AU777772B2 - Control of wool growth in sheep and related animals - Google Patents

Description

WO 00/71089 PCT/AUOO/00487 1 CONTROL OF WOOL GROWTH IN SHEEP AND RELATED ANIMALS Field of the Invention: This invention relates to a method and composition for permanently reducing or preventing wool growth at a selected locality or localities in sheep and related animals. In a particular application of the invention, the breech and/or pizzle area of sheep are treated in order to prevent the incidence of blow-fly strike and/or balanitis.

Background of the Invention: Blow-fly strike and balanitis (pizzle rot) are significant problems for sheep and wool growers. It is estimated that in Australia alone, losses exceeding $150 million per annum are caused by blow-fly strike and its containment.

Current methods for containing blow-fly strike and balanitis include crutching periodic close shearing around the breech and hind legs), ringing of the pizzle and the widely practised treatment known as the Mules operation. The Mules operation or "mulesing" involves the removal of a large flap of skin from lambs below the tail and down the hind leg using shears and without anaesthetic. While providing effective life-long protection against blow-fly strike, the operation causes considerable trauma to the recipient animals and can be associated with long-term arthritic problems and is likely to be phased out or banned for these reasons. The scarring caused by the Mules operation may also reduce the sale price of the carcass and hide. Consequently, considerable research has been directed to the identification of non-surgical mulesing methods.

In Proc. Aust. Soc. Animal Prod. 11, 189-192 (1976) Pratt and Hopkins disclosed the use of a number of cryogenic, irritant, fixative and protein denaturizing agents. Of these, the protein denaturizing agents were the only compounds found to be successful and included 40% phenol and a mixture of 20% phenol and 50% orthocresol. Application was by painting 15-30ml onto the relevant area. In discussing the effectiveness of these agents, the authors noted a number of disadvantages arising from the toxic and irritant properties of phenol and cresol, such as the need to take special precautions to prevent injury to human operators and increased toxicity to lambs resulting from excess absorption through intact skin.

WO 00/71089 PCT/AU00/00487 2 In Wool Technology and Sheep Breeding 23 26-27 (1976) the same authors reported the successful use of a 40% phenol emulsion which was applied to the relevant areas in an amount of 10-20ml using a roll-on dispenser. In this paper it is stated that the phenol rapidly penetrates the superficial layers of the skin, destroying cells and paralyzing nerve endings to produce an almost painless effect, whilst the germicidal nature of the phenol prevented infection in the healing tissue. Again it is noted that operators need to observe rigorous safety precautions.

Australian patent specification No. 73793/91 describes a non-surgical mulesing method involving the application of a cationic compound such as a halogenated quaternary aimnonium compound didecylmethylamine ammonium chloride), which complexes with glycosaminoglycans present in the skin to form full skin thickness eschars which are ultimately sloughed to leave linear scars similar to those resulting from the Mules operation. While found to be effective in reducing blow-fly strike incidence, the method has not been widely practised because it appears to cause sheep a similar amount of pain to that of surgical mulesing. A further disadvantage is the risk of injury to operators.

Accordingly, there remains a need for an effective, non-surgical mulesing method which is safe in use and causes little or no trauma to the recipient animals. It is also desired that such a method avoids damage to the skin which might devalue the hide.

Summary of the Invention: The present invention provides, in one aspect, a method for the controlled ablation of wool follicles within skin of a wool bearing animal at one or more selected localities, comprising; treating said skin at said selected locality(ies) with a composition conprising at least one follicle-ablating agent and, subsequently, irradiating said skin at said selected locality(ies) with electromagnetic radiation of an intensity and/or wavelength to bring about the photodynamic ablation of at least a portion of the wool follicles present in the skin at said selected locality(ies).

WO 00/71089 PCT/AU00/00487 3 Detailed disclosure of the Invention The invention is hereinafter described in reference to the preferred treatment of the wool follicles of sheep, although the present invention is suitable for treatment of the wool follicles of other wool bearing animals such as goats and alpacas.

