MX2011004235A

MX2011004235A - Compositions and methods for treating thymic stromal lymphopoietin (tslp)-mediated conditions.

Info

Publication number: MX2011004235A
Application number: MX2011004235A
Authority: MX
Inventors: Anthony B Wood; Richard L Watson; Gregory J Archambeau
Original assignee: Revalesio Corp
Priority date: 2008-10-22
Filing date: 2009-10-22
Publication date: 2011-06-24
Also published as: CN102256607B; AU2009308362B2; IL212277A0; BRPI0920201A2; AU2009308362A1; AU2009308302B2; JP5688370B2; EP2364154A4; CN102257130A; WO2010048455A1; JP2015044869A; CN102256607A; IL212309A; CN102257130B; JP2012506453A; MX337035B; JP2012506451A; EP2364154A1; WO2010048425A1; CA2741336A1

Abstract

Provided are methods for treating a TSLP-mediated or TSLPR-mediated disease or condition, comprising administration of an electrokinetically altered aqueous fluid comprising an ionic aqueous solution of charge-stabilized oxygen-containing nanostructures substantially having an average diameter of less than about 100 nanometers and stably configured in the ionic aqueous fluid in an amount sufficient for treating a TSLP-mediated or TSLPR-mediated disease or condition. The charge-stabilized oxygen-containing nanostructures are preferably stably configured in the fluid in an amount sufficient to provide for modulation of cellular membrane potential and/or conductivity. Certain aspects comprising modulation or down-regulation of TSLP expression and/or activity have utility for treating TSLP-mediated or TSLPR-mediated diseases or conditions as disclosed herein (e.g., disorders of the immune system, allergic inflammation, allergic airway inflammation, DC-mediated inflammatory Th2 responses, atopic dermatitis, atopic eczema, asthma, obstructive airways disease, chronic obstructive pulmonary disease, and food allergies, inflammatory arthritis, rheumatoid arthritis, psoriasis, IgE-mediated disorders, and rhino-conjunctivitis).

Description

COMPOSITIONS AND METHODS TO TREAT THE CONDITIONS CAUSED BY THYMETIC STRIPPING LYMPHOPOYETIN (TSLP) FIELD OF THE INVENTION The present invention relates generally to thymic lymphopoietin thymus (TSLP) and conditions mediated by TSLP, and more particularly to TSLP and conditions mediated by the TSLP receptor (e.g., immune system disorders, allergic inflammation, inflammation allergic respiratory tract, inflammatory Th2 responses mediated by DC, atopic dermatitis, atopic eczema, allergic asthma, asthma, obstructive respiratory disease, chronic obstructive pulmonary disease, and food allergies, inflammatory arthritis, rheumatoid arthritis, psoriasis, IgE-mediated disorders, and rhino-conjunctivitis). Particularly preferred aspects relate to the modulation (for example, treatment) of the TSLP and conditions mediated by the TSLP receptor, by administering a therapeutic composition comprising at least one electrokinetically generated fluid (including the electrokinetically enriched gas generated fluids (e.g. , enriched with oxygen)) as described herein, and in other aspects of combination therapy with the administration of those electrokinetic fluids in conjunction with at least one additional therapeutic agent.

BACKGROUND OF THE INVENTION Lymphopoietin. stroma.1 tímlca (TSLP). Thymic stromal lymphopoietin (TSLP) is a cytokine similar to IL-7 that triggers Th2-type inflammatory responses mediated by dendritic cells and is considered a master switch for allergic inflammation. TSLP is an integral growth factor for the development and maturation of B and T cells. Particularly, murine TSLP supports B lymphopoiesis and is required for the proliferation of B cells. The murine TSLP plays a crucial role in the control of transposition of the gamma locus of the T cell receptor (TCRy) and has a substantial stimulating effect on the thymocytes and mature T cells. See, for example, Friend and others, Exp. Hematol. , 22: 321-328, 1994; Ray et al., Eur. J. Immunol. , 26: 10-16, 1996; Candeias et al., Immunology Letters, 57: 9-14, 1997.

TSLP possesses cytokine activity similar to IL-7. For example, TSLP can replace IL-7 in stimulating B cell proliferation responses (Friend et al., Supra). Although TSLP and IL-7 mediate similar effects on target cells, they appear to have different signaling pathways and also vary in their biological response. For example, although TSLP modulates the activity of STAT5, it fails to activate any Janus member of the tyrosine kinase family (Levin et al., J. Immunol., 162: 677-683, 1999).

Effects of TSLP on dendritic cells and the production of TNF. After the human TSLP and the human TSLP receptor were cloned in 2001, it was discovered that human TSLP potently activated immature CDllc + myeloid dendritic cells (mDC) (see, for example, Reche et al., J. Immunol., 167: 336-343, 2001 and Soumelis et al., Nat. Immunol., 3: 673-680, 2002). Th2 cells are generally defined in immunology texts and literature as CD4 + T cells that produce IL-4, IL-5, IL-13, and IL-10. And Thl cells such as CD4 + T cells produce IFN-y and sometimes TNF. When TSLP-DC is used to stimulate virgin allogenic CD4 + T cells in vitro, a unique type of Th2 cell is induced which produces the classical Th2 cytokines IL-4, IL-5, and IL-13, and a large amount of TNF, but little or no IL-10 or interferon-? (Reche et al., Supra) (see also, for example, Soumelis et al., Nat. Immunol., 3: 673-680, 2002). TNF is not typically considered a Th2 cytokine. However, TNF is prominent in asthmatic airways and genotypes that correlate with increased TNF secretion are associated with an increased risk of asthma. See Shah et al., Clin. Exp. Allergy. , 25: 1038-1044, 1995 and Moffatt, M.F. and Cookson, W.O., Hum. Mol. Genet , 6: 551-554, 1997.

TSLP induces human mDGs to express the protein of the TNF superfamily OX40L at the level of the mRNA and the protein (Ito et al., J. Exp. Med., 202: 1213-1223). The expression of OX40L by TSLP-DC is important for the elaboration of inflammatory Th2 cells. Thus, the DC activated by TSLP create a microenvironment. permissive to Th2 by over-regulation of OX40L without inducing the production of Thi-polarizing cytokines. Id.

Expression of TSLP, allergen-specific responses and asin. Studies showed early that TSLP mRNA was highly expressed by human primary skin keratinocytes, bronchepithelcells, smooth muscle cells, and lung fibroblasts (Soumelis et al., Nat. Immunol., 3: 673-680, 2002) . Because TSLP is expressed mainly in the keratinocytes of the apical epidermal layers, this suggests that the production of TSLP is a characteristic of completely differentiated keratinocytes. The expression of TSLP in patients with atopic dermatitis was associated with the migration and activation of Langerhans cells in situ, which suggests that TSLP can directly contribute to the activation of these cells which could later migrate in suppurating lymph nodes and attenuate the responses specific allergen. Id. In a more recent study, it was shown by in situ hybridization, that the expression of TSLP increased in the airways of asthmatics and correlated with the expression of the Th2-attractant chemokine and with the severity of the disease which provides a link between TSLP and asthma (Ying et al., J. Immunol., 174: 8183-8190, 2005).

. TSLP receptor (TSLPR) and allergic asthma. The TSLP receptor (TSLPR) is a protein of approximately 50 kDa and has significant similarity to the β-chain. common. The TSLPR is a new type 1 cytokine receptor, which, combined with IL-7Ra (CD127), constitutes a receptor for the TSLP complex as described, for example, in Pandey et al., Nat. Immunol., 1 : 59-64, 2000. The TSLPR has a tyrosine residue close to its carboxyl terminal, which can be associated with phosphorylated STAT5 and mediates multiple biological functions when linked to TSLP (Isaksen et al., J. Immunol., 168 : 3288-3294, 2002).

The human TSLPR is expressed by monocytes and CDllc + dendritic cells, and the binding of TSLP induces the expression of TH2 cell attractant chemokines CCL17 and CCL22. Furthermore, as stated above, the activation induced by the TSLPR of the dendritic cells results indirectly in the increased secretion of the TH2 cytokines IL-4, -5 and -13, which may be necessary for the regulation of cell homeostasis. CD4 + T. In mice, TSLPR deficiency has no effect on lymphocyte numbers. However, a deficiency of the TSLPR and chain-? common results in fewer lymphocytes compared to mice deficient in the chain-? common alone. See Reche et al., J. Immunol., 167: 336-343, 2001 and Soumelis et al., Wa. Immunol., 3: 673-680, 2002.

The studies found that TSLP and TSLPR play a critical role in the initiation of allergic diseases in mice. In one study, it was shown that mice modified to overexpress TSLP in the skin developed atopic dermatitis that was characterized by ecezamatose lesions of the skin containing inflammatory infiltrates, a dramatic increase in circulating Th2 cells and elevated IgE in the serum (Yoo and others, J. Exp. Med., 202: 541-549, 2005). The study suggested that TSLP can directly activate DCs in mice. In another study, led by Li and others, the group confirmed that transgenic mice that overexpress TSLP in the skin developed atopic dermatitis that solidifies the link between TSLP and the development of atopic dermatitis.

Another set of studies demonstrated that TSLP is required for the initiation of allergic airway inflammation in mice in vivo. In a study, Zhou et al. Demonstrated that the specific lung expression of a TSLP transgene induced allergic inflammation of the airways (asthma) that was characterized by massive infiltration of leukocytes (including Th2 cells), Goblet cell hyperplasia, and fibrosis. subepithelial, and increased IgE levels in the serum (Zhou et al., Nat. Immunol., 6: 1047-1053, 2005). However, in contrast, mice lacking TSLPR failed to develop asthma in response to inhaled antigens (Zhou et al., Supra and Al-Shami et al., J. Exp. Med., 202: 829-839, 2005). . Thus, these studies together demonstrated that TSLP is required for the initiation of allergic airway inflammation in mice.

Moreover, in a study conducted by Yong-Jun and others, it was shown that TSLP derived from epithelial cells triggers inflammatory responses of Th2 mediated by DC in humans which suggests that TSLP represents a master switch of allergic inflammation in the DC-epithelial cell interface (Yong-Jun et al., J. Exp. Med., 203: 269-273, 2006).

In a recent study, it was shown that the modulation of DC fusion by inhibition of TSLPR decreased the severity in mice (Liyun Shi et al., Clin.Immunol., 129: 202-210, 2008). In another set of studies, it was demonstrated that TSLPR was not expressed only in DC, but also in macrophages, mast cells, and CD4 + T cells (Rochman et al., J. Immunol., 178: 6720-6724, 2007 and Omori M and Ziegler S., J. Immunol., 178: 1396-1404, 2007). To regulate the direct effects of the neutralization of TSLPR on CD4 + T cells or other effector cells in allergic inflammation, Liyun Shi and others performed experiments in which OVA-loaded DCs were treated in vitro with anti-TSLPR prior to adoptive transfer to the airways of virgin mice. It was previously found that OVA-DC triggered a strong inflammation of the eosinophilic airway and accompanied by mass production of Th2 cytokines such as IL-4 and IL-5 (Sung et al., J. Immunol., 166: 1261-1271 and Lambrecht et al., J. Clin. Invest., 106: 551-559, 2000). However, the pretreatment of OVA-DC with anti-TSLPR resulted in a significant reduction of eosinophil and lymphocyte infiltration as well as the levels of IL-4 and IL-5, further clarifying the role played by the TSLPR in allergic disease primed by DC. This result supports, furthermore, that blockage of the TSLPR in the DCs would help to control the inflammation of the airways (Liyun Shi et al., Supra).

There is a growing body of experiments involving the role of TSLP / TSLPR in various physiological and pathological processes. The physiological roles of TSLP include modulation of the immune system, particularly in stimulating the proliferation, development, and maturation of B and T cells. TSLP plays a vital role in the pathobiology of allergic asthma and local antibody mediated receptor blockade of the SLTS function to alleviate allergic diseases. Thus, the interaction between the TSLP and the TSLP receptor is considered important in many physiological disease processes such as: allergic inflammation, skin lesions of patients with atopic dermatitis or atopic eczema, allergic asthma and asthma.

SUMMARY OF THE INVENTION The particular aspects provide a method for the treatment of a condition or disease mediated by TSLP or TSLPR, comprising administering to a mammal in need thereof a therapeutically effective amount of an electrokinetically altered aqueous fluid comprising an aqueous ionic solution of nanostructures. containing stabilized oxygen per charge, which have substantially an average diameter of less than about 100 nanometers and which are stably configured in the aqueous ionic fluid in an amount sufficient to treat a condition or disease mediated by the TSLP or TSLPR. In certain aspects, the charge-stabilized oxygen-containing nanostructures are stably configured in the aqueous ionic fluid in an amount sufficient to provide, upon contact of a living cell with the fluid, the modulation of at least one of the cell membrane potential and of the conductivity of the cell membrane.

In certain aspects of the method, the nanostructures containing charge-stabilized oxygen are the main species of nanostructures that contain gas stabilized by charge in the fluid. In particular embodiments, the percentage of dissolved oxygen molecules present in the fluid as nanostructures containing stabilized oxygen per charge, is a percentage selected from the group consisting of greater than: 0.01%; 0.1%; 1%; 5%; 10%; fifteen%; twenty%; 25%; 30%; 35%; 40%; Four. Five%; fifty%; 55%; 60%; 65%; 70%; 75%; 80%; 85%; 90% and 95%. In particular aspects, the total dissolved oxygen is substantially present in charge-stabilized oxygen-containing nanostructures. In certain embodiments, the charge-stabilized oxygen-containing nanostructures substantially have an average diameter smaller than a size selected from the group consisting of: 90 nm; 80 nm; 70 nm; 60 nm; 50 nm; 40 nm; 30 nm; 20 nm; 10 nm; and less than 5 nm.

In particular aspects of the method, the aqueous ionic solution comprises a saline solution. In certain aspects, the electrokinetically altered aqueous fluid is superoxygen.

In particular aspects of the method, the electrokinetically altered aqueous fluid comprises a solvated electron form.

In certain aspects, the alteration of the electrokinetically altered aqueous fluid comprises the exposure of the fluid to localized, hydrodynamically induced electrokinetic effects. In certain embodiments, exposure to localized electrokinetic effects includes exposure to at least one of voltage pulses and current pulses. In particular aspects, the exposure of the fluid to electrokinetic effects localized, induced hydrodynamically, includes the exposure of the fluid to the structural characteristics that induce the electrokinetic effect of a device that is used to generate the fluid.

In particular aspects, the condition or disease mediated by the TSLP or TSLPR comprises a disease or disorder of the immune system, including but not limited to allergic inflammation. In particular aspects, allergic inflammation comprises at least one of allergic inflammation of the respiratory tract, inflammatory Th2 responses mediated by DC, atopic dermatitis, atopic eczema, asthma, obstructive airways disease, chronic obstructive pulmonary disease, mid-life disorders for IgE, rhino-conjunctivitis and food allergies. In certain embodiments, the condition or disease mediated by the TSLP or TSLPR comprises inflammatory arthritis, for example comprising at least one of rheumatoid arthritis and psoriasis.

In certain aspects, the method further comprises combination therapy, wherein at least one additional therapeutic agent is administered to the patient. In particular embodiments, the at least one additional therapeutic agent is selected from the group consisting of short-acting ~ 2 agonists, long-acting agonists-2, anticholinergics, corticosteroids, systemic corticosteroids, mast cell stabilizers, leukotriene modifiers, methylxanthines, and combinations thereof. In certain aspects, the at least one additional therapeutic agent is selected from the group consisting of: bronchodilators consisting of agonists-^ which include albuterol, levalbuterol, pirbuterol, artformoterol, formoterol, salmeterol, and anticholinergics such as ipratropium and tiotropium; corticosteroids including beclomethasone, budesonide, flunisolide, fluticasone, mometasone, triamcinolone, methylprednisolone, prednisolone, prednisone; leukotriene modifiers including montelukast, zafirlukast, and zileuton; mast cell stabilizers including cromolin and nedocromil; methylxanthines including theophylline, combination drugs including ipratropium and albuterol, fluticasone and salmeterol, budesonide and formoterol; antihistamines including hydroxzine, diphenhydramine, loratadine, cetirizine, and hydrocortisone; drugs that modulate the immune system that include tacrolimus and pimecrolimus; cyclosporin; azathioprine; mycophenolatemofetil; and combinations thereof. In particular aspects, the at least one additional therapeutic agent is an antagonist of TSLP and / or TSLPR, and in particular embodiments, the TSLP antagonist and / or TSLPR is selected from the group consisting of neutralizing antibodies specific for TSLP and the receptor of the TSLP, soluble TSLP receptor molecules, and TSLP receptor fusion proteins, which include immunoglobulin Fe-TSLPR molecules or polypeptides that encode the components of more than one receptor chain.

In particular aspects of the method, the modulation of at least one of the cell membrane potential and the cell membrane conductivity comprises altering at least one of the structures or functions of the cell membrane, comprising altering at least one of a conformation, the activity of binding to the ligand, and a catalytic activity of a protein or component associated with the membrane. In certain aspects, the membrane-associated protein comprises at least one selected from the group consisting of receptors, transmembrane receptors, ion channel proteins, intracellular binding proteins, cell adhesion proteins, integrins, etc. In certain embodiments, the transmembrane receptor comprises a receptor coupled to the G protein (GPCR). In particular aspects, the G protein-coupled receptor (GPCR) interacts with the a subunit of the G protein, for example, in which the a subunit of the G protein comprises at least one selected from the group consisting of Gas, GOÍÍ , Gaq and Goi2, and in certain embodiments the at least one a subunit of the G protein is Gaq.

In particular aspects of the method, the modulation of at least one of the cell membrane potential and the cell membrane conductivity comprises the modulation of the conductance of the whole cells, for example, in which the modulation of the conductance of the cell Complete is composed of the modulation of at least one of the linear and non-linear contributions dependent on the conductance voltage of the whole cells.

In certain aspects of the method, the modulation of at least one of the cell membrane potential and the cell membrane conductivity comprises the modulation of a calcium-dependent cellular messaging system or pathway. In certain aspects of the method, the modulation of at least one of the cell membrane potential and the cell membrane conductivity comprises the modulation of phospholipase C activity. In certain aspects of the method, the modulation of at least one of the potential of the cell membrane and the conductivity of the cell membrane comprises the modulation of the activity of adenylate cyclase (AC). In certain aspects of the method, the modulation of at least one of cell membrane potential and cell membrane conductivity comprises the modulation of intracellular signal transduction associated with at least one condition or symptom selected from the group consisting of diseases or disorders of the immune system. , allergic inflammation, allergic inflammation of the respiratory tract, inflammatory responses of Th2 mediated by DC, atopic dermatitis, atopic eczema, asthma, obstructive airway disease, chronic obstructive pulmonary disease, IgE-mediated disorders, rhino-with untivitis, food allergies, inflammatory arthritis, rheumatoid arthritis and psoriasis.

Particular aspects of the method include the administration of the electrokinetic fluid to a network or cell layer, and subsequently include the modulation of an intercellular junction therein. In certain embodiments, the intracellular junction comprises at least one selected from the group consisting of narrow junctions, junctions of clefts, zone adhesins and desmosomes. In particular aspects, the cellular networks or layers comprise at least one selected from the group consisting of the pulmonary epithelium, the bronchial epithelium, and the intestinal epithelium.

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

In certain aspects of the method, the electrokinetically altered aqueous fluid comprises at least one of solvated electrons and electrokinetically modified or charged oxygen species, for example, in which the form of solvated electrons or electrokinetically modified or charged oxygen species are present in an amount of 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 aspects, the electrokinetically altered aqueous fluid comprises a form of solvated electrons stabilized by molecular oxygen.

In certain aspects, the ability of the electrokinetically altered fluid to modulate at least one of the cell membrane potential and the cell membrane conductivity persists for at least two, at least three, at least four, at least five, at least 6, at least 12 months, or longer, in a closed container that is gas-proof.

In certain aspects, the amount of oxygen present in the stabilized oxygen-containing nanostructures by charging the electrokinetically altered 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 least 50 ppm, or at least 60 ppm oxygen at atmospheric pressure.

In particular aspects, the treatment comprises administration by at least one of the topical, inhalation, intranasal and intravenous routes.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 shows that the electrokinetically generated fluid of the invention (e.g., Revera 60 and Solas) reduced the expression of the TSP receptor induced by DEP in bronchial epithelial cells (BEC) by approximately 90% and 50%, respectively, whereas normal saline solution (NS) had only a marginal effect. In addition, the non-electrokinetic PP60 control autoclave fluid resulted in approximately 50% reduction in the expression of the TSP receptor induced by DEP.

