CA2435876A1 - The treatment of sexual dysfunction and cardiovascular disease with quinolinone enantiomers - Google Patents

Abstract

Methods for treating specific patient groups for sexual dysfunction are provided. The methods of the present invention comprise the utilization of enantiomers of flosequinan, and pharmaceutical compositions comprising flosequinan enantiomers, in patients who are free of cardiac disease, who have not been treated with a drug that causes hypotensive effects, and/or who have not been given organic nitrites or nitrates. Flosequinan enantiomers, and pharmaceutical compositions comprising flosequinan enantiomers are administered to patients intranasally, orally, topically, and/or through respiratory inhalation. Additionally, the present invention is directed to compositions and methods for treating cardiovascular disease. In a preferred embodiment, the present invention contemplates using the enantiomers of flosequinan (and formulations comprising flosequinan) in subjects not concurrently treated with nitrites or nitrates.

Description

THE TREATMENT OF SEXUAL DYSFUNCTION AND
CARDIOVASCULAR DISEASE WITH QUINOLINONE ENANTIOMERS
FIELD OF THE INVENTION
The present invention relates to methods for the treatment of sexual dysfunction in males and females (including but not limited to erectile dysfunction in males) in particular treatment groups. The methods of the present invention comprise the utilization of pharmaceutical compounds and compositions to patients who are free of symptoms of cardiac disease and who have not been treated with drugs which cause hypotensive effects, such as nitrites and nitrates. The present invention also relates to compositions and methods for the treatment of cardiovascular disease.
BACKGROUND
A. Sexual Dysfunction Impotence or erectile insufficiency is a widespread disorder that is thought to affect about twelve percent of adult men under age forty-five, about twenty percent of men at age sixty, and about fifty-five percent of men at age seventy-five.
There is more than one cause of erectile dysfunction. For example, erectile dysfunction can be psychological, resulting from anxiety or depression, with no apparent somatic or organic impairment. Such erectile dysfunction, which is referred to as "psychogenic," is responsible for about fifteen to twenty percent of cases of impotence. In other cases, the erectile dysfunction is associated with atherosclerosis of the arteries supplying blood to the penis; such dysfunction is referred to as "arteriogenic" or "atherosclerotic." About forty to sixty percent of cases of impotence are arteriogenic in origin.
In still other cases, there is leakage from veins in the penis such that sufficient pressure for an erection can be neither obtained nor maintained. This dysfunction is referred to as "venous leakage," or "abnormal drainage." This condition is often exacerbated by the presence of some arteriogenic dysfunction whereby the supply of blood to the penis is impaired. In still other cases, the dysfunction is associated with a neuropathy, such as nerve damage arising from, for example, surgery or a pelvic injury, in the nervous system affecting the penis. Such a dysfunction is referred to as "neurogenic" and this accounts for about ten to fifteen percent of cases of impotence.
There is also a high incidence of erectile insufficiency among diabetics, particularly those with insulin-dependent diabetes mellitus. Erectile dysfunction in diabetics is often classified as "diabetogenic," although the underlying dysfunction is usually neurogenic associated with neuropathy, but may be arteriogeuc or neurogenic and arteriogenic. About half of diabetic males suffer from erectile insufficiency, and about half of the cases of neurogenic impotence are in diabetics.
Additionally, erectile insufficiency is sometimes a side effect of certain drugs, such as beta-blockers that are administered to reduce blood pressure in persons suffering from hypertension, or drugs administered to treat depression or anxiety.
Excessive alcohol consumption has also been linked to erectile insufficiency.
Circumcision in males, which removes highly sensitive penile tissue and subjects the glans of the penis to desensitization through keratinization of the remaining tissue, is also believed to be a contributiilg factor to some forms of erectile insufficiency. These forms of erectile insufficiency may be regarded as a subset of neurogenic or psychogenic insufficiency.
A number of methods to treat impotence are available. These treatments include pharmacological treatments, surgery and, in cases of psychogenic dysfunction, psychological counseling is sometimes effective. Psychogenic impotence often can be cured by counseling coupled with a demonstration to the patient that he is capable of having a full erection by inducing such an erection once or a few times in the patients.
Insufficiency due to excessive alcohol consumption is sometimes cured by reducing or elimination such consumption.
In rare cases, where the insufficiency is physical because of venous leakage, surgery can usually be employed to repair the venous lesion and thereby either cure the insufficiency or, if there remains an erectile insufficiency after repair of the venous lesion, render the insufficiency amenable to treatment by pharmacological methods.
Also, penile implants, which provide a mechanical means to produce an erection _2_ sufficient for vaginal penetration, are widely used to treat impotence. In recent years, implants have been employed, especially in cases where pharmacological intervention is ineffective, which are usually cases of severe atherogenic impotence.
Treatment of impotence with penile implants, however, entails serious disadvantages. Such treatment requires surgery and necessitates total destruction of the erectile tissues of the penis, forever precluding normal erection.
Pharmacological methods of treatment are also available. Such methods, however, have not proven to be highly satisfactory and can be accompanied by severe side-effects. Papaverine is now widely used to treat impotence, although papaverine is ineffective in overcoming impotence due, at least in part, to severe atherosclerosis.
Papaverine is effective in cases where the dysfunction is psychogenic or neurogenic and severe atherosclerosis is not involved. Injection of papaverine, a smooth muscle relaxant, or phenoxybenzamine, a non-specific blocker and hypotensive, into a corpus cavernosum has been found to cause an erection sufficient for vaginal penetration.
. Also, in cases where severe atherosclerosis is not a cause of the dysfunction, intracavernosal injection of phentolamine, an a-adrenergic blocker, causes an erection sufficient for vaginal penetration. The resulting erection is one of significantly shorter duration than that induced by intracavernosal injection of papaverine or phenoxybenzamine and is of such short duration that satisfactory sexual relations are difficult or impossible.
Treatment of impotence with papaverine or phenoxybenzamine often results in priapism, a locking-up of an erection for a long period of time, typically a few hours and sometimes longer than twenty-four hours. Priapism is a serious, deleterious side effect of treatment of erectile insufficiency with these drugs. Beyond the embarrassment that may be caused for some men, priapism is usually painful, irreversibly damages erectile tissue, and, to be relieved, requires bleeding or pharmacological intervention, such as injection of a sympathomimetic drug, such as adrenaline.
Even if priapism does not occur with use of papaverine, such use is associated with a painful, burning sensation in the first two or so minutes after the injection and there are indications that repeated use of papaverine causes undesirable, extensive intracavernous fibrosis. Further, as indicated above, impotence arising from severe atherosclerosis is not susceptible to treatment with papaverine, phenoxybenzamine, phentolamine or papaverine together with phentolamine. In any case, phenoxybenzamine is not suitable for use in treating impotence because it is a carcinogen.
Sildenafil citrate (Viagra) has also been utilized as a pharmacological treatment for impotence. However, sildenafil citrate has a lack of specificity for its target, enzyme phosphodiesterase S (PDES), and exerts a definite inhibition on the enzyme phosphodiesterase 6 (PDE6), located in the retina. It has been shown that the inhibition of PDE6 results in color vision defects as a side effect of treatment with sildenafil citrate. Furthermore, side effects such as flushing, headache, nasal congestion, and dyspepsia (heartburn) have also been associated with sildenafil citrate treatment of impotence. (S'ee, Moreira et al., "Side-effect profile of sildenafil citrate (Viagra) in clinical practice," UYOIog~, 56(3): 474-76 (2000)).
Thus, although impotence is a ubiquitous problem, there are few satisfactory methods available for treating this disorder. Because of the relatively invasive intervention involved and the high failure rate of penile prostheses, surgical approaches provide unattractive alternatives. A safe pharmacological approach to the treatment of impotence is still to be achieved.
B. Cardiovascular Disease Cardiovascular disease is the number one cause of death in the United States.
Medical Sciences Bulletin, No. 238; p. 1 (1997). While cardiovascular disease presents many different clinical manifestations, hypertension and congestive heart failure (CHF) are major components of this disease state. Uncontrolled hypertension can result in myocardial infarction and stroke. Congestive heart failure, if untreated, is an ultimately fatal disease that kills more than half its victims within five years of initial diagnosis. CHF affects about 3 million people in the United States and about 15 million worldwide. Currently, an estimated 400,000 new cases are diagnosed in the United States each year, and CHF is responsible for about 900,000 hospitalizations a year.
The current treatment approaches involve anti-hypertensive compounds, such as: beta-blockers, calcium channel blockers (especially dihydropyrimidines), angiotensin-converting enzyme (ACE) inhibitors, diuretics, and alpha-blockers.
However, many patients fail to respond to (or tolerate poorly) these compounds.
For example, many patients do not respond to diuretics (with or without digitalis). Moreover, many patients cannot tolerate (or respond poorly to) ACE
