JP5688370B2 - Compositions and methods for treating thymic stromal lymphogenic factor (TSLP) mediated conditions - Google Patents

Description

The present invention relates generally to thymic stromal lymphogenic factor (TSLP) and TSLP-mediated conditions, and more specifically to TSLP and TSLP receptor-mediated conditions (eg, immune system diseases, allergic inflammation, allergic Airway inflammation, DC-mediated inflammation Th2 response, atopic dermatitis, atopic eczema, asthma, obstructive airway disease, chronic obstructive pulmonary disease, and food allergies, inflammatory arthritis, rheumatoid arthritis, psoriasis, IgE-mediated disease, and rhinitis Conjunctivitis). Particularly preferred embodiments include at least one electrokinetically generated fluid disclosed herein, including gas enriched (eg, oxygen enriched) electrokinetically generated fluids, It relates to the modulation (eg, treatment) of TSLP and TSLP receptor mediated conditions by administering a therapeutic composition.
This application is related to U.S. Provisional Application Nos. 61 / 107,480 and 61 / 107,453, both filed October 22, 2008, and filed October 24, 2008. No. 12 / 258,210, which claims the benefit of priority, both of which are hereby incorporated by reference in their entirety.

  Thymic stromal lymphocyte neogenesis factor (TSLP). Thymic stromal lymphogenic factor (TSLP) is an IL-7-like cytokine that causes a dendritic cell-mediated Th2-type inflammatory response and is considered a master switch for allergic inflammation. TSLP is an integrated growth factor into the development and maturation of both B and T cells. In particular, mouse TSLP supports B lymphocyte production and is required for B cell proliferation. Murine TSLP plays an important role in controlling rearrangement of the T cell receptor gamma (TCRγ) locus and has a substantial stimulatory effect on thymocytes and mature T cells. For example, see Non-Patent Document 1, Non-Patent Document 2, and Non-Patent Document 3.

  TSLP has the same cytokine activity as IL-7. For example, TSLP can replace IL-7 in stimulating B cell proliferative responses (see Non-Patent Document 1, above). TSLP and IL-7 mediate similar effects on target cells, but they appear to have different transmission pathways and their biological responses may be different. For example, TSLP modulates the activity of STAT5 but does not activate any Janus family tyrosine kinase members (Non-Patent Document 4).

  Effect of TSLP on dendritic cells and TNF production. After human TSLP and human TSLP receptor were cloned in 2001, it was found that human TSLP strongly activated immature CD11c + myeloid dendritic cells (see, for example, Non-Patent Document 5 and Non-Patent Document 6). See Th2 cells are generally defined in immunology textbooks and literature as CD4 + T cells that produce IL-4, IL-5, IL-13, and IL-10. In addition, Th1 cells such as CD4 + T cells produce IFN-γ and sometimes TNF. When TSLP-DC is used to stimulate naïve allogeneic CD4 + T cells in vitro, a unique type of Th2 cell is produced, which is the classic Th2 cytokine IL-4, IL-5, and IL-13, As well as producing large amounts of TNF but little or no IL-10 or interferon-γ (see Non-Patent Document 5, supra) (see also, for example, Non-Patent Document 6). TNF is typically not considered a Th2 cytokine. However, TNF is prominent in the asthmatic airways, and genotypes that correlate with increased TNF secretion are associated with asthma risk. See Non-Patent Document 7 and Non-Patent Document 8.

  TSLP induces human mDCs to express the TNF superfamily protein OX40L at both mRNA and protein levels (9). Expression of OX40L by TSLP-DC is important for assimilation of inflammatory Th2 cells. Thus, TSLP-activated DCs create a Th2-permissive microenvironment by up-regulating OX40L without inducing the production of Th1-polarizing cytokines. Same as above.

  TSLP expression, allergen specific response, and asthma. Early studies showed that TSLP mRNA was highly expressed by human primary skin keratinocytes, bronchial epithelial cells, smooth muscle cells, and lung fibroblasts s. Since TSLP is mainly expressed in keratinocytes in the apical layer of the epidermis, this suggests that TSLP production is characteristic of fully differentiated keratinocytes. TSLP expression in patients with atopic dermatitis is associated with in situ Langerhans cell migration and activation, which activates these cells that can subsequently migrate to draining lymph nodes , And suggest that it may contribute directly to major allergen specific reactions. Same as above. In a more recent study, in situ hybridization increased TSLP expression in the asthmatic airways, correlating with both Th2-induced chemokine expression and disease severity, providing an association between TSLP and asthma (Non-patent Document 10).

  TSLP receptor (TSLPR) and allergic asthma. The TSLP receptor (TSLPR) is a protein of about 50 kDa and has significant similarities to the common gamma chain. TSLPR is a novel type 1 cytokine receptor and, when combined with IL-7Ra (CD127), constitutes a TSLP receptor complex as described in Non-Patent Document 11, for example. TSLPR has a tyrosine residue near its carboxyl terminus, which, when associated with TSLP, associates with phosphorylated STAT5 and can mediate multiple biological functions (12). .

Human TSLPR is expressed by monocytes and CD11c + dendritic cells and TSLP binding induces the expression of T _H 2 cell attracting chemokines CCL17 and CCL22. Further, as described above, TSLPR-induced activation of dendritic cells may be necessary for the regulation of CD4 + T cell homeostasis, _{T H} 2 cytokines IL-4, -5, and indirectly the increased secretion -13 Bring. In mice, lack of TSLPR does not affect lymphocyte count. However, lack of TSLPR and common γ chain results in a lower lymphocyte count compared to mice lacking only the common γ chain. See Non-Patent Document 5 and Non-Patent Document 6.

  Research has found that TSLP and TSLPR play an important role in the initiation of murine allergic disease. In one study, mice engineered to overexpress TSLP in the skin have atopic skin characterized by eczema-like skin lesions including inflammatory infiltrates, a dramatic increase in circulating Th2 cells, and elevated serum IgE. It has been demonstrated to develop a flame (Non-patent Document 13). Studies have suggested that TSLP can directly activate DCs in mice. In another study conducted by Li et al., The group found that a transgenic mouse that overexpresses TSLP in the skin strengthens the association between TSLP and the development of atopic dermatitis. It was confirmed that dermatitis developed.

  Another series of studies demonstrated that TSLP is required for the onset of murine allergic airway inflammation in vivo. In one study, Zhou et al. Featured lung-specific expression of TSLP transgenes characterized by massive leukocyte infiltration (including Th2 cells), goblet cell hyperplasia, and subepithelial fibrosis, and increased serum IgE levels. It has been demonstrated to induce allergic airway inflammation (asthma) (Non-patent Document 14). However, in contrast, mice lacking TSLPR did not develop asthma that responded to the inhaled antigen (Non-Patent Document 14, see above, and Non-Patent Document 15). Together, these studies demonstrate that TSLP is required for the initiation of murine allergic airway inflammation.

  In addition, studies conducted by Yong-Jun et al. Demonstrated that epithelial cell-derived TSLP elicited human DC-mediated inflammatory Th2 responses, indicating that TSLP is allergic at the epithelial cell-DC interface. This suggests that it corresponds to a master switch of sexual inflammation (Non-patent Document 16).

  Recent studies have shown that modulation of DC function by inhibiting TSLPR reduces the severity of mice (Non-patent Document 17). In another series of studies, it was demonstrated that TSLPR is not only expressed in DC but also on macrophages, mast cells, and CD4 + T cells (Non-patent Document 18 and Non-patent Document 19). To rule out the direct effect of TSLPR neutralization on CD4 + T cells or other effector cells in allergic inflammation, Liyun Shi et al. Treated OVA-loaded DCs with anti-TSLPR in vitro before adoptive transfer into the airways of naive mice. Experiments were carried out using these. It has already been found that OVA-DC induces strong eosinophilic airway inflammation and is accompanied by mass production of Th2 cytokines such as IL-4 and IL-5 (Non-patent Document 20 and Non-patent Document 21). . However, pretreatment of OVA-DC with anti-TSLPR resulted in eosinophil and lymphocyte infiltration, and a significant reduction in IL-4 and IL-5 levels, in allergic diseases where TSLPR was sensitized by DC. The role played is further clarified. This result also confirms that blocking TSLPR on DCs helps control airway inflammation (see Non-Patent Document 17, supra).

  An increasing number of experiments consider that the role of TSLP / TSLPR is implicated in various physiological and pathological processes. The physiological role of TSLP includes the regulation of the immune system, in particular in stimulating B and T cell proliferation, development, and maturation. TSLP plays an important role in the pathobiology of allergic asthma and local antibody-mediated blockade of TSLP receptor function to alleviate allergic diseases. Thus, the interaction between TSLP and the TSLP receptor is important in many physiological disease processes such as skin lesions, allergic asthma, and asthma in patients with allergic inflammation, atopic dermatitis or atopic eczema. It is believed that there is.

Friend et al. , Exp. Hematol, 22: 321-328, 1994. Ray et al. , Eur. J. et al. Immunol. , 26: 10-16, 1996 Candias et al. , Immunology Letters, 57: 9-14, 1997. Levin et. al. , J .; Immunol. 162: 677-683, 1999. Reche et al. , J .; Immunol. 167: 336-343, 2001 Soumelis et al. Nat. Immunol. 3: 673-680, 2002. Shah et al. , Clin. Exp. Allergy. , 25: 1038-1044, 1995 Moffatt, M.M. F. and Cookson, W.M. O. , Hum. MoI. Genet. , 6: 551-554, 1997 Ito et al. , J .; Exp. Med. , 202: 1213-1223 Ying et al. , J .; Immunol. , 174: 8183-8190, 2005. Pandey et al. Nat. Immunol. 1: 59-64, 2000 Isaksen et al. , J .; Immunol. 168: 3288-3294, 2002 Yoo et al. , J .; Exp. Med. 202: 541-549, 2005. Zhou et al. Nat. Immunol. , 6: 1047-1053, 2005. AAl-Shami et al. , J .; Exp. Med. 202: 829-839, 2005. Yong-Jun et al. , J .; Exp. Med. , 203: 269-273, 2006. Liyun Shi et al. , Clin. Immunol. 129: 202-210, 2008. Rochman et al. , J .; Immunol. 178: 6720-6724, 2007. Omori M.M. and Ziegler S. , J .; Immunol. 178: 1396-1404, 2007 Sung et al. , J .; Immunol. 166: 1261-1271 Lambrecht et al. , J .; Clin. Invest. 106: 551-559,2000.

  Certain embodiments substantially have an average diameter of less than about 100 nanometers and are stably configured in an ionic aqueous fluid in an amount sufficient to treat a TSLP-mediated or TSLPR-mediated disease or condition. TSLP-mediated or TSLPR-mediated disease comprising administration of a therapeutically effective amount of an electrokinetically modified aqueous fluid comprising a charge-stabilized oxygen-containing nanostructured ionic aqueous solution to a mammal in need thereof Or provide a method for treating a condition. In certain embodiments, the charge-stabilized oxygen-containing nanostructure is ionic aqueous in an amount sufficient to provide modulation of at least one of cell membrane potential and cell membrane conductivity when contacted by a fluid with living cells. Stable in the fluid.

  In certain method aspects, the charge-stabilized oxygen-containing nanostructure is the primary charge-stabilized gas-containing nanostructure species in the fluid. In certain embodiments, as a charge-stabilized oxygen-containing nanostructure, the percentage of dissolved oxygen molecules present in the fluid is 0.01%, 0.1%, 1%, 5%, 10%, 15 %, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, and 95% The ratio is selected from the group consisting of super. In certain embodiments, all dissolved oxygen is substantially present in the charge-stabilized oxygen-containing nanostructure. In certain embodiments, the charge-stabilized oxygen-containing nanostructure has an average diameter smaller than a size selected from the group consisting of less than 90 nm, 80 nm, 70 nm, 60 nm, 50 nm, 40 nm, 30 nm, 20 nm, 10 nm, and 5 nm. Substantially.

  In certain method aspects, the aqueous ionic solution comprises a saline solution. In certain embodiments, the electrokinetically modified aqueous fluid is superoxygenated.

  In certain method aspects, the electrokinetically modified aqueous fluid comprises a solvated electron form.

  In certain aspects, the electrokinetically modified aqueous fluid modification comprises fluid exposure to a hydrodynamically induced localized electrokinetic effect. In certain embodiments, exposure to localized electrokinetic effects includes exposure to at least one of a voltage pulse and a current pulse. In certain aspects, exposure of a fluid to a hydrodynamically induced localized electrokinetic effect causes the fluid to undergo structural properties that induce the electrokinetic effect of the device used to generate the fluid. Includes exposure.

  In certain aspects, a TSLP-mediated or TSLPR-mediated disease or condition includes a disease or disorder of the immune system, including but not limited to allergic inflammation. In certain embodiments, the allergic inflammation is allergic airway inflammation, DC-mediated inflammation Th2 response, atopic dermatitis, atopic eczema, asthma, obstructive airway disease, chronic obstructive pulmonary disease, IgE-mediated disease, rhinitis conjunctivitis, And at least one of food allergies. In certain embodiments, the TSLP-mediated or TSLPR-mediated disease or condition comprises inflammatory arthritis, including, for example, at least one of rheumatoid arthritis and psoriasis.

In certain embodiments, the method further comprises a combination therapy, and at least one additional therapeutic agent is administered to the patient. In certain embodiments, the at least one additional therapeutic agent is a short-acting β ₂ agonist, a long-acting β ₂ agonist, an anticholinergic agent, a corticosteroid, a systemic corticosteroid, a mast cell stabilizer. , A leukotriene modifier, methylxanthine, and combinations thereof. In some embodiments, the at least one additional therapeutic agent is an albuterol, levalbuterol, pyrbuterol, alformoterol, formoterol, salmeterol, and beta ₂ agonists, including anticholinergics such as ipratropium and tiotropium; beclomethasone, budesonide, flunisolide Corticosteroids, including fluticasone, mometasone, triamcinolone, methiprednisolone, prednisolone, prednisone; leukotriene modifiers, including montelukast, zafirlukast, and zileuton; mast cell stabilizers, including cromolyn and nedocromil; theophylline, ipratropium, Including concomitant medications including fluticasone and salmeterol, budesonide and formoterol, Tilxanthine; consisting of hydroxyzine, diphenhydramine, loratadine, cetirizine, and hydrocortisone; antihistamines; immune system modifiers including tacrolimus and pimecrolimus; cyclosporine; azathioprine; mycophenolate mofetil; Selected from the group. In certain aspects, the at least one additional therapeutic agent is a TSLP and / or TSLPR antagonist, and in certain embodiments, the TSLP and / or TSLPR antagonist comprises two or more receptor chain components. A TSLPR immunoglobulin Fc molecule or polypeptide that encodes is selected from the group consisting of neutralizing antibodies specific for TSLP and TSLP receptors, soluble TSLP receptor molecules, and TSLP receptor fusion proteins.

In certain method aspects, modulation of at least one of cell membrane potential and cell membrane conductivity alters at least one of a membrane bound protein or component conformation, ligand binding activity, or catalytic activity. Modifying at least one of the cell membrane structure or function. In some embodiments, the membrane-bound protein comprises at least one selected from the group consisting of receptors, transmembrane receptors, ion channel proteins, intracellular adhesion proteins, cell adhesion proteins, integrins and the like. In certain embodiments, the transmembrane receptor comprises a G protein coupled receptor (GPCR). In certain aspects, the G protein coupled receptor (GPCR) interacts with, for example, the G protein a subunit, the G protein a subunit comprising the group consisting of Ga _s, Ga _i, Ga _q , and Ga _12. In certain embodiments, at least one G protein a subunit is Ga _q .

  In certain method aspects, modulation of at least one of cell membrane potential and cell membrane conductivity includes, for example, modulating total cell conductivity, wherein modulating total cell conductivity is a measure of total cell conductivity. Adjusting at least one of the linear or non-linear voltage-dependent contributions.

  In certain method aspects, the modulation of at least one of cell membrane potential and cell membrane conductivity comprises modulation of a calcium-dependent cell transmission pathway or system. In certain method aspects, the modulation of at least one of cell membrane potential and cell membrane conductivity comprises modulation of phospholipase C activity. In certain method aspects, the modulation of at least one of cell membrane potential and cell membrane conductivity comprises modulation of adenylate cyclase (AC) activity. In certain method aspects, modulation of at least one of cell membrane potential and cell membrane conductance is a disease or disorder of the immune system, allergic inflammation, allergic airway inflammation, DC mediated Th2 response, atopic dermatitis, atopy At least one condition or symptom selected from the group consisting of eczema, asthma, obstructive airway disease, chronic obstructive pulmonary disease, IgE-mediated disease, rhinitis conjunctivitis, food allergy, inflammatory arthritis, rheumatoid arthritis, and psoriasis Related modulation of intracellular signal transduction.

  Certain method aspects include administration of electrokinetic current bodies to a cell network or layer, and further include modulation of intercellular binding therein. In certain embodiments, the intercellular junction comprises at least one selected from the group consisting of tight junctions, gap junctions, adhesion bands, and desmosomes. In certain embodiments, the cell network or layer comprises at least one selected from the group consisting of lung epithelium, bronchial epithelium, and intestinal epithelium.

  In certain method aspects, the electrokinetically modified aqueous fluid is oxygenated and the oxygen in the fluid is at least 8 ppm, at least 15 ppm, at least 25 ppm, at least 30 ppm, at least 40 ppm, at least at atmospheric pressure. Present in an amount of oxygen of 50 ppm, or at least 60 ppm.

  In certain method aspects, the electrokinetically modified aqueous fluid comprises at least one of, for example, solvated electrons, and electrokinetically modified or charged oxygen species; The form of solvated electrons or electrokinetically modified or charged oxygen species is at least 0.01 ppm, at least 0.1 ppm, at least 0.5 ppm, at least 1 ppm, at least 3 ppm, at least 5 ppm, at least 7 ppm. , At least 10 ppm, at least 15 ppm, or at least 20 ppm. In certain embodiments, the electrokinetically modified aqueous fluid comprises a solvated electron form stabilized by molecular oxygen.

  In certain embodiments, the electrokinetically modified fluid's ability to modulate at least one of cell membrane potential and cell membrane conductivity is at least 3 months for at least 2 months in a sealed airtight container. It lasts for a period of at least 4 months, at least 5 months, at least 6 months, at least 12 months, or longer.

  In certain embodiments, the amount of oxygen present in the electrostatically modified fluid charge-stabilized oxygen-containing nanostructure is at least 8 ppm, at least 15 ppm, at least 20 ppm, at least 25 ppm, at least 30 ppm at atmospheric pressure. , At least 40 ppm, at least 50 ppm, or at least 60 ppm oxygen.

