NZ249598A - Pharmaceutical compositions and their use in treating zinc deficiency disorders - Google Patents

Description

New Zealand No. 249598 International No.

TO BE ENTERED AFTER ACCEPTANCE AND PUBLICATION Priority dates: 19.02.1992;14.04.1992; Complete Specification Filed: 17.02.1993 Classification:^) A61K33/30; A61K47/44.46 Publication date: 22 September 1997 Journal No.: 1420 NEW ZEALAND PATENTS ACT 1953 COMPLETE SPECIFICATION Title of Invention: Pharmaceutical compositions and their use for treating zinc deficient disorders Name, address and nationality of applicant(s) as in international application form: COLBY CORPORATION LIMITED, a New Zealand company of 5th Floor, BDO House, 18 London Street, Hamilton, 2001, New Zealand New Zealand No. International No. 249598 TO BE ENTERED AFTER ACCEPTANCE AND PUBLICATION Priority dates: International filing date: Classification: Publication date: Journal No.: NEW ZEALAND PATENTS ACT 1953 COMPLETE SPECIFICATION Title of invention: -Compos it-i nnrr anrl ariirn m sing ginc rasponsiva—disorders. fn/tfrtflCiufiCAu &«\fbSi-T,or43 AAJO Jnt.fi U51 &A ZssJC QinCftrJT fy$oA0£A& , Name, address and nationality of applicant(s) as in international application form: COLBY CORPORATION LIMITED of 5th Floor, BDO House, 18 London Street, Hamilton 2001, New Zealand, a New Zealand company. f ft C I WO 93/16708 £* 6 PCT/NZ93/0000 PHARMACEUTICAL COMPOSmONS AND THEIR USE FOR TREATING ZINC DEFICIENT DISORDERS Technical This invention relates to compositions and their administration, for addressing zinc responsive disorders. More specifically, the invention seeks to establish a lasting zinc 5 depot in an animal," so as to increase its resistance to zinc responsive disorders such as facial eczema. The establishment of a lasting zinc depot also appears to infer resistance to other disorders. In addition, various aspects of the present invention may also be used as a therapeutic or remedial treatment for an animal inflicted with these disorders.

BAPKOROUNn ART Disorders affecting the well-being of livestock can seriously effect a farmer's profitability. On a national scale, losses in monetary terms can be substantial.

For example, facial eczema is a disorder rising through the ingestion of the spores of Pithomyces chartarum, which are found in great numbers on dead plant litter during warm, damp weather conditions. Present in the spores is the toxin sporidesmin which 15 causes liver damage in an animal if ingested. Symptoms of facial eczema include photosensitivity and a tendency for animals to mutilate themselves by rubbing against standing objects. However not all animals exhibit these symptoms, and it is generally accepted that when 5% of a flock of lambs exhibit photo-sensitivity during an outbreak, about 50% will exhibit liver damage at slaughter.

As appreciated, facial eczema occurs predominantly among grazing animals including sheep, cattle, deer, goats and more exotic animals such as alpacas. In New Zealand, it has been recently stated that facial eczema costs New Zealand's fanners between NZ$80-120 million each year, while the effective cost to the nation when the inability to farm sheep effectively in many areas, is in excess of NZ$500 million. These figures relate only to the 25 sheep industry.

Facial eczema, which is not specific to New Zealand, can be addressed. One technique is to adopt farm management practices which reduce proliferation of the fungus. Spore counting to identify hazardous pastures and areas, serve to warn farmers to routinely transfer stock to safer sites to limit their total spore intake. Apart from improved farming 30 practices, it has been discovered that some control of facial eczema may be obtained through the regular administration of zinc to animals. However the effectiveness of this control relies on regular administration and to date there has been no treatment or preparation discovered which is able to impart long term, or even short term, resistance or immunity to the condition by the animal. Despite considerable and widespread research, a 35 solution addressing the disorder has eluded scientists. 1 SUBSTITUTE SHEET WO 93/16708 "f v' W v PCT/NZ93/0000* There clearly exists a need for a preparation which is able to impart short, medium or long term resistance to facial eczema. For stock such as dairy cattle which gather to a single point every day, it is relatively easy to administer existing preparations to the cattle on a daily or regular basis during high eczema risk seasons. However, beef, sheep, deer and 5 many other animals are not mustered or gathered on a frequent basis and high country stock mustering may occur only once a year. Consequendy it is either impractical or impossible to treat these other animals, which also succumb to the effects of facial eczema, on the regular basis required by existing preparations.

Continuing research in the area of zinc administration, and facial eczema, has also 10 illustrated that other disorders afflicting animals can be addressed by zinc. Further research by the inventors have extended the list of disorders which appear to respond to the regular administration of zinc, and will be discussed more fully herein.

Virtually all research on the administration of zinc has centered on the most obvious and also the easiest method of administration, which is through the digestive system. Typically 15 dosing of animals is conducted through dissolving a suitable zinc compound in a water supply. Research is also being conducted on a slow release bullet which is placed in the stomach or elsewhere in the digestive system of an animal. However, early work by the inventors has found the digestive system to be unreliable and inefficient For instance, the rates of zinc absorption by different animals, and under different conditions, can vary 20 significandy. The o . cr administration of zinc can be toxic to an animal, and thus if zinc levels are maintained at a low enough level not to cause toxicity for a majority of the herd, the levels may also be too low to provide any lasting effects. Also, conditions within the digestive system can vary significantly so that a slow release administration could release relatively quickly within another animal. The research of the inventors have therefore 25 centered upon an alternative method of administration, and compositions useful for non-digestive administration.

Disclosure of Invention It is an object of the present invention to address the foregoing problems or at least to provide the public with a useful choice. It shall also be seen that other advantages or 30 features may be offered by the invention.

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

According to one aspect of the present invention there is provided a method for addressing zinc responsive disorders in animals by providing a depot of zinc through the -) SUBSTITUTE SHEET 0 WO 93/16708 €=, ^ w w PCT/NZ93/00005 administration of a zinc substance exhibiting slow-release characteristics within the said animal, by its introduction via a non-digestive route.

According to another aspect of the present invention there is provided a method of treating zinc-responsive disorders comprising the administration of an effective yet non-lethal 5 amount of one or more zinc substances having a low aqueous solubility, via a non-digestive route.

According to another aspect of the present invention there is provided a composition of fluid or paste-like consistency for use in addressing zinc-responsive disorders in animals other than humans, comprising a zinc substance exhibiting slow release characteristics 10 within said animal, disbursed in a pharmacologically acceptable carrier.

According to another aspect of the present invention there is provided a composition as described above, which includes at least one of: i) a selenium containing compound or mineral; ii) a magnesium containing compound or mineral; iii) a fatty acid or a solution or dispersion thereof; iv) a plant derived oil; v) sulphur or sulphide compound; vi) a vitamin.

According to a further aspect of the present invention there is provided a bolus for 20 administration by introduction into at least one of i) the bloodstream, ii) muscle, and iii) subcutaneous tissue, of an animal other than a human, said bolus comprising a zinc substance and at least one of: i) a selenium containing compound or mineral; ii) a magnesium containing compound or mineral; iii) a fatty acid or a solution or dispersion thereof, iv) a plant derived oil; v) sulphur or sulphide compound; vi) a vitamin.

