The present invention provides methods and pharmaceutical compositions of the human glycoprotein fetuin, or .alpha.2-HS glycoprotein, or fragments thereof to mitigate tissue damage associated with ischemia, particularly in stroke or in myocardial infarction.

SUMMARY OF THE INVENTION

"Fetuin" as used herein refers, in the context of the human protein, to the glycoprotein referred to variously as ".alpha.2-HS-glycoprotein" or ".alpha.2-Z-globulin" or "human fetuin" or "human fetuin glycoprotein," and in broader context to any of the fetuin family of proteins, with members occurring in various species and closely related in sequence to bovine fetuin and human .alpha.2-HS. Two common alleles are known for .alpha.2-HS: one has threonine at position 248 and 256, the other has methionine at 248 and serine at 256. Use of "fetuin" or ".alpha.2-HS" according to the teachings herein shall correspondingly include use of allelic variants, glycosylation, sulfation and phosphorylation variants, reduced and native forms, precursor and proteolytically processed forms, and sequence variants substantially homologous to the polypeptides described by SEQ ID NO.'s 1 6 or to the fetuins of other (non-human) species, and fragments of any of the above. Such variants, whether naturally occurring, intentionally introduced or spontaneously arising, are conveniently tested for activity (and thereby evaluated for clinical utility) in accordance with the Detailed Description and Examples described herein.

The present invention arose out of a series of experiments wherein brain damage (cell death) subsequent to induced focal ischemia was found to be ameliorated by treatment with the glycoprotein .alpha.2-HS. The present invention identifies for the first time the cellular and tissue protective effects of administering .alpha.2-HS in the setting of ischemia. The present invention further provides methods and pharmaceutical compositions for preventing tissue damage in ischemia, particularly brain damage attendant to stroke or cerebral ischemia, comprising administering an effective amount of an .alpha.2-HS glycoprotein. Preferably, the .alpha.2-HS glycoprotein is a human .alpha.2-HS glycoprotein comprising a primary sequence according to SEQ ID NO. 1 through SEQ ID NO. 5 or a shorter fragment thereof. Highly homologous sequence variants are also useful in this regard, particularly such homologous glycoproteins as have effects quantitatively indistinguishable from .alpha.2-HS in the assays described herein. Such variant glycoproteins are conveniently produced according to techniques of molecular biology well-known in the art and are readily compared to human .alpha.2-HS glycoprotein in the assays described herein, or in comparable assays for cellular or tissue protection in the setting of ischemia.

The utility of the methods and compositions involving .alpha.2-HS glycoproteins as taught herein are directly extended to other instances of tissue ischemia, particularly heart attack or myocardial infarction. Stroke (ischemia and associated tissue damage) is well-known to arise from a variety of distal causes, giving rise to such clinical characterizations as: stroke, cerebral infarction, cerebrovascular accident, thrombotic stroke, embolic stroke, occlusive cerebrovascular lesion, apoplexy (of various types), apoplectic stroke, paralytic stroke, intracranial hemorrhage, hemorrhagic stroke, ruptured aneurysm, post-traumatic stroke, transient ischemic attack, and stroke syndrome. Likewise, heart attack may arise variously, giving rise to such representative clinical characterizations as: cardiac infarction, myocardial infarction (various types), coronary artery occlusion, coronary thrombosis, coronary embolism, periarteritis nodosa, and obliterating endarteritis. Similarly, other organs and tissues may become afflicted by ischemia involving, among other conditions, anemic, pale, white or bland infarction, various embolic disorders including embolic infarction, various thrombotic disorders including thrombotic infarction, hemorrhagic or red infarction, and coagulation necrosis. Any of these conditions of ischemia, and their clinical relations, is amenable to treatment according to the methods and with the pharmaceutical compositions taught herein.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the discovery that a human plasma glycoprotein, .alpha.2-HS, is beneficial in preventing tissue damage associated with ischemia, and specifically in treating stroke. Although fetuin was discovered more than 50 years ago as a component of fetal bovine serum, and subsequently found to share high homology with a human .alpha.2-HS counterpart (.alpha.2-HS-glycoprotein), no role for .alpha.2-HS in the natural history, etiology or treatment of stroke (cerebral infarction, cerebral ischemia, brain attack) or other tissue ischemia had been suspected. After recognizing the activity of .alpha.2-HS to potentiate the anti-inflammatory activity of certain low molecular weight compounds and metabolites, we tested for the occurrence and activity of .alpha.2-HS in a predictive animal model of human stroke, i.e., in experimentally induced focal cerebral ischemia in rats. We discovered that .alpha.2-HS occurs in increasing amounts in the areas of brain damage following permanent focal cerebral ischemia, and that administration of .alpha.2-HS alone, even after the onset of ischemia, dose-dependently decreased the volume of total brain damage in stroke. Asialofetuin (.alpha.2-HS glycoprotein treated to remove sialic acid residues) was ineffective or exacerbative in this regard.

The present invention provides novel methods (.alpha.2-HS administration as adjunctive or monotherapy) and pharmaceutical compositions (comprising .alpha.2-HS or glycoprotein sequence variants) to treat and mitigate against tissue damage in ischemia, particularly in stroke and heart attack. Effective doses of the therapy are determined by routine procedures in the art with reference to the findings described herein, and may be formulated in suitable pharmacological carriers for administration by any appropriate means including, but not limited to, injection (such as, intravenous, intramuscular, intrathecal, and intracranial) and other means available within the pharmaceutical arts. Treatment may be accomplished by administration of the .alpha.2-HS glycoprotein alone or in a pharmaceutical composition where it is mixed with suitable carriers or excipients to treat tissue ischemia or mitigate against ischemic tissue damage. Preferably, administration of .alpha.2-HS is systemic to provide organ sites of treatment including the brain, CNS, myocardium, or other organ site experiencing ischemia. A therapeutically effective dose refers to that amount of the active agent sufficient to treat tissue ischemia or to mitigate against ischemic tissue damage. Therapeutically effective doses may be administered alone or as adjunctive therapy in combination with other treatments or supportive measures for tissue ischemia, particularly for stroke or for heart attack. In particular, .alpha.2-HS may be co-administered with spermine or otherwise in accordance with the teachings of U.S. Ser. No. 08/780,311 (filed), the disclosure of which is incorporated herein by reference in its entirety. Techniques for the formulation and administration of the compounds of the instant application may be found in "Remington's Pharmaceutical Sciences," Mack Publishing Company, Easton, Pa., latest edition.
 

Claim 1 of 6 Claims

1. A method of treating a subject having tissue ischemia, the method comprising administering human .alpha.2-HS glycoprotein to the subject, wherein the human .alpha.2-HS glycoprotein comprises the amino acid sequence of a) SEQ ID. NO:1, or b) both SEQ ID NO: 3 and SEQ ID NO: 4.
 

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