Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving transferase
Reexamination Certificate
1998-06-22
2001-06-19
Marschel, Ardin H. (Department: 1617)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving transferase
C435S194000
Reexamination Certificate
active
06248549
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to methods for modulating muscle contraction, and particularly, to methods for modulating smooth muscle contraction in the absence of calcium, and for increasing the calcium sensitivity of both smooth muscle and cardiac muscle.
BACKGROUND OF THE INVENTION
p21-activated kinase (PAK) proteins are serine/threonine protein kinases homologous to the yeast Ste20 kinase. Several distinct members of the PAK family have been identified in mammalian cells, including PAK1, PAK2, PAK3, and PAK65. It has been shown that PAK proteins can be activated by the small Rho-family GTP-binding proteins Cdc42 and Rac1, which are known to regulate assembly of the actin cytoskeletal structure (Zhang et al. 1995
, J. Biol. Chem
. 270:23934-36).
Smooth muscle, in contrast to striated muscle (cardiac and skeletal muscle), is capable of maintaining force at low levels of intracellular calcium. One member of the small Rho-family of GTP-binding proteins, RhoA, has been shown recently to induce smooth muscle contraction independently of calcium. RhoA activates a serinet reonine kinase named ROK (RhoA associated kinase or p160ROK) which can cause smooth muscle calcium-independent contraction. Cardiac muscle, on the other hand, requires increased levels of intracellular calcium for contraction.
Smooth muscle is found in blood vessels, the airways of the lungs, the gastro-intestial tract, the uterus and the urinary tract. The uncontrolled contraction of smooth muscle in such tissues is involved in states such as hypertension (a known risk factor for heart disease), asthma, irritable bowel syndrome, incontinence or menstrual cramps. Hypertension or high blood pressure, is the most common disease affecting the heart and blood vessels. Statistics indicate that hypertension afflicts one out of every five American adults. Asthma is a chronic disease characterized by airway hyperactivity, it occurs in 5-8% of the U.S. population, and is an extraordinarily common cause of pulmonary impairment. Irritable bowel syndrome is a common syndrome characterized by frequently alternating constipation and diarrhea, usually with abdominal pain. Often stress induced, it is also caused by such physical factors as spicy foods, lack of dietary fiber, and excessive caffeine consumption. Incontinence is the lack of voluntary control over micturition. In infants it is normal because neurons to the external sphincter muscle are not completely developed. In the adult it may occur as a result of unconsciousness, injury to the spinal nerves controlling the urinary bladder, irritation due to abnormal constituents in urine, disease of the urinary bladder and inability of the detrusor muscle to relax due to emotional stress. Menstrual cramping is a painful spasmodic contraction of the uterine muscles.
Abnormal contraction in cardiac muscle is the basis of many heart diseases. Heart failure (HF) is a common heart disease caused mainly by coronary artery disease and hypertension. Because specific treatment is not possible for most patients, current therapies are designed to ameliorate the symptoms and/or forestall myocardial damage. This includes increasing myocardial contractility by increasing cardiac myofilament sensitivity to calcium (Nielsen, J. E. et al. 1995
, J. Cardiovasc. Phormacol
. 26:S77-S84). Changes in calcium sensitivity also occur with acute disease states including myocardial stunning. Stunning is a reversible mild form of ischemic (no blood flow) damage, and may be induced by physiological insult such as heart surgery. Severe ischemic damage occurs with myocardial infarction and results in changes to cardiac muscle function.
To date, treatments of the above mentioned states in smooth and cardiac muscle have not been completely effective.
SUMMARY OF THE INVENTION
The present invention is based, at least in part, on the discoveries that PAK induces contraction of smooth muscle in the absence of calcium and changes calcium sensitivity of cardiac muscle. These discoveries led to the methods of the invention.
By one aspect, the present invention provides methods for treating states characterized by smooth muscle contraction. These states characterized by smooth muscle contraction include states characterized by inappropriate smooth muscle contraction. The treatment can involve the reduction of or inhibition of inappropriate muscle contraction. Examples of such states include hypertension, asthma, irritable bowel syndrome, incontinence and menstrual cramps.
This invention pertains to a method of modulating smooth muscle contraction. The method involves administering a PAK modulating agent, e.g., PAK inhibitor, to a subject such that modulation of smooth muscle contraction occurs. A PAK inhibitor may be, for example, an agent which binds to, or blocks, either or both of the kinase domain of PAK and the p21 (e.g., Cdc42 or Rac1) binding domain of PAK, or the autophosphorylation sites of PAK, The smooth muscle is present preferably in a blood vessel, the airways of the lungs, the gastro-intestinal tract, the uterus or the urinary tract.
Another aspect of the invention pertains to a method of treating a subject having a state characterized by smooth muscle contraction. The method involves administering to a subject a therapeutically effective amount of a PAK modulating agent, e.g., PAK inhibitor, such that treatment of the state characterized by smooth muscle contraction occurs. In one embodiment, the smooth muscle has a high basal tone. In another embodiment, the state characterized by the contraction of smooth muscle involves abnormal or inappropriate contraction of smooth muscle. In yet another embodiment, the state characterized by the contraction of smooth muscle involves abnormal or inappropriate relaxation of smooth muscle. Finally, the state characterized by smooth muscle contraction is preferably hypertension, asthma, irritable bowel syndrome, incontinence or menstrual cramps.
Yet another aspect of the invention relates to a method of treating a subject having a heart condition associated with, or that could lead to, cardiac contractile dysfunction, for example, heart failure (HF). The method involves administering to a subject a therapeutically effective amount of a PAK modulating agent, e.g., a PAK stimulator or inhibitor, such that treatment of the heart condition occurs.
In yet another aspect, the invention provides assays, e.g., screening tests, to identify PAK or PAK modulating agents. For example, screening tests of the invention can be used to quantify the amount of PAK or PAK mRNA present, i.e., PAK expression, within smooth muscle or cardiac muscle. Insofar as PAK expression can be used as an indicator of a condition, such as hypertension or HF, screening tests according to the invention are therefore usefull in assessing, for example, the disease state of an individual.
Screening tests of the invention are also useful for detecting or identifying, for example, modulating agents which are inhibitors, or alternatively, stimulators, of PAK kinase expression or activity. In a preferred embodiment, the screening assay identifies agents that modulate the kinase activity of a PAK protein. For example, the invention provides a method involving providing an indicator composition comprising a PAK protein having PAK kinase activity. The method further includes contacting the indicator composition with a test agent, and determining the effect of the test agent on PAK kinase activity in the indicator composition to thereby identify a compound that modulates the kinase activity of a PAK protein. A statistically significant change, such as a decrease or increase, in the level of PAK kinase activity in the presence of the test agent (relative to what is detected in the absence of the test agent) is indicative of the test agent being a PAK modulating agent. The indicator composition can be, for example, a smooth muscle cell, a smooth muscle cell extract or a detergent-skinned smooth muscle fiber bundle system. Screening tests in accordance with the invention can involve identifying the p
Côté Graham P.
Mak Alan S.
Van Eyk Jennifer E.
Laccotripe Zacharakis Maria
Marschel Ardin H.
Miernicki Steeg Carol
Moran Marjorie A.
Queen's University at Kingston
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