AU2005248974B8 - Method of deactivating dust mite allergens - Google Patents

Description

AUSTRALIA

Patents Act 1990 RECKITT BENCKISER (UK) LIMITED; UNIVERSITY OF SOUTHAMPTON COMPLETE SPECIFICATION STANDARD PATENT Invention Title: Method of deactivating dust mite allergens The following statement is a full description of this invention including the best method of performing it known to us:- 0 METHO OFa DECIVTN DUST MITE ALLERGENS heduthawr dectiain douste mite allergen rspne bti wa Varyiu alergen0s~ areswnwhchr arecrasted O~I throughedtha the aifigraes uma ratieon.e Fort eaphads beenysu (known or a og ie ththus utntrigger allergeic re actons in th oyhmasbuc oivn as s ashm and l rhi ni Iasereporsyteds. eAya Cl 10 rv1928 thi was thends i in Eeiete adus tatere the pray soorcoe the allergenic responsesuti whas onlyen the v9c0um theat sraesarch appeted t coti bevdus thatn thei faeces th ou dus d mi nbte Demthgois ffarie (kowsng ias t) on envrmtphagds teryssipndan (kon ase Deff-p)nc trigger the o immun lresos fcthe bodheeb givieenrset well knn dnteh allergenicsmtos frepe f thsige n Exerien dntat adpplied Oneaiwayrto oercosmeahese aaernic rspnse hadeen toc vauumcea sroufces, anasuch aspa care atat cnand ths dstra me and therfee thnoshly and fchite bt thateidoh.iecnsmn ithst beTregulredn to esureane alrenaicbfree ance aenatie mseto of creatingaanollrg en-c 10/06 2008 TUE 10:17 FAX +61 2 8231 1099 FBRice Co 12006/017 00 2 p results in a low collision rate between the allergen ;Z denaturant and the airborne-allergens. The practical C consequence of such a low collision rate is that the allergen denaturant must be used in a high amount in order to be effective. There may be other consequences such as, in the case where the aerosol spray composition includes a Sperfume or fragrance, a strong perfume smell or a limited j00 fragrance choice.

PCT/GB98/02863 describes a method for deactivating 0 10 allergens derived from the Der-f and/or Der-p dust mite C( species, which comprises contacting the allergen with a deactivating amount of one or more of a variety of 28 deactivants as described. The deactivants which are specified for use include cedarwood oil, hinoki oil and thymol (6-isopropyl-m-cresol).

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.

We have now discovered a group of novel allergen denaturants for the house dust mite Der-p allergen which are derived from natural oils and can be delivered as a vapour to deactivate the allergens.

Accordingly, the present invention provides a method of deactivating a Der-p and/or Der-f allergen which comprises volatilizing into a space to be treated a deactivating amount of a volatile oil selected from cajeput oil (tea tree oil) or an oil comprising one or more terpene hydrocarbons.

According to an embodiment of the present invention there is provided a method of deactivating a Der-p and/or COMS ID No: ARCS-193714 Received by IP Australia: Time 10:23 Date 2008-06-10 10/06 2008 TUE 10:18 FAX +61 2 8231 1099 FBRice Co 2007/017 00 3 SDer-f allergen which comprises delivery of a vapour and ;Z dispersal into the air in a room to be treated, a deactivating amount of a volatile oil which is selected from cajeput oil (tea tree oil) or an oil comprising one or more terpene hydrocarbons, the volatile oil being heated in order to deliver it as a vapour into the air.

C According to a further embodiment there is provided 00 0 use of a volatile oil selected from cajeput oil (tea tree C oil) or an oil comprising one or more terpene hydrocarbons in deactivating a Der-p allergen and/or Der-f allergen Cq comprising delivering as a vapour and dispersal into an air in a room to be treated against said allergens, said volatile oil by heat-assisted vaporization.

Suitable oils comprising one or more terpene hydrocarbons which may be used in the present invention are those which are generically referred to as pinol such as these sold under the names Unitene D® and Unitene LE® (Bush Boake Allen). The main component of both Unitene D and Unitene LE comprise limonene as its major constituent.