inhibitors. In addition the use of beta blockers has been associated with loss of glycemic control. Studies on the Glycemic and Lipidemic Effect of Atenolol and Propranolol in Normal and Diabetic Rats, (Abstract), Arzneimittelforschung, 44(4):
496-501 (April, 1994).
What is needed is a pharmaceutical that is effective in the treatment of sexual disfunction in males and females but lacking in significant side effects.
Additionally, what is needed is a pharmacological intervention for cardiovascular disease (including, but limited to, hypertension and CHF) that is less disruptive to the patient and is be better tolerated in comparison to existing treatment modalities.
SUMMARY OF THE INVENTION
The present invention relates to methods for the treatment of sexual dysfunction in males and females (including but not limited to erectile dysfunction in males) in particular treatment groups. Additionally, the present invention relates to compositions and methods and for the treatment of cardiovascular disease.
A. Sexual Dysfunction The methods of the present invention comprise the utilization of pharmaceutical compounds and compositions to patients who suffer from symptoms of sexual dysfunction and are free of symptoms of cardiac disease and who have not been treated with drugs which cause hypotensive effects, such as nitrites and nitrates. The compositions comprise purified enantiomers of flosequinan, including derivatives thereof. In one embodiment, said purified enantiomer of flosequinan is a (+) enantiomer. In another embodiment, said composition is substantially free of the (-) enantiomer of flosequinan.
In one embodiment, the method comprises a) providing: i) a patient (whether male or female) suffering from one or more symptoms of sexual dysfunction; and ii) a pharmaceutical composition comprising a purified enantiomer of flosequinan, or a pharmaceutically acceptable salt thereof; and b) administering said pharmaceutical composition to the patient such that at least one of said one or more symptoms are reduced. A variety of such symptoms are contemplated, including but not limited to, poor blood flow to the sexual organs and/or failure to achieve orgasm. In one embodiment, the present invention contemplates administering said flosequinan to said male or female under conditions such that blood flow to the sexual orgms of said male or female is improved.
It is not intended that the methods of the present invention be limited to a patient who is free from cardiac disease. However, in one embodiment, the patient is free from cardiac disease. In another embodiment, said patient is not free from cardiac disease. In one embodiment, the patient is male. In another embodiment, the patient is female. In one embodiment, said administering step is selected from the group consisting of intranasal and respiratory inhalation.
It is not intended that the methods of the present invention be limited to a patient who has been, or is being, treated with a drug that causes hypotensive effects.
However, in one embodiment, the method comprises a) providing: i) a patient (whether male or female) suffering from one or more symptoms of sexual dysfunction who is not being treated with a drug that causes hypotensive effects, and ii) a pharmaceutical . composition comprising a purified enantiomer of flosequinan, or a pharmaceutically acceptable salt thereof; and b) administering flosequinan to the patient such that such at least one of said one or more symptoms are reduced. A variety of such symptoms are contemplated, including but not limited to, poor blood flow to the sexual organs and/or failure to achieve orgasm. In one embodiment, the present invention contemplates administering said flosequinan to said male or female under conditions such that blood flow to the sexual organs of said male or female is improved.
In one embodiment, the patient is free from cardiac disease. In one embodiment, the patient is male. In another embodiment, the patient is female. In one embodiment, said administering step is selected from the group consisting of intranasal and respiratory .
inhalation. In one embodiment, said patient is being, or has been, treated in the past with a drug that causes hypotensive effects.
It is not intended that the methods of the present invention be limited to a patient who is being, or has been, treated with a nitrate or nitrite. However, in another embodiment, the method comprises a) providing: i) a patient (whether male or female) suffering from symptoms of sexual dysfunction who. is not being treated with a nitrite or nitrate, and ii) a pharmaceutical composition comprising a purified enantiomer of flosequinan, or a pharmaceutically acceptable salt thereof; and b) introducing flosequinan to the patient such that such symptoms (or a subset thereof) are reduced.
A variety of such symptoms are contemplated, including but not limited to, poor blood flow to the sexual organs and/or failure to achieve orgasm. In one embodiment, the present invention contemplates administering said flosequinan to said male or female under conditions such that blood flow to the sexual organs of said male or female is improved. In one embodiment, the patient is free from cardiac disease. In one embodiment, the patient is male. In another embodiment, the patient is female.
In one embodiment, said administering step is selected from the group consisting of intranasal and respiratory inhalation. In one embodiment, said patient has been, or is being, treated with a nitrite or nitrate. In one embodiment, said nitrate is selected from the group consisting of glyceryl trinitrate, isosorbide dinitrate, isosorbide-5'-mononitrate, and erythrityl tetranitrate.
In one embodiment, the method comprises providing: i) a male or female subject with erectile dysfunction, and ii) a purified enantiomer of flosequinan, or a pharmaceutically acceptable salt thereof; and introducing flosequinan to the male or female subj ect such that an erection (i. e. penile or clitoral) is produced.
In another embodiment, the method comprises providing: i) a male or female subject with erectile dysfunction, and ii) a pharmaceutical composition comprising a purified enantiomer of flosequinan, or a pharmaceutically acceptable salt thereof; and introducing the pharmaceutical composition to the male or female subject such that an erection (i.e. penile or clitoral) is produced.
It is not intended that the present invention be limited by the method of introduction of a purified enantiomer of flosequinan, or a pharmaceutically acceptable salt thereof. In one embodiment, the enantiomer of flosequinan is introduced into the male or female orally. It is believed that an oral dosage up to approximately milligrams of a racemic mixture of flosequinan is an effective oral dosage. It is also believed that the oral administration of a purified of enantiomer of flosequinan is effective at even lower dosages (e.g. less than 200 mg). In one embodiment, the male or female is an adult human and the oral dosage of a purified enantiomer of flosequinan is in a single dose per day of up to approximately two hundred milligrams, more preferably, between approximately fifty to approximately seventy-five milligrams. In an even more preferred embodiment, a purified enantiomer of flosequinan is administered in a single oral dose per day of between approximately twenty and approximately fifty, and even more preferably, between approximately ten and approximately twenty milligrams per day. The administration of multiple dosages of a purified enantiomer of flosequinan are also contemplated.
In other embodiments, enantiomers of flosequinan are introduced cutaneously, transurethrally, by standard injection, intracavernosally, intranasally or through respiratory inhalation. In other embodiments, pharmaceutical compositions comprising a purified enantiomer of flosequinan, or a pharmaceutically acceptable salt thereof, is introduced cutaneously, transurethrally, by standard injection, intracavernosally, intranasally or through respiratory inhalation.
The present invention is not limited by the degree of response by the male subject. In one embodiment, the erection induced is sufficient for vaginal penetration.
Likewise, the present invention also contemplates the use of sexual stimulation in addition to the application of a pharmaceutical composition. For example, one embodiment comprises a) providing: i) a male, having a penis, with erectile dysfunction, ii) a purified enantiomer of flosequinan, or a pharmaceutically acceptable _g_ salt thereof, and iii) sexual stimulation; and b) introducing said flosequinan and sexual stimulation to the male such that an erection is produced.
In another embodiment, the method comprises a) providing: i) a male, having a penis, with erectile dysfunction, ii) a pharmaceutical composition comprising a purified enantiomer of flosequinan; or a pharmaceutically acceptable salt thereof, and iii) sexual stimulation; and b) introducing said pharmaceutical composition and sexual stimulation to the male such that an erection is produced.
In another embodiment, the present invention also contemplates the use of sexual stimulation in addition to the application of a pharmaceutical composition. For example, one embodiment comprises a) providing: i) a female, having a clitoris, with erectile dysfunction, ii) a purified enantiomer of flosequinan, or a pharmaceutically acceptable salt thereof, and iii) sexual stimulation; and b) introducing said flosequinan and sexual stimulation to the female such that an erection is produced.
In another embodiment, the method comprises a) providing: i) a female, having a clitoris, with erectile dysfunction, ii) a pharmaceutical composition comprising a purified enantiorner of flosequinan, or a pharmaceutically acceptable salt thereof, and iii) sexual stimulation; and b) introducing said pharmaceutical composition and sexual stimulation to the female such that an erection is produced.
Likewise, the present invention is not limited by the nature of the sexual stimulation. In one embodiment, the sexual stimulation is sexually explicit media. In another embodiment, the sexual stimulation involves manipulation of the penis, such as with vibration. In another embodiment, the sexual stimulation involves manipulation of the clitoris, such as with vibration or digital stimulation.
It is not intended that the present invention be limited by the nature of the formulation. In one embodiment, the present invention contemplates a formulation comprising a purified enantiomer of flosequinan, or derivative thereof, in a mixture comprising lactose.