Figure 2 shows that the electrokinetically generated fluid of the invention (e.g., Revera 60 and Solas) inhibited levels of cell-bound MMP9 induced by DEP in bronchial epithelial cells by approximately 80% and 70%, respectively, whereas normal saline solution (NS) only had a marginal effect. Additionally, the non-electrokinetic PP60 control autoclave fluid resulted in the reduction of approximately 30% of the cell surface bound MMP9 levels induced by DEP.

Figures 3A, 3B, and 3C demonstrate the results of a series of membrane-binding experiments that evaluated the effects of the electrokinetically generated fluid (e.g., RNS-60 and Solas) on the membrane polarity of epithelial cells and the activity of the ion channels at two time points (15 min (panels on the left) and 2 hours (panels on the right)) and on different voltage protocols.

Figures 4A, 4B, and 4C show, with respect to the experiments related to Figures 3A, 3B, and 3C, the graphs resulting from the subtraction of the Solas current data from the RNS-60 current data. in three voltage protocols (stepped A. from zero mV, B. stepped from -60 mV, C. stepped from -120 mV) and the two time points (15 min. (open circuits) and 2 hours (closed circuits) ).

Figures 5A, 5B, 5C, and 5D show the results of a series of membrane fixation experiments that evaluated the effects of electrokinetically generated fluid (eg, Solas (panels A. and B.) and RNS-60 (panels C. and D.)) on the polarity of the membrane of the epithelial cells and the activity of the ion channels using different external salt solutions and in the different voltage protocols (panels A and C show the staging from zero mV; panels B and D show the staging from -120 mV).

Figures 6A, 6B, 6C, and 6D present, with respect to the experiments related to Figures 5A, 5B, 5G, and 5D, the graphs resulting from the subtraction of the CsCl current data (shown in FIG. Figure 5) of the current data of 20 mM CaCl2 (diamonds) and 40 mM CaCl2 (filled squares), in two voltage protocols (panels A. and C, stepped from zero mV, B. and D. stepped from -120 mV) for Solas (panels A. and B.) and Revera 60 (panels C. and D.).

Figures 7A and 7B show the results of a membrane fixation experiment that evaluated the effects of dilution of the electrokinetically generated fluid (eg, RNS-60) on the polarity of the epithelial cell membrane and the activity of the channels of ions.

DETAILED DESCRIPTION OF THE INVENTION Methods are provided for the treatment of a condition or disease mediated by TSLP or TSLPR, which comprises the administration of an electrokinetically altered aqueous fluid comprising an ionic aqueous solution of charge-stabilized oxygen-containing nanostructures, which have substantially a smaller average diameter that approximately 100 nanometers and that are stably configured in the aqueous ionic fluid in an amount sufficient to treat a condition or disease mediated by the TSLP or TSLPR. The nanostructures containing charge-stabilized oxygen are preferably stable in the fluid, in an amount sufficient to provide modulation of the potential and / or conductivity of the cell membrane. Some aspects that comprise modulation or down regulation of TSLP expression and / or activity are useful for treating diseases or conditions mediated by TSLP or mediated by TSLPR as described herein (e.g., immune system disorders, allergic inflammation. , allergic inflammation of the respiratory tract, inflammatory responses of Th2 mediated by DC, atopic dermatitis, atopic eczema, asthma, obstructive respiratory disease, chronic obstructive pulmonary disease, and food allergies, inflammatory arthritis, rheumatoid arthritis, psoriasis , disorders mediated by IgE, and rino-conj untivitis).

Electrokinetically generated fluids: "Electrokinetically generated fluid", as used herein, refers to the electrokinetically generated fluids of Applicants' invention, for the purposes of the practical examples herein, by the exemplary mixing device described in detail herein ( see also US 200802190088 and WO 2008/052143, both incorporated herein by reference in their entirety). The electrokinetic fluids, as shown by the data disclosed and presented herein, represent new and fundamentally different fluids in relation to the non-electrokinetic fluids of the prior art, even in relation to the oxygenated non-electrokinetic fluids of the prior art (e.g. , oxygenated fluids in autoclave and the like). As disclosed in several aspects herein, the electrokinetically generated fluids have new and unique physical and biological properties, including but not limited to the following: In particular aspects, the electrokinetically altered aqueous fluid comprises an ionic aqueous solution of charge stabilized oxygen-containing nanostructures substantially having an average diameter of less than about 100 nanometers and which are stably configured in the aqueous ionic fluid in an amount sufficient to provide , upon contact of a living cell with the fluid, the modulation of at least one of the potential of the cell membrane and the conductivity of the cell membrane.

In particular aspects, the fluids generated electrokinetically refer to the fluids generated in the presence of electrokinetic effects (for example, voltage / current pulses), located (for example, non-uniform with respect to the volume of the fluid in general) hydrodynamically induced, such as the localized effects of the characteristics of the device as described herein. In particular aspects, said localized, hydrodynamically induced electrokinetic effects are in combination with the effects of the double layer related to the surface and / or the flow of the current as described and discussed herein.

In particular aspects, the electrokinetically altered aqueous fluids are suitable for modulating the line width of the C13 NMR of the reporter solutes (for example, trehalose) dissolved in them. The effects of NMR linewidth are in the indirect method of measurement, for example, of the 'fall' of the solute in a test fluid as described herein, in particular, in the practical examples.

In particular aspects, electrokinetically altered aqueous fluids are characterized by at least one of: distinctions of the distinctive square wave voltammetric peak at either -0.14V, -0.47V, -1.02V and -1.36V; the polarographic peaks at -0.9 volts; and the absence of polarographic peaks at -0.19 and -0.3 volts, which are unique for the electrokinetically generated fluids as described herein, in particular, in the practical examples.

In particular aspects, the electrokinetically altered aqueous fluids are suitable for modifying the conductivity of the cell membrane (eg, a voltage-dependent contribution of whole cell conductance as measured in the membrane binding studies disclosed herein).

In particular aspects, the electrokinetically altered aqueous fluids are oxygenated, in which the oxygen in the fluid is present in an amount of 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 of oxygen dissolved at atmospheric pressure. In particular aspects, electrokinetically altered aqueous fluids have less than 15 ppm, less than 10 ppm dissolved oxygen at atmospheric pressure, or about ambient oxygen levels.

In particular aspects, the electrokinetically altered aqueous fluids are oxygenated, in which the oxygen in the fluid is present in an amount between about 8 ppm and about 15 ppm, and in this case they are sometimes referred to herein as "Solas" .

In particular aspects, the electrokinetically altered aqueous fluid comprises at least one of solvated electrons (for example, stabilized by molecular oxygen), and modified and / or electrokinetically charged oxygen species, and which, in certain embodiments of the solvated electrons and / or modified and / or electrokinetically charged oxygen species are present in an amount of 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 particular aspects, electrokinetically altered aqueous fluids are suitable for modifying the structure or function of the cell membrane (e.g., alteration of a conformation, ligand-binding activity, or a catalytic activity of a membrane-associated protein) sufficient to provide modulation of intracellular signal transduction, in which, in particular aspects, the membrane-associated protein comprises at least one selected from the group consisting of transmembrane receptors, receptors (eg, the receptor coupled to the Protein G (GPCR), the TSLP receptor, the beta 2 adrenergic receptor, the bradykinin receptor, etc.), the ion channel proteins, the intracellular junction proteins, the cell adhesion proteins, and the integrins . In certain aspects, the Protein G Coupled Receptor (GPCR) interacts with the a subunit of the G protein (e.g., Gas, Gai, G q and In particular aspects, electrokinetically altered aqueous fluids are suitable for modulating the transduction of intracellular signals, comprising the modulation of a calcium-dependent cellular messaging system or pathway (eg, modulation of phospholipase C activity, or modulation of adenylate cyclase (AC) activity).

In particular aspects, the electrokinetically altered aqueous fluids are characterized by various biological activities (eg, the regulation of cytokines, receptors, enzymes and other proteins and intracellular signaling pathways) described herein.

In particular aspects, the electrokinetically altered aqueous fluids deploy synergy with albuterol, and with budesonide, as shown herein.

In particular aspects, the electrokinetically altered aqueous fluids reduce the expression of the TSPP receptor induced by DEP in the bronchial epithelial cells (BEC) as shown in the practical examples herein.

In particular aspects, the electrokinetically altered aqueous fluids inhibit the levels of M P9 bound to the cell surface induced by DEP in bronchial epithelial cells (BEC), as shown in the practical examples herein.

In particular aspects, the biological effects of the electrokinetically altered aqueous fluids are inhibited by the diphtheria toxin, which indicates that the beta blockade, the GPCR blockade and the blocking of the Ca channels affect the activity of the altered aqueous fluids. electrokinetically (for example, depending on the regulatory T cells) as shown herein.

In particular aspects, the physical and biological effects (for example, the ability to alter the structure of the cell membrane or function, sufficient to provide modulation of intracellular signal transduction) of the electrokinetically altered aqueous fluids persists for at least two , at least three, at least four, at least five, at least 6 months, or for a longer time, in a closed container (for example, in a closed container that is gas-proof).

Therefore, additional aspects provide said electrochenetically generated solutions and methods of producing an electrokinetically altered oxygenated aqueous fluid or solution comprising: providing a flow of a fluid material between two surfaces spaced in relative motion and defining a volume of mixture between them , in which the residence time of a single pass of the fluid material flowing in and through the mixing volume is greater than 0.06 seconds or greater than 0.1 seconds, and the introduction of oxygen (02) into the flowing fluid material within the volume of mixing under appropriate conditions to dissolve at least 20 ppm, at least 25 ppm, at least 30, at least 40, at least 50, or at least 60 ppm oxygen in the material, and to electrokinetically alter the fluid or the solution. In certain aspects, oxygen is infused into the material in less than 100 milliseconds, less than 200 milliseconds, less than 300 milliseconds, or less than 400 milliseconds. In particular embodiments, the ratio between the surface area and the volume is at least 12, at least 20, at least 30, at least 40, or at least 50.

In still further aspects, there is provided a method of producing an electrolytically altered oxygenated fluid or aqueous solution, comprising: providing a flow of a fluid material between two surfaces spaced in relative motion and defining a volume of mixture between them, and introducing oxygen in the material flowing into the mixing volume under appropriate conditions to infuse at least 20 ppm, at least 25 ppm, at least 30, at least 40, at least 50, or at least 60 ppm oxygen in the material in less than 100 milliseconds, less than 200 milliseconds, less than 300 milliseconds, or less than 400 milliseconds. In certain aspects, the residence time of the material flowing within the mixing volume is greater than 0.06 seconds or greater than 0.1. In particular embodiments, the ratio between the surface area and the volume is at least 12, at least 20, at least 30, at least 40, or at least 50.

In particular aspects, the electrokinetically altered fluids of the invention comprise oxygen-containing nanostructures stabilized by charge in an amount sufficient to provide modulation of at least one of the potential of the cell membrane and the conductivity of the cell membrane. In certain embodiments, the electrokinetically altered fluids are superoxygen (e.g., RNS-20, RNS-40 and RNS-60, comprising 20 ppm, 40 ppm and 60 ppm dissolved oxygen, respectively, in standard saline). In particular embodiments, the electrokinetically altered fluids are not superoxygen (e.g., RNS-10 or Solas, which comprises 10 ppm (eg, about the ambient levels of dissolved oxygen in standard saline.) In certain aspects, salinity, the sterility, pH, etc., of the electrokinetically altered fluids of the invention is established at the time of the electrokinetic production of the fluid, and the sterile fluids are administered by an appropriate route.Alternatively, at least one of the salinity, sterility , pH, etc., of the fluids is properly adjusted (eg, using sterile saline or appropriate diluents) to be physiologically compatible with the route of administration prior to fluid administration.Preferably, saline solutions and / or diluents and / or the buffered compositions used to adjust at least one of the salinity, sterility, pH, etc., of the fluids, are also fluid s electrokinetic, or otherwise compatible.

In particular aspects, the electrokinetically altered fluids of the invention comprise saline solution (for example, one or more dissolved salt (s), for example, salts based on alkali metals (Li, Na, K, Rb, Cs, etc. .), salts based on alkaline earth metals (for example, Mg, Ca), etc., salts based on transition metals (for example, Cr, Fe, Co, Ni, Cu, Zn, etc.,), together with any suitable anion / counterion component). Particular aspects comprise electrokinetic fluids based on mixed salt (for example, Na, K, Ca, Mg, etc., in various combinations and concentrations). In particular aspects, the electrokinetically altered fluids of the invention comprise standard saline solution (eg, about 0.9% NaCl, or about 0.15 M NaCl). In particular aspects, the electrokinetically altered fluids of the invention comprise saline solution at a concentration of at least 0.0002 M, at least 0.0003 M, at least 0.001 M, at least 0.005 M, at merlos 0.01 M, at least 0.015 M, at least 0.1 M, at least 0.15 M, or at least 0.2 M. In particular aspects, the conductivity of the electrokinetically altered fluids of the invention is at least 10 pS / cm, at least 40 μ? / Cm, at least 80 S / cm , at least 100 pS / cm, at least 150 yS / cm, at least 200 pS / cm, at least 300 S / cm, or at least 500 yS / 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, at least 300 mS / cm, or at least 500 mS / cm. In particular aspects, any salt can be used to prepare the electrokinetically altered fluids of the invention, with the proviso that they allow the formation of biologically active salt-stabilized nanostructures (e.g., salt-stabilized oxygen-containing nanostructures) as described in the present.

According to particular aspects, the biological effects of the compositions of the fluids of the invention comprise nanostructures containing gas stabilized by charge, they can be modulated (for example, increase, decrease, adapt, etc.) by altering the ionic components of the fluids, as for example described above, and / or by altering the gas component of the fluid. In preferred aspects, oxygen is used in the preparation of the electrokinetic fluids of the invention. In further aspects, mixtures of oxygen are used together with at least one of the other gases which are selected from nitrogen, oxygen, argon, carbon dioxide, neon, helium, krypton, hydrogen and xenon.

Preferred illustrative modalities: The particular aspects provide a method for the treatment of a condition or disease mediated by TSLP or TSLPR, which comprises administering to a mammal in need thereof a therapeutically effective amount of an electrochemically altered aqueous fluid comprising an aqueous ionic solution of charge-stabilized oxygen-containing nanostructures, which have substantially an average diameter of less than about 100 nanometers and which are stably configured in the aqueous ionic fluid in an amount sufficient to treat a condition or disease mediated by the TSLP or TSLPR. In certain aspects, the charge-stabilized oxygen-containing nanostructures are stably configured in the aqueous ionic fluid in an amount sufficient to provide, upon contact of a living cell with the fluid, the modulation of at least one of the cell membrane potential and of the conductivity of the cell membrane.

In certain aspects of the method, the nanostructures containing charge-stabilized oxygen are the main species of nanostructures that contain gas stabilized by charge in the fluid. In particular embodiments, the percentage of dissolved oxygen molecules present in the fluid as oxygen-containing nanostructures stabilized by charge is a percentage selected from the group consisting of greater than: 0.01%; 0.1%; 1%; 5%; 10%; fifteen%; twenty%; 25%; 30%; 35%; 40%; Four. Five%; fifty%; 55%; 60%; 65%; 70%; 75%; 80%; 85%; 90% and 95%. In particular aspects, the total dissolved oxygen is substantially present in charge-stabilized oxygen-containing nanostructures. In certain embodiments, the charge-stabilized oxygen-containing nanostructures substantially have an average diameter smaller than a size selected from the group consisting of: 90 nm; 80 nm; 70 nm; 60 nm 50 nm; 40 nm; 30 nm; 20 nm; 10 nm; and less than 5 nm.

In particular aspects, the condition or disease mediated by the TSLP or TSLPR comprises a disease or disorder of the immune system, including but not limited to allergic inflammation. In particular aspects, allergic inflammation comprises at least one of allergic inflammation of the respiratory tract, inflammatory Th2 responses mediated by DC, atopic dermatitis, atopic eczema, asthma, obstructive airways disease, chronic obstructive pulmonary disease, mid-life disorders for IgE, rhino-conj, and food allergies. In certain embodiments, the condition or disease mediated by the TSLP or TSLPR comprises inflammatory arthritis, for example, comprising at least one of rheumatoid arthritis and psoriasis.

In certain aspects, the method further comprises combination therapy, wherein at least one additional therapeutic agent is administered to the patient. In particular embodiments, the at least one additional therapeutic agent is selected from the group consisting of short-acting agonists-2, long-acting agonists-2, anticholinergics, corticosteroids, systemic corticosteroids, mast cell stabilizers, leukotriene modifiers, methylxanthines, and combinations thereof. In certain aspects, the at least one additional therapeutic agent is selected from the group consisting of: bronchodilators consisting of p2-agonists including albuterol, levalbuterol, pirbuterol, artformoterol, formoterol, salmeterol, and anticholinergics such as ipratropium and tiotropium; corticosteroids including beclomethasone, budesonide, flunisolide, fluticasone, mometasone, triamcinolone, methylprednisolone, prednisolone, prednisone; leukotriene modifiers including montelukast, zafirlukast, and zileuton; mast cell stabilizers including cromolin and nedocromil; methylxanthines including theophylline, combination drugs including ipratropium and albuterol, fluticasone and salmeterol, budesonide and formoterol; antihistamines including hydroxzine, diphenhydramine, loratadine, cetirizine, and hydrocortisone; drugs that modulate the immune system that include tacrolimus and pimecrolimus; cyclosporin; azathioprine; mycophenolate and combinations thereof. In particular aspects, the at least one additional therapeutic agent is an antagonist of TSLP and / or TSLPR, and in particular embodiments, the TSLP antagonist and / or TSLPR is selected from the group consisting of neutralizing antibodies specific for TSLP and the receptor of the TSLP, soluble TSLP receptor molecules, and TSLP receptor fusion proteins, which include the immunoglobulin Fe-TSLPR molecules or polypeptides that encode the components of more than one receptor chain.

In particular aspects of the method, the modulation of at least one of the cell membrane potential and the cell membrane conductivity comprises altering at least one of the structures or functions of the cell membrane, comprising altering at least one of a conformation , the activity of binding to the ligand, and a catalytic activity of a protein or component associated with the membrane. In certain aspects, the membrane-associated protein comprises at least one selected from the group consisting of receptors, transmembrane receptors, ion channel proteins, intracellular binding proteins, cell adhesion proteins, integrins, etc. In certain embodiments, the transmembrane receptor comprises a receptor coupled to the G protein (GPCR). In particular aspects, the G protein-coupled receptor (GPCR) interacts with the a subunit of the G protein, for example, in which the oi subunit of the G protein comprises at least one selected from the group consisting of Gas, Go¡i, Goíq and Go¡i2, and in certain embodiments the at least one subunit a of the G protein is Gaq.

In certain aspects of the method, the modulation of at least one of the cell membrane potential and the cell membrane conductivity comprises the modulation of a calcium-dependent cellular messaging system or pathway. In certain aspects of the method, the modulation of at least one of the cell membrane potential and the cell membrane conductivity comprises the modulation of phospholipase C activity. In certain aspects of the method, the modulation of at least one of the potential of the cell membrane and the conductivity of the cell membrane comprises the modulation of adenylate cyclase (AC) activity. In certain aspects of the method, the modulation of at least one of cell membrane potential and cell membrane conductivity comprises the modulation of intracellular signal transduction associated with at least one condition or symptom selected from the group consisting of diseases or disorders of the immune system. , allergic inflammation, allergic inflammation of the respiratory tract, inflammatory responses of Th2 mediated by DC, atopic dermatitis, atopic eczema, asthma, obstructive airways disease, chronic obstructive pulmonary disease, IgE-mediated disorders, rhino-conjunctivitis, allergies to food, inflammatory arthritis, rheumatoid arthritis and psoriasis.

In certain aspects of the method, the electrokinetically altered aqueous fluid comprises at least one of solvated electrons and electrokinetically modified or charged oxygen species, for example, in which the form of electrons solvated or of electrokinetically modified or charged oxygen species are present in an amount of 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 aspects, the electrokinetically altered aqueous fluid comprises a form of solvated electrons stabilized by molecular oxygen.

In certain aspects, the amount of oxygen present in the oxygen-containing nanostructures stabilized by electrokinetically altered fluid charge 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 least 50 ppm, or at least 60 ppm of oxygen at atmospheric pressure.

Relevant molecular interactions e Listen: Conventionally, it is believed that quantum properties belong to elementary particles of less than 10 ~ 10 meters, while the macroscopic world of our everyday life is referred to as classical, because it behaves according to Newton's laws of motion .