In certain embodiments, treatment includes administration by at least one of topical, inhalation, intranasal, and intravenous.
In a preferred embodiment of the present invention, for example, the following is provided:
(Item 1)
Charge-stabilized oxygen substantially having an average diameter of less than about 100 nanometers and stably constructed in an ionic aqueous fluid in an amount sufficient to treat a TSLP-mediated or TSLPR-mediated disease or condition A therapeutically effective amount of an electrokinetically modified aqueous fluid, including administration of an electrokinetically modified aqueous fluid containing a nanostructured ionic aqueous solution, to a mammal in need thereof A method for treating a TSLP-mediated or TSLPR-mediated disease or condition comprising administration.
(Item 2)
The charge-stabilized oxygen-containing nanostructure is in an amount sufficient to provide modulation of at least one of cell membrane potential and cell membrane conductivity when in contact with living cells by the fluid. The method of item 1, wherein the method is stably configured therein.
(Item 3)
2. The method of item 1, wherein the charge-stabilized oxygen-containing nanostructure is a major charge-stabilized gas-containing nanostructure species in the fluid.
(Item 4)
As the charge-stabilized oxygen-containing nanostructure, the percentage of dissolved oxygen molecules present in the fluid is 0.01%, 0.1%, 1%, 5%, 10%, 15%, 20% , 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, and more than 95% The method according to item 1, wherein the ratio is a ratio selected from:
(Item 5)
Item 2. The method of item 1, wherein all dissolved oxygen is substantially present in the charge-stabilized oxygen-containing nanostructure.
(Item 6)
The charge stabilized oxygen-containing nanostructure has an average diameter substantially smaller than a size selected from the group consisting of less than 90 nm, 80 nm, 70 nm, 60 nm, 50 nm, 40 nm, 30 nm, 20 nm, 10 nm, and 5 nm. The method according to Item 1.
(Item 7)
The method of item 1, wherein the aqueous ionic solution comprises a saline solution.
(Item 8)
Item 2. The method of item 1, wherein the fluid is hyperoxygenation.
(Item 9)
Item 2. The method of item 1, wherein the fluid comprises a form of solvated electrons.
(Item 10)
The method of claim 1, wherein the electrokinetically modified aqueous fluid modification comprises hydrodynamically induced exposure of the fluid to a localized electrokinetic effect.
(Item 11)
12. The method of item 10, wherein the exposure to the localized electrokinetic effect comprises exposure to at least one of a voltage pulse and a current pulse.
(Item 12)
Exposing the fluid to a hydrodynamically induced localized electrokinetic effect reduces the exposure of the fluid to structural properties that induce the electrokinetic effect of the device used to generate the fluid. The method according to item 10, comprising.
(Item 13)
13. The method of any one of items 1-12, wherein the TSLP-mediated or TSLPR-mediated disease or condition comprises an immune system disease or disorder.
(Item 14)
14. The method of item 13, wherein the immune system disease or disorder comprises allergic inflammation.
(Item 15)
The allergic inflammation includes allergic airway inflammation, DC-mediated inflammation Th2 response, atopic dermatitis, atopic eczema, asthma, obstructive airway disease, chronic obstructive pulmonary disease, IgE-mediated disease, rhinitis conjunctivitis, and food allergy 15. A method according to item 14, comprising at least one of them.
(Item 16)
13. The method of any one of items 1-12, wherein the TSLP-mediated or TSLPR-mediated disease or condition comprises inflammatory arthritis.
(Item 17)
The method according to item 16, wherein the inflammatory arthritis comprises at least one of rheumatoid arthritis and psoriasis.
(Item 18)
13. The method of any one of items 1-12, comprising combination therapy, wherein at least one additional therapeutic agent is administered to the patient.
(Item 19)
The at least one additional therapeutic agent includes a short-acting β ₂ agonist, a long-acting β ₂ agonist, an anticholinergic agent, a corticosteroid, a systemic corticosteroid, a mast cell stabilizer, a leukotriene modifying agent, 19. A method according to item 18, selected from the group consisting of methylxanthine, and combinations thereof.
(Item 20)
Said at least one additional therapeutic agent comprises a beta ₂ agonist comprising albuterol, levalbuterol, pyrbuterol, alformoterol, formoterol, salmeterol, and anticholinergics such as ipratropium and tiotropium ; beclomethasone, budesonide, flunisolide, fluticasone, Corticosteroids, including mometasone, triamcinolone, methiprednisolone, prednisolone, prednisone; leukotriene modifiers, including montelukast, zafirlukast, and zileuton; mast cell stabilizers, including cromolyn and nedocromil; , Methylxanthone, including complex drugs including budesonide and formoterol Antihistamines, including hydroxyzine, diphenhydramine, loratadine, cetirizine, and hydrocortisone; immune system modifiers, including tacrolimus and pimecrolimus; cyclosporine; azathioprine; mycophenolate mofetil; and a bronchodilator consisting of combinations thereof The method according to item 18, selected from:
(Item 21)
19. The method of item 18, wherein the at least one additional therapeutic agent is a TSLP and / or a TSLPR antagonist.
(Item 22)
The TSLP and / or TSLPR antagonist comprises a TSLPR immunoglobulin Fc molecule or polypeptide that encodes a component of two or more receptor chains, a neutralizing antibody specific for TSLP and the TSLP receptor, soluble 22. The method of item 21, wherein the method is selected from the group consisting of a TSLP receptor molecule and a TSLP receptor fusion protein.
(Item 23)
Modulation of at least one of cell membrane potential and cell membrane conductivity comprises altering at least one of a membrane bound protein or component conformation, ligand binding activity, and catalytic activity, a cell membrane structure or function 3. A method according to item 2, comprising modifying at least one of the above.
(Item 24)
24. The method of item 23, wherein the membrane-bound protein comprises at least one selected from the group consisting of a receptor, a transmembrane receptor, an ion channel protein, an intracellular adhesion protein, a cell adhesion protein, an integrin and the like.
(Item 25)
25. The method of item 24, wherein the transmembrane receptor comprises a G protein coupled receptor (GPCR).
(Item 26)
26. A method according to item 25, wherein the G protein-coupled receptor (GPCR) interacts with a G protein a subunit.
(Item 27)
Wherein G protein a _{subunit, Ga s, Ga i, Ga} q, and comprises at least one selected from the group consisting of _{Ga 12,} The method of claim 26.
(Item 28)
28. The method of item 27, wherein the at least one G protein a subunit is Ga _q .
(Item 29)
3. The method of item 2, wherein the modulation of at least one of cell membrane potential and cell membrane conductivity comprises modulating total cell conductivity.
(Item 30)
30. The method of item 29, wherein adjusting total cell conductivity comprises adjusting at least one of said linear and non-linear voltage dependent contributions of said total cell conductivity.
(Item 31)
3. The method of item 2, wherein the modulation of at least one of cell membrane potential and cell membrane conductivity comprises modulation of a calcium-dependent cell transmission pathway or system.
(Item 32)
3. The method of item 2, wherein the modulation of at least one of cell membrane potential and cell membrane conductivity comprises modulation of phospholipase C activity.
(Item 33)
3. The method of item 2, wherein the modulation of at least one of cell membrane potential and cell membrane conductivity comprises modulation of adenylate cyclase (AC) activity.
(Item 34)
Modulation of at least one of cell membrane potential and cell membrane conductivity is a disease or disorder of the immune system, allergic inflammation, allergic airway inflammation, DC-mediated inflammation Th2 response, atopic dermatitis, atopic eczema, asthma, obstructive Of intracellular signal transduction associated with at least one condition or symptom selected from the group consisting of airway disease, chronic obstructive pulmonary disease, IgE-mediated disease, rhinitis conjunctivitis, food allergy, inflammatory arthritis, rheumatoid arthritis, and psoriasis Item 3. The method according to Item 2, comprising adjustment.
(Item 35)
13. A method according to any one of items 1 to 12, comprising administration of said electrokinetic current body to a cell network or layer, further comprising modulation of intercellular binding therein.
(Item 36)
36. The method of item 35, wherein the intercellular bond comprises at least one selected from the group consisting of tight junction, gap junction, adhesion band, and desmosome.
(Item 37)
36. The method of item 35, wherein the cell network or layer comprises at least one selected from the group consisting of lung epithelium, bronchial epithelium, and intestinal epithelium.
(Item 38)
The electrokinetically modified aqueous fluid is oxygenated and the oxygen in the fluid is at least 8 ppm, at least 15 ppm, at least 25 ppm, at least 30 ppm, at least 40 ppm, at least 50 ppm, or at least at atmospheric pressure 13. A method according to any one of items 1 to 12, present in an amount of 60 ppm oxygen.
(Item 39)
Any of items 1-12, wherein the electrokinetically modified aqueous fluid comprises at least one of solvated electrons and electrokinetically modified or charged oxygen species.
The method according to claim 1.
(Item 40)
The form of the solvated electron, or electrokinetically modified or charged oxygen species is at least 0.01 ppm, at least 0.1 ppm, at least 0.5 ppm, at least 1 ppm, at least 3 ppm, at least 5 ppm, at least 40. The method of item 39, wherein the method is present in an amount of 7 ppm, at least 10 ppm, at least 15 ppm, or at least 20 ppm.
(Item 41)
41. A method according to item 40, wherein the electrokinetically modified aqueous fluid comprises a solvated electron form stabilized by molecular oxygen.
(Item 42)
Said ability to modulate at least one of cell membrane potential and cell membrane conductance is at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 in a sealed airtight container. 3. The method of item 2, wherein the method lasts for a period of at least 12 months or more.
(Item 43)
The amount of oxygen present in the charge-stabilized oxygen-containing nanostructure of the electrokinetically modified fluid is at least 8 ppm, at least 15 ppm, at least 20 ppm, at least 25 ppm, at least 30 ppm, at least 40 ppm at atmospheric pressure, 13. The method according to any one of items 1 to 12, wherein the method is at least 50 ppm, or at least 60 ppm oxygen.
(Item 44)
13. A method according to any one of items 1 to 12, wherein the treatment comprises administration by at least one of topical, inhalation, intranasal and intravenous.

Electrokinetically generated fluids of the present invention (eg, Revera 60 and Solas) reduce DEP-induced TSLP receptor expression in bronchial epithelial cells (BEC) by approximately 90% and 50%, respectively On the other hand, saline (NS) shows that there was only a marginal effect. Furthermore, the non-electrokinetic control pressurized pot fluid PP60 resulted in an approximately 50% reduction in DEP-induced TSLP receptor expression. While the electrokinetically generated fluids of the present invention (eg, Revera 60 and Solas) inhibit DEP-induced cell surface-bound MMP9 levels in bronchial epithelial cells at about 80% and 70%, respectively Saline (NS) indicates that there was only a marginal effect. Furthermore, the non-electrokinetic control pressurized pot fluid PP60 resulted in an approximately 30% reduction in DEP-induced cell surface bound MMP9 levels. Electrokinetically generated fluids for epithelial cell membrane polarity and ion channel activity at two time points (15 minutes (left panel) and 2 hours (right panel)) and with different voltage protocols (eg, RNS-60 and The results of a series of patch clamp experiments evaluating the effect of Solas) are shown. Electrokinetically generated fluids for epithelial cell membrane polarity and ion channel activity at two time points (15 minutes (left panel) and 2 hours (right panel)) and with different voltage protocols (eg, RNS-60 and The results of a series of patch clamp experiments evaluating the effect of Solas) are shown. Electrokinetically generated fluids for epithelial cell membrane polarity and ion channel activity at two time points (15 minutes (left panel) and 2 hours (right panel)) and with different voltage protocols (eg, RNS-60 and The results of a series of patch clamp experiments evaluating the effect of Solas) are shown. 3A-C, three voltage protocols (stepping from A.0 mV, stepping from B.-60 mV, stepping from C.-120 mV) and two time points (15 minutes (open circles)) And 2 hours (black circles)) shows a graph resulting from subtraction of Solas current data from RNS-60 current data. 3A-C, three voltage protocols (stepping from A.0 mV, stepping from B.-60 mV, stepping from C.-120 mV) and two time points (15 minutes (open circles)) And 2 hours (black circles)) shows a graph resulting from subtraction of Solas current data from RNS-60 current data. 3A-C, three voltage protocols (stepping from A.0 mV, stepping from B.-60 mV, stepping from C.-120 mV) and two time points (15 minutes (open circles)) And 2 hours (black circles)) shows a graph resulting from subtraction of Solas current data from RNS-60 current data. With different external saline and with different voltage protocols (Panels A and C show stepping from 0 mV, Panels B and D show stepping from -120 mV) for epithelial cell membrane polarity and ion channel activity Figure 2 shows the results of a series of patch clamp experiments that evaluated the effects of electrokinetically generated fluids (eg, Solas (panels A and B) and RNS-60 (panels C and D)). With different external saline and with different voltage protocols (Panels A and C show stepping from 0 mV, Panels B and D show stepping from -120 mV) for epithelial cell membrane polarity and ion channel activity Figure 2 shows the results of a series of patch clamp experiments that evaluated the effects of electrokinetically generated fluids (eg, Solas (panels A and B) and RNS-60 (panels C and D)). With different external saline and with different voltage protocols (Panels A and C show stepping from 0 mV, Panels B and D show stepping from -120 mV) for epithelial cell membrane polarity and ion channel activity Figure 2 shows the results of a series of patch clamp experiments that evaluated the effects of electrokinetically generated fluids (eg, Solas (panels A and B) and RNS-60 (panels C and D)). With different external saline and with different voltage protocols (Panels A and C show stepping from 0 mV, Panels B and D show stepping from -120 mV) for epithelial cell membrane polarity and ion channel activity Figure 2 shows the results of a series of patch clamp experiments that evaluated the effects of electrokinetically generated fluids (eg, Solas (panels A and B) and RNS-60 (panels C and D)). 5A-D, with respect to Solas (panels A and B) and Revera 60 (panels C and D), two voltage protocols (stepping from panels A and C.0 mV, B and D.D.). Steps from -120 mV), graphs resulting from subtraction of CsCl current data (shown in FIG. 5) from 20 mM CaCl ₂ (diamonds) and 40 mM CaCl ₂ (black squares) current data. 5A-D, with respect to Solas (panels A and B) and Revera 60 (panels C and D), two voltage protocols (stepping from panels A and C.0 mV, B and D.D.). Steps from -120 mV), graphs resulting from subtraction of CsCl current data (shown in FIG. 5) from 20 mM CaCl ₂ (diamonds) and 40 mM CaCl ₂ (black squares) current data. 5A-D, with respect to Solas (panels A and B) and Revera 60 (panels C and D), two voltage protocols (stepping from panels A and C.0 mV, B and D.D.). Steps from -120 mV), graphs resulting from subtraction of CsCl current data (shown in FIG. 5) from 20 mM CaCl ₂ (diamonds) and 40 mM CaCl ₂ (black squares) current data. 5A-D, with respect to Solas (panels A and B) and Revera 60 (panels C and D), two voltage protocols (stepping from panels A and C.0 mV, B and D.D.). Steps from -120 mV), graphs resulting from subtraction of CsCl current data (shown in FIG. 5) from 20 mM CaCl ₂ (diamonds) and 40 mM CaCl ₂ (black squares) current data. FIG. 6 shows the results of a patch clamp experiment evaluating the effect of dilution of electrokinetically generated fluid (eg, RNS-60) on epithelial cell membrane polarity and ion channel activity. FIG. 6 shows the results of a patch clamp experiment evaluating the effect of dilution of electrokinetically generated fluid (eg, RNS-60) on epithelial cell membrane polarity and ion channel activity.

  Charge-stabilized oxygen substantially having an average diameter of less than about 100 nanometers and stably constructed in an ionic aqueous fluid in an amount sufficient to treat a TSLP-mediated or TSLPR-mediated disease or condition Methods are provided for treating a TSLP-mediated or TSLPR-mediated disease or condition comprising the administration of an electrokinetically modified aqueous fluid comprising a nanostructured ionic aqueous solution containing. The charge-stabilized oxygen-containing nanostructure is preferably stably configured in the fluid in an amount sufficient to provide modulation of cell membrane potential and / or conductivity. Certain embodiments involving the modulation or down-regulation of TSLP expression and / or activity may include TSLP-mediated or TSLPR-mediated diseases or conditions (eg, immune system diseases, allergic inflammation, allergic airways) as disclosed herein. Inflammation, DC-mediated inflammatory Th2 response, atopic dermatitis, atopic eczema, asthma, obstructive airway disease, chronic obstructive pulmonary disease, and food allergy, inflammatory arthritis, rheumatoid arthritis, psoriasis, IgE-mediated disease, and rhinitis conjunctivitis Have utility for treating).

Electrokinetically generated fluid:
As used herein, “electrokinetically generated fluid” refers to Applicant's generated for the examples herein by the exemplary mixing device detailed herein. Refers to the electrokinetically generated fluid of the present invention (see also US200802190088 and WO2008 / 052143, both of which are hereby incorporated by reference in their entirety). As shown by the data disclosed and shown herein, the electrokinetic current body includes an oxygenated non-surface electrodynamic current body associated with the prior art (eg, an oxygenated fluid in a pressurized pot, etc.) A novel radically different fluid, which is a non-electrokinetic galvanic body relevant to technology, is shown. As disclosed in various aspects herein, electrokinetically generated fluids have unique and novel physical and biological properties including, but not limited to: .

  In certain embodiments, the electrokinetically modified aqueous fluid has an average diameter that is substantially less than about 100 nanometers, and is one of cell membrane potential and cell membrane conductivity when contacted with living cells by the fluid. A charge-stabilized, oxygen-containing, nanostructured, ionic aqueous solution that is stably configured in an ionic aqueous fluid in an amount sufficient to provide at least one of the following:

  In certain aspects, electrokinetically generated fluids are hydrodynamically induced localizations (eg, for total fluid volume, such as device functional localization effects described herein). Non-uniform) refers to fluid generated in the presence of electrokinetic effects (eg, voltage / current pulses). In certain embodiments, the hydrodynamically induced localized electrokinetic effect is combined with a surface related bilayer and / or charged current effect, as disclosed and discussed herein.

In certain embodiments, the electrokinetically modified aqueous fluid is suitable for adjusting the ¹³ C-NMR linewidth of the reporter solute (eg, trehalose) dissolved therein. The NMR linewidth effect, in certain examples, is an indirect method described herein, for example, for measuring the “rotation” of a solute in a test fluid.

  In certain embodiments, the electrokinetically modified aqueous fluid has a unique square wave at any one of -0.14V, -0.47V, -1.02V, and -1.36V. Volatmetric peak differences; polarographic peaks at −0.9 volts; and at least one absence of polarographic peaks at −0.19 and −0.3 volts, which are specific examples In particular, it is unique to electrokinetically generated fluids as disclosed herein.

  In certain embodiments, the electrokinetically modified aqueous fluid has cell membrane conductivity (eg, a voltage dependent contribution of total cell conductivity, as measured in the patch clamp test disclosed herein). It is suitable for modifying.

  In certain aspects, the electrokinetically modified aqueous fluid is oxygenated and the oxygen in the fluid is at least 15 ppm, at least 25 ppm, at least 30 ppm, at least 40 ppm, at least 50 ppm, or at least 60 ppm at atmospheric pressure. Present in an amount of dissolved oxygen. In certain embodiments, the electrokinetically modified aqueous fluid has less than 15 ppm and less than 10 ppm of dissolved oxygen at atmospheric pressure or near atmospheric oxygen levels.

  In certain aspects, the electrokinetically modified aqueous fluid is oxygenated and the oxygen in the fluid is present in an amount from about 8 ppm to about 15 ppm, in which case, optionally, herein, It is called “Solas”.

  In certain embodiments, the electrokinetically modified aqueous fluid comprises solvated electrons (eg, stabilized by molecular oxygen) and electrokinetically modified and / or charged oxygen species. In some embodiments, the solvated electrons and / or electrokinetically modified or charged oxygen species are at least 0.01 ppm, at least 0.1 ppm, at least 0. Present in an amount of .5 ppm, at least 1 ppm, at least 3 ppm, at least 5 ppm, at least 7 ppm, at least 10 ppm, at least 15 ppm, or at least 20 ppm.

In certain embodiments, the electrokinetically modified aqueous fluid alters cell membrane structure or function sufficient to provide modulation of intracellular signal transduction (eg, membrane bound protein conformation, ligand binding). In certain embodiments, the membrane-bound protein is a receptor, a transmembrane receptor (eg, a G protein coupled receptor (GPCR), a TSLP receptor, β2 Adrenergic receptor, bradykinin receptor, etc.), ion channel protein, intracellular adhesion protein, cell adhesion protein, and at least one selected from the group consisting of integrins. In some embodiments, the affected G protein coupled receptor (GPCR) interacts with a G protein a subunit (eg, Ga _s, Ga _i, Ga _q , and Ga ₁₂ ).

  In certain embodiments, the electrokinetically modified aqueous fluid is suitable for modulating intracellular signal transduction and modulating calcium-dependent cellular pathways or systems (eg, modulating phospholipase C activity, Or modulation of adenylate cyclase (AC) activity).

  In certain embodiments, electrokinetically modified aqueous fluids are used in various biological activities (eg, cytokines, receptors, enzymes and other proteins, and intracellular signal pathways described herein). Adjustment).

  In certain embodiments, the electrokinetically modified aqueous fluid exhibits a synergistic effect with albuterol and budesonide, as shown herein.

  In certain embodiments, the electrokinetically modified aqueous fluid reduces DEP-induced TSLP receptor expression in bronchial epithelial cells (BECs), as shown in the Examples herein.

  In certain aspects, the electrokinetically modified aqueous fluid inhibits DEP-induced cell surface-bound MMP9 levels in bronchial epithelial cells (BECs), as shown in the Examples herein.

  In certain aspects, the biological effects of electrokinetically modified aqueous fluids are such that beta blockers, GPCR blockers, and calcium channel blockers are (eg, controlled) as shown herein. Inhibited by diphtheria toxin, which shows an effect on the activity of electrokinetically modified aqueous fluids (in sex T cell function).

  In certain aspects, the physical and biological effects of an electrokinetically modified aqueous fluid (eg, the ability to alter cell membrane structure or function sufficient to provide modulation of intracellular signal transduction) Persist in a sealed container (eg, in a sealed, airtight container) for a period of at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, or longer .

Accordingly, a further aspect provides a method of producing the electrokinetically generated solution and electrokinetically modified oxygenated fluid or solution, the method comprising two spaced apart in relative motion Providing a flow of fluid material between defined surfaces and defining a mixing volume therebetween, wherein a single pass residence time of flowing fluid material in and through the mixing volume is greater than 0.06 seconds Or at least 20 ppm, at least 25 ppm, at least 30, at least 40, at least 50, or at least 60 ppm of oxygen dissolved in the substance to electrokinetically modify the fluid or solution Introducing oxygen (O ₂ ) into the flowing fluid material in a mixing volume under conditions suitable for In some embodiments, oxygen is injected into the material in less than 100 milliseconds, less than 200 milliseconds, less than 300 milliseconds, or less than 400 milliseconds. In certain embodiments, the ratio of surface area to volume is at least 12, at least 20, at least 30, at least 40, or at least 50.

  A still further aspect provides a method of producing an electrokinetically modified oxygenated aqueous fluid or solution, the method providing a flow of fluid material between two spaced surfaces and between them And at least 20 ppm, at least 25 ppm, at least 30, at least 40, at least 50, or at least less than 100 milliseconds, less than 200 milliseconds, less than 300 milliseconds, or less than 400 milliseconds Introducing oxygen into the flowing fluid in a mixing volume under conditions suitable for injecting at least 60 ppm of oxygen. In some embodiments, the residence time of the flowing fluid material within the mixing volume is 0.06 seconds or more, or 0.1 seconds or more. In certain embodiments, the ratio of surface area to volume is at least 12, at least 20, at least 30, at least 40, or at least 50.

  In certain embodiments, the administered electrokinetically modified fluid of the present invention has a charge stabilized oxygen in an amount sufficient to provide modulation of at least one of cell membrane potential and cell membrane conductivity. Containing nanostructures. In certain embodiments, the electrokinetically modified fluid is hyperoxygenated (eg, RNS-20, RNS-40, and RNS-60, including 20 ppm, 40 ppm, and 60 ppm dissolved oxygen, respectively). is there. In certain embodiments, the electrokinetically modified fluid is non-superoxygenated (eg, RNS-10 or Solas, including 10 ppm (eg, ambient level dissolved oxygen in standard saline)). . In certain embodiments, the salinity, neutrality, pH, etc. of the electrokinetically modified fluid of the present invention are determined during the electrokinetic production of the fluid, and the sterile fluid is administered by an appropriate route. Alternatively, at least one of fluid salinity, neutrality, pH, etc. is suitably adjusted (eg, sterile saline or appropriate) to be physiologically compatible with the route of administration prior to fluid administration. Using a suitable diluent). Preferably, the diluent and / or saline solution and / or buffer composition used to adjust at least one of the salinity, neutrality, pH, etc. of the fluid is also an electrokinetic current, or Otherwise it is compatible.

