- Abstract
- Introduction
- Metabolism in the possum gut
- Absorption across possum gut wall
- Table 1 Basal permeability properties of possum hindgut
- **Overview
- Summary
Oral Delivery of Bioactives to Possums - Protecting Bioactives & Enhancing their Uptake
BJ McLeod1, J Wen2, IG Tucker2, N Davies2, R Ledger2, Butt AG3
1AgResearch, Invermay, 2School of Pharmacy and 3Department of Physiology, University of Otago
Abstract
At least in the short to medium term, new possum biocontrol methods will require that the bioactive control agent be delivered via the oral route. Prerequisite for the development of oral delivery systems for possums is information specific to the possum on metabolism of the bioactive in the possum gut and absorption of the bioactive across the possum gut wall. We determined the rates of metabolism of a model protein and a model peptide in various regions of the possum gut, and the relative contribution of luminal and mucosal enzymes to this degradation. For both the protein and the peptide the greatest rate of metabolism occurs in the small intestine, due mainly to the action of luminal enzymes and primarily to chymotrypsin and, to a lesser degree, elastase. Selected metabolic inhibitors can block the rate of degradation by over 95 percent.
As the rate of metabolism is much lower in the possum hindgut, we investigated absorption across the epithelial lining of this region, using small hydrophilic compounds to establish a baseline, and compared a number of permeation enhancers for their ability to promote absorption. Permeability properties of the caecum and colon are very similar, and both these tissues respond in a similar way to permeation enhancers. This means that a delivery system could target the entire hindgut, giving the advantage of targeting a region with a large surface area and long retention time.
Introduction
In the previous paper, Ian Tucker has given an overview of the generic requirements to successfully develop a formulation for delivering bioactives via the oral route. For oral delivery to possums, we will need information that is specific to the possum on, among other things, the metabolism of the bioactive in the possum gut (the enzymatic barrier to delivery), and absorption of that bioactive across the possum gut wall (the physical barrier to delivery). These are the two aspects of oral delivery to possums that are addressed in this paper.
Metabolism in the possum gut
Using bovine serum albumin (BSA) and Luteinising hormone Releasing hormone (LHRH) as a model protein and peptide respectively, we determined rates of metabolism across all regions of the possum gut. In addition, for each gut region, the relative contribution that the enzymes contained within the lumen contents, and those associated with the gut mucosal tissue (contained within the brush border of the gut wall) made to this metabolism was assessed. This is important as in some species it has been shown that luminal enzymes are primarily responsible for metabolism of peptides and proteins in one part of the gut, whereas mucosal enzymes dominate metabolism in another.
The relative rates of degradation by luminal enzymes and by mucosal enzymes did vary between regions of the possum gut. However, degradation of both BSA and LHRH occurred mainly in the small intestine and the luminal enzymes played by far the greatest role. To make decisions on which compounds to include in any formulation to prevent this degradation, we also need to know specifically which proteolytic enzymes are involved. The pancreatic enzymes, chymotrypsin, trypsin and elastase are the major enzymes involved in breakdown of proteins and peptides within the lumen contents. Using purified enzyme preparations, we determined the contributions that each of these enzymes make to LHRH metabolism in lumen contents from the possum jejunum. Chymotrypsin degraded the peptide very rapidly, with no LHRH remaining after 10 minutes. In contrast, when incubated with trypsin, more than 90 percent of the peptide still remained after 3 hours. The activity of elastase was intermediate (see Figure 1). Therefore, to block LHRH degradation in the jejunum, a formulation must include compounds that inhibit the action of chymotrypsin, and to a lesser extent, those of elastase. The relative activity of the proteolytic enzymes is not uniform throughout the possum gut, so the enzyme inhibitors to be included in a formulation will depend on the region of the gut that is being targeted.
Another point to note is that the activity of both the luminal enzymes and the mucosal enzymes are very pH sensitive and quite small changes in pH can result in large increases in the rate of degradation (Figure 2). Thus, another strategy could be to include a buffering component in a formulation to control pH in the microenvironment surrounding the bioactive.
