NZ250163A - Pour-on application of trace metals to animals - Google Patents

Description

Patents Form # 5 25016J COONATED APPLICATION #250163/260638 based on Application # 250163 of 8 November 1993 and Application # 260638 of 31 May 1994 l4-1994 ' - / ... < > r •• NEW ZEALANP Fatents Act 1951 COMPLETE SPECIFICATION TITLE: Application of Trace Elements to Animals We, Ashmont Holdings Limited, a New Zealand company, of 48 Diana Drive, Glenfield, Auckland, New Zealand, hereby declare the invention, for which we pray that a patent xnay be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement: /tfJieNP60wtfwr8>c<iw>u..SS....i..otthe Patent Art JllZJZZ assistant COMMISSIONER PF-flgEMIS PF05.JWP FEE CODE - 1050 25016 FIELD This invention relates to the application of trace elements to animals.

BACKGROUND The provision of trace elements to animals in order to supplement their diet, has been common practice. In some cases it is desirable to provide farm animals with trace elements, and in other cases it is essential if there is a mineral deficiency in the soil. Additional levels of trace elements have been included in animals diet or dosed by means of oral or parenteral administration. They have also been administered by means of various slow release boluses and long acting injectibles.

There is a need to provide an easy and effective means of administering trace elements to animals, preferably without the need to inject the animal, or to administer the material orally.

The present invention seeks to provide an improved means of administering trace elements to animals, or one which will at least provide farmers and veterinarians with a useful choice.

STATEMENT OF INVENTION Surprisingly it has been discovered that trace elements can be administered to animals by means of pour-on formulations applied to the surface of an animal's skin, hide, or fleece.

In one aspect, the invention provides a pour-on formulation containing a therapeutically effective amount of a trace element in a pharmaceutically acceptable carrier.

In another aspect the invention provides a method of applying a trace element to an animal by topical application to achieve therapeutic serum levels of the trace element OBJECT 697&CS7.N94/JP/at 1 - CP ~ 250163 Preferably the phaxmaceutically acceptable carrier is an alcohol, water, or combinations thereof.

Preferably the trace element is chosen from the group containing selenium, copper, cobalt and iodine.

More preferably the trace element is a water soluble trace element, and is present in a formulation containing water, a sticking agent, a wetting agent, and a dyestuff.

Alternatively, the formulation may comprise a co-solvent formulation containing a water soluble trace element such as sodium selenate, together with butyl dioxitol and water. In addition the formulation may contain an endoparasiticide, such as levamisole, synthetic pyrethroids, avermectin, abamectin, or the like, vitamins, such as Vitamin 15 B12, dyestuff or other minor components. It may also contain at least one vitamin.

Other formulations can include non-aqueous formulations wherein the liquid carrier is an organic liquid. Examples include the alcohols, glycols or glycol esters; hydrocarbons such as xylene, paraffin or vegetable oils; isopropyl myristate; an ester of 20 a fatty acid; an alkylamide of a fatty acid; an ethoxylated block polymer.

In another aspect the invention provides a non-aqueous formulation containing one or more oxides of selenium or copper or cobalt dissolved in a solvent chosen from the group comprising ethanol, monopropylene glycol, and butyl dioxitol. Preferably the 25 trace element is present as selenium dioxide or selenium trioxide.

DRAWINGS: These and other aspects of the invention, will become apparent from the following 30 description, which is given by way of example only, with reference to the accompanying drawings in which: Figure 1 is a graph showing serum selenium levels, formulation 1, in animals; Figure 2 is a graph showing mean serum selenium levels, formulation 2, in animals; G97SCS7.N94/JP/U 250163 Figure 3 is a graph showing selenium blood levels, formulation 2, in animals.

Figure 4 is a graph showing blood selenium levels in Angus cattle during trial 5, 5 using formulation 2 over a 12 week period..

PREFERRED EMBODIMENTS Example 1 Formulation 1 % w/w Sodium selenate 2.0 Keltrol(Xanthum Gum BP) 0.3 Wetting agent 0.2 Water to 100 Example 2 Formulation 2 % w/w Sodium selenate 1.2 Butyl dioxitol 30.0 Water to 100 Example 3 Formulation 3 % w/w Copper Chloride 2H20 22.8 Nonidet 620P (Block polymer detergent) 10.0 MPG (Mono propylene glycol) 10.0 Formalin 0.2 Water 57.0 100 6975QCS7,596/HC/jq 250?65 Formulation 3 is prepared by the following method: To a clean dry mixing vessel, add the water, and with stirring, add Chloride 2^0 and stir until dissolved. With stirring, add Nonidet 620P and MPG and stir until fully dispersed and lastly add Formalin.