Recently it has been described that molecules form clusters that increase in size with dilution. These clusters measure several micrometers in diameter, and have been reported to increase in size non-linearly with dilution. It has been postulated that coherent quantum domains measuring 100 nanometers in diameter originate in pure water, and the collective vibrations of water molecules in the coherent domain can eventually become phase locked for fluctuations in the electromagnetic field, providing stable oscillations in water, and providing a form of "memory" in the form of excitation of long-lasting coherent oscillations, specifies for substances dissolved in water that change the collective structure of water, which can in turn determine coherent oscillations you specify that they develop. When these oscillations are stabilized by the coupling of the phases of the magnetic field, the water when diluted can still carry coherent oscillations 'seeds'. Because a grouping of molecules increases in size, their electromagnetic signature is amplified correspondingly, reinforcing the coherent oscillations carried by the water.

Despite variations in the size of the grouping of the dissolved molecules and the detailed microscopic structure of the water, there may nevertheless be a specificity of the coherent oscillations. A model for considering changes in water properties is based on considerations that are involved in crystallization.

A grouping of simplified protonated water, forming a nanoscale cage, is shown in the previous patent application of the applicants: O 2009/055729. A grouping of protonated water typically takes the form of H + (H20) n. Some clusters of protonated water are produced naturally, such as in the ionosphere. Without adhering to any particular theory, and according to particular aspects, other types of water groupings or structures (clusters, nanojails, etc.) are possible, including the structures comprising oxygen and stabilized electrons imparted to the materials resulting from the invention. Oxygen atoms can get trapped in the resulting structures. The chemistry of the semi-linked nanojaula allows oxygen and / or stabilized electrons to remain dissolved for extended periods of time. Other atoms or molecules, such as medicinal compounds, can be caged for sustained release purposes. The specific chemistry of the solution material and the dissolved compounds depends on the interactions of those materials.

The fluids processed by the mixing device have been previously shown, through the experiments to exhibit different structural features that are consistent with a fluid analysis in the context of a grouping structure. See, for example, WO 2009/055729.

Nanostructures stabilized by charge (for example, nanostructures containing oxygen stabilized by charge): As previously described in applicants' document WO 2009/055729, the "Effect of the Double Layer", the "Residence Time", the "Infusion Rate", and the "Bubble Size Measurements", the Electrokinetic mixing device creates, in a matter of milliseconds, a single dynamic interaction of the non-linear fluid of the first material and the second material with dynamic, complex turbulence, which provides the complex mixture in contact with a really huge surface area (including the of the device and that of the exceptionally small gas bubbles of less than 100 nm), which provides the novel electrokinetic effects described herein. Additionally, the electrokinetic effects localized by the characteristics (voltage / current) were demonstrated using a specially designed mixing device, which comprises the characteristics of the isolated rotor and stator.

As it is well known in the state of the art, it is known that the redistributions of solvated charges and / or electrons are very unstable in aqueous solution. According to particular aspects, the applicants' electrokinetic effects (for example, redistributions of charges, including, in particular aspects, solvated electrons) are surprisingly stabilized in the output material (eg, salt solutions, ionic solutions). : In fact, as described herein, the stability of the properties and the biological activity of the electrokinetic fluids of the invention (eg, RNS-60 or Solas) can be maintained for months in a gas-tight container , which indicates the participation of the dissolved gas (for example, oxygen) to help generate and / or maintain, and / or mediate the properties and activities of the solutions of the invention. Significantly, the redistributions of solvated charges and / or electrons are stably configured in the ionic aqueous ionic fluidic fluids of the invention, in an amount sufficient to provide, in contact with a living cell (eg, a mammalian cell) by the fluid, the modulation of at least one of the cell membrane potential and the cell membrane conductivity (see, for example, Practical Example 23 of cell membrane binding of WO 2009/055729 and as disclosed herein).

As described herein, under the term "Molecular Interactions," to refer to the stability and biological compatibility of the electrokinetic fluids of the invention (e.g., electrokinetic salt solutions), applicants have proposed that the interactions between the water molecules and the molecules of substances (eg, oxygen) dissolved in water, change the collective structure of water and provide nanoscale cage clusters, including nanostructures comprising oxygen and / or stabilized electrons transmitted to the resulting materials of the invention. Without adhering to the mechanism, the configuration of the nanostructures, in particular aspects, is such that: they comprise (at least for the formation and / or the stability and / or the biological activity) dissolved gas (for example, oxygen); allow electrokinetic fluids (eg, RNS-60 or Solas saline fluids) to modulate (eg, impart or receive) charges and / or effects of charges upon contact with a cell membrane or a related constituent of the same; and in particular aspects, they provide for the stabilization (eg, carrying, harboring, capturing) of solvated electrons in a biologically relevant form.

According to particular aspects, and as supported by the present disclosure, in saline solutions (eg, standard saline solution, NaCl) or ionic solutions, the nanostructures of the invention comprise nanostructures stabilized by charge (eg, of smaller average diameter). 100 nm), which may comprise at least one molecule of dissolved gas (eg, oxygen) within a hydration envelope stabilized by charge. According to other aspects, the hydration cover stabilized by charge may comprise a host cage or vacuum of at least one molecule of dissolved gas (e.g., oxygen). According to further aspects, by virtue of the provision of adequate charge-stabilized hydration coatings, charge-stabilized nanostructures and / or nanostructures containing charged stabilized oxygen may additionally comprise a solvated electron (for example, a stabilized electron solvated).

Without adhering to a particular mechanism or theory, after the present priority date, microbubbles stabilized by charge, stabilized by the ions in the aqueous fluid in equilibrium with the environmental gas (atmospheric) have been proposed (Bunkin et al., Journal of Experimental and Theoretical Physics, 104: 486-498 of 2007, incorporated herein by reference in its entirety). In accordance with particular aspects of the present invention, the novel electrokinetic fluids of the applicants comprise a new, biologically active form of nanostructures containing charged stabilized oxygen, and may additionally comprise novel matrices, clusters or associations of such structures.

According to the charge-stabilized microburst model, the short-range molecular order of the water structure is destroyed by the presence of a gas molecule (for example, a molecule of dissolved gas, initially complexed with a non-ion adsorptive provides a defect of short range order), providing the condensation of the ionic droplets, in which the defect is surrounded by a first and second spheres of coordination of water molecules, which are alternatively filled by adsorptive ions (e.g. , acquisition of a cover of Na + ion detection to form a double electric layer) and not adsorptive ions (for example, Cl ions "occupying the second sphere of coordination) that occupy six and 12 seats, respectively, in the spheres of coordination In ionic solutions not completely saturated (for example, saline solutions not completely saturated), this hydrated "core" remains stable until the first and second spheres are occupied by six adsorptive and five non-adsorptive ions, respectively, and then the Coulomb explosion suffers, creating an internal vacuum containing the gas molecule, in the which adsorptive ions (for example, those of Na + ions) adsorb to the surface of the resulting vacuum, while non-adsorptive ions, (or some portion thereof) diffuse into the solution (Bunkin et al., supra). In this model, the vacuum in the nanostructure is prevented from collapse by the Coulombic repulsion between the ions (for example, the Na + ions) adsorbed on its surface. The stability of the vacuum-containing nanostructures is postulated to be due to the selective adsorption of dissolved ions with equal charges on top of the vacuum / bubble surface and the diffusive equilibrium between the dissolved gas and the gas inside the bubble, where the pressure Electrostatic negative outward exerted by the resulting double electric layer, provides stable compensation for surface tension, and the pressure of the gas inside the bubble is balanced by the ambient pressure. According to the model, the formation of such microbubbles requires an ionic component, and in certain aspects, the associations mediated by collision between the particles, can lead to the formation of higher order groupings (matrices) (Id).

The charge-stabilized microbubble model suggests that the particles may be microbubbles of gas, but only contemplate the spontaneous formation of such structures in ionic solution in equilibrium with ambient air, is not characterized and is silent as to whether oxygen it is able to form these structures, and is equally silent as to whether the solvated electrons could be associated and / or stabilized by these structures.

According to particular aspects, the electrokinetic fluids of the invention comprising charge-stabilized nanostructures and / or nanostructures containing charge-stabilized oxygen are novel and fundamentally different from the non-electrokinetic atmospheric charge-stabilized microburst structures postulated in accordance with the model of the microbubbles. Significantly, this conclusion is unavoidable, deriving at least in part, from the fact that saline control solutions do not have the biological properties disclosed herein, while the nanostructures stabilized by applicants loading provide a novel biologically active form of nanostructures. They contain stabilized charge oxygen.

In accordance with particular aspects of the present invention, the novel electrokinetic device and applicants' methods provide electrokinetically novel altered fluids comprising significant amounts of stabilized nanostructures by loading in excess of any amount that may or may not occur spontaneously in the ionic fluids in equilibrium with air, or in any non-electrokinetically generated fluid. In particular aspects, the nanostructures stabilized by load comprise nanostructures containing oxygen stabilized by charge. In additional aspects, the charge-stabilized nanostructures are all, or substantially all nanostructures containing charge-stabilized oxygen, or the nanostructures containing charge-stabilized oxygen, the main species of oxygen-containing nanostructures stabilized by charge in the electrokinetic fluid.

According to other additional aspects, charge-stabilized nanostructures and / or charge-stabilized oxygen-containing nanostructures can comprise or receive a solvated electron, and thereby provide a novel stabilized solvated electron carrier. In particular aspects, charge-stabilized nanostructures and / or charge-stabilized oxygen-containing nanostructures provide a new type of electride (or inverted electride), which, unlike conventional solute electrons, have a single organically coordinated cation more that a plurality of cations stably ordered with respect to a vacuum or a void containing an oxygen atom, in which the ordered sodium ions are coordinated by the hydration covers of water, rather than by the organic molecules. According to particular aspects, a solvated electron can be accommodated by the hydration cover of the water molecules, or it can preferably be accommodated within the void of the nanostructure, distributed through all the cations. In certain aspects, the nanostructures of the invention provide a novel structure of "super electride" in the solution, since it not only provides the distribution and stabilization of the solvated electron through multiple ordered sodium cations, but also provides the association or the partial association of the solvated electrons with the oxygen molecule (s) caged in the vacuum of the solvated electrons are distributed through a matrix of sodium atoms and at least one oxygen atom. According to particular aspects, therefore, "solvated electrons" as disclosed herein in association with the electrokinetic fluids of the invention, can not be solved in the traditional model that comprises direct hydration through water molecules. On the other hand, in limited analogy with the dried electrolysis salts, the electrons solvated in the electrokinetic fluids of the invention can be distributed through multiple nanostructures stabilized by charge, to provide a "grid glue" to stabilize the order matrices. greater in aqueous solution.

In particular aspects, the load-stabilized nanostructures of the invention and / or the nanostructures containing charge-stabilized oxygen are capable of interacting with cell membranes or their constituents, or proteins, etc., to mediate the activities biological In particular aspects, the load-stabilized nanostructures of the invention and / or the nanostructures containing charge-stabilized oxygen that host a solvated electron, are capable of interacting with cell membranes or with their constituents, or proteins, etc., to mediate biological activities.

In particular aspects, the load-stabilized nanostructures of the invention and / or the nanostructures containing charged stabilized oxygen interact with cell membranes or with constituents thereof, or proteins, etc., as a load and / or a donor effect (delivery) of cargo and / or as a load and / or a load receptor effect to mediate biological activities. In particular aspects, the load-stabilized nanostructures of the invention and / or the nanostructures containing charge stabilized oxygen that host a solvated electron, interact with the cell membranes as a load and / or as a load donor effect and / or as a load and / or as a load receptor effect to mediate biological activities.

In particular aspects, the stabilized nanostructures by charge of the invention and / or nanostructures the stabilized charge containing oxygen are consistent with, and give an account of the stability observed and the biological properties of the electrokinetic fluids of the invention and, in addition, provide a novel electrolyte (or inverted electride) which provides solvated electrons stabilized in aqueous ionic solutions (eg, saline solutions, NaCl, etc.).

In particular aspects, the nanostructures containing oxygen stabilized by charge substantially comprise, taking the form of, or can give rise to, nanobubbles containing oxygen stabilized by charge. In particular aspects, the groups containing oxygen stabilized by filler provide the formation of relatively larger matrices of oxygen-containing nanostructures stabilized by charge, and / or nanobubbles containing oxygen stabilized by charge or matrices thereof. In particular aspects, the nanostructures containing oxygen stabilized by charge can provide the formation of hydrophobic nanobubbles after contact with a hydrophobic surface.

In particular aspects, charged stabilized oxygen-containing nanostructures substantially comprise at least one oxygen molecule. In certain aspects, charged stabilized oxygen-containing nanostructures substantially comprise 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, minus 100, or more oxygen molecules. In particular aspects, charged stabilized oxygen-containing nanostructures comprise or cause nanoburbutes (eg, hydrophobic nanobubbles) of about 20 nm x 1.5 nm, comprise about 12 oxygen molecules (eg, based on the size of a molecule of oxygen). oxygen (approx 0.3 nm by 0.4 nm), assumption of an ideal gas and application of n = PV / RT, where P = l atm, R = 0.082 D057 D1 atm / mol K, T = 295K; V = pr2h = 4.7xl0-22 L, where r = 10xl0-9 m, h = 1.5xl0-9 m, and n = l .95xl0 ~ 22 moles).

In certain aspects, the percentage of oxygen molecules present in the fluid that are in those nanostructures, or matrices thereof, which have a stabilized configuration with charge in the aqueous ionic fluid is a selected percent amount of the group consisting of greater than : 0.1%, 1%; 2%; 5%; 10%; fifteen%; twenty%; 25%; 30%; 35%; 40%; Four. Five%; fifty%; 55%; 60%; 65%; 70%; 75%; 80%; 85%; 90%; and greater than 95%. Preferably, this percentage is greater than about 5%, greater than about 10%, greater than about 15%, or greater than about 20%. In additional aspects, the substantial size of charge-stabilized oxygen-containing nanostructures, or matrices thereof, which have a stabilized configuration with charge in the aqueous ionic fluid is a size selected from the group consisting of less than: 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 1 nm. Preferably, this 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 certain aspects, the electrokinetic fluids of the invention comprise solvated electrons. In additional aspects, the electrokinetic fluids comprise charge-stabilized nanostructures and / or load-stabilized oxygen-containing nanostructures, and / or matrices thereof, which comprise at least one of: solvated electron (s), and distributions Single load (polar load distribution, symmetric, asymmetric). In certain aspects, the stabilized nanostructures by charge and / or the nanostructures containing charge-stabilized oxygen, and / or matrices thereof, have paramagnetic properties.

In contrast, in relation to the electrokinetic fluids of the invention, the control of the oxygenated fluids by autoclave (non-electrokinetic fluids) and the like, do not comprise such biologically active nanostructures stabilized by charge generated electrokinetically, and / or nanostructures containing stabilized oxygen. biologically active charge and / or matrices thereof, capable of modulating at least one of the potential of the cell membrane and the conductivity of the cell membrane.

Systems for making fluids enriched with gas The system and methods described herein allow the gas (eg, oxygen) to be stably enriched at a high concentration with minimum passive losses. This system and methods can be effectively used to enrich a wide variety of gases at increased rates in a wide variety of fluids. By way of example only, deionized water at room temperature that typically has levels of about 2-3 ppm (parts per million) of dissolved oxygen can achieve dissolved oxygen levels that are in the range of at least about 5 ppm, at less about 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 higher value or which is between these using the systems and / or method described. According to a particular illustrative embodiment, water enriched with oxygen can be generated with levels of approximately 30-60 ppm of dissolved oxygen.

Table 1 illustrates several measurements of the partial pressure taken in a cure of lesions treated with an oxygen-enriched saline solution (Table 1) and in samples of the oxygen enriched gas-enriched saline solution of the present invention.

TABLE 1 TSLP and conditions mediated by the TSLP TSLPR and TSLP agonists / antagonists: An agent has affinity for and stimulates physiological activity in cellular receptors normally stimulated by substances of natural origin, thus triggering a biochemical response. A receptor of the TSLP agonist has affinity for the TSLP receptor and stimulates an activity induced by the binding of the TSLP with its 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 a cellular receptor normally stimulated by a substance of natural origin. Correspondingly, a TSLP / TSLP receptor antagonist binds to TSLP or the TSLP receptor and inhibits the binding of TSLP to the TSLP receptor and / or inhibits an activity normally induced by the binding of TSLP to its receptor. For example, a TSLP / TSLP receptor antagonist can bind to TSLP or the TSLP receptor and decrease or prevent binding, for example, blocking the junction, from TSLP to the receiver of the TSLP. Alternatively, a TSLP / TSLP receptor antagonist can bind to the TSLP receptor and decrease or prevent downstream signaling that would normally be induced by the binding of TSLP to its receptor. Agonists and antagonists can include a variety of classes of molecules including polypeptides, such as ligand-like polypeptides, antibodies, and fragments or subsequences thereof. Agonists and antagonists may also include fusion polypeptides, antibodies, peptides (such as peptide less than about 20 amino acids in length), and small molecules. Exemplary antagonists include: neutralizing antibodies specific for TSLP and the TSLP receptor, soluble TSLP receptor molecules, and TSLP receptor fusion proteins, such as immunoglobulin Fe-TSLPR molecules or polypeptides that encode overlapping components of a receptor chain, thereby mimicking a heterodimer of the physiological receptor or oligomer of higher order. If the receptor includes more than one polypeptide chain, a single chain fusion can be used.

Antibody: A polypeptide ligand comprising at least one variable region of light chain or heavy chain immunoglobulin that specifically recognizes and binds an epitope (eg, as an antigen, such as TSLP or a fragment thereof, or a receptor) of the TSLP of a fragment of this one). This includes intact immunoglobulins and variants and portions thereof well known in the art, such as Fab 'fragments, F (ab) 1. sub.2 fragments, single chain Fv ("scFv") proteins, and stabilized Fv proteins. with disulfide ("dsFv"). A scFv protein is a fusion protein in which a light chain variable region of an immunoglobulin and a heavy chain variable region of an immunoglobulin are linked by a linker, while in dsFvs, the chains are mutated to introduce a disulfide bond to stabilize the association of the chains. The term also includes genetically modified forms such as chimeric antibodies (eg, humanized murine antibodies), heteroconjugated antibodies (eg, bispecific antibodies). See also, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, 111.); Kuby, J., Immunology, 3rd Ed., W.H. Freeman & Co. , New York, 1997. Typically, an immunoglobulin has a light and a heavy chain. Each light and heavy chain contains a constant region and a variable region, (the regions are also known as "domains"). In combination, the light and heavy chain variable regions specifically bind to the antigen. The light and heavy chain variable regions contain a "framework" region interrupted by three hypervariable regions, also called "complementarity determining regions" or "CDRs." The extent of the framework region and the CDRs were defined (see, Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1991, which is incorporated herein by reference). The Kabat database is now maintained online. The sequences of the framework regions of different light or heavy chains are relatively conserved within the species. The framework region of an antibody, which is the combined framework regions of the light and heavy chains of the constituent, serves to position and align the CDRs in the three-dimensional space. Antibodies include monoclonal antibodies, humanized antibodies, etc.

TSLP antagonists include small molecule antagonists, antibodies to TSLP, antibodies to the TSLP receptor, and TSLP receptor fusion proteins, such as immunoglobulin Fe-TSLPR molecules or polypeptides that encode components of more than one chain. of the receptor, which thus mimic a heterodimer of the physiological receptor or oligomer of higher order, among others. It was shown that TSLP binds directly to a member of the type I cytokine receptor superfamily (which are also known as members of the hematopoietin receptor superfamily), TSLPR. The TSLPR was cloned. The functional high affinity receptor for TSLP was shown to include two polypeptides, TSLPR and the alpha chain of the IL-7 receptor. Thus, TSLP and IL-7 share IL-7Ralfa as a component of their receptors. However, these receptors are distinctive in that the TSLP receptor additionally contains the TSLPR while the IL-7 receptor additionally contains the gamma chain of the common cytokine receptor, which is a component of signal transduction of several cytokine receptors. . The TSLPR (and Fe fusions of this receptor chain) are described, for example, in the United States patent application published no. 2002/0160949, which is incorporated herein by reference.

Antibodies to TSLP polypeptides are known in the art. Additionally, anti-TSLPR antibodies are commercially available (R &D Systems, Minneapolis, Minn., Cat.No. MAB981; DNAX Research, Inc., Palo Alto, Calif.). The antibodies are also prepared against the TSLP or TSLP receptor by immunization with specified epitopes, such as the regions of increased antigenicity determined by the Welling tracing of Vector NTI.RTM. Suite (Informax, Inc, Bethesda, Md.). The sequence of the TSLP receptor, and regions of increased antigenicity in the human TSLP receptor are described in U.S. patent application no. 2003/0186875. The pharmaceutical compositions (see above) generally include a therapeutically effective amount of a TSLP antagonist, and may also include additional agents. The preparation of pharmaceutical compositions is described above.

