AU699587B2 - Addressing bait aversion by disrupting associative learning - Google Patents

Description

~nI- Giii WO 95128081 PCT/NZ95/00030 ADDRESSING BAIT AVERSION BY DISRUPTING ASSOCIATIVE LEARNING TECHNICAL FIELD The present invention relates to techniques addressing bait aversion exhibited by animals.

Typically, the baits in question will be poisoned baits and the target animals vermin such as rodents, rabbits and hares, possums, birds, insects, arthropods, reptiles and amphibians etc. A preferred technique relies on disrupting the associative learning process. This may comprise affecting glutamate neurotransmitter functions. This is believed to disrupt the normal associative learning process of an animal, which is considered to be at least partially responsible for aversion developed in animals to baits.

The invention may also find use in compositions other than baits.

SBACKGROUND ART Poisoned baits are used for controlling a wide number of animal species. A bait will comprise a substance which an animal finds palatable or attractive and into which has been added a toxic substance or substance otherwise having an effect on the animal e.g a contraceptive. However, many animals will only sample a small quantity of a new or unknown food stuff before returning at a later time to consume a larger amount. If the animal becomes sick or suffers any ill effects (whether it was a result of the bait, foodstuff or not) then it will associate the discomfort with the new foodstuff and fail to return, no matter how palatable or attractive the bait may be made.

Animals which sample unknown foodstuffs do not ingest sufficient toxin (or other substance) at a first sampling for it to be effective. Return of the animal for subsequent feeding is generally required incorporat;ng high levels of poisons so the first sampling is lethal can be expensive and dangerous. Unfortunately an animal whih has developed an aversion will from that time on will avoid any foodstuff having the same physical attributes that it associates with the illness or discomfort, including a particular foodstuff, a particular colouring, a particular smell, or taste. The result is that a particular attractant or lure incorporated into the bait will no longer be effective against that animal and will even act as a warning to the animal that the bait or foodstuff is dangerous. Selective evolutionary pressures associated with aversion may favour the greater expression of cautious feeding strategies.

I

SUBSTITUTE SHEET L~~L II_ aa~sl-~ 'WO 95128081 PCTINZ95/00030 The development of aversion to poison-bait compounds represents a major problem in pest control. Indeed the effectiveness of all commercially available rodenticides and feral poisons is endangered by increasing development of population aversion. Aversion is a conditioned learning process and as such has a dependence on peripheral and central nervous system components. These in turn communicate via chemical messengers neurotransmitters.

Aversion has been documented for all poisons used to date, including recently anticoagulant and coagulant compounds in rats. The proportion of population developing/exhibiting aversion differs somewhat depending upon the poison, ranging from up to 60% in cyanide to 20-30% for anticoagulants such as warfarin.

Most research in the past has been directed to the development of new attractants and lures which will increase the palatability of a bait. The hope is that an animal will consider that the bait resembles a foodstuff with which it is already familiar, or is so appealing that the animal may be tempted to consume a significant portion at the first sampling. To a certain degree this is effective as many baits still exhibit a useful degree of effectiveness on pest populations, and the introduction of new lures provides an alternative to the old lures of which many animals may be wary. However none of the art has identified or directly tackled the question pertaining to the association that an animal makes between a bait, foodstuff or substance which makes it ill and the illness itself.

It is an object of the present invention to address the foregoing problems or at least to provide the public with a useful choice.

Further aspects and advantages of the present invention will become apparent from the ensuing description which is given by way of example only.

DISCLOSURE OF INVENTION According to one aspect of the present invention there is provided a method of addressing bait aversion comprising the inclusion into bait of a substance which disrupts the associative learning process, of a targeted animal species, at least insofar as it contributes to bait aversion.

According to a further aspect of the present invention there is provided a method of addressing bait aversion, substantially as described above, comprising the inclusion of a JL _I WO 95/28081 PCTINZ95/00030 substance blocking or disrupting glutamate neurotransmitter pathways, of a targeted animal species, into a bait.

According to yet a further aspect of the present invention there is provided a method of addressing bait aversion, substantially as described above, comprising the inclusion of at least one glutamate antagonist or agonist into a bait.

According to an even further aspect of the present invention there is provided a method substantially as described above which comprises the inclusion into a bait of at least one of the following: i) one or more cholecystokinin atagonists or agonists; ii) one or more neuropeptide Y antagonists or agonists; iii) one or more serotonin antagonists or agonists; iv) one or more calcitonin gene related peptide antagonists or agonists, and v) one or more doparmne antagonists or agonists, to the extent that glutamate release for neurotransmitter use in a target animal is affected.

According to a further aspect of the present invention there is provided a bait which includes a substance disrupting the associative learning proc(es, of a targeted animal species, at least insofar as it contributes to bait aversion.

According to another aspect of the present invention there is provided a bait substantially as described above which includes at least one of the following: i) one or more glutamate antagonists or agonists; ii) one or more nitrous oxide antagonists or agonists; iii) one or more cholecystokinin atagonists or agonists; iv) one or more neuropeptide Y an' agonists or agonists; v) one or more serotonin antagonists or agonists; vi) one or more calcitonin gene related peptide antagonists or agonists, and vii) one or more dopamine antagonists or agonists.