Example 4 Formulation 4 % w/w Ammonium Sulphate 0.50 Citric Acid 0.15 Vitamin B12 (ex Roche) 1.0 Sodium Selenate 1.18 Butyl Dioxitol .0 Water 87.17 100.00 Example 4 is prepared by the following method: Water was measured into a clean tank and ammonium sulphate and citric acid were added with the stirring. The vitamin B12 was added and stirred until fully dissolved. Sodium selenate was then added and stiired until fully dissolved. Lastly was added the butyl dioxitol and the solution was mixed until uniform.

Sample g Formulation 5 % w/v Selenium Dioxide (Se02) 0.71 Butyl Dioxitol to 100 mL To produce a selenium pour-on containing 5 grams of selenium per litre, the selenium dioxide is stirred into the butyl dioxitol until fully dissolved (approximately 30 minutes). This makes a non-aqueous pour-on formulation. 6975C37.N94/JP/at now amendedI 25 0 t 0 3 Other non-aqueous formulations can be made using a solvent chosen from a comprising monopropylene glycol, ethanol, and butyl dioxitol. We have found selenium dioxide, selenium trioxide, and H2Se04 are all soluable at this c solvents, and they are all useful solvents which can be used as pour-on formulations.

Trace elements, can be added to pour-on formulations containing^other active ingredients, for example pour-ons containing anthelmintics such as npxidectin, or any of the avermectins, for example ivamectin.

Formulation 6 has been the subject of a product stability trial, a6d this is shown in table 6. This table shows that the product is stable, and contains Refill amounts of selenium after three months.

TRIAL 1 Formulation 1 was trialled on two friesian bulls^ith the result shown in Table 1 and Figure 1.

This trial of Formulation 1 was conducted/to determine whether sodium selenate can be 20 successfully absorbed through the^Ain following the application of a topical ("pour-on") formulation.

Materials and Methods Two Friesian yearling bulls wfere weighed and examined to confirm the absence of any 25 back skin defects. Using^critical trial format whereby each animal acted as its own control, the two bulls w/we then treated with a pour-on formulation of the test product. This was administered along the back mid-line with a graduated syringe. The bulls were grazed normally over the trial period.

The test product was a 0.8% solution of elemental selenium (ie. sodium selenate) at 8 mg/mi in/accordance with Formulation 1. The normal oral dose is 20-30 mg/kg of seleniunr Accordingly, it was decided to apply the test product at double the adult dose, cm* 60 mg/kg of selenium. This equated to a volume of 7.5 ml of test product.

Tne two bulls were bled twice prior to treatment, and the serum selenium levels are shown in Table 1, and plotted in Figure 1. Both were treated on the same start date 6S75C37.N94/JP/at 250163 as amended COGNATE Nos.

Other non-aqueous formulations can be made using a solvent chosen from a group comprising monopropylene glycol, ethanol, and butyl dioxitol. We have found that selenium dioxide, selenium trioxide, and H2Se04 arc all soluable at this class of solvents, and they are all useful solvents which can be used as pour-on formulations.

Trace elements, can be added to pour-on formulations containing other active ingredients, for example pour-ons containing anthelmintics such as moxidectin, or any of the avermectins, for example ivamectin.

In the following trials, trials 1 and 3 are examples of trials including formulations similar to those of the invention, but not as claimed by the invention.

IS TRIAL 1 A formulation containing sodium selenate was trialled on two friesian bulls with the result shown in Table 1 and Figure 1.

This trial of was conducted to determine whether sodium selenate can be successfully absorbed through the skin following the application of a topical ("pour-on") formulation.

Materials and Methods Two Friesian yearling bulls were weighed and examined to confirm the absence of any 25 back skin defects. Using a critical trial format whereby each animal acted as its own control, the two bulls were then treated with a pour-on formulation of the test product. This was administered along the back mid-line with a graduated syringe. The bulls were grazed normally over the trial period.

