epithelia in the brushtail possum compared to those in eutherian mammals. These differences include: (i) electrogenic ion transport in the intestine of the brushtail possum differs significantly from that in eutherian species [72]; (ii) amiloride-sensitive sodium channels (those that classically play a major role sodium balance) are found throughout the entire length of the hindgut in brushtail possums, whereas they are restricted to the distal colon in eutherians [73] and in the brushtail possum these sodium channels are not regulated by the same mechanism that controls their activity in eutherian species [64]. Furthermore, (iii) the colon of the possum does not respond to secretagogues as eutherian mammals do [72], while (iv) in the ileum, cAMPdependent secretagogues such as forskolin and prostaglandin stimulate electrogenic bicarbonate ion secretion [74] rather than electrogenic Cl− secretion as is seen in eutherians [75]. 4. Pharmacology 4.1. Metabolism of ingested toxins

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

Brushtail possums are generalist feeders that have evolved metabolic pathways that are able to process the wide range of plant toxins ingested as part of their diet. In particular, Eucalyptus leaves contain an array of plant secondary metabolites (PSMs) such as phenols, tannins and cyanogenic compounds that retard digestion and are energetically expensive for small mammals to detoxify [2,17,76]. Herbivores must be able to remove 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 are extensively metabolised in the liver and that the cytochrome P450 (CYP) family of enzymes are responsible for the Phase I biotransformation of the majority of ingested drugs [78]. McLean and Duncan [79] provide an excellent review on how PSMs can be viewed as chemical defences that contribute to diet choice of herbivores just as drugs, many of which are

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PSMs or their derivatives (morphine and codeine, respectively), are studied in relation to their therapeutic effects in humans. The impetus for recent studies of the pharmacology of the brushtail possum has been to understand how this animal utilises a diet of Eucalyptus leaves that contain an array of PSMs (e.g. phenolics and tannins) and what factors determine diet choice. The determinants of diet choice and intake for marsupial herbivores are complex and continue to be an area of active research using behavioural feeding studies of captive animals [80]. The mechanism for removal of ingested toxins via detoxification appears to vary within the group of mammalian herbivores. Specialist herbivores, such as the koala (P. cinereus), use oxidation reactions to produce water-soluble metabolites that are easily excreted in urine (Phase 1 detoxification) [81]. The latter has been illustrated by Boyle et al. who identified a novel, extensively oxidized metabolite, 4-(1,2-dihydroxy-1methylethyl)-benzoic acid, in the urine of koalas fed a diet containing the dietary terpene, p-cymene [82]. Generalist feeders, such as the brushtail possum, rely on conjugation reactions to remove toxins (Phase 2 detoxification) [81], e.g. glucuronic acid [83]. The detoxification pathways utilised by mammalian herbivores such as the brushtail possum can be modified to deal with higher toxin loads encountered in their diet. An example of this, is the capacity of endemic herbivores in Western Australia to tolerate higher levels of the compound fluoroacetate than their conspecifics in the east of Australia [84]. Some native plants in Western Australia (e.g. the legumes Oxylobium species and Gastrolobium species) contain high concentrations of sodium monofluoroacetate (Compound 1080) and so in order to utilize these plants for food, common brushtail possums (and other indigenous marsupials) have developed a tolerance to this toxin [85]. Similarly, the brushtail possum is able to induce metabolic enzymes when exposed to a xenobiotic in their diet, for example 1,8-cineole [86]. As in humans, xenobiotics are detoxified by the cytochrome P450 enzymes and the CYP450 enzyme has