According to yet a further aspect of the present invention there is provided a composition 30 for addressing zinc-responsive disorders comprising, in percentage by weight and to a ratal of 100%: 0.01 - 80% a zinc substance as herein defined; 0-20% a selenium containing compound or mineral; 0-20% a magnesium containing compound or mineral; 0-80% a fatty acid; 0-95% a plant derived oil; 3 SUBSTITUTE SHEET 0-20% sulphur or sulphide compound; 0-5% a vitamin; 0-95% non-aqueous carrier.

According to another aspect of the present invention there is provided a method of 5 treatment and/or prevention, substantially as described above, comprising the incorporation of a substance enhancing the activity of the zinc, with the zinc compound.

According to another aspect of the present invention there is provided a method of treatment and/or prevention, substantially as described above, directed to address facial eczema and/or fading elk disease.

According to another aspect of the present invention there is provided a method of administering zinc for addressing facial eczema, fading elk disease and/or zinc-remediable afflictions comprising the subcutaneous, non-oral, non-anal administration of a substantially insoluble zinc releasing substance.

The term 'zinc substance', where used within this specification, is defined as being any 15 zinc containing substance including, for example, zinc metal, zinc complexes, zinc minerals and salts, zinc organic and organozinc compounds. Preferably, for use of the present invention, the zinc substance chosen should not be excessively toxic to an animal or else its use reduced to less than toxic levels. consisting of A 'preferred inorganic-zinc compound' is a member of the group^ zinc aluminate, zinc carbonate, zinc fluoride, zinc iodate, zinc oxide, zinc hydroxide, zinc orthophosphate, other zinc phosphates having a solubility of O.Olg/ml (H2O at 35°C), zinc selenate, zinc selenide, zinc silicate, and zinc sulphide.

A 'zinc organic salt' is a compound of zinc with an organic acid, though their method of synthesis need not be from an acid-base reaction. Examples of members of this 25 group include zinc acetate, zinc benzoate, zinc butyrate, zinc citrate, zinc ^/-lactate, zinc laurate, zinc oleate, zinc oxalate, zinc salicylate, zinc stearate, zinc tartrate, and zinc valerate. It should be appreciated that this list is not exhaustive and many other salts which could be said to be compounds of zinc with an organic acid are also envisaged.

The term 'fatty acid' shall be used in its normal sense though it is envisaged that readily 30 available acids such as linoleic acid and linolenic acid which occur in many vegetable oils will be used for convenience.

The term 'alkaline earth element' wherever used throughout this specification shall refer to a member of Group II of the periodic table, though more preferably Magnesium and Calcium. 2^9 5 598 The term 'zinc responsive disorder' shall refer to any affliction, disease, condition or deficiency which responds favourably, or may be treated with, zinc or zinc substances.

An 'enhancer' is a substance which increases or enhances the activity or increases the effectiveness of zinc with respect to the condition it has been applied to treat. This does not 5 necessarily mean improving the release of zinc from the compound used in the preparation - for instance, a soluble zinc compound would more readily release zinc to the animal but at the detriment of long term slow release, and possible toxic zinc effects. According to the present invention it appears that some compounds and substances, such as those of magnesium, enhance the effectiveness of the zinc in some instances.

These 'enhancers' should be substantially non-toxic at the proportions and quantities administered and not normally be totally insoluble in the blood stream or animal tissue. Examples of some compounds include the sulphates, carbonates, oxides, chlorides etc. of alkaline earth metals such as calcium and magnesium. Chelated forms may also be suitable. Trials have indicated that administrations of the present invention are more 15 . effective when the animal is not suffering from some other form of mineral or vitamin deficiency. For instance, selenium is deficient in many New Zealand soils and the co-admininstration of selenium appears to increase the effectiveness of the invention in some instances. The administration of sulphur, either in the elemental form, as a sulphide or sulphate, also appears to enhance the effectiveness of the present invention in addressing 20 zinc responsive disorders in some cases. The administration of 'enhancers' may be simultaneous (site and timing) with administration of a zinc composition or administered separately. The site may be different or nearby and the timing may be, for instance, at the same time, within a given period (e.g. within 12 hours before or after) or regularly throughout the life of the zinc composition. Administration of enhancers at a different site, 25 to increase their effectiveness or to prevent possible site reactions, is also considered. While the full pharmacological mechanism of the use of enhancers is not fully understood, it is believed that a healthy animal is better able to make use of the zinc provided by the present invention.

As is apparent.from the preceding description, the preferred main active component of the 30 present invention is a zinc substance. Ideally, for a lasting zinc depot for the animal, the chosen substance should exhibit slow release characteristics at the introduced site. The choice of site will have a bearing - for instance a substance introduced into the bloodstream may be more likely to dissolve than when subcutaneously located. The animal itself, and its environment, will affect its physiology which can indirectly affect the rate of release of 35 zinc and other minerals. However, as a general guide the solubility of the chosen zinc substance can be used. Typically, this should be a solubility of less than or equal to lOg per 1 litre water at 35°C, or more preferably less than or equal to lg per litre at 35°C. It SUBSTITUTE SHEET 24 9598 WO 93/16708 £ ^ V* PCT/NZ93/000«i; should be appreciated however that the present invention would also allow for ..ie substitution of a more soluble zinc compound or for the inclusion of several zinc compounds of differing degrees of solubility. Soluble zinc complexes which allow only the slow exchange or release of complexed or a chelated zinc would also be a very 5 acceptable choice.

The inclusion of various zinc compounds and substances which release zinc at different rates or times within a blood stream, can be used. Rather than having a zinc release profile which is substantially flat or gradually tails off over time, it is possible to have a more complex zinc release profile. This may be used for preparations which more accurately 10 tailor zinc release with an animals needs. For example, a remedial/treatment preparation having a relatively high initial zinc release (e.g. a more soluble or active zinc compound) may be included to provide an initial boost for animal already afflicted with a disorder. An intermediary zinc-rclease compound may also be included to ensure a therapeutically high zinc level for a short-medium term to help the animal recover while a third long-life, slow 15 release zinc compound may be used to continue a low level of zinc release for the continuing treatment and some immunity from recurrence of the affliction.

Of course the foregoing is but one example and many other variations are possible. It can be appreciated that by suitably selecting zinc compounds and substances of various solubilities, zinc contents and activities, the profile of zinc release and/or activity may be 20 altered with respect to time. This may be useful in determining pie-season, mid-season or late-season formulations for addressing certain disorders such as facial eczema. It can also be useful in customising preparations for the needs of individual species or animals. It is likely that several preparations, according to the present invention, may be provided which are combined according to the requirements of the animal, before its administration. 25 Furthermore, the chemistry and biological activity (notably in the blood stream of different species of animals) can vary and thus different formulations may be desirable to 'fine tune' a preparation to an animals specific requirements. It is also possible that different preparations are administered as a series over time and/or at different locations on the animal. Various zinc organic salts and preferred inorganic zinc compounds have .differing 30 solubilities, the solubility data for which are readily available and can be used as a guide to tailoring release rates.

