NZ572035A - Wax bolus containing zinc particles of a defined particle size - Google Patents

Abstract

Disclosed is a bolus including zinc particles wherein the zinc particles have a size between 45 microns in the shortest dimension and 150 microns in the longest dimension, and wherein the zinc particles are substantially evenly dispersed within a wax matrix. Also disclosed is the use of zinc having low particle size and wax for manufacture of a bolus for the prevention of conditions of zinc responsive skin disorders, fungal infections and/or facial eczema.

Description

0056611631 * 72 0 35 PATENTS FORM NO. 5 Fee No. 4: $250.00 PATENTS ACT 1953 COMPLETE SPECIFICATION In the first instance James & Wells Ref: 43331/3 IMPROVEMENTS IN THE PREVENTION OF FACIAL ECZEMA l/WE Bomac Research Limited, of 102 Wiri Station Road, Manukau City, Auckland, New Zealand, a New Zealand company; and James John BENNISON, a British citizen of Agrimin Ltd, The Flarepath, Elsham, Wold Industrial Estate, Brigg, North Lincolnshire, DN20 0SP, England hereby declare the invention for which I/We pray that a patent may be granted to me/us, and the method by which it is to be performed to be particularly described in and by the following statement: James & Wells Ref: 43331/3 IMPROVEMENTS IN THE PREVENTION OF FACIAL ECZEMA TECHNICAL FIELD The invention relates to improvements in the prevention of facial eczema. More specifically, the invention relates to the prevention of facial eczema in animals such 5 as sheep by administration of an intra-ruminal bolus made from zinc.

BACKGROUND ART Facial eczema is a disease caused by animals ingesting toxic spores from the fungus Pithomyces chartarum. This fungus grows in the dead litter layer of pasture. In favourable conditions (usually warm and humid weather), the fungus produces large 10 number of spores that contain a toxin called sporidesmin. This toxin causes severe and irreversible liver damage and may be fatal for sheep and damage the health of cattle. One reference estimates that the cost to New Zealand farmers per year is in the order of millions per year and in favourable weather, $65 to 75 million per year.

Affected animals are either in one of two categories, sub-clinical or clinical facial 15 eczema. It is desirable to have methods or treatments to treat or prevent both categories of facial eczema.

Existing treatments centre around prevention of facial eczema in the animal by supplementation with zinc or zinc based alloys before climatic conditions occur that cause growth of spores and hence production of sporidesmin.

One method of determining the facial eczema status of an animal is to measure the animal's liver function by testing for gamma-glutamyl transferase (GGT) enzyme levels. Normal levels for sheep are reported as being in the order of 30 to 55. Levels above this indicate problems with liver function including the presence of at least some degree of facial eczema. One measure of the success of a facial eczema 2 James & Wells Ref: 43331/3 treatment or preventative is to reduce or prevent an increase in GGT enzyme levels outside the normal range.

Zinc supplements are currently used to prevent and/or treat facial eczema in animals such as cows and sheep. These zinc supplements are either drenches or boluses.

Zinc boluses are typically in the form of zinc oxide. Their mode of action is to raise metabolised zinc levels in the animal, which appears to help in resisting facial eczema. The zinc supplementation is typically metabolised via blood serum, as evidenced by the fact that animals taking existing zinc supplements have significantly increased levels of zinc in their serum.

EP1225238A1 describes a zinc based alloy bolus for veterinary use. The specification teaches of a bolus comprising a metal alloy including by weight: (a) at least 50 wt% of zinc; (b) from 30 to 49 wt% of magnesium; and (c) up to 6 wt% of copper, as well as the use of this bolus in the preparation of a medicament for the treatment or prevention of facial eczema. The alloy and method of this specification 15 does not describe use of boluses containing less than 70% zinc. Of note is that this patent specification states that boluses containing more than 70% zinc are not of use, as the zinc is either insoluble or only sparingly soluble.

W09316708A1 describes a zinc depot treatment or prevention method for animals that may be established through the introduction of a zinc substance exhibiting slow-20 release characteristics for the physiology of the animal, preferably via a non-digestive route. The specification describes that a fluid or paste-like composition, or a bolus, may be introduced through preferred routes such as into the bloodstream, intramuscularly, or subcutaneously. Preferred zinc substances are described as including zinc organic salts and a specified range of preferred zinc inorganic 25 compounds. The specification describes that the provision of a lasting zinc depot in an animal is useful for addressing zinc responsive disorders, both in a preventative and remedial sense. Examples of zinc-responsive disorders include facial eczema. 3 James & Wells Ref: 43331/3 This specification is specific to administration to non-digestive routes and not via the rumen or digestive tract. In addition, the formulations described in this specification use various alloy combinations, none of which disclose more than 80% zinc.

