BRPI0920201A2 - compositions and methods for treating lymphopoietin-mediated conditions of the thymic stroma (tslp) - Google Patents

Abstract

"Compositions and Methods for Treating Thymic Stromal Lympopoietin (tslp) Mediated Conditions" The present invention provides methods for treating a tslp or tslpr mediated disease or condition comprising administering an electrokinetically altered aqueous fluid comprising an aqueous solution ionization of charge-stabilized oxygen-containing nanostructures having substantially a mean diameter less than about 100 nanometers and stably configured in the aqueous ionic fluid in sufficient quantity to treat a tslp-mediated or tslpr-mediated disease or condition. charge-stabilized oxygen-containing nanostructures are preferably configured in the fluid in sufficient quantities to permit modulation of cell membrane potential and / or conductivity. Certain embodiments comprising modulation or down-regulation of tslp expression and / or activity are useful for treating tslp-mediated or tslpr-mediated diseases or conditions as described herein (eg, immune system disorders, allergic inflammation, allergic inflammation airway, th2-type inflammatory responses, dc-mediated, atopic dermatitis, atopic eczema, asthma, obstructive airway disease, chronic obstructive pulmonary disease and food allergies, inflammatory arthritis, rheumatoid arthritis, psoriasis, lge-mediated disorders and rhinoconjunctivitis ).

Description

“COMPOSITIONS AND METHODS TO TREAT CONDITIONS MEDIATED BY TYPICAL STROMA LYMPHOPOETIN (TSLP)”

FIELD OF THE INVENTION

The present invention relates, in general, to thymic stromal lymphopoietin (TSLP) and to Conditions mediated by TSLP and, more specifically, to conditions mediated by TSLP and the TSLP receptor (for example, disorders of the immune system, allergic inflammation, allergic airway inflammation, DC-mediated inflammatory Th2 responses, atopic dermatitis, atopic eczema, allergic asthma, asthma, airway obstructive disease, chronic obstructive pulmonary disease and food allergies, inflammatory arthritis, arthritis rheumatoid, psoriasis, IgE-mediated disorders and rhinoconjunctivitis). Especially preferred embodiments refer to the modulation (for example, treatment) of conditions mediated by TSLP and TSLP receptor, administering a therapeutic composition comprising at least one electrokinetically generated fluid (including electrokinetically generated fluids from enriched gas (for example, enriched oxygen)), as described in this patent application and, additionally, modalities for therapies combined with the administration of these electrokinetic fluids in combination with at least one additional therapeutic agent.

CROSS REFERENCE TO RELATED PATENT APPLICATIONS

This application claims priority to Application Provisional Serial U.S. Patent No. 61 / 107,480 and 61 / 107,453, both filed October 22 , 2008, and Application Serial U.S. Patent Utility No. 12 / 258,210 , filed on October 24, 2008, both of which are incorporated herein, in their entirety, by reference in this patent application.

BACKGROUND OF THE INVENTION

Thymic stromal lymphopoietin (TSLP). Thymic stromal lymphopoietin (TSLP) is an IL-7-type cytokine that triggers Th2-type inflammatory responses mediated by dendritic cells, being considered the master key ”for allergic inflammation. TSLP is an integral growth factor for the development and maturation of B and T cells. Specifically, murine TSLP supports B lymphopoiesis and is necessary for B cell proliferation. Murine TSLP participates fundamentally in the control of receptor locus rearrangement T cell range (TCRy) and exhibits substantial stimulatory effect on mature thymocytes and T cells. See, for example, Friend etal., Exp. Hematol., 22: 321-328, 1994; Ray et al., Eur. J. Immunol., 26: 10-16, 1996; Candeias etal., Immunology Letters, 57: 9-14, 1997.

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

Effects of TSLP on dendritic cells and TNF production. After human TSLP and the human TSLP receptor were cloned in 2001, human TSLP was found to strongly activate immature myeloid CD11c + dendritic cells (mDCs) (see, for example, Reche etal., J. Immunol., 167: 336-343 , 2001 and Soumelis et ak, Nat. Immunol., 3: 673-680, 2002). Th2 cells are generally defined in immunology textbooks and in the literature as IL-4, IL-5, IL-13 and IL-10 producing CD4 + T cells. And Th1 cells, such as CD4 + T cells, produce IFN-γ and, sometimes, TNF. When TSLPDCs are used to stimulate CD4 + virgin allogeneic T cells in vitro, a unique type of Th2 cell is induced, which produces the classic Th2 cytokines, IL-4, IL-5 and IL-13, and large amounts of TNF, but little or no IL-10 or interferon-γ is produced (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, the presence of TNF is prominent in the airways of asthmatics, 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 mDCs to express the OX40L protein of the TNF superfamily at both the mRNA and protein levels (Ito et al., J. Exp. Med., 202: 1213-1223). The expression of OX40L by TSLP-DCs is important for the development of inflammatory Th2 cells. Thus, TSLP-activated DCs create a favorable Th2 microenvironment by positively regulating OX40L without inducing the production of cytokines that polarize Th1. Id.

TSLP expression responses to specific allergens and asthma. Initial studies demonstrated the high expression of TSLP mRNA by primary human cutaneous keratinocytes, bronchial epithelial cells, smooth muscle cells and pulmonary fibroblasts (Soumelis ef. Al., Nat. Immunol., 3: 673-680, 2002). TSLP is expressed mainly in keratinocytes of the apical layers of the epidermis, thus suggesting that the production of TSLP is a characteristic of completely differentiated keratinocytes. TSLP expression in patients with atopic dermatitis was associated with migration and activation in situ of Langerhans cells, which suggests that TSLP may directly contribute to the activation of these cells, which would subsequently migrate and penetrate into the lymph nodes of the lymphatic system and initiate specific responses to allergens. Id. In a more recent study, it was demonstrated by in situ hybridization that the expression of TSLP was increased in the airways of asthmatics and that it correlated not only with the expression of attractive Th2 chemokines but also with the severity of the disease, the which reinforced the existence of 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 common γ chain. TSLPR is a new type of cytokine receptor 1, which, combined with IL-7Ra (CD127), forms a TSLP receptor complex as described, for example, by Pandey et a., Nat. Immunol., 1:59 -64, 2000. TSLPR contains a tyrosine residue near its terminal carboxyl, which can be associated with phosphorylated STAT5 and mediate multiple biological functions when occupied by TSLP (Isaksen et al., J. Immunol., 168: 3288-3294, 2002).

Human TSLPR is expressed by monocytes and CD11c + dendritic cells, and when bound to TSLP it induces the expression of the chemokines CCL17 and CCL22 that attract T _H 2 cells. In addition, as stated above, activation of TSLPR-induced dendritic cells indirectly results in secretion increased cytokines IL-4, 5 and 13 by T _H 2, which may be necessary for the regulation of homeostasis by CD4 + T cells. In mice, TSLPR deficiency has no effect on the number of lymphocytes. However, TSLPR and common γ chain deficiency results in fewer lymphocytes when compared to mice with only the common γ chain deficiency. See Reche et a!., J. Immunol., 167: 336-343, 2001 eSoumelis et a /., Nat. Immunol., 3: 673-680, 2002.

Studies have found that TSLP and TSLPR play a key role in the initiation of allergic diseases in mice. In one study, it was shown that mice built to overexpress TSLP in the skin developed atopic dermatitis, characterized by eczematous skin lesions containing inflammatory infiltrates, marked increase in circulating Th2 cells and elevated serum IgE (Yoo et al., J. Exp. Med. , 202: 541-549, 2005). The study suggested that TSLP can directly activate DCs in mice. In another study, conducted by Li et al., The group confirmed that transgenic mice overexpressing TSLP in the skin developed atopic dermatitis, as it solidified the link between TSLP and the development of atopic dermatitis.

Another set of studies has shown that TSLP is necessary for the initiation of allergic inflammation of the airways in mice in vivo. In one study, Zhou et al. demonstrated that the specific expression in the lung of a TSLP transgene induced allergic inflammation of the airways (asthma), characterized by massive leukocyte infiltration (including Th2 cells), goblet cell hyperplasia and subepithelial fibrosis, in addition to increased levels of serum IgE ( Zhou etal., Nat. Immunol., 6: 1047-1053, 2005). However, on the other hand, mice devoid of TSLPR did not 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 have jointly demonstrated that TSLP is necessary for the initiation of allergic inflammation of the airways in mice.

- In addition, in a study conducted by Yong-Jun et al., It was shown that TSLP derived from epithelial cells triggers, in humans, Th2 type inflammatory responses, mediated by DC, suggesting that TSLP represents a master key 5 for inflammation allergic at the epithelial cell-DC interface (Yong-Jun et al., J. Exp. Med., ... 203: 269-273,2006).

In a recent study, modulation of DC function by inhibiting TSLPR has been shown to decrease severity in mice (Liyun Shi et al., Clin. Immunol., 129: 202-210, 2008). In another set of studies, TSLPR was shown to be ex10pressed not only in DCs, but also in 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). In order to rule out the direct effects of TSLPR neutralization on CD4 + T cells or other effector cells in allergic inflammation, Liyun Shi et al. carried out experiments in which DCs loaded with OVA were treated in vitro with anti-TSLPR before the adoptive transfer to the airways of virgin mice. It was previously found that OVA-DCs triggered strong eosinophilic inflammation in the airways, accompanied by massive production of Th2 cytokines, such as IL-4 and IL-5 (Sung et al., J. Immunol., 166: 1261-1271 and Lambrecht et a., J. Clin. Invest., 106: 551-559, 2000). However, pretreatment of OVA-CDs with anti-TSLPR resulted in a significant reduction in infiltration 20 by eosinophils and lymphocytes, as well as levels of IL-4 and IL-5, further clarifying the role that TSLPR plays in allergic disease initiated This result also reinforces the suggestion that blocking TSLPR over DCs will help to control airway inflammation (Liyun Shi et al., supra).

There is a growing set of experiments involving the participation of 25 TSLP / TSLPRO in various physiological and pathological processes. The physiological participation of TSLP includes modulating the immune system, especially by stimulating the proliferation, development and maturation of B and T cells. TSLP plays a vital role in the pathobiology of allergic asthma and in mediating local blockade of antibodies against the receptor function of TSLP to relieve allergic diseases. Thus, it is believed that the interaction between TSLP and the TSLP receptor is important in many physiological disease processes such as: allergic inflammation, skin lesions of patients with atopic dermatitis or atopic eczema, allergic asthma and asthma.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows that the electrokinetically generated fluid of the invention (for example, Reverá 60 and Solas) reduced DEP-induced TSLP receptor expression in bronchial epithelial cells (BEC) by approximately 90% and 50%, respectively, normal saline solution (NS) had only a marginal effect. In addition, the non-electrokinetic PP60 fluid from the control pressure cooker resulted in a reduction of approximately 50% in the expression of the TSLP receptor induced by DEP.

Figure 2 shows that the electrokinetically generated fluid of the invention (for example, Reverá 60 and Solas) inhibited DEP-induced cell surface-bound MMP9 levels in bronchial epithelial cells by approximately 80% and 70%, respectively, while saline normal (NS) had only a marginal effect. Additionally, the non-electrokinetic PP60 fluid from the pressure cooker resulted in a reduction of approximately 30% in the levels of MMP9 attached to the cell surface induced by DEP.

Figures 3 AC demonstrate the results of a series of patch clamping experiments that evaluated the effects of electrokinetically generated fluid (for example, RNS-60 and Soles) on epithelial cell membrane polarity and ion channel activity in two moments (15 min (panels on the left) and 2 hours (panels on the right)) and in different voltage protocols.

Figures 4 A-C show, in relation to the experiments related to Figures 3 A-

C, the graphs resulting from subtracting the Solas current data from the RNS-60 current data into three voltage protocols (A. steps starting from zero mV; B. steps starting from -60 mV; C. steps starting from -120 mV) and at both times (15 minutes (light circles) and 2 hours (dark circles)).

Figures 5 AD demonstrate the results of a series of patch clamping experiments that evaluated the effects of electrokinetically generated fluid (for example, Soles (panels A. and B.) and RNS-60 (panels C. and D.) on epithelial cell membrane polarity and ion channel activity, using different external salt solutions and at different voltage protocols (panels A. and C. show the steps starting from zero mV; panels B. and D. show the steps starting from -120 mV).

Figures 6 A-D show, in relation to the experiments related to Figures 5 A-

D, the graphs resulting from subtracting the current data from CsCI (shown in Figure 5) from the current data from 20 mM CaCI ₂ (diamonds) and 40 mM CaCh (dark squares) in two voltage protocols (panels A. and C steps starting from zero mV; B. and D. steps starting from -120 mV) for Solas (panels A. and B.) and Reverá 60 (panels C. and D ·).

Figures 7A and B demonstrate the results of a patch clamping experiment that evaluated the effects of dilution of the electrokinetically generated fluid (for example, RNS-60) on epithelial cell membrane polarity and ion channel activity.

SUMMARY OF EXAMPLE MODALITIES

A specific modality provides a method for treating a disease or condition mediated by TSLP or mediated by TSLPR, comprising administering to a mammal in need thereof an effective amount of an electrokinetically altered aqueous fluid, comprising an aqueous ionic solution of charge-stabilized nanostructures, substantially less than approximately 100 nanometers in diameter and stably configured in the aqueous ionic fluid in sufficient quantity to treat a TSLP-mediated or TSLPR-mediated disease or condition. In certain embodiments, the charge-stabilized oxygen-containing nanostructures are stably configured in the aqueous ionic fluid in sufficient quantity to allow, when in contact with a living cell by the fluid, the modulation of at least one between cell membrane potential and cell membrane conductivity .

In certain modalities of the method, charge-stabilized oxygen-containing nanostructures represent the main type of charge-stabilized gas-containing nanostructure in the fluid. In special modalities, the percentage of dissolved oxygen molecules present in the fluid, such as charge-stabilized oxygen-containing nanostructures, is a percentage selected from the group consisting of above: 0.01%, 0.1%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% , 90% and 95%. In special modalities, total dissolved oxygen is substantially present in charge-stabilized oxygen-containing nanostructures. In certain embodiments, charge-stabilized oxygen-containing nanostructures have an average diameter of less 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 special embodiments of the method, the aqueous ionic solution comprises a saline solution. In certain embodiments, the electrokinetically altered aqueous fluid is superoxygenated.