The test product was a 0.8% solution of elemental selenium (le. sodium selenate) at 8 mg/ml The normal oral dose is 20-30 mg/kg of selenium. Accordingly, it was decided to apply the test product at double the adult dose, or 60 mg/kg of selenium. This equated to a volume of 7.5 ml of test product. jj The two bulls were bled twice prior to treatment, and the serum selenium levels are shown in Table 1, and plotted in Figure 1. Both were treatedfofl:4he same^stlrt date A>V ""x-X 'v\ «i3c.7.NM/»/.t H 0 2 OCT BBS Jj V'- f^'7 250163 (time 0 in Figure 1) and then re-bled at one, two and three weeks post treatment. The sera was removed and frozen and subsequently analysed for selenium levels.

Results The test material (coloured blue) was very viscous and it seemed not to penetrate the hair mat to reach the skin surface. The following day the test material still seemed to remain on top of the hair mat.

Table 1 gives the results of the changes in seium levels of selenium for both trial animals. The selenium status of both animals was significantly raised (P,0.01) by the treatment. Normal serum selenium levels are typically greater than 150 nmol/1, whereas the selenium levels in Table 1 and shown graphically in Figure 1 were significantly above this level for animal 239.

Discussion The application of the test product markedly raised the serum selenium levels of both 30 treated animals in comparison to their pre-treatment level. Although each animal was treated with the same volume of material and each was approximately the same weight, the serum responses vary markedly in magnitude, as well as time to peak. These variations in rate and absorption may relate to the viscosity of the test product and the individual physical hair factors. The weather throughout the trial period was warm and 35 dry for the time of year. Certainly no rain fell on the animals for at least four days post-treatment. Prior to treatment and for 24 hours both bulls were under cover to Table 1 Animal Number Weight (kg) Volume of Test Product (ml) Serum Selenium Levels at Sampling Dates (nmol/1)* 27.7 Day -7 3.8 Day 0 .8 Day 7 17.8 Day 14 24.8 Day 21 229 335 7.5 79 68 120 120 130 239 315 7.5 93 89 420 310 280 ^Normal serum selenium levels are >150 nmol/1 6975CS7.H94/JP/ftt i now amended] 8- 25016, 9 ensure this aspect of coat dryness.

This pilot trial demonstrates that sodium selenate can be formulated to be successful, absorbed through the skin following topical application.

JO TRIAL 2 A trial was carried out on a number of animals to compare the seruraflevels from a selenium injection (Se-Hypo as the control) with formulation (2) at pates of 12 ml and 18 ml of pour-on (equivalent to 60 mg/kg and 90 mg/kg of seleniunrt respectively).

Materials and Method Twelve friesian weaned bulls having an average weighyfof 231 kgs and having low 15 serum selenium levels were randomly allocated to erne of three groups. The four animals in one group were each injected with 30 mg/kg of Se-Hypo. Animals in the other two groups were treated with formulation (2) at 60 mg/kg and 90 mg/kg respectively.

Serum selenium levels were measurecLprior to treatment and then weekly for nine weeks. On day 0 there was no significant difference between the three groups (means range 70-85 nmol/1). After seven days the mean serum selenium levels of the groups receiving topical application wera4iigher than for the control group (535 and 700 nmol/1 respectively compared to 513 nmol/1 in the control group).

Table 2 gives the results/tt the changes in serum selenium levels from day 0 (treatment day) to day 63.

Discussion From Table Yit can be seen that the serum selenium levels peaked on day 7 and declined steadily until day 35 when all three groups reached a plateau, - see (Figure 2).

None/6f the animals receiving topical application displayed any sensitivity or toxicity towards the pour-on formulation. The pour-on formulation is easy to apply to the lals and overcomes the need to inject individual animals. 6975CS7.N94/JP/*t As AMENDilD - 8- 250163 COGNATE Nos. ensure this aspect of coat dryness.

This pilot trial demonstrates that sodium selenate can be formulated to be successfully 5 absorbed through the skin following topical application.

TRIAL 2 A trial was carried out on a number of animals to compare the serum levels from a 10 selenium injection (Se-Hypo as the control) with formulation (i) at rates of 12 ml and 18 ml of pcur-on (equivalent to 60 mg/kg and 90 mg/kg of selenium respectively).