WO 2005/051487 describes a slow release bolus including zinc metal particles and 5 an oil wax. This product addresses a number of the problems in the prior art. However, the results shown in the PCT specification include a marked increase in zinc serum levels measured over time. Unfortunately, this is not desirable because increased zinc levels in the serum may result in flushing out of other valuable minerals from the blood stream. This can also hinder when the bolus is to be re-10 administered.

For example, it is normal practice to wait for the level of zinc to decrease in the serum before the zinc bolus is re-administered. Unfortunately the time between when the levels are tested and when levels drop in the animal may be sufficient for diseases to progress such as facial eczema - the very disease that the bolus is intended to treat.

Obviously it would be desirable if there can be provided a treatment of a better transfer of the zinc into the animal rather than loss of the blood stream.

In the inventor's experience, one problem with traditional zinc treatments such as those described above is that high zinc serum levels tend to result in flushing out of other valuable minerals.

A further problem is that typically, two zinc treatments are required per season for traditional methods of treatment. To avoid overdosing the animal, the second treatment can only be applied when the animal zinc serum levels decrease to a near normal level. This is a dangerous time as animals may become susceptible to facial eczema as the zinc levels reduce. 4 James & Wells Ref: 43331/3 It is an object of the present invention to address the foregoing problems or at least to provide the public with a useful choice.

All references, including any patents or patent applications cited in this specification are hereby incorporated by reference. No admission is made that any reference 5 constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents form part of the common general knowledge in the art, in 10 New Zealand or in any other country.

It is acknowledged that the term 'comprise' may, under varying jurisdictions, be attributed with either an exclusive or an inclusive meaning. For the purpose of this specification, and unless otherwise noted, the term 'comprise' shall have an inclusive meaning - i.e., that it will be taken to mean an inclusion of not only the listed 15 components it directly references, but also other non-specified components or elements. This rationale will also be used when the term 'comprised' or 'comprising' is used in relation to one or more steps in a method or process.

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 bolus including zinc having low particle size distributed within a wax matrix.

According to a further aspect of the present there is provided a method of preventing zinc-responsive disorders by administration of a bolus substantially as described 25 above to a ruminant animal.

James & Wells Ref: 43331/3 According to a further aspect of the present invention there is provided a method of preventing fungal infection in a ruminant animal by administration of a bolus substantially as described above.

According to a further aspect of the present invention there is provided a method of 5 preventing liver damage caused by fungal infection in a ruminant animal by administration of a bolus substantially as described above.

In preferred embodiments, fungal infections are those associated with Pithomyces species. More preferably, Pithomyces species include Pithomyces chartarum.

According to a further aspect of the present invention there is provided a method of 10 preventing facial eczema in a ruminant animal by administration of a bolus substantially as described above.

According to a further aspect of the present invention there is provided the use of zinc having low particle size and wax in the manufacture of a bolus for prevention of the conditions substantially as described above.

In preferred embodiments, the bolus is administered to sheep. It should be appreciated by those skilled in the art that sheep, at least in temperate climates, are susceptible to facial eczema particularly when grazed on pasture during spring conditions. This should not be seen as limiting, as it should be appreciated by those skilled in the art that the invention may also be used for other ruminant animals such 20 as cows and deer.

Preferably, at least one bolus is administered using a standard applicator known in the art, via the digestive tract of the animal.

In preferred embodiments, the zinc particles make up approximately 90% by weight of the bolus. 6 James & Wells Ref: 43331/3 Preferably, the zinc particles are in a variety of different shapes.

Preferably, the zinc portion of the bolus is approximately 99.995 % pure. Remaining metal components within the formulation include: aluminium (<10 ppm); copper (<10 ppm), cadmium (<10ppm), iron (<20ppm), lead (<50ppm), and tin (<10ppm). Amounts 5 are provided by way of indication only and are based on ICP-AES analysis.

The term low particle size is intended to identify a particle size that is sufficiently small to be gut active within the animal and to only enter the blood stream in minimum levels if at all.