In a preferred form, the present invention provides a method for the controlled ablation of wool follicles within skin of sheep at one or more selected localities, comprising; treating said skin at said selected locality(ies) with a composition comprising at least one follicle-ablating agent and, subsequently, irradiating said skin at said selected locality(ies) with electromagnetic radiation of an intensity and wavelength to bring about the photodynamic ablation of at least a portion of the wool follicles present in the skin at said selected locality(ies).

The follicle-ablating agent(s) may be selected from photosensitisers and substances which induce the formation and/or accumulation of an endogenous photosensitiser(s). Suitable photosensitisers include porphyrin derivatives and analogues haematoporphyrin (HpD) derivatives such as di-haematoporphyrin ether). It is, however, preferred to use a substance which induces the formation and/or accumulation of an endogenous photosensitiser(s).

Most preferably, the method comprises treating the skin at said selected locality(ies) by applying 5-aminolevulinic acid (5-ALA) or a derivative esterified 5-ALA or 5-ALA conjugated to one or more fatty acids may show enhanced skin permeability) or analogue thereof. While not wishing to be bound by theory, it is believed that 5-ALA, which is readily absorbed by the skin, induces the biosynthesis in skin of an excess of protoporphyrin IX (PpIX). Accumulated PpIX, when irradiated with electromagnetic radiation of a photosensitising wavelength white light or broad-spectrum red light), causes phototoxic damage or death (i.e.

ablation) of skin cells including cells of wool follicles.

The composition is preferably applied topically or intradermally/subcutaneously by hydraulic or pneumatic applicators).

The composition preferably comprises a suitable carrier. For topical application, the carrier is preferably selected from diethylene glycol WO 00/71089 PCT/AU00/00487 4 monoethyl ether (DGME) and mixtures including propylene glycol and glycerol. Other suitable carriers include ethanol and isopropyl myristate.

The composition may further comprise one or more chelating agents EDTA and 2,2-dipyridyl) and/or other agent(s) chlorophyll and prooxidants) to augment the effect of the follicle-ablating agents.

Prior to application of the composition, the wool at the skin locality(ies) to be treated may be removed, wholly or in part. Methods for removing the wool include shearing, close clipping with shears to a residual height of 1-2 mm), defleecing with epidermal growth factor (EGF), shaving and depilation. Degreasing of the wool and skin at the locality(ies) to be treated with soap or an alcohol wash is also advisable.

Following treatment of the skin with the composition, it is preferred that a period of time within 24 hours) sufficient to allow for the substantial completion of absorption and metabolism of the follicle-ablating agent be allowed to pass before being irradiated with electromagnetic radiation of an intensity and/or wavelength to bring about the photodynamic ablation of wool follicles. Presently, it is believed that the irradiation should be conducted between 2 and 15 hours, more preferably between 3 and hours, after treating the skin with the said composition.

Irradiation may be conducted using any suitable source of electromagnetic radiation. The electromagnetic radiation will be of an intensity and wavelength to bring about the photodynamic ablation of at least a portion of the wool follicles by sensitising the absorbed follicle-ablating agent and/or accumulated exogenous photosensitiser(s). The irradiation may be conducted using a monochromatic or polychromatic light source.

Preferably, the irradiation is conducted using a source of light of wavelength 600 to 700 n at a dose intensity) of 100-175 (preferably, 150) J/cm 2 and a fluence rate of less than 150 mW/cm 2 however, other sources of light may be used including ambient sunlight.

By varying the composition, the method provides for the controlled ablation of a selected percentage of follicles within the selected locality(ies).