The ■r-.c substance used for a particular embodiment should be relatively non-toxic at the normal dosage rates to be described below. Mild toxicity at these dosages may also be acceptable if a useful treatment results. Zinc oxide is one zinc compound useful in the 35 present invention, being aqueously insoluble, plentiful and relatively non-toxic other than possible zinc toxicity through over-administration. It is also amphoteric and likely to slowly release into alkaline or acid environments. Other possibilities include hydroxides, 6 SUBSTITUTE SHEET <n £ 0 K Q J (U. ;•'$ 3 WO 93/16708 £=, " ^ v PCT/NZ93/00005 carbonates, selenides arid sulphides of zinc, preferred inorganic zinc compounds, and zinc organic salts though the specific toxicity of each (or in combination with other included zinc substances) with respect to the animal should be tested for the particular animal species first. Many naturally occuring zinc minerals may also be suitable.

Other components in a preparation may include sources of trace elements and vitamins, especially those which may be deficient as a result of the condition or which improve the effectiveness of the zinc. Other pharmaceutical preparations or remedies may also be included to act in conjunction with the zinc compound. For example, flowers of sulphur have been included in some embodiments for specifically addressing eczema.

A preparation may be administered in a variety of ways. For instance, while oral administration is possible, not all the zinc may be absorbed and the preparation will normally pass through the animal's digestive tract in a relatively short period of time, thus providing no lasting results. The acidity of the stomach may also convert alkaline or amphoteric zinc compounds to other more soluble zinc salts which may end up being 15 flushed relatively quickly from the system. Oral, or suppository, administration may be useful as an initial, shock, or top-up application but according to the present invention this is not the preferred method of administration unless it can repeated on a regular basis (e.g. for dairy stock rather than high-country stock).

An intravenous, intramuscular or subcutaneous injection or implant is more effective, 20 introducing the preparation in a manner providing a theoretical 100% release into the body. This allows the preparation to be more accurately administered as the differences in different animal's metabolisms and digestive characteristics are not as significant Further, it has been found that this method of administration ensures the components remain within the animal's system for a much greater period of time. Preliminary trials suggest effective 25 treatment periods of 3 months (before a further dose is required) is not out of the question, and up to 6 months or longer may be realised.

Brief T)rsf kiptthn of Drawings Further aspects of the present invention will now be discussed by way of example only with reference to the accompanying diagrams in which: Figure 1 is a graph of the relationship between GGT levels and days on treatment for trials with mixed sex lambs - Sporidesmin dose rate of 0.2mg/kg body weight; Figure 2 is a graph of the relationship between GGT levels and days on treatment for trials with mixed sex lambs — Sporidesmin dose rate of 0.3mg/kg body 35 weight, and 7 SUBSTITUTE SHEET WO 93/16708 Figure 3 1 8 is a graph of weight gains during the same trials.

Bfst Modes for Carrying ntrr thf. Invention Most trials of the present invention have centred on the use of zinc oxide as being a representative of preferred zinc substances. This compound has many desirable attributes, 5 such as being cheap and plentiful, available in a finely divided form, being insoluble in water and for the purposes of tests, free from any other components which could influence the trial results. However, while the oxide is useful for testing, other zinc substances may be used in various embodiments of the present invention. Compounds such as zinc sulphide and zinc selenide, which also have low solubilities in water, can help supply other 10 elements which are commonly deficient. Pre-onions of these compounds (or others which can be a source of another element) may be combined with other zinc compounds to prevent toxicity by the additional element. Other members of the group of preferred inorganic compounds also exhibit low solubility rates, as do many zinc organic salts. Consequently most embodiments of the present invention will rely on a member of one of 15 these groups, or a combination thereof, as the main zinc substance.

Preferred embodiments of the present invention will comprise one or more members of the groups of preferred inorganic zinc compounds and zinc organic salts. Preferably members having a solubility of 0.01 g/ml H2O at 35°C, or less will be chosen. Even more preferably, a solubility of O.OOlg/ml H2O at 35°C will often be chosen as the main zinc 20 substance.

For fluid or paste-like embodiments, the one or more zinc substances will usually be dispersed in a pharmacologically acceptable carrier which will comprise at least one of: i) a plant derived oil; ii) a fatty acid or a solution or dispersion thereof; iii) a non-aqueous fluid.

Typical plant derived oils are vegetable oils such as sunflower, linseed etc though soya oil appears to impair the efficiency of the invention. A fatty acid may be present in the plant derived oil, or added to a carrier. Trials indicate that the use of oils high in fatty acid appear to enhance the efficiency of the invention. Other non-aqueous fluids (typically oily) 30 may be used as carriers or bulking agents. Typically the zinc substance will be insoluble in the carrier and the composition will comprise a dispersion of a finely divided form of the insoluble components.

As an alternative to providing fluid or paste-like compositions, the present invention may take the form of a bolus, which shall be defined as including within its scope any 8 SUBSTITUTE SHEET WO 93/16708 24 9 5 98 PCT/NZ93/00005 substantially solid form, lump or capsule of a composition. Capsules having small pores to control release of components may also be adopted. Most bolus embodiments of the present invention will include at least one zinc substance, such as would be used for a fluid or paste-like embodiment, but often include a higher amount of solid material. This may 5 also include substances such as gums to bind the other ingredients together. A proportion of fatty acids or oils may also be included.

Other fillers or binders may also be used, including at least one of: i) a calcium containing compound or mineral; ii) a magnesium containing compound or mineral; iii) a selenium containing compound or mineral; iv) a fatty acid; v) sulphur or a sulphide or sulphur containing compound.

Acceptable fillers and binders need not be restricted to this group, though the aforementioned group includes substances which appear from trials to enhance the 15 effectiveness of the present invention.

Some various examples, and trial results, of the present invention will now be discussed by way of example only.

Examples 1 Early tests have been performed with a preparation comprising zinc oxide in conjunction 20 with a sunflower or vegetable oil carrier in a ratio of 100 mg zinc oxide per 1 ml of oil. This may be varied considerably though will affect the quantity of preparation administered to the animal.

The tests were based on a dose of 20 ml of the preparation (100 mg/ml) per 200 kg of beast weight These tests have exhibited remedial effects in disorders such as Facial Eczema and 25 Fading Elks Disease (FED). Possible resistance to further attacks was also indicated.