The inventor has found that low zinc particles with a particle distribution that primarily 10 ranges from particles less than 150micron (140 mesh) to greater than 45micron (325 mesh) achieves this desired effect.

In one preferred embodiment for a particular grade of zinc metal the particle range is approximately as shown below in Table 1: Sieve Analysis Actual example0/® Typical spec % >250mm (>60 mesh) 0.3 3 max >150mm (>100 mesh) 1.0 6 max >106mm (>140 mesh) 21.2 12-32 >75mm (>200 mesh) 38.0 -50 >45mm (>325 mesh) 33.8 -45 <45mm (<325 mesh) .8 max Table 1: Sieve Analysis Results for Bolus Zinc Particles Preferably, the zinc particles are substantially evenly dispersed within the wax matrix.

James & Wells Ref: 43331/3 An alternate range of particle sizes for another grade is: +150 um £ 5% max 45um to 150 um 50% - 80% Less than 45 um < 40% max In preferred embodiments, the wax portion of the bolus accounts for approximately 12% (10-14%) of the total weight of the bolus.

Preferably, the wax portion of the bolus includes one, two or three paraffin waxes.

In preferred embodiments, there are two waxes of an approximately even split by weight of two different grades of hardness waxes.

In one preferred embodiment, the hard wax portion has a melting point of approximately 57 °C, and conforms to the European Pharmocopoeia monograph for 10 hard paraffin waxes, whilst the soft wax portion has a melting point of approximately 45 °C,. This soft wax is characterised by having unsaturated carbon bonds evident by a single absorbance observed at approximately at 1470 and 720 cm"1.

In preferred embodiments, zinc particles are evenly dispersed within wax during manufacture and subsequently pressed into a bolus shape.

In one variation to the above method, the zinc and wax mixture is further coated in wax or other coatings to assist in administration of the bolus to the animal digestive tract.

In preferred embodiments, it is the applicant's experience that administration of a bolus of the present invention will provide protection from fungal diseases, including 20 facial eczema for a time period of up to 77 days. It is the inventor's experience that at the very least, the length of protection from fungal diseases is at least 28 days. 8 James & Wells Ref: 43331/3 In preferred embodiments of the present invention, two, three or four boli are administered in one application. It should be appreciated that the number of boli to be administered may vary depending on factors including but not limited to factors such as the risk of fungal disease, the animal to be administered, individual animal 5 metabolism and the like.

In one preferred embodiment for application to sheep, the bolus size is roughly cylindrical in shape with a diameter of 20mm and a length of approximately 30mm. It should be appreciated that the dimensions may be varied to suit the desired application, dose and animal to be administered without departing from the scope of 10 the invention. In addition multiple boli may be administered.

Optionally, the above bolus may include other fillers and excipients.

It should be appreciated from the above description that there is provided a novel bolus formulation that includes a pure form of zinc having low particle dispersed within a wax matrix. It is the inventor's experience that the formulation not only prevents 15 fungal diseases with ruminant animals, but also does this in a different mechanism to existing treatments that provides a number of advantages. The mechanism appears to be that the formulation is gut-active and does not pass through the bloodstream of the animal. Trial results completed by the applicant have found that zinc levels are not raised within the serum of the animal after administration of the bolus even after 20 extended periods of time. By contrast, existing methods of treatment of animals are known to increase the serum level of zinc within the animal.

Because the zinc serum level is not altered by the bolus of the present invention, a number of advantages are provided. For example, elevated levels of zinc within the serum of the animal may result in the flushing out of other valuable minerals from the 25 animal's bloodstream. In the case of the present invention, it is anticipated that 9 James & Wells Ref. 43331/3 valuable minerals will not be flushed out of bloodstream, as serum zinc levels are not altered.

A further advantage of not altering the zinc serum level, is that it is possible to administer a repeat dosage of the bolus of the present invention at any stage during 5 the animal life cycle, rather than having to wart for zinc serum levels to reduce first. One problem with existing treatments is that it is necessary to withhold further treatments until zinc serum levels reduce to an acceptable level. This is to avoid dosing the animal with an unacceptable level of zinc and therefore causing metal poisoning of the animal. A disadvantage of having to wait until zinc serum levels 10 reduce, is that the animal may become susceptible to fungal infection while zinc serum levels are at a lower level. By being able to administer multiple doses without having to wait for zinc serum levels to reduce, it is possible to avoid this risk area where zinc levels become lowered and the animal becomes susceptible to infection.