In special methods of the method, the electrokinetically altered aqueous fluid comprises a form of solvated electrons.

In certain embodiments, the alteration of the electrokinetically altered aqueous fluid comprises exposure of the fluid to hydrodynamically induced localized electrokinetic effects. In certain embodiments, exposure to localized electrokinetic effects comprises exposure to at least one between voltage pulses and current pulses. In special embodiments, fluid exposure to hydrodynamically induced localized electrokinetic effects comprises exposure of the fluid to structural features of a device used to generate the fluid, inducing an electrokinetic effect.

In special embodiments, the TSLP-mediated or TSLPR-mediated disease or condition comprises a disease or disorder of the immune system, including, but not limited to, allergic inflammation. In special modalities, allergic inflammation comprises at least one of allergic inflammation of the airways, DC-mediated inflammatory Th2 responses, atopic dermatitis, atopic eczema, asthma, airway obstructive disease, chronic obstructive pulmonary disease, IgE-mediated disorders , rhinoconjunctivitis and food allergies. In certain embodiments, the TSPL-mediated or TSPLR-mediated disease or condition comprises inflammatory arthritis, for example, comprising at least one between rheumatoid arthritis and psoriasis.

In certain embodiments, the method further comprises combination therapy, in which at least one additional therapeutic agent is administered to the patient. In certain embodiments, the at least one additional therapeutic agent is selected from the group consisting of β ₂ -agonists, short - acting agonists β ₂ prolonged anticholinergic action, corticosteroids, systemic corticosteroids, stabilizers of mast cells, leukotriene modifiers, methylxanthines and combinations of these. In certain embodiments, the at least one additional therapeutic agent is selected from the group consisting of: bronchodilators consisting of β ₂ agonists, including albuterol, levalbuterol, pyrbuterol, artformoterol, formoterol, salmeterol, and anticholinergics such as ipatropium and tiotropium; corticosteroids including beclomethasone, budesonide, flunisolide, fluticasone, mometasone, triamcinolone, methylpredmsolone, prednisolone, prednisone; leukotriene modifiers including montelukast, zafirlukast and zileuton; mast cell stabilizers including chromoline and nedocromil; methylxanthines including theophylline, combined drugs including ipatropium and albuterol, flucatisone and salmeterol, budesonide and formoterol; antihistamines including hydroxyzine, diphenhydramine, loratadine, cetirizine and hydrocortisone; immune-modulating drugs including tacrolimus and pimecrolimus; cyclosporine; azothioprine; mycophenolate mofetil; and combinations of these. In special embodiments, the at least one additional therapeutic agent is TSLP and / or TSLPR antagonist and, in special embodiments, the TSLP and / or TSLPR antagonist is selected from the group consisting of specific neutralizing antibodies against TSLP and the receptor for TSLP, soluble TSLP receptor molecules, TSLP receptor fusion proteins, including TSLPR-Fc immunoglobulin molecules or polypeptides that encode components of more than one receptor chain.

In special embodiments of the method, modulating at least one between cell membrane potential and cell membrane conductivity comprises altering at least one between cell membrane structure or function, comprising changing at least one between conformation, ligand-binding activity and catalytic activity of protein associated with the membrane or constituent. In certain embodiments, 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 Protein G Coupled Receptor (GPCR). In special embodiments, the Protein G Coupled Receptor (GPCR) interacts with a subunit of a G protein, for example, where the subunit of a G protein comprises at least one selected from the group consisting of Ga _s , Ga ,, Ga _q and Ga ₁₂ and, in certain embodiments, the at least single subunit of a G protein is Ga _q .

In special embodiments of the method, modulation of at least one between cell membrane potential and cell membrane conductivity comprises modulating the entire cell conductance, for example, where modulating the entire cell conductance comprises modulating at least one between contribution to linear and nonlinear voltage dependence of the entire cell conductance.

In certain embodiments of the method, modulation of at least one between cell membrane potential and cell membrane conductivity comprises calcium-dependent cell pathway or messaging modulation. In certain embodiments of the method, modulation of at least one between cell membrane potential and cell membrane conductivity comprises modulation of phospholipase C activity. In certain embodiments of the method, modulation of at least one between cell membrane potential and cell conductivity. cell membrane comprises modulation of adenylate cyclase (AC) activity. In certain embodiments of the method, modulation of at least one between cell membrane potential and cell membrane conductivity comprises 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 airways, Th2 type inflammatory responses measured by CD, atopic dermatitis, atopic eczema, asthma, airway obstructive disease, chronic obstructive pulmonary disease, IgE-mediated disorders, rhinoconjunctivitis, food allergies, inflammatory arthritis , rheumatoid arthritis and psoriasis.

Special modalities of the method include the administration of the electrokinetic fluid to a cell network or layer and also comprise the modulation of an intercellular junction in it. In certain modalities, the intracellular junction comprises at least one selected from the group consisting of occlusive (tight) junctions, communicating junctions (gap) and adherent zone and desmosomes. In special modalities, the cell network or layers comprise at least one selected from the group consisting of pulmonary epithelium, bronchial epithelium and intestinal epithelium.

In certain methods of the method, the electrokinetically altered aqueous fluid is oxygenated, where 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 50 ppm or at least 60 ppm of oxygen at atmospheric pressure.

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

In certain embodiments, the ability of the electrokinetically altered aqueous fluid to modulate at least one between cell membrane potential and 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 for longer periods in a gas-tight closed container.

In certain embodiments, the amount of oxygen present in 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.

In special modalities, treating comprises administration by at least one among topical, inhalation, intranasal and intravenous.

DETAILED DESCRIPTION OF THE INVENTION

Methods are provided to treat a TSLP-mediated or TSLPR-mediated disease or condition, comprising administering an electrokinetically altered aqueous fluid, comprising an aqueous ionic solution of charge-stabilized oxygen-containing nanostructures, with substantially an average diameter of less than approximately 100 nanometers and stably configured in the aqueous ionic fluid in sufficient quantity to treat a TSLP-mediated or TSLPR-mediated disease or condition. The charge-stabilized oxygen-containing nanostructures are preferably stably configured in the fluid in sufficient quantity to allow modulation of cell membrane potential and / or conductivity. Certain modalities comprising modulation or negative regulation of TSLP expression and / or activity are useful for treating TSLP-mediated or TSLPR-mediated diseases or conditions, as described herein (for example, immune system disorders, allergic inflammation, allergic airway inflammation , DC-mediated inflammatory Th2 responses, atopic dermatitis, atopic eczema, asthma, obstructive airway disease, chronic obstructive pulmonary disease and food allergies, inflammatory arthritis, rheumatoid arthritis, psoriasis, IgE-mediated disorders and rhinoconjunctivitis).

Electrokinetically generated fluids:

Electrokinetically generated fluids ”, in this specification, refers to electrokinetically generated fluids of the Applicants' invention, generated, for the purposes of the present Examples of work, by the exemplary Mixing Device described in detail in this patent application (see also the document US200802190088 and W02008 / 052143, both of which are incorporated herein in their entirety by reference in this patent application). Electrokinetic fluids, as demonstrated by the data described and presented herein, represent new fundamentally distinct fluids with respect to prior art non-electrokinetic fluids, including, in relation to the prior art, oxygenated non-electrokinetic fluids (for example, oxygenated fluids from pressure cooker and the like).

According to various embodiments of the present invention, electrokinetically generated fluids exhibit new and unique physical and biological properties, including, among others, the following:

In special embodiments, the electrokinetically altered aqueous fluid comprises an aqueous ionic solution of charge-stabilized oxygen-containing nanostructures, with substantially an average diameter of less than approximately 100 nanometers and stably configured in the aqueous ionic fluid in sufficient quantity to allow, when in contact with a cell live by the fluid, modulation of at least one between cell membrane potential and / or cell membrane conductivity.

In special modalities, electrokinetically generated fluids refer to fluids generated in the presence of electrokinetic effects (eg voltage / current pulses) located (eg, not uniform with respect to the total fluid volume) and hydrodynamically induced, such as localized effects by device characteristics as described here. In special modalities, said hydrodynamically induced localized electrokinetic effects are combined with current flow and / or surface-related double layer effects, as described and discussed here.

In special embodiments, the electrokinetically altered aqueous fluids are suitable for modulating ¹³ C-NRM line widths of reporter solutes (eg trehalose) dissolved in them. The NRM line width effects represent an indirect method for measuring, for example, “spinning” solute in a test fluid, as described here, in specific working examples.

In special modalities, electrokinetically altered aqueous fluids are characterized by at least one of: distinguishable differences in peak square wave voltammetry at any voltage between -0.14 V, -0.47 V, -1.02 V and -1 , 3 V; polarographic peaks at -0.9 volts and absence of polarographic peaks at -0.19 V and -0.3 volts, which are unique to the electrokinetically generated fluids, as described here in specific working Examples.

In special embodiments, electrokinetically altered aqueous fluids are suitable for altering cell membrane conductivity (eg, contribution to voltage dependence on whole cell conductance as measured in patch clamping studies described here).

In special embodiments, electrokinetically altered aqueous fluids are oxygenated, where 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 in atmospheric pressure. In special modalities, electrokinetically altered aqueous fluids have less than 15 ppm, less than 10 ppm of dissolved oxygen at atmospheric pressure, or at approximately ambient oxygen levels.

In special modalities, electrokinetically altered aqueous fluids are oxygenated, in which the oxygen in the fluid is present in an amount between approximately 8 ppm and approximately 15 ppm and, in this case, is sometimes referred to in this specification as “Soles.

In special modalities, the electrokinetically altered aqueous fluid comprises at least one among solvated electrons (for example, stabilized by molecular oxygen) and electrokinetically modified and / or charged oxygen species and in which, in certain modalities, the solvated electrons and species of electrokinetically modified and / or charged oxygen are present in quantities of at least 0.01 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 special embodiments, electrokinetically altered aqueous fluids are suitable for altering cell membrane structure or function (for example, change in conformation, ligand-binding activity or catalytic activity of a membrane-associated protein), sufficiently to permit transduction modulation of intracellular signal, in which, in special modalities, the membrane-associated protein comprises at least one selected from the group consisting of receptors, transmembrane receptors (for example, Protein G Coupled Receptor (GPCR), TSLP receptor, beta receptor adrenergic 2, bradykinin receptor, etc.), ion channel proteins, intracellular binding proteins, cell adhesion proteins and integrins. In certain embodiments, the affected Protein G Coupled Receptor (GPCR) interacts with a subunit of a G protein (for example, Ga _s , Ga ,, Ga _q and Ga- | ₂ ).

In special embodiments, electrokinetically altered aqueous fluids are suitable for modulating intracellular signal transduction, comprising modulation of a calcium-dependent cellular pathway or message system (for example, modulation of phospholipase C activity or modulation of adenylate cyclase (AC) activity )).

In special embodiments, electrokinetically altered aqueous fluids are characterized by various biological activities (for example, regulation of cytokines, receptors, enzymes and other proteins and intracellular signaling pathways) described herein.

In special modalities, electrokinetically altered aqueous fluids exhibit synergy with Albuterol and Budesonide as shown in this specification.

In certain embodiments, electrokinetically altered aqueous fluids reduce DEP-induced TSLP receptor expression in bronchial epithelial cells (BEC), as shown in Examples of work in this specification.

In special modalities, electrokinetically altered aqueous fluids inhibit DEP-induced levels of cell-bound MMP9 in bronchial epithelial cells (BEC), as shown in Examples of work in this specification.

In special modalities, the biological effects of electrokinetically altered aqueous fluids are inhibited by diphtheria toxin, indicating that beta blocking, GPCR blocking and Ca channel blocking affect the activity of electrokinetically altered aqueous fluids (for example, on T cell regulatory function ), as shown in this specification.

In special embodiments, the physical and biological effects (for example, the ability to alter sufficient cell membrane structure or function to allow modulation of intracellular signal transduction) of electrokinetically altered aqueous fluids persist for at least two, at least three, at least at least four, at least five, at least 6 months or for longer periods in a closed container (eg closed gas-tight container).

Therefore, additional modalities provide the aforementioned electrokinetically generated solutions and methods to produce electrokinetically altered oxygenated fluid or aqueous solution, comprising: providing a flow of fluid material between two distant surfaces in relative motion and defining a mixing volume between them, in which the residence time of a single passage of the flow of fluid material within and through the mixing volume is greater than 0.06 seconds or greater than 0.1 seconds; and introducing oxygen (O ₂ ) into the flow of the fluid material within the mixing volume under conditions suitable to dissolve 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 in the material, and electrokinetically change the fluid or solution. In certain embodiments, 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 special embodiments, the ratio of the surface area to the volume is at least 12, at least 20, at least 30, at least 40 or at least 50.

Still further modalities provide a method for producing an electrokinetically altered oxygenated aqueous fluid or solution, comprising: providing a flow of fluid material between two spaced surfaces, defining a mixing volume between them;

and introducing oxygen into the material flow within the mixing volume under conditions suitable to infuse at least 20 ppm, at least 25 ppm, at least 30 ppm, at least 40, at least 50 or at least 60 ppm of oxygen into the material in less 100 milliseconds, less than 200 milliseconds, less than 300 milliseconds or less than 400 milliseconds. In certain embodiments, the dwell time of the material flow within the mixing volume is greater than 0.06 seconds or greater than 0.1 seconds. In special embodiments, the ratio of the surface area to the volume is at least 12, at least 20, at least 30, at least 40 or at least 50.

In special embodiments, the electrokinetically altered administered fluids of the invention comprise charge-stabilized oxygen-containing nanostructures in sufficient quantity to allow modulation of at least one between cell membrane potential and cell membrane conductivity. In certain embodiments, electrokinetically altered fluids are superoxygenated (for example, RNS-20, RNS-40 and RNS-60, comprising 20 ppm, 40 ppm and 60 ppm of dissolved oxygen, respectively, in standard saline). In special modalities, electrokinetically altered fluids are not superoxygenated (for example, RNS-10 or Soles, comprising 10 ppm (for example, approximately environmental levels of oxygen dissolved in standard saline). In certain modalities, salinity, stability, the pH, etc., of the electrokinetically altered fluids of the invention are established when the electrokinetic production of the fluid is made, and the sterile fluids are administered by the appropriate route, alternatively at least one between the salinity, sterility, pH, etc. of the fluids is appropriately adjusted (for example, using sterile saline or appropriate diluents) to be physiologically compatible with the route of administration prior to administration of the fluid, preferably diluents and / or saline solutions and / or buffer compositions, used to adjust at least one between the salinity, sterility, pH, etc., of the fluids are also fluids and electrokinetic or otherwise compatible.