Unitene D contains significant quantities of cineole and terpinolene, whilst Unitene LE contains significant quantities of terpene alcohols.

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.

Cajeput oil, which is generally known as tea tree oil is obtained from the Melaleuca leucandra, Melaleuca quin quenervia or other Melaleuca species. The main components of tea tree oil are cineole and terpinene-4-ol.

There are various methods which can be used to volatilise the volatile oils into the air and these delivery methods are discussed below.

COMS ID No: ARCS-193714 Received by IP Australia: Time 10:23 Date 2008-06-10 10/06 2008 TUE 10!18 FAX +61 2 8231 1099 FBRice Co 0008/017 00 4 SThe volatile oil may be volatilised by the use of ;Z heat to vaporize the oil. For example the volatile oil may be floated on water in an oil burner or heated directly in an oil burner. Alternatively the volatile oil may be vaporized from a heated wick dipped into a reservoir of the volatile oil.

C The volatile oil may be used as such, or may be 00 j00 presented in the form of an emulsion. Generally, the CI emulsion will be an oil-in-water emulsion comprising up to

S

1 0 5% by weight of the oil. The formation of emulsions is generally well known in the art and is described, for example, in Modern Aspects of Emulsion Science, edited by Bernard P. Binks, The Royal Society of Chemistry, 1998 and Surfactant Science and COMS ID No: ARCS-193714 Received by IP Australia: Time 10:23 Date 2008-06-10 Technology, Second Edition, Drew Myers, 1992, VCH Publishers, Inc.

In a still further aspect of the present invention the volatile oil is incorporated into a candle which is burnt in the space to be treated. In carrying out this aspect the present invention the 0 candle which is burnt will generally comprise at least Cl 2% by weight of the volatile oil, preferably at least by weight of the volatile oil and more preferably Cl 10 at least 10% by weight of the volatile oil.

By the term "candle" as used herein is meant a solid, semi-solid ot gelled body of a combustible material which contains an axially embedded combustible fibrous wick. When the wick of a candle is lit, the heat so generated melts the combustible material and the resulting liquid flows up the wick by capillary action and is combusted.

Typically, the combustible body of the candle may be a blend of organic materials such as beeswax, paraffin wax, montan wax, carnauba wax, microcrystalline wax, fatty alcohols, fatty acids, fatty esters or natural and synthetic resins. Clear candles may comprise as the combustible material a gel comprising mineral oil containing blends of diblock and triblock copolymers based on synthetic thermoplastic rubbers or a gel obtained by combining a liquid base material of a hydrogenated polyolefin, a gelling agent and optionally a gel enhancing agent.

A wick normally extends longitudinally through the candle body. More than one wick may be used, if desired, but usually a single wick is centrally disposed in the candle body. When a candle wick is ignited, the wick is adapted to burn gradually so that both the wick and the candle body are consumed.

Typically, the weight of candle which is burnt in a particular space to be treated will depend upon the actual volume of the space, e.g. room, to be treated.

10/06 2008 TUE 10!19 FAX 461 2 8231 1099 FBRice Co 0009/017 00 S6 g An appropriate allergen denaturing effect can be c' obtained in accordance with the method of the invention by burning in a room of volume 25 to 30m 3 a candle of weight approximately 150g before testing containing 5% by weight of the volatile oil for a period of 5 hours. The amount of the volatile oil which is released from the burning OQ candle can be calculated by weighing the candle at 1 hour intervals.

nThe length of time for which the candle is burnt in the space to be treated will generally be for up to 2 CI hours, generally up to 5 hours, although in some circumstances the candle may be burnt for a longer period of time, such as 10 hours or more. However, it will be understood by those skilled in the art that an allergen denaturing effect will be obtained even if the candles containing the selected volatile oils are burnt for a lesser period of time.

The present invention will be further described with reference to the following Examples.