Preferably, the method results in the ablation of at least 50%, more preferably at least 70%, of the follicles within the selected locality(ies). For use with sheep, it may be preferred that the method results in the ablation of about 85% of the wool follicles in the selected locality(ies). The subsequent regrowth provided by the 15-30% viable non-ablated or regenerated) WO 00/71089 PCT/AUOO/00487 follicles is insufficient for the occurrence of blow-fly strike and provides an advantage in that it prevents the development of skin cancers which might otherwise occur if 100% of the follicles were ablated. The development of skin cancers, however, is not anticipated to be a problem with treatment of the breech and/or pizzle area of sheep since these areas are substantially shaded from the sun by the sheep's body.

1000/ ablation of wool follicles in sheep skin may be achieved with a composition comprising 10-20% 5-ALA in a suitable carrier at pH (unbuffered) or 4.0. Decreasing the amount of 5-ALA to 5-9% is expected to reduce the percentage of ablated follicles to 70-85%.

Varying the duration and/or intensity of the irradiation used is also expected to affect the percentage of ablated follicles. For example, with a composition comprising 10% 5-ALA in a suitable carrier at pH (unbuffered), an irradiation time of about 5 minutes (with white light at an intensity of 150J/cm 2 may be expected to achieve 100% ablation whereas an irradiation time of about 3 minutes may be expected to achieve 70-85% ablation.

In a further aspect, the present invention provides a method for nonsurgical mulesing of a sheep, the method comprising; treating the skin at the breech area of the sheep with a composition comprising at least one follicle-ablating agent and, subsequently, irradiating at least a portion of the treated area with electromagnetic radiation of an intensity and/or wavelength to bring about photodynamic ablation of at least a portion of said wool follicles in said treated skin.

While the invention offers an alternative to the Mules Operation, it may also be employed for pizzle ringing.

Accordingly, in yet a further aspect, the present invention provides a method for non-surgical pizzle ringing of a sheep, the method comprising; treating skin at or near the pizzle area of the sheep with a composition comprising at least one follicle-ablating agent and, subsequently, irradiating at least a portion of the treated area with electromagnetic radiation of an intensity and/or wavelength to bring about photodynamic ablation of at least a portion of said wool follicles in said treated skin.

Moreover, the application of the method of the invention is not limited to the containment of incidence of blow-fly strike and balanitis. That is, the method may also be used for "wigging", removal or reduction of wool from WO 00/71089 PCT/AU00/00487 6 areas around the face, "crutching" (to prevent blow-fly strike and dags) and branding of sheep or other wool bearing animals.

The method of the present invention may also be used in conjunction with other wool treatments. For example, the method may be used as an adjunct to biological defleecing EGF defleecing).

The terms "comprise", "comprises" and "comprising" as used throughout the specification are intended to refer to the inclusion of a stated step, component or feature or group of steps, components or features with or without the inclusion of a further step, component or feature or group of steps, components or features.

The invention will hereinafter be further described by way of reference to the following, non-limiting examples and accompanying figure.

Brief description of the accompanying figures: Figure 1 provides graphical results discussed in Example 2 hereinafter.

The figure shows that in skin samples taken from three sheep, the concentration of PpIX, a metabolite of 5-ALA thought to cause phototoxic damage or death to skin cells upon irradiation with electromagnetic radiation of a photosensitising wavelength, peaked between about 6 and 13 hours after application of 5-ALA. The results suggest that the optimal time for irradiation is between about 8 and 10 hours after application of however some other, empirical evidence from treated sheep indicates that these times are effective but that optimal levels in the follicles may be attained prior to this between about 3 and 4 hours).

Example 1: Ablation of wool follicles in sheep Preparation of active agent: acid (5-aminolevulinic acid; 5-amino-4-oxopentan6ic acid) (5-ALA) is combined with any suitable vehicle for topical application and absorption. Tests on sheep employed a mixture of 20% propylene glycol plus 80% commercial sorbolene cream containing glycerine (glycerol). 5-ALA was tested at 5% and 17% Preparation of skin surface for treatment: Skin was prepared for treatment by using a defleecing dose of epidermal growth factor, by shaving or by using a depilating agent such as WO OOM1089 PCT/AU00/00487 7 thioglycollate. The area was swabbed with 70% ethanol to degrease it and assist penetration of the cream.