A further advantage of the bolus of the present invention is that the effects noted for 15 the invention bolus give an increased duration of raised faecal zinc levels and suppressed GGT enzyme levels produced by the animal liver compared to traditional treatments. This suggests that the bolus of the present invention would be able to be administered on a less frequent basis than existing treatments.

A further advantage found from the inventor's research was that a better correlation to 20 reduced GGT enzyme levels was found for elevated faecal zinc levels compared to serum zinc levels. In fact, the applicant's research suggests that elevated serum zinc levels do not have any correlation with prevention of facial eczema as measured via GGT enzyme levels. Given the above, the bolus of the present invention places the focus in the appropriate place in that it only elevates faecal zinc levels.

James & Wells Ref: 43331/3 BRIEF DESCRIPTION OF DRAWINGS Further aspects of the present invention will become apparent from the following description which is given by way of example only and with reference to the accompanying drawings in which: Figure 1 shows a graph comparing serum zinc levels over time for a trial comparing different methods of zinc treatment; Figure 2 shows a graph comparing GGT levels over time for a trial comparing different methods of zinc treatment; Figure 3 shows a graph comparing faecal zinc levels over time for a trial 10 comparing different methods of zinc treatment; Figure 4 shows a survival plot graph comparing survivor function versus time; Figure 5 shows scatter plots of the relationship between faecal and serum zinc for each of the treatment groups; Figure 6 shows scatter plots of GGT (iu/l) versus time for each of the treatment 15 groups; Figure 7 shows scatter plots of serum GGT (iu/l) versus faecal zinc levels for each of the treatment groups; Figure 8 shows box and whisker plots of serum GGT (iu/l) by treatment group for each time period; Figure 9 shows box and whisker plots of serum GGT (iu/l) by treatment group for each time period; 11 James & Wells Ref: 43331/3 Figure 10 shows box and whisker plots of natural log GGT by treatment group for each time period; Figure 11 shows box and whisker plots of serum zinc (umol/l) by treatment group for each time period; Figure 12 shows box and whisker plots of serum zinc (umol/l) by sampling episode for each treatment group; Figure 13 shows box and whisker plots of Faecal Zinc levels (mg/kg) by sampling episode for each treatment group; and, Figure 14 shows the relationship between faecal or serum zinc and dose of zinc 10 administered.

BEST MODES FOR CARRYING OUT THE INVENTION The invention is now described with reference to a clinical field study completed to evaluate the comparative efficacy of the zinc bolus formulation of the present invention to prevent facial eczema in sheep and its effect on blood and faecal zinc 15 levels.

Study Objectives The study tests an alternative zinc bolus utilising a highly pure form of zinc in particulate form dispersed within a wax matrix for its effect on treatment of facial eczema.

Materials & Methods 79 Romney sheep of mixed age were held on a farm where regular facial eczema risk is known to occur. 6 days before commencement of the trial (day -6 as counted from the planned time of treatment), all sheep were given numbered ear tags and blood 12 James & Wells Ref: 43331/3 samples taken. Samples were analysed for serum zinc concentrations as well as GGT and glutamate dehydrogenase (GDH) levels.

Sheep were firstly screened using serum GDH values and sheep with values exceeding 100 iu/ml were excluded. Sheep exceeding 70 kg bodyweight were also 5 excluded. Following the above exclusions, a total of 72 sheep left for the trial. These sheep were ranked in terms of their serum GGT levels and assigned to four groups from the ranking such that each group containing sheep of roughly similar mean levels of GGT values (52.7, 48.6, 50.7, and 47.4 respectively).

Treatment Groups On day 0, each group of (18) sheep were given the assigned oral treatments as follows: Group 1 were treated with a known zinc treatment termed Time Capsule' as a positive control. One bolus was administered to each animal orally.

Group 2 were orally administered 3 boli each of the invention composition.

Group 3 were orally administered 4 boli each of the invention composition.

Group 4 were not provided with any supplement, being untreated negative controls.