Indications It is believed that TSLP and TSLPR have roles in many types of allergic conditions including, but not limited to, immune system disorders, allergic inflammation, allergic airway inflammation, inflammatory Th2 responses mediated by DC, atopic dermatitis, atopic eczema, allergic asthma, asthma, obstructive respiratory disease, chronic obstructive pulmonary disease (COPD), food allergies, inflammatory arthritis, rheumatoid arthritis, psoriasis, IgE-mediated disorders, and rhino-conjunctivitis. The involvement of TSLP in Th2-mediated inflammatory responses mediated by DCs was shown in many publications including a recent review by Ziegler and Liu (Ziegler and Liu, Wat.Immunol., 7: 709-714, 2005, which is incorporated in the present as a reference in its entirety).

Allergic inflammation of the respiratory tract. A recent study demonstrated that TSLP is required for the initiation of allergic airway inflammation in mice in vivo (Zhou et al., Nat. Immunol., 6: 1047-1053, 2005). In this study, Zhou and others demonstrated that specific lung expression of a TSLP transgene induced allergic inflammation of the airways (asthma) that was characterized by massive infiltration of leukocytes (which include Th2 cells), hyperplasia of Goblet cells , and subepithelial fibrosis, and increased IgE levels in serum. Additionally, a recent study showed that the allergenic challenge caused a rapid accumulation of TSLP in the airways 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 inflammation of the respiratory tract. Here the applicants showed that the electrokinetically altered fluids of the invention down-regulate the TSLP significantly. According to certain embodiments, the electrokinetically altered fluids of the invention have substantial utility for the treatment of allergic inflammation of the respiratory tract and similar conditions.

Allergic inflammation A recent review summarizes and describes the results of studies that confer the role of TSLP in allergic inflammation, for example, allergic inflammation of the skin. (Ziegler and Liu, 2008). In particular, studies showed that normal skin or skin without lesions in patients with atopic dermatitis does not have detectable TSLP protein, whereas, skin taken from acute and chronic atopic dermatitis lesions has a high expression of TSLP. In another study, mice lacking TSLPR were constructed and examined for effects on allergic skin inflammation. (He and others, PNAS, 105: 11875-11880, 2008, which is incorporated herein by reference in its entirety). He and others discovered that the inflammation of the skin due to an allergen in mice lacking TSLPR was significantly reduced when compared to the wild type. In another study, it was shown that mice modified to overexpress TSLP in the skin developed atopic dermatitis that was characterized by ecezamatose lesions of the skin containing inflammatory infiltrates, a dramatic increase in circulating Th2 cells and high IgE in the serum (Yoo and others, J. Exp. Med., 202: 541-549, 2005). The study suggested that TSLP can directly activate DCs in mice. In another study, led by Li and others, the group confirmed that transgenic mice that overexpress TSLP in the skin developed atopic dermatitis that solidifies the link between TSLP and the development of atopic dermatitis. These results indicate that TSLP plays an important role in the pathogenesis of allergic inflammation, for example, allergic inflammation of the skin (e.g., atopic dermatitis and eczema). Here, the applicants showed that the electrokinetically altered fluids of the invention down-regulate significantly the TSLP. According to certain embodiments, the electrokinetically altered fluids of the invention have substantial utility for the treatment of allergic inflammation, for example, allergic inflammation of the skin (e.g., atopic dermatitis and eczema) and similar conditions.

Psoriasis A recent study showed that TSLP had a 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, the applicants showed that the electrokinetically altered fluids of the invention down-regulate the TSLP significantly. According to certain embodiments, the electrokinetically altered fluids of the invention have substantial utility for the treatment of psoriasis and similar conditions.

Allergic asthma. Recently, one study showed that the allergenic challenge caused a rapid accumulation of TSLP in the airways of asthmatic mice. (Liyun Shi et al., Clin.Immunol., 129: 202-210, 2008). In the same study, it was demonstrated that the modulation of the function of the DC by the inhibition of TSLPR reduced the severity in mice. These results indicate that TSLP plays an important role in the pathogenesis of allergic asthma. Here, the applicants showed that the electrokinetically altered fluids of the invention down-regulate the TSLP significantly. According to certain embodiments, the electrokinetically altered fluids of the invention have substantial utility for the treatment of allergic asthma and similar conditions.

Obstructive disease of the respiratory tract. A recent study showed that COPD is associated with an elevated expression of the bronchial mucosa of TSLP (Ying et al., J Immunol, 181: 2790-2798, 2008). COPD is a type of obstructive airway disease. These results indicate that TSLP plays an important role in the pathogenesis of obstructive airway disease, for example, COPD. Here, the applicants showed that the electrokinetically altered fluids of the invention down-regulate the TSLP significantly. According to certain embodiments, therefore, the electrokinetically altered fluids of the invention have substantial utility for the treatment of obstructive airway disease, for example, COPD.

Allergies to food. It was found that the dendritic cells of the intestines stimulate the virgin T cells, deviating them to a TH2 response in a manner dependent on OX40L. (Blazquez AB, Berin MC, Gastrointestinal dendritic cells promote Th2 by deviating via OX40L, J Immunol, 180: 4441-4450, 2008, which is incorporated herein by reference in its entirety). Additionally, a recent review discusses the presence of TSLP in the intestines and their role in the regulation of immune homeostasis. (Iliev ID, Atteoli G, Rescigno M. The yin and yang of intestinal epithelial cells in controlling dendritic cell function, J Exp Med; 204: 2253-2257, 2007, which is incorporated herein by reference in its entirety). These results indicate that TSLP has a role in food allergies. Here, the applicants showed that the electrokinetically altered fluids of the invention down-regulate the TSLP significantly. According to certain embodiments, the electrokinetically altered fluids of the invention have substantial utility for the treatment of food allergies and similar conditions.

Inflammatory arthritis A recent study found increased levels of TSLP in synovial fluid samples derived from patients with rheumatoid arthritis (RA) when compared to synovial fluid obtained from patients with other forms of arthritis. (Koyama et al. Biochem and Biophyis Res Comm., 357: 99-104, 2007, which is incorporated herein by reference in its entirety). The same study found that the use of an anti-TSLP neutralizing antibody improved an experimental TNF-a-dependent arthritis induced by the anti-collagen type II antibody in mice. These results indicate that TSLP is a significant actor in inflammatory arthritis such as RA. At the moment, Applicants showed that the electrokinetically altered fluids of the invention down-regulate significantly the TSLP. According to certain embodiments, the electrokinetically altered fluids of the invention have substantial utility for the treatment of inflammatory arthritis and similar conditions, for example, RA.

Allergic rhinitis. In a recent investigative study, Ou et al, discovered that TSLP was present in mRNA and protein levels in the nasal mucosa of all tested patients who had suffered from allergic rhinitis (AR). (Mou et al., Acta Oto-laryngologica, 129: 297-301, 2009, which is incorporated herein by reference in its entirety). Additionally, the TSLP levels correlated closely with the severity of AR. This results . indicate that TSLP plays an important role in the pathogenesis of RA and / or rhino-conjunctivitis. In the present, applicants showed that the electrokinetically altered fluids of the invention down-regulated the TSLP significantly in a relevant model. According to certain embodiments, therefore, the electrokinetically altered fluids of the invention have substantial utility for the treatment of RA, allergic rhino-conjunctivitis and similar conditions.

Treatment methods The term "treating" refers to, and includes, reversing, alleviating, inhibiting the progress of, or preventing a disease, disorder or condition, or one or more symptoms thereof, and "treatment" and "therapeutic" refer to act of dealing, as defined herein.

A "therapeutically effective amount" is any amount of any of the compounds used in the course of practicing the invention provided herein, which is sufficient to reverse, alleviate, inhibit the progress of, or prevent a disease, disorder or condition. , or one or more symptoms thereof.

Certain embodiments of the present invention relate to therapeutic compositions and methods of treatment for a subject by preventing or mitigating at least one symptom of the inflammation associated with certain conditions or diseases. Many conditions or diseases associated with inflammatory disorders have been treated with steroids, methotrexate, immunosuppressive drugs including cyclophosphamide, cyclosporine, azathioprine, and leflunomide, non-steroidal anti-inflammatory agents such as aspirin, paracetamol, and COX inhibitors. -2, the gold agents and the treatments against malaria. Routes and forms of administration As used herein, "subject" can refer to any living creature, preferably an animal, more preferably a mammal, and even more preferably a human being.

In particular exemplary embodiments, the gas enriched fluid of the present invention may function as a therapeutic composition, alone or in combination with another therapeutic agent, such that the therapeutic composition prevents or alleviates at least one symptom of inflammation. The therapeutic compositions of the present invention include compositions that are capable of being administered to a subject in need thereof. In certain embodiments, the formulation of the therapeutic composition may also comprise at least one additional agent selected from the group consisting of: vehicles, adjuvants, emulsifying agents, suspending agents, sweeteners, flavors, perfumes, and binders.

As used herein, "pharmaceutically acceptable carrier" and "carrier" generally refer to an encapsulating, diluting, or filling, liquid, semi-solid or solid non-toxic, inert material or auxiliary formulation of any type. Some non-limiting examples of materials that can serve as pharmaceutically acceptable carriers are sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; jelly; talcum powder; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cotton oil; safflower oil; Sesame oil; olive oil; corn oil and soybean oil; glycols; such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents; such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline solution; Ringer's solution; ethyl alcohol, and phosphate buffer solutions, as well as other compatible non-toxic lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweeteners, flavorings and flavors, preservatives and Antioxidants may also be present in the composition, according to the formulator's judgment. In particular aspects, such vehicles and excipients may be the fluids or the gas enriched solutions of the present invention.

The pharmaceutically acceptable carriers described herein, for example, vehicles, adjuvants, excipients or diluents, are well known to those skilled in the art. Typically, the pharmaceutically acceptable carrier is chemically inert to the therapeutic agents and has no detrimental 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 fluids of the present invention, the therapeutic composition may additionally comprise inert diluents such as water or other solvents not enriched with additional gas, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, peanut, corn, germ, olive, castor oil, and of sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and esters of sorbitan fatty acids and mixtures thereof. As appreciated by the experts, a novel and improved formulation of a particular therapeutic composition, a therapeutic fluid enriched with novel gas, and a new method of administering the therapeutic fluid enriched with novel gas can be obtained by the replacement of one or more diluents. inert with a fluid enriched with gas of the identical, similar or different composition. For example, conventional water can be substituted or supplemented with a fluid enriched with gas produced by mixing oxygen in water or deionized water to provide the gas enriched fluid.

In certain embodiments, the fluid enriched with inventive gas can be combined with one or more therapeutic agents and / or used alone. In particular embodiments, the incorporation of the gas enriched fluid may include the replacement of one or more solutions known in the art, such as deionized water, saline, and the like, with one or more fluids enriched with gas, thus providing a therapeutic composition. improved for administration to the subject.

Certain embodiments provide the therapeutic compositions comprising a gas enriched fluid of the present invention, a pharmaceutical or other therapeutic agent or a pharmaceutically acceptable salt or solvate thereof, and at least one pharmaceutical carrier or diluent. These pharmaceutical compositions can be used in the prophylaxis and treatment of prior diseases or conditions and in therapies as mentioned above. Preferably, the vehicle should be pharmaceutically acceptable and must be compatible with, that is, not have a deleterious effect on the other components of the composition. The carrier may be a solid or a 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 (for example subcutaneous, intramuscular, intra-arterial, intraperitoneal, intracisternal, intravesical, intrathecal or intravenous), rectal, topical including transdermal, intravaginal, infraocular, intraotic, intranasal, inhalation and injection or insertion of implantable materials or devices.

Administration Routes Most of the suitable means of administration for a particular subject will depend on the nature and severity of the disease or condition to be treated, or on the nature of the therapy used, as well as on the nature of the therapeutic composition or of the additional therapeutic agent. In certain embodiments, oral or topical administration is preferred.

Formulations suitable for oral administration may be provided as discrete units, such as tablets, capsules, sachets, syrups, elixirs, chewing gum, lollipop formulations, microemulsions, solutions, suspensions, pills, gel-coated ampoules, each containing a predetermined amount of the active compound; as powders or granules, as solutions or suspensions in aqueous and non-aqueous liquids, or as oil in water or oil-in-water emulsions.

Appropriate formulations for transmucosal application methods, eg, sublingual or buccal administration, include patches, pills, tablets, and the like comprising the active compound and, typically, a flavored base, such as sugar and gum arabic or tragacanth and Pills comprising the active compound in an inert base, such as gelatin and glycerin or acacia and sucrose.

Formulations suitable for parenteral administration typically comprise sterile aqueous solutions containing a predetermined concentration of the fluid enriched with active gas and, possibly, another therapeutic agent, the solution is preferably isotonic with the blood of the subject recipient. Additional formulations suitable for parenteral administration include formulations containing co-solvents and / or agents that form physiologically appropriate complexes, such as surfactants and cyclodextrins. Oil-in-water emulsions may also be appropriate for formulations for parenteral administration of the gas enriched fluid. Although these types of solutions are preferably administered intravenously, they can also be administered by subcutaneous or intramuscular injection.

Formulations suitable for urethral, rectal or vaginal administration include gels, creams, lotions, aqueous or oily suspensions, dispersible powders or granules, emulsions, soluble solids, showers, and the like. The formulations are preferably provided in the form of unit dose suppositories comprising the active ingredient in one or more solid carriers forming the suppository base, for example, cocoa butter. Alternatively, colonic lavages with the gas enriched fluids of the present invention can be formulated for colonic or rectal administration.

Formulations suitable for topical, infraocular, intraotic, or intranasal application are ointments, creams, pastes, lotions, pastes, gels (such as hydrogels), aerosols, powders and dispersible granules, emulsions, nebulizers or aerosols using flowing propellants (such as such as liposomal aerosols, nasal drops, nasal sprays, and the like) and oils. Suitable carriers for such formulations include petrolatum, lanolin, polyethylene glycols, alcohols, and combinations thereof. Nasal or intranasal administration may include controlled doses of any of these or other formulations. Similarly, the intraotic or infraocular may include drops, ointments, irritation fluids and the like.

The formulations of the invention can be prepared by any suitable method, typically uniformly uniformly mixing the gas enriched fluid optionally with an active compound with finely divided liquid or solid carriers or both, in the required proportions and then, if necessary, the configuration of the resulting mixture in the desired shape.

For example, a tablet may be prepared by compressing an intimate mixture comprising a powder or granules of the active ingredient and one or more optional ingredients, such as a binder, lubricant, inert diluent, or surface active dispersing agent, or by molding an intimate mixture of the active ingredient in powder form and a gas enriched fluid of the present invention.

Formulations suitable for administration by inhalation include fine particle powder or vaporizations that can be generated by various types of aerosols, nebulizers, or pressurized insufflators of controlled doses. In particular, the powders or other compounds of the therapeutic agents can be dissolved or suspended in a fluid enriched with gas of the present invention.

For pulmonary administration through the mouth, the particle size of the powder or droplets is typically in the range of 0.5 to 10 μ, preferably of 1-5 μ, to ensure administration to the bronchial tree. For nasal administration, a particle size in the range of 10 to 500 μ is preferred to ensure retention in the nasal cavity.

The metered dose inhalers are pressurized aerosol dispensers, which typically contain a formulation-solution or suspension of a therapeutic agent in a liquefied propellant. In certain embodiments, as described herein, the gas enriched fluids of the present invention may be used additionally or in place of the standard liquefied propellant. During use, these devices discharge the formulation through a valve adapted to provide a controlled volume, typically 10 to 150 μ ?, to produce a fine particle aerosol containing the therapeutic agent and the gas enriched fluid. Suitable propellants include certain chlorofluorocarbon compounds, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane and mixtures thereof.

The formulation may additionally contain one or more co-solvents, for example, ethanol surfactants, such as oleic acid or sorbitan trioleate, antioxidants and suitable flavoring agents. Nebulizers are commercially available devices that transform the solutions or suspensions of the active ingredient into a therapeutic aerosol vaporization, either by accelerating a compressed gas (typically air or oxygen) through a narrow diffuser orifice, or by of ultrasonic agitation. Formulations suitable for use in nebulizers consist of another therapeutic agent in a fluid enriched with gas and comprise up to 40% w / w of the formulation, preferably less than 20% w / w. In addition, other vehicles may be used, such as distilled water, sterile water, or a dilute aqueous solution of alcohol, preferably isotonic with body fluids by the addition of salts, such as sodium chloride. Optional additives include preservatives, especially if the formula is not prepared sterile, and may include methyl hydroxybenzoate, antioxidants, flavoring agents, volatile oils, buffering agents and surfactants.

Formulations suitable for administration by insufflation include finely powdered powders which can be administered by means of an insufflator or taken in the nasal cavity in the manner of a snuff. In the insufflator, the powder is contained in capsules or cartridges, typically made of gelatin or plastic, which are either perforated or opened in situ and the powder is emitted by the air passing through the device after inhalation, or by means of a manually operated pump. The powder employed in the insufflator consists, either alone of the active ingredient or of a powder mixture containing the active ingredient, a suitable powder diluent, such as lactose, and an optional surfactant. The active ingredient typically 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, taking into account the type of formulation in question. For example, formulations suitable for oral administration may include flavoring agents and formulations suitable for intranasal administration may include perfumes.

The therapeutic compositions of the invention can be administered by any conventional method available for use in combination with drugs, either as individual therapeutic agents or in a combination of therapeutic agents.

The dosage administered, of course, will vary depending on known factors, such as the pharmacodynamic characteristics of the particular agent and its mode and route of administration, the age, health and weight of the recipient, the nature and extent of the symptoms, the type of concomitant treatment, the frequency of treatment, and the desired effect. A daily dose of the active ingredient can be expected to be from about 0.001 to 1,000 milligrams (mg) per kilogram (kg) of body weight, with the preferred dose being 0.1 to 30 mg / kg.

The dosage forms (compositions suitable for administration) contain about 1 mg to 500 mg of the active ingredient per unit. In these pharmaceutical compositions, the active ingredient will normally be present in an amount of about 0.5 to 95% by weight with respect to the total weight of the composition.

Ointments, pastes, foams, occlusions, creams and gels may also contain excipients such as starch, tragacanth, cellulose, silicones, bentonites and talc, or mixtures thereof. The powders and aerosols may also contain excipients such as lactose, talc, silica acid, aluminum hydroxide, and calcium silicates, or mixtures of these substances. The nanocrystalline antimicrobial metal solutions can be converted into aerosols or sprays by any of the commonly known means used for the manufacture of aerosolized drugs. In general, said methods comprise pressurizing or providing a means for pressurizing a solution container, usually with an inert carrier gas, and passing the pressurized gas through a small orifice. The aerosols may additionally contain customary propellants, such as nitrogen, carbon dioxide and other inert gases. In addition, the microspheres or nanoparticles can be used with the therapeutic compositions enriched with gas or the fluids of the present invention, in any of the routes required to deliver the therapeutic compounds to a subject.

Formulations for use as an injection may be presented in sealed unit dose or multidose containers, such as ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition, which requires only the addition of the sterile liquid carrier. , or the fluid enriched with gas, immediately before use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules and tablets. The requirements for effective pharmaceutical vehicles for injectable compositions are well known to those skilled in the art. See, for example, Pharmaceutics and Pharmacy Practice, J. b. Lippincott Co. , Philadelphia, Pa, Banker and Chalmers, Eds. , 238-250 (1982) and ASHP Handbook on Injectable Drugs, Toissel, 4th ed., 622-630 (1986).

Formulations suitable for topical administration include dragees comprising a gas enriched fluid of the invention and, optionally, a therapeutic agent and an additional flavor, usually sucrose and gum arabic or tragacanth; pellets comprising a gas enriched fluid and optionally additional therapeutic agent in an inert base, such as gelatin and glycerin, or sucrose and acacia, and oral rinses or oral rinses comprising a gas enriched fluid and optionally an additional therapeutic agent in a suitable liquid vehicle, as well as creams, emulsions, gels, and the like.

Additionally, formulations suitable for rectal administration may be presented as suppositories, by mixing with a variety of bases such as emulsifying bases or water soluble bases. Formulations suitable for vaginal administration may be presented as ovules, tampons, creams, gels, pastes, foams, aerosols or formulas containing, in addition to the active ingredient, such carriers as is known in the appropriate art.

Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, Mack Publishing Company, a standard reference text in this field.

The dose administered to a subject, especially to an animal, especially a human being, in the context of the present invention, should be sufficient to effect a therapeutic response in the animal for a reasonable period of time. One skilled in the art will recognize that the dose will depend on a variety of factors including the condition of the animal, the body weight of the animal, as well as the condition being treated. An appropriate dose is one that will result in a concentration of the therapeutic composition in a subject known to affect the desired response.

The size of the dose will also be determined by the route, time and frequency of administration, as well as the existence, nature and extent of adverse side effects that could accompany the administration of the therapeutic composition and the desired physiological effect.

It will be appreciated that the compounds of the combination can be administered: (1) simultaneously by combining the compounds in a co-formulation or (2) by alternating, i.e., administering the compounds in series, sequentially, in parallel or simultaneously in separate pharmaceutical formulations. In alternating therapy, the delay in the administration of the second, and, optionally, a third active ingredient, should not be such that the benefit of a synergistic therapeutic effect of the combination of the active ingredients is lost. According to certain modalities of any of the administration methods (1) or (2), the combination would ideally be administered to achieve the most effective results. In certain embodiments by any of the administration methods (1) or (2), it would be ideal if the combination were administered to achieve maximum plasma concentrations of each of the active ingredients. A regimen of a pill once a day, by administering a combination of co-formulation may be feasible for some patients suffering from inflammatory neurodegenerative diseases. According to certain embodiments, the maximum effective plasma concentrations of the active ingredients of the combination will be in the range of about 0.001 to 100 mM. Maximum optimal plasma concentrations can be achieved by a formulation and a prescribed dosage regimen for a particular patient. It will also be understood that the fluids of the invention and a glucocorticoid spheroid (e.g., budesonide) or the physiologically functional derivatives of any of them, whether presented simultaneously or sequentially, can be administered individually, pooled, or in any combination of these forms of administration. In general, during the alternating therapy (2), an effective dosage of each compound is administered in series, in which, in the co-formulation therapy (1), the effective dosages of two or more compounds are administered in a joint The combinations of the invention may conveniently be presented as a pharmaceutical formulation in unit dosage form. A convenient unit dosage formulation contains the active ingredients in any amount from 1 mg to 1 g each, for example, but not limited to, 10 mg to 300 mg. The synergistic effects of the fluids of the invention in combination with, for example, a glucocorticoid steroid (eg, budesonide) can be observed in a broad ratio, for example, from 1:50 to 50: 1 (inventive fluid: a steroid glucocorticoid (for example, budesonide)). In one embodiment, the ratio can vary from 1:10 to 10: 1. In another embodiment, the weight / weight ratio of the fluid of the invention with respect to a glucocorticoid steroid (e.g., budesonide) in a co-formulated combination dosage form, such as a pill, tablet, tablet or capsule, will be approximately 1, ie, an approximately equal amount of the inventive fluid and a glucocorticoid steroid (eg, budesonide) . In other exemplary co-formulations, there may be more or less inventive fluid and a glucocorticoid steroid (eg, budesonide). In one embodiment, each compound will be used in the combination in an amount at which it exhibits anti-inflammatory activity when used alone. Other ratios and amounts of the compounds of said combinations are contemplated within the scope of the invention.

A unit dosage form may further comprise the inventive fluid and, for example, a glucocorticoid steroid (eg, budesonide), or physiologically functional derivatives of any of them, and a pharmaceutically acceptable carrier.

It will be appreciated by those skilled in the art that the amount of active ingredients in the combinations of the invention required for use in the treatment will vary according to a variety of factors, including the nature of the condition to be treated and the age and condition of the patient, and will ultimately be at the discretion of the treating physician or health professional. Factors to be considered include the route of administration and the nature of the formulation, the body weight of the animal, the age and general condition and the nature and severity of the disease to be treated.

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

According to particular aspects, the electrokinetically altered fluids of the invention have a substantial utility for the treatment of conditions mediated by TSLP and / or TSLPR, including but not limited to the illustrative gender of the indications described herein. According to other aspects, the electrokinetically altered fluids of the invention have utility for the treatment of several subgenres of the exemplary genus, where at least one indication of the genus is excluded from each of the subgenres.

EXAMPLE 1 (The synergistic effects of the electrokinetically altered fluids of the invention and the albuterol) General information. The electrokinetically altered fluids of the invention provided prolonged synergistic effects (e.g., suppression of bronchoconstriction) with albuterol in vivo in an animal model recognized in the art of human bronchoconstriction (asthma model in humans)) and therefore it allows a decrease in the use of albuterol in a patient. The results described in this Example are also disclosed in applicants' document WO 2009/055729.

First experiment In a first experiment, sixteen guinea pigs were evaluated for the effects of bronchodilators on the function of the respiratory tract in relation to methacholine-induced bronchoconstriction. After determination of the optimal dose, each animal was dosed with 50 μg / ml to administer the target dose of 12.5 g of albuterol sulfate in 250 μ? per animal. The study was a completely randomized design blocked for the weight and baseline values of the Penh. Two groups (A and B) received an intratracheal instillation of 250 μ? of 50 μg / ml of albuterol sulfate in one or two diluents: in group A it was deionized water that had passed through the inventive device, without the addition of oxygen, while Group B was water enriched with inventive gas. Each group was administered intratracheally with solutions using a Penn Century Microsprayer. In addition, the animals were stratified in all units of the Buxco plethysmograph so that each treatment group is equally represented within the feed of the nebulizers of the plethysmographs and the recording units. Animals that showed at least 75% of their reference Penh value at 2 hours after administration of albuterol were not included in the data analysis. This exclusion criterion is based on past studies in which failure to observe bronchoprotection with bronchodilators may be associated with dosing errors. As a result, one of the animals of the control group was discarded from the analyzes of the data. Once the animal had more than 50% bronchoconstriction, the animal was considered not protected. The results indicate that 50% of the animals in group B were protected from bronchoconstriction at 10 hours (at which time the test was terminated).

Second experiment An additional set of experiments was performed using a larger number of animals to evaluate the protective effects of the electrokinetically generated fluids of the invention (e.g., RDC1676-00, RDC1676-01, RDC1676-02 and RDC1676-03) against the methacholine-induced bronchoconstriction, when administered alone or as diluents for albuterol sulfate in male guinea pigs.

Materials and methods. Guinea pigs (Cavia porcellus) were Hartley albinos, Crl: (HA) BR from Charles River Canada Inc. (St. Constant, Quebec, Canada). Weight: Approximately 325 ± 50 g at the beginning of the treatment, the number of groups was 32, with 7 males per group (plus 24 additional from the same batch of animals). Diet: all animals had free access to a standard certified granulated commercial laboratory diet (PMI Certified Guinea Pig 5026, PMI Nutrition International Inc.), except during designated procedures. The route of administration was intratracheal instillation through a Penn Century Microsprayer and the challenge with methacholine through the inhalation of the whole body. The intratracheal route was selected to maximize the exposure of the lung to the test article / control solution. The challenge of whole-body inhalation has been selected for the methacholine challenge with the aim of eliciting a hypersensitivity response of the upper respiratory tract (ie, bronchoconstriction). The duration of the treatment was one day.

Experimental design. All animals were subjected to inhalation exposure of methacholine (500 μg / ml), 2 hours after TA / control administration. All animals received a dose volume of 250 μ? . Therefore, albuterol sulfate was diluted (in the control article and the 4 test articles) to concentrations of 0, 25, 50 and 100 and g / ml. Thirty minutes before administration, albuterol sulfate solutions of the 4 different concentrations (0, 25, 50 and 100 and g / ml) were formed in a stock solution lOx (500 and g / ml) in each of these four solutions of the test items (RDC1676-00, RDC1676-01, RDC1676-02, and RDC1676-03). These concentrations of albuterol sulfate were also formed in the non-electrokinetically generated control fluids (control 1). Dosing solutions were prepared by proper dilution of each stock solution. All the mother and dosage solutions were kept on ice once prepared. The dosage was completed within one hour after the test / control articles were prepared. A methacholine solution (500 μg / ml) was prepared on the same day of dosing.

Each animal received an intratracheal instillation of the test item or control with a Penn Century Microsprayer. The animals were deprived of food during the night and anesthetized using isoflurane, the larynx was visualized with the help of a laryngoscope (or the appropriate alternative) and the tip of the microsprayer was inserted into the trachea. A dose volume of 250 μ? / Animal of the test or control article was administered. The methacholine aerosol was generated in the air inlet of a mixing chamber using Aeroneb ultrasonic nebulizers, supplied with air from a Buxco polarized flow pump. This mixing chamber, in turn, feeds four individual plethysmographs without limits of the whole body, each operated under a slight negative pressure maintained by means of a gate valve located in the exhaust duct. A vacuum pump was used to exhaust the inhalation chamber at the required flow rate.

Before the start of the main phase of the study, 12 additional animals were assigned to the three groups (n = 4 / group) to determine the maximum exposure period at which animals may be exposed to methacholine to cause acute, severe bronchoconstriction but not mortal. Four animals were exposed to methacholine (500 pg / ml) for 30 seconds and measurements of respiratory parameters were made up to 10 minutes after the start of the aerosol. The concentration of the methacholine nebulizer and / or the exposure time of the aerosolization was suitably adjusted to induce acute / reversible, severe but non-fatal bronchoconstriction, which is characterized by a transient increase in afflictions.

Once before the administration of the test article (day -1) and again at 2, 6, 10, 14, 18, 22 and 26 hours after the dose, the animals were placed in the chamber and the ventilatory parameters (tidal volume, respiratory rate, total lung ventilation) and the increased Penh pause were measured over a period of 10 minutes using the Buxco Electronics BioSystem XA system, following the beginning of the aerosol challenge for methacholine. Once the animals were found within the baseline of the chambers, the values were recorded for 1 minute, after which the animals were exposed to methacholine for 30 seconds, at the nebulizer concentration of 500 pg / ml, and they were exposed to the aerosol for an additional 10 minutes during which ventilation parameters over time were evaluated continuously. Penh was used as an indicator of bronchoconstriction; Penh is a derived value obtained from maximum inspiratory flow, peak expiratory flow and expiration time. Penh = (Maximum expiratory flow / Maximum inspiratory flow) * (expiratory time / time to expire 65% of expiratory volume -1).

Animals that did not show severe acute bronchoconstriction during the challenge with methacholine predosis were replaced. Any animal that showed at least 75% of its value PenhPenes of the baseline at 2 hours after the dose, were not included in the analysis of the data. The respiratory parameters were recorded as the averages of 20 seconds. The data considered as non-physiological were excluded from the subsequent analysis. Changes in the Penh were traced for a period of 15 minutes and the value of the Penh was expressed as the area under the curve. The numerical data were subjected to the calculation of the average group values and standard deviations (as applicable).

Results The results of this experiment showed that, in the absence of albuterol, the administration of the electrokinetically generated fluids of the invention had no apparent effect on average percent of the Penh values of the baseline, when measured over a period of 26 hours Surprisingly, however, the administration of albuterol (representative data are shown for the 25 g albuterol / animal groups) formulated in the electrokinetically generated fluids of the invention (at all levels of oxygen values tested; environment, 20 ppm, 40 ppm and 60 ppm) resulted in a remarkable prolongation of the anti-bronchoconstrictive effects of albuterol, as compared to the control fluid. That is, the results of methacholine showed a prolongation of albuterol bronchodilation of at least 26 hours. The applicants also demonstrated that there were consistent differences in all oxygen levels between RDC1676 and normal saline control. Combining all 4 RDC1676 fluids, the p value for the treatment difference in general from normal saline was 0.03.

According to particular aspects, therefore, the electrokinetically generated solutions of the invention provide synergistic prolongation effects with albuterol, thus leading to a decrease in the use of a patient's albuterol, which allows for a cost-effective drug use more efficient, fewer side effects, and increases the period during which a patient can be treated and respond to treatment with albuterol.

EXAMPLE 2 (The effects of the electrokinetically altered fluids of the invention on the expression of cytokines were determined) General information. The electrokinetically altered fluids of the invention decreased the production of pro-inflammatory cytokines (IL-β, TNF-α, IL-6, and GM-CSF), chemokines (IL-8, IP-la, RANTES and eotaxin ), inflammatory enzymes (iNOS, COX-2 and MMP-9), responses to allergens (HC class II, CD23, B7-1 and B7-2) and Th2 cytokines (IL-4, IL-13) and IL-5), compared to control fluid, and increased anti-inflammatory cytokines (e.g., IL-R-a, TIMP) compared to the control fluid. The results described in this Example are also disclosed in applicants' document WO 2009/055729.

In particular aspects, human mixed lymphocytes were stimulated with the T3 or PHA antigen in the Revalesium oxygen enriched fluid, or in the control fluid, and the changes in IL-? Β, IL-2, IL-4, were evaluated. IL-5, IL-6, IL-7, IL-8, IL-10, IL-12 (p40), IL-12 (p70), IL-13, IL-17, eotaxin, IFN- ?, GM- CSF, γ-γ, MCP-1, G-CSF, FGFb, VEGF, TNF-α, RANTES, leptin, TNF-β, TFG-β, and NGF. As demonstrated, the pro-inflammatory cytokines (IL-? ß, TNF-a, IL-6, and GM-CSF), the chemokines (IL-8, MIP-la, RANTES and eotaxin), the inflammatory enzymes (iNOS) , COX-2 and MMP-9), responses to allergens (MHC class II, CD23, B7-1 and B7-2), and Th2 cytokines (IL-4, IL-13 and IL-5) tested the control fluid was reduced in the test fluid compared. In contrast, the anti-inflammatory cytokines (e.g., ILlR-a, TIMP) tested were increased in the test fluid compared to the control fluid.

Additionally, the applicants used a model system recognized in the state of the art that involves sensitization to ovalbumin, to evaluate allergic hypersensitivity reactions. The final points studied were the particular cellular and cytological components of the reaction, as well as the serological measurements of the proteins and LDH. The analysis of the cytokines was performed, including the analysis of eotaxin, IL-1A, IL-1B, KC, MCP-1, MCP-3, IP-1A, RANTES, TNF-A, and VCAM.

Briefly, Brown Norway male rats were injected intraperitoneally with 0.5 ml of ovalbumin (OVA) Grade V (A5503-1G, Sigma) in solution (2.0 mg / ml) containing aluminum hydroxide (Al (OH) 3) (200 mg / ml) once each of days 1, 2 and 3. The study was a randomized trial with 2 x 2 factorial treatment (4 groups). After a waiting period of two weeks, to allow an immune reaction to occur, the rats were exposed or treated for a week, either with RDC1676-00 (sterile saline solution processed through the proprietary Revalesium device), and RDC1676-01 (sterile saline solution processed through the proprietary Revalesium with added oxygen). At the end of week 1 of the treatment, once a day, the two groups broke in half and 50% of them. rats from each group received saline or challenged with OVA by inhalation.

Specifically, fourteen days after the initial serialization, 12 rats were exposed to RDC 1676-1600 per inhalation for 30 minutes each day for 7 consecutive days. The speed of the air flow through the system was set at 10 liters / minute. A total of 12 rats were aligned in the circular chamber, with a single port for the nebulized material to enter and distribute equally to the 12 sub-chambers of the Aeroneb.

Fifteen days after the initial sensitization, 12 rats were exposed to RDC 1676-1601 by ultrasonic nebulization for 30 minutes each day for 7 consecutive days. The air flow was also set at 10 liters / minute, with the same nebulizer and chamber. The RDC 1676-1600 was nebulized first and the Aeroneb chamber was completely dried before the RDC 1676-1601 was nebulized.

Approximately two hours after the last nebulization treatment, 6 rats from the RDC group 1676-1600 were challenged again with OVA (1% in saline) administered by intratracheal instillation using a Penn Century Microsprayer (Model 1A-1B). The other six rats from the RDC group 1676-00 were challenged with saline in the control group, administered by intratracheal instillation. The next day, the procedure was repeated with the group of RDC 1676-01.

Twenty-four hours after the second challenge, all rats in each group were sacrificed by overdose with sodium pentobarbital. The whole blood samples were collected from the inferior vena cava, and placed in two different blood collection tubes: Qiagen PAXgene ™ Blood RNA Tube and Qiagen PAXgene ™ Blood DNA Tube. The organs of the lungs were processed to obtain a bronchoalveolar lavage (BAL) of the fluid and lung tissue for RT-PCR, to evaluate changes in markers of cytokine expression that are known to be associated with lung inflammation in this model. A unilateral washing technique was used to preserve the integrity of the four lobes on the right side of the lung. The left "big" lobe was washed, while the four right lobes were tied and immediately placed in TRI-zol ™, homogenized and sent to the laboratory for further processing.

Analysis of the BAL. Lung lavage was collected and centrifuged for 10 minutes at 4 C at 600-800 g to pellet the cells. The supernatants were transferred to fresh tubes and frozen at -80 ° C. The bronchial lavage fluid ("BAL") was separated into two aliquots. The first aliquot was centrifuged and the supernatant was quickly frozen on chopped dry ice, placed at -80 ° C, and sent to the laboratory for further processing. The amount of protein and LDH present indicate the level of protein in the blood serum (the protein is a component of the serum that filters through the membranes when challenged as in this experiment) and cell death, respectively. The proprietary test side showed slightly less protein than the control.

The second aliquot of the bronchial lavage fluid was evaluated for the total protein and LDH content, as well as underwent a cytological examination. The treated group showed that the total cells were larger than those in the control group with saline. In addition, there was an increase in eosinophils in the treated group compared to the control group. There were also slightly different polymorphonuclear cells for those treated compared to the control side.

Blood analysis The whole blood was analyzed by transferring 1.2 to 2.0 ml of blood into a tube, and allowing it to clot for at least 30 minutes. The remaining blood sample (approximately 3.5- 5.0 ml) was saved for RNA extraction using TRI-zol ™ or PAXgene ™. Next, the coagulated blood sample was centrifuged for 10 minutes at 1200 g at room temperature. The serum (supernatant) was removed and placed in two fresh tubes, and the serum was stored at -80 ° C.

For RNA extraction using Tri-Reagent (TB-126, Molecular Research Center, Inc.), 0.2 ml of whole blood or plasma was added to 0.75 ml of Tri-Reagent BD supplemented with 20 μ? of 5N acetic acid per 0.2 ml of whole blood or plasma. The tubes were shaken and stored at -80 ° C. Using PAXgene ™, the tubes were incubated for approximately two hours at room temperature. The tubes were then placed on their side and stored in the freezer at -20 ° C for 24 hours, and then transferred to -80 ° C for long-term storage.

Luminex analysis. Using the Luminex platform, a microsphere analysis was used as a substrate for a binding reaction of related antibodies, which is read in units of luminosity and can be compared with the quantified standards. Each blood sample was run as two samples concurrently. The units of measurement are units of luminosity and the groups were divided into the controls of those challenged with OVA, those treated with OVA and those of treatment with the proprietary fluid challenged with saline solution.

For the generation of the Agilent gene matrix data, the lung tissue was isolated and immersed in TRI Reagent (TR118, Molecular Research Center, Inc.). 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 the glass-TefIon ™ or Polytron ™ homogenizer. The samples were stored at -80 ° C.

Results from blood samples. Each blood sample was divided into two samples and the samples ran concurrently. The units of measurement are units of luminosity and the groups, going from left to right are: controls challenged with OVA, treatment with Revalesio challenged with OVA; followed by treatment with saline challenge, and treatment with Revalesio challenged with saline. To facilitate the review, both RDC1676-01 groups were highlighted with shaded backgrounds in gray, while the control saline treatment groups have shaded backgrounds.

Generally, when comparing the two groups on the left, while the dispersion of data from the RDC1676-01 group is somewhat higher, the levels of particular cytokines in the RDC1676-01 group as a whole are lower than in the group samples Control treaty, typically approximately 30% numerical difference between the 2 groups. Generally, when comparing the two groups to the far right, group RDC1676-01 has a slightly higher numerical value compared to group RDC1676-00.

Applicants determined that the level of RANTES (super family of IL-8) produced after treatment with the electrokinetically altered fluids of the invention was lower than that produced by groups exposed only to saline. Applicants demonstrated that the electrokinetically altered fluids of the invention caused MCP-1 to be produced at lower levels compared to that produced by groups exposed only to saline. Applicants determined that the level of TNF alpha produced after treatment with the electrokinetically altered fluids of the invention was less than that produced by the groups exposed only to the saline solution.