B. Cardiovascular Disease The present invention provides compositions and methods for the treatment of cardiovascular disease. In a preferred embodiment, the present invention is particularly suited to the treatment of CHF. The methods of the present invention comprise the administration of pharmaceutical compositions to subjects who, in a preferred embodiment, are not concurrently administered nitrites and nitrates.
Specifically, the compositions of the present invention comprise quinolinones, including derivatives thereof. Quinolinones are also known as quinolones and oxo-quinolines.
In another embodiment, the present invention contemplates halogenated quinolinones (e.g., fluoroquinolinone). In a preferred embodiment, the quinolinone is a thioquinolinone or a sulphinyl or suphonyl derivatives thereof. In one embodiment, the halogenated quinolinone is flosequinan [(+-)-7-fluoro-1-methyl-3-(methyl-sulphinyl)-4(1H)-quinolinone]; [7-Fluoro-1-methyl-3-(methylsufinyl)-4(1H)-quinolone].
In a preferred embodiment, an enantiomer [either (+) or (-)] of flosequinan is used.
Iri one embodiment, the present invention contemplates a method, comprising:
a) providing: i) a patient presenting at least one symptom of cardiovascular disease, and ii) a purified enantiomer preparation of flosequinan; and b) administering said preparation to said patient (e.g. such that said symptom is reduced). A
variety of such symptoms of cardiovascular disease are contemplated. It is not intended that the present invention be limited to the reduction of a particular symptom of cardiovascular disease. In a preferred embodiment, the a symptom of hypertension is reduced.
Hypertension is an abnormal increase of blood pressure in the arteries continuing over a period of time. It occurs when the arterioles, the small blood vessels that branch off from the arteries, become constricted. This constriction of the arterioles makes it difficult for blood to flow which increases pressure against the artery walls.
A blood pressure reading of approximately 110/60 to 140/90 is considered to be in the normal range. The first number (110) is the systolic pressure which measures the blood pressure in the arteries when the heart is contracting and pumping blood.

