# Patent

## Title
Processes for inhibiting development of allergic disease

## Abstract

A process for inhibiting development of allergic disease comprises exposing a neonatal or immature mammal or bird to irradiation-detoxified lipopolysaccharide derived from microbial, protozoan and/or fungal endotoxin.

## Background
FIELD OF THE INVENTION
The present invention is directed to processes for inhibiting development of allergic disease, particularly in mammals and birds. More specifically, the invention is directed to processes for inhibiting development of allergic disease by treatment of a neonatal or immature mammal or bird.
BACKGROUND OF THE INVENTION
A major health issue in the western world is the increase in allergic (atopic) diseases, e.g. asthma and allergies. It has been estimated that the incidence of asthma and allergies affects from about 15 to 30% of the population and continues to increase, with a major impact on health and productivity. Currently available remedies are typically primarily aimed at short-term treatment via antihistamines and topical nasal steroids. Preventive measures have generally not been very successful or well tolerated, e.g. allergy shots to common allergens. Accordingly, the need exists for additional preventive or inhibitive measures.
SUMMARY OF THE INVENTION
Accordingly, it is an object of the present invention to provide novel means for inhibiting allergy development. It is a further object to provide improved means for inhibiting allergy which overcome various disadvantages of prior art allergy treatments.
These and additional objects are provided by the present invention. In one embodiment, the invention is directed to a process for inhibiting development of allergic disease. The process comprises exposing a neonatal or immature mammal or bird to irradiation-detoxified lipopolysaccharide derived from microbial, protozoan and/or fungal endotoxin.
Additional embodiments of the invention are described in further detail below. The present inventors have discovered that safe application of lipopolysaccharide (LPS) into the natural environment for young children or animals, currently living in an “overly sterilized” environment, helps to restore the development of normal immune functions and inhibits the development of allergies later in life. Additional objects, embodiments and advantages of the invention will be more fully apparent and understood in view of the following detailed description.


DETAILED DESCRIPTION
The present invention is directed to processes for inhibiting development of allergic disease. The processes comprise exposing a neonatal or immature mammal or bird to irradiation-detoxified lipopolysaccharide derived from microbial, protozoan and/or fungal endotoxin. Within the context of the present specification, the term “inhibiting” encompasses both decreasing the development and, in certain embodiments, preventing the development, of allergy. Additionally, within the context of the present specification, the term “immature” refers to a mammal or bird which has not completed life cycle development to its adult stage. Further, within the context of the present specification, the term “allergic disease” encompasses allergic atopic disease, e.g., both allergies and asthma.
It has been recently reported that growing up on a farm versus the modern urban “sterile” environment protects against allergic sensitization as well as the development of childhood allergic disease. See, for example, Ernst, P. et al, Am J Resp Crit Care Med, 161: 1563-1566 (2000), Gereda, J. E. et al, JAMA, 284: 1652-1653 (2000), and Gehring, U. et al, Am J Respir Crit Care Med, 166:939-944 (2002). It appears that regular contact with farm animals confers a protective effect. The protective effect of this environment has been linked to increased concentrations of endotoxin from gram-negative bacteria, which seem to stimulate the immune system. See, for example, Braun-Fahrlander, C. et al, N Engl J Med, 347:869-877 (2002) and Martinez, F. D., et al, Lancet, 354 suppl 2: SII 12-15 (1999). A microbial burden has been suggested to be crucial during the first years of life for developing a non-atopic, protective immune response, Matricardi, P. M., et al, Allergy, 58: 461-471 (2003). The cell components from bacteria, which are primarily responsible for activating the immune response are the lipopolysaccharides (LPS). However, LPS have undesirable side effects such as pyrogenicity, hypotension, and the like which typically prevent or at least limit their use as an immune stimulant, Thorn, J., Inflamm. Res., 50: 254-261 (2001). Füst et al., Infect Immun., 16: 26-31 (1977) have demonstrated that irradiation of LPS decreases their toxicity. Bertók et al. found that detoxified LPS retain some of their beneficial properties in animal experiments, Immunopharmacology, 8: 13-17 (1984), and, furthermore, irradiated LPS are not toxic as tested in human volunteers, Orvosi Hetilap, 140: 819-827 (1999).
The present inventors have now determined that irradiated LPS, versus native LPS, have significantly less stimulatory effect on Il-1 production of the cells in peripheral heparinized blood, as set forth in Table 1.