An off-shoot of an eczema or FED treatment according to the present invention, is the addressing of some other zinc responsive disorders, some examples of which that trials have indicated will be responsive to the present invention including (apart from facial eczema and fading elks disease): mycotoxin poisoning, poisoning from alkaloid toxins, 30 and disorders relating to phyto-oestrogen levels. Each of these are disorders which occur predominantly from matter which livestock have ingested, in the same manner that zinc is able to provide resistance to an animal against poisoning by the mycotoxin sporidesmin, so too does the availability of zinc appear to increase the resistance of an animal to these other disorders. The provision of an available zinc depot within an animal will provide the 9 SUBSTITUTE SHEET ts^ p (• ^ y WO 93/16708 (L ^ J ^ O PCT/NZ93/00005 exhibited enhanced resistance to these disorders typical of animals receiving regular i dosing, for substantially the life of the zinc depot The role of zinc is not clear though current understanding suggests that it is an essential trace element which is readily depleted from animals .undergoing stress, which may be 5 from a variety of environmental causes. Animals which have low zinc levels exhibit a lesser resistance to many disorders and thus the provision of a lasting zinc depot, such as by the administration of a composition containing a zinc substance exhibiting slow release characteristics within the body of the animal, will also improve the health of the animal in other ways. As an example, several other areas in which animals provided with a lasting 10 zinc depot may excel other zinc deficient animals include: improved reproduction efficiency by way of reduced foetal absorption, extended gestation period, addressing physical shock, stress during labour, addressing complications from excessive blood loss during labour, reduced still or premature biiths, paper skins and Teduced follicle coverage, improved birth weights.

- Improved animal resistance to infectious diseases and enhanced immune response; Improved growth rates of animals, especially during summer months of high pasture production; Improved wool and fibre production; Improved milk productions in cows, sheep, goats etc; - Greater adaptability of the animal to environmental changes, such as heat, drought etc.

Examnle 2 Further trials have been performed to assess whether a preferred preparation according to the present invention confers, to an animal, resistance to sporidesmin toxin.

These trials have been performed by injection of a preparation according to the invention (see Preparation #2 below), into an animal. In some trials based on this preparation it has been indicated that subcutaneous injection appears to yield the greatest length of protection time for the quantity administered. Intramuscular injection would appear to give the next longest protection period, followed by intravenous. Oral administration of preparation #2 30 appeared to give the shonest protection period and be the most inefficient method of dosing.

It was also noted that subcutaneous injection gave the least site reaction with only minimal swelling at the injection site. Depending on body weight of the subject, an injection may often be up to 15 mis. This is a relatively large quantity to inject and thus possible site 35 reaction needs to be considered if a single injection to administer the preparation is used SUBSTITUTE SHEET WO 93/16708 ^ t.M ^98 PCT/NZ93/00005 (multiple smaller injections at different iites are a possibility). This is one reason a subcutaneous injection is generally preferred over intramuscular injection. The use of a bolus, which will tend to have a reduced mass due to the lesser proportion of carrier present (i.e. the oil in preparation 2) may be preferable in some cases. A method of 5 administering multiple, smaller boluses, to different sites may also be considered in some cases.

Specific Preparation #2 (fluid composition) The preparation (#2) referred to above, expressed by weight, is as follows: 20g Zinc Oxide 2g Magnesium Sulphate 4g Flowers of Sulphur 74g Sunflower Oil General Preparation #3(fluid composition) A broader general preparation which is suitable for most applications involving farmed 15 domestic stock is as follows: -30% oxide of zinc, preferred inorganic zinc compound and/or zinc organic salt 0-8% sulphate, oxide and/or hydroxide of magnesium 0-8% flowers of sulphur to a total of 100% suitable carrier such as sunflower oil Caution should be exercised when including alkaline or acidic components. Inclusion of large quantities of components such as alkaline magnesium oxide or hydroxide has the potential to alter the pH and other chemistry of tissue surrounding the injection site. A large or single intravenous dose could affect blood chemistry. Consequently it may be 25 preferable, for some applications of the invention, to predominantly use substantially neutral components or to administer a preparation in smaller, tolerable, doses.

It should be appreciated that according to the invention, the proportions of a preparation may differ from general preparation #3 which is given by way of example only. An example of a "broader preparation, covering a greater range of types of application is as 30 follows: 11 SUBSTITUTE SHEET 9 A Q ^ Q WO 93/16708 fc *{ « u » W PCT/NZ93/000M General preparation #4 0.001-99.99% oxide of zinc and/or comparable zinc compound(s) 0-25% sulphate, oxide and/or hydroxide of magnesium and/or calcium 0-15% flowers of sulphur 0-50% inert filler or binder 0-25% mineral or vitamin supplements 0%-trace amounts nicotinic acid to a total of 100% suitable carrier(s) such as vegetable oil, fatty acid and/or nonaqueous carrier, and/or other filler or binder A preparation according to preparation #4 may take many forms. For instance it may comprise a paste or substantially dry form which, while could be used for less preferred routes af oral or suppository type application, is generally intended to be used as a concentrate for dilution and injection. A suitable carrier, such as a vegetable oil is typically used for subsequent dilution. The proportion of diluent in the injectable composition 15 should be sufficient for the composition to pass through the needle and be taken up by the animal. Typically, total carrier or diluent proportions of 60% or greater would be used for many applications.

Current trials with preparation #2 indicates that this preparation is working well for test animals artificially dosed with Sporidesmin toxin and it has been extrapolated that a 20 protection period of 100 days plus is being offered. At lower dose rates protection is of shorter duration and not able to withstand as high a challenge of Sporidesmin toxin, suggesting that the dose rate is best calculated according to animal size (within a particular species).

Early further testing with respect to Fading Elks Disease based on preparation #2 without 25 the flowers of sulphur showed promising results.

These early trials also highlighted that certain oils are not particularly suitable. For instance, while soya bean oil could be used, it not found to be particularly suitable. From this early research, no apparent problems arose from the use of sunflower oil. This is thought due to its high levels of fatty acids and Vitamin E (and its antioxidant effects). 30 However it is envisaged that many other oils, both vegetable and mineral may be suitable. Other pharmacologically acceptable carriers and diluents (providing they do not react with the components) cc;:ld, undoubtedly, also bt incorporated or substituted. 12 SUBSTITUTE SHEET 9 A Q 5 Q C.-> s ^ ^ General preparation #5 (fluid and paste-like compositions)* 0.01-80% a zinc substance as herein defined; 0-20% a selenium containing compound or mineral; 0-20% a magnesium containing compound or mineral; 0-80% . a fatty acid; 0-95% a plant derived oil; 0-20% sulphur, sulphide or oxy-sulphur compound; 0-5% a vitamin; 0-95% non-aqueous carrier.

General preparation #6 (fluid and paste-like compositions)* -40% a zinc substance whose aqueous solubility is less than, or equal to, 0.01 g/ml of H2O at 35°C; 0-5% a selenium containing compound or mineral; 0-8% a magnesium containing compound or mineral; 60-90% vegetable oil; 0-8% sulphur, sulphide or oxy-sulphur compound. * all percentages by weight to a total of 100%.

Preparations #5 and #6 are based on those used in trials and are directed to facial eczema risk livestock. Preparation #5, and especially preparation #6, may include a higher 20 proporition of zinc than normally used for other zinc-responsive disorders. Preparation #2 and its dosage was in respect of a medium/high risk exzema site/season. Compositions for lower or higher risk areas may have correspondingly lower or higher zinc proportions or their dosages altered accordingly.