  In certain embodiments, the electrokinetically modified fluid of the present invention comprises saline (eg, one or more dissolved salts, such as alkali metal salts (Li, Na, K, Rb, Cs, etc.), alkaline earth salts (eg, Mg, Ca), etc., transition metal salts (eg, Cr, Fe, Co, Ni, Cu, Zn, etc.)). Certain embodiments include mixed salt electrokinetic bodies (eg, Na, K, Ca, Mg, etc. in various combinations and concentrations). In certain embodiments, the electrokinetically modified fluid of the present invention comprises a standard saline solution (eg, about 0.9% NaCl or about 0.15M NaCl). In certain aspects, the electrokinetically modified fluid of the present invention is at least 0.0002M, at least 0.0003M, at least 0.001M, at least 0.005M, at least 0.01M, at least 0.015M, at least Contains sodium chloride at a concentration of 0.1M, at least 0.15M, or at least 0.2M. In certain embodiments, the conductivity of the electrokinetically modified fluid of the present invention is at least 10 μS / cm, at least 40 μS / cm, at least 80 μS / cm, at least 100 μS / cm, at least 150 μS / cm, at least 200 μS / cm. cm, at least 300 μS / cm, or at least 500 μS / cm, at least 1 mS / cm, at least 5 mS / cm, 10 mS / cm, at least 40 mS / cm, at least 80 mS / cm, at least 100 mS / cm, at least 150 mS / cm, at least 200 mS / cm cm, at least 300 mS / cm, or at least 500 mS / cm. In certain embodiments, the electrokinetics of the present invention provided that it enables the formation of bioactive salt stabilized nanostructures (eg, salt stabilized oxygen-containing nanostructures) disclosed herein. Any salt may be used to prepare a chemically modified fluid.

  In accordance with certain embodiments, the biological effects of fluid compositions of the present invention comprising charge-stabilized gas-containing nanostructures can be achieved, for example, by modifying the ionic constituents of the fluid, as described above, and / or fluids Can be adjusted (eg, increased, decreased, tuned, etc.) by modifying the gas components. In a preferred embodiment, oxygen is used to prepare the electrokinetic current body of the present invention. In a further aspect, a mixture of oxygen is used with at least one other gas selected from nitrogen, oxygen, argon, carbon dioxide, neon, helium, krypton, hydrogen, and xenon.

Exemplary preferred embodiments:
Certain embodiments substantially have an average diameter of less than about 100 nanometers and are stably configured in an ionic aqueous fluid in an amount sufficient to treat a TSLP-mediated or TSLPR-mediated disease or condition. TSLP-mediated or TSLPR-mediated disease comprising administration of a therapeutically effective amount of an electrokinetically modified aqueous fluid comprising a charge-stabilized oxygen-containing nanostructured ionic aqueous solution to a mammal in need thereof Or provide a method for treating a condition. In certain embodiments, the charge-stabilized oxygen-containing nanostructure is ionic aqueous in an amount sufficient to provide modulation of at least one of cell membrane potential and cell membrane conductivity when contacted by a fluid with living cells. Stable in the fluid.

  In some method embodiments, the charge-stabilized oxygen-containing nanostructure is the primary charge-stabilized gas-containing nanostructure species in the fluid. In certain embodiments, as a charge-stabilized oxygen-containing nanostructure, the percentage of dissolved oxygen molecules present in the fluid is 0.01%, 0.1%, 1%, 5%, 10% 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, and It is a ratio selected from the group consisting of more than 95%. In certain embodiments, all dissolved oxygen is substantially present in the charge-stabilized oxygen-containing nanostructure. In certain embodiments, the charge-stabilized oxygen-containing nanostructure has an average diameter that is less than a size selected from the group consisting of less than 90 nm, 80 nm, 70 nm, 60 nm, 50 nm, 40 nm, 30 nm, 20 nm, 10 nm, and 5 nm. Has substantially.

  In certain method aspects, the aqueous ionic solution comprises a saline solution. In some embodiments, the electrokinetically modified aqueous fluid is superoxygenated.

  In certain method aspects, the electrokinetically modified aqueous fluid comprises a solvated electron form.

  In certain embodiments, the electrokinetically modified aqueous fluid modification comprises fluid exposure to a hydrodynamically induced localized electrokinetic effect. In certain embodiments, exposure to the localized electrokinetic effect includes exposure to at least one of a voltage pulse and a current pulse. In certain aspects, exposure of a fluid to a hydrodynamically induced localized electrokinetic effect causes the fluid to undergo structural properties that induce the electrokinetic effect of the device used to generate the fluid. Includes exposure.

  In certain aspects, a TSLP-mediated or TSLPR-mediated disease or condition includes a disease or disorder of the immune system, including but not limited to allergic inflammation. In certain embodiments, the allergic inflammation is allergic airway inflammation, DC-mediated inflammation Th2 response, atopic dermatitis, atopic eczema, asthma, obstructive airway disease, chronic obstructive pulmonary disease, IgE-mediated disease, rhinitis conjunctivitis, And at least one of food allergies. In certain embodiments, the mediated or TSLPR mediated disease or condition comprises inflammatory arthritis, including, for example, at least one of rheumatoid arthritis and psoriasis.

In certain embodiments, the method further comprises a combination therapy, and at least one additional therapeutic agent is administered to the patient. In certain embodiments, the at least one additional therapeutic agent is a short-acting β ₂ agonist, a long-acting β ₂ agonist, an anticholinergic agent, a corticosteroid, a systemic corticosteroid, a mast cell stabilizer. , A leukotriene modifier, methylxanthine, and combinations thereof. In some embodiments, the at least one additional therapeutic agent is an albuterol, levalbuterol, pyrbuterol, alformoterol, formoterol, salmeterol, and beta ₂ agonists, including anticholinergics such as ipratropium and tiotropium; beclomethasone, budesonide, flunisolide Corticosteroids, including fluticasone, mometasone, triamcinolone, methiprednisolone, prednisolone, prednisone; leukotriene modifiers, including montelukast, zafirlukast, and zileuton; mast cell stabilizers, including cromolyn and nedocromil; theophylline, ipratropium, Including concomitant medications including fluticasone and salmeterol, budesonide and formoterol, Tilxanthine; consisting of hydroxyzine, diphenhydramine, loratadine, cetirizine, and hydrocortisone; antihistamines; immune system modifiers including tacrolimus and pimecrolimus; cyclosporine; azathioprine; mycophenolate mofetil; Selected from the group. In certain aspects, the at least one additional therapeutic agent is a TSLP and / or TSLPR antagonist, and in certain embodiments, TSLP and / or TSLPR antagonist comprises two or more receptor chain components. A TSLPR immunoglobulin Fc molecule or polypeptide that encodes is selected from the group consisting of neutralizing antibodies specific for TSLP and TSLP receptors, soluble TSLP receptor molecules, and TSLP receptor fusion proteins.

In certain method aspects, modulation of at least one of cell membrane potential and cell membrane conductivity alters at least one of a membrane bound protein or component conformation, ligand binding activity, and catalytic activity. Modifying at least one of the cell membrane structure or function. In some embodiments, the membrane-bound protein comprises at least one selected from the group consisting of receptors, transmembrane receptors, ion channel proteins, intracellular adhesion proteins, cell adhesion proteins, integrins and the like. In certain embodiments, the transmembrane receptor comprises a G protein coupled receptor (GPCR). In certain aspects, the G protein coupled receptor (GPCR) interacts with, for example, the G protein a subunit, the G protein a subunit comprising the group consisting of Ga _s, Ga _i, Ga _q , and Ga _12. In certain embodiments, at least one G protein a subunit is Ga _q .

  In certain method aspects, modulation of at least one of cell membrane potential and cell membrane conductivity includes, for example, modulating total cell conductivity, wherein modulating total cell conductivity is a measure of total cell conductivity. Adjusting at least one of the linear or non-linear voltage-dependent contributions.

  In certain method aspects, the modulation of at least one of cell membrane potential and cell membrane conductivity comprises modulation of a calcium-dependent cell transmission pathway or system. In certain method aspects, the modulation of at least one of cell membrane potential and cell membrane conductivity comprises modulation of phospholipase C activity. In certain method aspects, the modulation of at least one of cell membrane potential and cell membrane conductivity comprises modulation of adenylate cyclase (AC) activity. In certain method aspects, modulation of at least one of cell membrane potential and cell membrane conductance is a disease or disorder of the immune system, allergic inflammation, allergic airway inflammation, DC-mediated inflammation Th2 response, atopic dermatitis, atopic Associated with at least one condition or symptom selected from the group consisting of eczema, asthma, obstructive airway disease, chronic obstructive pulmonary disease, IgE-mediated disease, rhinitis conjunctivitis, food allergy, inflammatory arthritis, rheumatoid arthritis, and psoriasis The regulation of intracellular signal transduction.

  Certain method aspects include administration of electrokinetic current bodies to a cell network or layer, and further include modulation of intercellular binding therein. In certain embodiments, the intercellular junction comprises at least one selected from the group consisting of tight junctions, gap junctions, adhesion bands, and desmosomes. In certain embodiments, the cell network or layer comprises at least one selected from the group consisting of lung epithelium, bronchial epithelium, and intestinal epithelium.

  In some method aspects, the electrokinetically modified aqueous fluid is oxygenated and the oxygen in the fluid is at least 8 ppm, at least 15 ppm, at least 25 ppm, at least 30 ppm, at least 40 ppm, at least 50 ppm at atmospheric pressure. Or in an amount of oxygen of at least 60 ppm.

  In some method aspects, the electrokinetically modified aqueous fluid comprises, for example, at least one of solvated electrons and electrokinetically modified or charged oxygen species, and the solvent The form of the sum electron, or electrokinetically modified or charged oxygen species is at least 0.01 ppm, at least 0.1 ppm, at least 0.5 ppm, at least 1 ppm, at least 3 ppm, at least 5 ppm, at least 7 ppm, It is present in an amount of at least 10 ppm, at least 15 ppm, or at least 20 ppm. In some embodiments, the electrokinetically modified aqueous fluid comprises a solvated electron form stabilized by molecular oxygen.

  In certain embodiments, the electrokinetically modified fluid's ability to modulate at least one of cell membrane potential and cell membrane conductance is at least 2 months, at least 3 months, in a sealed airtight container. For at least 4 months, at least 5 months, at least 6 months, at least 12 months, or longer.

  In some embodiments, the amount of oxygen present in the electrokinetically modified fluid charge-stabilized oxygen-containing nanostructure is at least 8 ppm, at least 15 ppm, at least 20 ppm, at least 25 ppm, at least 30 ppm at atmospheric pressure, At least 40 ppm, at least 50 ppm, or at least 60 ppm oxygen.

  In certain embodiments, treatment includes administration by at least one of topical, inhalation, intranasal, and intravenous.

Exemplary related intermolecular interactions:
Usually, quantum properties are considered to belong to elementary particles less than ^10-10 meters, while the macroscopic world of daily life is classical in that elementary particles move according to Newton's law of motion. Called to be.

  Recently, molecules have been described to form clusters that increase in size with dilution. These clusters are reported to measure several micrometers in diameter and increase in size nonlinearly with dilution. Quantum coherent domains measuring 100 nanometers in diameter are assumed to occur in pure water, and collective oscillations of water molecules in the coherent domains can eventually become a phase that is fixed to electromagnetic field fluctuations, In the form of long-lasting coherent vibration excitation specific to dissolved matter in water that provides stable oscillations and changes the aggregate structure of water, it provides a form of “memory”, which also develops singularities A coherent oscillation can be determined. If these oscillations are stabilized by the connecting magnetic phase, the water upon dilution can further carry “seed” coherent oscillations. As the size of a molecular cluster increases, its electromagnetic characteristics are correspondingly amplified and enhance the coherent vibrations carried by water.

  Despite changes in the cluster size of dissolved molecules and the detailed microscopic structure of water, the specificity of coherent oscillations can still exist. One model for taking into account changes in water properties is based on considerations involved in crystallization.

A simple protonated water cluster that forms a nanoscale cage is shown in Applicant's previous patent application, WO 2009/055729. Protonated water clusters generally take the form of H ⁺ (H ₂ 0) _n . Some protonated water clusters occur naturally in the ionosphere and the like. While not being bound by any particular theory, according to particular embodiments, water clusters or other types of structures (clusters, nanocages, etc.) are possible and are provided for the output material of the present invention. , Structures containing oxygen and stabilized electrons. Oxygen atoms can be incorporated into the resulting structure. The chemical reaction of the semi-bonded nanocages can leave oxygen and / or stabilized electrons dissolved for long periods of time. Other atoms or molecules such as pharmaceutical compounds can be caged for sustained release purposes. Specific chemical reactions of solution substances and dissolved compounds depend on the interaction of these substances.

  It has already been shown through experiments that fluids processed by a mixing device exhibit different structural properties consistent with fluid analysis in a cluster structure. See, for example, International Publication No. 2009/055729.

Charge-stabilized nanostructures (eg, charge-stabilized oxygen-containing nanostructures):
As already described in Applicants' International Publication No. WO 2009/055729, “Double Layer Effect”, “Residence Time”, “Injection Rate”, “Bubble Size Measurement”, an electrokinetic mixing device is a composite. A unique, non-linear fluid dynamic interaction between the first material and the second material, as described herein, effectively providing an enormous surface area (device area and 100 nm, which provides the novel electrokinetic effect) Dynamic turbulence is created in approximately milliseconds, providing a composite that contacts and mixes (including less than an exceptionally small bubble area). The functionally localized electrokinetic effect (voltage / current) was also demonstrated using a specially designed mixing device including an insulated rotor and stator.

  As is well recognized in the art, charge redistribution and / or solvated electrons are known to be extremely unstable in aqueous solutions. According to certain embodiments, the electrokinetic effects of the application (eg, charge redistribution, including solvated electrons in certain embodiments) are surprisingly stable within the output material (eg, saline solution, ionic solution). It becomes. In practice, the properties and biological activity stability of the inventive electrokinetic current bodies (eg, RNS-60 or Solas) described herein are maintained for several months in an airtight container. It demonstrates the involvement of dissolved gases (eg, oxygen) in helping to generate and / or maintain and / or mediate the properties and activities of the solutions of the present invention. Significantly, charge redistribution and / or solvation electrons are sufficient to provide at least one modulation of cell membrane potential and cell membrane conductance when contacted by a fluid with a living cell (eg, a mammalian cell). By volume, it is stably constructed in the electrokinetic ionic aqueous fluid of the invention (see, eg, Example 23 of a cell patch clamp from WO 2009/055729, and disclosed herein) To be).

  To describe the stability and biocompatibility of the electrokinetic current bodies (eg, electrokinetic saline solution) of the present invention, as described herein in the “Intermolecular Interactions” section, Applicants have proposed that interactions between water molecules and molecules of substances dissolved in water (eg, oxygen) change the aggregate structure of the water, providing a nanoscale cage cluster. Includes nanostructures containing oxygen and / or stabilized electrons that are provided to the output material of the present invention. While not being bound by a mechanism, in certain embodiments, the structures of the nanostructures allow them to dissolve gas (eg, oxygen) (at least for formation and / or stability and / or biological activity). An electrokinetic body (eg, RNS-60 or Solas saline fluid) modulates charge and / or charge effects (eg, gives or accepts) when in contact with a cell membrane or its associated component. As well as, in certain embodiments, provide stabilization (eg, carry, possess, capture) of solvated electrons in a biologically relevant form.

  According to certain embodiments, as supported by this disclosure, in ionic or saline (eg, standard saline, NaCl) solutions, the nanostructures of the present invention are at least 1 in a charge-stabilized hydration shell. It includes a charge-stabilized nanostructure (eg, an average diameter of less than 100 nm) that can contain one dissolved gas molecule (eg, oxygen). According to additional aspects, the charge-stabilized hydration shell can include a cage or void with at least one dissolved gas molecule (eg, oxygen). According to a further aspect, by providing a suitable charge-stabilized hydration shell, charge-stabilized nanostructures and / or charge-stabilized oxygen-containing nanostructures can be solvated electrons (eg, stabilized solvents). (Sum sum).

  Without being bound by mechanism or particular theory, after this priority date, charge-stabilized ultrafine bubbles stabilized by ions in an aqueous liquid in equilibrium with the surrounding (atmospheric) gas have been proposed. (Bunkin et al., Journal of Experimental and Theoretical Physics, 104: 486-498, 2007; incorporated herein by reference in its entirety). In accordance with certain aspects of the present invention, Applicants' novel electrokinetic current bodies include novel biologically active forms of charge-stabilized oxygen-containing nanostructures, and novel arrays of such structures, It may further comprise a cluster or an association.

According to the charge-stabilized ultrafine bubble model, the short-range molecular order in the water structure is destroyed by the presence of gas molecules (for example, dissolved gas molecules initially complexed with non-adsorbed ions have short-range order defects). This provides concentration of ionic droplets and the defect is surrounded by the first and second coordination spheres of water molecules, occupying 6 and 12 vacancies, respectively, in the coordination sphere Replaced by adsorbed ions (eg, capture of a “screening shell” of Na ⁺ ions to form an electric double layer) and non-adsorbed ions (eg, Cl ⁻ ions occupying a second coordination sphere) Filled. In an unsaturated ion solution (eg, an unsaturated saline solution), this hydrated “cell nucleus” is filled with first and second coordination spheres with six adsorbed ions and five non-adsorbed ions, respectively. Until it remains stable and then undergoes a Coulomb explosion creating an internal void containing gas molecules, adsorbed ions (eg, Na ⁺ ions) are adsorbed on the surface of the resulting void, while non-adsorbed ions (Or some portion thereof) diffuse into the solution (see Bunkin et al., Supra). In this model, voids in the nanostructure are not collapsed by a Coulomb explosion between ions adsorbed on the surface (eg, Na ⁺ ions). The stability of void-containing nanostructures is assumed to be due to the selective adsorption of dissolved ions with the same charge to the void / bubble surface and the diffusion equilibrium between the dissolved gas and the gas inside the bubble, The static voltage (external electrostatic voltage provided by the resulting electric double layer) provides stable compensation for interfacial tension, and the gas pressure inside the bubble is balanced by the ambient pressure. According to the model, the formation of such ultrafine bubbles requires ionic constituents, and in certain embodiments, collision-mediated association between particles can provide the formation of larger ordered clusters (arrays). ).

  The charge-stabilized ultrafine bubble model suggests that the particles can be gas ultrafine, but only contemplates the spontaneous formation of such a structure in an ionic solution in equilibrium with the surrounding atmosphere, where oxygen As to whether such a structure can be formed, it has not been characterized and remains unspecified, as well as solvated electrons can be associated and / or stabilized by such a structure. Whether or not it remains unspecified.

  According to a particular embodiment, the electrokinetic current bodies of the present invention comprising charge-stabilized nanostructures and / or charge-stabilized oxygen-containing nanostructures are novel and according to a superfine bubble model Basically, it is different from the electric and atmospheric charge stabilization ultrafine bubble structure. Of note, the control saline solution does not have the biological properties disclosed herein, while Applicant's charge-stabilized nanostructures are charge-stabilized oxygen-containing nanostructures. This conclusion, which derives at least in part from the fact that it provides a novel biologically active form, is unavoidable.

  In accordance with certain aspects of the present invention, Applicants' novel electrokinetic devices and methods are spontaneously produced in an ionic fluid in equilibrium with the atmosphere, or any non-electrokinetically generated fluid. Novel electrokinetically modified fluids are provided that contain significant amounts of charge-stabilized nanostructures beyond any amount that may or may not occur. In certain embodiments, the charge-stabilized nanostructure comprises a charge-stabilized oxygen-containing nanostructure. In additional embodiments, the charge-stabilized nanostructures are all or substantially all charge-stabilized oxygen-containing nanostructures, or the charge-stabilized oxygen-containing nanostructures are in the electrokinetic current body. The main charge-stabilized gas-containing nanostructure species.

  According to a still further aspect, the charge-stabilized nanostructure and / or the charge-stabilized oxygen-containing nanostructure comprises or can have solvated electrons, thereby providing a novel stabilized solvated electron carrier I will provide a. In certain embodiments, charge-stabilized nanostructures and / or charge-stabilized oxygen-containing nanostructures provide a new type of electrode (or reverse electrode) and traditional solute electrodes with a single organic coordination cation Rather, it has a plurality of cations that are stably arranged around the voids or voids containing oxygen atoms, and the arranged sodium ions are caused by water hydration shells rather than by organic molecules. Coordinated. According to certain embodiments, solvated electrons can be accommodated by a hydration shell of water molecules, or preferably within nanostructured voids distributed across all cations. In some embodiments, the nanostructures of the present invention not only provide for the distribution / stabilization of solvated electrons (solvated electrons distributed over a series of sodium atoms and at least one oxygen atom) across a plurality of arranged sodium cations. By providing an association or partial association of oxygen molecules and solvated electrons in the voids, a new “superelectrode” in solution is provided. Thus, according to a particular embodiment, the “solvated electrons” currently disclosed in connection with the electrokinetic current bodies of the invention cannot be solvated in conventional models involving direct hydration by water molecules. Alternatively, in a limited example using dry electrode salts, solvated electrons in the electrokinetic current bodies of the present invention can be “lattice green” to stabilize highly ordered arrays in aqueous solutions. Can be distributed across a plurality of charge-stabilized nanostructures.

  In certain embodiments, the charge-stabilized nanostructures and / or charge-stabilized oxygen-containing nanostructures of the present invention interact with cell membranes or components thereof, proteins, etc. to mediate biological activity. Is possible. In certain embodiments, the charge-stabilized nanostructures and / or charge-stabilized oxygen-containing nanostructures with solvated electrons of the present invention are cell membranes or components thereof, or mediate biological activity, or It is possible to interact with proteins and the like.

  In certain embodiments, the charge-stabilized nanostructures and / or charge-stabilized oxygen-containing nanostructures of the present invention can serve as a charge and / or charge effect provider (delivery) to mediate biological activity. And / or as a charge and / or charge effect receptor, interact with a cell membrane or a component thereof, a protein, or the like. In certain embodiments, the charge-stabilized nanostructures and / or charge-stabilized oxygen-containing nanostructures with solvated electrons of the present invention provide charging and / or charging effects to mediate biological activity. Interact with the cell membrane as a person and / or as a charged and / or charged effector.

  In certain embodiments, the charge-stabilized nanostructures and / or charge-stabilized oxygen-containing nanostructures of the present invention are consistent with the observed stability and biological properties of the electrokinetic current bodies of the present invention, And a novel electrode (or reverse electrode) that provides stabilized solvated electrons in an aqueous ionic solution (eg, saline solution, NaCl, etc.).

  In certain embodiments, the charge-stabilized oxygen-containing nanostructure can substantially comprise, take, or generate a charge-stabilized oxygen-containing nanobubble. In certain embodiments, charge-stabilized oxygen-containing clusters provide for the formation of relatively large arrays of charge-stabilized oxygen-containing nanostructures and / or charge-stabilized oxygen-containing nanobubbles or arrays thereof. In certain embodiments, charge-stabilized oxygen-containing nanostructures can provide for the formation of hydrophobic nanobubbles when contacted with a hydrophobic surface.