Control Pre-treatment Allergen Level When using house dust for allergen denaturing tests an inherent difficulty is the variability of the amount of allergen in each small sample, even when taken from the same dust reservoir. The amount of dust in the pretreatment sample must be accurately estimated in order to determine the extent of any COMS ID No: ARCS-193714 Received by IP Australia: Time 10:23 Date 2008-06-10 0~ 7 0 allergen denaturing. In these tests the dust sample o was applied to the test exposure surface and then one c half of this surface dust was removed to measure the control pre-treatment allergen level of that specific sample. Each control was directly relevant to each sample, which gave the best possible estimate of the 00 0j- level of allergen in the sample before exposure to possible denaturant.

o The following Examples all measure the reduction C< 10 of the house dust mite (Dermatophagoides pteronyssinus) allergen Der pi.

EXAMPLE 1 House dust was passed through a number of sieves and the fraction smaller than 53 micrometres was collected. 0.lg of dust was placed in a. small sieve to distribute it evenly over the test surface. The test surface was an aluminium tray 0.6m x Im. The dust was applied to the tray by moving the sieve continuously over the surface. One half of the dust was then removed by suction onto an in-line filter and the weight recorded, this was the pre-treatment control. The tray was then placed in a plastic lined booth 0.8m x 0.8m x 1.5m. An oil burner containing 8001 of the test sample floated on 6ml of distilled water was placed in the booth, and the booth was sealed. The oil burner candle was lit and allowed to burn until all the liquid had been vaporised (approx.

1 hour). The candle was then smothered and the dust was left exposed in the booth. After 24 hours the tray was removed, the dust was collected from it and its weight recorded. The booth was washed with strong detergent between tests on the same chemical; the booth lining was changed between test chemicals.

Test samples evaluated were: Hinoki Oil (comparative) q 8 o 0 Citronella Oil (comparative) o Tea Tree Oil c Pinol (Unitene

D)

Pinol (Unitene LE) 3 5 The test samples were assayed for Der pl using an 0 ELISA (Enzyme linked immunosorbent assay) to determine 00 the allergen content. This was then related to the Ic weight of dust that had been present in each sample.

o All of the samples were multiplied up to compare the ^C 10 amount of allergen expected to be present in a 0.lg sample of dust. The percentage difference between the control sample and the exposed sample was then obtained and is presented in Figure 1.

The difference in the amount of allergen reduction after exposure to any of the volatile oils released from the oil burner when compared to the inherent loss in sampling was significant when compared in a two-tailed t-test. Therefore, in conditions of the test, exposure to the above oils released from an oil burner resulted in a significant reduction in the allergen contained in the dust samples.

EXAMPLE 2 House dust was passed through a number of sieves and the fraction smaller than 53 micrometres was collected. O.lg of dust was placed in a small sieve to distribute it evenly over the test surface. The test surface was an aluminium tray 0.6m x Im. The dust was applied to the tray by moving the sieve continuously over the surface. One half of the dust was then removed by suction onto an in-line filter and the weight recorded, this was the pre-treatment. The tray was then placed in a plastic lined booth 0.8m x 0.8m x For control tests dust was distributed on the tray, the pre-treatment control collected and the dust o 9left in the booth for 24 hours. The tray was then removed, the dust was collected from the tray and M weighed. In subsequent tests 800ul of volatile oil was added to 150ml of distilled water in the nebuliser. The tests were then completed as in the control tests. The booth was washed with strong O0 detergent between tests. The samples evaluated were: C Tea Tree Oil o Pinol (Unitene D) o 10 Pinol (Unitene LE) The collected dust samples were assayed for Der pl using an ELISA to determine the allergen content.

This was then related to the weight of dust that had been present in each sample. All of the samples were multiplied up to compare the amount of allergen expected to be present in a 0.lg sample of dust. The percentage difference between the control sample and the exposed sample was then obtained and is presented in Figure 2.

The difference in the amount of allergen reduction after exposure .either tea tree oil or Unitene D released from the nebuliser when compared to the loss in sampling control was significant (P<0.05) when compared on a two tailed t-test. Therefore, in the conditions of the test, exposure to either tea tree oil or Unitene D released from a nebuliser resulted in a significant reduction in the allergen contained in the dust samples.

EXAMPLE 3 Dust was collected from vacuum cleaner bags and passed through a series of sieves down to 53 microns.