TABLE 1







In vitro comparison of the interleukin 1 (IL-1) released by native


endotoxin (LPS) and irradiation-detoxified LPS (RDTX-LPS)


from heparinized human peripheral blood cells.











IL-1 (pg/ml) released



CELLS
(mean + SD)







Non-stimulated (control) cells
9.12 + 8.6



Cells stimulated with native LPS
172.26 + 26.7 



Cells stimulated with RDTX-LPS
98.22 + 19.4










(Average values of 3 experiments. Measurement of IL-1 by ELISA in the supernatants). It should be noted that the values of SD for native LPS stimulated cells compared to SD values for RDTX-LPS stimulated cells are different for p<0.001.

Accordingly, it is advantageous to inactivate the detrimental components of LPS derived from microbial (including bacterial), protozoan or fungal sources from a farm or other natural origin and then to apply the detoxified LPS fraction for stimulation of the Th-1 arm of the immune system. The safe application of LPS into the environment for young children or animals, for example, mammals or birds, currently living in an “overly sterilized” environment, will help to restore the development of normal immune function and prevent or decrease the development of allergies later in life.
In a more specific embodiment, the present processes provide a safe, irradiation detoxified LPS to young mammals in order to stimulate an immune response by stimulating the Th-1 arm of the immune system, thereby inhibiting, by preventing or minimizing, the development of allergic (atopic) disease such as common allergies and asthma. More specifically, the present process through irradiation inactivates the undesirable effects of the endotoxins from microbes, including bacteria, protozoan or fungi and while retaining their immune stimulatory antiallergenic properties. In one embodiment, the endotoxins are extracted from bacteria, protozoan or fungi. In a more specific embodiment, the endotoxins are extracted from bacterial, protozoan or fungal sources in a farming or other natural environment. In a further embodiment, the endotoxins are extracted from, for example, E. coli. 
Any suitable extraction method known in the art may be employed, an example of which is a solvent extract system, preferably with polarity adjustment to maximize yield. Such solvent extraction systems may employ a phenol-water system, other alcohol-water systems, an acetonitrile-water system, or the like. The extracted endotoxins may then be irradiated with sufficient radiation to detoxify the toxins therein, for example radiation in an amount of from about 25 to about 150 kGy. The irradiation changes the structure of the endotoxin, removing toxicity, while maintaining its positive immune effect. Typically, the endotoxins may be irradiated in an aqueous solution. The neonatal or immature mammal or bird is exposed to the resulting detoxified LPS, whether through environmental exposure or by direct contact with the animal. As detailed below, exposure can be effected by various means, inducing the long-lasting stimulation of the immune system without any dangerous side-effects.
In one embodiment, the subject is an infant human. In a further embodiment, the subject is a human of 1 month to 2 years of age. In an alternate embodiment, the subject is a primate of 2 weeks to 12 months of age. In yet further embodiments, the subject is a dog or cat of 1 week to 12 months of age. In additional embodiments, the subject is a farm animal, for example a cow, pig, goat, horse, chicken or turkey of 2 days to 12 months of age.