In special embodiments, the electrokinetically altered fluids of the invention comprise saline solution (for example, one or more dissolved salts; for example, alkali metal salts (Li, Na, K, Rb, Cs, etc.), alkaline earth metal salts (eg Mg, Ca), etc., transition metal salts (eg Cr, Fe, Co, Ni, Cu, Zn, etc.), together with any suitable anionic / counterionic components). Special modalities comprise electrokinetic fluids based on mixed salts (for example, Na, K, Ca, Mg, etc., in various combinations and concentrations). In special embodiments, the electrokinetically altered fluids of the invention comprise standard saline (for example, approximately 0.9% NaCI or around 0.15 M NaCI). In special embodiments, the electrokinetically altered fluids of the invention comprise saline solution in concentration of at least 0.0002 M, at least 0.0003 M, at least 0.001 M, at least

0.005 M, at least 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 special embodiments, the conductivity of the electrokinetically altered fluids of the invention is at least 10 pS / cm, at least 40 pS / cm, at least 80 pS / cm, at least 100 pS / cm, at least 150 pS / cm, at least 200 pS / cm, 300 pS / cm or at least 500 pS / cm, at least 1 mS / cm, at least 5 mS / cm, at least 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 special embodiments, any salt can be used to prepare the electrokinetically altered fluids of the invention, as long as they allow the formation of biologically active salt-stabilized nanostructures (for example, salt-stabilized oxygen-containing nanostructures), as described herein.

According to special modalities, the biological effects of the fluid compositions of the invention, comprising nanostructures containing charged stabilized gas, can be modulated (for example, increased, decreased, adjusted, etc.), changing the ionic components of the fluids as , for example, described above, and / or by changing the gaseous component of the fluid. In preferred embodiments, oxygen is used to prepare the electrokinetic fluids of the invention. In additional modalities, mixtures of oxygen together with at least one other gas selected from nitrogen, oxygen, argon, carbon dioxide, neon, helium, krypton, hydrogen and xenon.

Preferred exemplary modalities:

A special modality provides a method for treating a TSLP-mediated or TSLRP-mediated disease or condition, comprising administering to a mammal in need thereof, a therapeutically effective amount of an electrokinetically altered aqueous fluid, comprising an aqueous ionic solution of oxygen-containing nanostructures charge stabilized, with substantially an average diameter of less than approximately 100 nanometers and stably configured in the aqueous ionic fluid in sufficient quantity to treat a TSLP-mediated or TSLRP-mediated disease or condition. In certain embodiments, the charge-stabilized oxygen-containing nanostructures are stably configured in the aqueous ionic fluid in sufficient quantity to allow, when in contact with a living cell by the fluid, the modulation of at least one between cell membrane potential and cell membrane conductivity .

In certain modalities of the method, charge-stabilized oxygen-containing nanostructures represent the main type of charge-stabilized gas-containing nanostructure in the fluid. In special embodiments, the percentage of dissolved oxygen molecules present in the fluid, in the form of charge-stabilized oxygen-containing nanostructures, is a percentage selected from the group consisting of above: 0.01%, 0.1%, 1% , 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,

75%, 80%, 85%, 90% and 95%. In special modalities, total dissolved oxygen is substantially present in charge-stabilized oxygen-containing nanostructures. In certain embodiments, charge-stabilized oxygen-containing nanostructures have an average diameter of less 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 special embodiments of the method, the aqueous ionic solution comprises a saline solution. In certain embodiments, the electrokinetically altered aqueous fluid is superoxygenated.

In special methods of the method, the electrokinetically altered aqueous fluid comprises a form of solvated electrons.

In certain embodiments, the alteration of the electrokinetically altered aqueous fluid comprises exposure of the fluid to hydrodynamically induced, localized electrokinetic effects. In certain embodiments, exposure to localized electrokinetic effects comprises exposure to at least one between voltage pulses and current pulses. In special modalities, the fluid's exposure to hydrodynamically induced localized electrokinetic effects comprises exposure of the fluid to structural characteristics that induce the electrokinetic effect of a device to generate the fluid.

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

In certain embodiments, the method further comprises combination therapy, in which at least one additional therapeutic agent is administered to the patient. In special embodiments, the at least one additional therapeutic agent is selected from the group consisting of: short-acting β2 agonists, long-acting β ₂ agonists, anticholinergics, corticosteroids, systemic corticosteroids, mast cell stabilizers, leukotriene modifiers, methylxanthines and combinations of these. In certain embodiments, the at least one additional therapeutic agent is selected from the group consisting of: bronchodilators consisting of β ₂ agonists, including albuterol, levalbuterol, pirbuterol, artformoterol, formoterol, salmeterol, and anticholinergics such as ipatropium and tiotropium; corticosteroids including beclomethasone, budesonide, flunisolide, fluticasone, mometasone, triamcinolone, methylprednisolone, prednisolone, prednisone; leukotriene modifiers including montelukast, zafirlukast and zileuton; mast cell stabilizers including chromoline and nedocromil; methylxanthines including theophylline, combined drugs including ipatropium and albuterol, flucatisone and salmeterol, budesonide and formoterol; antihistamines including hydroxyzine, diphenhydramine, loratadine, cetirizine and hydrocortisone; immune-modulating drugs including tacrolimus and pimecrolimus; cyclosporine; azothioprine; mycophenolate mofetil; and combinations of these. In special embodiments, the at least one additional therapeutic agent is TSLP and / or TSLPR antagonist and, in particular embodiments, the TSLP and / or TSLPR antagonist is selected from the group consisting of neutralizing antibodies specific for TSLP and the receptor for TSLP, soluble TSLP receptor molecules, TSLP receptor fusion proteins, including TSLPR-Fc immunoglobulin molecules or polypeptides that encode components of more than one receptor chain.

In special embodiments of the method, modulating at least one between cell membrane potential and cell membrane conductivity comprises altering at least one between cell membrane structure or function, comprising changing at least one between conformation, ligand-binding activity and catalytic activity of protein associated with the membrane or constituent. In certain embodiments, 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 Protein G Coupled Receptor (GPCR). In special embodiments, the Protein G Coupled Receptor (GPCR) interacts with a subunit of a G protein, for example, where the subunit of a G protein comprises at least one selected from the group consisting of Ga _s , Gai, Ga _q and Gai ₂ and, in certain embodiments, the at least single subunit of a G protein is Ga _q .

In special embodiments of the method, modulation of at least one between cell membrane potential and cell membrane conductivity comprises modulating the entire cell conductance, for example, where modulating the entire cell conductance comprises modulating at least one between contribution to linear and nonlinear voltage dependence of the entire cell conductance.

In certain embodiments of the method, modulation of at least one between cell membrane potential and cell membrane conductivity comprises calcium-dependent cell pathway or messaging modulation. In certain embodiments of the method, modulation of at least one between cell membrane potential and cell membrane conductivity comprises modulation of phospholipase C activity. In certain embodiments of the method, modulation of at least one between cell membrane potential and cell conductivity. cell membrane comprises modulation of the activity of adenylate cycse (AC). In certain embodiments of the method, the modulation of at least one between cell membrane potential and cell membrane conductivity comprises 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 airway inflammation, Th2 type inflammatory responses measured by CD, atopic dermatitis, atopic eczema, asthma, airway obstructive disease, chronic obstructive pulmonary disease, IgE-mediated disorders, rhinoconjunctivitis, food allergies, arthritis inflammatory disease, rheumatoid arthritis and psoriasis.

Special modalities of the method include the administration of the electrokinetic fluid to a cell network or layer and also comprise the modulation of an intercellular junction in it. In certain modalities, the intracellular junction comprises at least one selected from the group consisting of occlusive (tight) junctions, communicating junctions (gap) and adherent zone and desmosomes. In special modalities, the cell network or layers comprise at least one selected from the group consisting of pulmonary epithelium, bronchial epithelium and intestinal epithelium.

In certain methods of the method, the electrokinetically altered aqueous fluid is oxygenated, where 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 50 ppm or at least 60 ppm of oxygen at atmospheric pressure.

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

In certain embodiments, the ability of the electrokinetically altered aqueous fluid to modulate at least one between cell membrane potential and 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 for longer periods in a gas-tight closed container.

In certain embodiments, the amount of oxygen present in 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.

In special modalities, treating comprises administration by at least one between topical, inhalation, intranasal and intravenous.

Relevant exemplary molecular interactions:

Conventionally, quantum properties are believed to belong to elementary particles of less than 10 ^'10 meters, while the macroscopic world of our daily life is referred to as classic while it behaves according to Newton's laws of motion.

Recently, molecules have been described as clusters (clusters) in formation that increase in size with dilution. These clusters measure several micrometers in diameter and, according to what has been reported, increase in size in a non-linear manner with dilution. It has been postulated that quantum coherent domains measuring 100 nanometers in diameter arise in pure water, and collective vibrations of water molecules in the coherent domain may eventually close in phase against fluctuations in the electromagnetic field, providing stable oscillations in water, creating a type of memory in the form of excitation of lasting coherent oscillations, specific to substances dissolved in water that alter the collective structure of the water, which, in turn, determines the specific coherent oscillations that develop. When these oscillations are stabilized by magnetic field phase coupling, the water, when diluted, can still carry seeds of coherent oscillations. As a cluster of molecules grows in size, its electromagnetic signature is correspondingly amplified, reinforcing the coherent oscillations transmitted by water.

Despite variations in the cluster size of dissolved molecules and the detailed microscopic structure of the water, a specificity of coherent oscillations may still exist. A model for considering changes in water properties is based on aspects related to crystallization.

A simplified cluster of protonated water forming a nanoscale cage is shown in the Applicants' previous patent application: WO 2009/055729. A protonated water cluster typically takes the form of H ⁺ (H ₂ O) _n . Some protonated water clusters occur naturally, such as in the ionosphere. Without being bound by any particular theory, and according to special modalities, other types of water clusters or structures (clusters, nanogays, etc.) are possible, including structures comprising oxygen and stabilized electrons transmitted to the materials obtained by the invention. Oxygen atoms can be trapped in the resulting structures. The chemistry of the semi-bound nanogage allows oxygen and / or stabilized electrons to remain dissolved for extended periods of time. Other atoms or molecules, such as medicinal compounds, may be trapped for the purpose of sustained release. The specific chemistry of the solution material and dissolved compounds depends on the interactions of these materials.

Fluids processed by the mixing device have previously been demonstrated by means of experiments to exhibit different structural characteristics that are consistent with an analysis of the fluid in the context of cluster structure. See, for example, WO 2009/055729.

Charge-stabilized nanostructures (for example, charge-stabilized oxygen-containing nanostructures):

As previously described in Applicants' WO 2009/055729, “double layer effect”, “dwell time”, “infusion rate” and “bubble size measurements”, the electrokinetic mixing device creates, in a matter of milliseconds , a non-linear fluid dynamic interaction of the first material and the second material with dynamic complex turbulence, providing a complex mixture in contact with an effectively huge surface area (including that of the device and the exceptionally small gas bubbles below 100 nm) that give rise to the electrokinetic effects described in this patent application. In addition, electrokinetic effects localized by characteristics (voltage / current) were demonstrated using a specially designed mixing device, comprising isolated rotor and stator characteristics.

As well recognized in the prior art, charge redistribution and / or solvated electrons are known to be highly unstable in aqueous solutions. According to special modalities, the applicants' electrokinetic effects (for example, charge redistributions, including, in special modalities, solvated electrons) are surprisingly stabilized within the material obtained (for example, saline solutions, ionic solutions). In fact, according to this description, the stability of the properties and biological activity of the electrokinetic fluids of the invention (for example, RNS-60 or Soles) can be kept for months in a gas-tight container, indicating involvement of dissolved gas (for example , oxygen) that helps to generate and / or maintain and / or mediate the properties and activities of the solutions of the invention. Significantly, charge redistributions and / or solvated electrons are stably configured in the aqueous electrokinetic ionic fluids of the invention in sufficient quantity to allow, when in contact with a living cell (for example, mammalian cell) by the fluid, modulation of at least one between cell membrane potential and cell membrane conductivity (see, for example, Example 23 of cell clamping patch work from WO 2009/055729 and as described herein).

According to the description under “Molecular Interactions in this report, to justify the stability and biological compatibility of the electrokinetic fluids of the invention (for example, electrokinetic saline solutions), the Applicants proposed that interactions between the water molecules and the molecules of the substances ( for example, oxygen) dissolved in water change the collective structure of the water and allow the formation of nano-scale gai20 ola clusters, including nanostructures comprising oxygen and / or stabilized electrons, which are transmitted to the materials obtained by the invention. Without being attached to a mechanism, the configuration of nanostructures, in special modalities, is such that they: comprise dissolved gas (at least for formation and / or stability and / or biological activity); allows electrokinetic fluids (for example, RNS-60 or Soline fluids with saline) to modulate (for example, transmit or receive) charges and / or effects on charge when in contact with a cell membrane or a constituent related to it; and, in special modalities, they allow the stabilization (for example, carrying, harboring, trapping) solvated electrons in a biologically pertinent form.

q According to special modalities and reinforced by this specification, in ionic or saline solutions (for example, standard saline, NaCI), the nanostructures of the invention comprise charge-stabilized nanostructures (for example, mean diameter less than 100 nm) that can comprise at least one molecule of dissolved gas (e.g., oxygen) within the charge stabilized hydration sphere. According to additional modalities, the charge-stabilized hydration sphere may comprise a cage or space housing the at least one molecule of dissolved gas (for example, oxygen). According to other modalities, because suitable charge-stabilized hydration spheres are present, the charge-stabilized nanostructure and / or charge-stabilized oxygen-containing nanostructures may further comprise a solvated electron (e.g., stabilized solvated electron).