Facial Eczema Challenge All animals were grazed together on 'risk' pasture, as determined by location history, including known spore count reports from prior seasons. Pasture spore counts were 20 taken weekly from day -6 until day 77 following treatments. Spore counts on four randomly selected individual faecal samples were also reported weekly from day 35 to 77 (excepting day 42). 13 James & Wells Ref: 43331/3 Samples collected Blood samples were collected weekly from day -6, until day 77 (except at day 0). On day 21 an amendment was made to the study plan, whereby faecal samples were collected weekly from all sheep (except group 1 at the final collection), until day 77. 5 On each sampling day, clotted blood samples, fresh faecal samples and a pasture sample were transported to the laboratory within 4 hours of collection for analysis.

Other Observations The sheep were examined and observed by the study veterinarian on day 0, and on each of the following blood sample days. During the study sheep were observed 10 frequently (usually daily) for any signs of adverse effects or ill thrift, by the farm stock manager. The results of these observations were recorded in a daily log.

Results Day -6 to Day 0 Results from initial blood samples showed that serum zinc levels were all at low levels 15 (6 to 14|xmol/l). However, GGT enzyme values indicated that a low level of prior liver challenge had occurred in many of the sheep. Statistical analysis confirmed that there were no significant variations between GGT and zinc values for all groups.

Thus initial randomisation of groups appeared satisfactory. 14 James & Wells Ref: 43331/3 Early Trends In the first 2 weeks after treatment, serum zinc changes in each group were as shown in Table 2.

Zinc Concentration [(jmol/l (±SD)] Day -7 Day 14 Positive Control 11.0 (+/- 2.0) 13.2 (+/- 4.2) Invention - 3 boli .3 (+/- 1.2) 9.7 (+/-1.6) Invention - 4 boli .3 (+/-1.6) 11.1 (+/- 1.7) Negative Control .8 (+/- 1.3) 9.2 (+/-1.4) Table 2: Serum Zinc Changes to Week 2.

For the positive control group, serum zinc increased modestly but with substantial variability. For the invention test product groups, increases were very small, and much less that expected with only a small variation.

Day 0 to Day 77 The results as shown in Figure 1 show that serum zinc levels increased significantly as expected after 14 days from administration of the positive control. Unexpectedly, the invention test products showed minimal change in serum zinc levels compared with the negative control over the duration of the trial.

To provide more data on the behaviour of the treatments during their dissolution 15 periods, faecal samples were collected concurrently with blood samples to allow testing for faecal zinc content, thus providing an indicator as to relative rates of dissolution. Faecal samples were taken for the duration of the starting at week 3.

James & Wells Ref: 43331/3 Faecal Zinc Levels From faecal analyses it was immediately apparent that for each treatment substantial dissolution of zinc into the gastrointestinal tract was occurring. This early indication gave some assurance that a facial eczema protective effect might be expected from 5 the test treatments (also supported by early GGT trends, discussed below). The rates of dissolution had substantially consistent rankings as shown in Table 3 below and Figure 3.

Zinc Concentration [mg/kg] Day 21 Day 28 Day 35 Day 42 Day 49 Day 56 Day 63 Day 70 Day 77 Positive Control 556 375 315 216 95 46 36 37 nd Invention - 3 boli 489 298 220 203 247 209 197 176 98 Invention - 4 boli 747 613 378 275 506 239 186 172 107 Negative Control 29 33 28 38 44 43 39 31 Table 3: Faecal Zinc Changes From Week 3.

It may be readily distinguished that for each treatment (positive control and invention treatments), substantial zinc dissolution into the gut is occurring, when compared to control mean values for faecal zinc that are consistently less than 50mg/kg zinc. The pattern of dissolution of the invention test products differ from the positive control. The positive control falls relatively rapidly from about day 42. Invention test products, 15 result in faecal zinc levels that are persistently above 200mg/kg until at least day 56 and then these levels only gradually decline, retaining faecal zinc levels well above both the positive and negative controls through to day 77 of the trial. 16 James & Wells Ref: 43331/3 Facial Eczema Challenge As indicated by single weekly pasture spore counts, dangerous facial eczema challenge (>100,000 spores per gram) occurred in weeks 2, 4, 7, 8, and 9 of the study.

Once faecal samples were collected for zinc analysis, faecal spore counts were also taken from four randomly selected faeces in most weeks. While due to limited prior data, the interpretation of these counts is not well defined, the mean values obtained showed less variability than pasture counts. On the basis of a >50,000 spores per gram threshold indicating facial eczema danger, faecal counts found indicated facial 10 eczema danger in weeks 7, 8, 9 and 10 of the study (days 49 to 70).

