Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
1998-01-30
2003-02-11
Brumback, Brenda (Department: 1654)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C514S601000, C514S602000, C514S604000, C530S327000, C564S001000, C564S080000, C564S084000, C564S090000, C564S091000
Reexamination Certificate
active
06518245
ABSTRACT:
BACKGROUND OF THE INVENTION
a) Field of the Invention
The present invention relates to methods for the treatment of arrhythmias by inhibition of a multifunctional calcium/calmodulin-dependent protein kinase (CaM kinase), pharmaceutical compositions useful in such treatments, and methods for identifying new agents useful for such treatments.
b) Description of Related Art
Arrhythmias are a leading cause of cardiac-related death in the United States. Prolongation of the cardiac action potential is an important predisposing condition for these arrhythmias. Many antiarrhythmic drugs directly prolong the action potential duration and so may further contribute to these arrhythmias (i.e. the proarrhythmic effects of antiarrhythmic drugs). Despite their cost, implantable cardiac defibrillators (ICDs) have become the treatment of choice for arrhythmias. In order to prevent painful shocks ~50% of patients with ICDs require additional treatment with antiarrhythmic drugs. Thus there is an important need to develop better antiarrhythmic drug therapies.
Early afterdepolarizations (EADs) are depolarizing oscillations in the action potential (AP) that occur during repolarization. One cause of EADs is inward L-type Ca
2+
current (I
Ca
). I
Ca
is present at cell membrane potentials (Vm) within the window of I
Ca
steady state activation and inactivation overlap; such as occur during action potential repolarization. Prolongation of action potential repolarization may increase the time that the Vm is in the window current range for I
Ca
and thus the likelihood of EADs. EADs are important because they are one probable cause of lethal arrhythmias associated with long QT intervals including torsade de pointes. A long QT interval reflects prolonged action potential repolarization in ventricular myocardium and is due to a wide variety of conditions including bradycardia and hypokalemia. One important cause of long QT intervals are antiaffhythmic drugs and the ventricular proarrhythmic effects of many antiarrhythmic agents are due to QT interval prolongation.
Intracellular Ca
2+
increases simultaneously with EADs in isolated ventricular myocytes (De Ferrari et al. (1995)
Circ
91:2510-2515). Elevation of intracellular Ca
2+
([Ca
2+
]
i
) has complex effects on I
Ca
including indirect enhancement through a multifunctional Ca
2+
/calmodulin-dependent protein kinase II pathway (Anderson et al. (1994)
Circ Res
75:854-861) and direct inactivation. The net effect of elevated [Ca
2+
]
i
in rabbit ventricular myocytes following flash photolysis of the photolabile Ca
2+
chelator Nitr−5 is 40-50% augmentation of peak I
Ca
that is mediated by a multifunctional Ca
2+
/calmodulin-dependent protein kinase II, hereafter referred to as CaM kinase II. CaM kinase II, like other multifunctional Ca
2+
/calmodulin-dependent protein kinases, is an ubiquitous serine-threonine kinase that is activated when Ca
2+
is bound to the Ca
2+
binding protein calmodulin. Once activated by Ca
2+
/calmodulin, CaM kinase II activation may be sustained by intersubunit enzyme autophosphorylation that confers Ca
2+
-independent activity, allowing for its activity to persist during the long diastolic intervals associated with QT interval prolongation and torsade de pointes. This Ca
2+
-independent activity is enhanced by long stimulating pulses (De Koninck and Schulman (1998)
Science
279:227-230), as occur with prolonged action potential repolarization. It is possible that EADs caused by I
Ca
may be enhanced by increased [Ca
2+
]
i
through the CaM kinase II pathway. However, not all EADs are due to I
Ca
, and EADs can occur in conditions adverse to CaM kinase activity such as enhanced [Ca
2+
]
i
buffering.
Delayed afterdepolarizations (DADs) are another cause of ventricular arrhythmias associated with intracellular calcium overload. DADs are caused by an inward current that follows completion of action potential repolarization. This inward current is a marker of intracellular calcium overload (Thandroyen et al. (1991)
Circ Res
69:810-819). Intracellular calcium overload is a central feature of many ventricular arrhythmias occurring during ischemia (Lee et al. (1988)
Circ
78:1047-1059) including ventricular fibrillation.