Without being bound by a specific mechanism or theory, after the present priority date, microbubbles stabilized by charge by ions in aqueous liquid in equilibrium with ambient (atmospheric) gas were proposed (Bunkin et a /., Journal of Experimental and Theoretical Physics, 104 : 486-498, 2007; incorporated herein, in its entirety, by reference in this patent application). According to specific embodiments of the present invention, the Applicants' new electrokinetic fluids comprise a new biologically active form of charge-stabilized oxygen-containing nanostructures, and may further comprise new arrangements, clusters or associations of such structures.

According to the charge-stabilized microbubble model, the short-range molecular order of the water structure is destroyed by the presence of a gas molecule (for example, gas molecule initially dissolved forming a complex with non-absorptive ion results in defect order short-range), enabling the condensation of ionic droplets, in which the defect is surrounded by the first and second spheres of coordination of water molecules, which are alternately filled by absorptive ions (for example, acquisition of a “screening shelf ( shielding sphere) of Na ions for a double electrical layer) and non-absorptive ions (for example, Cl 'ions occupying the second coordination sphere), occupying six to 12 vacant spaces, respectively, in the coordination spheres. In sub-saturated ionic solutions (for example, sub-saturated saline solutions), this hydrated core remains stable until the first and second spheres are filled with six absorptive and five non-absorptive ions, respectively, and then undergoes a Coulomb explosion. creating an internal space containing the gas molecule, in which the absorptive ions (for example, Na ⁺ ions) are adsorbed to the surface of the resulting space, while the non-absorptive ions (or some of these) diffuse into the solution (Bunkin et al ., supra). In this model, the space in the nanostructure is prevented from collaborating by Coulomb repulsion between the ions (for example, Na ⁺ ions) adsorbed to its surface. The stability of the nanostructures containing space is postulated as a result of the selective adsorption of dissolved ions with equal charges on the surface of the space / bubble and the diffusion balance between the dissolved gas and the gas inside the bubble, in which the resulting double electrical layer make it possible to stably compensate for surface tension, and the gas pressure inside the bubble is balanced by the ambient pressure. According to the model, the formation of these microbubbles requires an ionic component and, in certain aspects, associations mediated by collision between particles may allow the formation of clusters (arrangements) of greater order (/ d).

The charge-stabilized microbubble model suggests that the particles may be gaseous microbubbles, but only considers spontaneous formation of these structures in ionic solution in equilibrium with ambient air, does not characterize and omits whether oxygen is able to form these structures, in addition to similarly if solvated electrons could or could not be associated and / or stabilized by these structures.

According to special modalities, the electrokinetic fluids of the invention, comprising charge-stabilized nanostructures and / or charge-stabilized oxygen-containing nanostructures, are new and fundamentally different from the atmospheric charge-stabilized non-electrokinetic microbubble structures according to the microbubble model . Significantly, this conclusion stems, at least in part, from the fact that saline control solutions do not exhibit the biological properties described here, while claimants' oxygen-stabilized nanostructures offer a new biologically active form of oxygen-stabilized nanostructures. charge.

In accordance with special embodiments of the present invention, the Applicants' new electrokinetic device and methods provide new electrokinetically altered fluids, comprising significant amounts of charge-stabilized nanostructures, exceeding any amount that may or may not occur spontaneously in ionic fluids in equilibrium with air, or in any non-electrokinetically generated fluid. In special embodiments, charge-stabilized nanostructures comprise charge-stabilized oxygen-containing nanostructures. In additional embodiments, charge-stabilized nanostructures are all or substantially all charge-stabilized oxygen-containing nanostructures or charge-stabilized oxygen-containing nanostructures are the main type of charge-stabilized gas-containing nanostructure in the electrokinetic fluid.

According to additional modalities, the charge-stabilized nanostructures and / or the charge-stabilized oxygen-containing nanostructures may comprise or house a solvated electron and in addition this may offer a new type of stabilized solvated electron carrier. In special modalities, charge-stabilized nanostructures and / or charge-stabilized oxygen-containing nanostructures offer a new type of electret (electride) (or inverted electret), which, in contrast to conventional solute electrets containing a single organically coordinated cation, more precisely it contains a plurality of cations in a stable arrangement close to a space or a space containing an oxygen atom, in which the sodium ions in the arrangement are coordinated by water hydration spheres, rather than by organic molecules. According to special modalities, a solvated electron can be accommodated by the hydration sphere of water molecules or, preferably, be accommodated within the nanostructure space, distributed over all cations. In certain embodiments, the nanostructures of the invention offer a new "super electret" structure in solution, not only allowing the distribution / stabilization of the solvated electron over multiple sodium cations in the array, but also allowing the association or partial association of the solvated electron with the oxygen molecule (s) trapped in space - the solvated electron being distributed over an array of sodium atoms and at least one oxygen atom. According to special modalities, therefore, "solvated electrons", as presently described in association with the electrokinetic fluids of the invention, may not be solvated in the traditional model comprising direct hydration by water molecules. Alternatively, in limited analogy with dehydrated electret salts, electrons solvated in the electrokinetic fluids of the invention can be distributed over multiple charge-stabilized nanostructures, serving as a glue for distribution arrangement ”to stabilize higher order arrangements in aqueous solution.

In special modalities, charge-stabilized nanostructures and / or charge-stabilized oxygen-containing nanostructures are able to interact with cell membranes or their constituents or proteins, etc., and mediate biological activities. In special modalities, charge-stabilized nanostructures and / or charge-stabilized oxygen-containing nanostructures, housing solvated electrons, are able to interact with cell membranes or their constituents or proteins, etc., and mediate biological activities.

In special modalities, charge-stabilized nanostructures and / or charge-stabilized oxygen containing nanostructures interact with cell membranes or their constituents or proteins, etc., as a donor (release) of charge and / or charge effect and / or as load receptor and / or load effect to mediate biological activities. In special modalities, the charge-stabilized nanostructures and / or the charge-stabilized oxygen-containing nanostructures of the invention, housing solvated electrons, interact with cell membranes or their constituents or proteins, etc., as donor, charge and / or charge effect and / or as a load receptor and / or load effect to mediate biological activities.

In special embodiments, the charge-stabilized nanostructures and / or the charge-stabilized oxygen-containing nanostructures of the invention are compatible and responsible for the stability and observed biological properties of the electrokinetic fluids of the invention, and also offer a new electret (or inverted electret) which makes possible the presence of stabilized solvated electrons in aqueous ionic solutions (for example, saline solutions, NaCI, etc.).

In special modalities, charge-stabilized oxygen-containing nanostructures substantially comprise, assume the shape, or may give rise to charge-stabilized oxygen-containing nanoblocks. In special modalities, clusters containing oxygen stabilized by charge allow the formation of relatively larger arrays of nanostructures containing oxygen stabilized by charge and / or nanobubbles containing 20 oxygen stabilized by charge or arrangements thereof. In special modalities, charge-stabilized oxygen-containing nanostructures can enable the formation of hydrophobic nanobubbles upon contact with a hydrophobic surface.

In special embodiments, charge-stabilized oxygen-containing nanostructures comprise substantially at least one oxygen molecule. In certain embodiments, charge-stabilized oxygen-containing nanostructures substantially comprise at least 1, at least 2, at least 3, at least 5, at least 10, at least 15, at least 20, at least 50, at least 100 or more oxygen molecules. In special embodiments, charge-stabilized oxygen-containing nanostructures comprise or give rise to nanobubbles (for example, 30 hydrophobic nanobubbles) of approximately 20 nm x 1.5 nm, comprise around 12 oxygen molecules (for example, based on size oxygen molecule (approximately 0.3 nm to 0.4 nm), assuming an ideal gas and the application of n - PV / RT, where P = 1 atm, R = 0.0820571. atm / mol.K; T = 295 K; V = Póh = 4.7 x 10 ²² L, where r = 10 x 10 ^-9 m, h = 1.5 x 10 '( ⁹ men = 1.95 x 10' ²² moles).

In certain modalities, the percentage of oxygen molecules present in the fluid that are in these nanostructures, or arrangements thereof, with charge-stabilized configuration in the aqueous ionic fluid, is a percentage value selected from the group consisting of above: 0, 1%, 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45 / o, 50 / o, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or above 95%. Preferably, this percentage is above approximately 5%, above approximately 10%, above approximately 15% or above approximately 20%. In additional modalities, the substantial size of the charge-stabilized oxygen-containing nanostructures, or arrangements thereof, with charge-stabilized configuration in the aqueous ionic fluid, is that 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 approximately 50 nm, less than approximately 40 nm, less than approximately 30 nm, less than approximately 20 nm or less than approximately 10 nm.

In certain embodiments, the electrokinetic fluids of the invention comprise solvated electrons. In additional embodiments, the electrokinetic fluids of the invention comprise charge-stabilized nanostructures and / or charge-stabilized oxygen-containing nanostructures and / or arrangements thereof, which comprise at least one of: solvated electron (s) and unique charge distributions (polar, symmetric, asymmetric distribution). In certain embodiments, charge-stabilized nanostructures and / or charge-stabilized oxygen-containing nanostructures and / or arrangements of these exhibit paramagnetic properties.

On the other hand, in relation to the electrokinetic fluids of the invention, oxygenated control pressure cooker fluids (non-electrokinetic fluids) and the like do not include these biologically active charge-stabilized nanostructures and / or electrocinetically generated charge-stabilized oxygen-containing nanostructures , and / or such arrangements, capable of modulating at least one between cell membrane potential and cell membrane conductivity.

Systems for producing fluids enriched with gas

The system and methods presently described allow a gas (for example, oxygen) to be stably enriched in high concentration with minimal passive loss. This system and methods can be effectively used to enrich a wide variety of gases in high percentages in a wide variety of fluid. As an example only, deionized water at room temperature that practically exhibits levels around 2-3 ppm (parts per million) of oxygen can reach levels of dissolved oxygen ranging from at least approximately 5 pmm, at least approximately 10 pmm, at least at least approximately 15 pmm, at least approximately 20 pmm, at least approximately 25 pmm, at least approximately 30 pmm, at least approximately 35 pmm, at least approximately 40 pmm, 45 pmm, at least approximately 50 pmm, at least approximately 55 pmm, at least approximately 60 pmm, at least approximately 65 pmm, at least approximately 70 pmm, at least approximately 75 pmm, at least approximately 80, pjmm>. 85 pmm, at least approximately 90 pmm, at least approximately 95 pmm, at least approximately 100 pmm or any higher or intermediate value thereof, using the systems and / or methods described. According to a particularly exemplary modality, -water enriched with oxygen can be generated at levels of approximately 30-60 ppm dissolved oxygen.

Table 1 illustrates various measures of partial pressure obtained in a wound treated with an oxygen enriched solution (Table 1) and in samples of the oxygen enriched saline solution of the present invention.

TABLE 1

TISSUE OXYGEN MEASUREMENTS

--- Z082BO probe In the air: 171 mmHg (22.8 kPa) 23 ° C Column Partial Pressure (mmHg) B1 32-36 B2 169-200 B3 20-180 * B4 40-60 * minimum wound depth, majority> 150, occasional 20 s

TSLP and TSLP-mediated conditions

TSLP and TSLPR agonists / antagonists: Agent with affinity and that stimulates physiological activity in cellular receptors normally stimulated by natural substances, thus triggering a biochemical response. A TSLP receptor agonist has an affinity for the TSLP receptor and stimulates an activity induced by the binding of TSLP to its receptor. For example, a TSLP agonist / TSLP receptor is a molecule that binds to the TSLP receptor and induces intracellular signaling. On the other hand, an “antagonist” is an agent that inhibits the activity of a cell receptor normally stimulated by a natural substance. Thus, a TSLP / TSLP receptor antagonist binds to TSLP or the TSLP receptor and inhibits TSLP binding to the TSLP receptor and / or inhibits an activity normally induced by TSLP binding to its receptor. For example, a TSLP antagonist / TSLP receptor can bind to TSLP or the TSLP receptor and slow or prevent binding, for example, by blocking TSLP binding to the receptor

TSLP. Alternatively, a TSLP antagonist / TSLP receptor can bind to the TSLP receptor and decrease or prevent downward signaling that would normally be induced by TSLP binding to its receptor. Agonists and antagonists can include a variety of classes of molecules, including peptides, such as ligand-type peptides, antibodies and fragments or subsequences thereof. Agonists and antagonists may also include fusion polypeptides, antibodies, peptides (such as peptides less than approximately 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 TSLLP receptor fusion proteins such as TSLPR-Fc molecules of immunoglobulins or polypeptides that encode components of more than one receptor chain therefore mimic a higher order physiological receptor or oligomer heterodimer. If the receptor includes more than one polypeptide chain, fusion of a single chain can be used.

Antibody: A polypeptide linker comprising at least one variable region of the immunoglobulin light chain or heavy chain, which specifically recognizes and binds to an epitope (for example, as an antigen, such as TSLP or a fragment thereof or a TSLP receptor on a fragment thereof). These include intact immunoglobulins and their variants and parts thereof, well known in the art, such as Fab 'fragments, F (ab)' sub.2 fragments, single chain Fv proteins (“scFv) and Fv proteins stabilized with disulfide ("dsFV"). ScFV protein is a fusion protein in which a variable region of an immunoglobulin light chain and a variable region of an immunoglobulin heavy chain are linked by a linker, whereas in dsFVs, the chains have been mutated to introduce a disulfide bond in a way to stabilize the chain association. The term also includes genetically engineered forms such as chimeric antibodies (e.g., humanized murine antibodies), heteroconjungant antibodies (e.g., bispecific antibodies). See also, Pierce Catalog and Handbook, 1994 - 1995 (Pierce Chemical Co., Rockford, IL.); Kuby, J., Immunology, 3 ^rd Ed., WH Freeman & Co., New York, 1997. Typically, an immunoglobulin has a heavy and a light chain. Each light chain contains a constant region and a variable region (the regions are also known as "domains"). Combined, the variable regions of the heavy and light chains bind specifically to the antigen. The variable regions of the light and heavy chain contain a framework region interrupted by three hypervariable regions, also called complementarity determining regions or CDRs. The extent of the framework region and CDRs has been defined (see, Kabat et a!., Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, 1991, which is incorporated herein by reference in this patent application). The Kabat database is currently maintained online. The sequences of the framework regions of different heavy or heavy chains are relatively conserved within a species. The framework region of an antibody, that is, the combined framework regions of the constituent chains, light and heavy, serves to position and align the CDRs three-dimensionally. Antibodies include monoclonal antibodies, humanized antibodies, etc.