Preparations #5 and #6 are directed towards livestock under New Zealand conditions, the 25 inclusion of selenium addressing a deficiency readily recurring amongst livestock in this country. Typical forms of selenium include zinc selenide, zinc selenate or other selenium compounds used for veterinary use. The proportion will depend on the calculated rate of release within an animal so as to avoid selenium poisoning. The sulphur component may commonly comprise elemental sulphur, magnesium sulphate or zinc sulphide but need not 30 be restricted to these compounds.

Preparation #7 Preparations #5 and #6, as well as other preparations, are also suitable for the preparation of a bolus though the percentage of fluid components may be reduced. The formation of a bolus may be by pressing or other pill manufacturing methods. A gum or resinous material 35 may also be used to bind components together. Many natural and synthetic gums (and equivalent materials) are known which may be used. The rate of dissolution of the binder 13 SUBSTITUTE SHEET WO 93/16708 e) £ Q K Q Q PCT/NZ93/00005 may also influence release of the zxttc ctriisottierif/enabling zinc compounds of gre solubility (and often mobility) to be used. This may be useful for non-intravenous sites.

Preparation #8 This involves adjusting zinc release profiles and is based on any one of the preceding 5 preparations but includes, as a proportion of a zinc substance, at least one other zinc substance having a higher solubility than the main zinc component. Typically the predominant zinc component will have a low aqueous solubility (e.g. less than or equal to 0.01 g/ml of H2O at 35°C, or typically less than or equal to 0.001 g/ml of H2O at 35°C), whilst the additional zinc component(s) will have a solubility exceeding 0.01 g/ml of H2O 10 at 3S°C. Often these additional components will not be excessively soluble (e.g. solubility in the range 0.01-0.1 g/ml of H2O at 35°Q and many zinc organic compounds are suitable. Yet additional higher solubility zinc components may be included, typically as an additional shock treatment.

An example of a preparation is as follows: 1-40% zinc substance(s) having solubilities of less than or equal to O.Olg/ml of H2O at 35°C (typically a preferred inorganic zinc compound) 0-25% zinc substance(s) having solubilities in the range (exclusive) of 0.01-0.1 g/ml of H2O at 35°C (typically a zinc organic compound) 20 0-15% zinc substance(s) having solubilities equal to or exceeding 0.1 g/ml of H2O at 35°C (typically a soluble inorganic zinc compound) Remainder to 100% comprising other components such as present in preparation #l-#7 Example 3 25 Summary Preparation #2 was given by subcutaneous injection to 60 Romney-cross, mixed-sex lambs (20-30 kg live weight).

Treatment groups (5 animals per group) received various amounts of preparation #2, as well as sporidesmin toxin.

In comparison with an untreated control, results show that preparation #2 has protected sheep against damage caused by sporidesmin, and that the best treatment provided protection for 100 days. 14 SUBSTITUTE SHEET WO 93/16708 / ' H y Q PCI7NZ93/0000? w'.u u ^ vii.' >*»r W Material 40 Romney-cross, mixed-sex lambs were randomly divided into 8 groups.

* Group 1 was untreated and the control for gamma glutamyltransferase or (GGT).

* Group 2 control sporidesmin dosed only.

* Groups 3, 4 and 5 treated with preparation #2 at a rate of 3, 6 and 9 ml/animal respectively.

* Groups 6,7 and 8 contained animals that were given individual rates of 10, 20, 30, 50 and 100 ml/animal respectively of preparation #2.

* The group of lambs, 2 and 6 received a sporidesmin dose at 0.2 mg/kg body weight 10 inira-ruminal.

* Groups 7 and 8 received preparation #2 only.

* A further 20 Romney-cross, mixed-sex lambs (weight 20-30 kg) were added to the trial with group 9 sporidesmin dosed only.

* Group 10, 11 and 12 treated with preparation #2 at 3, 6 and 9 ml/animal 15 respectively, and all groups dosed with sporidesmin at 0.3 mg/kg intra-ruminal.

* All lambs in groups 2 to 12 were treated with preparation #2 by subcutaneous injection in the neck.

Method * Day 1 _ All animals were randomly selected, weighed and tagged and treated with preparation #2, and blood samples were taken for GGT levels.

* Day 7 Respective groups dosed with sporidesmin.

Day 17 Blood samples taken for GGT levels.

* Day 28 Animals weighed and blood samples taken for GGT levels.

* Day 30 Animals in the appropriate groups dosed with 0.2 mg/kg body weight, of sporidesmin.

All animals were grazed together on improved pasture at all times, with access to a plentiful supply of running water and natural shade.

Animals were observed on a daily basis and yarded as necessary.

Regular inspections were made by members of an approved Ethics Committee, a 30 veterinarian and a scientist.

SUBSTITUTE SHEET 91. Q R Q v v.. ^ 8 PCT/N Z93/00005 Results The measurements of the blood enzyme (gamma glutamyltransferase) GGT which is concentrated in the liver when the liver becomes damaged and the GGT leaks into the blood stream, are in proportion to the amount of damage to the liver.

Figure 1 illustrated the movements of the GGT levels from day to to day 90.

The animals described in Figure 1, were dosed with sporidesmin on day 0, (animals in groups 3, 4, 5 and 6 had previously been injected with preparation #2 7 days previously). Dose rate of sporidesmin 0.2 mg/kg body weight. On day 20, repeat doses of sporidesmin were given at similar dose rates.

Figure 2 describes lambs that had been pre-treated with preparation #2 7 days "lead in" then dosed with sporidesmin at the rate 0.3 mg/kg body weight.

Figure 3 shows the toxic effects of sporidesmin dosing by a dramatic reduction in growth rates of lambs. The protective effect of preparation #2 at various dose rates to reach adequate levels is also illustrated. The growth enhanced effect of preparation #2 is 15 apparent in group 6.

Carcass Acceptability Results to date from animals that have been slaughtered both in post-mortem situation and in export abattoirs have passed New Zealand Ministry of Agriculture & Fisheries export inspections with an excellent report. This in turn indicates that the methodology is 20 acceptable to New Zealand's Border Protection Services.

Discussion of Trial Results Several interesting aspects emerged from this trial.

In Figure 1, group 6 treated with 10 ml preparation #2 has given a consistent "straight line" effect in maintaining protection against sporidesmin intoxication.

Groups 4 and 5 with 6 and 9 ml preparation #2 likewise have given an element of protection especially to the 50 day stage.

Group 3 treated with 3 ml preparation #2 while given good protection to day 50, appear to be unprotected subsequently.

Figure 2 also shows a similar result with the straight line effect of protection in group 12 at 30 a 9 ml treatment with preparation #2.

It is to be noted in this situation a much higher challenge of toxin was used. 16 SUBSTITUTE SHEET ("j p" Q WO 93/16708 ^ J y O PCT/N Z93/00005 Visual observation of the lambs in these groups, showed that group 2 has very obvious signs of eczema, swollen about the eyes and ears, red and inflamed, loss of skin and wool in pans exposed to the sun, while lambs in group 3 (and in all the other groups) do not show these symptoms. This would suggest that even at low dose rates of preparation #2 5 there is an element of protection from sporidesmin intoxication for short durations.