According to an even further aspect of the present invention there is provided the use of a substance disrupting the associative learning process of a targeted animal species, at least insofar as it relates to aversion, in compositions to be ingested by or administered to said targeted animal species.

According to yet a further aspect of the present invention there is provided a composition for administration to a targeted animal species, said composition including components I I~ WO 95/28081 PCTINZ95/00030 having adverse affects on said targeted animal species, said composition including at least one substance which disrupts the associative learning process of the targeted animal species, at least insofar as it contributes to aversion.

Bait aversion, in the primary source the animal that samples) is dependent upon associated learning correlating the ill effects with the sample or sample characteristics.

Previous trials by others in the art suggest that the associative learning process is dependent upon neurotransmitter function within the hippocampus and amygdala of the brain. Trials with rats in mazes has indicated that the neurotransmitter of prime importance appears to be glutamate. However, prior art trials studying the effect of glutamate neurotransmitter function in relation to associative learning has been restricted predominantly to mazes and various paradigms. No research has apparently been directed to practical applications of the largely experimental results.

The present invention is directed to the association between the associative learning process and substance aversion in many animals. Accordingly, most embodiments of the present invention seek to disrupt the associative learning process in an animal and thereby inhibit any bait aversion tendencies before they form. Typically, disruption of the associative learning process will be achieved through the interference of neurotransmitter function.

The present invention provides baits, and compositions which may be included in baits or administered to animals, which will block or disrupt certain neurotransmitter functions. Most embodiments will be characterised by including a glutamate antagonist which can affect neurotransmitter function within the hippocampus. Other embodiments and methods according to the present invention may take a less direct path to disrupt neurotransmitter function. This will be discussed later within the specification.

A variety of glutamate antagonists and agonists, which among other uses are commonly used for addressing certain forms of epilepsy, are known, some examples of which are as follows: 5,7-dichloro kynurenate CGP 37849 7-chloro-5-iodo kynurenic acid NK-801 4C3 HPG CPP CGS 19755 MNQX L689560 I I WO 95/28081 PCT/NZ95/00030 phencyclidine 7-chlorothio kynurenic acid CNQX dextrorphen APV dizocilipine NBQX L-l-amino-3-phosphono propanoic acid GYK152466 L-2-amino-4-phosphono butanoic acid HA966 ST2 EAB-515

APH

It is also noted that other glutamate antagonists may also be used though the above examples represent a selection currently being considered by the applicant. Of the above examples, the antagonists being given the most serious consideration, and the subject of ongoing trials are: SD2 EAB-515 APH APV L-AP4 MK-801 It is noted that some glutamate antagonists may be more effective in particular animal species than others, though this will largely depend on the target animal's physiology.

Generally it is desirable that a glutamate antagonist having a high activity is used, though increased amounts of less active antagonists could also be used where practical.

Some embodiments of the present invention may include the use of synergistic agents which can enhance the effectiveness of the glutamate antagonists.

Indirect routes, rather than directly targeting glutamate receptors, may also be relied upon. Generally the associative learning process may be affected merely by disrupting neurotransmitter function without the necessity for substantial or total blockage.

Accordingly, agents which may indirectly act to disrupt neurotransmitter function may also be relied upon and fall within the scope of the present invention. Figure 1 is, a schematic view of various pathways associated with glutamate receptors and indicates (af) possible sites of drug action to affect glutamate neurotransmitter pathways.

The process of aversion involves inputs from the peripheral nervous system (and central nervous system) that signal the ill effects associated with the poison. These may be modified by other neurotransmitter systems in the brain on the way to the hippocampus (or within the hippocampus itself). The release of glutamate will then relate to the overall integrated level of stimulus. If it is strong enough nitric oxide (NO) may also be 3, 1 ~I r~aap~p M-M P-MMPIT"*-l WO 95/28081 PCT/NZ95/00030 synthesised and this is likely to potentiate the aversion development.

With reference to figure 1, can be seen pathways for CCK (Cholecystokinin) and NPY (Neuropeptide Y) which both are capable of feeding glutamate driving neurones to prompt a release of glutamate. Antagonists or agonists which disrupt or block CCK and/or NPY will have an effect on subsequent glutamate relaease and thus antagonists to CCK and NPY can be considered an alternative to, or in addition to, glutamate blocking antagonists.

(Serotonin) modulates CCK glutamate interactions and thus 5HT antagonists or agonists may be considered for disrupting the link between glutamate and bait aversion Similarly GABA (Gamma-4-amino butyric acid) modulates the NPY glutamate pathway and thus GABA antagonists or agonists may also be considered for indirectly disrupting the link between glutamate release and bait aversion.

GABA, and also DA (dopamine), affect the calcitonin gene related peptide (CGRP) and thus GABA and DA antagonists and agonists may be considered for indirectly disrupting the link between glutamate neurotransmitter function and substance aversion.

An alternative arrangement is to block the parallel pathway linking glutamate with bait aversion and involving nitrous oxide release. Nitrous oxide is released only when very high glutamate release levels are attained and act to potentate glutamate effects. It is considered by the applicant that the nitrous oxide release may be just as important as glutamate release when certain animals develop aversion tendencies. Accordingly it is considered that nitrous oxide antagonists may be useful to minimise or eliminate the contribution of this pathway in the role of developing bait aversion tendencies in animals.