In addition, the applicants demonstrated that the level of MIP-1 alpha produced after treatment with the electrokinetically altered fluids of the invention was "less than that produced by the groups exposed only to saline." Applicants demonstrated that the electrolytically altered fluids of The invention caused IL-1 alpha to be produced at lower levels compared to that produced by groups exposed only to saline.The Applicants observed that the Vcam level produced after treatment with the electrokinetically altered fluids of the invention was less than that produced by the groups exposed only to saline The Applicants observed that the level of beta IL-1 produced after treatment with the electrokinetically altered fluids of the invention was lower than that produced by the groups exposed only to the solution The applicants proved that Electrokinetically altered fluids of the invention caused eotaxin and MCP-3 to be produced at lower levels compared to those produced by groups exposed only to saline.

In summary, this standard assay of inflammatory reaction to a known sensitization produced, at least in the blood samples, a marked clinical and serological affectation. Additionally, while a significant number of control animals were physiologically stressed and almost died in the process, none of the group treated with RDC1676-01 showed such clinical stress effects. This was then reflected in the circulating levels of cytokines, with approximately 30% differences between the treated RDC1676-01 and RDC1676-01 treated groups in the OVA-challenged groups. In contrast, there were small and relatively insignificant changes in the cytokine, cell and serological profiles between treated RDC1676-01 and RDC1676-01 treated groups in the non-challenged groups with OVA, which probably only represent minimal changes of the baseline fluid itself.

EXAMPLE 3 (The effects of the electrokinetically altered fluids of the invention on the modulation of T cell proliferation were determined) General information. The electrokinetically altered fluids of the invention improved the function of the regulatory T cells, as shown by the relatively reduced proliferation. The results described in this Example are also disclosed in applicants' document WO 2009/055729.

The ability of the particular modalities disclosed herein to regulate T cells was studied by irradiation of antigen-presenting, antigen-introducing cells, and T cells. Typically, these stimulated T cells proliferate. However, after the introduction of regulatory T cells, the usual proliferation of T cells is suppressed.

Methods Briefly, the anti-CD25 antibody conjugated with FITC (ACT-1) used in the classification, was purchased from DakoCytomation (Chicago, IL). The other antibodies used were the following: CD3 (HIT3a for soluble conditions), GITR (conjugated with PE), CD4 (Cy-5 and conjugated with FITC), CD25 (conjugated with APC), CD28 (clone CD28.2), CD127 - APC, Granzima A (conjugated with PE), FoxP3 (BioLegend), mouse IgGl (isotype control), and XCL1 antibodies. All antibodies were used according to the manufacturer's instructions.

CD4 + T cells were isolated from peripheral whole blood with the Rosette Kit CD4 + (Stemcell Technologies). The CD4 + T cells were incubated with anti-CD127-APC, anti-CD25-PE and anti-CD4-FITC antibodies. The cells were sorted by flow cytometry using an Aria FACS in CD4 + CD25 + CD25hiCD1271o / nTreg and CD4 + CD25-T responding cells.

The suppression assays were carried out in 96-well microtitre plates with rounded bottoms. 3.75 x 103 CD4 + CD25neg responding T cells, 3.75 x 103 autologous T-regulatory, 3.75 x 104 CD3-depleted irradiated allogenic PBMC were added as indicated. All wells were supplemented with anti-CD3 (HIT3a clone at 5.0 ug / ml). T cells were cultured for 7 days at 37 ° C in RPMI 1640 medium supplemented with 10% fetal bovine 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 the Tomtec cell harvester and the incorporation of 3H-thymidine was determined using a Perkin Elmer scintillation counter. The antigen presenting cells (APC) consisted of peripheral blood mononuclear cells (PB C) depleted T cells using StemSep human CD3 + T cell depletion (Stemcell Technologies), followed by 40 Gy of radiation.

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

The statistical analysis was performed using the GraphPad Prism software. Comparisons between the two groups were made using the two-tailed, unpaired Student's t-test. The comparisons between the three groups were made using a one-way ANOVA. The values of p < 0.05 were considered significant (two-tailed). The correlation between two groups was determined to be statistically significant through the Spearman coefficient if the value of r was greater than 0.7 or less than -0.7 (two-tailed).

Results The proliferation of regulatory T cells was studied by stimulating the cells with particulate matter from combustion gases (PM, from EPA). Applicants determined that cells stimulated with PM (without Rev, without Solas) resulted in a decrease in secreted IL-10, whereas cells exposed to PM in the presence of the fluids of the present disclosure ("PM + Rev") resulted in a sustained or slightly decreased production of IL-10 in relation to the saline and media controls (without PM). In addition, the diphtheria toxin (DT390, a truncated diphtheria toxin molecule, 1:50 dilution of the standard commercial concentration) was titrated in the fluid samples of the invention, and blocked the Rev-mediated effect of the increase in the IL-10. It should be noted that treatment with Rev resulted only in higher levels of IL-10 relative to the saline and media controls. Similar results were obtained with GITR, granzyme A, XCL1, pStat5 and Foxp3, respectively.

The applicants also obtained data from ?? PBMC, obtained from an allergic asthma profile (AA) of peripheral blood mononuclear cells (PBMC), evaluating tryptase. AA PBMC data were consistent with previous data from regulatory T cells, since cells that were stimulated with particulate material (PM) showed high levels of tryptase, while cells treated with PM in the presence of fluids of the present disclosure ("PM + Rev") resulted in significantly lower tryptase levels, similar to those of the saline and media controls. Consistent with the data of regulatory T cells, exposure to DT390 blocked the effect mediated by Rev on the levels of tryptase, resulting in an elevated level of tryptase in the cells, as observed for the PM only (less Rev, without Rev , without Solas). Treatment with Rev alone resulted in lower tryptase levels relative to the saline and media controls.

In summary, the applicants observed a decrease in proliferation in the presence of PM and Rev in relation to the PM in the control fluid (without Rev, without Solas), indicating that the electrokinetically generated regenerative fluid Rev improved the function of the regulatory T cells, as shown by the relatively reduced proliferation in the assay. Moreover, the evidence indicates that beta blockade, GPCR blockade and blockade of Ca channels affect the activity of Rev as a function of Treg.

EXAMPLE 4 (The synergistic effects between the electrokinetically altered fluids of the invention and the budesonide) General information. The electrokinetically altered fluids of the invention provided synergistic anti-inflammatory effects with budesonide in vivo in an animal model recognized in the state of the art for allergic asthma. The results described in this Example are also disclosed in applicants' document WO 2009/055729.

Applicants initially conducted experiments to evaluate the anti-inflammatory properties of the airways of the electrokinetically generated fluids of the invention (eg, RDC-1676-03) in an ovalbumin sensitization model in the Brown Norway rat. The Brown Norway rat is a recognized model in the state of the art for the determination of the effects of a test material on the function of the respiratory tract and this strain has been widely used, for example, as a model of allergic asthma. The biochemical changes and airway pathology induced by the sensitization of ovalbumin in this model resemble those observed in man (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 exposure of the lung to the test material or to the control solution. Animals sensitized with ovalbumin were treated with budesonide alone or in combination with the test material RDC 1676-1603 for 7 days before challenge with ovalbumin. 6 and 24 hours after the challenge, the total blood count and the levels of various pro and anti-inflammatory cytokines, as well as various respiratory parameters were measured to estimate any beneficial effect of the administration of the test material on various inflammatory parameters.

Materials and methods: Brown Norway rats of strain Bn / Crl were obtained from Charles River Kingston, with an approximate weight of 275 + 50 g at the beginning of the experiment. All animal studies were carried out with the approval of the PCS-MTL Institutional Animal Care and Use Committee. During the study, the use and care of the animals were carried out in accordance with the guidelines of the USA National Research Council, as well as the Canadian Council of Animal Care.

Sensitization . On day 1 of the experiment, the animals (14 animals in each treatment group) were sensitized by administering an intraperitoneal injection of 1 ml of a freshly prepared solution of 2 mg ovalbumin / 100 mg of aluminum hydroxide per 1 ml of chloride of sodium at 0.9%, followed by the repetition of the injection on day 3.

Treatment Fifteen days after initial sensitization, animals were subjected to nebulized control exposure (normal saline) or test solutions (electrokinetically generated fluids RDC1676-00, RDC1676-02 and RDC-1676-03), either administered alone or in combination with budesonide, once a day for 15 minutes for 7 consecutive days. The animals were dosed in a full-body chamber of approximately 201, and the test atmosphere was generated in the air inlet of the chamber using Aeroneb ultrasonic nebulizers, supplied with air from a Buxco polarized flow pump. The air flow rate was set at 10 liters / min.

Challenge with ovalbumin. On day 21, two hours after treatment with the test solutions, all animals were inoculated with a 1% ovalbumin nebulized solution for 15 minutes (in a full-body chamber at 21 / min airflow).

Sampling . At the time points of 6 and 24 hours after challenge with ovalbumin, blood samples were taken for total and differential blood cell count, as well as to measure the levels of various pro and anti-inflammatory cytokines. In addition, immediately after, and at 6 and 24 hours after challenge with ovalbumin, the increased Penh pause and tidal volume were measured over a period of 10 minutes using the Buxco Electronics BioSystem XA system.

Results: Eosinophil count: As expected, treatment with budesonide ("NS + budesonide 750 g / Kg" - densely shaded bar graph) reduced the total eosinophil count in the challenged animals relative to the normal saline treatment control "NS" only. Additionally, while the treatment with the inventive fluid "RDC1676-03" alone did not significantly reduce the eosinophil count, it nonetheless showed a substantial synergy with budesonide in the reduction of the eosinophil count ("RDC1676-03 + budesonide 750 g / kg) Similarly,% of eosinophils also reflected a similar trend, while RDC1676-03 or budesonide 750 ug / kg alone did not have a significant effect on the% eosinophil count in the challenged animals, the two in combination significantly reduced the% of eosinophils.

Therefore, the applicants determined, according to particular aspects, that the electrokinetically generated fluids of the invention (e.g., RDC1676-03) have a substantial utility, from the synergistic point of view, in combination with budesonide to significantly reduce the eosinophil count ("% eosinophils" and total count) in a rat model recognized in the prior art for human allergic asthma.

Respiratory parameters: The applicants also demonstrated the observed effect of the test fluids on the Penh and the tidal volume immediately measured, at 6 and 24 hours after challenge with ovalbumin. The Penh is a derived value obtained from the maximum inspiratory flow, the maximum expiratory flow and the expiration time, and the decrease in the value of the Penh reflects a favorable result for lung function.

Penh = (maximum expiratory flow / maximum inspiratory flow) * (expiratory time / time to expire 65% expiratory volume - 1).

Treatment with budesonide (both at 500 and 750 ug / kg) alone or in combination with any of the test fluids, did not significantly affect the Penh values immediately after the challenge. However, six hours after the challenge, the animals treated with RDC1675-03 alone or in combination with budesonide at 500 or 750 ug / kg, showed a significant fall in the Penh values. Although the magnitude of this drop was less than 24 hours after the challenge, the tendency of a synergistic effect of the RDC fluid and budesonide was still observed at this point in time.

The tidal volume is the volume of air in the lungs during inspiration with respect to the final expiratory position, which leaves the lungs passively during expiration in the course of quiet breathing. Animals treated with budesonide alone did not show changes in tidal volume immediately after challenge. However, RDC1676-03 only had a significant stimulating effect on tidal volume, even at this early time point. And again, RDC1676-03 in combination with budesonide (both at 500 and 750 ug / kg) had an even more pronounced effect on tidal volume measurements at this time point. Six hours after the challenge, RDC1676-03 was sufficient to cause a significant increase in tidal volume and the addition of budesonide to the treatment regimen, either alone or in combination, had no added effect on tidal volume. Any effect observed in these two previous time points, however, lost by the time point of 24 hours.

Taken together, these data demonstrate that RDC1676-03, alone or in combination with budesonide, provides significant relief to airway inflammation as evidenced by the increased tidal volume and the decrease in Penh values at 6 o'clock. hours after the challenge.

Analysis of cytokines: To analyze the mechanism of the effects observed in the physiological parameters previously discussed, a number of pro and anti-inflammatory cytokines were measured in blood samples taken at 6 and 24 after the challenge, performing the physiological measurements immediately afterwards.

The applicants observed that Rev 60 (or RDC1676-03) alone significantly decreased the level of eotaxin in blood, both at 6 and at 24 hours after the challenge. Budesonide at 750 ug / kg also reduced levels; of eotaxin in blood at these two time points, while budesonide at 250 ug / kg only had a noticeable effect at the later time point. However, the Rev 60 test solution alone showed effects that are significantly more potent (in reducing eotaxin blood levels) than both budesonide concentrations, at both time points. Eotaxin is a small C-C chemokine known to accumulate in and attract eosinophils to the lungs of asthmatics and other tissues in allergic reactions (for example, in the intestine in Crohn's disease). Eotaxin binds to a G protein coupled to the CCR3 receptor. CCR3 is expressed by a number of cell types such as Th2 lymphocytes, basophils and mast cells, but the expression of this receptor by Th2 cells is of particular interest since these cells regulate the recruitment of eosinophils. Several studies demonstrated an increased production of eotaxin and CCR3 in the asthmatic's lung, as well as a link between these molecules and airway hyperreactivity (reviewed in Eotaxin and the attraction of eosinophils to the asmatic lungs, Dolores M Conroy and Timothy J. Williams in Respiratory Research 2001, 2: 150-156).

Taken together, these results strongly indicate that treatment with RDC1676-03 alone or in combination with budesonide can significantly reduce the total eosinophil count and the% in blood 24 hours after challenge with ovalbumin. This correlates with a significant decrease in the levels of eotaxin in the blood observed at 6 hours after the challenge.

The blood levels of the two main key anti-inflammatory cytokines, IL-10 and interferon gamma, are also significantly higher at 6 hours after exposure as a result of treatment with Rev 60 alone or in combination with budesonide. The applicants observed such effects on interferon gamma and IL-10, respectively. Rev 60 alone or Rev 60 in combination with budesonide at 250 ug / kg significantly increased the blood level of IL-10 in challenged animals up to 6 hours after challenge. Similarly, Rev 60 alone or in combination with budesonide at 250 ug / kg or 750 ug / kg significantly increased blood levels of IFN gamma at 6 hours after challenge. The increase of these anti-inflammatory cytokines can explain, at least in part, the beneficial effects observed in the respiratory physiological parameters observed at 6 hours after the challenge. The effect on these cytokines was no longer observed at 24 hours after challenge (data not shown).

Rantes or CCL5 is a cytokine that is expressed by circulating T cells and is chemotactic for T cells, eosinophils and basophils and has an active role in the recruitment of leukocytes at sites of inflammation. Rantes also activates the release of eosinophils, for example, the cationic eosinophilic protein. This changes the density of the eosinophils and makes them hypodense, which is thought to represent a state of generalized cellular activation. It is also a potent activator of oxidative metabolism specific to eosinophils.

The applicants observed that the systemic levels of Rantes were significantly reduced at 6 hours, but not at 24 hours after the challenge in the animals treated with Rev 60 alone or in combination with budesonide at 250 ug / kg or 750 ug / kg. Once again, there was a clear synergistic effect of budesonide at 750 ug / kg and Rev 60. A similar downward trend was observed for a number of other pro-inflammatory cytokines, such as KC or IL8, MCP3, IL1B, GCSF, TGFb , as well as NGF, observed both at 6 or 24 hours after challenge, in animals treated with Rev 60 alone or in combination with budesonide.

EXAMPLE 5 (The effects of the electrokinetically altered fluids of the invention on the intercellular tight junctions were determined)) General information. The electrokinetically altered fluids of the invention showed that they modulate the intercellular tight junctions. The results described in this Example are also disclosed in the applicants document O 2009/055729.

According to particular aspects, the therapeutic fluids processed by the inventive diffuser have a substantial utility for modulating narrow intercellular junctions, including those related to pulmonary and systemic administration and bioavailability of the polypeptides, including the exemplary salmon calcitonin polypeptide (sCT ).

General example Salmon calcitonin (sCT) is a peptide of 32 amino acids with a molecular weight of 3,432 daltons. The pulmonary administration of calcitonin has been studied extensively in model systems (eg model systems in rodents, rat model systems, etc.) to investigate methods for improving drug delivery at the pulmonary level (eg, intratracheal drug administration). In accordance with particular exemplary aspects, the therapeutic fluid processed by the inventive diffuser has substantial utility for modulating (eg, increasing) the intercellular tight junctions, for example those associated with pulmonary and systemic administration and the bioavailability of the sCT in a system model in rat.

Methods: Intratracheal drug administration. According to particular embodiments, the sCT was formulated into the inventive therapeutic fluid and administered to rats using an intratracheal drug delivery device. In certain aspects, a Penn Century Micro-Sprayer device designed for the administration of intratracheal drugs to rodents is used, which allows good administration to the lung, but as seen in the state of the art, with relatively low alveolar deposition, resulting in poor systemic bioavailability of the peptides. According to particular aspects, this model system recognized in the state of the art. it was used to confirm that the therapeutic fluid processed by the inventive diffuser has substantial utility for modulating (eg, increasing) the intercellular tight junctions, including those associated with the administration and pulmonary and systemic bioavailability of the polypeptides.

Groups of animals and dosage. In certain aspects, the rats are assigned to one of the 3 groups (n = 6 per group): a) sterile saline solution; b) basic solution without enrichment of 02 ("basic solution"), or c) therapeutic fluid processed by the inventive diffuser ("solution based on the enriched of the invention"). The inventive enriched based solution is formed, for example, by the infusion of oxygen in the 0.9% saline solution. Preferably, the basic solution comprises approximately 0.9% saline, to minimize the chances of hypo-osmotic disruption of the epithelial cells. In certain embodiments, the sCT is reconstituted separately in the basic solution and the inventive enriched based solution and the respective solutions are administered to the respective groups of animals by intratracheal instillation within 60 minutes (10 iq sCT in 200 μ? Per animal) .

Essays. In particular aspects, blood samples were collected (for example, 200 μm) and placed in EDTA coated tubes before administration, and at 5, 10, 20, 30, 60, 120 and 240 minutes after dosing . The plasma is collected and stored at = -70 ° C until analyzed for sCT using the ELISA technique.

For the generation of the Agilent gene matrix data, the lung tissue was isolated and immersed in TRI Reagent (TR118, Molecular Research Center, Inc.). Briefly, approximately 1 ml of TRI Reagent was added to 50-100 mg of tissue in each tube. The samples were homogenized in TRI Reagent, using the glass-TefIon ™ or Polytron ™ homogenizer. The samples were stored at -80 ° C.

Results: Increase of the narrow joints. Applicants noted that RDC1676-01 (sterile saline solution processed through the proprietary device of the present with added extra oxygen, electrokinetically enriched gas generated fluid (Rev) of the present disclosure), decreased systemic administration and bioavailability of sCT . According to particular aspects, the systemic decrease in administration results in decreased adsorption of sCT, most likely as a result of the increase in tight pulmonary junctions. RDC1676-00 represents a sterile saline solution processed according to the presently disclosed methods, but without oxygenation.

Additionally, according to particular aspects, the proteins related to the tight junctions were regulated positively in the lung tissue. The applicants showed positive regulation of the adhesion molecules of the JAM 2 and 3, GJA1, 3, 4 and 5 junctions (adhesions of the junctions), OCLN (occludin), claudins (for example, CLDN 3, 5, 7, 8, 9, 10), TJP1 (narrow binding protein 1), respectively.

EXAMPLE 6 (The effects of the electrokinetically altered fluids of the invention on the conductance of whole cells were determined) General information. The electrokinetically altered fluids of the invention decreased the conductance of the whole cells, as demonstrated by the analysis of membrane fixation conducted in the bronchial epithelial cells (BEC). The analysis of membrane fixation conducted on bronchial epithelial cells (BEC) perfused with the electrokinetically-altered altered fluid (RNS-60) revealed that exposure to RNS-60 resulted in a decrease in the conductance of whole cells. In addition, stimulation with a cAMP-stimulating "cocktail", which dramatically increased the conductance of whole cells, also increased the drug-sensitive portion of the whole-cell conductance, which was ten times greater than that observed in basal conditions. The results described in this Example are also disclosed in applicants' document WO 2009/055729.

The membrane binding studies were carried out to further confirm the utility of the electrokinetically generated fluids of the invention to modulate intracellular signal transduction by modulating at least one of the membrane structures, the membrane potential or the conductivity of the membrane, membrane receptors or proteins, and cellular messaging systems dependent on calcium channels and ion channels.

