The General Hospital Corporation

Bion Inc.

Garner T Haupert, Jr

Rex T Gallagher

Michael N Margolies

Behnaz Parhami-Seren

The present invention relates to rapid, quantitative, specific, high through-put methods for screening test substances for their ability to inhibit activity of an ouabain-resistant Na&#x2014;K-ATPase involved in a variety of biological processes such as regulation of osmotic balance and cell volume, maintenance of the resting membrane potential, establishment of the ionic composition of cerebrospinal fluid and aqueous humor, electrical activity of muscle and nerve, and receptor-mediated endocytosis, cardiac muscle contractility, neurotransmitter metabolism and vascular muscle cell contraction. These methods can be employed to identify compounds for use in therapeutic applications for disease processes in which dysfunction of the Na&#x2014;K-ATPase contributes to a pathological process. The present invention also includes kits which are used in the methods provided herein. The present invention further includes methods of treating or preventing diseases or disorders which are associated with dysfunction of the Na&#x2014;K&#x2014;ATPase.

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The invention relates to a monoclonal antibody or antigen binding fragment thereof having binding specificity for ouabain, wherein the antibody or antigen binding fragment does not crossreact with digoxin. Preferably the anti-ouabain monoclonal antibody can bind ouabain with an affinity of at least about 10M, preferably 10M, and more preferably 10M. The invention also relates to diagnostic and therapeutic uses of the monoclonal antibodies described herein.

Ouabain-specific monoclonal antibodies

The present invention relates to a method of isolating and/or purifying hypothalamic inhibitory factor (HIF) from a sample (e.g., tissue fluid) containing HIF. The present invention provides for isolation of large amounts of HIF using diafiltration, solid phase extraction and immunoaffinity techniques. In one embodiment, the invention relates to a method of purifying hypothalamic inhibitory factor from a sample containing hypothalamic inhibitory factor comprising subjecting the sample to diafiltration, solid phase extraction and immunoaffintiy chromatography. In a particular embodiment, the invention relates to subjecting the sample to diafiltration to produce a diafiltrate of HIF; subjecting the diafiltrate to a first solid phase extraction (SPE) to produce a first fraction of HIF; subjecting the first fraction to immunoaffinity chromatography, wherein an antibody which binds to HIF is coupled to an immunoaffinity column, to produce a second fraction of HIF; subjecting the second fraction of HIF to reverse phase HPLC chromatography to produce a third fraction of HIF; and recovering purified HIF from the third fraction.

Process for large-scale isolation and purification of hypothalamic inhibitory factor

The present invention relates to rapid, quantitative, specific, high through-put methods for screening test substances for their ability to inhibit activity of an ouabain-resistant Na-K-ATPase involved in a variety of biological processes such as regulation of osmotic balance and cell volume, maintenance of the resting membrane potential, establishment of the ionic composition of cerebrospinal fluid and aqueous humor, electrical activity of muscle and nerve, and receptor-mediated endocytosis, cardiac muscle contractility, neurotransmitter metabolism and vascular muscle cell contraction. These methods can be employed to identify compounds for use in therapeutic applications for disease processes in which dysfunction of the Na-K-ATPase contributes to a pathological process. The present invention also includes kits which are used in the methods provided herein. The present invention further includes methods of treating or preventing diseases or disorders which are associated with dysfunction of the Na-K-ATPase.

Methods for screening HIF like ouabain-resistant Na+-K+-ATPase agents

A method of treating cardiac malfunction by administering a positive inotropic effect-producing amount of Hypothalamic Inhibitory factor (HIF).

Method for treating cardiac malfunction

The present invention is based on the unexpected discoveries that hypothalamic inhibitory factor (HIF) has a positive inotropic effect in a whole organ preparation, such as an isolated perfused heart, and that HIF, to a greater extent than ouabain, dilates the coronary arteries in the isolated perfused heart as manifested in increased coronary flow in hearts treated with HIP. The present invention relates to a method for increasing coronary perfusion in a mammalian host comprising administering to the host in need thereof an effective amount of hypothalamic inhibitory factor (HIF). The invention also relates to a method for producing an increased coronary vasodilatory effect in a mammalian host comprising administering to a mammalian host in need thereof an effective amount of HIF. The method of the present invention can be used to prevent and/or treat ischemic cardiac malfunction and coronary artery restenosis.

Treatment of ischemic cardiac malfunction

The present invention is based on the unexpected discoveries that hypothalamic inhibitory factor (HIF) has a positive inotropic effect in a whole organ preparation, such as an isolated perfused heart, and that HIF, to a greater extent than ouabain, dilates the coronary arteries in the isolated perfused heart as manifested in increased coronary flow in hearts treated with HIF. The present invention relates to a method for increasing coronary perfusion in a mammalian host comprising administering to the host in need thereof an effective amount of hypothalamic inhibitory factor (HIF). The invention also relates to a method for producing an increased coronary vasodilatory effect in a mammalian host comprising administering to a mammalian host in need thereof an effective amount of HIF. The method of the present invention can be used to prevent and/or treat ischemic cardiac malfunction and coronary artery restenosis.

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Littleton, MA, United States of America

Garner T Haupert, Jr