· TSLP antagonists include small molecule antagonists, antibodies against TSLP, antibodies against TSLP receptor and TSLP receptor fusion proteins, such as TSLPR-Fc molecules of immunoglobulins or polypeptides that encode a physiological receptor heterodimer or order oligomer highest, among others. TSLP has been shown to bind directly to a member of the type I cytokine receptor superfamily (which are also known as members of the hematopoietin receptor superfamily), TSLPR. TSLPR has been cloned. The high affinity functional receptor for TSLP has been shown to include two polypeptides, TSLPR and the alpha chain of the IL-7 receptor. Thus, both TSLP and IL-7 share IL-7Ralfa as components of their receptors. However, these receptors are different in that the TSLP receptor additionally contains 15 TSLPR, while the IL-7 receptor additionally contains the gamma chain of common cytokine receptors, which is a component for signal transduction of various cytokine receptors. . TSLPR (and Fc fusions from this receptor chain) is described, for example, in U.S. Patent Application Publication No. 2002/0160949, which is incorporated herein by reference in this patent application.

Antibodies to TSLP polypeptides are known in the art. In addition, anti-TSLPR antibodies are commercially available (R & D Systems, Minneapolis, Minn., Cat. Ref. MAB981; DNAX Research, Inc., Palo Alto, California). Antibodies are also prepared against the TSLP receptor or against TSLP by immunization with specific epitopes, such as regions of greater antigenicity, determined by the Welling plot of the Vector NTI.RTM.Suite program (Informax, Inc., Bethesda, Md.). The sequence of the TSLP receptor and regions of greatest antigenicity in human TSLP receptor are described in U.S. Patent Publication No. 2003/0186875. Pharmaceutical compositions (see above) generally include a therapeutically effective amount of a TSLP antagonist, and may also include additional agents. The preparation of 30 pharmaceutical compositions is described above.

Indications

TSLP and TSLPR are believed to participate in many types of allergic conditions, including, but not limited to, immune system disorders, allergic inflammation, allergic airway inflammation, DC-mediated inflammatory Th2 responses, atopic dermatitis, atopic eczema, allergic asthma, asthma, obstructive airway disease, chronic obstructive pulmonary disease (COPD), food allergies, inflammatory arthritis, rheumatoid arthritis, psoriasis, IgE-mediated disorders and rhinoconjunctivitis. The involvement of TSLP in DC-mediated inflammatory Th2 responses has been demonstrated in several publications, including a recent review by Ziegler and Liu (Ziegler and Liu, Nat. Immunol., 7: 709714, 2005, which is incorporated here, in in its entirety, by reference in this patent application).

Allergic inflammation of the airways. A recent study demonstrated that TSLP is necessary for the onset of allergic inflammation of the airways in mice in vivo (Zhou et al., Nat. Immunol., 6: 1047-1053, 2005). In this study, Zhou et al. demonstrated that the specific pulmonary expression of a TSLP transgene induced allergic airway inflammation (asthma), characterized by massive leukocyte infiltration (including Th2 cells), goblet cell hyperplasia and subepithelial fibrosis, in addition to increased levels of serum IgE. In addition, a recent study showed that allergenic challenge caused 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 airway inflammation. In this specification, Applicants show that the electrokinetically altered fluids of the invention significantly negatively regulated TSLP. According to certain embodiments, the electrokinetically altered fluids of the invention are considerably useful for treating allergic inflammation of the airways and similar conditions.

Allergic inflammation. A recent review summarizes and describes the results of studies investigating the role of TSLP in allergic inflammation, for example, allergic skin inflammation (Ziegler and Liu, 2008). In particular, studies have shown that normal skin or non-lesional skin in patients with atopic dermatitis does not have detectable TSLP protein, while skin obtained from acute and chronic lesions of topical dermatitis exhibits high expression of TSLP. In another study, mice lacking TSLPR were constructed and examined for effects on allergic skin inflammation (He et al, PNAS, 105: 11875-11880, 2008), which is incorporated herein in its entirety by reference in this application patent). He et al. found that skin inflammation caused by an allergen in mice lacking TSLPR was significantly reduced when compared to wild type mice. In yet another study, it was demonstrated that mice built to overexpress TSLP in the skin developed atopic dermatitis, characterized by eczematous skin lesions containing inflammatory infiltrates, a marked increase in circulating Th2 cells and elevated serum IgE (Yoo et al., J. Exp. Med. , 202: 541-549, 2005). The study suggested that TSLP can directly activate DCs in mice. In another study, conducted by Li et al., The group confirmed that transgenic mice overexpressing TSLP in the skin developed atopic dermatitis, which solidified 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 skin inflammation (for example, atopic dermatitis and eczema). In this specification, Applicants show that the electrokinetically altered fluids of the invention significantly negatively regulated TSLP. According to certain modalities, the electrokinetically altered fluids of the invention are considerably useful for treating allergic inflammation, for example, allergic skin inflammation (for example, atopic dermatitis and eczema). and similar conditions.

Psoriasis. A recent study showed that TSLP had a substantially higher expression in skin biopsies obtained from patients with acute psoriasis (GuttmanYassky, 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. In this specification, Applicants show that the electrokinetically altered fluids of the invention significantly negatively regulated TSLP. According to certain embodiments, therefore, the electrokinetically altered fluids of the invention are considerably useful for treating psoriasis and similar conditions.

Allergic asthma. Recently, a study showed that allergenic challenge caused 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 shown that modulating the function of DCs by inhibiting TSLPR decreases severity in mice. These results indicate that TSLP plays an important role in the pathogenesis of allergic asthma. In this specification, Applicants show that the electrokinetically altered fluids of the invention significantly negatively regulated TSLP. According to certain embodiments, therefore, the electrokinetically altered fluids of the invention are considerably useful for treating allergic asthma and similar conditions.

Obstructive airway disease. A recent study has shown that COPD is associated with elevated expression of TSLP in the bronchial mucosa (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. In this specification, Applicants show that the electrokinetically altered fluids of the invention significantly negatively regulated TSLP. According to certain embodiments, therefore, the electrokinetically altered fluids of the invention are considerably useful for treating obstructive airway disease, for example, COPD.

Food allergies. Dendritic cells in the intestines have been shown to stimulate virgin T cells, diverting them to a TH2 type response in an OX40L-dependent manner (Blazquez AB, Berin MC. Gastrointestinal dendritic cells promote Th2 skewing via OX40L. J. Immunol., 180: 4441-4450, 2008, incorporated herein in its entirety, by reference in this patent application). In addition, a recent review discusses the presence of TSLP in the body and its role in regulating immune homeostasis (Kiev ID, Matteoli G, Reseigno M. The yin and Yan of intestinal epithelial cells in controlling dendritic cell function. J Exp Med; 204 : 2253-2257, 2007, which is incorporated herein, in its entirety, by reference in this patent application). These results indicate that TSLP plays an important role in food allergies. In this specification, Applicants show that the electrokinetically altered fluids of the invention significantly negatively regulated TSLP. According to certain embodiments, therefore, the electrokinetically altered fluids of the invention are considerably useful for treating food allergies and similar conditions.

Inflammatory arthritis. A recent study revealed increased levels of TSLP in samples of synovial fluid derived from patients with rheumatoid arthritis (RA), when compared to synovial fluid from patients with other forms of arthritis (Koyama et al., Biochem and Biophys Res Comm., 357: 99-104, 2007, which is incorporated herein, in its entirety, by reference in this patent application). The same study found that the use of neutralizing anti-TSLP antibody improved experimental TNF-a-dependent arthritis induced by type II anti-collagen antibody in mice. These results indicate that TSLP is an important participant in inflammatory arthritis, such as RA. In this specification, Applicants show that the electrokinetically altered fluids of the invention significantly negatively regulated TSLP. According to certain embodiments, therefore, the electrokinetically altered fluids of the invention are considerably useful for treating inflammatory arthritis and similar conditions, for example, RA.

Allergic rhinitis. In a recent research study, Mou et al. found that TSLP was present at both the mRNA and protein levels in the nasal mucosa of all tested patients who suffered from allergic rhinitis (RA) (Mou ef al., Acta Oto-laryngologica, 129: 297-301, 2009, incorporated here , in its entirety, by reference in this patent application). In addition, TSLP levels correlated strictly with the severity of AR. These results indicate that TSLP plays an important role in the pathogenesis of RA and / or rhinoconjunctivitis. In this specification, Applicants show that the electrokinetically altered fluids of the invention significantly negatively regulated TSLP. According to certain embodiments, therefore, the electrokinetically altered fluids of the invention are considerably useful for treating RA, allergic rhinoconjunctivitis and similar conditions.

Treatment methods

The term "treat" refers to, and includes, reversing, relieving, inhibiting the evolution or preventing a disease, disorder or condition, or one or more of its symptoms, and treatment and "therapeutically" refer to the act of treating , as defined in this specification.

"Therapeutically effective amount" is any amount of any of the compounds used in the course of practicing the invention provided herein, sufficient to reverse, alleviate, inhibit evolution or prevent a disease, disorder or condition, or one or more of its symptoms.

Certain embodiments of the present invention relate to therapeutic compositions and modes of treatment for a subject, preventing or alleviating at least one symptom of inflammation associated with certain conditions or diseases. Many conditions or diseases with inflammatory disorders have been treated with steroids, mettrexate, immunosuppressive drugs, including cyclophosphamide, cyclosporine, azothioprine and leflunomide, non-steroidal anti-inflammatory agents such as aspirin, acetominafen and COX-2 inhibitors, agents with gold and antimalarial treatments.

Routes and forms of administration

In this specification, “subject can refer to any living creature, preferably an animal, more preferably a mammal and even more preferably a human being.

In specific exemplary embodiments, the gas-enriched fluid of the present invention can function as a therapeutic composition, alone or in combination with another therapeutic agent, so that the therapeutic composition prevents or alleviates at least one symptom of inflammation. Therapeutic compositions of the invention include compositions 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, flavoring agents, perfumes and binding agents.

In this specification, "pharmaceutically acceptable carrier" and "carrier generally refer to non-toxic, inert, solid, semi-solid or liquid filler, diluent, encapsulating material or auxiliary formulation of any kind. 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 carbomethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatine; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil; Sesame oil; olive oil; corn oil and soy oil; glycols: as propylene glycol; esters such as ethyl oleate and ethyl laurate; 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, release promoting agents, coating agents, sweetening, flavoring and perfume agents, preservatives and antioxidants may also be present in the composition, according to the opinion of the person responsible for the formulation. In special embodiments, these vehicles and excipients can be fluids or solutions enriched with gas of the present invention.

The pharmaceutically acceptable vehicles 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 therapeutic agents and without unfavorable side effects or toxicity under the conditions of use. Pharmaceutically acceptable carriers can include polymeric polymers and matrices, nanoparticles, microbubbles, and the like.

In addition to the gas-enriched therapeutic fluid of the present invention, the therapeutic composition may further comprise inert diluents, such as additional water not enriched with gas or other solvents, solubilizing and emulsifying agents, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, acetate ethyl, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (specifically, cottonseed oil, peanuts, corn, germ, olive, castor and sesame), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and sorbitan fatty acid esters, and mixtures thereof. As is appreciated by those skilled in the art, a new and better formulation of a therapeutic composition in particular, a new therapeutic fluid enriched with gas, and a new method for releasing the new therapeutic fluid enriched with gas can be obtained by substituting one or more inert diluents with a fluid enriched with gas of identical, similar or different composition. For example, conventional water can be replaced or supplemented by a fluid enriched with gas, produced by mixing oxygen in water or deionized water to provide fluid enriched with gas.

In certain embodiments, the gas-enriched fluid of the invention can be combined with one or more therapeutic agents and / or used alone. In special embodiments, incorporating the gas-enriched fluid may include replacing one or more solutions known in the art, such as deionized water, saline and the like, with one or more gas-enriched fluids, thereby providing a therapeutic composition improved to be released to the subject.

Certain embodiments provide pharmaceutical compositions comprising a gas-enriched fluid of the present invention, a pharmaceutical composition or other therapeutic agent or 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 the preceding diseases or conditions and in therapies as mentioned above. Preferably, the vehicle must be pharmaceutically acceptable and must be compatible, that is, without harmful effect on the other ingredients in the composition. The carrier can be solid or liquid and is preferably formulated in unit dose formulation, for example, a tablet that can contain from 0.05 to 95% by weight of the active ingredient.

Possible routes of administration include oral, sublingual, buccal, parenteral (e.g., subcutaneous, intramuscular, intraarterial, intraperitoneal, intracisternal, intravesical, intrathecal or intravenous), rectal, topical including.transdemic, intravaginal, intraoeular, intraoptic, inhalation, and injection or insertion of implantable devices or materials.

Routes of administration

The most suitable means of administration for a given subject will depend on the nature and severity of the disease or condition being treated, or the nature of the therapy being used, as well as the nature of the therapeutic composition or the additional therapeutic agent. In certain embodiments, oral or topical administration is preferred.

Formulations suitable for oral administration can be supplied in separate units, such as tablets, capsules, sachets, syrups, elixirs, chewing gum, "lollipop" formulations, microemulsions, solutions, suspensions, lozenges, or gel-coated ampoules, each one containing a predetermined amount of the active compound; in the form of powder or granules; in the form of solutions or suspensions in aqueous liquids or not or in the form of oil in water or water in oil emulsions.

Formulations suitable for transmucosal methods, such as, for example, sublingual or buccal administration, include adhesive lozenges, tablets and the like, comprising the active compound and typically a flavored base, such as, for example, sugar and acacia or tragacanth, and lozenges comprising the active compound on an inert base, such as gelatin and glycerin or acacia, sucrose.

Formulations suitable for parenteral administration typically comprise sterile aqueous solutions, containing a pre-determined concentration of the gas-enriched active fluid and possibly another therapeutic agent; the solution is preferably isotonic with the blood of the intended recipient. Additional formulations suitable for parenteral administration include formulations containing physiologically suitable co-solvents and / or complexing agents such as surfactants and cyclodextrins. Oil-in-water emulsions may also be suitable for formulations intended for parenteral administration of fluid enriched with gas. Although preferably administered intravenously, these solutions 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, dissolvable solid materials, 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 lavage solutions with the gas-enriched fluids of the present invention can be formulated for colonic or rectal administration.