been shown to be induced rapidly when brushtail possums are exposed to terpenes as part of the diet [87]. Tissue distribution of CYP3A has been investigated in brushtail possums fed diets with a mixture of terpenes. It was found that there is a differential distribution of CYP3A-like isoforms in a variety of tissues (brain, testes, adrenal gland and gut tissue). The jejunum only produces CYP3A P1; ileum, kidney, testes and adrenal tissues produce CYP3A P2 and all 3 isoforms were identified in the liver and the duodenum. Such data support the importance of these enzymes to the ability of brushtail possums to tolerate a wide range of PSMs [88]. Marsh et al. [89] have provided evidence that the dietary strategy of the brushtail possum to eat a variety of plant foods, with a range of PSMs, is to enable the animal to utilize a number of different detoxification pathways. Brushtail possums use a combination of oxidation, hydrolysis and conjugation to detoxify PSMs [89]. Furthermore, it is thought that the saturation of the detoxification pathways is the mechanism by which PSMs limit foliage intake by marsupial herbivores [90]. However, an alternative theory has been proposed by Sorensen and Dearing [91] that efflux transporters in the GI tract (Pglycoproteins) determine the absorption of PSMs that in turn regulate the intake and diet choice of herbivores. 4.2. Drug pharmacokinetics in possums The pharmacokinetics of drugs used for humans has been extensively characterised both in vivo and in vitro. In contrast, there is a paucity of information about the metabolism of drug compounds in the brushtail possum. Recent and ongoing work in our laboratory is the first time that the pharmacokinetics of insulin has been investigated in this species (A. McDowell, unpubl. data) (Table 4). However, some pharmacokinetic information for paracetamol is known for this species. Following intravenous administration, paracetamol was rapidly absorbed in the brushtail possum (Tmax 1–2 h) and the plasma

Table 4 Comparison of pharmacokinetic variables for compounds investigated in the brushtail possum and eutherian mammals Compound

Route

Possums

Reference

Route

Humans

Reference

Paracetamol

Oral

Eason et al. [92]

Oral

i.v. Oral

Eason et al. [92] Eason et al. [92]

i.v. Oral

tmax = 0.3–1 h t1/2 = 2 h t1/2 = 12 h t1/2 = 37 h

Forrest et al. [100]

Antipyrine Warfarin Insulin Fluorescein

i.v. Perfused GI tract

tmax = 1–2 h t1/2 = 5.2–12.9 h t1/2 = 1.3 h tmax = 6 min t1/2 = 11.9 h t1/2 = 14.2 min t1/2 = 13.5 min Cl = 4.3 ml/min/kg Vd = 82 ml/kg

A. McDowell (unpubl. data) McLeod et al. [71]

i.v. –

t1/2 = 2–4 min –

Duckworth et al. [103] –

Compound

Route

Possums

Reference

Route

Rats

Reference

LHRH

Perfused GI tract

t1/2 = 18 min Cl = 42 ml/min/kg Vd = 344 ml/kg

McLeod et al. [71]

i.v.

t1/2 = 2.6 min Cl = 54.4 ml/min/kg Vd = 954.2 ml/kg

Berger et al. [104]

Compound

Route

Possums

Reference

Route

Cats

Reference

Iophenoxic acid

Oral

tmax = 7 h t1/2 = 0.8 d

Eason et al. [105]

Oral

tmax = 12 h t1/2 = 107 d

Eason et al. [105]

Branch et al. [101] Chan et al. [102]

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elimination half-lives were similar to those reported for eutherian species (Table 4) [92]. However, in contrast to eutherians, the highest paracetamol dose tested (2000 mg kg− 1) did not saturate plasma clearance in the brushtail possum and there were no signs of hepatic damage from the formation of toxic metabolites due to excess paracetamol in the liver [92]. The in vivo pharmacokinetics of antipyrine and warfarin was also investigated in the same study by Eason et al. [92]. The half-life for antipyrine was 1.2 h and warfarin 11.9 h was within the range recorded for eutherian mammals (t1/2 = 1–1.4 h and 10.9–12.9 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 and 11 min, respectively [93], similar to that reported in rats [104] (Table 4). Generally, the pharmacokinetics of the compounds tested to date has been similar in the brushtail possum to that in eutherian animals. However, there are instances (e.g. paracetamol) in which possums appear to be able to tolerate very high doses of a drug, which may be indicative of the presence of specialised efflux transporters in this species of marsupial. 5. Conclusions This review gives an overview of information on the brushtail possum that is pertinent to the development of oral delivery formulations. In this instance, the brushtail possum is not being investigated as a potential animal model, but is the species being targeted. Consequently, information obtained on the delivery strategies and therapeutic outcomes using this species is directly applicable [94]. For any new biological control program for the brushtail possum to be successful, it must be underpinned by the ability to effectively administer the control agent(s) to the target population. Given that the oral route is the preferred route of administration, it is essential that the features and processes of the GI tract of the brushtail possum are well characterised and understood. In addition, it is essential to have information on the uptake and fate of the bioactive and of the delivery system in vivo. In the development of oral delivery systems for brushtail possums, an understanding of the mechanisms by which these animals metabolise and/or excrete the plant toxins that they ingest, may be of relevance. How can the development of oral delivery strategies for the brushtail possum be of significance to the development of therapeutic outcomes for humans? The information presented in this review encompasses a major proportion of all the data available for any marsupial species. As marsupials diverged from placental mammals some 130 million years ago [1] and have developed some different strategies for vital physiological mechanisms [74], such studies may lead to novel methods for therapeutic oral delivery systems that would not have become evident from studies using standard eutherian animal models. Another important difference in using wild-caught brushtail possums as an animal model is that this is an outbred population that will have considerable genetic variation in those mechanisms that influence the efficacy of an oral formulation dose between individuals. This contrasts with the use of standard