It is noted that many of the pathways (specifically comprising a-g in figure 1) are indirect and blocking one of these pathways will typically serve to limit rather than totally block the role of glutamate in the animal's neuralprocesses. Where disruption rather than total blockage is desired, these alternative pathways (such as the examples illustrated in figure 1) may be relied upon The use of antagonists which block parallel pathways also associated with the development of bait aversion, such as the illustrated nitrous oxide example, may also be relied upon. While it is considered that such pathways do play a role in bait aversion, their contribution for specific animals is not specifically known and thus the blocking of "M WAN WO 95/28081 PCT/NZ9S/00030 these alternative pathways may not always be the equivalent to the use of direct glutamate antagonists. However the availability for blocking such alternative pathways provides the user with more flexibility and control, and is useful in addressing any resistance which may develop amongst certain targeted animals.

Following are examples of antagonists and agonists against various of the specific compounds illustrated in figure 1.

Cholecystokinin, (CCK): Neuropeptide Y, (NPY): Nitrous oxide, (NO): Serotonin, (SHT): Gamma-4-amino butyric acid, (GABA): Benzotript, RB211, CI988, L365260 Lorglumidc sodium salt, PD 135, 138, N-methyl-D-glucamine salt, proglumide, proglumide sodium salt.

Leu 3 1

PRO

3 4 Neuropeptide Y, Fragments 18-36 NPY, Methyl-Tyr NPY

L-N

5 ,-(1-Iminoethyl) ornithine HCL, NG- Monomethyl-L-argininc acetate, N 4 -nitro-L-arginine (L- NOARG), NG-nitro-L-arginine methyl ester HCL.

cinanserin, MDL-72222, LY-278, 584 maleate, metergoline, methysergidc maleate.

phaclofen, bicuculline, SR-95531, B-IHydrastine, 2- Hydroxysaclofen, baclofen, muscimol, THIP HCL Example 3d gives a possible composition based on the pathways of figure 1.

It is generally considered that the glutamate antagonist can be introduced into a bait during its preparation. For some baits it may be appropriate to include a glutamate antagonist or antagonists, and other active agents, during manufacture of the bait.

However, this may not always be feasible and thus the present invention also provides preparations which may be applied to previously prepared baits and foodstuffs.

Antagonists may also be administered or introduced to the animal in other manners than in a bait or foodstuff, though it is currently considered that inclusion in matters consumed by the animal is perhaps the most feasible and effective manner to introduce the antagonist to the animal. However, other methods of introduction to the animal are also possible. It is possible that a dose of a suitable preparation could be injected into the WO 95/28081 PCT/NZ95/00030 animal. For instance some bait stations may include a mechanism in which a needle or sharp object can prick the animal and introduce an effective dose of an active preparation.

It may also be a needle that an animal may scratch themselves against thereby introducing the compound. Antagonists which may be absorbed through the skin or by inhalation (perhaps of a vapour or mist) are further possibilities and it is envisaged that some g embodiments of preparations may provide for these alternatives if this is the preferred method of administration for the user of the product.

As previously mentioned, the amount of a glutamate antagonist or antagonists which need to be administered to an animal can vary. The amount required will be partially dependent on the animal's physiology. However, when the glutamate antagonists are included in a bait or foodstuff, the quantity of foodstuff likely to be consumed by a targeted animal during their first sampling must also be taken into account. Where an animal is likely to take only a small nibble then it is apparent that a higher concentration of glutamate antagonist must be present. An alternative would be to selectively increase the concentration of glutamate antagonists in various portions of the bait. For instance, the outermost portion of a bait or piece of foodstuff could be given a higher concentration of glutamate antagonists rather than being uniformly dispersed theretrough. The use of preparations which can be sprayed over the bait or foodstuff is one manner by which this may be achieved.

Various other considerations in the preparation of a bait or composition is the presence of substances which can inhibit the effectiveness of the glutamate antagonists, or which may react with the antagonist thereby reducing its effectiveness. Manufacturer's data sheets for specific glutamate, antagonists can generally be consulted to determine which substances should be avoided in baits and preparations.

Various other substances may be included in baits, these including attractants, flavour enhancers, substances increasing palatability of the bait, masking agents which can cover any unpleasant taste or smells associated with the glutamate antagonists, etc.

BRIEF DESCRIPTION OF DRAWINGS Further aspects of the present invention will become apparent from the ensuing description which is given by way of example only and with reference to the accompanying drawings in which: Figure 1 is a schematic view of pathways and points of actions of various C~ YI WO 95/28081 PCT/NZ95/00030 substances in the glutamate neurotransmission process.

Figure 2 is a schematic diagram depicting grouping of trial animals in example 1 Figure 3 is a schematic diagram of experimental protocols of example 1 Figure 5-8 are graphs of example 1 trial results BEST MODES FOR CARRYING OUT THE INVENTION Example 1 Pharmacological antagonism or agonism of various neurotransmitter points within the scheme of Figure 1 offers potential for disrupting the aversion process.

A combination of various antagonists and agonists acting at multiple sites may also be more efficacious than a single site manipulation. The window of time for potential disruption of the aversion development will, of course, differ depending upon the site (or sites) chosen for targeting.

The pharmacological combination must possess certain features to be useful. These include: absorption through the GI system to peripheral and central sites.

nR tim of r.tinn thit nrrlldes develnnpment of aversinn (this will diffpr depnnrlinc on the site of action).

palatability to the targeted species.

ability to be compounded into bait.

shelf and field "life" appropriate to usage.

economic viability.

