Patent Application
20050008579
It has been known for a long time that house dust can trigger allergenic reactions in humans, such as asthma and rhinitis. It was reported, as early as 1928, that it was the dust mites in the dust that were the primary source of the allergenic response but it was only in the 1960's that researchers appreciated its significance.
It is believed that the faeces of two particular house dust mite species, Dermatophagoides farinae (known as Der-f) and Dermatophagoides pteronyssinus (known as Der-p) trigger the immune responses of the body, thereby giving rise to well known allergenic symptoms.
A review of this is given in Experimental and Applied Acarology, 10 (1991) p. 167-186 in an article entitled “House dust-mite allergen”: A review by L. G. Arlian.
Both the Der-f and Der-p species are found throughout the world. In some areas, Der-f will be the sole Dermatophagoides species. In other areas Der-p will be the sole species. In still other areas, the two species are both present through, generally, one or the other will predominate.
One way to overcome these allergenic response has been to vacuum surfaces, such as carpets, that contain the dust mites and their faeces thoroughly and often, but that is both time consuming (i.e. has to be regularly done if one wants to make an allergenic free environment) and is very dependant on the efficiency of vacuum cleaner and filter bag used e.g. micron filter bag or 2-layer vacuum bags.
An alternative method of creating an allergen-free environment has been to denature the allergen, for example as described in U.S. Pat. No. 4,806,526. The only effective method however of which we are aware is to apply tannic acid to the allergen. However, tannic acid can cause staining, and this is a particularly acute problem for light coloured carpets (e.g. white and light beige carpets) and other textile surfaces as tannic acid leaves a deep brown stain.
Therefore, we have been looking for allergenic denaturants which will not stain susceptible surfaces such as carpets and still deactivate the allergen.
We have discovered a number of allergen deactivants which are effective against both the Der-f and the Der-p species. Quite surprisingly, we have discovered that some of these deactivants are specific to the type of dust mite allergen being treated. For example an effective Der-f allergen deactivants will not automatically work effectively as a Der-p allergen deactivant.
According to the invention there is provided a method for deactivating allergens derived from the Der-f and/or Der-p dust mite species, which comprises contacting the allergen with a deactivating effective amount of one or more of deactivants (herein after defined as the deactivant).
The deactivants effective against one or both of Der-f allergens and Der-p allergens are:
A compound of formula I is commercially available as Aerosol OT.
The compound of formula II is commercially available as parsley camphor.
Hinoki oil is a mixture of thujan-3-one, 2-pinene, 3,5,7,3′,4′-pentahydroflavanone and 1,3,3-trimethyl-2-norcamphanone.
Hydrogenated Hop Oil is the potassium salt of tetrahydroiso humulinic acid (also known as reduced isomerised hop extract).
Propylene glycol alginate is
Chlorohexadine is 1,1′-hexamnethylenebis [5-(4-chlorophenyl)]-biguanide.
Hinokitol is β-thujaplicin, a compound of the formula
Germall II is diazolidinylurea.
Thymol is 6-isopropyl-m-cresol.
Cedarwood oil contains α- and β-cedrene (ca 80%), cedrol (3-14%) and cedrenol. Other sesquiterpenes and some monoterpenes are also present.
Polyquaternium-10 is a polymeric quaternary ammonium salt of hydroxyethyl cellulose reacted with a trimethyl ammonium substituted epoxide commercially available as Polymer JR-125.
Silica gel is also known as colloidal silica or silicic acid and is commercially available as Kent.
“Immobilised tannic acid” is tannic acid on polyvinyl pyrrolidone beads. Immobilised Tannic Acid was prepared as follows: 100 mg of tannic acid was dissolved in water; 50 mg of Polyclar 10 (ISP, Guildford Surrey) polyvinyl pyrrolidone beads were added and stirred for one hour; the beads were filtered off the solution and washed with a few mls of iced water until no colour was seen in the washings; they were then dried in the oven at 50° C.