In certain embodiments, the charge-stabilized oxygen-containing nanostructure substantially comprises at least one oxygen molecule. In some embodiments, the charge-stabilized oxygen-containing nanostructures are substantially at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20 At least 50, at least 100, or more oxygen molecules. In certain embodiments, the charge-stabilized oxygen-containing nanostructures comprise or generate about 20 nm × 1.5 nm nanobubbles (eg, hydrophobic nanobubbles) and have about 12 oxygen molecules (eg, Based on size (approximately 0.3 nm × 0.4 nm), ideal gas assumptions, and n = PV / RT application, where P = 1 atm, R = 0.082 □ 057 □ l.atm / mol. K; T = 295K; V = pr ² h = 4.7 × 10 ⁻²² L, where r = 10 × 10 ⁻⁹ m, h = 1.5 × 10 ⁻⁹ m, and n = 1.95 × 10 ⁻²² mol).

  In some embodiments, the percentage of oxygen molecules present in fluids in such nanostructures or arrays thereof having a charge stabilizing structure in an ionic aqueous fluid is 0.1%, 1%, 2% 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85 %, 90%, and more than 95%. Preferably, this proportion is about 5% or more, about 10% or more, about 15% or more, or about 20% or more. In additional embodiments, the substantial size of charge-stabilized oxygen-containing nanostructures having a charge-stabilizing structure in an ionic aqueous fluid, or their arrangement, is 100 nm, 90 nm, 80 nm, 70 nm, 60 nm, 50 nm , 40 nm, 30 nm, 20 nm, 10 nm, 5 nm, 4 nm, 3 nm, 2 nm, and a size selected from the group consisting of less than 1 nm. Preferably, the size is less than about 50 nm, less than about 40 nm, less than about 30 nm, less than about 20 nm, or less than about 10 nm.

  In one embodiment, the electrokinetic current body of the present invention contains solvated electrons. In a further aspect, the electrokinetic current body of the present invention comprises charge-stabilized nanostructures and / or charge-stabilized oxygen-containing nanostructures, and / or arrays thereof, which are solvated electrons, and It includes at least one of unique charge distributions (polar, symmetric, asymmetric charge distribution). In some embodiments, the charge-stabilized nanostructures and / or charge-stabilized oxygen-containing nanostructures and / or arrays thereof are paramagnetic.

  In contrast, in connection with the electrokinetic fluid of the present invention, a control pressurized pot oxygenated fluid (non-electrokinetic fluid) or the like is used for such electrokinetically generated charge-stabilized biology. Active nanostructures and / or biologically active charge-stabilized oxygen-containing nanostructures, and / or arrays thereof, can be adjusted for at least one of cell membrane potential and cell membrane conductivity.

Systems for generating gas-enriched fluids The presently disclosed systems and methods are capable of stably enriching gases (eg, oxygen) at high concentrations with minimal passive losses. This system and method can be effectively used to enrich a wide variety of gases at a high rate in a wide variety of fluids. By way of example only, deionized water having a level of about 2-3 ppm (parts per million) of dissolved oxygen at room temperature is typically at least about 5 ppm, at least about about 1 ppm using the disclosed systems and / or methods. 10 ppm, at least about 15 ppm, at least about 20 ppm, at least about 25 ppm, at least about 30 ppm, at least about 35 ppm, at least about 40 ppm, at least about 45 ppm, at least about 50 ppm, at least about 55 ppm, at least about 60 ppm, at least about 65 ppm, at least about 70 ppm, At least about 75 ppm, at least about 80 ppm, at least about 85 ppm, at least about 90 ppm, at least about 95 ppm, at least about 100 ppm, or any value or more It is possible to achieve a level of dissolved oxygen in the range between. According to certain exemplary embodiments, oxygen enriched water may be produced at a level of about 30-60 ppm of dissolved oxygen.

  Table 1 shows various partial pressure measurements obtained on healing wounds treated with oxygen-enriched saline solution (Table 1) and samples of the gas-enriched oxygen-enriched saline solution of the present invention.

ＴＳＬＰおよびＴＳＬＰ媒介状態
ＴＳＬＰおよびＴＳＬＰＲアゴニスト／拮抗剤：自然発生的な物質により通常刺激される細胞受容体に対する親和性を有し、かつ細胞受容体における生理学的活性を刺激し、それによって、生化学的反応を誘発する薬剤。ＴＳＬＰ受容体アゴニストは、ＴＳＬＰ受容体に対する親和性を有し、ＴＳＬＰのその受容体との結合により誘発される活性を刺激する。例えば、ＴＳＬＰ／ＴＳＬＰ受容体アゴニストは、ＴＳＬＰ受容体に結合し、かつ細胞内シグナル伝達を誘発する分子である。対照的に、「拮抗剤」は、自然発生的な物質により通常刺激される細胞受容体の活性を阻害する薬剤である。したがって、ＴＳＬＰ／ＴＳＬＰ受容体拮抗剤は、ＴＳＬＰまたはＴＳＬＰ受容体に結合し、ＴＳＬＰのＴＳＬＰ受容体への結合を阻害する、および／またはＴＳＬＰのその受容体との結合により通常誘発される活性を阻害する。例えば、ＴＳＬＰ／ＴＳＬＰ受容体拮抗剤は、ＴＳＬＰまたはＴＳＬＰ受容体に結合し、例えば、ＴＳＬＰのＴＳＬＰ受容体への結合を遮断することにより、結合を軽減または防止することができる。あるいは、ＴＳＬＰ／ＴＳＬＰ受容体拮抗剤は、ＴＳＬＰ受容体に結合し、ＴＳＬＰのその受容体との結合により通常誘発され得る、下流シグナル伝達を軽減または防止することができる。アゴニストおよび拮抗剤は、リガンド様ポリペプチド、抗体、およびその断片または部分配列等のポリペプチドを含む、種々のクラスの分子を含むことができる。アゴニストおよび拮抗剤はまた、融合ポリペプチド、抗体、ペプチド、（約２０アミノ酸未満の長さのペプチド等）、および小分子を含むこともできる。例示的な拮抗剤としては、ＴＳＬＰおよびＴＳＬＰ受容体に特異的な中和抗体、可溶性ＴＳＬＰ受容体分子、ならびに２つ以上の受容体鎖の構成成分をコード化し、それによって、生理学的な受容体二量体または高次オリゴマーを模倣する、ＴＳＬＰＲ免疫グロブリンＦｃ分子またはポリペプチド等の、ＴＳＬＰ受容体融合タンパク質が挙げられる。受容体が２つ以上のポリペプチド鎖を含む場合、一本鎖融合を利用することができる。

TSLP and TSLP-mediated states TSLP and TSLPR agonist / antagonist: have affinity for and stimulate physiological activity at cell receptors normally stimulated by naturally occurring substances and thereby biochemistry A drug that induces a physical response. A TSLP receptor agonist has an affinity for a TSLP receptor and stimulates the activity elicited by binding of TSLP to that receptor. For example, a TSLP / TSLP receptor agonist is a molecule that binds to the TSLP receptor and induces intracellular signaling. In contrast, an “antagonist” is an agent that inhibits the activity of cellular receptors normally stimulated by naturally occurring substances. Thus, a TSLP / TSLP receptor antagonist binds to TSLP or TSLP receptor, inhibits binding of TSLP to TSLP receptor, and / or exhibits activity normally induced by binding of TSLP to that receptor. Inhibit. For example, TSLP / TSLP receptor antagonists can reduce or prevent binding by binding to TSLP or TSLP receptor, eg, blocking binding of TSLP to TSLP receptor. Alternatively, a TSLP / TSLP receptor antagonist can bind to a TSLP receptor and reduce or prevent downstream signaling that can normally be triggered by binding of TSLP to that receptor. Agonists and antagonists can include various classes of molecules, including polypeptides such as ligand-like polypeptides, antibodies, and fragments or subsequences thereof. Agonists and antagonists can also include fusion polypeptides, antibodies, peptides (such as peptides of less than about 20 amino acids in length), and small molecules. Exemplary antagonists include TSLP and neutralizing antibodies specific for TSLP receptors, soluble TSLP receptor molecules, and components of two or more receptor chains, thereby providing physiological receptors TSLP receptor fusion proteins, such as TSLPR immunoglobulin Fc molecules or polypeptides that mimic dimers or higher order oligomers. If the receptor contains more than one polypeptide chain, single chain fusion can be utilized.

Antibody: A polypeptide ligand comprising at least one light chain or heavy chain immunoglobulin variable region that specifically recognizes and binds an epitope (eg, an antigen such as TSLP or a fragment thereof, or a TSLP receptor of the fragment). This is the Fab ′ fragment, F (ab) ′. sub. Includes complete immunoglobulins and variants and portions thereof well known in the art, such as two fragments, single chain Fv protein ("scFv"), and disulfide stabilized Fv protein ("dsFv") . The scFv protein is such that the immunoglobulin light chain variable region and the immunoglobulin heavy chain variable region are joined by a linker, whereas in dsFv, the chain introduces disulfide bonds to stabilize the chain association. It is a fusion protein mutated to The term also includes genetically engineered forms such as chimeric antibodies (eg, humanized mouse antibodies), heteroconjugated antibodies (eg, bispecific antibodies), and the like. Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, I11.), Kuby, J. et al. ^, Immunology, 3 rd Ed. , W .; H. Freeman & Co. See also, New York, 1997. Typically, an immunoglobulin has a heavy chain and a light chain. Each heavy and light chain contains a constant region and a variable region (regions are also known as “domains”). In combination, the heavy and light chain variable regions bind specifically to the antigen. Light and heavy chain variable regions contain a “framework” region that is blocked by three hypervariable regions, also called “complementarity determining regions” or “CDRs”. Framework regions and CDR ranges have been defined (see Kabat et al., Sequences of Proteins of Immunological Interstitut, U.S. Department of Health and Human Services, 1991, incorporated herein by reference). ). The Kabat database is currently managed online. The sequences of the different light or heavy chain framework regions are relatively conserved among species. The framework region of an antibody, ie, the complex framework region of light and heavy components, serves to position and align the CDRs in three-dimensional space. Examples of antibodies include monoclonal antibodies and humanized antibodies.

  TSLP antagonists inter alia encode small molecule antagonists, antibodies to TSLP, antibodies to TSLP receptors, and components of two or more receptor chains, whereby physiological receptor dimers or TSLP receptor fusion proteins such as TSLPR immunoglobulin Fc molecules or polypeptides that mimic higher order oligomers. TSLP has been shown to bind directly to the type I cytokine receptor superfamily member (also known as the erythropoiesis factor receptor superfamily member), TSLPR. TSLPR has been cloned. Functional high affinity receptors for TSLP have been demonstrated to contain two polypeptides, TSLPR and IL-7 receptor alpha chain. Thus, both TSLP and IL-7 share IL-7Rα as a component of their receptors. However, these receptors contain a common cytokine receptor gamma chain, whereas the TSLP receptor further contains TSLPR, while the IL-7 receptor also contains signaling components of various cytokine receptors. It is unique in that point. TSLPR (and Fc fusion of this receptor chain) is described, for example, in published US Patent Application No. 2002/0160949, which is incorporated herein by reference.

  Antibodies against TSLP polypeptides are known in the art. In addition, anti-TSLPR antibodies are commercially available (R & D Systems, Minneapolis, Minn., Cat. No. MAB981, DNAX Research, Inc., Palo Alto, Calif.). Antibodies are also available from Vector NTLRTM. Prepared against TSLP receptor or TSLP by the application of specific epitopes, such as increased antigenic regions as determined by Suite's Welling plot (Informax, Inc, Bethesda, Md.). The sequence of the TSLP receptor and the increased antigenic region in the human TSLP receptor are disclosed in US Patent Publication No. 2003/0186875. A pharmaceutical composition (see above) generally comprises a therapeutically effective amount of a TSLP antagonist and can also comprise additional agents. The preparation of the pharmaceutical composition is disclosed above.

Signs TSLP and TSLPR are diseases of the immune system, allergic inflammation, allergic airway inflammation, DC-mediated inflammation Th2 reaction, atopic dermatitis, atopic eczema, allergic asthma, asthma, obstructive airway disease, chronic obstructive lung It is believed to be involved in many types of allergic conditions, including but not limited to disease (COPD), food allergies, inflammatory arthritis, rheumatoid arthritis, psoriasis, IgE-mediated diseases, and rhinitis conjunctivitis. The involvement of TSLP in DC-mediated inflammatory Th2 responses has been shown in several references, including a recent review by Ziegler and Liu (Ziegler and Liu, Nat. Immunol., 7: 709-714, 2005, Which is incorporated herein by reference in its entirety).

  Allergic airway inflammation. Recent studies have demonstrated that TSLP is required for the initiation of murine allergic airway inflammation in vivo (Zhou et al., Nat. Immunol., 6: 1047-1053, 2005). In one study, Zhou et al. Featured lung-specific expression of TSLP transgenes characterized by massive leukocyte infiltration (including Th2 cells), goblet cell hyperplasia, and subepithelial fibrosis, and increased serum IgE levels. It has been demonstrated to induce allergic airway inflammation (asthma). In addition, recent studies have shown that allergen stimulation causes rapid accumulation of TSLP in the respiratory tract of asthmatic mice (Liyun Shi et al., Clin. Immunol., 129: 202-210, 2008). These results indicate that TSLP plays an important role in the pathogenesis of allergic airway inflammation. Here, Applicants show that the electrokinetically modified fluid of the present invention significantly down-regulated TSLP. According to certain embodiments, the electrokinetically modified fluids of the present invention have substantial utility for treating allergic airway inflammation and similar conditions.

  Allergic inflammation. A recent review summarizes and describes the results from studies investigating the role of TSLP in allergic inflammation, such as allergic dermatitis (Ziegler and Liu, 2008). Specifically, studies have shown that normal and non-lesional skin in patients with atopic dermatitis do not have detectable TSLP protein, while skin collected from acute and chronic atopic dermatitis lesions is It shows having high expression. In another study, mice lacking TSLPR were constructed and tested for effects on allergic dermatitis (He et al., PNAS, 105: 11875-11880, 2008, hereby incorporated by reference in its entirety. Incorporated into). He et al. Found that allergen-induced dermatitis in mice lacking TSLPR was significantly reduced compared to wild-type. In yet another study, mice engineered to overexpress TSLP in the skin were atopy characterized by eczema-like skin lesions including inflammatory infiltrates, a dramatic increase in circulating Th2 cells, and elevated serum IgE. It has been demonstrated to develop dermatitis (Yoo et al., J. Exp. Med., 202: 541-549, 2005). Studies have suggested that TSLP can directly activate DCs in mice. In another study conducted by Li et al., The group found that a transgenic mouse that overexpresses TSLP in the skin strengthens the association between TSLP and the development of atopic dermatitis. It was confirmed that dermatitis developed. These results indicate that TSLP plays an important role in the pathogenesis of allergic inflammation, such as allergic dermatitis (eg, atopic dermatitis and eczema). Here, Applicants show that the electrokinetically modified fluid of the present invention significantly down-regulated TSLP. According to certain embodiments, the electrokinetically modified fluid of the present invention is for treating allergic inflammation, such as allergic dermatitis (eg, atopic dermatitis and eczema), and similar conditions. Has substantial utility.

  psoriasis. Recent studies have shown that TSLP had substantially higher expression in skin biopsies taken from patients with acute psoriasis (Guttman-Yassky, et al., J. Allergy and Clinical Immunology 119: 1210). -1217, 2007). This result indicates that TSLP plays an important role in the pathogenesis of psoriasis. Here, Applicants show that the electrokinetically modified fluid of the present invention significantly down-regulated TSLP. Thus, according to certain embodiments, the electrokinetically modified fluids of the present invention have substantial utility for treating psoriasis and similar conditions.

  Allergic asthma. Recently, studies have shown that allergen stimulation causes rapid accumulation of TSLP in the respiratory tract of asthmatic mice (Liyun Shi et al., Clin. Immunol., 129: 202-210, 2008). In the same study, modulation of DC function by inhibiting TSLPR was shown to reduce the severity of mice. These results indicate that TSLP plays an important role in the pathogenesis of allergic asthma. Here, Applicants show that the electrokinetically modified fluid of the present invention significantly down-regulated TSLP. Thus, according to certain embodiments, the electrokinetically modified fluid of the present invention has substantial utility for treating allergic asthma and similar conditions.

  Obstructive airway disease. Recent studies have demonstrated that COPD is associated with increased bronchial mucosal expression of TSLP (Ying et al., J Immunol., 181: 2790-2798, 2008). COPD is a type of obstructive airway disease. This result indicates that TSLP plays an important role in the pathogenesis of obstructive airway diseases such as COPD. Here, Applicants show that the electrokinetically modified fluid of the present invention significantly down-regulated TSLP. Thus, according to certain embodiments, the electrokinetically modified fluids of the present invention have substantial utility for treating obstructive airway diseases, such as COPD.

  Food allergies. Intestinal dendritic cells were found to stimulate naïve T cells and bias them to TH2 responses in an OX40L-dependent manner (Blazquez AB, Berin MC. Gastrointestinal dendritic cells promote Th2 skewing via OX40L. 180: 4441-4450, 2008, which is incorporated herein by reference in its entirety). In addition, recent reviews have examined the presence of TSLP in the gut and its role in regulating immune homeostasis (Iliev ID, Mattelioli G, Resigno M. Theyin and yang of epithelial cells in controll. J Exp Med; 204: 2253-2257, 2007, which is incorporated herein by reference in its entirety). These results indicate that TSLP plays an important role in food allergy. Here, Applicants show that the electrokinetically modified fluid of the present invention significantly down-regulated TSLP. Thus, according to certain embodiments, the electrokinetically modified fluid of the present invention has substantial utility for treating food allergies and similar conditions.

  Inflammatory arthritis. Recent studies have found increased levels of TSLP in synovial fluid samples obtained from patients with rheumatoid arthritis (RA) compared to synovial fluid obtained from patients with other forms of arthritis (Koyayama et al. Biochem and Biophys Res Comm., 357: 99-104, 2007, which is incorporated herein by reference in its entirety). The study found that the use of anti-TSLP neutralizing antibodies improved TNF-a-dependent experimental arthritis induced by anti-type II collagen antibodies in mice. These results indicate that TSLP plays an important role in inflammatory arthritis such as RA. Here, Applicants show that the electrokinetically modified fluid of the present invention significantly down-regulated TSLP. Thus, according to certain embodiments, the electrokinetically modified fluids of the present invention have substantial utility for treating inflammatory arthritis and similar conditions, such as RA.

  allergic rhinitis. In recent studies, Mou et al. Found that TSLP was present at both mRNA and protein levels in the nasal mucosa of all study patients suffering from allergic rhinitis (AR) (Mou et al., Acta). Oto-laryngologica, 129: 297-301, 2009, which is incorporated herein by reference in its entirety). Furthermore, TSLP levels were closely correlated with AR severity. These results indicate that TSLP plays an important role in the pathogenesis of AR and / or rhinitis conjunctivitis. Here, Applicants show that the electrokinetically modified fluid of the present invention significantly down-regulated TSLP in the relevant model system. Thus, according to certain embodiments, the electrokinetically modified fluids of the present invention have substantial utility for treating AR, allergic rhinitis conjunctivitis, and similar conditions.

The term method of treatment "treatment (Treating)" are diseases, disorders, or conditions or normalizing one or more symptoms thereof, mitigation refers to inhibiting the progress of, or preventing, and meaning of these And “treatment” and “therapeutically” refer to the act of treatment, as defined herein.

  A “therapeutically effective amount” is provided herein that is sufficient to normalize, alleviate, inhibit or prevent the progression of a disease, disorder, or condition, or one or more of these symptoms. Any amount of any compound utilized in the course of practicing the invention.

  Certain embodiments herein relate to therapeutic compositions and methods for subjects by preventing or alleviating at least one symptom of inflammation associated with a condition or disease. Many conditions or diseases associated with inflammatory diseases include immunosuppressants including steroids, methotrexate, cyclophosphamide, cyclosporine, azathioprine, and leflunomide, non-steroids such as aspirin, acetaminophen and COX-2 inhibitors Are treated with anti-inflammatory drugs, gold drugs, and antimalarial drugs.

Routes of Administration and Forms As used herein, “subject” can refer to any organism, preferably an animal, more preferably a mammal, and even more preferably a human.

  In certain exemplary embodiments, the gas-enriched fluids of the present invention are used alone or in combination with another therapeutic agent so that the therapeutic composition prevents or alleviates at least one symptom of inflammation. May function as a therapeutic composition. The therapeutic compositions of the present invention include compositions that can be administered to a subject in need thereof. In certain embodiments, the therapeutic composition formulation may also include at least one additive selected from the group consisting of carriers, adjuvants, emulsifiers, suspending agents, sweeteners, flavors, fragrances, and binders.

  As used herein, “pharmaceutically acceptable carrier” and “carrier” are generally non-toxic, inert solid, semi-solid, or liquid fillers, diluents, encapsulating materials, or Refers to all types of formulation aids. Some non-limiting examples of substances that can serve as pharmaceutically acceptable carriers include sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose, and derivatives thereof. Sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; tragacanth powder; malt; gelatin; talc; excipients such as cocoa butter and suppository; peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil Oils such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Re Gel solutions; ethyl alcohol, and phosphate buffer solutions, and other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, and according to the inventor's judgment, colorants, release agents, coating agents, Sweetening agents, flavoring agents, and flavoring agents, preservatives and antioxidants may also be present in the composition. In certain embodiments, such carriers and excipients can be gas-enriched fluids or solutions of the present invention.

  The pharmaceutically acceptable carriers described herein are known to those skilled in the art, for example, vehicles, adjuvants, excipients, or diluents. In general, pharmaceutically acceptable carriers are chemically inert to the therapeutic agents and do not have deleterious side effects or toxicity under the conditions of use. Pharmaceutically acceptable carriers can include polymers and polymer matrices, nanoparticles, microbubbles, and the like.