A number of compounds that meet most of these requirements were trialed in rats and to a limited extent in possums as described below.

Example la Methods and Materials Rat Trials Experimental Animals Rattus norwegicus (laboratory albino white rat) were used in the trials.

Both male and female rats, body weights 300-500 g were used. Rats were individually WO 95/28081 PCT/NZ95/00030 housed with continued access to water and subjected to a 12 hour light 12 hour dark cycle. Ambient temperature was controlled at Feed Regimes Each rat was fed, daily, at 0900 hours, a controlled amount of two feeds. Standard rat pellets were provided at a total weight of 40 g/day and COCO POPSTM (a chocolate cereal product based on expanded rice) were provided at a total weight of 20 g/day. Each of these was wet with 2 ml of water.

All rats received this regime for four days (Days 1-4).

Poison and Drug Regimes On days 5-8 animals were randomly assigned into one of four groups.

o Group 1 continued as above with 40 g standard pellets and 20 g COCO POPST.

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S Group 2 received 40 g of standard pellets and 20 g of COCO POPSTM containing one of two doses of the poison sodium monofluoroacetate (1080). Dose 1 was 0.01 mg 1080 per kg rat bodyweight, while dose 2 was 0.001 mg 1080/kg rat bodyweight. Rats received either dose 1 or dose 2 on day 5 and day 6 and they all received 0.001 mg 1080/kg bodyweight on day 7.

o Group 3 received 40 g of standard pellets and 20 g of COCO POPSTM containing one of the test drug mixtures. This was repeated on day 6 and 7.

a Group 4 received 40 g of standard pellets and 20 g of coco POPSTM containing one of the test drug mixes and 0.01 mg/kg bodyweight of 1080 on day 5, 6 and 7.

On days 9-12 all groups received feeding as in day 1-4. On day 14 and day 28, groups 2 and 4 received the identical protocols to day 5 for the respective groups. On day 15 and 29, groups 2 and 4 were fed as for days 1-4. Figures 2 and 3 summarise these regimes.

Drugs Seven different formulations, each at 3 different concentrations were trialed. Two of these proved notably successful at the concentrations trialed. These were: Drug Formulation A a mixture of NO inhibitors and glutamate antagonists. This was given at a mix concentration of 40, 60 or 80 mg per g COCO POPS

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~fi~L~RJ~a~Y~ WO 95/28081 PCTINZ95/00030 Drug Formulation B a mixture of NO inhibitors, glutamate antagonists and CCK antagonists. Again given at a mix concentration of 40, or 80 mg per 20 g COCO POPSTM.

Both drugs and 1080 were made up in a 2 ml solution added to absorb into 20 g of COCO

POPS

T

Feeding of drug/poisoned COCO POPS TM occurred on the same 24 hours, 0900 hrs delivery, schedule as per normal feeding.

Behavioural Observations and Aversion Measurement Behavioural observations were made every 15 minutes, for 1 hour, immediately following feeding at 0900 hrs and again at 1500 hrs. Seven types of behaviour were frequency recorded. These were: exploring or moving, grooming, eating, drinking, sleeping, lying and sitting.

Each day, immediately prior to feeding, animals were weighed and amount of pellets and COCO POPS T M consumed in the previous 24 hours calculated. Amount of water drunk was also measured.

The first four days served to establish a baseline of grams of COCO POPS TM and pellets consumed by an animal every 24 hours. Rats have an "innate" preference for COCO POPSTM because of their sweetness.

When poisons or drugs were added, aversion was measured as the change in relative consumption of the two sources. With aversion, animals will consume more pellets and less COCO POPSTM* Results Rat Trials Over the first four days of trials, a consistent pattern of consumption was observed. As rat bodyweight varied, coco pop and pellet consumption data has been normalised to of bodyweight.

On days 1-4, trial animals consumed 2.9 0.7% (mean standard deviation) bodyweight of pellets and 4.0 0.4% bodyweight COCO POPSTM. Water consumed was 42.3 9.4 ml.

Group 1 animals maintained this consistent pattern throughout the trial, with a trial average of 2.9 0.5% bodyweight pellets, 4.0 0.1% bodyweight COCO POPSTM and 43 1 10.2 ml water (Figure 4).

./2 WO 95/28081 PCT/NZ95/00030 Group 1 animals that received 0.01 mg/kg bodyweight 1080, showed an overall dramatic decreases in all food and water consumption on day 5. On day 6 and 7, the consumption of COCO POPSTM was extremely low (less than 0.1% bodyweight) and that of pellets rose slightly (greater than 5% bodyweight). With resumption of normal feeding regimes animals eventually increased their consumption of COCO POPSTM, returning to become close to day 1-4 baselines by day 12. One of this group, rate 8, however, did not resume eating of COCO POPSTM in the trial. On days 14 and 28, with re-addition of 1080 these rats again would not eat COCO POPSTM, but on days 15 and 29, with the exception of rat 8, Coco POPSTM without 1080 were eaten as per normal. Figure 5 summarises this data.

Group 2 animals which received 0.001 mg/kg bodyweight 1080 showed a slight fall in coco pop consumption on day 5, but a return to normal baseline levels on day 6 which did not subsequently change (Figure 6).