The second number (60) is the diastolic pressure which measures the blood pressure in the arteries when the heart is at rest. Hypertension adds to the workload of the heart and arteries. Over time, this can lead to heart and blood vessel damage which causes hardening of the arteries, heart failure, stroke, kidneys problems, blindness, and brain damage. In one embodiment of the present invention, a symptom of cardiovascular disease comprises a measured blood pressure of approximately 140/90 or higher.
In a preferred embodiment, the diagnosis of said hypertensive blood pressure (e.g.
approximately 140/90 or higher) is confirmed by a plurality of measurements of approximately 140190 or higher spaced over the period of at least two weeks.
It is not intended that the present invention be limited by the means by which blood pressure is measured. Moreover, additional symptoms of hypertension also include, but are not limited to, tiredness, cbnfusion, nausea, vomiting, anxiety, excessive perspiration, muscle tremor, chest pain, nosebleed, and buzzing in the ears.
In another embodiment, the present invention contemplates compositions and methods to reduce the symptoms of CHF (also referred to as 'heart failure').
CHF is characterized by an inadequacy of the heart so that, as a pump, it fails to maintain the circulation of blood, such that congestion and edema develop in the tissues of the heart are reduced. Symptoms of CHF include, but are not limited to, shortness of breath, pitting edema, an enlarged and tender liver, engorged neck veins, and pulmonary rates in various combinations.
It is not intended that the present invention be limited by the method by which CHF is diagnosed. CHF may be diagnosed based on a medical history and complete physical examination, which may include a blood pressure check, listening to the subject's heart through a stethoscope and taking the subject's pulse. At physical exam.
a Health Care Provider (including, but not limited to, Physicians, Nurse Practitioners, or Physician's Assistants) may look for the symptoms of CHF (as listed above).
If a Health Care Provider does not find enough symptoms to make a diagnosis, but is still suspicious that the subj ect has CHF, then her or she may order further tests.
These test include, but are not limited to, blood tests to assess for anemia and thyroid function, urine tests to measure sugar, an Electrocardiogram (EKG), an exercise stress test, an Echocardiogram, a stress echocardiogram, radionuclide imaging tests (such as a radionuclide ventriculogram).
More invasive exploratory tests may also be ordered in conjunction with, or instead of the above. These tests include a coronary angiogram, in which a contrast dye is delivered by catheter to the coronary arteries to visualize the blood vessels and identify heart damage or dysfunction.
The present invention is not limited by the degree of response by the subject.
It is expected that the administration of quinolinone enantiomers will reduce the symptoms associated with cardiovascular diseases including, but not limited to, angina pectoris, myocardial infarction, congestive heart failure, cardiomyopathy, hypertension, arterial stenosis, and venous stenosis. In a preferred embodiment, the enantiomers of flosequinan are administered to reduce the symptoms associated with hypertension.
In another preferred embodiment, the enantiomers of flosequinan are administered to reduce the symptoms of CHF. The symptoms of CHF include, but are not limited to, shortness of breath, pitting edema, an enlarged and tender liver, engorged neck veins, and pulmonary rates in various combinations.
Symptoms are "reduced" when the magnitude (e.g. intensity) or frequency of symptoms is reduced. It is not intended that the present invention be limited only to cases where the symptoms are eliminated. The present invention specifically contemplates treatment such that symptoms are reduced (and the condition of the subject is thereby "improved"), albeit not completely eliminated. Moreover, it is sufficient if one or more (e.g., a subset) of symptoms are reduced.
In a preferred embodiment, the subject is a human and the oral dosage of either the (+) or (-) enantiomer of flosequinan is in a single dose per day of up to approximately two hundred milligrams. In another embodiment said dosage is between approximately twenty-five to approximately seventy-five milligrams. In another embodiment, the (+) or the (-) enantiomer is administered in a single oral dose per day of between approximately one hundred and twenty-five and approximately two hundred milligrams. In another embodiment, the administration of said enantiomers of flosequinan comprises three daily doses, before meals, each dose of up to approximately two hundred milligrams per dose. In another embodiment, said daily doses comprise between approximately twenty-five to approximately seventy-five milligrams per dose. In another embodiment, said daily doses comprise between approximately one hundred and twenty-five and approximately two hundred milligrams per dose.
In selected embodiments said enantiomers of flosequinan are introduced orally, cutaneously, by standard injection (e.g. intravenously), or intranasally.
In one embodiment, the method comprises a) providing: i) a patient suffering from symptoms of cardiac disease who is not being administered nitrates or nitrites;
and ii) an enantiomer of flosequinan; and b) introducing said enantiomer of flosequinan to said patient such that said symptoms of cardiac disease are reduced.
In one embodiment, said substantially purified enantiomer of flosequinan is a (+) enantiomer. In another embodiment, said composition is substantially free of the (-) enaiitiomer of flosequinan.
In one embodiment, the method comprises a) providing: i) a subject suffering from symptoms of cardiovascular disease; and ii) a purified enantiomer preparation of flosequinan, or a pharmaceutically acceptable salt thereof; and b) administering said preparation to the subject such that symptoms are reduced. In one embodiment, said cardiovascular disease is selected from the group consisting of hypertension, angina , pectoris, myocardial infarction, and congestive heart failure. Said administering step is selected from the routes consisting of intranasal and respiratory inhalation.
In another embodiment, the method comprises a) providing: i) a subject suffering from symptoms of cardiovascular disease who is not being treated with a drug that causes hypotensive effects, and ii) a purified enantiomer preparation of flosequinan, or a pharmaceutically acceptable salt thereof; and b) administering said preparation to the subject such that such symptoms are reduced.
In another embodiment, the method comprises a) providing: i) a subject suffering from symptoms of cardiovascular disease who is not being treated with a nitrite or nitrate, and ii) a purified enantiomer preparation of flosequinan, or a pharmaceutically acceptable salt thereof; and b) introducing said preparation to the subject such that such symptoms are reduced. Said nitrate is selected from the group consisting of glyceryl trinitrate, isosorbide dinitrate, isosorbide-5'-mononitrate, and erythrityl tetranitrate.
It is not intended that the present invention be limited by the method of introduction of a purified enantiomer preparation of flosequinan, or a pharmaceutically acceptable salt thereof. In one embodiment, the enantiomer of flosequinan is introduced orally. In a preferred embodiment, an adult human is provided an oral dosage as a single dose per day of 10 to 200 milligrams. In other embodiments, enantiomers of flosequinan are introduced cutaneously, by standard injection, intranasally, or through respiratory inhalation.
The present invention is not limited by the degree of response by the subject.
In one embodiment, relief of pain from angina pectoris is sufficient.
It is not intended that the present invention be limited by the nature of the formulation. In one embodiment, the present invention contemplates a mixture of a purified enantiomer of flosequinan and a Garner, i.e. a mixture comprising lactose.
In one embodiment, the enantiomers recited in the present invention are introduced into said subject by oral administration or cutaneous administration. In another embodiment, said subject is an adult human and said oral administration comprises up to approximately 200 milligrams of flosequinan.
In a preferred embodiment, the enantiomers of flosequinan recited in the present invention are administered to a subject who has not been treated in the past with a drug that causes hypotensive effects. In a more preferred embodiment, the enantiomers of flosequinan recited in the present invention are administered to a subject who is not being treated with a nitrite or nitrate. In one embodiment said nitrate is selected from the group consisting of glyceryl trinitrate, isosorbide dinitrate, isosorbide-5-mononitrate and erythrityl tetranitrate.
The present invention also contemplates a method, comprising: a) providing: i) a subject suffering from a symptom of a cardiovascular disease selected from the group of hypertension and congestive heart failure and; ii) a purified enantiomer preparation of flosequinan and; b) administering said preparation to said subject under conditions such that said symptom is reduced. In a preferred embodiment, said purified enantiomer of flosequinan is the (+) enantiomer. In another preferred embodiment, said purified enantiomer of flosequinan is the (-) enantiomer.
In one embodiment, the enantiomer preparation introduced into said subject by oral or cutaneous administration. In one embodiment said subject is an adult human and said oral administration, of said enantiomer preparation, comprises up to approximately 200 milligrams of flosequinan.
The present invention also contemplates a purified flosequinan enantiomer preparation comprising a carrier.
DESCRIPTION OF THE DRAWINGS
Figure 1 depicts the respective HPLC column retention times and optical rotations of the enantiomers of flosequinan separated by the method provided in Example 2.
Figure 2 depicts the chemical structure of a quinolinone (top) and 16 C-7 substituents (bottom).
DEFINITIONS
As used herein, the term "enantiomer" refers to stereoisomers of molecules that are non-superimposable mirror images of each other. Enantiomers have identical physical properties, such as melting points and boiling points, and also have identical spectroscopic properties. Enantiomers differ from each other with respect to their interaction with plane-polarized light and with respect to biological activity.
As used herein, the term "stereoisomer" refers to compounds that have their atoms connected in the same order but differ in the arrangement of their atoms in space. (e.g. cis-2-butane and traps-2-butane).
As used herein, the term "diastereoisomers" refers to stereoisomers that are not mirror images of each other.
As used herein, the term "quinolinone" refers to chemical compositions comprising quinolinone as set forth in the following structure (2-quinolone):