  In addition to the therapeutic gas-enriched fluid of the present invention, the therapeutic composition comprises additional inert diluents such as additional non-gas enriched water or other solvents, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, Benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oil (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol, And solubilizers and emulsifiers, such as fatty acid esters of sorbitan, and combinations thereof. As will be apparent to those skilled in the art, new and improved formulations of specific therapeutic compositions, new gas-enriched therapeutic fluids, and new methods of delivering new gas-enriched therapeutic fluids are identical, similar Or by replacing one or more inert diluents with a gas-enriched fluid of a different composition. For example, conventional water can be replaced or supplemented by a gas-enriched fluid produced by mixing oxygen with water or deionized water to provide a gas-enriched fluid.

  In certain embodiments, the gas-enriched fluids of the present invention can be combined with one or more therapeutic agents and / or used alone. In certain embodiments, conjugating the gas-enriched fluid includes replacing one or more solutions known in the art, such as deionized water, saline solution, etc., with one or more gas-enriched fluids. , Thereby providing an improved therapeutic composition for delivery to a subject.

  Certain embodiments comprise a therapeutic composition comprising a gas-enriched fluid, pharmaceutical composition, or other therapeutic agent of the invention, or a pharmaceutically acceptable salt or solvent thereof, and at least one pharmaceutical carrier or diluent. I will provide a. These pharmaceutical compositions can be used in the prevention and treatment of the aforementioned diseases or conditions, and in therapy as described above. Preferably, the carrier must be pharmaceutically acceptable and should be compatible, ie, have no deleterious effects on other ingredients in the composition. The carrier can be solid or liquid and is preferably formulated as a unit dose formulation, for example a tablet which may contain from 0.05 to 95% by weight of the active ingredient.

  Possible routes of administration include oral, sublingual, buccal, parenteral (eg, subcutaneous, intramuscular, intraarterial, intraperitoneal, intracisternal, intravesical, intrathecal, or intravenous), rectal, topical ( Transdermal, intravaginal, intraocular, intraauricular, intranasal, inhalation, and injection or insertion of implantable devices or substances).

The optimal route of administration to a particular subject depends on the nature and severity of the disease or condition being treated, or the nature of the treatment used and the nature of the therapeutic composition or additional therapeutic agent. In certain embodiments, oral or topical administration is preferred.

  Formulations suitable for oral administration are tablets, capsules, cachets, syrups, elixirs, chewing gums, “lollipop” agents, microemulsions, solutions, suspensions, troches, each containing a predetermined amount of the active compound. Or as individual units such as gel-coated ampoules, as powders or granules, as solutions or suspensions in aqueous or non-aqueous liquids, or as oil-in-water or water-in-oil emulsions .

  Formulations suitable for transmucosal methods, such as sublingual or buccal administration, include lozenges, patches, tablets, etc., and gelatin and glycerin or sucrose acacia containing active compounds and generally flavor bases such as sugar and acacia or tragacanth. Such pastels contain the active compound in an inert base.

  Formulations suitable for parenteral administration generally comprise a sterile aqueous solution containing a pre-determined concentration of active gas-enriched fluid and possibly another therapeutic agent, the solution preferably comprising the blood of the intended recipient. Isotonic solution. Additional formulations suitable for parenteral administration include formulations containing physiologically suitable cosolvents and / or complexing agents such as surfactants and cyclodextrins. Oil-in-water emulsions may also be suitable for formulations for parenteral administration of gas-enriched fluids. Such a solution is preferably administered intravenously, but may be administered by subcutaneous or intramuscular injection.

  Formulations suitable for urethral, rectal or vaginal administration include gels, creams, lotions, aqueous or oil suspensions, dispersible powders or granules, emulsions, absorbable solid substances, irrigation and the like. The formulation is preferably provided as a unit dose suppository containing the active ingredient in one or more solid carriers forming a suppository base, for example cocoa butter. Alternatively, colon lavage using the gas-enriched fluids of the present invention can be formulated for colon or rectal administration.

  Formulations suitable for topical, intraocular, intraaural or intranasal administration include ointments, creams, pastes, lotions, gels (hydrogels, etc.), sprays, dispersible powders and granules, emulsions, fluid propellants Sprays or aerosols (eg, liposomal sprays, nasal drops, nasal sprays, etc.), as well as oils. Suitable carriers for such formulations include petrolatum, lanolin, polyethylene glycol, alcohol, and combinations thereof. Nasal or intranasal administration may include quantitative amounts of these formulations or any other. Similarly, the ear or eyeball may contain drops, ointments, irrigation fluids and the like.

  The formulations of the present invention are optionally mixed in a uniform and intimate manner with the gas enriched fluid in any suitable manner, generally a liquid or finely divided solid carrier, or both, and the gas-enriched fluid in the required ratio. Then, if necessary, it can be prepared by shaping the resulting mixture into the desired shape.

  For example, a tablet may compress a homogeneous mixture comprising a powder or granules of the active ingredient, and one or more optional ingredients such as a binder, lubricant, inert diluent or surfactant dispersant, or a powder active ingredient And can be prepared by shaping a homogeneous mixture of the gas-enriched fluid of the present invention.

  Formulations suitable for administration by inhalation include particulate powders or mists that can be generated using various types of metered pressure aerosols, nebulizers, or inhalers. In particular, therapeutic agent powders or other compounds may be dissolved or suspended in the gas-enriched fluid of the present invention.

  For pulmonary administration by mouth, the particle size of the powder or droplets is generally 0.5-10 μM, preferably 1-5 μM, to ensure delivery to the bronchial tree. For nasal administration, a particle size of 10-500 μM is preferred to ensure retention in the nasal cavity.

  A metered dose inhaler is a pressurized aerosol dispenser that generally contains a suspension or solution formulation of a therapeutic agent in a liquefied propellant. In certain embodiments, as disclosed herein, the gas-enriched fluid of the present invention may be used in addition to or instead of a standard liquefied propellant. In use, these devices release the formulation via a valve adapted to deliver a metered amount, generally 10-150 μL, to produce a fine particle spray containing the therapeutic agent and a gas-enriched fluid. Suitable propellants include certain chlorofluorocarbon compounds such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, and mixtures thereof.

  The formulation may further contain one or more co-solvents such as ethanol surfactants such as oleic acid or sorbitan trioleate, antioxidants, and suitable flavors. A nebulizer is a commercially available device, which is a solution or suspension of an active ingredient, either by acceleration of a compressed gas (typically air or oxygen) through a narrow venturi hole, or by ultrasonic agitation. Is a device that transforms into a therapeutic aerosol mist. A formulation suitable for use in a nebulizer consists of another therapeutic agent in a gas-enriched fluid and comprises up to 40% w / w, preferably less than 20% w / w of the formulation. In addition, other carriers may be made isotonic with body fluids, preferably using distilled water, sterile water, dilute aqueous alcohol solution, or the like, for example, by the addition of a salt such as sodium chloride. Optional additives include preservatives, particularly when the formulation is not sterile prepared, and may include methylhydroxy-benzoates, antioxidants, flavoring agents, volatile oils, buffering agents, and surfactants.

  Formulations suitable for administration by inhalation include finely divided powders that can be delivered by inhaler or taken into the nasal cavity by inhalation through the nose. In an inhaler, powders are placed in capsules or cartridges, typically made of gelatin or plastic, which are pierced or opened in situ and the powder is delivered by air drawn from the device during inhalation or by a manual pump . Powders employed in inhalers consist of the active ingredient alone or of a powder blend containing the active ingredient, a suitable powder diluent such as lactose, and optional surfactants.

  The active ingredient generally comprises from 0.1 to 100 w / w of the formulation. In addition to the ingredients specifically mentioned above, the formulations of the present invention may include other agents known to those skilled in the art in view of the type of formulation in question. For example, formulations suitable for oral administration can include flavorings, and formulations suitable for nasal administration can include flavorings.

  The therapeutic compositions of the present invention can be administered by any conventional method usable with pharmaceutical preparations, either as individual therapeutic agents or in combination with therapeutic agents.

  Of course, the dose administered will depend on the pharmacodynamic properties of the particular drug, as well as the mode and route of administration thereof; the age, health status, and weight of the recipient; the nature and range of symptoms; the type of co-treatment; The frequency of treatment; as well as known factors such as the desired effect may vary. The daily dose of active ingredient is about 0.001 to 1000 milligrams (mg) per kilogram (kg) of body weight, preferably 0.1 to about 30 mg / kg can be expected. .

  Dosage forms (suitable compositions for administration) include from about 1 mg to about 500 mg of active ingredient per unit. In these pharmaceutical compositions, the active ingredient may usually be present in an amount of about 0.5 to 95% by weight, based on the total weight of the composition.

  Ointments, pastes, foams, occlusions, creams, and gels are also excipients such as starch, tragacanth, cellulose derivatives, silicon, bentonite, silica acids, and talc, or mixtures thereof May also be included. Powders and sprays can also contain excipients such as lactose, talc, silica acid, aluminum hydroxide, and calcium silicate, or mixtures of these substances. Nanocrystalline antibacterial metal solutions can be converted to aerosols or sprays by any of the known means routinely used to make aerosol pharmaceuticals. In general, such methods usually involve using an inert carrier gas to pressurize the container of the solution, or provide a means for pressurization, and to pass the pressurized gas through a small hole. Including. The spray may further contain conventional propellants such as nitrogen, carbon dioxide, and other inert gases. In addition, the microspheres or nanoparticles can be employed with the gas-enriched therapeutic composition or fluid of the present invention in any route required to administer the therapeutic compound to the subject.

  Injectable formulations may be in unit or multi-dose sealed containers such as ampoules and vials, freeze-dried (just prior to use with the addition of sterile liquid excipients or gas-enriched fluids) It can be stored in a (lyophilized) state. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets. The requirements for effective pharmaceutical carriers for injectable compositions are well known to those of ordinary skill in the art. See, for example, Pharmaceuticals and Pharmacy Practice, J. et al. B. Lippincott Co. , Philadelphia, Pa. , Banker and Chalmers, Eds. , 238-250 (1982) and ASHP Handbook on Injectable Drugs, Toissel, 4th ed. 622-630 (1986).

  Suitable formulations for topical administration include the gas-enriched fluids of the invention and optionally a troche containing additional treatment and flavor, usually sucrose and acacia or tragacanth; an inert base such as gelatin and glycerin or sucrose and acacia Pastel containing gas-enriched fluid and optionally additional therapeutic agent; mouthwash or gargle containing gas-enriched fluid and optionally additional therapeutic agent in a suitable liquid carrier; and creams, emulsions, gels Agents and the like.

  Additionally, formulations suitable for rectal administration can be presented as suppositories by mixing with a variety of bases such as emulsifying bases or water-soluble bases. Formulations suitable for intravaginal administration include pessaries, tampons, creams, gels, pastes, foams, or active ingredients plus carriers known to be appropriate in the art Can be shown as a spray.

  Suitable pharmaceutical carriers are described in the standard reference text in the field “Remington's Pharmaceutical Sciences (Mack Publishing Company)”.

  In the context of the present invention, the dose administered to a subject, particularly an animal, specifically a human, should be sufficient to produce an animal therapeutic response over an appropriate time frame. One skilled in the art will appreciate that dosages can vary depending on a variety of factors, including the condition of the animal, the weight of the animal, and the condition to be treated. A suitable dose is one that can result in a concentration of the therapeutic composition in the subject that is known to affect the desired response.

  The size of the dose can also be determined by the route, timing and frequency of administration, as well as the presence, nature, and extent of any side effects that can entail administration of the therapeutic composition and the desired physiological effect.

  The compounds of the combination can be (1) delivered simultaneously by a combination of compounds in a co-formulation, or (2) delivered in separate pharmaceutical formulations in alternation, ie sequentially, sequentially, in parallel, or simultaneously. It will be appreciated that it can be administered. In alternating treatment, delayed administration of the second and optionally third active ingredient should not lose the synergistic therapeutic benefit of the active ingredient combination. According to certain embodiments according to the administration method of either (1) or (2), ideally the combination should be administered to achieve the most effective results. In certain embodiments according to either (1) or (2) method of administration, ideally the combination should be administered to achieve the respective peak plasma concentration of the active ingredient. One tablet once-per-day regimen once daily by administration of the combination co-formulation may be feasible for some patients suffering from inflammatory neurodegenerative diseases. According to certain embodiments, the effective peak plasma concentration of the active ingredients in the combination can range from about 0.001 to 100 μM. The optimal peak plasma concentration can be achieved by the formulation and dosing regimen prescribed for a particular patient. The fluids and glucocorticoid steroids (eg, budesonide) or any of these physiologically functional derivatives of the present invention, whether given simultaneously or sequentially, individually, in multiples, or any It will also be understood that these combinations can be administered. In general, during alternation therapy (2), an effective dosage of each compound is administered sequentially, and in co-formulation therapy (1), an effective dosage of two or more compounds is administered together.

  The combination of the present invention can be appropriately represented as a pharmaceutical preparation in a unit dosage form. Convenient unit dosage formulations each contain the active ingredient in any amount from 1 mg to 1 g (eg, but not limited to 10 mg to 300 mg). For example, the synergistic effect of the fluid of the present invention in combination with a glucocorticoid steroid (eg, budesonide) can be, for example, 1: 50-50: 1 (fluid: glucocorticoid steroid (eg, budesonide) of the present invention over a wide ratio. ). In one embodiment, the ratio can range from 1:10 to 10: 1. In another embodiment, the weight / weight ratio of the fluid of the invention to a glucocorticoid steroid (eg, budesonide) in a co-formulation combination dosage form such as a pill, tablet, caplet, or capsule is about 1 That is, approximately equal amounts of the fluid of the invention and a glucocorticoid steroid (eg, budesonide). In other exemplary co-formulations, there may be more or less fluid of the present invention and glucocorticoid steroid (eg, budesonide). In one embodiment, each compound may be utilized in combination in amounts that exhibit anti-inflammatory activity when used alone. Other ratios and amounts of the combinations of compounds are contemplated within the scope of the present invention.

  Unit dosage forms may further comprise a fluid of the invention and, for example, a glucocorticoid steroid (eg, budesonide), or a physiologically functional derivative of any of these, and a pharmaceutically acceptable carrier. .

  The amount of active ingredient in the combination of the invention required for use in therapy varies according to various factors, including the nature of the condition being treated and the age and condition of the patient, and ultimately Those skilled in the art will recognize that this is at the discretion of the attending physician or health care provider. Factors to be considered include the route of administration and the nature of the formulation, the animal's weight, age and general condition, and the nature and severity of the disease to be treated.

  It is also possible to combine any two active ingredients in unit dosage form for simultaneous or sequential administration with a third active ingredient. The combination of the three parts can be administered simultaneously or sequentially. When administered sequentially, the combination can be administered in two or three doses. According to certain embodiments, the combination of the three portions of the fluid of the invention and a glucocorticoid steroid (eg, budesonide) can be administered in any order.

  In accordance with certain aspects, the electrokinetically modified fluids of the invention treat TSLP and / or TSLPR mediated conditions, including but not limited to the exemplary genera of indications disclosed herein. Has substantial utility for. According to a further aspect, the electrokinetically modified fluid of the present invention has utility for treating various subgeneras of exemplary genera, wherein at least one indication of the genera is subgenus Excluded from each.

Example 1
(Showed synergistic effect of electrokinetically modified fluid and albuterol of the present invention)
wrap up. The electrokinetically modified fluid of the present invention can be used in vivo in animal models recognized in the art of human bronchoconstriction (human asthma model) using synergistic prolongation effects (eg bronchial) using albuterol in vivo. (Suppression of contraction), and therefore a reduction in the patient's albuterol usage. The results disclosed in this example are also disclosed in Applicant's International Publication No. 2009/055729.

  First experiment. In the first experiment, 16 guinea pigs were evaluated for the effect of bronchodilators on airway function along with methacholine-induced tracheal contraction. After determining the optimal dose, each animal was dosed at 50 μg / mL to deliver a target dose of 12.5 μg albuterol sulfate at 250 μL per animal. The study was a randomized block design for body weight and baseline PenH values. Two groups (A and B) were given 250 μL intratracheal infusion of 50 μg / mL albuterol sulfate in one or two diluents: Group A was a device of the invention without the addition of oxygen , While Group B was the gas-enriched water of the present invention. Each group was dosed intratracheally with the solution using a Penn Century Micro sprayer. In addition, the animals were stratified across the BUXCO plethysmographic units so that each treatment group is shown uniformly in the nebulizer that supplies the plethysmograph and recording unit. Animals that showed at least 75% of their baseline PenH value 2 hours after albuterol administration were not included in the data analysis. This exclusion criterion is based on past studies where defects to observe bronchoprotection with bronchodilators can be associated with dosing errors. As a result, one animal from the control group was excluded from the data analysis. Animals were considered unprotected if they had more than 50% bronchoconstriction. 50% of the animals in group B were protected from bronchoconstriction for up to 10 hours (end of study).

  Second experiment. In male guinea pigs, when administered albuterol sulfate alone or as a diluent, electrokinetically generated fluids of the present invention against methacholine-induced bronchoconstriction (eg, RDC 1676-00, RDC 1676-01, RDC 1676). In order to evaluate the protective effect of -02, and RDC1676-03), an additional series of experiments was performed using large numbers of animals.

  Materials and methods. Guinea pigs (Caviar porcellus) are available from Charles River Canada Inc. Hartley-based albino from (St. Constant, Quebec, Canada), Crl: (HA) BR. Body weight: about 325 ± 50 g at the start of treatment. The number of groups was 32, with 7 male animals per group (plus 24 spares form the same batch of animals). Diet; All animals were fed ad libitum with a standard certified pelleted commercial experimental diet (PMI Certified Guinea Pig 5026; PMI Nutrition International Inc.) except at designated treatments. The route of administration was methacholine stimulation via intra-airway infusion via Penn Century Microsprayer and systemic inhalation. The endotracheal route was selected to maximize pulmonary exposure to the test substance / control solution. Systemic inhalation stimulation has been selected for methacholine stimulation to cause an upper airway hypersensitivity reaction (ie bronchoconstriction). The duration of treatment was one day.

  Experimental design. All animals received inhalation exposure to methacholine (500 μg / mL) 2 hours after TA / control administration. All animals received a dose volume of 250 μL. Thus, albuterol sulfate was diluted to concentrations of 0, 25, 50, and 100 μg / mL (in the control and 4 test substances). Thirty minutes prior to dosing, four different concentrations (0, 25, 50, and 100 μg / mL) of albuterol sulfate solution were added to these four test substance solutions (RDC1676-00, RDC1676-01, RDC1676-02, and RDC1676). -03) in 1 Ox stock (500 μg / mL). These concentrations of albuterol sulfate were also made in a non-electrokinetically generated control fluid (Control 1). Dosing solutions were prepared by making appropriate dilutions of each stock solution. All stock and dosing solutions were kept on ice as soon as they were prepared. Dosing was completed within 1 hour of creating the test / control substance. A solution of methacholine (500 μg / mL) was prepared on the day of administration.

  Each animal received an intratracheal infusion of test or control substance using a Penn Century microsprayer. The animals were fasted overnight, anesthetized with isoflurane, the larynx visualized under a laryngoscope (or a suitable alternative) and the tip of a microsprayer inserted into the trachea. A dose volume of test substance or control substance of 250 μL / animal was administered. The methacholine aerosol was generated at the air inlet of the mixing chamber using an Aeroneb ultrasonic nebulizer supplied with air from a Buxco bias flow pump. Similarly, this mixing chamber supplied four individual whole body unrestrained plethysmographs, each operated under slightly negative pressure, maintained by a gate valve located in the exhaust line. A vacuum pump was used to evacuate the suction chamber at the required flow rate.

  Prior to the start of the main phase of the study, 12 preliminary animals are assigned to 3 groups (n = 4 / group) so that the animals induce severe but non-fatal acute bronchoconstriction. The maximum exposure period that could be exposed to methacholine was determined. Four animals were exposed to methacholine (500 μg / mL) for 30 seconds and respiratory parameters were measured until 10 minutes after starting the aerosol. The methacholine nebulizer concentration and / or aerosolization exposure time was appropriately adjusted to induce severe but non-fatal acute / reversible bronchoconstriction as characterized by a transient increase in Penh .

  Prior to test substance administration (Day 1) and at 2, 6, 10, 14, 18, 22, and 26 hours after dosing, animals are placed in the chamber and aerosol stimulation to methacholine begins. Later, ventilation parameters (tidal volume, respiratory rate, induced minute excretion) and enhanced pause Penh were measured for 10 minutes using a Buxco Electronics BioSystem XA system. At the time the animal is in the chamber, the baseline value is recorded for 1 minute, and then a methacholine nebulizer concentration of 500 μg / mL is aerosolized for 30 seconds when the animal continuously evaluates the ventilation parameters. It was exposed to aerosol for an additional 10 minutes. Penh is used as an index of bronchoconstriction, and Penh is an induced value obtained from peak inspiratory flow, peak expiratory flow, and expiratory time. Penh = (peak expiratory flow / peak inspiratory flow) * (expiration time / time-1 to exhale 65% of expiratory volume).

  Animals that did not show severe acute bronchoconstriction upon prior administration of methacholine stimulation were replaced. None of the animals that showed at least 75% of their baseline PenhPenes values 2 hours after dosing were included in the data analysis. Respiration parameters were recorded as a 20 second method. Data deemed to be non-physiological was further excluded from the analysis. The change in Penh was plotted over 15 minutes and the Penh value was expressed as the area under the concentration curve. For the numerical data, group mean values and standard deviations (as specified) were calculated.

  result. The results from this experiment show that in the absence of albuterol, the administration of the electrokinetically generated fluid of the present invention had no apparent effect on the baseline% PenH value when measured up to 26 hours. Showed that. However, surprisingly, administration of albuterol formulated in the electrokinetically generated fluid of the present invention (representing representative data for 25 μg albuterol / animal group) (all tested At ambient oxygen levels; ambient, 20 ppm, 40 ppm, and 60 ppm) resulted in a significant prolongation of the anti-bronchoconstrictive action of albuterol compared to the control fluid. That is, methacholine results showed a prolonged bronchodilation of albuterol at least 26 hours. Applicants also showed that there was a consistent difference in all oxygen levels between RDC1676 and saline controls. When all four RDC1676 fluids were synthesized, the p-value, which is the difference between all treatments and saline, was 0.03.