Group 3 animals showed a slight decrease in coco pop consumption on day 5 with all the trialed formulations, but this returned to baseline value on day 6 and stayed at this level for the remainder of the trial (Figure 7).

In group 4 animals, all formulations used in combination with 0.01 mg/kg bodyweight 1080 showed some degree of reduction of aversion as compared to animals in group 2.

Two formulations, A and B, however stood out as being most efficacious, with 40 and mg concentrations producing similar effects.

On day 5, a reduction in COCO POPSTM consumption, similar to that in group 3, was seen. With formulations A and B days 6 and 7 showed similar consumption of COCO POPSTM to that in baseline days 1-4, as did days 9-12. Days 14, 15, 28 and 29 were also unchanged from baselines. Figure 8 demonstrates these data.

Examples 2 Methods and Materials Possum Trials Six possums (Trichosurus vulpecula Australian bushtail) were captured near feeders, tagged and released into the wild. On subsequent nights these possums returned to feeders and established a normal pattern of feeding on 3 g cinnamon laced cereal pellets.

After establishment of a normal, nightly, pattern of feeding, the animals were exposed to r0 pellets of 4 varieties: 12 pCT/NZ95000 3

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WO 95/28081 WO 95/28081 PCT/NZ95/00030 normal 3 g cinnamon pellets, 3 g pellet containing 3 mg 1080, 3 g cinnamon pellets containing 1080 and 80 mg of drug formulations A from the rat trial, or 3 g cinnamon pellets containing 80 mg of drug formulation A.

A further three possums were captured and tagged and subject to the same regime in a later trial.

Aversion was measured through the choice of pellets following consumption of 1080 and/or drug laced pellets or presence at feeders, but absence of consumption of any pellets following drug/1080 exposure.

Animals that did not return to the feeders could not be judged as showing aversion, but speculatively this is a possibility.

Results: Possum Trials In the first trial 2 animals that consumed pellets and 1080/drugs showed no aversion whereas 2 animals that consumed pellets and 1080 alone developed an aversion to pellets.

In the second trial, 1 animal consumed pellets and 1080/drugs and showed a subsequent aversion as did 1 animal that consumed pellet and 1080 alone.

In this second trial the drugs had been sitting within the pellets at 25ooC for 6 weeks and in the field with adverse weather conditions for a number of weeks. The temperature and time stability of the drug formulations is not yet known.

Discussion of Examples 1 and 2 When faced with the addition of a new compound, be it 1080 or 1080 and drugs or drugs alone, the first response of the rats is to reduce their food intake. This may be a "sample and see" survival behaviour. In the case of 1080, only, this reduction continued past the first day. It is possible that the initial reduction of feed with 1080 related to "ill effects" from 1080 and these triggered subsequent aversion development.

The development of bait-1080 aversion was quite clear in possums and in rats. In rats a dose of 0.01 mg/kg bodyweight, but not 0.001 mg/kg bodyweight, 1080 was needed to develop aversion. In the majority of rats the aversion appeared to the 1080 rather than the bait per se.

WO 95/28081 PCTINZ95/00030 The aversion may be the smell of 1080. This was supported by behavioural characteristics that the rnts exhibited following aversion. Rats would pick up coco POPSTM individually and sniff them. If they contained 1080, they were thrown away in the cage. If they did not contain 1080, they were consumed. The aversion was obviously long lasting as it could be demonstrated 14 and 28 days after the start of trial (or 7 and 21 days following the last exposure to 1080). This aversion was prevented by the combination of certain drug formulations with the 1080 in the COCO POPSTM.

Drug formulations that contained glutamate antagonists exhibited the greatest effectiveness in preventing the development of aversion to 1080 and the most efficacious of the formulations also contained CCK antagonists. Whether the drugs are acting peripherally, centrally or both is not known from this study, but their time course of distribution to active site, following oral consumption, is obviously within a window of time that allows antagonism of aversion development.

All the concentrations of formulations A and B used proved effective in disrupting aversion and it is possible that these concentrations were at the upper end of the dose response curve. Further testing of different concentrations is needed to construct a full dose response curve and also provide the economically viable rarge for each formulation.

In terms of commercial applications it is interesting to note that formulation A had a unit cost of 5 cents (NZ) per 20 g of bait-poison. Such a low cost makes it economically viable for addition to standard rodenticides.

In rats both cholecystokinin and glutamate would appear to be crucial to the development of aversion (although other actions of the drugs cannot be ruled out) and NO also would appear to have a contributing effect.

Speculatively CCK may "feed" into the glutamate pathway relaying "unpleasant" sensory information concerning the effects of 1080. CCK may also cause anxiety in the animals following its activation by 1080 further enhancing the aversion process. Glutamate and NO may be the agents that allow the conditioning association and subsequent aversion memory.

In the first trial the 2 animals receiving the drug formulations showed a clear disruption of aversion whereas in the second trial the 1 animal that received the drug formulation did not show an obviius disruption of the aversion process. In the second trial there were a -number of other uncontrolled variables. The drugs, incorporated with 1080, in the i WO 95/28081 PCTINZ95/00030 pellets were stored at 25oeC for a considerable period of time and then subsequently exposed to adverse weather conditions. The drug formulations appear relatively stable at room temperatures for several weeks.