s HN
s as well as other forms of quinolinone, (e.g., isoquinolone):
O
s 7 ° NH
I ...-i As used herein, the phrase "derivatives of quinolinone" refers to chemical compositions comprising quinolinone with a chemical group attached, including (but not:limited to) halogenated quinolinones.
As used herein, the phrase "methylsulphinyl derivatives of quinolinone" refers to chemical compositions comprising quinolinone with a methylsulphinyl group attached. Examples include flosequinan (7-fluoro-1-methyl-3-(methylsulphinyl)-4(1H)-quinolone; 7-fluoro-1-methyl-3-(methylsufinyl)-4(1H)-quinolinone):

's ~a~, and sulfone metabolites of flosequinan:
'' ~-~Ha N. Cs CHa As used herein, the terms "purified enantiomer" and "purified enantiomer preparation" are meant to indicate a preparation (e.g. derived from a racemic mixture or synthesized de fzovo) wherein one enantiomer has been enriched over the other, and more preferably, wherein the other enantiomer represents less than 10%, and more preferably less than 5%, and still more preferably, less than 2% of the preparation.
As used herein, the term "racemic mixture" refers to a mixture of the two enantiorners of one compound. An ideal racemic mixture is one wherein there is a 50:50 mixture of both enantiomers of a compound such that the optical rotation of the (+) enantiomer cancels out the optical rotation of the (-) enantiomer.
As used herein, the phrase "pharmaceutically acceptable salts" or "a pharmaceutically acceptable salt thereof' refer to salts prepared from pharmaceutically acceptable non-toxic acids or bases including inorganic acids and bases and organic acids and bases. Since the compound of the present invention is basic, salts may be prepared from pharmaceutically acceptable non-toxic acids. Suitable pharmaceutically acceptable acid addition salts for the compound of the present invention include acetic, benzenesulfonic (besylate), benzoic, camphorsulfonic, citric, ethenesulfonic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric, isethionic, lactic, malefic, malic, mandelic, methanesulfonic, muck, nitric, pamoic, pantothenic, phosphoric, succinic, sulfuric, tartaric acid, p-toluenesulfonic, and the like. Preferred acid addition salts are the chloride and sulfate salts.
As used herein, a patient who is "free from cardiac disease" and a patient who is "free from symptoms of cardiac disease" indicate that the patient has not been diagnosed with angina, myocardial infarction, congestive heart failure and that symptoms of angina, ischemia, myocardial infarction, and/or congestive heart failure have not been detected.
As used herein, "symptoms of cardiovascular disease" refers to any clinical manifestation of a disease state associated with the heart and vasculature.
For example, said clinical manifestation include: angina pectoris, myocardial infarction, congestive heart failure, cardiomyopathy, hypertension, arterial stenosis, and venous stenosis. The present invention specifically contemplates treatment such that symptoms are reduced (and the condition of the subject is thereby "improved"), albeit not completely eliminated.
As used herein, "congestive heart failure" is a specific cardiovascular disease which is characterized, but not limited to, the following symptoms: shortness of breath, pitting edema, an enlarged and tender liver, engorged neck veins, and pulmonary rates in various combinations.
The terms "lower alkyl", "lower alkoxy", "lower alkanoyl", and "lower alkythio" denote such groups containing 1-8 carbon atoms, especially 2-4 carbon atoms for lower alkanoyl and 1-4 carbon atoms for the other groups. Examples of such groups include methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-heptyl, n-octyl, methoxy, ethoxy, propoxy, n-butoxy, isobutoxy, acetyl, propionyl, butyryl, methylthio, ethylthio, propylthio and n-butylthio.
As used hereinafter, the term "active compound" denotes a pyridinone compound of general formula I (as illustrated and described below in the section entitled "Other Compounds Useful In The Treatment of Cardiovascular Disease"), or a quinolinone or quinolinone derivative as illustrated and described above.