Photodyncnic treatment: mixture was rubbed into the area briefly by hand and the animals then left for a period of 3 hours to allow the 5-ALA to be absorbed and metabolised in the skin and follicles. After 3 hours the treatment area was irradiated with a white light source a modified slide projector) containing a heat filter. Light, 600 700nm, was administered at a dose of 125J/cm 2 at a fluence rate of less than 150 mW/cnm. In the case of the slide projector (containing a 300W bulb) the lens was held 10 cm from the skin for no more than 10 mins. Spray anaesthetic was sometimes applied after the light was removed.

Results: Following treatment there is some slight localised edema which persists for about 48 hours. There is some discolouration then crusting of the skin in the treated area. After 4-5 weeks the skin regains its normal appearance but wool regrowth is either sparse or non-existent in the treated area. Tests with mixtures containing 5% and 17% 5-ALA were found to be effective. The treatment appears to be relatively painless with no side effects or chemical residues in the animal.

Example 2: 5-ALA metabolism time profiles in sheep skin samples Tests were conducted to determine the period of time, following application of 5-ALA, required to achieve peak concentrations of the metabolite PpIX in skin. Since this metabolite is believed to cause phototoxic damage or death to skin cells upon irradiation with electromagnetic radiation of a photosensitising wavelength, it was reasoned that more consistent ablation of wool follicles in sheep could be achieved by irradiating the 5-ALA treated skin at a time when the peak concentration of PpIX was expected.

Test method: was formulated as a 10% aqueous solution in 90% (w/w) DGME in the presence of various test compounds to measure their effectiveness in enhancing PpIX formation. These formulations were applied to clipped, mid-flank skin of several sheep after swabbing with 70% ethanol.

Skin biopsies were commnenced immediately thereafter using a sterile 0.8cm WO 00/71089 PCTr/AUO/00487 8 human biopsy punch. Samples were taken at 2 hourly intervals for times up to about 20 hours. Skin samples were immediately frozen in dry ice and stored at -70C until processed. Skin samples were then thawed, homogenised in aqueous buffer and PpIX extracted into an acid solution and then into an organic phase of ethyl acetate. PpIX levels were measured in a spectrofluorimeter using a set of standards of known PpIX concentration.

The excitation wavelength was 430nm and emission was measured at 595nm.

Results: The results of the tests are shown in Figure 1. The formulations used for the treatment of the three sheep were: Animal 4347 10% 5-ALA, 90% DGME Animal 4365 10% 5-ALA, 90% DGME, 10mM EDTA Animal 4468 10% 5-ALA, 90% DGME, 20mM 2,2-dipyridyl.

The profiles of PpIX formation shown in Figure 1 suggest that the optimal treatment time (for irradiation with electromagnetic radiation) would be about 8 to 10 hours after 5-ALA application. A similar conclusion has been made from PplX formation profiles obtained using other formulations (data not shown).

Discussion: Since obtaining the PpIX formation profiles shown in Figure 1, follicle -ablation experiments have been conducted on a further 8 sheep using the formulation: 10% 5-ALA, 90% DGME, 10mM EDTA (applied to close-clipped breech), followed 10 to 14 hours later with a 5 minute irradiation. The light source used for the irradiation was a bank of five 24V 250W tungsten halogen projector lamps at a distance to yield a dose of about 150J/cm (heat, and any excess UV, was shielded with a glass panel treated with an infra-red reflective coating).

Initial observations indicated that the treated animals showed a more consistent level of follicle ablation. Subsequent observations (up to 2 years after treatment) have generally indicated that animals which are bare in the treated area 2 months after treatment, remain so permanently.