Enzyme Responses Serum gamma glutamyl transferase (GGT) enzyme levels were measured weekly for the period of the study as shown in Table 4 below and Figure 2.

GGT enzyme Concentration Day -6 Day 7 Day 14 Day 21 Day 28 Day 35 Day 42 Day 49 Day 56 Day 63 Day 70 Day 77 [iu/l] Positive Control 53 64 93 95 118 114 106 103 102 150 202 192 Test - 3 tablets 49 49 48 48 55 61 79 121 157 239 369 370 Test - 4 tablets 51 52 54 53 58 59 59 74 103 191 293 313 Negative Control 47 51 50 62 100 115 142 194 283 423 593 637 Table 4: Mean serum GGT level changes 17 James & Wells Ref; 43331/3 In the negative control group, mean GGT values were largely unchanged until day 21. Mean GGT levels then increased progressively past 10Oiu/l at day 28, 194iu/l at day 49, 283iu/l at day 56 and 637iu/l at day 77. Therefore, enzyme levels in the unprotected sheep indicate that an increasing facial eczema challenge occurred 5 during the study period.

As early as day 14, mean GGT levels increased for the positive control group, to a mean of 93iu/l, which was 1.9 fold greater than the negative control group mean.

This change is understood by the inventor's to not be indicative of facial eczema challenge, as a change in only one group (i.e., not being reflected even in the control 10 group), indicates that small variations between groups can occur due to individual differences in challenge conditions experienced. Mean GGT levels were then relatively stable for the positive control group at <110iu/I until day 56, after which it advanced to exceed 200iu/l at day 70 indicating a reduced protection effect.

Enzyme levels remained relatively low for extended periods in sheep administered the 15 invention test products, being <100iu/l until day 42 for the 3-boli treatment and day 49 for the 4-boli treatment. These values then progressively increased to mean levels of 369iu/l (3 boli) and 299iu/l (4 boli) respectively at 70 days. Through the period from day 28 until day 77, mean levels for these treatment groups were at ratios of 0.4-0.6 less than both the positive and negative control group mean levels.

In each group, small numbers of sheep showed substantial enzyme responses, having dramatic effects on the arithmetic means. For example, 10 individual values exceeded 1000iu/l. To reduce the impact of such extreme values, log-transformed data were used in the statistical analysis.

Clinical Responses Almost no clinical signs of facial eczema, such as photosensitivity, dermal irritation or loss of body condition were observed. One exception noted were mild signs of 18 James & Wells Ref: 43331/3 photosensitivity in one sheep from group 1 at about day 63. Photosensitivity is one symptom of facial eczema in sheep.

In view of the substantial spore counts and serum GGT levels recorded, the iack of clinical signs was unexpected. It appears that the mature sheep used in the study 5 had substantial clinical or acute tolerance to facial eczema, while at the same time clear sub-clinical or sub-acute responses (hepatic enzyme release) were recorded.

All sheep were weighed at the beginning and end of the study. There were no significant changes in mean bodyweights, either between initial and final weighing, or between groups. This appears to be consistent with the absence of clinical signs of 10 disease.

Necropsies and Histopathology At the conclusion of the study, two sheep from each group were euthanized and necropsies were performed. All of the sheep euthanized were found to be in good condition with 1-2 cm of subcutaneous fat cover. In most sheep, fat colour was 15 slightly yellow, suggestive of a mildly icteric condition. Each animal had livers with rounded borders and fibrous changes to the capsule. No obvious fibrosis of bile ducts was seen grossly.

Histopathology changes reported were also similar for each sample, and were described as chronic proliferative cholangitis with marked fibrosis, acute neutrophilic 20 hepatitis and cholestasis. Changes were typical of sporidesmin toxicity (facial eczema challenge) of moderate severity.

Statistical Evaluation The data was initially ordered and analysed using a variety of software packages and subsequently imported into the statistical program SYSTAT. 19 James & Wells Ref: 43331/3 Figures 5 to 7 show scatter plots from the analysis which suggest a favourable response to treatment with the invention treatments. From the plots it is apparent that the GGT data is skewed (not normally distributed).

Because of the structure of the study, a 'repeated measures analysis' of variance was 5 used to take account of the expected within subject variation, i.e., results for the same animal are likely to be similar for measurements taken close in time.