Clean petri dishes were labelled with the chemical to be tested and lined with filter paper. 0-.3g of dust was added to each dish and spread evenly over the 0 o- 10 C) filter paper. 0.lg of dust was then removed from the filter paper for a control sample. The remaining dust c was then redistributed evenly over the filter paper.

2 .4g 0.2g of test chemical was.sprayed onto the dust sample. The dust sample was left open to the air until the filter paper was dry. The dust was 00 collected into eppendorfs and the weight of dust C recovered was measured. Iml of 1% Bovine Serum o Albumin Phosphate Buffered Saline Tween (BSA-PBS- T) was added to the control samples. iml of 5% BSA- PBS-T was added to the test samples. The samples were left overnight in the fridge and then centrifuged for minutes at 13,000 rpm. The supernatant was pipetted into an eppendorf for assay by Der pi ELISA.

The test liquids were: Distilled water 2% Tea Tree Oil in distilled water (Plus 0.1% Tween) 2% Citronella Oil in distilled water (Plus 0.10% Tween) 1% Thymol in distilled water (Plus 0.8% Tween) 2% Hinoki Oil in distilled water (Plus 0.1% Tween) 2% Tannic Acid Replicates were completed for each test liquid.

The allergen content of the controls for each replicate was compared with the test sample allergen.

The percentage reduction in allergen between the control and the test was determined for each replicate. The average allergen reduction of all replicates is presented in Figure 3.

The water tests showed an average allergen reduction of 34.2%. The addition of Tea Tree Oil to the dust reduced the allergen by another 29.6%, the difference was significant when compared on a t test Thymol reduced the allergen by 23.6% more than the water alone tests, the difference was significant when compared on a t test The addition of tannic acid to the dust reduced the 11 allergen by an average of 99.15% in the tests.

When taking the reduction of allergen in the water samples into account, some of the test liquids still significantly reduced the allergen content in the dust samples. Tannic acid was used as a positive control as it is known to denature allergen, and its effect was recorded in the tests. Tea tree oil significantly reduced the allergen content in the dust samples.

I0 EXAMPLE 4 Method The tests were completed in 28m 3 test rooms with no windows and a door that was closed throughout the duration of the test. The rooms did not contain any furniture and had easily cleaned floors of nonreactive resin. Six test areas 0.7 x 0.7m were marked out on the floor of each room with tape. Each test area was divided into two halves. Test dust had been obtained from household vacuum cleaner bags. House dust was passed through a number of sieves and the fraction smaller than 53 microns was collected. 0.1g of dust was placed in a small sieve to distribute it evenly over the test surface. The dust was applied by moving the sieve continuously over the surface. Dust was removed from half of each of the 6 test areas by suction of 201/min through an in-line glass fibre filter (2.5cm diameter) and the weight recorded.

These were the pre-treatment controls. The selected test candles of approximately 150g before testing were lit and placed in the rooms for 5 hours. The candles were then smothered and the dust was left exposed in the rooms for 16 hours. The dust was then collected as for the controls and weighed.

The collected samples were assayed by Der pl ELISA to determine the allergen content. This was o 12then related to the weight of dust that had been present in each sample. All the samples were Cc multiplied up to compare the amount of allergen expected to be present in a O.lg sample of dust. The percentage differences between the control samples and the exposed samples were then obtained and presented 00 in Figure 4.

C During the 5 hour burn period approximately 27g o of each of the candles was burnt. For candles B and C o 10 detailed below this equated to a release rate of 270pl of essential oil per hour.

Tests completed were: Test Description A Unfragranced candle, room relative humidity (rh) B 5% W/W Tea Tree oil candle, room rh C 5% w/w Unitene LE candle, room rh M No Treatment, room rh The room rh recorded during the tests was between 50 and 57%.

Results It can be seen from Figure 4 that there is a significant reduction (P<0.05) Der pl allergen content of dust exposed to both the tea tree oil and Unitene LE candle as compared to the no treatment control 3.19 and 2.38 respectively).

Discussion The results indicate that a significant reduction in allergen can be achieved in a room environment by burning candles containing either tea tree oil or Unitene LE for 5 hours.