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laboratory animal models, which are usually highly inbred populations. In this case, there can be gross underestimation of between-animal variation. There are a number of challenges to be met when attempting to develop oral delivery formulations for possums. For example, the formulation itself must be extremely robust and stable under a wide range of environmental conditions. Firstly, the formulation must withstand processing conditions when it is incorporated into an oral bait. Secondly, it must withstand the rigours that it will be exposed to in the field where it may be stored for long periods of time under uncontrolled environmental conditions. At this time and again when distributed to the possum population, the formulation will be exposed to a wide range of temperatures, to changes in humidity, to ultraviolet light and to degradation by microorganisms. The microorganisms that inhabit the GI tract play an important role in the metabolism of ingested substances, including therapeutic compounds. To date records of the microflora from the GI tract of the brushtail possum are general and very few species have been identified. While there have been advances in the characterisation of microflora from humans using molecular techniques [95], identifying the bacteria in the brushtail possum gut may be challenging because of the limited database of reference sequences for herbivores. Furthermore, to characterise the enzymes produced by the gut flora, it is necessary to use traditional culture techniques to grow the bacteria, which is particularly difficult for anaerobic species. An additional challenge for oral formulations that are distributed in the field for the control of free-ranging animals is that there is no control over the dose taken by individual animals or the frequency of dosing. Ideally, the bioactive agent would be extremely potent such that consumption a single bait pellet, would achieve the desired effect. Exposure to a sub-lethal dose of a toxin that causes nausea for example has disastrous effects on possum control operations because possums have a highly-developed bait aversion response referred to as “baitshyness” [12]. A major purpose for reducing possum numbers in New Zealand is to reduce their impact on native fauna. Therefore, if the action of the bioactive is not possum-specific, then the formulation needs to be such that the bioactive is unavailable to non-target species, or at least unattractive to them [96]. References [1] M.A. Nilsson, U. Arnason, P.B. Spencer, A. Janke, Marsupial relationships and a timeline for marsupial radiation in South Gondwana, Gene 340 (2004) 189–196. [2] S.J. Cork, W.J. Foley, in: R.T. Palo, C.T. Robbins (Eds.), Plant Defences Against Mammalian Herbivory, CRC Press, Boca Raton, Florida, 1991, pp. 133–166. [3] K. Brown, J. Innes, R. Shorten, Evidence that possums prey on and scavenge birds' eggs, birds and mammals, Notornis 40 (1993) 169–177. [4] P.E. Cowan, A. Moeed, Invertebrates in the diet of brushtail possums, Trichosurus vulpecula, in lowland podocarp broadleaf forest, Orongorongo Valley, Wellington, New Zealand, N.Z. J. Zool. 14 (1987) 163–177. [5] R. Sadleir, in: T.L. Montague (Ed.), The Brushtail Possum. Biology, Impact and Management of an Introduced Marsupial, Manaaki Whenua Press, Lincoln, New Zealand, 2000, pp. 126–131.

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