As used herein, "drugs that have hypotensive effects" are those drugs which, when administered, cause the patient's end-diastolic blood pressure to be reduced.
Nitrates are commonly used drugs which have hypotensive effects.
As used herein, "nitrates" are compounds that contain the -N03- moiety.
Nitrates typically used in the clinic are shown in Table 1.
As used herein, "nitrites" are compounds that contain the -NOZ- moiety.
Nitrites typically used in the clinic are shown in Table 1.
As used herein, the term "erectile dysfunction" refers to certain disorders of the cavernous tissue of the penis and the associated facia which produce impotence, the inability to attain a sexually functional erection.
As used herein, "symptoms of erectile dysfunction" refers to any two of the following symptoms: penile flaccidity, lack of penile tumescence, lack of penile rigidity, and inability to produce an erection sufficient for vaginal penetration.
Symptoms are "reduced" when the magnitude (e.g. intensity) or frequency of symptoms is reduced. It is not intended that the present invention be limited only to cases where the symptoms are eliminated. The present invention specifically contemplates treatment such that one or more symptoms are reduced (and the condition of the patient is thereby "improved"), albeit not completely eliminated.
As used herein, "symptoms of sexual dysfunction" includes, but is not limited to, poor blood flow to the sexual organs and/or failure to achieve orgasm.
Symptoms are "reduced" when the magnitude (e.g. intensity) or frequency of symptoms is reduced. It is not intended that the present invention be limited only to cases where the symptoms are eliminated. The present invention specifically contemplates treatment such that one or more symptoms are reduced (and the condition of the patient is thereby "improved"), albeit not completely eliminated.
As used herein, an "erection" refers to the condition of a penis whereby it is at least semi-rigid as opposed to being in a flaccid state.
As used herein, the term "subject" refers to both humans and animals.
As used herein "standard injection" refers to the placement of a pharmaceutical composition into a subject (e.g., with a hypodermic needle). For example, such NAMPS AND TRADE CHEhZIChL PREPARATIONS, USUAL DOSES, ttlD
NAMES STRUCTURE ..~KO~S OF AD~STR~.TION*
Amyl nitrite H'Cw Inh: O.I8 or 0.3 ml, inhalation (ixoarriyl nitrite) H3C~CHCH2CHzONO
Nitroglycerin T 0.15 to 0.6 mg as needed (glyecryl trinitratc;HZC-O-NOZ S: 0.4 mg per spray as needed t>rrl:o am, NrrROSTwT,HC=O-NO C: 2.5 to 9 mg two to 2 four tames daily - x~~- ~Q-D~~ H2G-o-NOz ,S: I mg ovary 3 to 5 h O: L25 to 5 cm (1!i to 2 in.), topically to skin ovcry 4 to 8 h D; I disc (2.5 to 1S Trig) avery.?tl h N: 5 pglmin; increments of 5 ~,glmin ~osoxbidc dinitratcHzC T: _ 2.5 to 1~f mg overy 2 to 3 h (tea., soEZSrrRt.Tn,HC-O-NOz T(C): 5 to 10 mg ovcry 2 to 3 h ~u~ATE, others> ~ T(o~. to to 40 mg G~~ry 6 r-CH ~ h ~

40 to 80 mg every 8 to I2 h o2N-O-~soxorbide-5-tnonanitrate . - T: 10 to 40 mg twine daily lsrao, others) C: 60 mg daily .
~ythtityl tctranitratc H2e'-o-NOQ T: S to 10 mg as needed HC-O-NOz T(O): 10 mg three times daily N~~-O.-N02 H2c-a-NO2 13 6txcal (tratismueocal) tablt~ C, suctafned-crlease capsule or tabteG D. ~
Vie; Iah, inhalant; IV iaccaveacus injcctiaa; O, oinaacaq S. hagnat spray; T, tablet for iublingttai ttsc; T(C). chtwable tablet; T(O~.
vm1 tablet or capsalc.

injection can be made subcutaneously, intravenously, intramuscularly, intracavernosally, etc.
As used herein, "intracavernosal" injection is injection into the corpus cavernosum of the penis.
As used herein, "intranasally" refers to the introduction of a pharmaceutical composition within the nasal cavity.
As used herein, "respiratory inhalation" refers to the introduction of a pharmaceutical composition within the respiratory tract.
As used herein, "by oral administration" refers to the introduction of a pharmaceutical composition into a subject by way of the oral cavity (e.g., in aqueous liquid or solid form).
As used herein, "cutaneously" refers to the introduction of a pharmaceutical composition into a subject by application to the surface of the skin such that the composition is absorbed into the subject.
As used herein, "transurethrally" refers to the introduction of a pharmaceutical composition to the urethra of a subject such that the composition is absorbed into the subj ect.
As used herein, "intranasally" refers to the introduction of a pharmaceutical composition within the nasal cavity.
As used herein, "respiratory inhalation" refers to the introduction of a pharmaceutical composition within the respiratory tract.
As used herein, "sufficient for vaginal penetration" refers to the state of a~1 erection such that the penis is capable of entering a vagina without manual manipulation.
As used herein, "sexual stimulation" refers to activity that would induce an erection in a male without erectile dysfunction (e.g., sexually explicit media, manual manipulation, vibration, live erotic entertainment, etc. ) As used herein, "sexually explicit media" refers to films, videos, books, magazines, etc. that depict sexual activity.