S-13 t EXAMPLE Method British (containing Der pl) or American.

J 5 (containing Der fl) house dust was passed through a number of sieves and the fraction smaller than 53 00 microns was collected. O.lg of dust of the selected origin was placed in a small sieve and distributed o evenly over the test surface. The test surface was an S 10 aluminium tray 0.6m x Im, which could be easily cleaned with strong detergent. The dust was applied to the tray by moving the sieve continuously over the surface. Half of the dust was then removed by suction of 20L/min through an in-line glass fibre filter (2.5cm diameter) and the weight recorded. This was the pre-treatment control. The tray was then placed in a plastic booth 1 x 0.7m x 0.7m.

The candle to be tested of approximately 150g weight was placed in the booth. The candle was lit and the booth door closed. After approximately 2 hours the temperature and humidity in the booth was measured; the candle was allowed to burn for a total of 5 hours. The candle was then smothered and the dust was left exposed in the booth for 17 hours.. The tray was then removed and the booth ventilated. The dust was vacuumed from the tray onto a filter and weighed.

Test candles evaluated were: Control candle Tea Tree Oil candle Pinol (Unitene LE) candle Six single exposure replicates were completed for each candle. The collected samples were assayed by Der pi or Der fl ELISA to determine the allergen 14 content. This'was then related to the weight of dust 0that had been present in each sample. All the samples were multiplied up to compare the amount of allergen expected to be present in a 0.lg sample of dust. The percentage difference between the control sample and the exposed sample was then obtained.

00 The results for Der pl are presented in Figure C and the results for Der fl are presented in Figure 6.

I1 The reduction of Der pl allergen concentration in 1 0 the dust was significant after exposure to either the tea tree oil or Unitene LE candles and the reduction in Oer fl allergen concentration in the dust was significant after exposure to the tea tree oil candle.

EXAMPLE 6 The general procedure of Example 5 was repeated but with three repeated exposures to a candle containing 5% tea tree oil burnt for five hours (i.e.

total 15 hours burn) as compared to a single exposure to a candle containing 5% tea tree oil burnt for hours or to a control candle. Six replicate experiments were completed.

The results are given in Figure 7. It will be noted that repeated exposure further reduces the Der pl allergen concentration of dust on a surface.

EXAMPLE 7 Experiments were completed using the same method as described in Example 5 except that dust samples were exposed in each booth at the same time. 0.025g of dust was distributed evenly over a 0.32m 2 aluminium tray. Half of this was then removed as a control sample as described in Example 5. The tray was placed in the booth. 5 other trays were prepared in this way c 15 o Sand placed in the booth. The 6 trays containing the o test dust samples were exposed in the booth to a ee n hour burn of the selected candle. The trays were left exposed in the booth for a further 17 hours, the test dust samples were then collected and assayed by the appropriate ELISA. Figure 8 show a comparison of the 00 Der pl allergen reduction after exposure to clear In .gel candles containing 0% (control) or 5% tea tree Soil.

C< 10 The reduction of allergen concentration in the dust was significant after exposure to the gel candle containing tea tree oil.

EXAMPLE 8 Experiments were completed using the same method as described in Example 4. However, instead of burning a candle, a nebuliser was used to deliver the volatile oils.

The ultra-sonic jet nebuliser used in Example 2 was used in these room tests. When the nebuliser was activated a jet of cold, ultra-fine mist was expelled from the top of the reservoir. Tests were completed with 5ml of either tea tree oil or Unitene D floated on top of 1.50 ml deionised water in the nebuliser.

The nebuliser was activated for 3 hours. It is not known exactly how much of the .volatile oil was released as some of the water/oil mixture remained in the nebuliser at the end of the test. Controls were completed with deionised water alone in the nebuliser.

The results are given in Figure 9.

There was a significant reduction of the allergen content of the dust after exposure to the tea tree oil or Unitene D.