General information. The applicants showed that the binding of bradykinin to the B2 receptor was concentration dependent, and the binding affinity was increased in the electrokinetically generated fluid (eg, Rev, the electrokinetically enriched gas generated fluid) of the present disclosure in comparison with normal saline. Additionally, the applicants showed in the context of regulatory T cells stimulated with particulate material (PM), that there was a decrease in the proliferation of regulatory T cells in the presence of PM and Rev in relation to PM in the control fluid (without Rev, without Solas), which indicates that the electrically generated inventive Rev fluid improved the function of Regulatory T cells, for example, as evidenced by relatively low proliferation in the assay. Moreover, exposure to the fluids of the invention resulted in a sustained or slightly decreased production of IL-10 relative to the saline and media controls (without PM). Similarly, in the context of allergic asthma (AA) profiles of peripheral blood mononuclear cells (PBMC) stimulated with particulate matter (PM), the data showed that exposure to fluids of the present disclosure ("PM + Rev ") resulted in significantly lower levels of tryptase, similar to those of the saline and media controls. Additionally, the effects of diphtheria toxin (DT390) indicate that beta blockade, GPCR blockade and blockade of Ca channels affect the activity of electrolytically generated fluids on the function of Treg and PBMC. In addition, the applicants demonstrated, in accordance with additional aspects, after exposure to the fluids of the invention, that the proteins related to the tight junctions were positively regulated in the lung tissue. The applicants showed positive regulation of the adhesion molecules of the JAM 2 and 3, GJA1, 3.4 and 5 (binding adhesins), OCLN (occludin), claudin (for example, CLDN 3, 5, 7, 8, 9) , 10), TJP1 (narrow binding protein 1), respectively. The membrane-binding studies were performed to further investigate and confirm these utilities.

Materials and methods: The bronchial epithelial line Calu-3 was used in membrane fixation studies. Calu-3 bronchial epithelial cells (ATCC # HTB-55) were cultured in a 1: 1 mixture of Ham F12 and DMEM medium that was supplemented with 10% FBS on glass coverslips until the time of the experiments. In summary, a voltage-clamping device in whole cells is used to measure the effects on Calu-3 cells exposed to the electrokinetically generated fluids of the invention (eg, RNS-60, normal saline solution electrokinetically treated with 60 ppm of dissolved oxygen, sometimes referred to as "drug").

The membrane fixation techniques were used to evaluate the effects of the test material (RNS-60) on the polarity of the membrane of the epithelial cells and the ion channel activity. Specifically, voltage fixation in whole cells was performed in the bronchial epithelial line Calu-3 in a bath solution consisting of: 135 mM NaCl, 5 mM KC1, 1.2 mM CaC12, 0.8 mM MgC12, and 10 mM HEPES (pH adjusted to 7.4 with N-methyl-D-glucamine). The basal currents were measured, after which the RNS-60 was perfused in the cells.

More specifically, the membrane pipettes were extracted from the borosilicate glass' (Garner Glass Co, Claremont, California) with a two-stage Narishige PB-7 vertical extractor and then a resistance between 6-12 Mohms was polished to the fire. a microform Narishige MF-9 (Narishige International USA, East Meadow, NY). The pipettes were filled with an intracellular solution containing (in mM): 135 KC1, 10 NaCl, 5 EGTA, 10 Hepes, the pH was adjusted to 7.4 with NMDG (N-methyl-D-glucamine).

Cultured Calu-3 cells were placed in a chamber containing the following extracellular solution (in mM): 135 NaCl, 5 KC1, 1.2 CaC12, 0.5 MgC12 and 10 HEPES (free acid), the pH was adjusted to 7.4 with NMDG.

The cells were observed using the 40X CID objective of an Olympus 1X71 microscope (Olympus Inc., Tokyo, Japan). After a high-resistance (gigaseal) seal attached to the cell was established, a gentle suction was applied to burst, and to achieve complete cell configuration. Immediately after the irruption, the cell was subjected to voltage fixation at -120, -60, -40 and 0 mV, and was stimulated with voltage steps of ± 100 mV (500 ms / step). After collecting the currents of the whole cells in the control condition, the same cell was perfused through a bath with the test fluid comprising the same extracellular solutes and the pH as for the fluid of the previous control, and the currents of whole cells at different exploitation potentials were recorded with the same protocols.

Electrophysiological data were acquired with an Axon Patch 200B amplifier, low pass filtered at 10 kHz and digitized with 1400A Digidata (Axon Instruments, Union City, CA). The software pCLAMP 10.0 (Axon Instruments) was used to acquire and analyze the data. The relationships of current (I) -a -voltage (V) (conductance of whole cells) was obtained by graphically plotting the value of the actual current at approximately 400 msec in the step, compared to the potential of. exploitation (V). The slope of the I / V ratio is the conductance of the whole cells.

Drugs and chemical products. Whenever indicated, cells were stimulated with a cA P stimulator cocktail with 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 stock solution of 25 mM in H20. Forskolin (Sigma) and IBMX (Sigma) were used from a DMSO solution containing both 10 mM forskolin and 200 mM of the IBMX stock solution.

Results of membrane fixation: The applicants determined the currents of the whole cells under basal conditions (without cAMP), with a stepped protocol from zero mV with an exploitation potential at +/- 100 mV. The representative traces (control, followed by traces of whole cells, while perfused with the test solution) were performed on an average of n = 12 cells. The "delta" plots of the compound were obtained by subtracting the average values of the test from those under the control conditions. The conductance of the whole cells, obtained from the current-to-voltage ratios, was highly linear under both conditions, and reflected a modest though significant change in conductance, due to the test conditions. The contribution to the conductance of the whole cells, that is, the component inhibited by the drug (electrokinetically generated inventive fluid) was also linear, and the regression potential was close to zero mV. There was a decrease in the conductance of whole cells under hyperpolarization conditions.

In addition, the applicant determined the currents of the whole cells under basal conditions, with a stepped protocol from -40 mV of exploitation potential to ± 100 mV. The representative traces (control, followed by traces of whole cells, while perfused with the test solution) were performed on an average of n = 12 cells. The "delta" plots of the compounds were obtained by subtracting the average values of the test, from those under control conditions. The conductance of the whole cells, obtained from the current-to-voltage ratios, was highly linear under both conditions, and reflected a modest though significant change in conductance, due to the test conditions. The contribution to the conductance of the whole cells, that is, the component inhibited by the drug (electrokinetically generated inventive fluid) was also linear, and the regression potential was close to zero mV. The values were comparatively similar to those obtained with the mV zero protocol.

The applicants determined the currents of the whole cells under basal conditions, with a stepped protocol from -60 mV with a potential of ± 100 mV. The representative traces (control, followed by traces of whole cells, while perfused with the test solution) were performed on an average of n = 12 cells. The "delta" plots of the compound were obtained by subtracting the average values of the test, from those under control conditions. Conductance of whole cells, obtained from current-to-voltage ratios, was highly linear under both conditions, and reflected a minor but significant change in conductance, due to the test conditions. The contribution to the conductance of the whole cells, that is, the component inhibited by the drug (electrokinetically generated inventive fluid) is also linear, and the regression potential was close to zero mV. The values were comparatively similar to those obtained with the mV zero protocol.

The applicants also determined the currents of the whole cells under basal conditions, with a stepped protocol from -120 mV with a potential of ± 100 mV. The representative traces (control, followed by traces of whole cells, while perfused with the test solution) were performed on an average of n = 12 cells. The "delta" plots of the compound were obtained by subtracting the average values of the test, from those under control conditions. Conductance of whole cells, obtained from current-to-voltage ratios, was highly linear under both conditions, and reflected a minor but significant change in conductance, due to the test conditions. The contribution to the conductance of whole cells, ie, the component inhibited by the drug (electrokinetically generated inventive fluid) is also linear, and the regression potential 'was close to zero mV. The values were comparatively similar to those obtained with the mV zero protocol.

In addition, the applicants determined the currents of the whole cells under conditions of stimulation with cA P, obtained with staggered protocols from several exploitation potentials at ± 100 mV. The representative traces are the average of n = 5 cells. Representative traces (control, followed by traces of whole cells after stimulation with cAMP, followed by perfusion with the solution containing the drug) were made at an average of n = 12 cells. The delta traces of the compound (corresponding to the voltage protocols at zero mV, and at -40 mV) were obtained by subtracting the average values of the test on the drug + cAMP from those under the control conditions (cAMP alone) . The conductance of whole cells obtained from the current-to-voltage relationships was highly linear under all conditions, and reflected a change in conductance due to the test conditions.

The applicants demonstrated the currents of the whole cells under conditions of stimulation with cAMP, obtained with staggered protocols from several potential exploitation at ± 100 mV. Representative traces (control, followed by traces of whole cells after stimulation with cAMP, followed by perfusion with the solution containing the drug) were made at an average of n = 5 cells. The delta traces of the compound (voltage protocols at -60 mV and -120 mV) were obtained by subtracting the average values of the test on the drug + cAMP from those under control conditions (cAMP alone). The conductance of whole cells, obtained from the current-to-voltage relationships, was highly linear under all conditions, and reflected a change in conductance due to the test conditions.

The applicants also demonstrated the effect of the exploitation potential on currents activated with cAMP. The effect of the drug (the electrokinetically generated fluids of the invention, RNS-60, electrokinetically treated normal saline solution comprising 60 ppm of dissolved oxygen) on the conductance of whole cells was observed under protocols of different voltages (0, -40, -60, -120 mV of exploitation potentials). Under the basal conditions, the current of the drug sensitive cells was identical to all the exploitation potentials (contribution of the insensitive voltage). Under the conditions of activation by cAMP, however, the drug-sensitive currents were much higher, and sensitive to the applied voltage protocol. Current-to-voltage relationships are highly nonlinear. This was observed in the subtracted currents, where the contribution of the conductance of whole cells to zero mV was subtracted additionally for each protocol (n = 5).

Summary of the example. According to particular aspects, therefore, the data indicate that there is a moderate but constant effect of the drug (the electrokinetically generated fluids of the invention, RNS-60, normal saline solution electrokinetically treated with 60 ppm of dissolved oxygen) under the conditions basal To increase the effect of the drug on the conductance of the whole cells, experiments were also carried out by perfusing the drug after stimulation with a "cocktail" of stimulating cAMP, which dramatically increased the conductance of the whole cells. Interestingly, this protocol also increased the drug-sensitive portion of whole-cell conductance, which was ten times greater than that observed under baseline conditions. Furthermore, in the presence of cAMP stimulation, the drug showed different effects with respect to the various voltage protocols, which indicates that the electrokinetically generated fluids affect a voltage-dependent contribution of the conductance of whole cells. There was also a decrease in a linear component of the conductance, which further suggests at least one contribution of the drug to the inhibition of another pathway (e.g., ion channel, voltage-dependent cation channels, etc.) In particular aspects, and without adhering to the mechanism, the data of the applicants are consistent with the electrokinetically generated fluids of the invention (for example, RNS-60, normal saline solution electrokinetically treated with 60 ppm of dissolved oxygen), producing a change already either on a channel (s), that is blocked or that is recovered from the plasma membrane.

In conjunction with other data from the applicants, particular aspects of the present invention provide compositions and methods for modulating intracellular signal transduction, including modulation of at least one of the membrane structure, membrane potential or conductivity. of the membrane, membrane proteins or receptors, ion channels, and calcium-dependent cellular signaling systems, which comprise the use of the electrokinetically generated solutions of the invention to impart conformational and / or electrochemical changes in the membrane structures ( for example, membrane, and / or membrane proteins, receptors and other components), which include but are not limited to GPCR and / or g proteins, and TSLP. According to additional aspects, these effects modulate the gene expression, and may persist, depending, for example, on the half-life of the components of the individual messaging, etc.

EXAMPLE 7 (The effects of the electrokinetically altered fluids of the invention on the conductance of whole cells were determined) General information. The membrane binding assay carried out on Calu-3 cells perfused with the electrokinetically generated fluids of the invention (RNS-60 and Solas) revealed that (i) exposure to RNS-60 and Solo reed in an increase in conductance of the whole cells, (ii) that the exposure of the cells to RNS-60 produced an increase in the non-linear conductance, evident at 15 minutes of the incubation times, and (iii) that the cells were exposed to RNS -60 produces a saline RNS-60 effect on the calcium permeable channels. Applicants conducted membrane-binding studies to further confirm the utilities, as described herein, of the electrokinetically generated saline fluids of the invention (RNS-60 and Solas), including utility for modulating whole cell currents. Two series of experiments were carried out.

The summary of the data from the first series of experiments indicates that the conductance of the whole cells (current-to-voltage ratio) obtained with the Solas saline solution is highly linear, both for incubation times (15 min, 2 hours) , and for all voltage protocols. However, it is evident that the greater incubation (2 hours) with Solas increased the conductance of the whole cells. The exposure of the cells to RNS-60 produced an increase in non-linear conductance, as shown in the delta currents (subtraction Rev-Sol), which is only evident at 15 minutes of incubation time. The effect of RNS-60 in this non-linear current disappears, and is instead highly linear in the two-hour incubation time. The contribution of the non-linear complete cell conductance, as previously observed, was voltage sensitive, although it was present in all voltage protocols.

The summary of the data from the second series of experiments indicates that there is an effect of saline RNS-60 on a non-linear current, which became evident at the high level of calcium in the external solution. The contribution of the conductance of the non-linear complete cells, although sensitive to voltage, was present in both voltage protocols, and indicates a saline RNS-60 effect on the calcium permeable channels.

First series of experiments (increase in conductance and activation of a conductance regulated by non-linear voltage) Methods for the first series of experiments. See above for general methods of membrane fixation. In the next first series of experiments, membrane binding studies were carried out to further confirm the utility of the electrokinetically generated saline fluids of the invention (RNS-60 and Solas) to modulate whole cell currents, using cells Calu-3 under basal conditions, with staggered protocols, either with a zero potential mV, -120 mV, or -60mV.

The conductance of the whole cells in each case was obtained from the current-to-voltage ratios obtained from the incubated cells either for 15 minutes or for two hours. In this study, groups were obtained at any given time, either for Solas or RNS-60 saline solutions. The obtained data are expressed as the mean + SEM of the whole cell current for 5-9 cells.

Res Figures 3 AC show the res of a series of membrane-binding experiments that evaluated the effects of the electrokinetically generated fluid (e.g., RNS-60 and Solas) on the membrane polarity of epithelial cells and the activity of the channels of ions in two time points (15 min (panels on the left) and 2 hours (panels on the right)) and in the protocols of different voltages (A, intensifying from zero mV; B, intensifying from -60 mV, and C, intensifying from -120 mV). The res indicate that RNS-60 (filled circles) has a greater effect on the conductance of whole cells than Solas (open circles). In the experiment similar res were observed in the three voltage protocols and both at the incubation time points of 15 minutes and two hours.

The graphs of Figures 4 AC show as a result of the subtraction of the Solas current data from the RNS-60 current data in the three voltage protocols ("Delta currents") (A, intensifying from zero mV B, intensifying from -60 mV, and C, stepped from -120 mV) and from the two time points (15 minutes (open circles) and 2 hours (filled circles)). These data indicated that at the 15-minute time point with RNS-60, there was a non-linear voltage-dependent component that is absent at the 2-hour time point.

As in previous experiments, the data with "normal" saline solution gave a very consistent and time-independent conductance used as a reference. The present results were obtained by comparing the groups, either with Solas or with RNS-60 saline, and indicate that the exposure of Calu-3 cells to RNS-60 saline under basal conditions (without cAMP, or any other stimulus), produces time-dependent effects, consistent with the activation of a regulated conductance with the voltage at shorter incubation times (15 min). This phenomenon was not so evident at the two-hour incubation point. As described elsewhere herein, the linear component is more evident when the conductance is increased by stimulation with the "cocktail" of cAMP. However, the incubation time of two hours showed a greater linear conductance, both for RNS-60 and for the Solas saline solution, and in this case, the RNS-60 saline doubled the conductance of the whole cells, in comparison with Solas alone. This evidence indicates that at least two contributions to the conductance of whole cells are affected by saline RNS-60, namely, the activation of a conductance regulated by non-linear voltage, and a linear conductance, which is more evident to longer incubation times.

Second series of experiments (effect on the calcium permeable channels) Methods for the second series of experiments. See above for general methods of membrane fixation. In the next second series of experiments, membrane fixation studies were carried out to further confirm the utility of the electrokinetically generated saline fluids of the invention (RNS-60 and Solas) to modulate whole cell currents, using cells Calu-3 under basal conditions, with staggered protocols, either from zero mV exploitation potentials or from -120 mV.

The conductance of the whole cells in each case was obtained from the current-to-voltage ratios obtained from the cells incubated for 15 min with saline. To determine if there is a contribution of the calcium-permeable channels to the conductance of the whole cells, and if this part of the conductance of the whole cell is affected by the incubation with RNS-60 saline, the cells were patched in saline normal after the incubation period (carries a high external solution of NaCl, while the internal solution contains high KC1). The external saline solution was then replaced with a solution where the NaCl is replaced by CsCl to determine if there is a change in the conductance by substituting the main external cation. Under these conditions, the same cell was then exposed to increasing concentrations of calcium, so that? An entry step of calcium becomes more evident.

Results: Figures 5 AD show the results of a series of membrane fixation experiments that evaluated the effects of the electrokinetically generated fluid (for example, Solas (panels A and B) and RNS-60 (panels C and D)) on the polarity of the epithelial cell membrane and the activity of the ion channels using different solutions of external salts and in the different voltage protocols (panels A and C show staggered from zero mV, while panels B and D show staggered from -120 mV). In these experiments a time point of 15 minutes was used. For Solas (panels A and B) the results indicate that: 1) the use of CsCl (square symbols) instead of NaCl in the external solution, increased the conductance of the whole cells with a linear behavior in comparison with the control (symbols diamonds), and 2) CaCl2 both at 20 mM CaCl2 (circular symbols) and at 40 mM · CaCl2 (triangular symbols) increased the conductance of whole cells in a non-linear manner. For RNS-60 (panels C and D), the results indicate that: 1) the use of CsCl (square symbols) instead of NaCl in the external solution had little effect on the conductance of whole cells, compared to the control (diamonds symbols), and 2) 40 mM CaCl2 (triangular symbols) increased the conductance of whole cells in a non-linear manner.

Figures 6 AD show the graphs resulting from the subtraction of the current data in CsCl (shown in Figure 5) from the current data in 20 mM CaCl 2 (diamond symbols) and 40 mM CaCl 2 (symbols square) of two voltage protocols (panels A and C, stepped from zero mV, and B and D, stepped from -120 mV) of Solas (panels A and B) and RNS-60 (panels C and D). The results indicate that both Solas and RNS-60 solutions activated the conductance of whole cells induced by calcium in a non-linear manner. The effect was greater with RNS-60 (indicating a response capacity to the dosage), and with RNS-60 it was only increased to the highest concentrations of calcium. Furthermore, the non-linear calcium-dependent conductance at the highest calcium concentration was also increased by the voltage protocol.

The data from this second series of experiments also indicate an effect of Saline RNS-60 and Saline Solas for conductance data of whole cells obtained in Calu-3 cells. The data indicate that incubation for 15 minutes with any saline solution produces a different effect on the conductance of the whole cells, which is more evident with RNS-60 and when the external calcium is increased, and also indicates that RNS-60 Saline increases a non-linear calcium-dependent component of the conductance of whole cells.

The accumulated evidence suggests the activation by Revalesio salina of the ion channels, which contributes differently to the basal cellular conductance.

In conjunction with other data from the applicants (e.g., data from applicants other than the practical examples), particular aspects of the present invention provide compositions and methods for modulating intracellular signal transduction, including modulation of at least one of membrane structures, membrane potential or membrane conductivity, membrane proteins or receptors, ion channels, lipid components, or intracellular components that are interchangeable by the cell (eg. signaling pathways, such as calcium-dependent cellular signaling systems), which comprise the use of the electrokinetically generated solutions of the invention to impart electrochemical and / or conformational changes in the membranous structures (e.g., the membrane, and / or membrane proteins, receptors or other components of the membrane), q which includes but is not limited to GPCR and / or proteins g. According to additional aspects, these effects modulate the gene expression, and may persist, depending, for example, on the average life of the components of the individual messaging, etc.

EXAMPLE 8 (The effects of the electrokinetically altered fluids of the invention on the conductance of the whole cells were investigated, and a dose-response curve was generated) General information: In this experiment, the applicants evaluated the effect of dilutions of the electrokinetically altered fluid ( for example, RNS-60) on the polarity of the membrane of the epithelial cells and the activity of the ion channels.