Materials and Method Twelve friesian weaned bulls having an average weight of 231 kgs and having low 15 serum selenium levels were randomly allocated to one of three groups. The four animals in one group were each injected with 30 mg/kg of Se-Hypo. Animals in the other two groups were treated with formulation (1) at 60 mg/kg and 90 mg/kg respectively.

Serum selenium levels were measured prior to treatment and then weekly for nine weeks. On day 0 there was no significant difference between the three groups (means range 70-85 nmol/1). After seven days the mean serum selenium levels of the groups receiving topical application were higher than for the control group (535 and 700 nmol/1 respectively compared to 513 nmol/1 in the control group).

Table 2 gives the results of the changes in serum selenium levels from day 0 (treatment day) to day 63.

PiSCUSSiQl) From Table 2 it can be seen that the serum selenium levels peaked on day 7 and declined steadily until day 35 when all three groups reached a plateau, - see (Figure 2).

None of the animals receiving topical application displayed any sensitivity or toxicity towards the pour-on formulation. The pour-on formulation is easy to apply to the 35 animals and overcomes the need to inject individual animals. ...v0 v;\ «»7SC*7.HM,JF/.t ij q g OCT .'.'I vr- / \ - -o / now amended ^30163 / Over the 63 day trial period the 60 mg/kg pour-on produced a serum response that was bio equivalent to the 30 mg/kg dose of injected Se-Hypo. The 90 mg/kg pour-on/vas seen to be superior to the control.

It can be concluded that at both 60 mg/kg and 90 mg/kg the pour-on formulation (2) was very effective in raising serum selenium levels. / Table 2 Animal No.

Weight (kg) Treatment* Serum Selenium (nmoj//)- Days Post-Treatment 0 7 14 21 /28 42 49 56 63 6 235 Se-Hypo 86 490 340 2 270 210 220 220 220 180 223 Se-Hypo 76 520 34°/ '280 320 210 210 210 190 180 12 245 Se-Hypo 69 480 rfo 250 240 190 180 180 190 170 7 195 Se-Hypo 86 56 9/ 350 300 270 190 230 220 230 220 mean 79 /l3 335 280 285 200 210 208 208 188 29 222 A12 / 550 300 240 220 160 200 180 250 160 42 247 A12 A 500 290 230 240 200 210 190 190 170 27 236 A12 / 64 590 320 250 280 190 210 210 190 170 23 271 Al^/ 82 500 270 210 200 140 180 160 180 160 mean / 70 535 295 233 235 173 200 185 203 165 194 / 7 A18 76 770 400 270 260 190 190 200 190 180 9 245/ A18 120 660 400 330 370 240 290 230 260 140 ^7 A18 63 650 340 300 290 220 230 200 190 200 / ^232 A18 79 720 400 300 300 240 240 230 220 190 / mean 85 700 385 300 305 223 238 215 215 178 Se-Hypo given at 6 ml (30 mg). Formulation (2) test product at rates of 12 and 18 ml equivalent to 60 and 90 mg respectively. 6975CS7.N94/JP/at as amended 0 163 COGNATE Nos Over the 63 day trial period the 60 mg/kg pour-on produced a serum response that was bio equivalent to the 30 mg/kg dose of injected Se-Hypo. The 90 mg/kg pour-on was seen to be superior to the control.

It can be concluded that at both 60 mg/kg and 90 mg/kg the pour-on formulation (1) was very effective in raising serum selenium levels.

Table 2 Animal Weight Treat Serum Selenium (nmolII). Days Post-Treatment No. (kg) ment* 0 7 14 21 28 42 49 56 63 6 235 Se-Hypo 86 490 340 290 270 210 220 220 220 180 223 Se-Hypo 76 520 340 280 320 210 210 210 190 180 12 245 Se-Hypo 69 480 310 250 240 190 180 180 190 170 7 195 Se-Hypo 86 560 350 300 270 190 230 220 230 220 mean 79 513 335 280 285 200 210 208 208 188 29 222 A12 61 550 300 240 220 160 200 180 250 160 42 247 A12 71 500 290 230 240 200 210 190 190 170 27 236 A12 64 590 320 250 280 190 210 210 190 170 23 271 A12 82 500 270 210 200 140 180 160 180 160 mean 70 535 295 233 235 173 200 185 203 165 194 A18 76 770 400 270 260 190 190 200 190 180 9 245 A18 120 660 400 330 370 240 290 230 260 140 227 A18 63 650 340 300 290 220 230 200 190 200 232 A18 79 720 400 300 300 240 240 230 220 190 mean 85 700 385 300 305 223 238 215 215 178 * Se-Hypo given at 6 ml (30 mg). Formulation (2) test product at rates of 12 and 18 ml equivalent to 60 and 90 mg respectively. •. • ; ft?. QC!Vi33 «»15CJ7.N»4/JP/«t \ now amended] - 0163 TRIAL 3 Formulation 3 was trialled on jersey bulls with the results shown in Table 3.