Analysis and graphing shows that most of the facial eczema challenge during the study occurred in the latter part of the study. Assuming a lag phase of two to three weeks between challenge and GGT response, the challenge period appears to have 10 commenced in early March and steadily built up over the following weeks.

By day 14 of the study, a noticeable difference was observed in serum GGT levels between the treated and untreated groups (Figure 5). Conversely, up to day 56 of the study, no obvious difference between the treatment groups was observed. For the remaining three weeks of the study post day 56, the treatment groups appear to 15 diverge in apparent effectiveness at controlling GGT levels and by inference, preventing facial eczema.

While the study was planned with the expectation of observing a relationship between serum zinc and serum GGT, the data presented in Table 5 below provides surprising and new information about the relationship between serum, faecal zinc, and GGT 20 response.

James & Wells Ref: 43331/3 Dose given Serum GGT Serum Zinc Faecal Zinc Dose given 1.000 Serum GGT -0.156 1.000 Serum Zinc 0.042 -0.071 1.000 Faecal Zinc 0.445 -0.201 0.295 1.000 Table 5: Pearson correlation matrix, all data The negative relationship observed between serum GGT and either serum zinc or faecal zinc confirmed that zinc supplementation is protective, as expected. It is 5 evident that for this data there is some correlation (29.5%) between serum and faecal zinc. Surprisingly, the relationship between dose rate and serum zinc is very weak (4%), while the relationship between dose rate and faecal zinc is quite strong (44.5%).

More importantly, the relationship to GGT is stronger for faecal zinc (20%) than serum zinc (7%).

Correlations were also calculated for separate treatment groups as shown below in Figure 6. These correlations were calculated to compare GGT response to zinc level two weeks previously, i.e., lagged by 14 days. To maximise the number of observations used, faecal zinc was left out of the first set of correlations for each group. This is because the SYSTAT software excludes the rows associated with no 15 early faecal zinc observations. The number of observations quantifies this rate of exclusion. 21 James & Wells Ref: 43331/3 Group GGT Serum Zinc Faecal Zinc Number of Observations 1 GGT 1.000 Serum Zinc 0.002 1.000 216 1 GGT 1.000 Serum Zinc -0.098 1.000 Faecal Zinc -0.095 1.000 123 2 GGT 1.000 Serum Zinc 0.021 1.000 216 2 GGT 1.000 Serum Zinc -0.001 1.000 Faecal Zinc -0.238 -0.068 1.000 125 3 GGT 1.000 Serum Zinc -0.077 1.000 216 3 GGT 1.000 Serum Zinc -0.161 1.000 Faecal Zinc -0.213 0.498 1.000 124 4 GGT 1.000 Serum Zinc -0.026 1.000 210 4 GGT 1.000 Serum Zinc 0.014 1.000 Faecal Zinc 0.339 0.074 1.000 120 Table 6: Pearson Correlation Matrix by Group 22 James & Wells Ref: 43331/3 The general trends of these correlations, when GGT is lagged two weeks, are: • Serum zinc has a trivial correlation with serum GGT; • Faecal zinc is negatively associated with serum GGT i.e. higher faecal zinc levels are associated with lower serum GGT levels.

Negative control group 4 is a marked exception to this general rule. This result is consistent with there being a threshold level of faecal zinc before the protective effects are observed.

As an alternate method of presenting the data, survival analysis was tested at a number of thresholds. Thresholds chosen include 150 % of initial value; and 75, 250 10 and 550 iu/l. From the Wilcoxon test for significance, results at the 75 iu/l threshold indicate: • Group 1 (positive control) is not significantly different from any other group; • Group 2 (invention test product with 3 tablets) is significantly different from negative control group 4 (p = 0.043).

• Group 3 (invention test product with 4 tablets) is most significantly different from negative control group 4 (p= 0.068).

Discussion and Conclusions The study was located and timed so that the sheep could be exposed to facial eczema challenge and the results found show that mild to moderate facial eczema 20 challenge occurred from week 2, and severe challenge occurred from week 7.

The invention treatments are intended to provide high doses of zinc continuously for a period of weeks. It has been indicated for the positive control that the mode of action relies on sustained elevation of serum zinc levels. The serum levels produced by the 23 James & Wells Ref: 43331/3 positive control (Time Capsule) in this study were increased to a limited and variable extent that was statistically very poorly related to dose. For the positive control, correlations of serum and faecal zinc to GGT response (14 days later) were about equal (10 %).