Formulations suitable for topical, intraocular, intraoptic or intranasal application include ointments, creams, pastes, gels (such as hydrogels), sprays, powders and dispersible granules. emulsions, sprays or aerosols using flow propellants (such as liposomal sprays, nasal drops, nasal sprays and the like) and oils. Suitable vehicles for these formulations include petroleum gelatin, lanolin, polyethylene glycols, alcohols and combinations thereof. Nasal or intranasal delivery may include metered-dose inhalers of any of these formulations or others. Likewise, intraoptic or intraocular can include drops, ointments, non-irritating solutions and the like.

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

For example, a tablet can be prepared by compressing a uniform mixture comprising powder or granules of the active ingredient and one or more optional ingredients, such as an aggregate, lubricant, inert diluent or surface-active dispersing agent, or by molding a uniform mixture of powdered active ingredient and a gas-enriched fluid of the present invention.

Formulations suitable for administration by inhalation include fine particulate powders or mists, which can be generated by various types of metered-dose pressurized aerosols, nebulizers or insufflators. In particular, powders or other compounds of therapeutic agents can be dissolved or suspended in a fluid enriched with gas of the present invention.

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

Metered inhalers are pressurized aerosol dispensers, typically containing a suspension formulation or solution of a therapeutic agent in liquefied propellant. In certain embodiments, according to this specification, the gas-enriched fluids of the present invention can be used in addition to or instead of the standard liquefied propellant. During use, these devices discharge the formulation through a valve adapted to release a metered volume, typically 10 to 150 pL, to produce a fine particle spray 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 solutions or suspensions of the active ingredient into a therapeutic aerosol cloud either by accelerating a compressed gas (typically air or oxygen), through a narrow venturi orifice, or by ultrasonic agitation. Suitable formulations for use in nebulizers consist of another therapeutic agent in a fluid enriched with gas and comprising up to 40% w / w of the formulation, preferably less than 20% w / w. In addition, other vehicles can be used, such as distilled water, sterile water or diluted aqueous alcohol solution, preferably made isotonic with body fluids by the addition of salts, such as sodium chloride. Optional additives include 15 preservatives, especially if the formulation is not prepared sterile, and may include methylhydroxybenzoate, antioxidants, flavoring agents, volatile oils, buffering agents and surfactants.

Formulations suitable for administration by insufflation include finely divided powders that can be released via insufflator or delivered to the nasal cavity in the manner of aspiration. In the insufflator, the powder is contained in capsules or cartridges, typically made of gelatin or plastic, which are ajar or opened in situ, and the powder is released by air removed through the device upon inhalation or by means of a manually operated pump. The powder used in the insufflator consists exclusively of the active ingredient or powder mixture comprising 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 cited 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 method available for use in conjunction with pharmaceutical drugs, either in the form of individual therapeutic agents or in the form of combined therapeutic agents.

the dose administered will, of course, 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 active ingredient can be expected to be around 0.001 to 1000 milligrams. . (mg) per kilogram (kg) of body weight, with a preferred dose of 0.1 to around 30 mg / kg. ,,

Presentation forms (compositions suitable for administration) contain from approximately 1 mg to approximately 500 mg of active ingredient per unit. In these pharmaceutical compositions, the active ingredient will usually be present in an amount of around 0.5 - 95% by weight based on in the total weight of the composition.

Ointments, pastes, foams, occlusions, creams and gels may also contain excipients, such as starch, tragacanth, cellulose derivatives, silicones, bentonites, silica acid and talc, or mixtures thereof. Powders and sprays may also contain excipients such as lactose, talc, silica acid, aluminum hydroxide and calcium silicates, or mixtures of these substances. Solutions of antimicrobial nanocrystalline metals can be converted into aerosols or sprays by any of the known means routinely used to produce aerosol pharmaceuticals. In general, these methods comprise pressurizing or providing a means for pressurizing a container containing the solution, usually with inert carrier gas, and causing the pressurized gas to pass through a small orifice. Sprays can contain customary propellants, such as nitrogen, carbon dioxide and other inert gases. In addition, microspheres or nanoparticles can be used with the therapeutic compositions enriched with gas or fluids of the present invention in any of the ways necessary to administer the therapeutic compounds to a subject.

Formulations for injectable use can be presented in sealed single-dose or multiple-dose containers, such as ampoules and vials, and can be stored in lyophilized condition, requiring only the addition of the sterile liquid excipient, or fluid enriched with gas, immediately before of use. Extemporaneous injectable solutions and suspensions can be prepared from sterile powders, granules or 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, JB Lippincott Co., Philadelphia, Pa., Banker and Chalmers, Eds., 238 - 250 (1982) and ASHP Handbook on Injectable Drugs, Toissel, 4 ^th ed „622-630 ( 1986).

Formulations suitable for topical administration include lozenges comprising a fluid enriched with gas and, optionally, an additional therapeutic and flavoring agent, usually sucrose and acacia or tragacanth; tablets comprising a fluid enriched with gas and optional additional therapeutic agent on an inert basis, such as gelatin and glycerin, or sucrose and acacia; and mouth rinses or oral wash solutions comprising a fluid enriched with gas and optional additional therapeutic agent in a suitable liquid carrier; as well as creams, emulsions, gels and the like.

In addition, formulations suitable for rectal administration can be presented in the form of suppositories, mixing a variety of bases, such as 5 emulsifying bases or water-soluble bases. Formulations suitable for vaginal administration may be presented in the form of weights, tampons, creams, gels, pastes, foams or spray formulas containing, in addition to the active ingredient, those vehicles known in the art as suitable.

Suitable pharmaceutical vehicles are described in Remington’s Pharmaceutical 10 Sciences, Mack Publishing Company, a standard reference text in this field.

The dose administered to a subject, especially an animal, particularly a human, in the context of the present invention should be sufficient to obtain a therapeutic response in the animal on a reasonable schedule. 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. Adequate dose is that which will result in the concentration of the therapeutic composition in a subject, known to obtain the desired response.

The dose size will also be determined by the route, time and frequency of administration, as well as the existence, nature and extent of any 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) alternately, that is, releasing the compounds in series, in sequence, in parallel or simultaneously in separate pharmaceutical formulations. In alternate therapy, the delay in administering the second and, optionally, the third active ingredient, should not be such as to cause the loss of the benefit of a synergistic therapeutic effect of the combination of the active ingredients. According to certain modalities by any method of administration (1) or (2), ideally the combination should be administered to achieve the most effective results. In certain modalities by any method of administration (1) or (2), ideally the combination should be administered to achieve maximum plasma concentrations of each of the active ingredients. A once-a-day pill regimen by administering a combined co-formulation may be feasible for some patients suffering from neurodegenerative inflammatory diseases. According to certain modalities, 35 the maximum effective plasma concentrations of the active ingredients of the combination will vary in the range of approximately 0.001 to 100 μΜ. Optimal maximum plasma concentrations can be achieved by a formulation and administration regimen prescribed for a given patient. It will also be understood that the fluids of the invention and a glucocorticoid steroid (e.g., Budesonide) or the physiologically functional derivatives of any of these, whether presented simultaneously or in sequence, can be administered individually, in multiples or in any combination thereof. In general, during alternate therapy (2), an effective dose of each compound is administered in series, in which co-formulation therapy (1), effective doses of two or more compounds are administered together.

The combinations of the invention can be conveniently presented in pharmaceutical formulation in unit dose form. A convenient unit dose formulation contains active ingredients in any amount from 1 mg to 1 g each, for example, among others, 10 mg to 300 mg. The synergistic effects of the fluid of the invention in combination with, for example, a glucocorticoid steroid (for example, Budesonide), can be obtained over a wide range of proportions, for example, 1:50 to 50: 1 (fluid of the invention; steroid glucocorticoid (eg Budesonide)). In one embodiment, the ratio can range from 1:10 to 10: 1. In another embodiment, the weight / weight ratio of the fluid of the invention to a glucocorticoid steroid (eg Budesonide), in the form of co-formulated combination administration, such as a pill, tablet, caplet or capsule, will be around 1, that is, an approximately equal amount of fluid of the invention and glucocorticoid steroid (e.g., Budesonide). In other exemplary coformulations, there may be more or less fluid of the invention and glucocorticoid steroid (for example, Budesonide). In one embodiment, each compound will be used in the combination in quantity in which it exhibits anti-inflammatory activity when used alone. Other proportions and quantities of the compounds of said combinations are contemplated within the scope of the invention.

A unit dose form may further comprise fluid of the invention and, for example, a glucocorticoid steroid (for example, Budesonide), or physiologically functional derivatives of any of these, in addition to a pharmaceutically active carrier.

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

It is also possible to combine any two active ingredients in unit dose form for simultaneous or sequential administration with a third active ingredient. The triple combination can be administered simultaneously or in sequence. When administered in sequence, the combination can be performed in two or three administrations. According to certain embodiments, the triple combination of fluid of the invention and a glucocorticoid steroid (e.g., Budesonide) can be administered in any order.

According to special modalities, the electrokinetically altered fluids of the invention are considerably useful for treating conditions mediated by TSLP and / or TSLPR, including, among others, the exemplary kind of indications described in this report. According to special modalities, the electrokinetically altered fluids of the invention are useful for treating several subgenres of the exemplary genus, in which at least one indication of the genus is excluded from each of the said subgenera.

EXAMPLE 1 (Synergistic effects of electrokinetically altered fluids of the invention and Albutrol have been demonstrated)

Overview. The electrokinetically altered fluids of the invention provided prolonged synergistic effects (for example, suppression of bronchoconstriction) with Albuterol in vivo in an animal model recognized in the state of the art of human bronchoconstriction (human asthma model) and, thus, makes it possible to decrease the use of albuterol in a patient. The results described in this Example are also described in Applicants' WO 2009/055729.

First experiment. In a first experiment, sixteen guinea pigs were evaluated for the effects of bronchodilators on airway function in conjunction with methacholine-induced bronchoconstriction. After determining the ideal dose, each animal was administered a dose of 50 pg / mL to release the target dose of 12.5 pg of albuterol sulfate in 250 pL per animal. The study had the design of blocks randomized by weight and baseline PenH values. Two groups (A and B) received intratracheal instillation of 250 pL of 50 pg / ml of albuterol sulfate in one or two diluents: Group A: deionized water was passed through the device of the invention, without the addition of oxygen, while in Group B, was gas-enriched water of the invention. Each group was administered intrathecal doses of solutions using Pen Century's Microsprayer micro-spray. Additionally, the animals were stratified in units of the BUXCO plethysmograph, so that each treatment group is represented equally in the nebulizers feeding the plethysmographs and the recording units. Animals that exhibited at least 75% of their baseline PenH value within 2 hours after albuterol administration were not included in the data analyzes. This exclusion criterion is based on past studies, in which failure to observe bronchoprotection with brondilators can be associated with dose errors. As a result, one animal in the control group was discarded from the data analysis. As soon as an animal obtained more than 50% bronchoconstriction, the animal was considered unprotected. The results indicate that 50% of the animals in Group B were protected from bronchoconstriction for up to 10 hours (at which point, the test was completed).

Second experiment. An additional set of experiments was conducted using a larger number of animals to evaluate the protective effects of the electrokinetically generated fluids of the invention (for example, RDC1676-00, RDC1676-01, -RDC167.6-02 and RDC1676-03) against induced bronchoconstriction methacholine, when administered alone or as diluents for albuterol sulfate in male guinea pigs.

Materials and methods. Guinea pigs (Cavia porceilus) were albino Hartley, Crl: (HA) BR, from Charles River Canada Inc. (St. Constant, Quebec, Canada). Weight: Approximately 325 ± 50 g at the beginning of the treatment; number of groups: 32 with 7 males per group (plus 24 spares form the same batch of animals). Diet: all animals had free access to a commercial laboratory diet in granules, with standard certificate (PMI Certified Guinea Pig 5026; PMI Nutrition International Inc.), except during designated procedures. The route of administration was intratracheal instillation with Penn Century Microsprayer and the challenge with methacholine via whole body inhalation. The intratracheal route was selected to maximize pulmonary exposure to the test item / control solution. The whole body inhalation challenge was selected for methacholine challenge in order to elicit an upper airway hypersensitivity response (ie, bronchodonstriction). The duration of treatment was one day.

Experimental draw. All animals were exposed to methacholine inhalation exposure (500 pg / mL), 2 hours after TA / Control administration. All animals received a dose volume of 250 pL. Therefore, albuterol sulfate was diluted (in the control item and in the 4 test items) to concentrations of 0, 25, 50 and 100 pg / mL. Thirty minutes after administration, albuterol sulfate solutions of 4 different concentrations (0. 25, 50 and 100 pg / mL) were formed in a stock of 1 Ox (500 pg / mL) in each of these four solutions of the test item (RDC1676-00, RDC1676-01, RDC167602 and RDC1676-03). These concentrations of albuterol sulfate were also made in control fluid not generated electrokinetically (control 1). The dose solutions were prepared by the appropriate dilution of each stock solution. All solutions, stock and with doses, were kept on ice, once prepared. Administration was completed within an hour after the test / control items were completed. A methacholine solution (500 pg / ml) was prepared on the day of dose administration.

Each animal received an intratracheal instillation of the item under test or control, using a Penn Century micro spray. The animals were deprived of food during the night and anesthetized with isoflurane, the larynx was visualized with the aid of a laryngoscope (or an appropriate alternative) and the tip of the micro-spray was inserted into the trachea. A dose with a volume of 250 pL / animal was administered for the test or control item. The methacholine aerosol was generated at the air outlet of a mixing chamber, using aerobic ultrasonic nebulizers, supplied with air from a Buxco constant bias flow pump. This mixing chamber, in turn, fed four unsealed full-body plethysmographs, each operated under slight negative pressure maintained by means of a regulating valve located in the exhaust pipe. A vacuum pump was used to exhaust the inhalation chamber at the required flow.