This trial of formulation 3 was conducted to determine whether a comber salt could be successfully and safely absorbed through the skin following the application of a topical ("pour-on") formulation, to raise liver copper levels.

Materials and Methods A group of 29 jersey bulls aged 18 months were weighed and randomly divided into three groups comprising 10, 9 and 10 animals. Eaclygroup was allocated one of the following treatments: — Cuprax (10) — Untreated controls (9) — Formulation 3 (10) The 29 animals were all treated at th^ same time and for the following week no rain was 20 recorded as possibly affecting the group 3 response. Each animal given Cuprax was treated with two lOg capsules according to the manufacturer's instructions.

Formulation 3 was adnjinistered at 1 ml per 20 kg poured along the midline back region.

All the animals were grazed normally for the three week duration of the trial.

Three weeky after treatment, the bulls were all slaughtered for human consumption and samples oi liver were removed from each animal. Each liver was separately sampled in 30 4 remote places. Two of the samples from each animal were analysed and the results averaged. The results were analysed statistically using a one-way analysis of variants id pair-wise comparison of means. «975CS7.N»«/JP/»t AS AMENDED 250163 - COGNATE Nos.

TRIAL 3 A formulation containing copper chloride was trialled on jersey bulls with the results shown in Table 3.

This trial was conducted to determine whether a copper salt could be successfully and safely absorbed through the skin following the application of a topical ("pour-on") formulation, to raise liver copper levels.

Materials and Methods A group of 29 jersey bulls aged 18 months were weighed and randomly divided into three groups comprising 10, 9 and 10 animals. Each group was allocated one of the following treatments: - Cuprax (10) — Untreated controls (9) The formulation containing copper chloride The 29 animals were all treated at the same time and for the following week no rain was 20 recorded as possibly affecting the group 3 response. Each animal given Cuprax was treated with two lOg capsules according to the manufacturer's instructions.

The formulation was administered at 1 ml per 20 kg poured along the midline back region.

All the animals were grazed normally for the three week duration of the trial.

Three weeks after treatment, the bulls were all slaughtered for human consumption and samples of liver were removed from each animal. Each liver was separately sampled in 30 4 remote places. Two of the samples from each animal were analysed and the results averaged. The results were analysed statistically using a one-way analysis of variants and pair-wise comparison of means.

//S" \ |:i QCT !:j €#75CS7.H84/Jp/at \ m ~^qy/^amenpepJ 0J6J 11 - $ Results Table 3 gives individual results and group means. There was no statistical differem recorded between any of the group means, but it is noteworthy that the highesuwean liver copper level was recorded for the group given formulation 3.

The topical formulation produced severe skin abrasion along the midline bpdk. This did not appear to cause the animals any distress and did not subsequently lead to any downgrading of the hide at slaughter time.

Table 3 Liver Copper Levels in Individual Animalsr and Group Means Animal No.

Control Animal No. ^prax Animal No.

Test Product 032 382 108 > / 702 010 298 148 606 078 / 788 164 1133 111 280 o 952 289 570 154 119 /290 310 101 632 342 786 / 212 1093 240 959 119 749 y 054 783 367 1378 089 487/ 022 867 139 293 170 359 278 153 884 272 /fl78 021 469 338 641 / 091 630 120 725 mean / 636 mean 687 mean 751 assion THe fact that the topical test formulation produced a higher mean liver copper level /within the first three weeks would suggest that topical application can provide animals successfully with supplementary copper. The main problem associated with formulation 3 was the toxicity at the site of application.

«S75CS7.*94/JP/tt AS AMENDC-U 250 163 -11- COGNATE Nos.

Results Table 3 gives individual results and group means. There was no statistical difference recorded between any of the group means, but it is noteworthy that the highest mean liver copper level was recorded for the group given the formulation.