The exposure may be achieved by various means. In one embodiment, a topical composition comprising the irradiation-detoxified lipopolysaccharide is applied to the subject. Any topical composition vehicle may be employed. For example, in one embodiment, the topical composition further comprises a powder vehicle or carrier, examples of which include, but are not limited to talcum powder, corn starch, beet starch, rice flour, oatmeal, or a mixture thereof. In an alternate embodiment, the topical composition is in the form of a topical cream, lotion or gel. Examples of suitable cream, lotion or gel base components include, but are not limited to, water, short and long chain alcohols, acids, esters and oils, alkylene glycols, glycerols, glycerides, petroleum jelly and the like. The compositions may also include additional components typically employed in topical compositions, for example, vitamins, fragrances, herbal extracts, humectants and the like.
The exposure may be achieved by contacting the subject with a cloth or film impregnated with, for example, a suspension or dispersion of the irradiation-detoxified lipopolysaccharide. Any type of woven or nonwoven natural or synthetic cloth may be employed. Suitably, the irradiation-detoxified lipopolysaccharide may be formulated in a water or water/alcohol base for impregnation. More specifically, the subject can be contacted with a wipe impregnated with a composition comprising the irradiation-detoxified lipopolysaccharide. In a further embodiment, an infant can be provided with a diaper impregnated with a composition comprising the irradiation-detoxified lipopolysaccharide.
Alternatively, the exposure may be achieved by administering an aerosol spray composition comprising the irradiation-detoxified lipopolysaccharide to surrounding environment or directly to the subject. Such a spray may contain an aqueous, organic or mixed vehicle and may be sprayed from a pressurized or manual pump spray container, in accordance with spray techniques known in the art. The aerosol spray may then be provided in a living area to achieve the desired exposure.
The exposure level should be that sufficient to obtain the desired inhibition effects. In a specific embodiment, the irradiation-detoxified lipopolysaccharide is delivered in a concentration from 0.01 ug/g to 100 ug/g of delivery vehicle, for example, topical composition, impregnated cloth, or aerosol spray. In a further embodiment, exposure to the irradiation-detoxified lipopolysaccharide is achieved shortly after birth and during the maturing life cycle of the mammal or bird. In a more specific embodiment, exposure to the irradiation-detoxified lipopolysaccharide is achieved on a daily or weekly basis during growth of the mammal or bird.
The following examples illustrate non-limiting specific embodiments of the compositions suitable for use in the processes of the invention.
EXAMPLE 1
This example demonstrates one embodiment for preparation of irradiated LPS. Other methods will be apparent to one of ordinary skill in the art and are equally suitable for use in connection with the present inventive processes.
From a fermented culture of Escherichia coli 0101 H, LPS is extracted by a modified “hot-phenol-water” method according to Westphal et al, Z. Naturforsch., 76:148-155 (1952). The resulting powdered LPS is suspended in distilled water (10 mg/ml) and irradiated in the cabin of NORTOM equipment (with cooling) by 60Co gamma irradiation (150 kGy). The irradiated material is lyophilized immediately. The detoxification of LPS is measured using a biological test in animals for residual toxicity and for endotoxin tolerance inducing capacity. The irradiated LPS preparation can preserve its biological activity for about 10 years in the lyophilized form.
EXAMPLE 2
This example demonstrates a pressurized aerosol formulation:
