We tested a range of metabolic inhibitors for their ability to block protein and peptide metabolism in the the possum gut, at a range of concentrations and in a number of different combinations. Several inhibitors can block the rate of degradation of peptides and proteins by over 95 percent. This demonstrates the potential that including appropriate metabolic inhibitors in a formulation can have on maintaining the potency of an orally delivered bioactive.
Absorption across possum gut wall
The second aspect that will control uptake of a bioactive following oral delivery is the rate of its absorption across the gut wall. As degradation of proteins and peptides is much lower in the hindgut (the caecum and colon) and this is likely to be the site for delivery of bioactives, we assessed absorption across this region of the possum gut. Firstly, we determined the permeability properties of the possum hindgut using small hydrophilic compounds. We found that rates of absorption across the caecum and the colon were very similar (Table 1), suggesting it may be possible to target the delivery of bioactives to both of these regions simultaneously.
Table 1 Basal permeability properties of possum hindgut | ||
Fluorescein |
| |
Colon |
10.6 _ 1.5 |
64 _ 5 |
Caecum |
11.2 _ 4.1 |
71 _ 10 |
Having established baseline absorption across the gut epithelium, we assessed the effectiveness of compounds known to increase uptake across the gut wall (permeation enhancers), at least in other species. Permeation enhancers can operate by acting on the mucus layer, thus allowing the bioactive to come in closer contact with the epithelial layer, or by interfering with the integrity of the epithelial cell layer, thus allowing more bioactive to cross.
For example, a mucolytic agent, dithiothreitol (DTT), had a marked effect, reducing the mucus layer by more than 50 percent in both the caecum and colon. One of the permeation enhancers that we tested, sodium deoxylcholic acid (SDA), had no effect on mucus. However, both of these compounds significantly increased permeability of the possum hindgut (Figure 3). When both agents were used in combination there was an additive effect on permeability.
We assessed the effectiveness of a number of permeation enhancers on possum gut tissue. Both colon and caecum tissues appear to respond in a similar way. An important consideration is the dose of permeation enhancer to include in a formulation. Because of their action on epithelial tissue the level of enhancer used is critical. It must be sufficient to promote uptake of the bioactive, without inducing severe damage to the tissue.
In summary, permeability properties of the possum caecum and colon are very similar and both of these tissues respond in a similar way to the permeation enhancers we have tested. This is a considerable advantage as it means that formulations can be targeted to the entire hindgut, rather than to a specific region. Another advantage is that the residence time in this region of the gut, especially the caecum, are long, maximising the opportunity for a bioactive to come in contact with the gut wall and be absorbed.
Currently, we are developing pelleted formulations for delivering peptides via the oral route - using model peptides and combining these with metabolic inhibitors, permeation enhancers and binding agents that enable the formulation to be compressed into pellets.
**Overview
Oral delivery of biocontrol agents to possums has a number of advantages and is likely to be the option of choice in the short to medium term. However, the delivery of a protein or peptide bioactive to the feral possum population in New Zealand via a bait, has inherent problems. The bait (and therefore the bioactive) will be subjected to a wide range of environmental conditions that may adversely affect its stability before being consumed. At the time of taking the bait, individual possums may be in a variety of physiological conditions that could influence the potency or the effectiveness of the bioactive. The amount of bait eaten, and therefore the dose of bioactive taken by the possum will be unknown and highly variable. All of these circumstances favour loss of potency of the bioactive before it is consumed and highly variable rates of metabolism after it is eaten. To maximise the chances of the bioactive invoking sterility, it must be highly potent so that even a small dosage will be effective, and it must be presented in a formulation that protects it from degradation in the environment and from metabolism in the possum gut and maximises its uptake.
Summary
To develop oral delivery of bioactives to possums we need to:
- understand the mechanisms of gut function in possums;
- know how the bioactive is degraded in the possum gut;
- decide which region of the gut is to be targeted for delivery;
- develop a formulation that will minimise degradation and maximise uptake of the bioactive.
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