  Thus, according to a particular embodiment, the electrokinetically generated solution of the present invention provides a synergistic prolongation effect with albuterol, thus providing a reduction in patient albuterol use and being more efficient Cost-effective drug use, allowing fewer side effects and increasing the period during which patients can be treated and respond to treatment with albuterol.

Example 2
(Shows the effect of the electrokinetically modified fluid of the present invention on cytokine expression)
wrap up. The electrokinetically modified fluids of the present invention, compared to the control fluid, are inflammatory cytokines (IL-1β, TNF-a, IL-6, and GM-CSF), chemokines (IL-8, MIP -1a, RANTES, and eotaxin), inflammatory enzymes (iNOS, COX-2, and MMP-9), allergen responses (MHC class II, CD23, B7-1, and B7-2), and Th2 cytokines (IL- 4, IL-13, and IL-5) were decreased and anti-inflammatory cytokines (eg, IL1R-α, TIMP) were increased compared to control fluids. The results disclosed in this example are also disclosed in the applicant's International Publication No. 2009/055729.

  In certain aspects, the human mixed lymphocytes are stimulated with T3 antigen or PHA in Revalesio oxygen-enriched fluid or control fluid, and IL-1β, IL-2, IL-4, IL-5, IL-6, IL -7, IL-8, IL-10, IL-12 (p40), IL-12 (p70), IL-13, IL-17, eotaxin, IFN-γ, GM-CSF, MIP-1β, MCP-1 , G-CSF, FGFb, VEGF, TNF-a, RANTES, leptin, TNF-β, TFG-β, and NGF were evaluated. As shown, tested inflammatory cytokines (IL-1β, TNF-a, IL-6, and GM-CSF), chemokines (IL-8, MIP-1a, RANTES, and eotaxin), inflammatory enzymes ( iNOS, COX-2, and MMP-9), allergen responses (MHC class II, CD23, B7-1, and B7-2), and Th2 cytokines (IL-4, IL-13, and IL-5) are Decrease in test fluid vs. control fluid. In contrast, tested anti-inflammatory cytokines (eg, IL1R-a, TIMP) were increased in the test fluid versus the control fluid.

  In addition, Applicants used a model system recognized in the art, including ovalbumin sensitization, to assess allergic hypersensitivity reactions. The endpoints studied were specific cytological and cellular components of the reaction, as well as serological measurements of protein and LDH. Cytokine analysis was performed, including analysis of eotaxin, IL-1A, IL-1B, KC, MCP-1, MCP-3, MIP-1A, RANTES, TNF-A, and VCAM.

Briefly, male Brown Norway rats were given a solution containing aluminum hydroxide (Al (OH) ₃ ) (200 mg / mL) once each on day 1, 2 and 3 (200 mg / mL). Intraperitoneal injection of 0.5 mL ovalbumin (OVA) grade V (A5503-1G, Sigma) in 2.0 mg / mL). The study was a 2 x 2 factorial randomized treatment (Group 4). After a 2-week waiting period until an immune response is generated, the rats are exposed or RDC1676-00 (sterile saline treated through Revalesio's proprietary device), or RDC1676-01 (added further) Treated with oxygen (sterile saline treated through Revalesio's proprietary device) for one week. At the end of the once-daily treatment for one week, the two groups were divided in half and in each group 50% of the rats received either saline or OVA stimulation by inhalation.

  Specifically, 14 days after initial serialization, 12 rats were exposed to RDC 1676-00 by inhalation for 30 consecutive days for 7 consecutive days. The airflow rate through the system was set at 10 liters / minute. A total of 12 rats were lined up in a pie chamber with a single port that placed the spray material into the 12 subchambers of Aeroneb and evenly distributed.

  Fifteen days after initial serialization, 12 rats were exposed to RDC1676-01 by ultrasonic nebulization for 7 consecutive days for 30 minutes. The airflow was also set at 10 liters / minute and the same nebulizer and chamber were used. First, RDC1676-00 was sprayed and the Aeroneb chamber was completely dried before spraying RDC1676-01.

  Approximately 2 hours after the final nebulization procedure, 6 rats from the RDC 1676-00 group were treated with OVA (1 in saline) delivered by intratracheal infusion using the Penn Century Microsprayer (Model 1A-1B). %). The other 6 rats from the RDC 1676-00 group were stimulated with saline as a control group delivered by intratracheal infusion. The next day, the procedure was repeated with RDC 1676-01 group.

  Twenty-four hours after restimulation, all rats in each group were euthanized by overdose of pentobarbital sodium. Whole blood samples were collected from the inferior vena cava and placed into two separate blood collection tubes, Qiagen PAXgene® Blood RNA Tube and Qiagen PAXgene® Blood DNA Tube. In this model, lung organs were treated to assess changes in markers of cytokine expression known to be associated with lung inflammation, bronchoalveolar lavage (BAL) fluid and RT-PCR for RT-PCR Got the organization. In order to maintain the integrity of the four lobes on the right side of the lung, a unilateral lavage technique was employed. The left “large” leaf was washed, while the four right lobes were tied and immediately placed with TRI-zol®, homogenized and sent to the laboratory for further treatment.

  BAL analysis. Lung lavage was collected and centrifuged at 4O <0> C at 600-800g for 10 minutes to precipitate the cells. The suspension was transferred to a new tube and frozen at -80 ° C. Bronchial lavage fluid (“BAL”) was aliquoted into two aliquots. The first aliquot was allowed to settle and the suspension was snap frozen on crushed dry ice, placed in -80 ° C and shipped to the laboratory for further treatment. The amount of protein and LDH present indicates the level of serum protein (protein is the serum component that leaks through the membrane when stimulated, as in this experiment) and cell death, respectively. The unique test side showed slightly less protein than the control.

  A second aliquot of bronchial lavage fluid was evaluated for total protein and LDH content and subjected to cytological examination. The treatment group showed that there were more total cells than the saline control group. Furthermore, there was an increase in eosinophils in the treated group relative to the control group. There were also slightly different polymorphonuclear cells in the treated group versus the control group.

  Blood analysis. Whole blood was analyzed by transferring 1.2-2.0 mL of blood into one tube and allowed to clot for at least 30 minutes. The remaining blood sample (approximately 3.5-5.0 mL) was stored for RNA extraction using TRI-zol® or PAXgene®. Next, the coagulated blood sample was centrifuged at 1200 g for 10 minutes at room temperature. Serum (float) was removed and placed in two new tubes and the serum was stored at -80 ° C.

  For RNA extraction utilizing Tri-Reagent (TB-126, Molecular Research Center, Inc.), 0.75 mL of TRI Reagent BD, supplemented with 0.2 μL of whole blood or 20 μL of 5N acetic acid per plasma, 0.2 mL whole blood or plasma was added. The tube was shaken and stored at -80 ° C. Using PAXgene®, the tubes were incubated for approximately 2 hours at room temperature. Tubes were then placed on their side and stored in a −20 ° C. freezer for 24 hours and then moved to −80 ° C. for long-term storage.

  Luminex analysis. With the Luminex platform, microbead analysis is utilized as a substrate for antibody-related binding reactions, which can be read in the photometric unit and compared to quantification standards. Each blood sample was examined as two samples simultaneously. The measurement unit was a luminosity unit and the groups were divided into OVA-stimulated control groups, OVA-stimulated treatment groups, and saline-stimulated unique fluid-treated groups.

  Lung tissue was isolated and submerged in TRI reagent (TR118, Molecular Research Center, Inc.) for generation of Agilent gene array data. Briefly, approximately 1 mL of TRI reagent was added to 50-100 mg of tissue in each tube. Samples were homogenized in TRI reagent using a glass Teflon® or Polytron® homogenizer. Samples were stored at -80 ° C.

  Results from blood samples. Each blood sample was divided into two samples and the samples were examined simultaneously. The units of measurement are light intensity units and groups, from left to right, OVA-stimulated controls, OVA-stimulated Revalesio treatment, then saline-stimulated treatment, and saline-stimulated Revalesio treatment It is. For ease of review, both RDC1676-01 groups are highlighted with a gray shaded background, while the saline control treatment group is an unshaded background.

  In general, compared to the two left groups, the spread of the RDC1676-01 group data is slightly larger, while overall, the specific cytokine levels in the RDC1676-01 group are smaller than the samples in the control treatment group, There is a numerical difference of about 30% between the two groups. In general, when comparing the two rightmost groups, the RDC1676-01 group has a slightly higher number compared to the RDC1676-00 group.

  Applicants believe that the level of RANTES (IL-8 superfamily) produced after treatment with the electrokinetically modified fluid of the present invention is higher than that produced by the group exposed to saline alone. Judged to be low. Applicants have shown that the electrokinetically modified fluid of the present invention produced MCP-1 at a lower level compared to that produced by the group exposed to saline alone. It was. Applicants determined that the level of TNFα produced after treatment with the electrokinetically modified fluid of the present invention was lower than that produced by the group exposed to saline alone.

  Furthermore, Applicants have found that the level of MIP-1α produced after treatment with the electrokinetically modified fluid of the present invention was lower than that produced by the group exposed to saline alone showed that. Applicants have shown that the electrokinetically modified fluid of the present invention produced IL-1α at a lower level compared to that produced by the group exposed to saline alone. It was. Applicants have observed that the level of Vcam produced after treatment with the electrokinetically modified fluid of the present invention was lower than that produced by the group exposed to saline alone. Applicants observed that the level of IL-1β produced after treatment with the electrokinetically modified fluid of the present invention was lower than that produced by the group exposed to saline alone. did. Applicant has determined that the electrokinetically modified fluid of the present invention produced eotaxin and MCP-3 at lower levels compared to those produced by the group exposed to saline alone. showed that.

  In summary, this standard assay of inflammatory response to known sensitization has produced significant clinical and serological effects, at least in blood samples. In addition, a significant number of control animals were physiologically stressed and critically dead in the process, but none of the RDC 1676-01 treated groups showed such clinical stress effects. This was then reflected in the blood levels of cytokines, which had an approximately 30% difference between the RDC1676-01 treated group and the RDC1676-01 treated group in the OVA-stimulated group. In contrast, there was a small amount of cytokines, cells, and serological profile, and very slight changes between the RDC1676-01 treated group and the RDC1676-01 treated group in the OVA unstimulated group, It is considered merely indicative of a minimal baseline change in the fluid itself.

Example 3
(Determined the effect of the electrokinetically modified fluid of the present invention to regulate T cell proliferation)
wrap up. The electrokinetically modified fluid of the present invention improved regulatory T cell function, as indicated by a relatively reduced proliferation. The results disclosed in this example are also disclosed in the applicant's International Publication No. 2009/055729.

  The ability of certain embodiments disclosed herein to control T cells was studied by irradiating antigen presenting cells and introducing antigen and T cells. In general, these stimulated T cells proliferate. However, normal T cell proliferation is suppressed upon introduction of regulatory T cells.

  Method. Briefly, the FITC-conjugated anti-CD25 (ACT-1) antibody used for selection was purchased from DakoCytomation (Chicago, IL). Other antibodies used were as follows. CD3 (HIT3a for soluble conditions), GITR (PE conjugated type), CD4 (Cy-5 and FITC conjugated type), CD25 (APC conjugated type), CD28 (CD28.2 clone), CD127-APC, Granzyme A (PE conjugated), FoxP3 (BioLegend), mouse IgG1 (isotype controlled), and XCL1 antibody. All antibodies were used according to manufacturer's instructions.

  CD4 + T cells were isolated from peripheral whole blood using a CD4 + Rosette kit (Stemcell Technologies). CD4 + T cells were incubated with anti-CD127-APC, anti-CD25-PE, and anti-CD4-FITC antibodies. Cells were sorted by flow cytometry using FACSAria on CD4 + CD25hiCD127lo / nTreg and CD4 + CD25 reactive T cells.

Inhibition assays were performed in round bottom 96 well microtiter plates. As indicated, 3.75 × 103 CD4 + CD25 negative reactive T cells, 3.75 × 103 self-T regulatory, 3.75 × 104 allogeneic irradiated CD3 depleted PBMC were added. All wells were supplemented with anti-CD3 (5.0 ug / mL, clone HIT3a). T cells were cultured for 7 days at 37 ° C. in RPMI 1640 medium supplemented with 10% fetal calf serum. Sixteen hours before the end of the incubation, 1.0 mCi of ³ H-thymidine was added to each well. Plates were harvested using a Tomtec cell harvester and ³ H-thymidine incorporation was determined using a Perkin Elmer scintillation counter. Antigen presenting cells (APC) consisted of StemSep human CD3 + T cell depletion (StemCell Technologies) and then peripheral blood mononuclear cells (PBMC) depleted from T cells using 40 Gy irradiation.

  Regulatory T cells were stimulated with anti-CD3 and anti-CD28 conditions and then stained with Live / Dead Red viability staining (Invitrogen) and surface markers CD4, CD25, and CD127. Cells were fixed in Lyze / Fix PhosFlow® buffer and permeabilized in denatured Permbuffer III®. The cells were then stained with antibodies against each particular selected molecule.

  Statistical analysis was performed using GraphPad Prism software. Comparison between the two groups was done by using a two-sided unpaired student t-test. Comparison between the three groups was performed by using one-way ANOVA. P values less than 0.05 were considered significant (two-sided). The correlation between the two groups was determined to be statistically significant (two-sided) via the Spearman coefficient if the r value was above 0.7 or below -0.7.

  result. Regulatory T cell proliferation was studied by stimulating cells with diesel exhaust particulate matter (PM from EPA). Applicants have noted that cells stimulated with PM (no Rev, no Solis) produced a decrease in secreted IL-10, while cells exposed to PM in the presence of the fluid of the present disclosure (“PM + Rev”). ") Was determined to have resulted in sustained or slightly reduced production of IL-10 compared to saline and media controls (no PM). In addition, diphtheria toxin (DT390, advanced diphtheria toxin molecule; standard commercial concentration 1:50 dilution) was titrated into the fluid sample of the present invention to block the Rev-mediated effect of increasing IL-10. Note that treatment with Rev alone resulted in higher IL-10 levels compared to saline and media controls. Similar results were obtained with GITR, Granzyme A, XCL1, pStat5, and Foxp3, respectively.

  Applicants also obtained AA PBMC data obtained from allergic asthma (AA) of peripheral blood mononuclear cells (PBMC) evaluating tryptase. AA PBMC data was consistent with the T regulatory cell data described above when cells stimulated with particulate matter (PM) showed high levels of tryptase, while in the presence of the fluids of the present disclosure, Cells treated with PM (“PM + Rev”) produced significantly lower tryptase levels, similar to those of saline and media controls. Consistent with data from T-regulatory cells, exposure to DT390 blocked Rev-mediated effects at tryptase levels, as seen in PM only (-Rev, no Rev, no Solas). Elevated levels of tryptase were produced. Treatment with Rev alone resulted in lower tryptase levels compared to saline and media controls.

  In summary, Applicants observed a decrease in proliferation in the presence of PM and Rev compared to PM in the control fluid (no Rev, no Solas) and indicated by a relatively reduced proliferation in the assay. As can be seen, Rev of the electrokinetically generated fluid of the present invention has improved regulatory T cell function. Furthermore, evidence indicates that β block, GPCR block, and Ca channel block affect the activity of Revera in Treg function.

Example 4
(The synergistic effect between the electrokinetically modified fluid of the present invention and budesonide was determined)
wrap up. The electrokinetically modified fluid of the present invention provided a synergistic anti-inflammatory effect using budesonide in vivo in an art recognized animal model for allergic asthma. The results disclosed in this example are also disclosed in the applicant's International Publication No. 2009/055729.

  Applicants first conducted experiments to evaluate the airway anti-inflammatory properties of the electrokinetically generated fluids of the invention (eg, RDC-1676-03) in a Brown Norway rat ovalbumin sensitization model. went. Brown Norway rats are a recognized model in the art for determining the effect of test materials on airway function, and this strain is widely used, for example, as a model for allergic asthma. Airway lesions and biochemical changes induced by ovalbumin sensitization in this model are similar to those observed in humans (Elwood et al., J Allergy Clin Immuno 88: 951-60, 1991, Sirois). & Bissonnette, Clin Exp Immunol 126: 9-15, 2001). The inhalation route was selected to maximize pulmonary exposure to the test material or control solution. Ovalbumin sensitized animals were treated with budesonide alone or in combination with test material RDC 1676-03 for 7 days prior to ovalbumin stimulation. Any time 6 and 24 hours after stimulation, whole blood counts and levels of several inflammatory and anti-inflammatory cytokines, as well as various respiratory parameters are measured and test material is administered at various inflammatory parameters The beneficial effect was estimated.

Materials and methods:
At the start of the experiment, a Brown Norway rat strain Bn / Crl, weighing approximately 275 ± 50 g, was obtained from Charles River Kingston. All animal studies were conducted with the approval of the PCS-MTL Institutional Animal Care and Use Committee. During the study, animal use and care was performed according to the guidelines of the USA National Research Council and the Canadian Council of Animal Care.

  Sensitization. On the first day of the experiment, animals (14 animals in each treatment group) received 1 mL intraperitoneal injections of a freshly prepared solution of 2 mg ovalbumin / 100 mg aluminum hydroxide per mL 0.9% sodium chloride. Immunized by dosing and then repeated injections on day 3.

  treatment. Fifteen days after initial treatment sensitization, animals are exposed to spray exposure to controls (saline) or test solutions (electrokinetically generated fluids RDC1676-00, RDC1676-02, and RDC-1676-03). And administered for 7 consecutive days, once daily for 15 minutes, alone or in combination with budesonide. Animals were dosed in an approximately 20 L whole body chamber and a test environment was created at the air inlet of the chamber using an Aeroneb ultrasonic nebulizer supplied with air from a Buxco bias flow pump. The air flow rate was set at 10 liters / minute.

  Ovalbumin stimulation. On day 21, 2 hours after treatment with the test solution, all animals were stimulated with 1% ovalbumin spray solution for 15 minutes (in the whole body chamber at 2 L / min airflow).

  Sample collection. At 6 and 24 hours after ovalbumin stimulation, blood samples were drawn to measure total and differential blood cell counts and levels of various inflammatory and anti-inflammatory cytokines. In addition, immediately after 6 and 24 hours after ovalbumin stimulation, enhanced pause Penh and tidal volume were measured using a Buxco Electronics BioSystem XA system for 10 minutes.

result:
Eosinophil count: As expected, treatment with budesonide (“NS + budesonide 750 μg / Kg”; fine hatched bar graph) was stimulated compared to treatment with saline “NS” alone control. The total eosinophil count in the animals was reduced. In addition, treatment with the fluid “RDC1676-03” alone of the present invention did not significantly reduce eosinophil counts, but nevertheless a substantial synergistic effect with budesonide in reducing eosinophil counts (“RDC1676-03 + Budesonide 750 μg / Kg”). Similarly, eosinophil percentage also showed a similar trend. RDC 1676-03 or budesonide 750 ug / kg alone had no significant effect on eosinophil rate in stimulated animals, but the two combined significantly reduced eosinophil rate.

  Accordingly, Applicants have determined that the electrokinetically generated fluids of the present invention (eg, RDC1676-03) are eosinophil counts (“eosinophils” in a rat model recognized in the art for human allergic asthma. In combination with budesonide, it was determined to have substantial synergistic utility so as to significantly reduce the “sphericity” and the total number).

Respiration parameters:
Applicant also showed the observed effect of test fluid on Penh and tidal volume, as measured immediately after 6 and 24 hours after ovalbumin stimulation. Penh is an induced value derived from peak inspiratory flow, peak expiratory flow, and expiratory time, and a decrease in penh value reflects a beneficial result of lung function.

  Penh = (peak expiratory flow / peak inspiratory flow) * (expiration time / time-1 to exhale 65% of expiratory volume).

  Treatment with budesonide (both 500 and 750 ug / kg) alone or in combination with either of the test fluids did not significantly affect the Penh values immediately after stimulation. However, 6 hours after stimulation, animals treated with RDC1676-03 alone or in combination with budesonide 500 or 750 ug / kg showed a significant reduction in Penh values. This range of decline disappeared 24 hours after stimulation, but a trend of synergistic effects of budesonide and RDC fluid was further observed at this point.

  Tidal volume is the volume of air that is drawn into the inhaling lung from the end of expiration, which is passively evacuated during exhalation during rest breathing. Animals treated with budesonide alone showed no change in tidal volume immediately after stimulation. However, RDC1676-03 alone had a significant promoting effect on tidal volume even at this early time point. Furthermore, RDC1676-03 in combination with budesonide (both 500 and 750 ug / kg) had an even more pronounced effect on tidal volume measurement at this point. Six hours after stimulation, RDC1676-03 alone is sufficient to produce a significant increase in tidal volume, and the addition of budesonide to the treatment regimen, either alone or in combination, can be further increased in tidal volume. There was no effect. However, any effects observed at these early time points disappeared by the 24 hour time point.

  Taken together, these data indicate that RDC1676-03 alone or in combination with budesonide, as evidenced by increased tidal volume and decreased Penh values 6 hours after stimulation Indicates that it provided significant mitigation.

Cytokine analysis:
In order to analyze the mechanism of effect seen in the physiological parameters discussed above, a number of inflammatory and anti-inflammatory cytokines were drawn 6 and 24 hours after stimulation and then immediately after physiological measurements. Measured in blood samples.

  Applicants observed that Rev 60 (or RDC 1676-03) alone significantly reduced eotaxin blood levels both at 6 and 24 hours after stimulation. Budesonide 750 ug / kg also reduced blood eotaxin levels at both of these time points, while budesonide 250 ug / kg alone had a significant effect at later time points. However, the test solution Rev 60 alone showed a significantly stronger effect (in reducing blood eotaxin levels) than both concentrations of budesonide at both time points. Eotaxin is a small amount of CC chemokine known to accumulate eosinophils and attract eosinophils in asthmatic lungs and other tissues in allergic reactions (eg, Crohn's gut). It is. Eotaxin binds to the G protein coupled receptor CCR3. CCR3 is expressed by many cell types such as Th2 lymphocytes, basophils, and mast cells, but the expression of this receptor by Th2 lymphocytes is when these cells regulate eosinophil recruitment. It becomes a specific target. Several studies have shown that in the asthmatic lung, increased production of eotaxin and CCR3 and the link between these molecules and airway hypersensitivity (Eotaxin and the attraction of the eosinophils to the asthmatic lang Reviews in Dolores M Conroy and Timothy J Williams Respiratory Research 2001, 2: 150-156).