Examples 3 A preparation including a glutamate antagonist may be prepared according to the following formulation: Example 3a To a total of 100% 0.0001-99.99% 0.01-99.999% Glutamate antagonist or antagonists Acceptable carrier or diluent or other components The above example la represents a broad range of preparations covering relatively diluted preparations more suitable for bulk spraying of a large amount of bait, through to concentrated preparations for more specific use or to be diluted before final use.

A composition which is more likely to fine more widespread use is as follows: Example 3b To a total of 100% 0.0001-10% 90-99.9999% Glutamate antagonist or antagonists Acceptable carrier or diluent or other components A further composition, which may find use for application to many preparations directed to rodents and possums, is as follows: Example 3c To a total of 100% 0.1-2% 98-99.9% Glutamate antagonist or antagonists Acceptable carrier or diluent In the above examples, a wide range of diluents or carriers or combinations thereof may be used. Where the glutamate antagonist is water soluble, then water may be conveniently used as a cheap carrier/diluent. However, in some cases, it may be inappropriate to use water in which case other solvents, carriers and diluents may he used.

WO 95/28081 PCT/NZ95/00030 For instance, a volatile solvent may be useful so that when it evaporates it leaves a crust or concentrated layer of glutamate antagonist behind. Other insoluble substances may be included so that in many respects the composition resembles a paint or lacquer so that once the volatile solvents have been removed a layer or matrix of material including glutamate antagonist remains.

Some further embodiments include preparations which are predominantly oil rather than aqueous based. The use of glycerol and similar types of carriers or diluents are also a possibility.

Previous preparations may be used as a liquid bait preparation though typically they will be applied to prepared baits and foodstuffs, or included in the preparation of baits. The preparation of baits is as according to existing techniques. Any poison may be already present in the bait or bait preparation, or alternatively included in the glutamate preparation for inclusion into a foodstuff or bait preparation.

Example 3d To a total of 100%- 0.0001-99.99% Glutamate neurotransmission disrupting substance, comprising at least one antagonist or agonist affecting at least one member of the group comprising CCK, NPY, NO, 5HT, GABA, DA and CGRP 0-99.99% A glutamate antagonist or antagonists other than CCK, NPY, NO, 5HT, GABA, DA and CGRP 0.01-99.999% Acceptable carrier or diluent Reference is made to figure 1 and the proceeding description within this specification indicating in more detail the considered pathways of action for compositions according to example Id. This figure may be relied upon to enable the user to tailor the method by which a composition acts on the target animal species. This may be useful in addressing resistance by target animals.

Example 3e As for any of examples la-lc except a nitrous oxide antagonist or antagonists are used instead of a glutamate antagonist or antagonists.

WO 95/28081 PCTINZ95/00030 Example 3f As for any of examples la-lc except that a composition includes 0.0001-99.9% of a nitrous oxide antagonist or antagonists.

Example 4 Example 2 relates to some possible bait preparations in accordance with the present invention. For example: Example 4a A preparation such as described in example 1 is applied by iras.-rsion or spray or another technique to a prepared comestible article, or an art:Ct: ti foodstuff such as fruit, vegetables, meat etc. Poison may be included in the preparation prior to its application or introduced to the foodstuff according to known techniques. Attractants, flavour masking agents, enhancers etc. may also be incorporated.

Example 4b A bait formulation, comprising to a total of 100%: 0.0001-10% Glutamate Antagonist 0-99.999% Foodstuff or palatable substance 0-99.999% Bulking agent 0-90% Toxic Substance 0-09% Contraceptive substance 0-90% Hormonal substance affecting targeted animal species 0-50% Attractant to the targeted animal species 0-50% Repellent to non targeted species Optional Other components as required Example 4c A bait formulation, comprising to a total of 100%: 0-10% Glutamate antagonist 0-10% Nitrous Oxide antagonist 0-10% CCK antagonist or agonist 0-10% NPY antagonist or agonist WO 95/28081 PCT/NZ95/00030 0-10% 5HT antagonist or agonist 0-10% GABA antagonist or agonist 0-10% DA antagonist or agonist 0-10% CGRP antagonist or agonist and wherein there is at least 0.0001% in total of antagonists or agonists, said antagonists or agonists comprising any one or combination of the foregoing, said composition also including: 0-99.999% Foodstuff or palatable substance 0-99.9- 3% Bulking agent 0-90% Poison 0-50% Attractant to the targeted animal species 0-50% Repellent to non targeted species Optional Other components as required The palatable substance will typically be a foodstuff or other agent which the targeted animals finds palatable. -This will depend largely upon the targeted animals in question.

The poison will typically comprise known poisoning agents and the prior art may be drawn upon here.

Bulking agents will typically comprise substances which the targeted animal may not necessarily value as a foodstuff or particularly palatable but on the other hand may not necessarily find unpleasant. Bulking agents may be used to extent the lifetime of products, which with certain foodstuffs could otherwise be quite short. A wide range of substantially inert bulking agents are known in the food industry and may be included in the present invention.

Attractants may comprise a range of substances. These substances may increase the palatability of the bait to a targeted animal species. Attractants may also arouse the targeted animal's interest in the bait and may perhaps even lure the animal to the bait.

The use of pheromones is one particular example which may be put into practice.