Before the main phase of the study was started, 12 spare animals were assigned to 3 groups (n = 4 / group) to determine the maximum exposure period in which animals can be exposed to methacholine to induce severe, but not fatal, acute bronchoconstriction. Four animals were exposed to methacholine (500? G / ml_) for 30 seconds, and respiratory parameters were measured for up to 10 minutes after the start of the aerosol. The concentration of methacholine in the nebulizer and / or exposure time to aerosolization were duly adjusted to induce severe but reversible acute / reversible bronchoconstriction, characterized by a transient increase in pens.

Once before administration of the test item (Day -1) and again at 2, 6, 10, 14, 18, 22 and 26 hours post-dose, the animals were placed in the chamber, and ventilatory parameters (tidal volume, frequency respiratory, derived from minute volume) and enhanced pause (enhanced pause) Penh were measured over a period of 10 minutes, using the Buxco Electronics BioSystem XA system, followed by the start of the methacholine aerosol challenge. Once the animals were inside the chambers, baseline values were recorded for 1 minute, after which, methacholine, in concentration in the 500 pg / mL nebulizer, was aerosolized for 30 seconds, and the animals were exposed to the aerosol for another 10 minutes, during which ventilatory parameters were continuously assessed. Penh was used as the indicator of bronchoconstriction; Penh is a derived value obtained from the maximum inspiratory flow, maximum expiratory flow and expiration time. Penh = (Maximum expiratory flow / Maximum inspiratory flow) * (Expiration time / time to exhale 65% of expiratory volume -1).

Animals that did not exhibit severe acute bronchoconstriction during the methacholine pre-dose challenge were replaced. Any animal that exhibited at least 75% of its baseline PenhPenes value within 2 hours after the dose was not included in the data analysis. Respiratory parameters were recorded in averages of 20 seconds. Data considered non-physiological were excluded from further analysis. Changes in Penh were inserted in a plot over a period of 15 minutes, and the Penh value expressed in area under the curve. Numerical data were submitted to the calculation of mean values and group standard deviations (as applicable).

Results. The results of this experiment revealed that in the absence of Albu42 terol, the administration of electrokinetically generated fluids of the invention had no apparent effect on the mean baseline percentage values of PenH, when measured over a 24 hour period. Surprisingly, however, the administration of albuterol (representative data for the 25-pg groups of albuterol / animal is shown) formulated in the electrokinetically generated fluids of the invention (at all tested oxygen level values; environment, 20 ppm, 40 ppm and 60 ppm) resulted in a marked prolongation of bronchoconstricting effects of albuterol, compared to the control fluid. That is, the results of methacholine revealed prolongation of albuterol bronchodilation up to at least 26 hours. The Applicants also showed that there were constant differences in all oxygen levels between RDC1676 and the normal saline control. Combining all 4 fluids RDC1676, the p-value for the overall treatment difference compared to saline was 0.03.

According to special modalities, the electrokinetically generated solutions of the invention provide synergistic effects of prolongation with Albuterol, thus allowing to decrease the use of albuterol in a patient, enabling more rewarding and efficient use of the drug, the occurrence of fewer effects side effects and increasing the period over which a patient can be treated and respond to treatment with albuterol.

EXAMPLE 2 (Effects of electrokinetically altered fluids of the invention on cytokine expression were determined)

Overview. The electrokinetically altered fluids of the invention decreased the production of pro-inflammatory cytokines (IL-1 ?, TNF-a, IL-6 and GM-CSF), chemokines (IL-8, MIP1a, RANTES and eotaxin), 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), when compared to control. The results described in this Example are also described in Applicants' WO 2009/055729.

In special modalities, mixed human lymphocytes were stimulated with T3 or PHA antigen in oxygen enriched Revalesio fluid, or control fluid, and changes in IL-1 β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12 (p40), IL-13, IL-17, Eotaxin, IFN-y, GM-CSF, ΜΙΡ-1β, MCP-1, G-CSF, FGFb , VEGF, TNF-a, RANTES, Leptin, TNF-β, TFG-β and NFG were evaluated. As shown, pro-inflammatory cytokines (IL-1 ?, TNF-a, IL-6 and GM-CSF), chemokines (IL-8, MIP-1a, RANTES and Eotaxin), 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 were reduced in the test fluid compared to that of control. On the other hand, anti-inflammatory cytokines (eg, IL1R-a, TIMPs) tested were increased in the test fluid compared to the control fluid.

In addition, the Applicants made use of a state-of-the-art model system, involving sensitization with ovalbumin, to assess allergic hypersensitivity reactions. The studied outcomes were specific cytological and cellular components of the reaction, as well as serological measurements of protein and LDH. Cytokine analysis was performed, including analysis of Eotaxin, 1L-1A, IL-1b, KC, MCP-1, MCP-3, MIP-1A, RANTES and VCAM.

Briefly, male brown Norwegian rats were injected intraperitoneally with 0.5 mL of Grade V Ovalbumin (OVA) (A5503-1G, Sigma) in solution (2.0 mg / mL) containing aluminum hydroxide (Al (OH ) ₃ ) (200 mg / mL), once each on Days 1,2 and 3. The study adopted a 2x2 randomized factorial arrangement of treatments (4 groups). After a two-week waiting period to allow an immune reaction to occur, the rats were exposed or treated for one week with RDC1676-00 (sterile saline processed through the proprietary Revalesio device) and RDC1676-01 (sterile saline processed through the proprietary Revalesio device with extra oxygen added); the end of ^the first week of treatment once a day, the 2 groups were broken in half and 50% of mice in each group were subjected to challenge with saline or OVA inhalation.

Specifically, fourteen days after the initial serialization, 12 rats were exposed to RDC 1676-00 by inhalation for 30 minutes, every day for 7 consecutive days. The air flow through the system was set at 10 liters / minute. In total, 12 rats were aligned in the sectorial chamber, with a single door for the entrance of nebulized material and uniformly distributed to the 12 sub-chambers of Aeroneb.

Fifteen days after the initial sensitization, 12 rats were exposed to RDC 167601 by ultrasonic nebulization for 30 minutes every day for 7 consecutive days. The air flow was also set to 10 liters / minute, using the same nebulizer and chamber. RDC 1676-00 was first fogged, and the Aeroneb chamber was carefully dried before RDC 1676-01 was fogged.

Approximately 2 hours after the last nebulization treatment, 6 rats from the RDC 1676-00 group were again challenged with OVA (1% in saline), released by intratracheal instillation using Penn Century Microsprayer (Model 1A-1B). The other 6 rats in the RDC 1676-00 group were challenged with saline as the control group, released by intratracheal instillation. The following day, the procedure was repeated with the group of 1676-01.

Twenty-four hours after the new challenge, all rats in each group were sacrificed by overdosing with sodium pentobarbital. 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 pulmonary organs were processed to obtain bronchoalveolar lavage fluid (BAL) and lung tissue for RT-PCR to assess changes in cytokine expression markers known to be associated with pulmonary inflammation in this model. A one-sided lavage technique was employed to preserve the integrity of the „4 lobes on the right side of the lung. The large left lobe was washed, while the 4 right wolves were tied and removed and immediately placed in TRI-zol ™ ·, - homogenized and sent to the laboratory for further processing.

BAL analysis The lung lavage was collected and centrifuged for 10 minutes at 4 ° C and 600 - 800 g to obtain cell granules. The supernatants were transferred to new tubes and frozen at -80 ° C. The bronchial lavage fluid (“BAL”) was separated into two aliquots. The first aliquot was precipitated by rotation, and the supernatant was immediately frozen on crushed dry ice, placed at - 80 ° C and sent to the laboratory for further processing. The amount of protein and LDH present indicates the level of serum blood protein (the protein is a serum component that leaks through the membranes when it is caused as in this experiment) and cell death, respectively. The tested side revealed slightly less protein than the control.

The second aliquot of the bronchial lavage fluid was evaluated for total protein and LDH content, as well as submitted to cytological examination. The treated group revealed the presence of a greater number of total cells compared to the control group with saline solution. In addition, they show an increase in eosinophils in the treated group, compared to the control group. Polymorphonuclear cells were also slightly different for the treated side compared to the control side.

Blood test. Whole blood was analyzed by transferring 1.2 - 2.0 mL of blood to a tube and allowing it to clot for at least 30 minutes. The rest of the blood sample (approximately 3.5 - 5.0 mL) was set aside for RNA extraction using Tri-zol ™ or PAXgene ™. Then, the sample with coagulated blood was centrifuged for 10 minutes at 1200 g at room temperature. The serum (supernatant) was removed and placed in two new tubes, and 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 pL of acetic acid 5 N 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. They were then placed in a lateral position and stored in the freezer at -20 ° C for 24 hours and then transferred to -80 ° C for prolonged storage.

Luminex analysis. With the Luminex platform, a microsphere analysis was used as a substrate for binding reaction to related antibody, which is read in units of luminosity and can be compared with quantified standards. Each blood sample was tested on 2 samples simultaneously. The units of measurement are luminosity units, and the groups are divided into controls challenged with OVA, treatment challenged with Ova and challenged with saline solution with proprietary fluid. '

To generate data from the Agilant gene array, lung tissue was isolated and submerged in TRI Reagent (TR118, Molecular Research Center, Inc.). Briefly, around 1 mL of TRI Reagent was added to 50 - 100 mg of tissue in each tube. The samples were homogenized in TRI Reagent, using Teflon ™ or Polytron ™ glass homogenizer. The samples were stored at 80 ° C.

Results of blood samples. Each blood sample was divided into 2 samples that were tested simultaneously. The units of measurement are units of luminosity, and the groups from left to right are: controls challenged with OVA, treatment with Revalesio challenged with OVA; followed by saline treatment challenged with saline solution, both groups of RDC1676-01 are highlighted with gray shaded background screens, while in the control saline treatment groups, the background screens are not shaded.

Generally, when comparing the two groups on the left, although the distribution of data from the RDC1676-01 group is slightly higher, specific cytokine levels in the RDC1676-01 group, as a whole, are lower than that of samples in the group control treaty. In general, when comparing the two groups on the right, the RDC1676-01 group has a slightly higher numerical value than the RDC167600 group.

The Applicants determined that the level of RANTES (IL-8 superfamily), produced after treatment with the electrokinetically altered fluids of the invention, was lower than that produced by groups exposed to saline only. The Applicants determined that the electrokinetically altered fluids of the invention caused MCP-1 to be produced at lower levels when compared to those produced by groups exposed only to saline. The 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 groups exposed to saline only.

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 groups exposed to saline only. The Applicants demonstrated that the electrokinetically altered fluids of the invention caused IL-1 to be produced at lower levels when compared to those produced by groups exposed only to saline. The Applicants noted that the level of Vcam produced after treatment with the electrokinetically altered fluids of the invention was lower than that produced by groups exposed to saline only. The Applicants demonstrated that the electrokinetically altered fluids of the invention caused Eotaxin and MCP-3 to be produced at lower levels when compared to those produced by groups exposed to saline only.

In short, this standard test of inflammatory reaction to a known sensitization produced, at least in blood samples, a marked clinical and serological effect. In addition, while significant numbers of control animals became physiologically stressed and nearly died in the process, no group treated with RDC176-01 exhibited such effects of clinical stress. This was then reflected in the circulating levels of cytokines, with approximately 30% differences between the group treated with RDC1767-01 and the groups treated with RDC1767-01 in the groups challenged with OVA. On the other hand, there were small and almost insignificant changes in cell and serological profiles of cytokines between the group treated with RDC1767-01 and the groups treated with RDC1767-01 in the groups not challenged with OVA, which possibly represents only minimal baseline changes in their own fluid.

EXAMPLE 3 (Effects of the electrokinetically altered fluids of the invention to modulate T cell proliferation were determined)

Overview. The electrokinetically altered fluids of the invention improved the regulatory function of T cells. The results described in this Example are also described in Applicants' WO 2009/055729.

The ability of specific modalities described in this report to regulate T cells has been studied by irradiating antigen presenting cells, and introducing antigen and T cells. Typically, these stimulated T cells proliferate. However, when regulatory T cells are introduced, the usual proliferation of T cells is suppressed.

Methods. Briefly, anti-CD25 antibody (ACT-1) conjugated to FITC, used in selection, was purchased from DakoCytomation (Chicago, IL). The other antibodies used were as follows: antibodies against CD3 (HIT3a for soluble conditions), GITR (conjugated with PE), CD4 (conjugated with Cy-5 and FITC), CD25 (conjugated with APC), CD28 (clone CD28.2) , CD127-APC, Granzyme A (conjugated to PE), FoxP3 (BioLegend), mouse IgG1 (isotype control) and XCI1. All antibodies were used according to the manufacturer's instructions.

CD4 + T cells were isolated from peripheral whole blood with the CD4 + Rosette Kit (Stemcell Technologies). CD4 + T cells were incubated with anti-CD127-APC, anti-CD25-PE and anti-CD4-FITC antibodies. The cells were classified by cytofluxome47 tria, using FACS Aria on CD4 + CD25hiCD127lo / nTreg and CD4 + CD25- responding T cells.

Suppression tests were performed on microtiter plates with 96 round bottom wells. 3.75 x 103 T cells responding to CD4 + CD25neg, 3.75 x 103 T cells reg. Autologous, 3.75 x 104 CD3-depleted allogeneic irradiated PBMC were added as indicated. All wells were supplemented with anti-CD3 (HIT3a clone at 5 µg / 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. The plates were harvested using a Tomtec cell harvester, and the incorporation of ³ H-thymidine determined with a Perkin Elmer scintillation counter. Antigen presenting cells (APC) consisted of peripheral blood mononuclear cells (PBMC) whose T cells had been depleted with human CD3 + T cell StemSep (StemCell Technologies), followed by 40 Gy of irradiation.

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

Statistical analysis was performed with the GraphPad Prism software. Comparisons between two groups were made using Student's unpaired bilateral T test. Comparisons between three groups were made using ANOVA one-way analysis of variance. P-values less than 0.05 were considered significant (bilateral). The correlation between two groups was determined to be statistically significant by the Spearman coefficient if the r-value was greater than 0.7 or less than -0.7 (bilateral).

