Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Carbohydrate doai
Reexamination Certificate
2000-02-11
2004-12-28
Winkler, Ulrike (Department: 1648)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Carbohydrate doai
C424S537000, C424S563000, C424S570000, C435S072000, C514S021800, C514S022000, C514S023000, C536S001110, C536S005000
Reexamination Certificate
active
06835715
ABSTRACT:
BACKGROUND OF THF INVENTION
1. Field of the Invention
This invention relates to the field of mammalian ouabain-like factors (OLF) and more particularly to a novel dihydroouabain like factor (Dh-OLF), and its use for the detection and treatment of conditions and diseases associated with regulation of the sodium-potassium pump, in particular for the detection and treatment of congestive heart failure. This invention also relates to those plant cardenolides that are cardiac glycosides. This invention also relates to antibodies and antibody fragments useful for the treatment of toxicity due to excess OLF or Dh-OLF.
2. Description of the Background
Digoxin and ouabain, plant cardenolides that are the two most commonly used cardiac gylcosides, are commonly administered to patients suffering from congestive heart failure because of their beneficial effect on cardiac contractility, that is, their positive inotropic effect. Positive inotropic effect generally refers to the enhancement of contractility of the cardiac cells in a dose-dependent manner. These drugs produce an increase in the force and velocity of ventricular contraction, and also a slowing of the heart rate. These two effects combine to provide a stronger heart beat. However, cardiac glycosides have narrow therapeutic indices and their use is frequently accompanied by toxic effects that can be severe or lethal.
The exact mode of action of cardiac glycosides is not completely known, but it is thought that the effects are mediated through regulation of the sodium pump. Abnormal sodium pump activity has been postulated to be involved in the pathophysiology of several diseases, including cardiovascular, neurological, renal, and metabolic disorders, among others. These complex effects may be related to the role of the pump in controlling the cellular ingress of other molecules.
The NA
+
,K
+
-ATPase enzyme or sodium pump is a membrane protein responsible for establishing an electrochemical gradient of Na
+
and K
+
ions across the plasma membrane of mammalian cells. The ion gradient formed by this enzyme is necessary for the active transport of essential nutrients into the cells, for regulation of osmotic balance and cell volume, and for maintaining the resting membrane potential in excitable cells. The Na
+
,K
+
-ATPase enzyme is the only known receptor for cardiac glycosides such as digitalis. The tight conservation of the digitalis binding site over many phyla, among other observations, suggests the existence of endogenous sodium pump inhibitors (SPIs) in mammals as well. These hypothetical mammalian inhibitors would be involved in modulating the activity of the sodium pump, and might be involved in vivo sodium homeostasis.
One of the side effects of administration of cardiac glycosides is arterial hypertension. It is thought that an excess of endogenous factors may also cause arterial hypertension, which is a risk factor in complications associated with various organs. Higher plasma levels of ouabain-like compounds (OLCs) were found in patients with primary aldosteronism and ectopic corticotrophin syndrome, two types of mineralocorticoid hypertension. In addition, 30-45% of patients with essential hypertension had increased plasma levels of OLCs and the blood pressures were statistically correlated with the OLC levels. Although presently available anti-hypertensive agents have proven beneficial, they still fall short of completely reversing the effects caused by high blood pressure. Accordingly, there still is a need for agents which are more specific and effective than those presently available in order to reverse the toxic effects of high levels of ouabain, whether administered or endogenous. It would be of further benefit to have a means of predicting the occurrence of hypertension in patients before the occurrence of organ damage due to sustained hypertension.
The kidneys have also been implicated in changes in blood pressure variations. A rise in blood pressure has been observed in clinical forms of hypertension, such as Liddle's syndrome, glucocorticoid-suppressible aldosteronism, and the syndrome of apparent mineralocorticoid excess, which appears to be closely related to a constitutive increase in sodium (Na
+
) reabsorption in the kidney. Essential hypertension is a heterogeneous disease which is thought to result from genetic and environmental factors that interact to increase blood pressure, thus, gene mutations may also contribute to hypertension, and since these genes were shown to converge on a final common pathway, the result may be increased Na
+
reabsorption and/or decreased Na
+
excretion in the kidney. Although still uncertain, however, why increased Na
+
reabsorption might lead to high blood pressure, salt retention and plasma volume expansion trigger the secretion of sodium pump inhibitors (SPIs), which restore extracellular fluid volume via natriuresis. An increased secretion of SPIs, thus, may also elevate cytosolic Ca
2+
and produce vasoconstriction, which will account for the development and perpetuation of hypertension. Whatever the mechanism, it has been empirically noted that hypertension is associated with abnormal function of sodium regulation.
The chronic administration of ouabain has been shown to cause the development of hypertension in rats. Immunization of these Dahl salt-sensitive (S) rats against ouabain prevented a reduction in renal mass-saline hypertension and Na
+
-induced hypertension.
Plant cardenolides, such as ouabain and digoxin, have been shown to bind specifically to highly conserved epitopes on the &agr;-subunit of the Na
+
,K
+
-ATPase enzyme (sodium pump) and to stabilize the phosphorylated intermediate. This effect leads to the inhibition of pump-associated transport of sodium, potassium and other important biologic compounds across cell membranes. It has furthermore been postulated that a hormonal-axis may regulate the activity by mammalian ligands similar to plant cardenolides of the sodium pump. Two types of mammalian compounds have been found to date with properties similar to those of plant cardenolides: (1) digoxin-like factors such as DLF or DLIF, and (2) ouabain-like factors such as OLF or HIF. The DLIF family includes a series of deglycosylated species and Dh-DLIF, a dihydrodigoxin-like isomer. Cytochrome P
450
was recently shown to mediate the conversion of dihydrodigoxin, a plant cardenolide of low DLIF immunoreactivity, to one with high DLIF immunoreactivity. This suggests a possible in vivo metabolic conversion of Dh-DLIF, a less active dihydro-species, to DLIF, a more biologically active species. The present inventors have recently reported the presence of a deglycosylated species of human OLF analogous to the plant ouabain. Dihydroouabain (dho), the chemically-reduced form of ouabain having a saturated lactone ring, has been used to study the biological activity of ouabain and its interaction with the sodium pump. The chemical formulas of ouabain and dihydroouabain are shown below.
Ouabain Dihydroouabain
C
29
H
44
O
22
C
29
H
46
O
12
MW 584 MW 586
Ouabain contains an aglycone consisting of a steroid nucleus with an unsaturated lactone ring attached at the C
17
position, a sole sugar molecule, a rhamnose, attached at the C
3
position. The only difference between ouabain and dihydroouabain (dho) is that the latter has a fully hydrogenated lactone ring.
In general, the reduced lactone ring forms of both digoxin and ouabain show relatively lower potencies than their oxidized counterparts for inhibiting Na
+
,K
+
pump catalytic activity. However, they have other biophysical properties, such as being rapidly washed out from tissues, etc. that are rather advantageous.
Antibodies to specific molecular structures may be made by immunization of animals such as mice or rabbits with the target structure by methods known in the art. Non-human antibodies, including polyclonal and monoclonal antibodies, raised against specific human molecules have been used for diagnosis and th
Qazzaz Hassan M. A. M.
Valdes, Jr. Roland
Fish & Richardson P.C. P.A.
University of Louisville Research Foundation Inc.
Winkler Ulrike
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