Sterile Water
100 ml



Irradiated LPS
 10 ug/ml










Butanol or Nitrogen (propellant) sufficient for aerosol delivery
EXAMPLE 3
This example demonstrates a manual pump aerosol formulation:
















Sterile Water
100 ml



Irradiated LPS
 15 ug/ml










EXAMPLE 4
This example demonstrates another manual pump aerosol formulation:
















5% isopropyl alcohol
100 ml



Irradiated LPS
 5 ug/ml










EXAMPLE 5
This example demonstrates a dry powder formulation:
















Talcum powder
100 grams



Dried irradiated LPS
 20 ug/gram talcum powder










EXAMPLE 6
This example demonstrates another dry powder formulation:
















Corn starch
100 grams



Dried irradiated LPS
 25 ug/gram corn starch










EXAMPLE 7
This example demonstrates another dry powder formulation:
















Beet starch and rice flour
100 grams



Dried irradiated LPS
 5 ug/gram beet starch










EXAMPLE 8
This example demonstrates another dry powder formulation, particularly advantageous for application to a subject via bath water:
















Oatmeal
100 grams



Dried irradiated LPS
 5 ug/gram oatmeal










EXAMPLE 9
A cloth such as a diaper is provided with irradiated LPS applied uniformly as powder in an amount of about 0.01 ug. In another embodiment, a diaper is provided with irradiated LPS applied uniformly as a liquid in an aqueous carrier in an amount of about 0.01 ug.
In a further embodiment, a single wipe cloth is impregnated with irradiated LPS in an amount of about 0.01 ug/wipe.
EXAMPLE 10
This example demonstrates a topical cream or lotion formulation:
A base mixture of purified water, canola oil, stearic acid, glycerol stearate, propylene glycol, glycerin, cetyl alcohol, lecithin, aloe vera gel, squalane (vegetable), avocado oil, vitamins A, D and E, D-panthenol, allantoin, chamomile extract, herbal fragrance, propylparaben, methylparaben, and triethanolamine, is combined with irradiated LPS in an amount of about 10 ug irradiated LPS per 50 ml of the base mixture.
EXAMPLE 11
This example demonstrates another topical cream or lotion formulation:
















Petroleum jelly
 100 grams



Irradiated LPS
 0.1 ug/gram petroleum jelly










EXAMPLE 12
This example demonstrates another topical cream or lotion formulation:
Base mixture of sweet almond oil, shea butter and vitamin E
















Irradiated LPS
2.5 ug per gram base mixture










The specific illustrations and embodiments described herein are exemplary only in nature and are not intended to be limiting of the invention defined by the claims. Further embodiments and examples will be apparent to one of ordinary skill in the art in view of this specification and are within the scope of the claimed invention.

## Claims

1. A process for decreasing development of allergic asthma, the process comprising exposing a neonatal or immature mammal to irradiation-detoxified lipopolysaccharide (IR-LPS) derived from extracted bacterial endotoxin and operable to stimulate the Th 1 arm of the mammal's immune system, wherein exposure comprises at least weekly administration during maturation of the mammal via application of the IR-LPS to a living environment of the mammal.

2. A process according to claim 1, wherein the irradiation-detoxified lipopolysaccharide is detoxified by exposure of the endotoxin to irradiation at a level of from about 25 to about 150 kGy.

3. A process according to claim 1, wherein the irradiation changes the structure of the endotoxin while maintaining its Th 1 stimulatory effect in the resulting irradiation-detoxified lipopolysaccharide.

4. A process according to claim 1, wherein an infant mammal is exposed.

5. A process according to claim 4, wherein the exposure is achieved by administering a topical composition comprising the irradiation-detoxified lipopolysaccharide to the infant mammal.

6. A process according to claim 5, wherein the topical composition further comprises a powder.

7. A process according to claim 6, wherein the powder comprises talcum powder, corn starch, beet starch, rice flour, oatmeal, or a mixture thereof.

8. A process according to claim 5, wherein the topical composition is in the form of a topical cream.

9. A process according to claim 1, wherein application is achieved by administering an aerosol spray composition comprising the irradiation-detoxified lipopolysaccharide.

10. A process according to claim 4, wherein the exposure is achieved by contacting the infant mammal with a wipe impregnated with a composition comprising the irradiation-detoxified lipopolysaccharide.

11. A process according to claim 4, wherein the exposure is achieved by contacting the infant mammal with a diaper impregnated with a composition comprising the irradiation-detoxified lipopolysaccharide.

12. A process according to claim 1, wherein the mammal is a human and during maturation is between 1 month and 2 years of age.

13. A process according to claim 1, wherein a primate of 2 weeks to 12 months of age is exposed.

14. A process according to claim 1, wherein a dog or cat of 1 week to 12 months of age is exposed.

15. A process according to claim 5, wherein the irradiation-detoxified lipopolysaccharide is delivered in a concentration from 0.01 ug/g to 100 ug/g of topical composition.

16. A process according to claim 1, wherein exposure to the irradiation-detoxified lipopolysaccharide is initiated shortly after birth and “during maturation” is throughout the maturing life cycle of the mammal.

17. A process according to claim 1, wherein administration is on a daily basis.

18. A process according to claim 1, wherein the mammal is a farm animal.

19. A process according to claim 18, wherein the farm animal is a cow, pig, goat, horse, chicken or turkey of 2 days to 12 months of age.