Possible Mode of action From these observations it would appear that the mode of action of the injectable product preparation #2 is such, that at small dose rates, the depot deposit runs out at about 50 days. To give adequate protection and to have sufficient slow release product stored in the 10 liver at any given time, it is essential to have a dose rate that releases sufficient product to achieve adequate liver storage levels. This would suggest, to give good protection beyond 50 days, a dose rate of 10 ml would be required.

Potentiation: It has been known for some time that animals exposed to small amounts of sporidesmin 15 toxin become sensitized to its accumulative effect. While even at comparatively low toxin levels at any one challenge, the cumulative effect leads to liver damage and subsequent clinical effect for the animal.

Figure 1 gives a clear illustration of this potentiation effect.

On day 1 animals received 0.2 mg/kg body weight of sporidesmin. GGT levels rose very 20 slightly in group 2, however, on re-dosing at the same dose rate, GGT levels rose dramatically in all groups except group 6 that gave adequate protection.

Weight gain Figure 3 illustrates a distinctive weight gain over the controls, along with the dramatic toxin effect on weight gain, and the close relationship to the levels of protection offered by the 25 various dose rates.

The results of the weight gain achieved in group 6 compared to the control that did not receive any preparation #2, would indicate for optimum growth rates, animals would benefit from receiving an injection of preparation #2 even outside the eczema season.

Toxicity of preparation #2 There appears to be no visible change of gross pathology or histopathology at approximately 4-5 times the recommended dose rates of 10 ml preparation #2/24 kg lamb. At 100 ml/24 kg body weight, or between 4 and 5 times the recommended dose rate. 17 SUBSTITUTE SHEET O li t?\ ra WO 93/16708 d, A? l) f) }4 <K PCT/NZ93/0000: At these levels there appears to be a good safety margin to work with in its practical application.

Conclusions from Example 3 trials The results to date give a clear demonstration that in adequate doses preparation #2 is 5 highly effective in controlling facial eczema in sheep for an extended period of time of approximately 100 days.

This evidence has been sustained at different challenge rates of sporidesmin intoxication.

In dosages that are effective for protection against the toxin, sheep have shown a weight gain in excess of 1.5 kg above the control group, whether they have been exposed to the 10 toxin or not.

It therefore appears from the results that the product also promotes growth, which implies the health of the sheep is enhanced by the product use.

. Examples 4 Results for preparation #2 and variations indicates protection to 130 days which would 15 cover any normal eczema season.

As a farming practice local fanners could for example, inject their animals mid-February to confer protection right through the New Zealand eczema season. This would be adjusted for other countries.

Trial results have also given good results and an additional effect of preparation#2 is in 20 increased weight gains over the base controls. This is still very apparent even at the 130 days mark. For sheep we are looking at an additional weight gain of 1 *£ kg per month which is an extra 53g per day (substantial) which is a useful additional effect from use of the present invention.

In trial work with deer, weight increases of twice as much weight for the treated mobs as 25 for the control mobs have been observed. In these trials animals have been restored firom apparent neardeath (due to FED) to a healthy productive animal.

The Fading Elks Disease affects both male and female. It is not as pronounced in fallow deer as in the elks and wapiti which are substantially larger and faster growing. It is considered that any adversity in their performance is exasperated by their higher metabolic 30 and growth rates.

In understanding the problems of Fading Elks Disease the animals will continue to feed. Often they are fed maze and other concentrates, which they will eat but due to their 18 SUBSTITUTE SHEET WO 93/16708 0 £. W ^ QO PCT/NZ93/0000? condition arc apparently unable to take advantage bf. TOis wentually leads to rapid body weight loss along with scouring.

From the animals that have been treated there is normally rapid improvement within one week. They will continue to gain weight to become quiet respectable animals within the 5 month, and back to complete health in two months.

As previously discussed, administration is by injection which avoids the stomach system -an inefficient and least preferred pathway for administration of the preparation.

TABLE 1 - Results of trials with preparation #2 with deer comparing weight gain in treated and non-treated animals.

Wapiti Cows Weight (leg) Weight (kg) after 29 days TAC 138 115.9 132.0 Tir,ated WHITE 129 156.0 164.5 Treated WHITE 136 138.5 147.5 Treated WHITE 121 138.5 147.5 Treated WHITE 140 144.5 152.0 Non-treated WHITE 143 112.0 117.0 Non-treated WHITE 146 127.0 135.0 Non-treated Average weight increase 13.8kg treated; 6.8kg non-treated Red Stags Weight (kg) Weight (kg) after 29 days W 12 86.0 111.0 Treated W 11 93.0 114.0 Treated W 14 82.0 101.0 Treated W 13 86.0 105.0 Treated W10 94.0 105.0 Non-treated W 16 80.0 101.0 Non-treated W 9 110.0 121.0 Non-treated Average weight increase 21kg treated; 11kg non-treated Example 5 - Toxicology nf Compositions Further tests were conducted to investigate the safety of the present invention for use on livestock. The preparation used in conducting the tests was as follows (expressed in percentage by weight): 19 SUBSTITUTE SHEET 24 Q 8 PCT /N Z93/00005 Preparation #9 % Zinc Oxide 2% Magnesium Sulphate 4% Flowers of Sulphur 74% Sunflower Oil Summary: Eighteen hoggets were injected with twice the normal dose (7.5ml) of preparation #9. Six additional hoggets acting as controls were given saline injections. The trial preparation was injected subcutaneously in two sites using both sides of the neck. The sheep injected 10 preparation #9 developed variable and palpable neck reactions but no pain or heat was associated. Three treated sheep and three controls were euthanased at both 21 and 56 days post-treatment. Neck lesions were present in a large proportion of the treated sheep at the site of injection. These were classified as granulomatous inflammatory lesions (granulomas).

Blood chemistry (Ca, Zn, Se and Cu levels) and tissue chemistry (liver and kidney levels of Se, Cu, and Zn) examinations at 21 and 56 days following treatment indicated that there were no significant differences between the control and treated sheep except for serum and tissue zinc levels and kidney copper levels. The treated sheep had significantly higher serum zinc levels than the control animals (P<0.001) on Days 21 and 56. Two randomly 20 selected groups of three treated and three control sheep separately slaughtered on Day 21 and 56 showed that liver and kidney zinc levels were significantly higher in the treated animals (P<0.01) and that these elevated levels were all at the lower end of the toxic range. Histopathology on the liver, pancreas and kidneys of these two groups of three treated sheep indicated that preparation #9 had induced no notable changes except on Day 56 when 25 two out of the three treated sheep showed severe multifocal fibrosis in 5-10% of the pancreas. No adverse weight changes were recorded in the treated sheep when the means of the two groups were compared (Day 56).

It can be concluded that the only adverse effect of preparation #9 was following treatment with twice the recommended dose. At the site of injection, sterile granulomatous reactions 30 of varying sizes appeared soon after treatment. None of these were painful upon pressure and were still palpable at 56 days post-treatment Materials and Methods Twenty four hoggets of similar weight and breed were randomly divided into two groups containing 18 and 6 animals each. The group of eighteen were all injected with double the SUBSTITUTE SHEET WO 93/16708 PCT/NZ93/00005 normal dose of preparation #9 using two sites, one on either side of the neck. The group of six hoggets designated as controls were similarly injected at two sites with saline.