The topical formulation produced severe skin abrasion along the midline back. This did not appear to cause the animals any distress and did not subsequently lead to any downgrading of the hide at slaughter time.

Table 3 Liver Copper Levels in Individual Animals and Group Means Animal No.

Control Animal No.

Cuprax Animal No.

Test Product 032 382 108 702 010 298 148 606 078 788 164 1133 111 280 016 952 289 570 154 119 290 310 101 632 342 786 212 1093 240 959 119 749 054 783 367 1378 089 487 022 867 139 293 170 1149 359 278 153 884 111 1178 021 469 338 641 091 630 120 725 mean 636 mean 687 mean 751 Discussion The fact that the topical test formulation produced a higher mean liver copper level within the first three weeks would suggest that topical application can provide animals successfully with supplementary copper. The main problem assoc^ted; with the formulation was the toxicity at the site of application. /'v r *.. / i •*. fo l J «»7SCS7.H9»/JP/»t V) i- v" ' howamended 250153 Formulation 3 delivered 5 mgs of copper per kg. The maximum comparable rate iorA parenteral copper product is approximately 1 mg per kg. The dose rate for topically applied chemicals is typically greater than that for oral or parenteral formulations but not usually by a factor of 5 (e.g ivomec x 2.5). It may therefore be possible loxombine a lower dose rate (mg/kg) and increased volume to reduce the toxic effects without jeopardising the effectiveness of the treatment. / In conclusion, the test product appeared to successfully raise livei/copper levels above those of the untreated controls. / TRIAL 4 / A trial was conducted was low blood selenium cattlgr Limousin heifers 12 months old). This trial compared the pour-on formulation (2) at i lower dose rate than in trial 2 with Se-Hypo. The results arc as shown in Table A./ Materials and Method / The pour-on formulation (2) was administered at a rate of 6 mis per 100 kg. This effectively applies 30 mg per kg or selenium on the backline of cattle. Se-Hypo was administered at the rate of 2 mls^per 100 kg. This effectively supplies lOmg per 100 kg subcutaneously. / The cattle were bled prior to the commencement of the trial and then placed in 3 groups on the basis of their hiood selenium levels. 1. Group contwfls (6) 2. Se-Hypa<7) 3. Formulation 2 of selenium (7) The/Cattle were bled at 2, 4 and 8 weeks post treatment. The blood levels were monitored and the cattle were retreated in the treatment group nine weeks after the /commencement of the trial. 697SCS7.N94/JF/at as amended 25 0 163 -12- COGNATE Nos.

The formulation delivered 5 mgs of copper per kg.The maximum comparable rate for a parenteral copper product is approximately 1 mg per kg. The dose rate for topically applied chemicals is typically greater than that for oral or parenteral formulations but 5 not usually by a factor of 5 (e.g ivomec x 2.5). It may therefore be possible to combine a lower dose rate (mg/kg) and increased volume to reduce the toxic effects without jeopardising the effectiveness of the treatment.

In conclusion, the test product appeared to successfully raise liver copper levels above 10 those of the untreated controls.

XRIAL4 A trial was conducted was low blood selenium cattle (Iimousin heifers 12 months old). 15 This trial compared the pour-on formulation (1) at a lower dose rate than in trial 2 with Se-Hypo. The results are as shown in Table 4.

Materials and Method The pour-on formulation (1) was administered at a rate of 6 mis per 100 kg. This 20 effectively applies 30 mg per kg of selenium on the backline of cattle. Se-Hypo was administered at the rate of 2 mis per 100 kg, This effectively supplies lOmg per 100 kg subcutaneously.