Example 3: Blow-fly strike prevention field trial In October 1999, a field trial was conducted in Armidale, NSW, Australia, to assess the effectiveness of the method of the present invention in preventing blow-fly strike in sheep.

WO 00/71089 PCT/AUO/00487 9 Field trial method: 191 Merino lambs (4-5 month old ewes and whethers) were divided into three groups of 66, 66 and 59 animals per group, with the latter group serving as an untreated control) group. 66 animals were subjected to conventional, surgical mulesing and the remaining 66 animals were treated with the method of the present invention, specifically application of a ALA formulation (10% 5-ALA in 80% DGME, 5% cineole, 200 mM 2,2-dipyridyl) to the breech area. Prior preparation of the breech consisted of close-clipping so that 1-2 mm of wool remained) and swabbing briefly with 70% ethanol. Animals were then treated with a minute exposure to a 1000 watt metal halogen light (estimated dose 120 J/cm 2 at a fluence rate of 150 mW/cm 2 10 hours after application of the ALA formulation. All animals were left to graze at pasture and were observed closely during the following seasons which were favourable for blow-fly strike.

Results: Breech-strike results as of 9 May 2000 (a period of 7 months) were as shown in Table 1.

TABLE 1: Animal Group No of animals observed with breech-strike Surgically mulesed group 1 treated group 5 Untreated group 19* (28.8%) *including 5 multiple or repeat strikes.

Discussion: The results demonstrate that the method of the present invention is effective in significantly reducing the incidence of breech-strike as compared to untreated sheep.

While not wishing to be bound by theory, it is believed that the success of the method of the present invention may, in part, also stem from an effect on skin structure, vasculature and other organs associated with the wool follicles in the treated area. Thus, the ablation of sebaceous glands and microvasculature may affect both the ability of wool follicles to regenerate as well as the likelihood of the skin becoming inflamed and damaged in wet conditions that normally predispose to, or attract, blow-fly strike.

Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

It will be appreciated by persons skilled in the art that numerous variations Sg and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly 20 described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

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Claims (31)

1. A method for the controlled ablation of wool follicles within skin of a wool bearing animal at one or more selected localities, comprising; treating said skin at said selected locality(ies) with a composition comprising at least one follicle-ablating agent and, subsequently, irradiating said skin at said selected locality(ies) with electromagnetic radiation of an intensity and/or wavelength to bring about the photodynamic ablation of at least a portion of the wool follicles in the skin at said selected locality(ies).

2. A method according to claim 1, wherein the said wool bearing animal is a goat, alpaca or sheep.

3. A method for non-surgical mulesing of a sheep, the method 15 comprising; treating the skin at the breech area of the sheep with a composition comprising at least one follicle-ablating agent and, subsequently, irradiating at least a portion of the treated skin with electromagnetic radiation of an intensity and/or wavelength to bring about photodynamic ablation of at least a portion of said wool follicles in said treated skin.

4. A method for non-surgical pizzle ringing of a sheep, the method comprising; treating skin at or near the pizzle area of the sheep with a composition se: 25 comprising at least one follicle-ablating agent and, subsequently, irradiating at least a portion of the treated skin with electromagnetic radiation of an intensity and/or wavelength to bring about photodynamic ablation of at least a portion of said wool follicles in said treated skin.

5. A method for crutching a sheep, the method comprising; treating skin in an area of the breech and hind legs of the sheep with a composition comprising at least one follicle-ablating agent and, subsequently, irradiating at least a portion of the treated skin with electromagnetic radiation of an intensity and/or wavelength to bring about photodynamic ablation of at least a portion of said wool follicles in said treated skin.

6. A method according to any one of the preceding claims, wherein the method results in the ablation of at least 50% of the wool follicles in the treated skin.

7. A method according to any one of the preceding claims, wherein the method results in the ablation of at least 70% of the wool follicles in the treated skin.

8. A method according to any one of the preceding claims, wherein the 10 method results in the ablation of 70-85% of the wool follicles in the treated skin.