  Taken together, these results strongly suggest that treatment with RDC1676-03 alone or in combination with budesonide can significantly reduce total eosinophil counts and blood levels 24 hours after ovalbumin stimulation. Suggest. This correlates with the significant decrease in blood eotaxin levels observed as early as 6 hours after stimulation.

  Blood levels of two major anti-inflammatory cytokines, IL10 and interferon gamma, are also significantly enhanced 6 hours after stimulation as a result of treatment with Rev 60 alone or in combination with budesonide. Applicants observed the effects on interferon γ and IL10, respectively. Rev60 alone or in combination with budesonide 250 ug / kg significantly increased IL10 blood levels in stimulated animals for up to 6 hours after stimulation. Similarly, Rev 60 alone or in combination with budesonide 250 ug / kg or 750 ug / kg significantly increased IFNγ blood levels 6 hours after stimulation. These increases in anti-inflammatory cytokines may well explain, at least in part, the beneficial effects seen in physiological respiratory parameters that are seen 6 hours after stimulation. The effects on these cytokines were no longer observed 24 hours after stimulation (data not shown).

  Rantes or CCL5 is a cytokine expressed by circulating T cells, is chemotactic for T cells, eosinophils and basophils and plays an active role in recruiting leukocytes to inflammatory sites. Fulfill. Rantes also activates eosinophils that release, for example, eosinophil cationic proteins. The density of eosinophils is changed to lower the concentration of eosinophils, which is thought to indicate a general state of cell activation. Rantes is also a potent activator of oxidative metabolism specific for eosinophils.

  Applicants indicated that systemic levels of Rantes were significantly reduced 6 hours after stimulation in animals treated with Rev 60 alone or in combination with budesonide 250 ug / kg or 750 ug / kg, but after 24 hours. Observed that it did not drop. As before, there was a clear synergistic effect of budesonide 750 ug / kg and Rev 60. Similar downward trends were observed in KC or many other inflammatory cytokines such as IL8, MCP3, IL1b, GCSF, TGFb, and NGF, and were stimulated in animals treated with Rev60 alone or in combination with budesonide. It was observed either after 6 hours or after 24 hours.

Example 5
(The effect of the electrokinetically modified fluid of the present invention on intercellular tight junctions was determined)
wrap up. The electrokinetically modified fluid of the present invention has been shown to modulate cell-cell tight junctions. The results disclosed in this example are also disclosed in the applicant's International Publication No. 2009/055729.

  According to certain embodiments, the diffusion treated therapeutic fluids of the present invention have substantial utility for modulating intercellular tight junctions, including those associated with pulmonary and systemic delivery, and The bioavailability of a polypeptide, including the exemplary polypeptide salmon calcitonin (sCT) is mentioned.

  Example summary. Salmon calcitonin (sCT) is a 32 amino acid peptide with a molecular weight of 3,432 daltons. Pulmonary delivery of calcitonin has been extensively studied to investigate methods for enhancing pulmonary drug delivery (eg, endotracheal drug delivery) in model systems (eg, rodent model systems, rat model systems, etc.). Has been. According to certain exemplary embodiments, the diffusion-treated therapeutic fluids of the present invention modulate intercellular tight junctions, such as those associated with pulmonary and systemic delivery, and sCT bioavailability in a rat model system Has substantial utility (eg, to strengthen).

Method:
Intratracheal drug delivery. According to certain embodiments, sCT is formulated in a therapeutic fluid of the present invention and administered to rats using an endotracheal drug delivery device. In one aspect, a Penn Century Micro-Sprayer device designed for rodent endotracheal drug delivery is used to enable good pulmonary delivery, but as understood in the art, There is relatively low alveolar deposition resulting in poor systemic bioavailability. According to certain embodiments, using this art-recognized model system, the diffusion treated therapeutic fluids of the present invention can be used for intercellular tight junctions (those associated with pulmonary and systemic delivery, as well as polypeptides). It has been found to have substantial utility for regulating (eg, enhancing) bioavailability (including bioavailability).

Animal group and dose. In some embodiments, the rats are assigned to one of the following three groups (n = 6 per group): a) sterile saline, b) basal solution not enriched in O ₂ (“basal solution”). Or c) a diffusion treated therapeutic fluid of the present invention (“enriched base solution of the present invention”). The enriched base solution of the present invention is formed, for example, by injecting oxygen in 0.9% saline. Preferably, the base solution contains about 0.9% saline so as to minimize the possibility of hypotonic collapse of the epithelial cells. In certain embodiments, sCT is reconstituted separately in the base solution and the enrichment-based solution of the present invention and delivered to each group of animals by intratracheal infusion within 60 minutes (in 200 μL per animal) Of 10 μg sCT).

  Assay. In certain embodiments, a blood sample (eg, 200 μL) is taken and inserted into an EDTA-coated tube before dosing and 5, 10, 20, 30, 60, 120, and 240 minutes after dosing. . Plasma was harvested and stored at −70 ° C. or lower until sCT assay using ELISA.

  Lung tissue was isolated and submerged in TRI reagent (TR118, Molecular Research Center, Inc.) for generation of Agilent gene array data. Briefly, approximately 1 mL of TRI reagent was added to 50-100 mg of tissue in each tube. Samples were homogenized in TRI reagent using a glass Teflon® or Polytron® homogenizer. Samples were stored at -80 ° C.

result:
Strengthen tight bonds. Applicant has identified RDC 1676-01 (sterilized saline treated through the unique device of the present disclosure with additional oxygen added, the gas-enriched electrokinetically generated fluid (Rev) of the present disclosure). It was observed that systemic delivery and sCT bioavailability were reduced. According to certain aspects, the reduction in systemic delivery results from a decrease in sCT adsorption, most likely from enhanced lung tight junctions. RDC 1676-00 represents sterile saline that has been treated according to the methods of the present disclosure but is not oxygenated.

  Additionally, according to certain embodiments, tight junction related proteins were upregulated in lung tissue. Applicants have found that the bond adhesion molecules JAM2 and 3, GJA1, 3, 4, and 5 (bond adhesion), OCLN (ocridin), claudin (eg CLDN3, 5, 7, 8, 9, 10), TJP1 ( Each upregulation of tight junction protein 1) was shown.

Example 6
(The effect of the electrokinetically modified fluid of the present invention on total cell conductivity was determined)
wrap up. The electrokinetically modified fluid of the present invention reduced total cell conductivity as shown by patch clamp analysis performed on bronchial epithelial cells (BEC). Patch clamp analysis performed on bronchial epithelial cells (BEC) perfused with the electrokinetically generated fluid (RNS-60) of the present invention shows that exposure to RNS-60 reduces total cell conductivity. Made it clear that In addition, stimulation with a cAMP-stimulated “cocktail” that dramatically increased total cell conductivity also increased the drug-sensitive portion of total cell conductivity, which is 10 times higher than that observed under basic conditions. It was twice as expensive. The results disclosed in this example are also disclosed in the applicant's International Publication No. 2009/055729.

  To perform patch clamp studies and regulate intracellular signal transduction by regulating at least one of membrane structure, membrane potential, or membrane conductivity, membrane proteins or receptors, ion channels, and calcium-dependent cell transmission systems The usefulness of the electrokinetically generated fluid of the present invention was further confirmed.

  wrap up. Applicants have shown that bradykinin binding to the B2 receptor is concentration dependent and binding affinity is compared to saline in electrokinetically generated fluids of the present disclosure (eg, Rev, gas, Enriched electrokinetically generated fluid). In addition, Applicants have found that in the context of T regulatory cells stimulated with particulate matter (PM), in the presence of PM and Rev compared to PM in the control fluid (No Rev, No Solas), There is a decrease in proliferation of T regulatory cells, for example, the electrokinetically generated fluid Rev of the present invention improves regulatory T cell function, as indicated by a relatively decreased proliferation in the assay. It showed that. Furthermore, exposure to the fluid of the present invention resulted in sustained or slightly reduced production of IL-10 compared to saline and media controls (no PM). Similarly, in the context of allergic asthma (AA) profiles of peripheral blood mononuclear cells (PBMC) stimulated with particulate matter (PM), the data show that exposure to the fluid of the present disclosure (“PM + Rev”) It was shown to result in significantly lower tryptase levels similar to those of water and medium controls. In addition, the diphtheria toxin (DT390) effect indicates that β-blocking, GPCR blocking, and Ca channel blocking affect the activity of electrokinetically generated fluids in Treg and PBMC function. Further, according to additional aspects, Applicants have shown that tight junction associated proteins were upregulated in lung tissue when exposed to the fluids of the present invention. Applicants have found that the bond adhesion molecules JAM2 and 3, GJA1, 3, 4, and 5 (bond adhesion), OCLN (ocridin), claudin (eg CLDN3, 5, 7, 8, 9, 10), TJP1 ( Each upregulation of tight junction protein 1) was shown. A patch clamp study was performed to further investigate and confirm the utility.

Materials and methods:
The bronchial epithelial cell line Calu-3 was used for patch clamp studies. Calu-3 bronchial epithelial cells (ATCC # HTB-55) were grown in a 1: 1 Ham F12 and DMEM medium mixture supplemented with 10% FBS on glass coverslips until the time of experimentation. . Briefly, a whole cell voltage clamping device is used to electrokinetically generate fluids of the invention (eg, RNS-60, electrokinetically treated saline containing 60 ppm dissolved oxygen). The effect on Calu-3 cells exposed to water, sometimes referred to as “drug” was measured.

  The effect of the test material (RNS-60) on epithelial cell membrane polarity and ion channel activity was evaluated using the patch clamp method. Specifically, the whole cell voltage clamp is an immersion liquid consisting of 135 mM NaCl, 5 mM KCl, 1.2 mM CaCl2, 0.8 mM MgCl2, and 10 mM HEPES (pH adjusted to 7.4 with N-methyl D-glucamine). In the bronchial epithelial cell line Calu-3. The basal current was measured after RNS-60 was perfused into the cells.

  More specifically, the patch pipette is pulled from borosilicate glass (Garner Glass Co, Clarmont, Calif.) Using a two-stage Narishige PB-7 vertical puller, and then the Narishige MF-9 microforge (Narishige International US). , East Meadow, NY) and fire polished to a resistance of 6-12 megohms. Pipettes were filled with an intracellular solution (in mM) containing 135 KCl, 10 NaCl, 5 EGTA, 10 Hepes and the pH was adjusted to 7.4 using NMDG (N-methyl-D-glucamine).

  Cultured Calu-3 cells are placed in a chamber containing the following extracellular solution (mM): 135 NaCl, 5 KCl, 1.2 CaCl2, 0.5 MgCl2, and 10 Hepes (free acid) The pH was adjusted to 7.4 using NMDG.

  Cells were viewed using a 40X DIC objective of an Olympus IX71 microscope (Olympus Inc., Tokyo, Japan). After constructing the cell-adhesive giga-seal, light aspiration was applied to invade the whole cell configuration and obtain a whole cell configuration. Immediately after invasion, the cells were voltage clamped at −120, −60, −40, and 0 mV and stimulated with voltage steps between ± 100 mV (500 ms / step). After collecting the total cell current under control conditions, the same cells are perfused through the bath with a test fluid containing the same extracellular solute and pH as the control fluid described above, and the whole cells at different holding potentials. Current was recorded using the same protocol.

  Electrophysiological data was acquired using an Axon Patch 200B amplifier, low pass filtered at 10 kHz, and digitized using a 1400A Digidata (Axon Instruments, Union City, Calif.). Data was acquired and analyzed using pCLAMP 10.0 software (Axon Instruments). The current (I) -voltage (V) relationship (whole cell conductivity) was obtained by plotting the actual current value versus holding potential (V) at about 400 milliseconds in a step. The slope of the I / V relationship is whole cell conductivity.

  Drugs and drugs. When indicated, cells were stimulated with a cAMP stimulation cocktail containing 8-Br-cAMP (500 mM), IBMX (isobutyl-1-methylxanthine, 200 mM), and forskolin (10 mM). The cAMP analog 8-Br-cAMP (Sigma Chem. Co.) was used from a 25 mM stock in H2O solution. Forskolin (Sigma) and IBMX (Sigma) were used from a DMSO solution containing both 10 mM forskolin and 200 mM IBMX stock solution.

Patch clamp results:
Applicants determined total cell currents using a protocol stepping from a holding potential of 0 mV to +/− 100 mV under basal (no cAMP) conditions. A representative transcript (control, then whole cell transcript during perfusion of test solution) was made with an average of n = 12 cells. A composite “difference” transcript obtained by subtracting the test mean from the value under control conditions was obtained. The whole cell conductivity obtained from the current-voltage relationship is highly linear under both conditions and reflects a significant change in conductivity with the test conditions, albeit in small amounts. The contribution to total cell conductivity, ie the component inhibited by the drug (electrokinetically generated fluid of the invention) was also linear and the reversal potential was approximately 0 mV. There was a decrease in whole cell conductivity under hyperpolarized conditions.

  In addition, Applicants determined total cell current using a protocol stepping from -40 mV holding potential to ± 100 mV under basal conditions. A representative transcript (control, then whole cell transcript during perfusion of test solution) was made with an average of n = 12 cells. A composite differential transcript was obtained by subtracting the test mean from the value under control conditions. The whole cell conductivity obtained from the current-voltage relationship was highly linear under both conditions and reflected a significant change in conductivity with test conditions, albeit in small amounts. The contribution to total cell conductivity, ie the component inhibited by the drug (electrokinetically generated fluid of the invention) was also linear and the reversal potential was approximately 0 mV. The values were relatively similar to those obtained using the 0 mV protocol.

  Applicants determined total cell current using a protocol stepping from -60 mV holding potential to ± 100 mV under basal conditions. A representative transcript (control, then whole cell transcript during perfusion of test solution) was made with an average of n = 12 cells. A composite differential transcript was obtained by subtracting the test mean from the value under control conditions. The total cell conductivity obtained from the current-voltage relationship was highly linear and trace in both conditions, but reflected a significant change in conductivity with test conditions. The contribution to total cell conductivity, ie the component inhibited by the drug, was also linear and the reversal potential was approximately 0 mV. The values were relatively similar to those obtained using the 0 mV protocol.

  Applicants also determined total cell current using a protocol stepping from a holding potential of −120 mV to ± 100 mV under basal conditions. A representative transcript (control, then whole cell transcript during perfusion of test solution) was made with an average of n = 12 cells. A composite differential transcript was obtained by subtracting the test mean from the value under control conditions. The total cell conductivity obtained from the current-voltage relationship was highly linear and trace in both conditions, but reflected a significant change in conductivity with test conditions. The contribution to total cell conductivity, ie the component inhibited by the drug, was also linear and the reversal potential was approximately 0 mV. The values were relatively similar to those obtained using the 0 mV protocol.

  In addition, Applicants determined total cell currents obtained using a protocol stepping from various holding potentials to ± 100 mV under cAMP-stimulated conditions. A representative transcript is the average of n = 5 cells. Representative transcripts (control, then cAMP stimulated and then perfused with drug-containing solution) were made with an average of n = 12 cells. Subtraction of the test mean value of drug + cAMP from the value under control conditions (cAMP only) gave a composite “difference” transcript (corresponding to the voltage protocol at 0 mV and −40 mV). The whole cell conductivity obtained from the current-voltage relationship was highly linear under all conditions, reflecting the change in conductivity with the test conditions.

  The application showed total cell currents using a protocol stepping from various holding potentials to ± 100 mV under cAMP-stimulated conditions. Representative transcripts (control, then cAMP stimulated and then perfused with drug-containing solution) were made with an average of n = 5 cells. Subtraction of the drug + cAMP test mean value from the value under control conditions (cAMP only) gave a composite “difference” transcript (corresponding to the voltage protocol at −60 mV and −120 mV). The whole cell conductivity obtained from the current-voltage relationship was highly linear under all conditions, reflecting the change in conductivity with the test conditions.

  Applicants have also shown the effect of holding potential on cAMP activation current. The effect of drugs (electrokinetically generated fluid of the present invention, RNS-60, electrokinetically treated saline containing 60 ppm dissolved oxygen) on total cell conductivity is shown by different voltage protocols ( 0, −40, −60, −120 mV holding potential). Under basal conditions, drug-sensitive whole cell currents were the same at all holding potentials (low voltage sensitivity contribution). However, under cAMP activation conditions, the drug sensitive current was even higher and sensitive to the applied voltage protocol. The current-voltage relationship is highly nonlinear. This was further observed in the subtracted current, and the contribution of total cell conductivity at 0 mV was further subtracted for each protocol (n = 5).

  Example overview. Thus, according to a particular aspect, the data was electrokinetically processed under basic conditions with drug (electrokinetically generated fluid of the invention, RNS-60, 60 ppm dissolved oxygen) A small amount of (saline) indicates a consistent effect. To enhance the effect of the drug on whole cell conductivity, the experiment was also performed by perfusing the drug after stimulation with a cAMP-stimulated “cocktail”, which dramatically increased the whole cell conductivity. It was. Interestingly, this protocol also increased the drug-sensitive portion of whole cell conductivity, which was 10 times higher than that observed under basal conditions. In addition, in the presence of cAMP stimulation, drugs have different effects on various voltage protocols, and electrokinetically generated fluids affect the voltage-dependent contribution of whole cell conductivity It shows that. There is also a decrease in the conductive linear component, which further suggests at least one contribution of the drug to the inhibition of alternative pathways (eg, ion channels, voltage-dependent cation channels, etc.).

  In certain embodiments, while not being bound by a mechanism, Applicant's data are electrokinetically generated according to the present invention that produce channel changes that are blocked or recovered from the plasma membrane. Consistent with the fluid produced (eg, RNS-60, electrokinetically treated saline containing 60 ppm dissolved oxygen).

  Taken together with Applicant's other data, certain aspects of the present invention include membrane structures, membrane potentials or conductivities, membrane proteins or receptors, ion channels, and calcium-dependent intracellular signaling systems. Compositions and methods are provided for modulating intracellular signal transduction, including at least one modulation, including but not limited to membrane structures (e.g., but not limited to GPCRs and / or g-proteins, and TSLP). , Membranes and / or membrane proteins, receptors, or other components), and the use of electrokinetically generated solutions of the present invention to impart conformational changes. According to additional aspects, these effects modulate gene expression and can persist, for example, depending on the half-life of the individual transfer components.

Example 7
(The effect of the electrokinetically modified fluid of the present invention on total cell conductivity was determined)
wrap up. Patch clamp analysis performed on Calu-3 cells perfused with electrokinetically generated fluids of the present invention (RNS-60 and Solas) showed that (i) exposure to RNS-60 and Solas (Ii) exposure of cells to RNS-60 resulted in an increase in non-linear conductivity, apparent at 15 minutes incubation time, and (iii) cells to RNS-60, which resulted in an increase in cell conductivity Showed that the effect of RNS-60 saline on calcium permeable channels was produced. Applicants have conducted patch clamp studies and described herein the electrokinetically generated saline fluids (RNS-60 and RNS-60), including the utility for modulating total cell currents. The usefulness of Solas) was further confirmed. Two sets of experiments were performed.

  A summary of the data for the first set of experiments shows that the total cell conductivity (current-voltage relationship) obtained with Solas saline is the same for both incubation times (15 minutes, 2 hours) and for all voltage protocols. On the other hand, it shows high linearity. However, it is clear that longer incubations with Solas (2 hours) increased total cell conductivity. Exposure of cells to RNS-60 results in an increase in non-linear conductivity, as shown by the delta current (Rev-Sol subtraction), which is only apparent with an incubation time of 15 minutes. The effect of RNS-60 on this non-linear current disappears and instead is highly linear with an incubation time of 2 hours. Although present in all voltage protocols, the nonlinear whole cell conductivity contribution was voltage sensitive, as observed above.

  A summary of the data from the second set of experiments showed that there is an effect of RNS-60 saline on non-linear currents, which was revealed at high calcium in the external fluid. The contribution of non-linear whole cell conductivity, although voltage sensitive, is present in both voltage protocols and indicates the effect of RNS-60 saline on calcium permeable channels.

First set of experiments (increased conductivity and activation of non-linear voltage regulated conductivity)
Method for the first set of experiments. See above for general patch clamp methods. In the first set of experiments below, a patch clamp study was performed using a protocol that stepped from either 0 mV holding potential, -120 mV, or -60 mV using Calu-3 cells under basal conditions, The utility of the electrokinetically generated saline fluids (RNS-60 and Solas) of the present invention for regulating total cell current was further confirmed.

  In all cases, total cell conductivity was obtained from the current-voltage relationship obtained from cells incubated for either 15 minutes or 2 hours. In this study, groups were obtained for Solas or RNS-60 saline solution at predetermined times. The data obtained is expressed as mean ± SEM total cell current for 5-9 cells.

  result. 3A-C show two time points (15 minutes (left panel) and 2 hours (right panel)) and different voltage protocols (stepping from A.0 mV, stepping from B.-60 mV, C.-120 mV). Shows the results of a series of patch clamp experiments that evaluated the effects of electrokinetically generated fluids (eg, RNS-60 and Solas) on epithelial cell membrane polarity and ion channel activity. The results show that RNS-60 (black circle) has a greater effect on whole cell conductivity than Solas (white circle). In the experiment, similar results were seen in the three voltage protocols and at the 15 minute and 2 hour incubation points.

  4A-C shows three voltage protocols ("delta current")) (stepping from A.0 mV, stepping from B.-60 mV, stepping from C.-120 mV), and two time points (15 minutes) (Open circles) and 2 hours (filled circles)) show graphs resulting from subtraction of Solas current data from RNS-60 current data. These data indicated that there was a non-linear potential dependent component that was absent at the 2 hour time point at the 15 minute time point using RNS-60.

  In previous experiments, etc., data with “physiological” saline yielded a highly consistent, time-dependent conductivity that was used as a reference. This result was obtained by matching the group with either Solas or RNS-60 saline and under basic conditions (no cAMP or any other stimulation) Calu-3 into RNS-60 saline Cell exposure produces a time-dependent effect, indicating that with a shorter incubation time (15 minutes), it is consistent with voltage-regulated conductivity activation. This phenomenon was not evident at the 2 hour incubation point. As described elsewhere herein, the linear component is more apparent as conductivity increases upon stimulation with a cAMP “cocktail”. Nevertheless, an incubation time of 2 hours shows a higher linear conductivity for both RNS-60 and Solas saline, in which case RNS-60 saline compared to whole cell conduction compared to Solas alone. Sex doubled. This evidence suggests that at least two contributions to total cell conductivity, namely non-linear voltage-regulated conductivity and linear conductivity activation, are affected by RNS-60 saline, which is further The incubation time is even more apparent.