On days 2,3,4,5, t 7,14 and 21 after treatment all twenty four sheep were examined for injection site reactions and records made of the observations. These observations 5 continued on a weekly basis for the 18 sheep remaining after Day 21. On Days 21 and 56 both groups were weighed and individuals bled for blood chemistry (serum calcium, zinc and copper levels and blood selenium). In addition, six pre-identified sheep from both groups (3 + 3) were euthanased and post-mortemed at both intervals (Days 21 and 56). Kidney and liver samples were removed for calculating selenium, zinc and copper levels. 10 Histopathology examinations were made on the liver, kidney, pancreas and injection sites of all six sheep slaughtered on Days 21 and 56.

Swabs of the granulomatous skin reactions of the slaughtered trial preparation sheep were taken for bacteriology. The trial preparation test material was also tested for sterility.

Results Following injection of the trial preparation a high proportion of sites developed visible and palpable reactions. These were classified into three grades - no response, moderate (<15mm) and large (>15mm). Table 1 illustrates the palpable responses.

None of the enlarged sites of the trial preparation injections were painful upon pressure or had associated heat Two animals had abscessed sites and in one animal this was attributed 20 to injection technique.

The twenty four trial animals were weighed on Days 0, 21 and the remaining 18 on Day 56. These data are recorded in Appendix I (raw data sheets). No significant difference between the means of the treated and control animals emerged over the trial period.

Table 1 Number of Palpable Responses to the Trial Preparation Injection* Response Days After Injection 2 3 4 6 7 14 21 28 None 22 23 21 21 22 23 29 26 Moderate 7 1 3 0 1 1 1 Large 7 8 13 7 9+ 14 14 * calculated over the 36 injection sites on 18 animals t three sites discharging 21 SUBSTITUTE SHEET PCT/NZ93/OO0O5 Group Zinc (umol/1) Calcium (mmol/1) Copper (|imol/l) Selenium (nmol/1) Day 21 Treated 29.7t 2.50 19.45 467 Control 14.3 2.49 ' 12.47 513 Day 56 Treated 23.5+ 2.61 9.71 483 Control 13.3 2.64 9.4 440 MAF Range Undosed FE Control Toxicity 9-20 18-34 27-92 2.40-3.10 (Normal range) 250-4000 (Normal range) * Group sizes: Day 21,18 treated and 6 control sheep - Day 56,15 treated and 3 control sheep f Differences between control and treated means P<0.001 Blood chemistry results from the Day 21 and 56 samples are recorded in Table 2 and Appendix I. These demonstrate that no significant differences exist between the control and treated animals for mean blood levels of calcium, copper and selenium. However, the mean serum levels of zinc on Days 21 and 56 in the treated animals are at the high end of the range expected in zinc-dosed animals, and significantly higher than the mean for the 10 control animals (P<0.001).

Tissue levels of copper, zinc and selenium were measured on liver and kidney samples trken from the six sheep euthanased on each of Days 21 and 56 and are displayed in Table 3. These analyses show no significant differences between the treated and control animals for copper and selenium on Day 21, but on Day 56 the three trial preparation-treated 15 animals had a significantly higher level of kidney copper (P<0.04) than the untreated controls. The liver and kidney levels of zinc in the three treated sheep are significantly higher (P<0.01) compared with the three control animals at both sampling intervals. These elevated levels arc at the low end of the toxic range as specified by New Zealand's Ruakura Animal Health Laboratory and illustrated in Table 3. 22 SUBSTITUTE SHEET WO 93/16708 ^ f -/ 5 C| 3 PCT/NZ93/00005 Table 3 Tissue Biochemistry Results (means of the groups) Group Liver Zinc (mg/kg) Liver Copper (Hmol/kg) Liver Selenium (nmol/kg) Kidney Zinc (mg/kg) Kidney Copper (Hmol/ke) Kidney Selenium Dav21 Treated 156.7* 877 783 86.7* 112 4133 Control 40.0 1580 1090 .. 24.0 88 4167 Day 56 Treated 166.7* 863 1427 129.7* 61.7+ 3867 Control 42.3 1140 1123 22.3 55.0 4367 MAF Range Undosed FE Control Toxicity 40-60 55-125 115-665 >95 (normal value) 450-1000 °-45 40-110 100-1200 >95 (normal value) Significant differences * between controls and treated, P<0.01 f. between controls and treated, P<0.04 Histopathological examination of liver, pancreas and kidney of the six sheep on day 21 revealed no changes indicative of a toxic or adverse effect in the treated sheep when compared with the controls. However, on Day 56, two of the three trial preparation-treated sheep showed small areas of fibrosis and infiltration of inflammatory cells (5 and 10% respectively) in the pancreas when compared with the controls.

Discussion These trials relate to the effects of the trial preparation treatment in sheep given double the recommended dosage. A number of parameters were investigated including blood and tissue levels of calcium, zinc, copper and selenium plus histopathological examination of key tissues including the pancreas. As the composition comprised a significant level of a 15 zinc compound, particular focus was placed on any histopathology in the pancreas of treated sheep. In all parameters there was nothing suggestive of an extreme adverse or toxic effect. The only adverse effect noticed was at the site of injection where the trial preparation produced visible and palpable reactions at a high proportion of sites. 23 SUBSTITUTE SHEET 9 A Q * WO 93/16708 C-:l g PCT/NZ93/0000- At post-monem on day 21, all six sites in the three treated sheep were found to be affected with granulomatous reactions despite four sites recording no palpable reactions in the live animals. These reactions were subcutaneous and able to be trimmed. There appeared to be no difference in the size of the lesions on Day 56 but on post-mortem ventral tracking of 5 the product along facial muscle planes in one animal with the production of serous fluid was discovered. On examination this was consistent with a chronic granulomatous inflammation accompanied by a bacterial secondary infection.

The six treated sheep examined on Days 21 and 56 displayed significantly higher kidney and liver zinc levels (P<0.001) compared with the controls. The kidney levels remained 10 within the Ruakura FE (Facial Eczema) control range while the lover levels were at the lower end of the Ruakura FE toxic range. The 18 treated sheep had significantly higher serum zinc levels than the six control animals (P<0.01).

The changes noted in the pancreas of two of the three treated animals (Day 56) of small areas of multifocal fibrosis was likely an effect of the high levels of zinc provided by the 15 trial preparation. It is considered highly unlikely that there would be any detrimental effect to the animal until at least 70-80% of the pancreas was damaged.

These results indicate that the trial preparation at double the normal dosage did not produce any serious adverse systemic or internal tissue reactions. The significance of the granulomatous skin reactions remains to be fully evaluated although at this time, the 20 absence of any pain on palpation suggests a wider consideration of welfare aspects is unnecessary. The raised tissue levels of zinc in the the trial preparation-treated animals is to be expected and illustrates that the depot form of zinc appears to be operating efficiently. Even at double the recommended dosage these tissue levels appear to be within a range unlikely to jeopardize the health of treated animals.