9. A method according to any one of the preceding claims, wherein the method results in the ablation of substantially all of the wool follicles within 15 the treated skin.

A method according to any one of the preceding claims, wherein the follicle-ablating agent(s) is a photosensitiser. 0 20

11. A method for treating or preventing blow-fly strike in a sheep, said method comprising; treating skin in an area of the breech and hind legs of the sheep with a composition comprising at least one photosensitiser and/or substance which induces the formation and/or accumulation of an endogenous photosensitiser(s) and, subsequently, irradiating at least a portion of the treated skin with electromagnetic radiation of an intensity and/or wavelength to sensitise the photosensitiser and/or endogenous sensitiser(s).

12. A method according to claim 10 or 11, wherein the photosensitiser is selected from porphyrin derivatives and analogues.

13. A method according to claim 10 or 11, wherein the photosensitiser is selected from 5-aminolevulinic acid

14. A method according to claim 10 or 11, wherein the photosensitiser is aminolevulinic acid (5-ALA) conjugated to one or more fatty acids.

A method according to claim 10 or 11, wherein the photosensitiser is esterified 5-aminolevulinic acid (5-ALA) conjugated to one or more fatty acids.

16. A method according to any one of the preceding claims, wherein the composition is applied topically to said skin.

17. A method according to claim 16, wherein the composition includes a carrier selected from diethylene glycol monoethyl ether (DGME) and mixtures including propylene glycol and glycerol. 15

18. A method according to any one of the preceding claims, wherein said composition further comprises one or more chelating agents.

19. A method according to any one of the preceding claims, wherein said composition further comprises chlorophyll or prooxidants.

A method according to any one of the preceding claims, wherein the skin to be treated is pre-treated to wholly or partially remove wool fibres.

21. A method according to claim 20, wherein the skin to be treated is pre- 25 treated by a method selected from shearing, close clipping with shears, defleecing with epidermal growth factor (EGF), shaving and depilation.

22. A method according to any one of the preceding claims, wherein said step of irradiating is conducted between 2 and 15 hours after treating said skin with the composition.

23. A method according to claim 22, wherein said step of irradiating is conducted between 3 and 10 hours after treating said skin with the composition. 14

24. A method according to claim 22, wherein said step of irradiating is conducted about 10 hours after treating the skin with the composition.

A method according to any one of the preceding claims, wherein said electromagnetic radiation is provided by a source of light of wavelength 600 to 700 nm.

26. A method according to any one of claims 22 to 24, wherein said electromagnetic radiation has an intensity of 100-175 J/cm 2 and a fluence rate of less than 150 mW/cm 2

27. A method according to any one of claims 1 to 5 or 11 composition comprising of an aqueous solution of 5-aminolevulinic acid (5-ALA) and 90% (w/w) diethylene glycol monoethyl ether (DGME).

28. A method according to any one of claims 1 to 5 or 11, wherein the composition comprises 10% of an aqueous solution of 5-aminolevulinic acid (5-ALA), diethylene glycol monoethyl ether (DGME) and 10 mM EDTA.

29. A method according to any one of claims 1 to 5 or 11, wherein the composition 2 comprises 10% of an aqueous solution of 5-aminolevulinic acid (5-ALA), 20 diethylene glycol monoethyl ether (DGME) and 20 mM 2,2-dipyridyl.

30. A method according to any one of claims 1 to 5 or 11, wherein the composition comprises 10% of an aqueous solution of 5-aminolevulinic acid (5-ALA), diethylene glycol monoethyl ether (DGME), 5% cineole and 200 mM 2,2- 25 dipyridyl.

31. A method for the controlled ablation of wool follicles within the skin of a wool bearing animal at one or more selected localities, substantially as hereinbefore described with reference to the Examples. Dated this sixth day of September 2004 Commonwealth Scientific and Industrial Research Organisation Patent Attorneys for the Applicant: F B RICE CO