0 -16 0EXAMPLE 9 rExperiments were completed as detailed in Example 1, but with American house dust. Test dust samples were exposed to oil burners in small booths containing h 8002l of tea tree oil floated on 6ml of distilled 00 m- water. These were compared dust lost in sampling.

ci Dust samples were collected after 24 hours and assayed by Der fl ELISA. The results are given in Figure ci 10 -There was a significant reduction of the allergen content of the dust after exposure to the tea tree oil.

EXAMPLE Experiments were completed using the same method as described in Example 4. However, instead of burning a candle oil burners were used to deliver the tea tree oil.

Two types of oil burners were used in the tests.

Small oil burners were used in the small booth tests (detailed in Example 4) and in one of the test room tests. The oil burners were ceramic with a small dish with a 15ml capacity to hold the water and volatile oil. A single tea candle was placed under the suspended dish to evaporate the water and tea tree oil." Large oil burners were used in the remaining tests completed in 28m 3 test rooms. These were also ceramic and had a 'large dish with a 35ml capacity and were wider in the base so that three tea candles could be placed under the dish to evaporate this larger amount of liquid more efficiently. The tea tree oil was always floated on water in the oil burners as this regulated the temperature and enabled a controlled release rate of the tea tree oil.

Two large oil burners were used in most of the room tests, as this was a much larger volume over o -17 o Swhich to deliver the water and tea tree oil. Two o large oil burners contained in total 65ml of deionised Cf water and where specified, 5ml of the tea tree oil.

This was not a direct translation of the small booth tests as it was found that this would have been unrealistic (336ml water and 44.8ml test chemical).

0- They were placed in the rooms and the candles burnt until all of the liquid had evaporated. Tests were completed with tea tree oil. Controls were conducted C< 10 with deionised water alone in the oil burners. To quantify any effect due to the candles, tests were conducted with 6 tea candles alone. One test was also completed with a small oil burner containing 6ml of water and 800 al of tea tree oil, so that a comparison could be made with the small booth tests.

The results are given in Figure 11 There was a significant reduction of the allergen content of the dust after exposure to the tea tree oil.

Claims (12)

1. A method of deactivating a Der-p and/or Der-f allergen which comprises delivery of a vapour and dispersal into the air in a room to be treated, a deactivating amount of a volatile oil which is selected from cajeput oil (tea tree oil) or an oil comprising one 00 or more terpene hydrocarbons, the volatile oil being C, heated in order to deliver it as a vapour into the air. 0 10

2. A method as claimed in claim 1 wherein the volatile oil is floated on water in an oil burner.

3. A method as claimed in claim 1 wherein the volatile oil is heated directly in an oil burner.

4. A method as claimed in claim 1 wherein the volatile oil is vaporized from a heated wick dipped into a reservoir of the volatile oil.

5. A method as claimed in any one of the preceding claims wherein the volatile oil is provided as a water-in- oil emulsion containing up to 5% by weight of the volatile oil.

6. A method as claimed in claim 1 wherein the volatile oil is incorporated into a candle which is burnt in the space to be treated.

7. A method as claimed in claim 6 wherein the candle which is burnt comprises at least 2% by weight of the volatile oil.

8. A method as claimed in claim 7 wherein the candle comprises at least 10% by weight of the volatile oil. COMS ID No: ARCS-194184 Received by IP Australia: Time 15:29 Date 2008-06-12 12/06 2008 THU 15:28 FAX +61 2 8231 1099 FBRice Co 2005/007 00 19

9. A method as claimed in any one of claims 6 to 8 wherein the candle is burnt for 2 hours or more.

A method as claimed in any one of the preceding claims wherein the oil comprising one or more terpene hydrocarbons is a pinol. 00

11. Use of a volatile oil selected from cajeput oil (tea O tree oil) or an oil comprising one or more terpene hydrocarbons in deactivating a Der-p allergen and/or Der-f allergen comprising delivering as a vapour and dispersal into an air in a room to be treated against said allergens, said volatile oil by heat-assisted vaporization.

12. A method of deactivating a Der-p and/or Der-f allergen substantially as hereinbefore described with reference to the examples, excluding comparative examples, COMS ID No: ARCS-194184 Received by IP Australia: Time 15:29 Date 2008-06-12