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. 24 SUBSTITUTE SHEET

Claims (1)

1. 249598 we claim is: A method for the treatment in non-human animals of facial eczema, mycotoxin poisoning, fading elks syndrome or alkaloid poisoning by providing a depot of zinc through the administration of a zinc substance, exhibiting a slow-release characteristics within said animal, in combination with a non-aqueous fluid comprising at least one of: (i) a planfderived oil; (ii) a fatty acid; administration being by introduction via a non-digestive route into said animal. A method as claimed in claim 1 wherein said fatty acid is in the form of a nonaqueous solution or dispersion. A method as claimed in claim 1 or 2 in which said zinc substance is in the form of a substantially solid bolus implanted-via~a-non-digestive route. A method as claimed in any one of claims 1, 2 or 3 in which the administration includes a pharmacologically acceptable portion comprising at least one of: (ii) a magnesium containing compound or mineral; (iii) a selenium containing compound or mineral; (iv) sulphur or sulphide compound. A method as claimed in any one of claims 1 through 4 wherein said zinc substance comprises a zinc organic salt (as herein defined) or a preferred inorganic zinc compound (as herein defined). A method as claimed in any one of claims 1 through 4 wherein the aqueous solubility of said zinc substance is less than or equal to 0.01 g/ml of HjO at 35°C. A method as claimed in any one of claims 1 through 6 in which.includes (i) a calcium containing compound or mineral; 249598 26 administration of at least one of the following substances within 12 hours; (i) a vitamin; (ii) a magnesium containing compound or mineral; (iii) a selenium containing compound or mineral; \ (iv) a fatty acidf) O ' (v) sulphur or sulphide compound. 8 A method as claimed in any one of claims 1 through 7 wherein the method of administration is by introduction into at least one of: (i) bloodstream; (ii) muscle; (iii) subcutaneous tissue; (iv) a site near the liver. 9 A method of treating non-human animals for facial eczema, mycotoxin poisoning, fading elks disease or alkaloid poisoning comprising the administration of an effective yet non-lethal amount of one/more zinc substances having low aqueous solubility in combination with a non-aqueous fluid comprising at least one of: (i) a plan}"derived oil; (ii) a fatty acid; administration being by introduction via a non-digestive route into said animal. 10 A method as claimed in claim 9 wherein said fatty acid is in the form of a nonaqueous solution or dispersion. 11 A method as claimed in claim 9 or 10 in which the zinc substance is coupled with at least one of: (i) a selenium containing compound or mineral; (ii) a magnesium containing compound or mineral; (iii) sulphur or sulphide compound. 12 A method as claimed in any one of claims 9 through 11 wherein a said zinc c .. " \'SJ{ -] 13 14 15 -16 17 18 24 Sr -*7 substance comprises a zinc organic salt (as herein defined) or a preferred inorganic zinc compound (as herein defined). A method as claimed in any one of claims 9 through 12 wherein the aqueous solubility of a said zinc substance is less than or equal to 0.01 g/ml of H40 at 35°C. A method as claimed in any one of claims 9 through 13 which includes an additional zinc substance having a solubility exceeding 0.01 g/ml of HjO at 35°C. A method as claimed in any one of claims 9 through 14 wherein the method of administration is by introduction into at least one of: (i) bloodstream; (ii) muscle; (iii) subcutaneous tissue; (iv) a site near the liver. A composition of fluid or paste-li Inconsistency for use in a method of treatment of non-human animals for facial eczema, mycotoxin poisoning, fading elks syndrome or alkaloid poisoning comprising a zinc substance exhibiting slow-release characteristics within said animal, dispersed in a non-aqueous fluid comprising at least one of: (i) a plarfderived oil; (ii) a fatty acid. A composition of fluid or paste-like consistency as claimed in claim 16 wherein said fatty acid is in the form of a non-aqueous solution or dispersion. A composition as claimed in claim 16 or 17 which includes an additional zinc substance having a solubility exceeding 0.01 g/ml of KaO at 35°C. A composition as claimed in any one of claims 16 through 18 which includes at ^ •' m? I i <4 2.8 least one of: (i) a selenium containing compound or mineral; (ii) a magnesium containing compound or mineral; (iii) sulphur or sulphide compound; (iv) a vitamin. 24 A composition as claimed in any one of claims 16 through 19 where in a zinc substance comprises a zinc organic salt (as herein defined) or a preferred inorganic zinc compound (as herein defined). A composition as claimed in any one of claims 16 through 20 wherein the aqueous solubility of a said zinc substance is less than or equal to 0.01 g/ml of HjO at 35°C. A composition as claimed in any one of claims 16 through 20 wherein the aqueous solubility of a said zinc substance is less than or equal to 0.001 g/ml of at 35°C. A bolus for use in a method of treatment of non-human animals for facial eczema, mycotoxin poisoning, fading elks syndrome or alkaloid poisoning for administration by introduction into at least one of (i) the bloodstream, (ii) muscle, and (iii) subcutaneous tissue of said animal, said bolus comprising a zinc substance, at least one of: (i) a plant derived oil; f'" (ii) a fatty acid; and at least one of: (i) a selenium containing compound or mineral; (ii) a magnesium containing compound or mineral; (iii) sulphur or a sulphide compound; (iv) a vitamin. A bolus as claimed in claim 23 wherein said fatty acid is in the form of ajpon- f -t? 249! aqueous solution or dispersion. 25 A bolus as claimed in claim 23 or 24 which includes a zinc substance whose aqueous solubility is less than, or equal to, 0.01 g/ml of H4© at 35°C. 26 A bolus as claimed in any one of claims 23 through 25 which includes at least one other zinc substance whose aqueous solubility exceeds 0.01 g/ml of H*0 at 35°C. 27 A composition for addressing zinc-responsive disorders comprising, in percentage by weight and to a total of 100%. 10-40%. a zinc substance whose aqueous solubility is less than, or equal to, 0.01 g/ml of Hk0 at 35°C; 0-5% a selenium containing compound or mineral; 0-8% a magnesium containing compound or mineral; 60-90% vegetable oil; 0-8% sulphur, sulphide or oxy-sulphur compound. 28 A method of treatment of non-human animals to improve fecundity and general reproductive health, the method comprising the administration of a zinc substance, exhibiting slow-release characteristics within/said animal, in combination with at least one of: (i) a plant derived oil; (ii) a fatty acid; administration being by introduction via a non-digestive route into said animals. 29 A method according to claim 28 wherein said fatty acid is in the form of a nonaqueous solution or dispersion. 30 A method for addressing zinc responsive disorders comprising the administration via a non-digestive route of a composition or bolus, substantially as described herein with reference to the contained examples. j J/-;; ; ^ i j 4 3o 31 A composition comprising a zinc substance, for addressing zinc responsive disorders, substantially as described herein with reference to the contained examples. 249598 1011:53 *j0O COLBY CORPORATION LIMITED By their Atttorneys PETER VERBOEKET AND COMPANY per: END OF CLAIMS O '■> Ml ^97