The cattle were bled prior to the commencement of the trial and then placed in 3 groups 25 on the basis of their blood selenium levels. 1. Group controls (6) 2. Se-#ypo (7) 3. Formulation X of selenium (7) The cattle were bled at 2, 4 and 8 weeks post treatment. The blood levels were monitored and the cattle were retreated in the treatment group nine weeks after the commencement of the trial. •• - f • \ (;:;i n 2 OCT <338 Table 4 2; 4 START FEB FEB MAR MAR 7/1/94 9 26 31 CONTROL Tag 15 260 300 210 340 520 Tag 16 240 220 200 290 460 Tag 21 200 200 290 320 410 Tag 24 240 250 300 400 410 Tag 29 240 250 190 370 490 Tag 33 240 260 310 310 450 Average 237 247 250 338 457 SELJECT Tag 13 290 560 4P0 670 1000 Tag 20 230 580 550 590 960 Tag 23 220 550 540 1600* 1100 Tag 27 250 580 490 620 910 Tag 28 180 550 440 590 Tag 32 180 530 460 520 850 Tag 36 220 560 420 570 920 Average 224 559 483 593 957 FORMULATION 2 Tag 17 330 500 580 490 820 Tag 19 230 790 650 1200 Tag 22 230 550 500 530 930 Tag 25 290 650 520 660 1000 Tag 26 230 900 710 870 1200 Tag 30 230 670 400 830 920 Tag 35 210 770 630 740 1000 Average 250 690 557 681 1010 ^Injected in error before bleeding (5 minutes) NOTE: All units above ae whole blood selenium as nmol/1 SS7SCJ?.ll»4/J?/at now amended 250163 - 14 TRIAL 5 A trial was conducted on twenty clinically healthy 18 months old Angys cattle.

Materials and Methods The cattle were bled prior to the commencement of the trial aiyf then randomly placed in one of the following groups: Group 1: Test animals treated with our seleniunyf>our-on formulation # 2 (as described in Example 2) (contains 5mg/ml selenium as sodium/&eJenate at the proposed label dose rate of l.om./50kg body weight) Group 2: Test animals treated with Se-Hypo (contains 5mg/ml selenium as sodium selenate at the label dose rate of 5nrf injection per cattle).

The cattle were bled at 4 and 12 W' Quality Management Laboratory Iflncoln. post treatment and samples sent to the MAF Assessment Assessment was made on t£6 basis of selenium blood levels (nmol/1) of treated animals.

Results The results are outlined in Table 5 and Figure 4.

Blood samples jvere blinded and selenium levels tested by MAF Quality Management Laboratory Lincoln.

Animals/Cheated with both our selenium pour-on and Se-Hypo obtained a rise in blood selenium levels when measured at 4 weeks post-treatment This rise was maintained and ^lightly increased at second testing (12 weeks) which indicates a long term benefit selenium pour-on and Se-Hypo treatments in selenium deficiency and related iseases in cattle. 6975C57.N94/J?/At AS AMENDED 0 1 14' COGNATE Nos.

TRIAL 5 A trial was conducted on twenty clinically healthy 18 months old Angus cattle.

Materials and Methods The cattle were bled prior to the commencement of the trial and then randomly placed in one of the following groups: Group 1: Test animals treated with our selenium pour-on formulation #1 (as described in Example 2) (contains 5mg/ml selenium as sodium selenate at the proposed label dose rate of 1.5m./50kg body weight) Group 2: Test animals treated with Se-Hypo (contains 5mg/ml selenium as sodium selenate at the label dose rate of 5ml injection per cattle).

The cattle were bled at 4 and 12 weeks post treatment and samples sent to the MAF 20 Quality Management Laboratory Lincoln.

Assessment Assessment was made on the basis of selenium blood levels (nmol/1) of treated animals.

Results The results are outlined in Table 5 and Figure 4.

Blood samples were blinded and selenium levels tested by MAF Quality Management Laboratory Lincoln.

Animals treated with both our selenium pour-on and Se-Hypo obtained a rise in blood selenium levels when measured at 4 weeks post-treatment. This rise was maintained and slightly increased at second testing (12 weeks) which indicates a long term benefit of our selenium pour-on and Se-Hypo treatments in selenium deficiency and related 35 diseases in cattle.

XT A- /o M7S«7.m/JP/»t ' f> 0 ftp'" : |rj y t J1" >1 V, /J.-v-r 1 - / 250 Table 5 Blood selenium levels in cattle in nmol/1. Reference values are: Responsive < 130; Marginal 130-250; Adequate 250-2000 (MAF Quality, Lincoln) Animal No.

Initial Results 8.4.

One month 12.5.