Second set of experiments (effect on calcium permeable channels)
Method for the second set of experiments. See above for general patch clamp methods. In the second set of experiments below, still further patch clamp studies were performed, using a protocol stepping from either 0 mV or -120 mV holding potential using Calu-3 cells under basal conditions, The utility of the electrokinetically generated saline fluids (RNS-60 and Solas) of the present invention for regulating total cell current was further confirmed.

  In all cases, total cell conductivity was obtained from the current-voltage relationship obtained from cells incubated for 15 minutes with either saline. To determine whether there is a calcium permeable channel contribution to total cell conductivity and whether this portion of total cell conductivity is affected by incubation with RNS-60 saline, After the incubation period, it was patched into saline (with high NaCl external solution, but the internal solution contains high KCl). The external saline was then replaced with a solution in which NaCl was replaced with CsCl, and the main external cation was replaced to determine if there was a change in conductivity. Under these conditions, the same cells were then exposed to increasing concentrations of calcium so that the calcium entry step was further revealed.

Results: FIGS. 5A-D, with different external saline, with different voltage protocols (panels A and C show stepping from 0 mV, panels B and D show stepping from −120 mV), epithelium A series of patch clamp experiments evaluating the effects of electrokinetically generated fluids (eg, Solas (panels A and B) and RNS-60 (panels C and D)) on cell membrane polarity and ion channel activity. Results are shown. In these experiments, a time point of 15 minutes was used. For Solas (panels A and B), the results are: 1) Increased total cell conductivity with linear behavior when compared to control (diamonds) using CsCl (squares) instead of NaCl as external solution 2) Both 20 mM CaCl ₂ (circles) and 40 mM CaCl ₂ (triangles) indicate that CaCl ₂ increased total cell conductivity in a non-linear manner. For RNS-60 (panels C and D), the results are: 1) The effect on total cell conductivity when compared to the control (diamond mark) using CsCl (square mark) instead of NaCl as the external solution. There was little, and 2) CaCl ₂ (triangle) at 40 mM showed increased total cell conductivity in a non-linear manner.

6A-D shows 20 mM with two voltage protocols (stepping from panel A and C.0 mV, stepping from B and D.-120 mV) for Solas (panels A and B) and Revera 60 (panels C and D). FIG. 6 shows a graph resulting from subtraction of CsCl current data (shown in FIG. 5) from CaCl ₂ (diamonds) and 40 mM CaCl ₂ (squares) current data. The results show that both Solas and RNS-60 solutions activated calcium-induced nonlinear whole cell conductivity. The effect of using RNS-60 (showing dose responsiveness) was greater, and increased with higher calcium concentrations only when RNS-60 was used. Furthermore, at higher calcium concentrations, non-linear calcium-dependent conductivity was also increased by the voltage protocol.

  The data for this second set of experiments further show the effect of RNS-60 saline and Solas saline on whole cell conductivity data obtained in Calu-3 cells. The data show that incubation for 15 minutes with either saline produces a clear effect on total cell conductivity, which is most evident when using RNS-60, with external calcium In increasing, RNS-60 saline further shows increasing the calcium-dependent nonlinear component of whole cell conductivity.

  Accumulated evidence suggests activation of ion channels by Revalesio saline, which makes a different contribution to basal cell conductivity.

  When combined with applicant's other data (e.g., applicant's other example data), certain aspects of the invention include membrane structure, membrane potential or conductivity, membrane protein or receptor, ion channel A composition for modulating intracellular signal transduction, comprising modulation of at least one of: a lipid component, or an intracellular component replaceable by a cell (eg, a signal pathway such as a calcium-dependent cell signaling system) and Methods, including but not limited to GPCRs and / or g-proteins, electrochemistry of membrane structures (eg, membranes and / or membrane proteins, receptors, or other membrane components) and And / or use of electrokinetically generated solutions of the present invention to impart conformational changes. According to additional aspects, these effects modulate gene expression and can persist, for example, depending on the half-life of the individual transfer components.

Example 8
(The effect of the electrokinetically modified fluid of the present invention on total cell conductivity was investigated and a dose response curve was generated)
wrap up. In this example, Applicants evaluated the effect of dilution of electrokinetically generated fluid (eg, RNS-60) on epithelial cell membrane polarity and ion channel activity.

  Method. See above for general patch clamp methods. In the following experiment, a patch clamp test was performed to further confirm the usefulness of the electrokinetically generated saline fluid (RNS-60) of the present invention for modulating total cell current. Specifically, experiments evaluated the effect of dilution of the electrokinetically generated saline fluid of the present invention. Solutions of the present invention in saline at concentrations of 100% (Rev), 75% (3: 4), 50% (1: 1), 25% (4: 3), and 0% (Sal) It was made by diluting electrokinetically generated saline fluid.

  result. FIGS. 7A and B show a series of results of a patch clamp experiment that evaluated the effect of diluted electrokinetically generated fluid (eg, RNS-60) on epithelial cell membrane polarity and ion channel activity. Panel A shows the total cell conductance voltage versus current for each diluted sample (Rev 3: 4, 1: 1, 4: 3, and Sal) as shown in the graph. Panel B shows the change in dilution versus current comparing the dilution to saline. The results show that the mechanism of action of the RNS-60 solution occurs in the form of a linear dose response.

Example 9
(Treatment of primary bronchial epithelial cells (BEC) with electrokinetically generated fluids of the present invention, as well as non-electrokinetically controlled pressurized pot fluids, is one of the two major respiratory inflammatory pathways, MMP9 and TSLP. Resulting in a reduction in protein expression and / or activity)
wrap up. Applicants here have (electro-generated in this disclosure compared to saline (using Bio-Layer Interferometric Biosensor, Octet Rapid Extended Detection (RED) (forteBio®)). In the presence of a modified fluid (eg, Rev; a gas-enriched electrokinetically generated fluid), bradykinin binding to the B2 receptor is concentration dependent, indicating increased binding affinity. In addition, in the context of T regulatory cells stimulated with diesel exhaust particulate matter (PM, standard commercial source), Applicants' data is compared to PM in the control fluid (No Rev, No Solis) Show a decrease in the proliferation of T regulatory cells in the presence of PM and Rev, eg The Rev of electrokinetically generated fluids of the present invention showed improved regulatory T cell function, as shown by the reduced proliferation of the cells. This resulted in sustained or slightly reduced production of IL-10 compared to saline and media controls (no PM), as well as peripheral blood mononuclear cells stimulated with particulate matter (PM) ( In the context of the allergic asthma (AA) profile of PBMC), the data show that exposure to the fluid of the present disclosure (“PM + Rev”) results in significantly lower tryptase levels similar to the levels of saline and media controls. In addition, diphtheria toxin (DT390, advanced diphtheria toxin molecule; 1:50 dilution of standard commercial concentration) is an electrokinetically generated stream to Treg and PBMC function. In addition, the Applicant's activity resulted in β-blocking, GPCR-blocking, and Ca-channel blocking, which, when exposed to fluids of the present invention, resulted in tight junction related proteins (eg, JAM2 and 3, 3, 4, and 5 (bond adhesion), OCLN (ocridin), claudin (eg CLDN3, 5, 7, 8, 9, 10), TJP1 (tight junction protein 1)) up in lung tissue In addition, as shown in the patch clamp test, the electrokinetically generated fluid of the present invention (eg, RNS-60) is a bronchial epithelial cell (BEC; eg, Calu- 3) affects the regulation of whole cell conductivity (eg under hyperpolarization conditions), and according to an additional aspect, the regulation of whole cell conductivity reflects the regulation of ion channels. The

  In this example, Applicants have expanded these findings by conducting additional experiments to measure the effects of the production of two major proteins of the airway inflammatory pathway. Specifically, MMP9 and TSLP were assayed in primary bronchial epithelial cells (BEC).

Materials and methods:
Commercial primary human bronchial epithelial cells (BEC) (HBEpC-c from Promocell, Germany) were used for these studies. About 50,000 cells were plated in each well of a 12 well plate until a confluency of about 80% was reached. Then, before raising for FACS analysis, at a 1:10 dilution (100 μL in 1 mL airway epithelial growth medium) with diesel exhaust particulate matter (DEP or PM), saline, control fluid Solas, Cells were treated for 6 hours with non-electrokinetic control pressurized pot fluid, or test fluid Revera 60. Both MMP9 and TSLP receptor antibodies were obtained from BD Biosciences and used as per manufacturer's specifications.

result:
In FIGS. 1 and 2, DEP indicates cells exposed to diesel exhaust particulate matter (PM, standard commercial source) alone, “NS” indicates cells exposed to saline alone, “DEP + NS "Refers to cells treated with particulate matter in the presence of saline," Revera 60 "refers to cells exposed only to the test material, and" DEP + Revera 60 "refers to the test material. Refers to cells treated with particulate matter in the presence of Revera 60. In addition, “Solas” and “DEP + Solas” refer to cells exposed to the control fluid Solas alone or in combination with particulate matter, respectively. “PP60” indicates cells exposed to non-electrokinetic control pressurized pot fluid, and “DEP + PP60” was treated with particulate matter in the presence of non-electrokinetic control pressurized pot fluid. Refers to cells (ie, has 60 ppm dissolved oxygen).

  FIG. 1 shows that the test material Revera 60 reduces DEP-induced TSLP receptor expression by about 90% in bronchial epithelial cells (BEC). Solas resulted in a 55% decrease in DEP-induced TSLP receptor expression, whereas saline did not produce a similar level of decrease in DEP-induced TSLP receptor expression (approximately 20% reduction). Furthermore, the non-electrokinetic control pressurized pot fluid PP60 resulted in a 50% reduction in DEP-induced TSLP receptor expression.

  The effects of the Revera 60, Solas, and PP60 solutions of the present invention that reduce the expression of TSLP receptors indicate that TSLP plays a pivotal role in the pathobiology of allergic asthma, and local antibody-mediated blockade of TSLP receptor function. Considering recent research results showing alleviation of allergic diseases, it is a significant finding (Liu, YJ, Thymic stromal lymphopoietin: Master switch for allergic inflammation, J Exp Med 203: 269-273, 2006, -Shami et al., A role for TSLP in the development of information in an asthma model, J Exp Med 202: 829-839, 2005, and Shi et al., Local block of of TSLP receptor alleleating allied irreducible air dendrite cells 29, 829-839, 2005, Shi et al.

  Similarly, FIG. 2 shows the effect of Revera 60, Solas, non-electrokinetic control pressurized pot fluid (PP60), and saline in increasing DEP-mediated MMP9. Specifically, Revera 60 inhibits about 80% of DEP-induced cell surface-bound MMP9 levels in bronchial epithelial cells, while Solas has an inhibitory effect of about 70%, while saline (NS ) Had a reduction of about 20% of the marginal effect. Furthermore, the non-electrokinetic control pressurized pot fluid PP60 resulted in an approximately 30% reduction in the expression of DEP-induced cell surface adhesion MMP9 levels. MMP-9 is one of the major proteinases involved in airway inflammation and bronchial remodeling in asthma. In recent years, MMP-9 levels have been significantly increased in patients suffering from stable asthma and higher in acute asthmatic patients compared to healthy control subjects. MMP-9 plays an important role in the induction of airway inflammatory cell invasion and airway hypersensitivity, indicating that MMP-9 may play an important role in inducing and retaining asthma (Vignola et al ., Sputum metalloproteinase-9 / tissue inhibitor of metalloproteinase-1 ratio correlates with airflow obstruction in asthma and chronic bronchitis, Am J Respir Crit Care Med 158:. 1945-1950,1998, Hoshino et al, Inhaled corticosteroids decrease subepithelial col agen deposition by modulation of the balance between matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 expression in asthma, J Allergy Clin Immunol 104:. 356-363,1999, Simpson et al, Differential proteolytic enzyme activity in eosinophilic and neutrophilic asthma, Am J Respir Crit Care Med 172: 559-565, 2005, Lee et al., A murine mo. el of toluene diisocyanate-induced asthma can be treated with matrix metalloproteinase inhibitor, J Allergy Clin Immunol 108:. 1021-1026,2001, and Lee et al, Matrix metalloproteinase inhibitor regulates inflammatory cell migration by reducing ICAM-1 and VCAM-1 expression in a murine model of toluene diisocynate-induced asthma, J Allergy Clin Immunol 2003; 111: 1278-1284).

  Thus, according to additional aspects, the electrokinetically generated fluid of the invention is for modulating (eg, reducing) expression of TSLP receptors and / or for expression of MMP-9 and / or It has substantial therapeutic utility for inhibiting activity, including, for example, for the treatment of inflammation and asthma.

  According to a further aspect, non-electrokinetic control pressurized pot fluid (ie, 60 ppm dissolved oxygen) to modulate (eg, reduce) expression of TSLP receptor and / or expression of MMP-9 And / or have substantial therapeutic utility to inhibit activity, including, for example, for the treatment of inflammation and asthma. Although not bound by mechanism, Applicants 'collected data show that non-electrokinetic control pressurized pot fluids in this system are different from Applicants' electrokinetically generated fluid mechanisms. Mediated by This is not only due to the relatively small effect, but the non-electrokinetic control pressurized pot fluid does not show activity in other assays that show activity by applicant's electrokinetically generated fluid. It is also because of the damage. Nonetheless, Applicants' discovery of the activity disclosed herein of non-electrokinetic control pressurized pot fluids in the system is related to asthma and related conditions as disclosed herein. Represents a novel use for such pressurized pot fluids.

  Accordingly, in accordance with certain aspects, the methods of the present invention comprising administration of Applicant's electrokinetically generated fluid are immune system diseases, allergic inflammation, allergic airway inflammation, DC-mediated inflammation Th2 response, atopy At least one disease or condition selected from the TSLP-mediated group consisting of atopic dermatitis, atopic eczema, asthma, obstructive airway disease, chronic obstructive pulmonary disease, and food allergy, inflammatory arthritis, rheumatoid arthritis, and psoriasis Provides modulation (down-regulation of TSLP expression and / or activity) applicable to the treatment of

  The results disclosed herein are in complete agreement with the art-recognized role of TSLP as a master switch for allergic inflammation in epithelial cell-DC interference (Yong-Jun et al. , J. Exp. Med., 203: 269-273, 2006) and is more consistent with the phenotype of mice lacking TSLPR (eg, does not develop asthma in response to inhaled antigens; Zhou et al. , See above, and Al-Shami et al., J. Exp. Med., 202: 829-839, 2005), the results were obtained from pretreatment of OVA-DC with anti-TSLPR (eg, , Resulting in eosinophil and lymphocyte infiltration, and significant reduction in IL-4 and IL-5 levels).

  The presently disclosed object further elucidates the role that TSLPR plays in DC-sensitized allergic diseases, and includes novel compositions and methods involving the administration of Applicant's electrokinetically generated fluids. provide.

Claims (31)

A composition comprising an electrokinetically modified aqueous fluid for treating a patient having a TSLP-mediated or TSLPR-mediated disease or condition of the immune system, the fluid having an average diameter of less than 100 nanometers A TSLP-mediated or TSLPR-mediated disease or condition of the immune system comprising allergic inflammation and inflammatory arthritis A composition comprising at least one selected from the group consisting of : The charge-stabilized oxygen-containing nanobubbles in the ionic aqueous fluid in an amount sufficient to provide at least one modulation of cell membrane potential and cell membrane conductivity when contacted by a living cell with the fluid. The composition according to claim 1, which is stably constituted. The composition of claim 1, wherein the charge-stabilized oxygen-containing nanobubbles are the main charge-stabilized gas-containing nanostructure species in the fluid. The composition of claim 1, wherein the proportion of dissolved oxygen molecules present in the fluid as the charge-stabilized oxygen-containing nanobubbles is greater than 0.01%. The composition of claim 1, wherein all dissolved oxygen is substantially present in the charge-stabilized oxygen-containing nanobubbles. The composition of claim 1, wherein the modification of the electrokinetically modified aqueous fluid comprises hydrodynamically induced exposure of the fluid to a localized electrokinetic effect. Exposure to the localized electrokinetic effect includes exposure to at least one of a voltage pulse and a current pulse; or a hydrodynamically induced fluid to the localized electrokinetic effect 7. The composition of claim 6, wherein exposure comprises exposure of the fluid to a structural property that induces an electrokinetic effect of a device used to generate the fluid. The composition of claim 1, wherein the aqueous ionic solution comprises a saline solution. The composition of claim 1, wherein the fluid is hyperoxygenated . The allergic inflammation comprises at least one of allergic airway inflammation, DC-mediated inflammation Th2 response, atopic dermatitis, atopic eczema, IgE-mediated disease, rhinitis conjunctivitis, and food allergy. The composition of any one of these . The composition of claim 1, wherein the inflammatory arthritis comprises at least one of rheumatoid arthritis and psoriasis. 10. Composition according to any one of the preceding claims, characterized in that the fluid is to be administered to the patient in combination with at least one additional therapeutic agent. The at least one additional therapeutic agent includes a short-acting β ₂ agonist, a long-acting β ₂ agonist, an anticholinergic agent, a corticosteroid, a systemic corticosteroid, a mast cell stabilizer, a leukotriene modifying agent, Β ₂ agonists including: methylxanthine, albuterol, levalbuterol, pyrbuterol, alformoterol, formoterol, salmeterol, and anticholinergics such as ipratropium and tiotropium; beclomethasone, budesonide, flunisolide, fluticasone, mometasone, triamcinolone, Corticosteroids, including prednisolone, prednisone; leukotriene modifiers, including montelukast, zafirlukast, and zileuton; cromolyn and nedocromi Mast cell stabilizers; including theophylline, ipratropium and albuterol, fluticasone and salmeterol, concomitant drugs including budesonide and formoterol, methylxanthines; including hydroxyzine, diphenhydramine, loratadine, cetirizine, and hydrocortisone; antihistamines; tacrolimus and pimecrolimus The composition of claim 12, wherein the composition is selected from the group consisting of: an immune system modifier; cyclosporine; azathioprine; mycophenolate mofetil; and combinations thereof . 13. The composition of claim 12, wherein the at least one additional therapeutic agent is a TSLP and / or TSLPR antagonist. The TSLP and / or TSLPR antagonist comprises a TSLPR immunoglobulin Fc molecule or polypeptide that encodes a component of two or more receptor chains, a neutralizing antibody specific for TSLP and the TSLP receptor, soluble 15. The composition of claim 14, wherein the composition is selected from the group consisting of a TSLP receptor molecule, and a TSLP receptor fusion protein. Modulation of at least one of cell membrane potential and cell membrane conductivity includes altering at least one of conformation, ligand binding activity, and catalytic activity of a membrane bound protein of a cell membrane structure or function Modifying at least one, wherein modulating at least one of cell membrane potential and cell membrane conductivity comprises modulating total cell conductivity;
Or regulation of at least one of cell membrane potential and cell membrane conductance may be a modulation of calcium-dependent cell transmission pathways or systems, or of phospholipase C activity, or of adenylate cyclase (AC) activity, or of the immune system Disease or disease, allergic inflammation, allergic airway inflammation, DC-mediated inflammation Th2 reaction, atopic dermatitis, atopic eczema, asthma, obstructive airway disease, chronic obstructive pulmonary disease, IgE-mediated disease, rhinitis conjunctivitis, food allergy 3. The composition of claim 2, comprising modulation of intracellular signal transduction associated with at least one condition or symptom selected from the group consisting of: inflammatory arthritis, rheumatoid arthritis, and psoriasis. The composition according to claim 16, wherein the membrane-bound protein comprises at least one selected from the group consisting of a receptor, a transmembrane receptor, an ion channel protein, an intracellular adhesion protein, a cell adhesion protein, and an integrin. 18. The composition of claim 17, wherein the transmembrane receptor comprises a G protein coupled receptor (GPCR). 19. The composition of claim 18, wherein the G protein coupled receptor (GPCR) interacts with a G protein α subunit. 20. The composition of claim 19, wherein the G protein [alpha] subunit comprises at least one selected from the group consisting of G [alpha] _s, G [alpha] _i, G [alpha] _q , and G [alpha] ₁₂ . 21. The composition of claim 20, wherein the at least one G protein α subunit is Gα _q . 17. The composition of claim 16, wherein modulating total cell conductivity comprises adjusting at least one of the linear and non-linear voltage dependent contribution of the total cell conductivity. The composition is intended to be administered to the cell network or layer, and is used for the regulation of cell-cell junctions therein, the composition according to any one of claims 1-9 Thing . 24. The composition of claim 23, wherein the intercellular bond comprises at least one selected from the group consisting of tight junctions, gap junctions, adhesion bands, and desmosomes. 25. The composition of claim 23 or 24, wherein the cell network or layer comprises at least one selected from the group consisting of lung epithelium, bronchial epithelium, and intestinal epithelium. The electrokinetically modified aqueous fluid is oxygenated and the oxygen in the fluid is present in an amount of at least 8 ppm at atmospheric pressure;
Or the electrokinetically modified aqueous fluid comprises at least one of solvated electrons and electrokinetically modified or charged oxygen species. The composition according to claim 1. 27. The composition of claim 26, wherein the solvated electrons, or electrokinetically modified or charged oxygen species are present in an amount of at least 0.01 ppm. 28. The composition of claim 27, wherein the electrokinetically modified aqueous fluid comprises a solvated electron form stabilized by molecular oxygen. Said ability to modulate at least one of cell membrane potential and cell membrane conductance is at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 in a sealed airtight container. 3. The composition of claim 2, wherein the composition lasts for a period selected from the group consisting of months and at least 12 months. 10. The amount of oxygen present in a charge-stabilized oxygen-containing nanovalve of the electrokinetically modified fluid is at least 8 ppm at atmospheric pressure. Composition . 10. The composition of any one of claims 1 to 9, wherein the composition is administered to the patient by at least one of topical, inhalation, intranasal, and intravenous means. A composition according to 1 .

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