The composite of silver chloride and TiO2 is made up of 20% wt/wt AgCl on 80% TiO2 3-5 μm porous beads.
In compositions containing the deactivant, the deactivant is present in an amount of from 0.01% to 7%, preferably from 0.01% to 3%.
In methods for treating rugs and carpets to deactivate allergents, the amount of deactivant present is from about 16 gm to about 170 gm per 10 square meters, preferably about 32 gm per 10 square meters.
Preferably the deactivant is selected from
Further according to the invention there is provided an aerosol composition containing
Preferably the amount of deactivant present in such a composition is from 0.01% to 7%, more preferably 0.01% to 3%,
Preferably the amount of propellant present in such a composition is from 4% to 50%, more preferably from 4% to 30%,
Preferably the amount of solvent present in such a composition is 0% to 99.95%, more preferably 0% to 90%, and most preferably from 20% to 90%.
Preferably the deactivant in such aerosol composition is selected from
Preferably the propellant is selected from those commercially available, for example C1-4 alkanes, chlorofluorocarbons and compressed gases such as nitrogen, air and carbon dioxide.
Preferably the solvent is selected from C1-6 alcohols (e.g. ethanol) or water.
In addition, the compositions of this invention may also contain one or more of the following:
It has been found that deactivants of the invention have as effective allergen deactivating properties as tannic acid but without the drawback of staining.
The invention will now be illustrated by the following Examples.
The test procedure in Examples 1 to 55 is as follows and is known as the ELISA protocol.
The ELISA protocol for Der-f and Der-p has been developed as follows as a measure of denaturing property for denaturants.
ELISA Protocol 1
1. Dust is collected from Hoover™ vacuum cleaner bags and passed through a series of sieves down to 63 microns.
2. Clean petri dishes are labelled with the chemical to be tested (on the base). Three replicates are used for each treatment.
3. Filter paper is used to line each dish and 0.2 g of dust is added to each dish onto the filter paper. The lid (or base, as dishes are inverted) is replaced and the dish is shaken to disperse the dust evenly over the filter paper.
4. 2% aqueous solutions of deactivant were used except for the silver chloride composite where 0.05% was used instead. Immobilised tannic acid was used as a 1% dispersion. The hydrogenerated hop end was used at the 2% level (in the form of a 10% solution). Water-insoluble deactivants were emulsified with a sorbitone oleate surfactant (i.e. Tween). Hinokitol was used at 0.5% not 2%.
5. The dust is sprayed with the corresponding treatment, 2 sprays are required for sufficient coverage(1 spray=1.5 ml).
6. Leave uncovered at room temperature, in well aerated room, until filter paper is dry. This can take up to 4 hours.
7. Empty dust in epindorfs labelled according to treatment.
8. Add 1 ml of 5% Bovine Serum Albumen Phosphate Butter Saline—Tween BSA-PBS-T to each epindorf (5 times the weight of dust) (20 ml of BSA-PBS-T=1 g of BSA in 20 ml of PBS-T).
9. Leave overnight in a refrigerator.
10. Centrifuge for 5 minutes at 13,000 rpm.
11. Decant the supernatant into a new epindorf labelled according to treatment.
12. Centrifuge again for 5 minutes at 13,000 rpm.
13. Make up dilutions of 1:10 and 1:100 by adding 100 μl of neat solution to 900 μl of 1% BSA-PBS-T (1:10). This is repeated using 100 μl of 1:10 dilution and add to 900 μl of 1% BSA-PBS-T for 1:100 dilution.
ELISA Protocol 2 for Der-f and Der-p: Indoor Biotechnologies
1. Prepare samples and dilutions as in protocol
2. Prepare 500 ml of 50 mM carbonate/bicarbonate buffer by dissolving 0.795 g Na2CO3 and 1.465 g NaHCO3 in 500 ml of distilled water. Check the pH is at 9.6. (This solution is kept in the refrigerator in a conical flask).