Three month 13.7. 31 1000 1420 1410 32 560 1120 1200 33 860 1370 1470 34 510 1060 960 610 1060 1130 36 500 1060 1380 •37 500 1180 1600 38 440 1260 1420 39 960 310 490 970 1180 Mean Selpor 643 1166 1305 STD 201 145 188 311 580 1220 1320 312 1060 313 380 1030 1440 314 930 1440 1810 315 830 1240 1300 316 490 317 570 1190 1320 318 560 960 1120 319 970 1530 1790 320 410 1010 1190 Mean Se-Hypo 678 1202 1411 STD 234 190 241 Animals 31-310 Selenium pour-on treatment Animals 310-320 Se-Hypo treatment 65is«j Conclusion The pour-on formulation when applied at the rate of 6mls per 100 kg gave blood 5 selenium levels that were similar in effect on low blood selenium cattle to the levels produced by treating cattle with Se-Hypo (see Figure 3).

Whilst these examples have been given as illustrative of the invention, the invention is not limited to the examples and other alterations and modifications can be made to the foregoing such as the addition of other adjunctive compounds that can be administered topically without departing from the scope of this invention as claimed. 30 €97SCS7>N94/JP/«t [now amended] 1 6 3 WHAT WE CLAIM IS 1. A pour-on formulation containing a therapeutically effective amount of aj/ace 5 element in a pharmaceutical^ acceptable carrier. 2. A formulation as claimed in claim 1 wherein the trace element is selected from the group: selenium, copper, cobalt and iodine.

JO A formulation as claimed in any one of the precedin^claims wherein the pharmaceutical carrier is an organic liquid.

A formulation as claimed in claims 1 and 2 wher^the pharmaceutical carrier is water or in combination v/ith water.

A formulation as claimcd in claim 3 or c}a\m 4 where the carrier is an alcohol, glycol or glycol ester. 6. A formulation as claimed in claim 3 where the carrier is selected from xylene, / 7. A formulation as claimed in claim 3 or 4 where the carrier is a liquid carrier isopropyl myristate. 8. A formulation z limed in claim 3 or 4 where the carrier is an ester of a fatty acid.

A fo dation as claimed in claim 8 where the carrier comprises an alkylamide of a y acid.

AA formulation as claimed in claim 3 or 4 where the carrier is an ethoxylated block polymer.

A formulation as claimed in claim 1 wherein the formulation is a non-aqueous formulation containing one or more oxides of selenium or copper or cobalt dissolved in a solvent selected from the group: ethanol, monopropylene glycol, July 1936/6975ICLM.795/hc/iv -8 JUL 1996 { NOW AMENDEPii 250163 and butyl dioxitol. 12. A formulation as claimed in claim 11 wherein the trace element is presen^ selenium dioxide or selenium trioxide. 13. A formulation as claimed in claim 1 wherein the formulation isjm aqueous formulation containing water and a wetting agent. 14. A formulation as claimed in any one of the preceding claijtfs further including at least one endoparasiticide.

. A formulation as claimed in any one of the preceding claims further including at least one vitamin. 16. A method of raising the serum level^of a trace element in animals by topical application to the animal of a poison formulation as claimed in any one of the preceding claims. 17. A method as claimed inydaim 8 wherein the trace element is selenium. 18. A process for thaf>reparation of a pour-on formulation as claimed in any one of claims 1 - 15 substantially as herein described with reference to the examples.

JAMES W PIPER & CO Au^rneys for the Applicant (hmont Holdings Limited 4 , 6975QCS7.596/HC/jq AS AMENDE 17 250163 COGNATE Nos.

Claims (6)

WHAT WE CLAIM IS:

1. A pour-on formulation containing a therapeutically effective amount of a trace element in a pharmaceutically acceptable carrier, wherein the formulation is a non-aqueous formulation containing one or more oxides of selenium or copper or cobalt dissolved in a solvent chosen from the group consisting of ethanol, monopropylene glycol and butyl dioxitol.

2. A pour-on formulation as claimed in claim 1 wherein the trace element is present as selenium dioxide or selenium trioxide, or sodium selenate.

3. A pour-on formulation as claimed in any of the preceding claims further including at least one endoparasiticide or at least one vitamin.

4. A method of raising the serum levels of a trace element in animals by topically applying to the animal a pour-on formulation as claimed in any one of claims 1 to 3.

5. A pour-on formulation as claimed in claim 1 substantially as herein described with reference to the examples.

6. A method of raising the serum levels of a trace element in an animal by topically applying to the animal a pour-on formulation as claimed in claim 1, substantially as herein described with reference to the examples. JAMES W PIPER & CO Attorneys for the Applicant Ashmont Holdings Limited rp\6975ecl4o98 .,\s /<C | 0 2 0« B3J *j