In many cases it is not desirable to indiscriminately kill animals thus there may be advantage in including substances which are repellent to certain animal species. These may include substances which are repellent to the animals so that they are not lured to the bait in the first place. This may also include substances which render the bait unpalatable to certain non-targeted animal species. This may prevent such animal fr:m consuming a WO 95/28081 PCT/NZ95/00030 sufficient amount of the bait to produce any substantially adverse effects. Unfortunately, the presence of the glutamate antagonist will also inhibit any associative process linking sickness with the bait in these non-targeted species.

By way of explanation it is noted that glutamate antagonists have been described for use in the present invention. It is noted that other substances which interfere with glutamate receptors, even though they may not specifically be termed within the art as being a glutamate antagonist, may also find use in various embodiments of the present invention.

It is also noted that combinations of glutamate antagonists, and incorporated synergistic agents, may find use in some embodiments.

The techniques of the present invention may be used in compositions other than toxic baits. For instance some medical or nutritional compositions may have unpleasant sideeffects or characteristics. Where administration by voluntary ingestion by an animal (for instance) is relied upon, addressing any developing aversion may be desirable. The administration by voluntary feeding of various substances to dairy and beef cattle, sheep and other livestock or domesticated animals is one example.

Aspects of the present invention have been described by way of example only and it should be appreciated that modifications and additions may be made thereto without departing from the scope thereof as defined in the appended claims.

Claims (21)

1. A method of addressing bait aversion comprising the inclusion into bait of a substance which disrupts the associative learning process, of a targeted animal species, at least insofar as it contributes to bait aversion.

2. A method of addressing bait aversion, as claimed in claim 1, comprising the inclusion of a substance blocking or disrupting glutamate neurotransmitter pathways, of a targeted animal species, into a bait or composition.

3. A method of addressing bait aversion, as claimed in claim 1, comprising the inclusion of at least one glutamate antagonist or agonist into a bait or composition.

4. A method as claimed in claim 2 or claim 3 in which an included substance is present in sufficient quantity so that a typical portion ingested by a target animal, when sampling the bait or composition at a first encounter, contains sufficient included substance to significantly disrupt the associate learning process, insofar as it relates to bait aversion, for a high proportion of the targeted animal species. A method as claimed in either claim 2 or claim 3 comprising the inciusion of at least one member of the following group: D-AP5; CGP 37849; NK-801; CPP; MNQX; phencyclidine; CNQX; APV; NBQX; GYK152466; HA966; APH; 5,7-dichloro kynurenate; 7-chloro-5-iodo kynurenic acid; 4C3 HPG;CGS 19755; L689560;

7-chlorothio kynurenic acid; dextrorphen; dizocilipine;L-l-amino-3-phosphono propanoic acid; L-2-amino-4-phosphono butanoic acid, and ST2 EAB-515. 6. A method as claimed in claim 3 which includes the inclusion of an additional agent enhancing the effectiveness of the included substance. 7. A method as claimed in claim 6 in 'which an included additional agent comprises the addition of at least one of: i) one or more cholecystokinin atagonists or agonists; ii) one or more neuropeptide Y antagonists or agonists; iii) one or more serotonin antagonists or agonists; iv) one or more calcitonin gene related peptide antagonists or agonists, and v) one or more dopamine antagonists or agonists. WO 95/28081 PCTINZ95/00030

8. A method as claimed in claim 1 which comprises the inclusion into a bait of at least one of the following: i) one or more cholecystokinin atagonists or agonists; ii) one or more neuropeptide Y antagonists or agonists; iii) one or more serotonin antagonists or agonists; iv) one or more calcitonin gene related peptide antagonists or agonists, and v) one or more dopamine antagonists or agonists, to the extent that glutamate release for neurotransmitter use in a target animal is affected.

9. A method as claimed in claim 8 in which: an included cholecystokinin atagonists or agonists comprises at least one member of the group comprising: Benzotript, RB211, CI988, L365260 Lorglumidc sodium PD 135, 138, N-methyl-D-glucamine salt, proglumide, and proglumide sodium salt; and wherein an included neuropeptide Y antagnoist or agonist comprises at least one membL. of the group comprising: Leu 3 1 PRO 3 4 Neuropeptide Y, Fragments 18-36 NPY, and Methyl-Tyr NPY; and wherein an included serotonin antagonist or agonist comprises at least one member of the group comprising: cinanserin, MDL-72222, LY-278, 584 maleate, metergoline, and methysergic maleate. A method as claimed in claim 8 wherein there is also included within the bait a glutamate antagonist or agonist.

11. A method as claimed in claim 10 wherein an included antagonist or agonist comprises at least one member of the group comprising: CGP 37849; NK-801; CPP; MNQX; phencyclidine; CNQX; APV; NBQX; GYK152466; HA966; APH; 5,7-dichloro kynurenate; kynurenic acid; 4C3 HPG;CGS 19755; L689560; 7-chlorothio kynurenic acid; dextrorphen; dizocilipine;L-l-amino-3-phosphono propanoic acid; L-2-amino-4-phosphono butanoic acid, and ST2 EAB-515.

12. A method as claimed in any one of claims 2, 3, or 8, which also includes the addition into a bait of a nitrous oxide antagonist or agonist. ~_11~1~

13. A method as claimed in claim 12 in which an included nitrous oxide antagonist or agonist comprises at least one member of a group comprising: L-NS-(1-Iminoethyl) omithine HC1, NG-Monomethyl-L-arginine acetate, N 4 -nitro-L-arginine (L-NOARG), and NG-nitro-L-arginine methyl ester HC1.