3. Monoclonal antibody (kept in the freezer) has to be added to the buffer using the following method, (1 μg per well; 11 ml is needed) applied to the ELISA plate
(Stored in freezer, epindorf in use is in the refrigerator) is added to the buffer. To ensure that all the antibody is removed from the tip, wash out the pipette tip by sucking up and down I the buffer solution, gently stirring to mix thoroughly.
4. Pipette 100 μl of the antibody solution into each well of the microtiter plate, cover with a plate sealer and leave overnight at 4° C.
5. Empty the plate by quickly inverting it over the sink, then dry by banging on a stack of paper towels.
6. Add 200 μl of wash buffer to each well: PBS/0/05% tween (PBS-T).
7. Repeat stages 5 and 6 once more (making a total of 2 washes).
8. Make sure all the wells are dry, then add 100 μl of 1% BSA-PBS-T. Replace the plate sealer and incubate for 1 hour at room temperature*.
9. Repeat steps 5 to 7 (2 washes).
10. *During the hour incubation period, prepare the allergen standards at dilutions between 125 and 1 μg/ml Der f 1 or Der p1:
11. Add 100 μl aliquots of the allergen sample to the plate along with the standard allergen samples for the reference curve in duplicate. The standards usually go in the first two columns on the left hand side, with the least concentrated on top. Incubate for 1 hour.
12. Follow stages 5 to 6, completing a total of 5 washes.
13. Pour 11 ml of 1% BSA-PBS-T(0.11 g of BSA to 11 ml of PBS-T) to the dispensing tray. Add 11 μl of the biotinylated monoclonal antibody (refrigerator) and mix thoroughly.
14. Pipette 100 μl into each well and incubate for 1 hour at room temperature.
15. Empty plate and wash as described in stage 12. (5 washes).
16. Add 11 μl of Streptavidin (freezer) to 11 ml of 1% BSA-PBS-T. Pipette 100 μl into each well and incubate for 30 minutes. Reserve any remaining solution in a vial.
17. Empty plate and wash as described in stage 12 (5 washes).
18. Make a solution of OPD, by putting the two tablets (in silver and gold foil) into 20 ml of distilled water (in a glass vial). Shake quite vigorously in the dark until the tablets have dissolved (Wrap the vial up either in tin foil or paper towel).
19. Add a small amount to the remaining solution from stage 16. Wait for a colour change (positive reaction). Add 200 μl to each well and incubate for a minimum of 30 minutes in the dark.
20. Read the plate at 450 nm/405 nm if filter not available.
The deactivants, as set out in the following table, were used against Der-f allergens according to the above procedure and the results are as given below. Tannic acid was used as a comparator. What was measured after treatment with deactivant and tannic acid was the amount of allergen remaining active after treatment. The ratio of amount of remaining active allergen after treatment with deactivant and tannic acid is also given.
The deactivants, as set out in the following table, were used against Der-p allergens according to the above procedure and the results are as given below. What was measured were the amount of allergens remaining after treatment with deactivant and the amount of allergens remaining after vacuuming with no deactivant treatment.
Further samples were tested as above and compared against tannic acid. The ratio of actives remaining after deactivant treatment and actives remaining after tannic acid treatment are given below:
The following formulations can be made up as carrier compositions for use in an aerosol for deactivating Der-f and Der-p allergens.
*= May replace with 95% Ethanol (SD Alcohol 40) at 61.755% by weight and 9.970% by weight Deionized water
*= May replace 95% Ethanol (SD Alcohol 40) at 61.755% by weight and 5.370% by weight Deionized water.
| Number | Date | Country | Kind |
|---|---|---|---|
| 9720275.8 | Sep 1997 | GB | national |
| 9720298.0 | Sep 1997 | GB | national |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 09509308 | May 2000 | US |
| Child | 10911895 | Aug 2004 | US |