For the invention test products, the correlation of serum zinc to GGT response is divergent for the two test doses (0.1% and 16%); but for faecal zinc the correlation is strong and relatively consistent (24%, 21%). Thus it appears that GGT values during sporidesmin challenge for the invention test products are better correlated with faecal zinc than with serum zinc.

The invention products present zinc as microfined elemental metal in a wax matrix. As it appears that zinc dissolved in the gut may be protective against sporidesmin regardless of assimilation into the bloodstream, the traditional relevant mode of action via the bloodstream is now open to new interpretation.

While GGTs have been reported initially as arithmetic means, statistical analysis as 15 log transformed values should be considered more relevant, firstly because the data is skewed due to dramatic responses of a few animals. In addition, GGT responses to sporidesmin are known to be strongly induced; thus an initial result is reflected in successive values for 2-3 months, irrespective of further challenge. As a result of this effect, progressively higher GGT values should be regarded as having diminishing 20 sensitivity and/or significance.

Bearing these considerations in mind, it is clear that all treatments provided a protective benefit to sheep during facial eczema challenge, when measured against the untreated control group. Differences persisted until the end of the study (11 weeks after treatments). GGT values eventually increased for all groups however, for 25 treated groups, this occurred mainly after a plateau extending to 56 days. 24 James & Wells Ref: 43331/3 A scale of severity has been used for the evaluation of rams when challenge tested, and that may be applied in this case, is: GGT of < 75 = normal; < 250 = mildly affected; 250 - 550 = moderately affected; >500 = severely affected by facial eczema.

On this scale, treated sheep were within a 'mildly affected' limit until about day 63, representing the protection provided at day 49.

Faecal zinc levels at this time were > 250 mg/kg for the test product groups.

Faecal zinc levels as recorded to 11 weeks show that for the invention products there is a slow decline in zinc released, not a precipitate change. During this phase an 10 augmenting dose could be expected to return faecal zinc levels above a protective threshold, such as 250 mg/kg. The possibility of partial, 'top-up' dosing is made more feasible by the form of the invention product, whereby standard doses are made up of 2 to 4 subunits (tablets).

In this field study, the test treatments have shown useful protection from facial 15 eczema challenge. The treatments tested appear to have comparable protective efficacy to the reference product, and with a suitable dose regime may achieve a longer period of protection.

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 20 departing from the scope of the appended claims.

Claims (5)

1. i 1.
2. 2.
3. 3.
4. 4. 5. 6. 7. 8. 9. WHAT l/WE CLAIM IS: A bolus including zinc particles wherein the zinc particles have a size between 45 microns in the shortest dimension and 150 microns in the longest dimension, and wherein the zinc particles are substantially evenly dispersed within a wax matrix. A bolus as claimed in claim 1 wherein the wax is paraffin. A bolus as claimed in either claim 1 or 2 which includes two waxes having two different grades of hardness. A bolus as claimed in claim 3 wherein one wax has a melting point of approximately 57°C, and the other wax has a melting point of approximately 45°C. A bolus as claimed in any one of claims 1 to 4 which has a coating. A method of preventing zinc responsive skin disorders by administration of one or more boli to ruminant animals as claimed in any one of claims 1 to 5. A method as claimed in claim 6 wherein the ruminant animal is a sheep. A method as claimed in any one of claims 6 or 7 wherein a number of boli are introduced into the animal at one time. A method of preventing fungal infection in ruminant animals by administration of a bolus as claimed in any one of claims 1 to 5. A method as claimed in claim 9 wherein the fungal infection is associated with Pithonyces species. 26 i 11. 12. 13. 14. 15. A method as claimed in claim 10 wherein the Pithonyces species include Pithonyces chartraum. A method as claimed in any one of claims 9 to 11 wherein the ruminant animal is sheep. A method of preventing facial eczema in a ruminant animal by administration of a bolus as claimed in any one of claims 1 to 5. A method as claimed in claim 13 wherein the ruminant animal is sheep. The use of zinc having low particle size and wax for manufacture of a bolus for the prevention of conditions of zinc responsive skin disorders, fungal infections and/or facial eczema. BOMAC RESEARCH LIMITED AND JAME
5. S JOHN BENNISON by their Attorneys JAMES & WELLS 27