14. A bait which includes a substance disrupting the associative learning process, of a targeted animal species, at least insofar as it contributes to bait aversion. A bait as claimed in claim 14 which includes a substance blocking or disrupting glutamate neurotransmitter pathways in a target animal.

16. A bait as claimed in either claim 14 or claim 15 which includes at least one of the following: i) one or more glutamate antagonists or agonists; ii) one or more nitrous oxide antagonists or agonists; iii) one or more cholecystokinin antagonists or agonists; o p p p Cr C P 0 p iv) one or more neuropeptide Y antagonists or agonists: v) one or more serotonin antagonists or agonists; vi) one or more calcitonin gene related peptide antagonists or agonists, and vii) one or more dopamine antagonists or agonists.

17. A bait as claimed in claim 16 wherein: an included glutamate antagonist or agonist comprises at least one member of the group P. p p f* P P C p p *a a 20 comprising: D-AP5; CGP 37849; NK-801; CPP; MNQX; phencyclidine; CNQX; APV; NBQX; GYK152466; HA966; APH; 5,7-dichloro kynurenate; 7-chloro-5-iodo kynurenic acid; 4C3 HPG; CGS 19755; L689560; 7-chlorothio kynurenic acid; dextrorphen; dizocilipine; L-l-amino-3-phosphono propanoic acid; L-2-amino-4-phosphono butanoic acid; and ST2 EAB-515; and wherein an included nitrous oxide antagonist or agonist comprises at least one member of the group comprising: L-Ns-(1-Iminoethyl) omithine HC1, NG-Monomethyl-L-arginine acetate, N 4 -nitro-L-arginine (L-NOARG), and NG-nitro-L-arginine methyl ester HC1; and wherein an included cholecystokinin antagonist or agonist comprises at least one member of the group comprising: Benzotript, RB211, CI988, L365260 Lorglumide sodium salt, PD 135, 138, N-methyl-D- glucamine salt, proglumide, and proglumide sodium salt; WX I I MONIPMOPK WO 95/28081 PCT/NZ95/00030 and wherein an included neuropeptide Y antagnoist or agonist comprises at least one member of the group comprising: Leu 3 1 PRO 34 Neuropeptide Y, Fragments 18-36 NPY, and Methyl-Tyr NPY; and wherein an included serotonin antagonist or agonist comprises at least one member of the group comprising: cinanserin, MDL-72222, LY-278, 584 maleate, metergoline, and methysergidc maleate.

18. A bait as claimed in claim 14 or claim 15 which includes a lure for one or more targeted animal species.

19. A bait as claimed in claim 14 or claim 15 which includes an effective amount of an included substance to disrupt the associative learning process, insofar as it relates to bait aversion, in a high proportion of a targeted animal species in a typical ingested portion the size of bait for that targeted animal species. A bait as claimed in claim 14 or claim 15 which includes at least one of: i) a toxin for said targeted animal species; ii) a contraceptive agent for said targeted animal species; iii) a hormonal substance affecting a targeted animal species, and iv) a repellent for a non-targett, d animal species.

21. The use of a substance disrupting the associative learning process of a targeted animal species, at least insofar as it relates to aversion, in compositions to be ingested by or administered to said targeted animal species.

22. A method as claimed in claim 21 in which the included substances comprises at least one of: i) one or more glutamate antagonists or agonists; ii) one or more nitrous oxide antagonists or agonists; iii) one or more cholecystokinin atagonists or agonists; iv) o e or more neuropeptide Y antagonists or agonists; v) one or more serotonin antagonists or agonists; vi) one or more calcitonin gene related peptide antagonists or agonists, and vii) one or more dopamine antagonists or agonists. I

23. A composition for administration to a targeted animal species, said composition including components having adverse effects on said targeted animal species, and said composition further including at least one substance which disrupts the associative learning process of the targeted animal species, at least insofar as it contributes to aversion, said components and substance(s) including at least one of: i) one or more glutamate antagonists or agordsts; ii) one or more nitrous oxide antagonists or agonists; iii) one or more cholecyptokinin antagonists or agonists; iv) one or more neuropeptide Y antagonists or agonists; v) one or more serotonin antagonists or agonists; vi) one or more calcitonin gene related peptide antagonists or agonists; and 24 vii) one or more dopamine antagonists or agonists.

24. A composition for administration to a targeted animal species, said composition 15 including components having adverse effects on said targeted animal species, and said i: composition further including at least one substance which disrupts the associative learning process of the targeted animal species, at least insofar as it contributes to aversion, said components including at least one of: i) a contraceptive agent for said targeted animal species; 20 ii) a hormonal substance affecting a targeted animal species; iii) a repellent for a non-targeted animal species, and iv) a medicinal or pharmaceutical substance.

25. A method of addressing bait aversion, substantially as described herein with reference to the accompanying drawings and contained examples.

26. A bait or composition, substantially as described herein with reference to the accompanying drawings and contained examples. DATED this 29 th day of September 1998 THE MEAT INDUSTRY RESEARCH INSTITUTE SOF NEW ZEALAD INC By its Patent Attorneys '-vADDERNS >424