Methods See above for general methods of membrane fixation. In the following experiment, membrane binding studies were carried out to further confirm the utility of the electrokinetically generated saline fluids of the invention (RNS-60) to modulate whole cell currents. In particular, the experiment evaluated the effect of dilutions of the electrokinetically generated saline fluid. The solutions were made by diluting the electrokinetically generated saline fluid in normal saline at concentrations of: 100% (Rev), 75% (3: 4), 50% (1: 1), 25% (4: 3) , and 0% (Salt).

Results Figures 7? and B show the results of a series of membrane fixation experiments that evaluated the effects of the electrokinetically diluted fluid generated (e.g., RNS-60) on the polarity of the membrane of epithelial cells and the activity of ion channels. Panel A shows the volts with respect to the conductance current of the whole cells for each diluted sample as indicated in the graph (Rev, 3: 4, 1: 1, 4: 3, and Sal). Panel B shows the amount of dilution with respect to the change in current by comparing the dilution with normal saline. The results indicate that the mechanism of action of the RNS-60 solution occurs in a linear dose-response form.

EXAMPLE 9 . { The treatment of the primary bronchial epithelial cells (BEC) with the electrokinetically generated fluids of the invention, as well as with the non-electrokinetic control autoclave fluid, resulted in the reduction of the expression and / or activity of two key proteins in the reactions Inflammatory pathways respiratory, MMP9 and TSLP) General information. Applicants have now demonstrated (using a Bio-Layer Interferometry biosensor, Octet Rapid Extended Detection (RED) (forteBio ™)), that in the presence of electrokinetically generated fluids (eg, Rev, electrochenetically enriched gas-enriched fluid) of the present disclosure compared to normal saline, binding of bradykinin to B2 receptor was concentration dependent, and binding affinity was increased. In addition, in the context of regulatory T cells stimulated with particulate matter from combustion gases (P, standard commercial source), the applicants' data showed a decrease in the proliferation of regulatory T cells in the presence of PM and Rev relative to PM in the control fluid (without Rev, without Solas), indicating that the electrochenetically generated Rev fluid improved the function of regulatory T cells, for example, as demonstrated by the relatively small proliferation in the assay. Moreover, exposure to the fluids of the invention resulted in a sustained or slightly decreased production of IL-10 relative to the saline and media controls (without PM). Similarly, in the context of allergic asthma (AA) profiles of peripheral blood mononuclear cells (PBMC) stimulated with particulate matter (PM), the data showed that exposure to fluids of the present disclosure ("PM + Rev ") resulted in significantly lower levels of tryptase, similar to those of the saline and media controls. Additionally, diphtheria toxin (DT390, a molecule of truncated diphtheria toxin, dilution 1:50 of the standard commercial concentration) resulted in beta blockade, GPCR blockade and blockade of Ca channels from the effects of activity of the fluids generated in electrokinetically on the function of Treg and PBMC. In addition, applicants have shown that after exposure to the fluids of the invention, the proteins related to the tight junctions (eg, JAM, 2 and 3, GJA1, 3, 4 and 5 (adhesion binding), OCLN ( occludin), claudins (eg, CLDN 3, 5, 7, 8, 9, 10), TJP1 (tight binding protein 1)) were positively regulated in the lung tissue. In addition, as shown in the membrane binding studies, the electrokinetically generated fluids of the invention (e.g., RNS-60) affect the conductance modulation of whole cells (e.g., under hyperpolarizing conditions) in Epithelial Cells Bronchial (BEC, for example, Calu-3), and according to additional aspects, the modulation of the conductance of the whole cells reflects the modulation of the ion channels.

In this example, the applicants have extended these discoveries by conducting additional experiments to measure the effects of the production of two key proteins of the inflammatory response of the respiratory tract. Specifically, MMP9 and TSLP were tested on primary bronchial epithelial cells (BEC).

Materials and methods: Commercially available primary bronchial epithelial cells (BEC) (HBEpC-c from Promocell, Germany) were used in these studies. Approximately 50,000 cells were seeded in each well of a 12-well plate until they reached -80% confluence. The cells were treated for 6 hours with normal saline, Solas control fluid, non-electrokinetic control autoclave fluid, or Revera 60 test fluid at a 1:10 dilution (100 ul in 1 ml of epithelial growth medium of airways), together with particulate matter from combustion gases (DEP or PM), before lifting them for FACS analysis. Both antibodies for the MMP9 and TSLP receptors were obtained from BD Biosciences and used according to the manufacturer's specifications.

Results: In Figures 1 and 2, DEP represents cells exposed to particulate matter of combustion gases (PM, of standard commercial origin) alone, "NS" represents cells exposed to a normal saline solution alone, "DEP + NS" represent the cells treated with the particulate material in the presence of normal saline, "Revera 60" refers to cells exposed only to the test material, "DEP + Revera 60" refers to cells treated with particulate material in the presence of the test material Revere 60. In addition, "Solas" and "DEP + Solas" represent cells exposed to the control fluid alone, alone or in combination with the particulate material, respectively. "PP60" represents the cells exposed to the non-electrokinetic control autoclave fluid, and "DEP + PP60" refers to the cells treated with particulate material in the presence of the non-electrokinetic control autoclave fluid (i.e. having 60 ppm of dissolved oxygen) .

Figure 1 shows that the Revera 60 test material reduces the expression of the TSPP receptor induced by DEP in the bronchial epithelial cells (BEC) by approximately 90%. It alone resulted in a 55% reduction in the expression of the receptor of TSLP induced by DEP, while normal saline did not produce the same level of reduction of TSLP receptor expression induced by DEP (approximately 20% reduction). Additionally, the non-electrokinetic PP60 control autoclave fluid resulted in approximately 50% reduction in the expression of the TSPP receptor induced by DEP.

The effect of the solutions of the invention Revera 60, Solas, and also of PP60, in the reduction of the expression of the TSLP receptor is a significant discovery in view of the recent findings that show that TSLP plays a fundamental role in the pathobiology of the Allergic asthma and blockade mediated by local antibodies to the function of the TSLP receptor of relieved allergic disease (Liu, YJ, Thymic stromal lymphopoietin: Master switch for allergic inflammation, J Exp Med 203: 269-273, 2006, Al-Shami and others, A role for TSLP in the development of inflammation in an asthma model, J Exp Med 202: 829-839, 2005; and Shi et al, Local blockade of TSLP receptor alleviated allergic disease by regulating airway dendritic cells, Clin Immunol 29 August, 2008 (electronic publication before printing)).

Similarly, Figure 2 shows the effect of Revera 60, Solas, the non-electrokinetic control autoclave fluid (PP60) and normal saline, on the increase of the MP-9 mediated by DEP. Specifically, Revera 60 inhibited cell surface bound MMP9 levels induced by DEP in bronchial epithelial cells by approximately 80%, and Solas had an inhibitory effect of approximately 70%, whereas normal saline (NS) had a marginal effect of approximately 20% reduction. Additionally, the non-electrokinetic PP60 control autoclave fluid resulted in the reduction of approximately 30% of the cell surface bound M P9 levels induced by DEP. MMP-9 is one of the major proteases involved in airway inflammation and bronchial remodeling in asthma. Recently, it has been shown that MMP-9 levels increased significantly in patients with stable asthma, and even more in patients with acute asthma, compared to healthy control subjects. MMP-9 plays a crucial role in the infiltration of inflammatory pathway cells and in the induction of airway hyperreactivity, which indicates that MMP-9 may have an important role in the induction and maintenance of the respiratory tract. asthma (Vignola et al., Sputum metalloproteinase-9 / tissue inhibitor of the metalloproteinase-1 ratio corset refers to airflow obstruction in asthma and chronic bronchitis, Am J Respir Crit Care Med 158: 1945-1950, 1998; Hoshino et al., Inhaled corticosteroids decrease subepithelial collagen 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 model 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 diisocyanate-induced asthma, J Allergy Clin Immunol 2003; 111: 1278-1284).

According to further aspects, therefore, the electrokinetically generated fluids of the invention have substantial therapeutic utility to modulate (e.g., reduce) the expression of the TSLP receptor and / or to inhibit the expression and / or activity of the MMP. -9, including, for example, for the treatment of inflammation and asthma.

In accordance with yet further aspects, the non-electrokinetic control autoclave fluid (i.e., having 60 ppm dissolved oxygen) has therapeutic utility for the modulation (eg, reduction) of TSLP receptor expression and / or for the inhibition of MMP-9 expression and / or activity, including, for example, for the treatment of inflammation and asthma. Without adhering to the mechanism, the collective data of the applicants indicate that the action of the non-electrokinetic control autoclave fluid in this system is mediated by a mechanism that is different from that of the electrokinetically generated fluids of the applicants. This is not only because the effects are relatively smaller, but also because the non-electrokinetic control autoclave fluid has not shown activity in other assays where activity has been shown with the electrokinetically generated fluids of the applicants. However, the discy of applicants for the activity disclosed herein in the non-electrokinetic control autoclave fluid in this system represents a new use for autoclaving fluid in the context of asthma and related conditions as described in I presented.

According to particular aspects, therefore, the methods of the invention comprising the administration of the electrokinetically generated fluids of the applicants provide the modulation (negative regulation of TSLP expression and / or activity) are applicable to the treatment of at least a disease or condition selected from those in the group that are mediated by TSLP which consist of immune system disorders, allergic inflammation, allergic inflammation of the airways, inflammatory Th2 responses mediated by DC, atopic dermatitis, eczema atopic, asthma, obstructive airways disease, chronic obstructive pulmonary disease and food allergies, inflammatory arthritis, rheumatoid arthritis and psoriasis.

The results disclosed herein are entirely consistent with the recognized role in the prior art of TSLP as a major switch of allergic inflammation at the epithelial DC cell interface (Yong-Jun et al., J. Exp. Med., 203: 269-273, 2006), and are further consistent with the phenotypes of mice lacking TSLPR (for example, they fail to develop asthma in response to inhaled antigens; Zhou et al., Supra and Al-Shami et al. J. Exp. Med., 202: 829-839, 2005), and with the results obtained from the pretreatment with OVA-DC with anti-TSLPR (for example, resulting in a significant reduction of eosinophils and lymphocyte infiltration). , as well as in the levels of IL-4 and IL-5).

The presently disclosed material further illuminates the role of TSLPR in allergic disease predisposed by DC, and provides n compositions and methods comprising the administration of the electrokinetically generated fluids of the applicants.

Claims

NOVELTY OF THE INVENTION Having described the present invention, it is considered as a novelty and, therefore, the content of the following is claimed as a priority: CLAIMS

1. A method for the treatment of a condition or disease mediated by TSLP or TSLPR, comprising administering to a mammal in need thereof a therapeutically effective amount of an electrokinetically altered aqueous fluid comprising an ionic aqueous solution of stabilized oxygen containing nanostructures per charge, which have substantially an average diameter of less than about 100 nanometers and which are stably configured in the aqueous ionic fluid in an amount sufficient to treat a condition or disease mediated by the TSLP or TSLPR.

2. The method of claim 1, wherein the charge-stabilized oxygen-containing nanostructures are stably configured in the aqueous ionic fluid in an amount sufficient to provide, upon contact of a living cell with the fluid, the modulation of at least one of the potential of the cell membrane and the conductivity of the cell membrane.

3. The method of claim 1, wherein charge-stabilized oxygen-containing nanostructures are the main species of nanostructures containing gas stabilized by charge in the fluid.

4. The method of claim 1, wherein the percentage of dissolved oxygen molecules present in the fluid as oxygen-containing nanostructures stabilized by charge is a percentage selected from the group consisting of greater than: 0.01%, 0.1%, 1%, 5%; 10%; fifteen%; twenty%; 25%; 30%; 35%; 40%; Four. Five%; fifty%; 55%; 60%; 65%; 70%; 75%; 80%; 85%; 90%; and 95%.

5. The method of claim 1, wherein the total dissolved oxygen is substantially present in charge-stabilized oxygen-containing nanostructures.

6. The method of claim 1, wherein the charge-stabilized oxygen-containing nanostructures substantially have an average diameter smaller than a size selected from the group consisting of: 90 nra; 80 nm; 70 nm; 60 nm; 50 nm; 40 nm; 30 nm; 20 nm; 10 nm; and less than 5 nm.

7. The method of claim 1, wherein the aqueous ionic solution comprises a saline solution.

8. The method of claim 1, wherein the fluid is superoxygenated.

9. The method of claim 1, wherein the fluid comprises a solvated electron form.

10. The method of claim 1, wherein the alteration of the electrokinetically altered aqueous fluid comprises the exposure of the fluid to localized, hydrodynamically induced electrokinetic effects.

11. The method of claim 10, wherein, exposure to localized electrokinetic effects comprises exposure to at least one of voltage pulses and current pulses.

12. The method of claim 10, wherein the exposure of the fluid to electrokinetic effects localized, induced hydrodynamically, comprises the exposure of the fluid to the structural characteristics that induce the electrokinetic effect of a device that is used to generate the fluid.

13. The method of any of claims 1-12, wherein the condition or disease mediated by the TSLP or TSLPR comprises a disease or disorder of the immune system.

14. The method of claim 13, wherein the disease or disorder of the immune system comprises allergic inflammation.

15. The method of claim 14, wherein the allergic inflammation comprises at least one of allergic inflammation of the respiratory tract, inflammatory Th2 responses mediated by DC, atopic dermatitis, atopic eczema, asthma, obstructive airway disease, lung disease. chronic obstructive, IgE-mediated disorders, rhino-conjunctivitis and food allergies.

16. The method of any of claims 1-12, wherein the condition or disease mediated by the TSLP or TSLPR comprises inflammatory arthritis.

17. The method of claim 16, wherein the inflammatory arthritis comprises at least one of rheumatoid arthritis and psoriasis.

18. The method of any of claims 1-12, further comprising combination therapy, wherein at least one additional therapeutic agent is administered to the patient.

19. The method of claim 18, wherein the at least one additional therapeutic agent is selected from the group consisting of the short acting ^ 2 agonists, long-acting ^ 2 agonists, anticholinergics, corticosteroids, systemic corticosteroids, mast cell stabilizers, modifiers of the leukotriene, methylxanthines, and combinations thereof.

20. The method of claim 18, wherein the at least one additional therapeutic agent is selected from the group consisting of: bronchodilators consisting of agonists - which include albuterol, levalbuterol, pirbuterol, artformoterol, formoterol, salmeterol, and anticholinergics such as ipratropium and tiotropium; corticosteroids including beclomethasone, budesonide, flunisolide, fluticasone, mometasone, triamcinolone, methylprednisolone, prednisolone, prednisone; leukotriene modifiers including montelukast, zafirlukast, and zileuton; mast cell stabilizers including cromolin and nedocromil; methylxanthines including theophylline, combination drugs including ipratropium and albuterol, fluticasone and salmeterol, budesonide and formoterol; antihistamines including hydroxzine, diphenhydramine, loratadine, cetirizine, and hydrocortisone; drugs that modulate the immune system that include tacrolimus and pimecrolimus; cyclosporin; azathioprine; mycophenolatemofetil; and combinations thereof.

21. The method of claim 18, wherein the at least one additional therapeutic agent is a TSLP and / or TSLPR antagonist.

22. The method of claim 21, wherein the TSLP and / or TSLPR antagonist is selected from the group consisting of neutralizing antibodies specific for TSLP and the TSLP receptor, soluble TSLP receptor molecules, and TSLP receptor fusion proteins, which include TSLPR-immunoglobulin Fe molecules or polypeptides that code for the components of more than one receptor chain.

23. The method of claim 2, wherein the modulation of at least one of the cell membrane potential and the cell membrane conductivity comprises altering at least one of the structures or functions of the cell membrane, comprising altering at least one of a conformation, the ligand-binding activity, and a catalytic activity of a protein or component associated with the membrane.

24. The method of claim 23, wherein the membrane-associated protein comprises at least one selected from the group consisting of receptors, transmembrane receptors, ion channel proteins, intracellular binding proteins, cell adhesion proteins, integrins, etc.

25. The method of claim 24, wherein the transmembrane receptor comprises a Receptor Coupled to Protein-G (GPCR).

26. The method of claim 25, wherein the G-Protein Coupled Receptor (GPCR) interacts with a subunit of the G protein.

27. The method of claim 26, wherein the a subunit of the G protein comprises at least one selected from the group consisting of Gas, GOÍÍ, Gaq, and Gai2.

28. The method of claim 27, wherein the at least one subunit oi of the G protein is Gaq.

29. The method of claim 2, wherein the modulation of at least one of the cell membrane potential and the conductivity of the cell membrane comprises the modulation of the conductance of the whole cells.

30. The method of claim 29 wherein the modulation of the conductance of the whole cells comprises modulating at least one of a linear voltage dependent and a non-linear contribution of the complete cell conductance.

31. The method of claim 2, wherein the modulation of at least one of the cell membrane potential and the cell membrane conductivity comprises the modulation of a calcium dependent cellular messaging system or pathway.

32. The method of claim 2, wherein the modulation of at least one of the cell membrane potential and the cell membrane conductivity comprises modulating the activity of phospholipase C.

33. The method of claim 2, wherein the modulation of at least one of the cell membrane potential and the cell membrane conductivity comprises the modulation of adenylate cyclase (AC) activity.

34. The method of claim 2, wherein the modulation of at least one of cell membrane potential and cell membrane conductivity comprises the modulation of intracellular signal transduction associated with at least one condition or symptom selected from the group consisting of diseases or disorders. of the immune system, allergic inflammation, allergic inflammation of the respiratory tract, inflammatory responses of Th2 mediated by DC, atopic dermatitis, atopic eczema, asthma, obstructive airways disease, chronic obstructive pulmonary disease, IgE-mediated disorders, rhino- conjunctivitis, food allergies, inflammatory arthritis, rheumatoid arthritis and psoriasis.

35. The method of any of claims 1-12, which comprises administering the electrokinetic fluid to a network or cell layer, and subsequently comprising modulating an intercellular junction therein.

36. The method of claim 35, wherein the intercellular junction comprises at least one selected from the group consisting of junctions of slits, zone adhesins and desmosomes.

37. The method of claim 35, wherein the cellular network or layer comprises at least one selected from the group consisting of pulmonary epithelium, bronchial epithelium, and intestinal epithelium.

38. The method of any of claims 1-12, wherein the electrokinetically altered aqueous fluid is oxygenated, and wherein the oxygen in the fluid is present in an amount of at least 8 ppm, at least 15 ppm, at least 25 ppm. , at least 30 ppm, at least 40 ppm, at least 12 ppm, or at least 60 ppm oxygen at atmospheric pressure.

39. The method of any of claims 1-12, wherein the electrokinetically altered aqueous fluid comprises at least one of solvated electrons, and oxygen species charged or electrokinetically modified.

40. The method of claim 39, wherein the form of solvated electrons or electrokinetically modified or charged oxygen species are present in an amount of 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.

41. The method of claim 40, wherein the electrokinetically altered aqueous fluid comprises a form of solvated electrons stabilized by molecular oxygen.

42. The method of claim 2, wherein the ability to modulate at least one of the cell membrane potential and the cell membrane conductivity persists for at least two, at least three, at least four, at least five, at least 6 , at least 12 months, or longer, in a closed container that is gas-proof.

43. The method of any of claims 1-12, wherein the amount of oxygen present in the stabilized oxygen-containing nanostructures by electrokinetically altered fluid charge 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 least 12 ppm, or at least 60 ppm oxygen at atmospheric pressure.

44. The method of any of claims 1-12, wherein the treatment comprises administration by at least one of the topical, inhalation, intranasal, and intravenous route. SUMMARY OF THE INVENTION Methods are provided for the treatment of a condition or disease mediated by TSLP or TSLPR, which comprises the administration of an electrokinetically altered aqueous fluid comprising an ionic aqueous solution of charge-stabilized oxygen-containing nanostructures, which have substantially a smaller average diameter that approximately 100 nanometers and that are stably configured in the aqueous ionic fluid in an amount sufficient to treat a condition or disease mediated by the TSLP or TSLPR. The nanostructures containing charge-stabilized oxygen are preferably stably configured in the fluid, in an amount sufficient to provide modulation of the potential and / or conductivity of the cell membrane. Some aspects comprising modulation or down regulation of TSLP expression and / or activity are useful for treating diseases or conditions mediated by TSLP or mediated by TSLPR as described herein (eg, immune system disorders, allergic inflammation , allergic inflammation of the respiratory tract, inflammatory responses of Th2 mediated by DC, atopic dermatitis, atopic eczema, asthma, obstructive respiratory disease, chronic obstructive pulmonary disease, and food allergies, inflammatory arthritis, rheumatoid arthritis, psoriasis , disorders mediated by IgE, and rino-conj untivitis.