Results. The proliferation of regulatory T cells was studied by stimulating cells with cross-linked material resulting from the burning of diesel (PM, from EPA). Applicants determined that cells stimulated with PM (without Rev, without Soles) resulting in a decrease in secreted IL-10, while cells exposed to PM in the presence of the fluids of the present invention (PM + Rev) resulted in maintained or slightly decreased production of IL-10 in relation to Saline and Media controls (without PM). In addition, diphtheria toxin (DT390, truncated diphtheria toxin molecule; 1:50 dilution of standard commercial concentration) was titrated on fluid samples of the invention and blocked the REV-mediated effect of IL-10 increase. Note that treatment with Rev alone resulted in higher levels of IL-10 compared to Saline and Media controls. Similar results were obtained with GITR, Granzima A, XCL1, pStatõ and Foxp3, respectively.

Applicants also obtained data on AA PBMC, obtained from an allergic asthma (AA) profile of peripheral blood mononuclear cells (PBMC) assessing tryptase. The PBMC data of AA was compatible with the above data on regulatory T cells, since cells stimulated with particulate matter (PM) exhibited high levels of tryptase, while cells treated with PM in the presence of fluids of the present invention (PM + Rev 'j resulted in significantly lower levels of tryptase, similar to those in the Saline and Media controls. According to data from regulatory T cells, exposure to DT390 blocked the Rev-mediated effect on tryptase levels, resulting in a high level of tryptase in the cells, as seen for only PM (minus Rev, without Rev, without Soles). Treatment with Rev only resulted in lower levels of tryptase compared to Saline and Media controls.

In short, the Applicants observed less proliferation in the presence of PM and Rev, compared to PM in control fluid (without Rev, without Soles), indicating that the electrokinetically generated fluid of the invention Rev, improved the function of regulatory T cells, as shown by relatively decreased proliferation in the assay. In addition, the evidence indicates that beta blocking, GPCR blocking and calcium channel blocking affect Rev activity on regulatory T function.

EXAMPLE 4 (Synergistic effects between the electrokinetically altered fluids of the invention and Budesonide were determined)

Overview. The electrokinetically altered fluids of the invention enabled the existence of synergistic anti-inflammatory effects with Budesonide In vivo in an animal model recognized in the state of the art of allergic asthma. The results described in this Example are also described in WO 2009/055729.

The Applicants initially performed experiments to evaluate the anti-inflammatory properties in the airways of the electrokinetically generated fluids of the invention (for example, RDC1676-03) in a sensitization model with brown rat ovaibumin from Norway. The brown rat from Norway is a model recognized in the state of the art to determine the effects of a test material on airway function, and this strain has been widely used, for example, as a model of allergic asthma. Airway pathology and biochemical changes induced by sensitization with ovaibumin in this model are similar to 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 inhaled route was selected to maximize lung exposure to the test material or the control solution. Animals sensitized with ovaibumin were treated with budesonide alone or combined with the test material RDC 1676-03 for 7 days, before the challenge with ovaibumin, 6 and 24 hours after the challenge, and whole blood count and levels of various pro cytokines and anti-inflammatory, as well as various respiratory parameters were measured to estimate any beneficial effect of administering the test material on various inflammatory parameters.

Materials and methods:

Brown Norwegian rats of the Bn / Crl strain were obtained from Charles River

Kingston, weighing approximately ^ pte 257 ± 5Q g at the beginning of the experiment. All animal studies were conducted with the approval of the PCS-MTL Institutional Animal Care and Use Committee. During the study, use and care of animals were conducted according to the guidelines of the National Research Council of the United States, as well as 10 as well as of the Council of Animal Care of Canada.

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

Treatment. Fifteen days after the initial sensitization, the animals were subjected to nebulized exposure to control solutions (normal saline) and test solutions (electrokinetically generated fluids, RDC1676-00, RDC1676-02 and RDC-1676-03), administered alone or combined with Budesonide, once a day for 15 minutes for 7 consecutive days. The animals received doses in a full body chamber of approximately 20 liters, and a test atmosphere was generated at the air inlet of the chamber, using aerobic ultrasonic nebulizers, supplied with air by a Buxco constant flow pump. The air flow was set at 10 liters / min.

Challenge with ovalbumin. On Day 21, 2 hours after treatment with test solutions, all animals were challenged with nebulized 1% ovalbumin solution for 15 25 minutes (in a full-body chamber at 2 L / min airflow).

Sample collection. At 6 and 24 hours after the ovalbumin challenge, blood samples were collected for total and differential blood cell counts, as well as to measure levels of various pro and anti-inflammatory cytokines. In addition, immediately after and after 6 and 24 hours of the ovalbumin challenge, the 30 Penh increased pause and tidal volume were measured over a period of 10 minutes using the Xux system from Buxco Electronics System.

Results:

Eosinophil count: As predicted, treatment with Budesonide (NS + Budesonide 750 pg / Kg ”; densely crossed bar graph) reduced the total eosinophilic count in challenged animals compared to treatment with normal NS control saline only . In addition, although treatment with the fluid of the invention “RDC1676-03” has not only significantly reduced the eosinophil count, it nevertheless exhibited considerable synergy with Budesonide in reducing the eosinophil count (“RDC1673-03 + Budesonide 750 pg / Kg'j. Similarly, the% eosinophils also reflected the same trend.While RDC1676-03 or Budesonide, alone, did not have a significant effect on the% eosinophils count in challenged animals, the two in combination reduced the% of eosinophils significantly.

'' Therefore, Claimants-determined, according to special modalities, that the electrokinetically generated fluids of the invention (for example, RDC1676-03) find considerable synergistic usefulness in combination with Budesonide to significantly reduce the count of eosinophils (“% eosinophils ”And total count) in a rat mode10 Io recognized in the state of the art of allergic asthma.

Respiratory parameters:

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

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

Treatment with Budesonide (in doses of 500 and 750 pg / kg), alone or in combination with any of the fluids under test, was unable to significantly affect Penh values immediately after the challenge. However, 6 hours after the challenge, animals treated with RDC1676-03, alone or combined with Budesonide 500 and 750 pg / kg demonstrated a significant drop in Penh values. Although the extent of this drop had decreased within 24 hours after the challenge, the tendency for a synergistic effect 25 of Budesonide and the RDC fluid was still observed at this time.

Tidal volume is the volume of air drawn into the lungs during inhalation from the final expiratory point, which passively leaves the lungs during exhalation in the course of quiet breathing. Animals treated with Budesonide alone did not show changes in tidal volumes immediately after the challenge. However, 30 RDC1676-03 alone had a significant stimulating effect on tidal volume, even at this early stage. And again, RDC1673-03 in combination with Budesonide (in doses of 500 and 750 pg / kg) had an even more pronounced effect on tidal volume measurements at this point. Six hours after the challenge, RDC1676-03 alone was sufficient to cause a significant increase in tidal volume and the addition of 35 Budesonide to the treatment regimen, either alone or in combination, had no additive effect on tidal volume. Any effect observed in these earlier moments was, however, lost at the time of 24 hours.

Claims (15)

1. Use of an electrokinetically altered aqueous fluid CHARACTERIZED because it is for the production of a medicament for the treatment of a disease or condition mediated by TSLP or mediated by TSLPR, where the medicament comprises a therapeutically effective amount of an electrokinetically altered aqueous fluid, comprising an aqueous ionic solution of charge-stabilized oxygen-containing nanostructures having substantially an average diameter of less than approximately 10 nanometers and stably configured in the aqueous ionic fluid for use in the production of the drug to treat a TSLP-mediated disease or condition mediated by the immune system . 2. Use, according to claim 1, CHARACTERIZED by the fact that the charge-stabilized oxygen-containing nanostructures are stably configured in the aqueous ionic fluid in an amount sufficient to allow, when in contact with a living cell by the fluid, modulation of at least one between cell membrane potential and cell membrane conductivity. 3. Use, according to claim 1, CHARACTERIZED by the fact that charge-stabilized oxygen-containing nanostructures are the main type of charge-stabilized gas-containing nanostructures in the fluid, preferably when the percentage of dissolved oxygen molecules present in the. fluid in the form of charge-stabilized oxygen-containing nanostructures is greater than 0.01%, preferably when total dissolved oxygen is substantially present in charge-stabilized oxygen-containing nanostructures. 4. Use, according to claim 1, CHARACTERIZED by the fact that the alteration of the electrokinetically altered aqueous fluid comprises exposure of the fluid to hydrodynamically induced, localized electrokinetic effects, preferably when exposure to the localized electrokinetic effects comprises exposure to at least one of pulses of voltage and current pulses, or when exposure of the fluid to hydrodynamically induced effects, localized electrokinetics comprise exposure of the fluid to structural characteristics inducing the electrokinetic effects of a device used to generate the fluid. 5. Use, according to claim 1, CHARACTERIZED by the fact that the aqueous ionic solution comprises a saline solution. 6. Use, according to claim 1, CHARACTERIZED by the fact that the fluid is superoxygenated. 7. Use according to any one of claims 1 to 6, CHARACTERIZED by the fact that the disease or immune system disorder comprises allergic inflammation, preferably when the allergic inflammation comprises at least one among allergic inflammation of the airways, inflammatory responses of the type Th2 mediated by CD, atopic dermatitis, atopic eczema, asthma, obstructive airway disease, chronic obstructive pulmonary disorder, IgE-mediated disorders, rhinoconjunctivitis and food allergies, preferably when the TSLP-mediated or TSLPR-mediated disease or disorder comprises inflammatory arthritis, preferably when such inflammatory arthritis comprises at least one rheumatoid arthritis and psoriasis. 8. Use according to any one of claims 1 to 6, CHARACTERIZED by further comprising a combined therapy, in which at least one additional therapeutic agent is administered to the patient, preferably where at least one additional therapeutic agent is selected from the group consisting of β ₂ -agonists, short - acting agonists β ₂ long - acting anticholinergics, corticosteroids, systemic corticosteroids, mast cell stabilizers, leukotriene modifiers, methylxanthines, β ₂ agonists, including albuterol, levalbuterol, pirbuterol, artformoterol, formoterol, salmeterol , and anticholinergics such as ipatropium and tiotropium; corticosteroids including beclomethasone, budesonide, flunisolide, fluticasone, mometasone, triamcinolone, methylprednisolone, prednisolone, prednisone; leukotriene modifiers including montelukast, zafirlukast and zileuton; mast cell stabilizers including chromoline and nedocromil; methylxanthines including theophylline, combined drugs including ipatropium and albuterol, fluticasone and salmeterol, budesonide and formoterol; antihistamines including hydroxyzine, diphenhydramine, loratadine, cetirizine and hydrocortisone; immune-modulating drugs including tacrolimus and pimecrolimus; cyclosporine; azothioprine; mycophenolate mofetil; and combinations thereof, or where at least one additional therapeutic agent is a TSLP and / or TSLPR antagonist, wherein such TSLP and / or TSLPR antagonist is selected from the group consisting of neutralizing antibodies specific against TSLP and the receptor of TSLP, soluble TSLP receptor molecules and TSLP fusion proteins, including TSLPR immunoglobulin Fc molecules or polypeptides that encode components of more than one receptor chain. 9. Use, according to claim 2, CHARACTERIZED by the fact that the modulation of at least one among cell membrane potential and cell membrane conductivity comprises altering at least one among cell membrane structure or function, comprising altering at least one among conformation , ligand-binding activity, and catalytic activity of a membrane-associated protein, preferably when the membrane-associated protein comprises at least one selected from the group consisting of receptors, transmembrane receptors, ion channel proteins, fixation proteins intracellular, cell adhesion proteins, integrins, preferably when the transmembrane receptor comprises Protein G Aco3 Receptor (GPCR), .. preferably when the Protein G Coupled Receptor (GPCR) interacts with an α subunit of the G protein, preferably when the α subunit of the G protein comprises at least one selected from the group consisting of Ga _s , Ga ,, Ga _q and Ga ₁₂ , “F preferably when at least one α subunit of the G protein is Ga _q , or where the modulation of at least one of cell membrane potential and cell membrane conductivity comprises modulating whole cell conductance, preferably when conductance modulation whole cell comprises modulating at least one of the voltage-dependent linear or non-linear contribution of the entire cell conductance, or when modulating at least one of cell membrane potential and cell membrane conductivity comprises modulation of a pathway or messaging system calcium dependent cells, or modulation of phospholipase C activity, or modulation of the activity of adenylate cyclase (AC), or 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 airway inflation, inflammatory responses of the type Th2 mediated by CD, atopic dermatitis, atopic eczema, asthma, obstructive airway disease, chronic obstructive pulmonary disease, IgE-mediated disorders, rhinoconjunctivitis, food allergies, inflammatory arthritis, rheumatoid arthritis and psoriasis. Use according to any one of claims 1 to 6, CHARACTERIZED by comprising the administration of the electrokinetic fluid to a network or cell layer and by further comprising modulation of an intercellular junction thereon, preferably when the intracellular junction comprises at least one selected from the group consisting of occlusive junctions, connecting junctions, adherent zone and desmosomes. 11. Use, according to claim 10, CHARACTERIZED by the fact that the cell network or layers comprise at least one selected from the group consisting of pulmonary epithelium, bronchial epithelium and intestinal epithelium. 12. Use, according to any one of claims 1 to 6, CHARACTERIZED by the fact that the electrokinetically altered aqueous fluid is oxygenated and the oxygen in the fluid is present in an amount of at least 8 ppm at atmospheric pressure, or the electrokinetically aqueous fluid altered comprise at least one among solvated electrons and electrokinetically modified or charged oxygen species, preferably when the solvated electrons or electrokinetically modified or charged oxygen species are present in an amount of at least 0.01 ppm, and / or where the aqueous fluid electrokinetically altered comprises a form of 5 solvated electrons stabilized by molecular oxygen. 13. Use according to claim 2, CHARACTERIZED by the fact that the ability to modulate at least one of the cell membrane potential and 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 for longer periods in 10 gas-tight closed container. 14. Use, according to any one of claims 1 to 6, CHARACTERIZED by the fact that the amount of oxygen present in nanostructures containing oxygen stabilized by charge of the electrokinetically altered fluid is at least 8 ppm at atmospheric pressure. 15. Use, according to any one of claims 1 to 6, CHARACTERIZED by the fact that the treatment of the disease or condition mediated by TSLP or mediated by TSLPR of the immune system comprises administration via, at least one among, topical, inhalation, intranasal and intravenous.