Inhibition of CaM kinase activity can be used to test for a facilitatory role of CaM kinase in EADs and DADs. There are several methods for blocking CaM kinase activity. Synthetic pseudo-substrate peptide inhibitors of CaM kinase provide a specific approach to CaM kinase inhibition and have been used in a variety of cell types including ventricular myocytes (Braun et al. (1995)
J. Physiol
488:37-55). The peptide sequence KKALHRQEAVDCL (SEQ ID NO:1), like other similar peptides, was found to inhibit CaM kinase in a highly specific manner. SEQ ID NO:1 is a much less efficient inhibitor of both protein kinase A (PKA) and protein kinase C (PKC), with an IC
50
value of at least 500 &mgr;mol/l for each. Myristoylated inhibitory peptides are cell membrane permeant and thus could also be effective when added extracellularly.
A variety of cell—membrane permeant organic CaM kinase and calmodulin inhibitors are available and widely used (Braun et al. (1995)
Ann Rev Physiol
57:417-445). A principle disadvantage for these inhibitors as experimental agents is that many of them directly block I
Ca
.
However, direct I
Ca
blockade by the CaM, kinase inhibitor KN-93 has not been previously reported. KN-93 (2-[N-(2-hydroxyethyl)-N-(4-methoxy-benzenesulfonyl)]-amino-N-(4-chlorocinnamyl)-N-methylbenzylamine) is a methoxybenzene sulfonamide derivative that competitively inhibits calmodulin binding to CaM kinase with a reported K
i
of 0.37 &mgr;mol/l. KN-93 has been shown to inhibit CaM kinase—dependent processes in PC12h cells, fibroblasts, and gastric parietal cells. There are four isoform groups of CaM kinase II (&agr;,&bgr;,&ggr;,&dgr;) and the &dgr;
B
and &dgr;
C
isoforms have been identified in myocardium. The catalytic and regulatory domains in CaM kinase are highly conserved in all known CaM kinase isoforms so inhibitors that interact with either of these domains are expected to work in all cell types including cardiac. KN-92 (2-N-(4-methoxybenzenesulfonyl)-amino-N-(4-chlorocinnamyl)-N-methylbenzylamine) is a congener of KN-93 without CaM kinase inhibitory activity and is used as an experimental control. Direct I
Ca
blockade by KN-92 has also not been previously reported, and neither KN-93 nor KN-92 have appreciable effects on other serine threonine kinases such as protein kinase A (PKA) or protein kinase C (PKC).
Two general approaches are currently used to suppress ventricular arrhythmias due to action potential prolongation, in addition to ICD implantation. One is to shorten the action potential using antiarrhythmic agents (e.g. mexilitine, pinacidil) or by increasing the heart rate using artificial pacing or the &bgr;-adrenergic agent isoproterenol. The second is to indirectly suppress protein kinase A (PKA), which enhances L-type calcium current and calcium release from the sarcoplasmic reticulum, through left stellate ganglionectomy or with &bgr;-adrenergic blocking drugs. Neither of these approaches is broadly applicable for two reasons: 1) action potential duration is governed by a number of different ionic currents and it is not typically known which current is critical in a given patient. Furthermore, specific antiarrhythmic drugs are not available for modification of many of these ionic currents. In addition, not all causes of action potential prolongation respond to pacing by action potential shortening. Isoproterenol also does not always shorten the action potential, can itself be arrhythmogenic, may cause ischemia, and can only be used in an acute setting. 2) At present, suppression of PKA is accomplished indirectly by stellate gangionectomy or by &bgr;-adrenergic antagonists. &bgr;-adrenergic antagonists are effective in reducing death from arrhythmias, but app
Anderson Mark E.
Braun Andrew P.
Schulman Howard
Sung Ruey J.
Brumback Brenda
Gupta Anish
The Board of Trustees of the Leland Stanford Jr. University
Townsend and Townsend / and Crew LLP
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