As used herein "single dosage" refers to a pharmaceutical composition of a formulation that is capable of achieving its intended effect in a single administration or application.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to methods for the treatment of sexual dysfunction in males and females (including but not limited to erectile dysfunction in males) in particular treatment groups. This will be reviewed in section (A) below. The present invention also relates to the treatment of cardiovascular disease. This will be reviewed in section (B) below.
A. The Treatment of Sexual Dysfunction The present invention relates to methods for the treatment of sexual dysfunction in males and females (including but not limited to erectile dysfunction in males) in particular treatment groups. The methods of the present invention comprise the utilization of pharmaceutical compounds and compositions to patients who are free of symptoms of cardiac disease and who have not been treated with drugs which cause hypotensive effects, such as nitrites and nitrates. The compositions comprise enantiomers of flosequinan, including derivatives thereof.
In one embodiment a purified enantiomer. of flosequinan is administered.
Importantly, flosequinan enantiomers may potentiate the hypotensive effects of nitrates, and its administration to patients who are concurrently using organic nitrates in any form may be contraindicated. It is contemplated that an enantiomer of flosequinan be achninistered cutaneously, transurethrally, by standard injection, intracavemosally, intranasally or through respiratory inhalation, although it is not intended that the methods of the present invention be limited to the mode of administration of an enantiomer of flosequinan.
In other embodiments, a pharmaceutical composition comprising a purified enantiomer of flosequinan is administered. It is contemplated that said pharmaceutical composition be administered cutaneously, transurethrally, by standard injection, intracavernosally, intranasally or through respiratory inhalation, although it is not intended that the methods of the present invention be limited to the mode of administration of said pharmaceutical composition.
In one embodiment, the present invention contemplates the use of compositions that are effective to induce an erection in a human male suffering from impotence of any origin, other than anatomical deficiencies (i.e., lacking a penis or a significant portion thereof) that preclude an erection sufficient for vaginal penetration.
In particular, these compositions may be used to induce an erection in a male suffering from impotence caused by severe atherosclerosis, and also impotence that is neurogenic or psychogenic in origin. The compositions utilized in the methods of the present invention comprise purified enantiomers of flosequinan, including derivatives thereof.
Although the present invention is not limited by a specific means of producing enantiomers of flosequinan, methods of producing a racemic mixture of flosequinan, are set forth in U.S. Patent Nos. 5,079,264 and 5,011,931 to MacLean et al., hereby incorporated by reference. Moreover, a means of resolving the enantiomers of flosequinan is set forth in Morita et al., "Synthesis and Absolute Configuration of the Enantiomers of 7-Fluoro-1-methyl-3-(methylsulfinyl)-4(lI~-quinolinone (Flosequinan)," Chem. Pharrn. Bull., 42(10): 2157-2160 (1994), hereby incorporated by reference.
1. Resolution of the (+) and (-) Enantiomers of Flosequinan The present invention contemplates the resolution of the (+) and (-) enantiomers of flosequinan. Many organic compounds, including flosequinan, exist in optically active forms (i. e., they have the ability to rotate the plane of plane-polarized light). In describing an optically active compound, the prefixes D and L or R
and S
are used to denote the absolute configuration of the molecule about its chiral center(s).
The prefixes d and 1 or (+) and (-) are employed to designate the sign of rotation of plane-polarized light by the compound, with (-) or 1 meaning that the compound is levorotatory (rotates to the left). A compound prefixed with (+) or d is dextrorotatory (rotates to the right). For a given chemical structure, these compounds, called "stereoisomers," are identical except that they are mirror images of one another. A
specific stereoisomer may also be referred to as an "enantiomer.
Stereochemical purity is of importance in the field of pharmaceuticals, where 12 of the 20 most prescribed drugs exhibit chirality. A case in point is provided by the L-form of the beta-adrenergic blocking agent, propranolol, which is known to be 100 times more potent than the D-enantiomer. Furthermore, optical purity is important since certain isomers may actually be deleterious rather than simply inert.
For example, it has been suggested that the D-enantiomer of thalidomide was a safe and effective sedative when prescribed for the control of morning sickness during pregnancy, while the corresponding L-enantiomer has been believed to be a potent teratogen.
The present invention is not limited by any specific means of resolving the (+) and (-) enantiomers of flosequinan to obtain a purified enantiomer of flosequinan. In one embodiment, said enantiomers are resolved as follows. A racemic mixture of flosequinan is subjected to high-performance liquid chromatography (HPLC) over a Chiracel OD column (Chiral Technologies, Exton, PA) at a flow rate of 1.0 ml/minute.
A mobile phase comprising methanol is utilized, resulting in the resolution of a distinct peak for each enantiomer. The resolved (+) and (-) enantiomers of flosequinan are eluted with methanol with an optical purity greater than 99%.
While it is not necessary to understand any particular mechanism to carry out the present invention, it is believed that in some circumstances, enantiomers of flosequinan can act as a direct-acting vasodilator to relax the corpus cavernosum smooth muscle cells, which in turn increases blood flow into the cavernosa space.
This then leads to increased cavernosa pressure to produce an erect penis.
The action of the enantiorners of flosequinan in the body are not precisely understood. Its activity in the body is attributed to flosequinan itself, as well as its sulfone metabolite. It has been reported to be useful to some degree in the treatment of heart failure. (See Kelso et al., J. Cardiovasc. Plaarmacol. 25: 376 (1995)).

However, its action appears to have little effect in patients with end-stage failure (Perreault et al., Br. J. Pharznacol. 106: 511 (1992)) and does not affect mortality or arrhythmias following coronary artery ligation (See, Jones et al., Bn. J.
Phannacol.
108: 1111 (1993)).
Likewise, flosequinan has been reported to be a selective inhibitor of phosphodiesterase III (PDE3). (See, Gristwood et al., Br. J. Pharnzacol., 105:

(1992); Frodsham et al., Eur. J. Pharmacol. 211: 383 (1992)). It has been found that inhibitors of PDE3 cause vasodilation leading to a concomitant reduction in arterial pressure. (See, Shiraishi et al., "Effect of cilostazol, a phosphodiesterase type III
inhibitor, on histamine-induced increase in [Ca2+]; and force in middle cerebral artery of the rabbit," Br. J. Plza>~znacol., 123: 869-878 (1998)). Moreover, it has also been shown that PDE3 is present in human corpus cavernosum tissue. (See, Ellis &
Terrett, PCT Application No. WO 94/28902). However, reports concerning phosphodiesterase inhibition of flosequinan, as relevant to its efficacy in heart failure, is questionable.
Thus, the application of flosequinan to particular purposes in the body is not well-characterized and must be determined empirically.
It is not intended that the present invention be limited to any particular mechanism to reduce symptoms of sexual dysfunction. In one embodiment, the (+) and (-) enantiomers of flosequinan were subjected to biochemical assays to determine their respective percent inhibition of PDE3. In one embodiment, it was shown that the (+) ~enantiomer of flosequinan has an eight-fold increase in PDE3 inhibition as compared to that of the (-) enantiomer of flosequinan at the same molar concentration.
Moreover, in the same embodiment, the (+) enantiomer of flosequinan exhibited greater inhibition of PDE3 than a racemic mixture of flosequinan at the same molar concentration.
2. Diagnosis of Male Erectile Dysfunction Determination whether a human male is suffering from impotence that is substantially only neurogenic or psychogenic is readily made by a person skilled in the art using a number of readily available diagnostic procedures. Thus, a male suffering from impotence can first be given a physical examination with particular attention to possible penile and scrotal pathology, whereby any anatomical deficiency precluding an erection sufficient for vaginal penetration can be detected. In the absence of such an anatomical deficiency, the male can be subjected to tests, whereby penile venous leakage or severe or untreatable atherosclerosis can be detected.
Such tests include determination of the penobrachial blood pressure index (PBPI), doppler investigation of the penile arteries, and a papaverine test.
The PBPI is the penile systolic blood pressure divided by the systolic blood pressure determined at one of the arms. These blood pressures can be determined by any number of standard techniques. Thus, the penile systolic blood pressure can be determined by i) placing an inflatable cuff around the base of the free part of the penis in the flaccid state which is capable of being used to apply variable pressure, readable from a gauge, to an object around which the cuff is placed, ii) localizing the penile arteries with a Doppler ultrasound probe (e.g., 8 MHz probe, such as the Mini Doplex D500 available from Huntleigh Technology, Luton, United Kingdom), and then iii) inflating and deflating the cuff and ascertaining the pressure at which the Doppler sound reappears.
The pressure at which the Doppler sound reappears is the penile systolic blood pressure. A male's penile blood pressure is regarded as normal if his PBPI is >0.80.
With regard to Doppler investigation, each of the two penile cavernous arteries is investigated distal to the aforementioned cuff using the Doppler ultrasound problem.
The function of each of the two arteries is assessed by Doppler ultrasound using an arbitrary scale of 0, 1, 2 or 3, where 0 means that the function is so deficient that the artery cannot be located and 3 means that the artery is well enough that maximal Doppler sound is observed.
In the papaverine test, a tourniquet is placed at the base of the free part of the penis and tightened and then, with the patient seated, 30 mg of papaverine in 1 ml of a physiologically acceptable fluid (e.g., physiological saline or phosphate-buffered saline) is injected into the penile cavernous body. In persons suspected of having impotence due to a suprasacral nerve lesion or a psychogenic dysfunction, only 15 mg of papaverine is administered, because of the high incidence of papaverine-induced priapism in such cases.
Five minutes after the injection, the tourniquet is removed and an ultrasound Doppler investigation of the penile cavernous arteries is carried out as described above.
S The function of the arteries is regarded as normal if both of them score a 3 on~the arbitrary scale. After the Doppler investigation, penile vibration, at about a 4 Hz with an amplitude of about 1.2 mm (carried out with, e.g., a Vibrector, from Multicept, Gentofte, Denmark) is carried out for five to ten minutes and then erectile response is evaluated.
Erectile response is classified as full rigidity, if the angle between the penis and the legs in the standing position is >90°, and tumescence or no response if the angle is less than or equal to 45°. An impotent male, who does not have an anatomical deficiency that would preclude having an erection sufficient for vaginal penetration, who has a PBPI >0.80, who has scores of 2 or 3 in Doppler ultrasound investigations of both of the cavernous arteries of the penis, after papaverine injection as described above, and who has a fully rigid erection after papaveriiie injection and vibration as described above, is suffering from impotence that is "substantially only neurogenic or psychogenic" in origin.
It is possible that atherosclerosis or venous leakage contributes to such impotence, and atherosclerosis likely does contribute if the score is less than 3 in the Doppler investigation of one or both of the cavernous arteries after papaverine injection; but any venous leakage or atherosclerosis in such impotence is not untreatable and, consequently, is not a substantial factor in the impotence and such atherosclerosis, if any, is less than severe.
Impotence, which is a side-effect of drugs such as beta-Mockers, is deemed to be neurogenic impotence in the present specification. Similarly, impotence which is a result of alcoholism or excessive consumption of alcohol, is deemed to be neurogenic or psychogenic impotence, for purposes of the present specification. Thus, a male who is diagnosed in accordance with the present specification as suffering from impotence that is "substantially only neurogenic or psychogenc" in origin is suffering from _27_ impotence that is substantially only neurogenic, psychogenic or neurogenic and psychogenic in origin, even though an underlying cause of the impotence has been identified as a side-effect of a drug, alcoholism or excessive consumption of alcohol.
Generally, a male with a PBPI less than about 0.60, with scores of 0 in Doppler investigations of both penile cavernous arteries (after papaverine injection as described above), and with a less than fully rigid erection after papaverine injection and vibration will have impotence caused by "untreatable" atherosclerosis.
Methods are available to ascertain whether impotence is untreatable because of venous leakage.
One method of ascertaining whether untreatable venous leakage is a cause of impotence is by cavernosometry, optionally supplemented with cavernosography.
(See, e.g., Delcour et al., Radiology 161: 799 (1986); Porst et al., J. Urol. 137:

(1987); Lue et al., J. Urol. 37: 829 (1987)). Cavernosometry can be done using, both before and after intracavernosal injection of 60 mg of papaverine (in 1 ml of physiological saline), infusion of physiological saline through a 19-gauge needle into one corpus cavernosum with a 21-gauge needle inserted into the other corpus cavernosum for measurement of intracorporal pressure (which is recorded on a plotter).
The infusion rates needed to induce and maintain an erection are measured. If the infusion rate needed to maintain an erection is greater than 50 ml/min before administration of the papaverine and greater than 1~5 ml/min after administration of the papaverine, untreatable venous leakage is present. As long as an erection can be achieved at some flow rate less than about 100 ml/min before injection of the papaverine and less than about 50 ml/min after the injection of papaverine, it might be possible, using cavernosography, to locate the venous lesion associated with the leakage, and thereby confirm the diagnosis based on cavernosometry and provide information for possible surgical correction for the leakage. In the cavernosography, the penis is X-rayed, both before and after intracavernosal injection of 60 mg papaverine (in 1 ml of physiological saline), while infusing contrast medium into the corpus cavernosum (e.g., through a 19-gauge needle) at a flow rate that maintains an erection during the X-ray. Numerous contrast media suitable for the procedure are available in the art; these are typically aqueous solutions of iodinated compounds that provide between about 180 mg/ml and about 360 mg/ml of iodine. Examples are a solution of iohexol providing 240 mg/ml of iodine sold by Winthrop Pharmaceuticals, New York, N.Y., USA, and a solution of iopamidol providing 300 mg/ml iodine sold by Astra Meditec, Goteborg, Sweden. Typically 50-100 ml of the contrast medium will be employed for each x-ray (i.e., before and then after the injection of papaverine). In the cavernosometry and cavernosography, 30 mg papaverine (in 1 ml physiological saline) coupled with stimulation by vibration can be employed in place of 60 mg papaverine (in 1 ml physiological saline).
3. Diagnosis of Female Erectile Dysfunction Females have sexual dysfunction. Post-menopausal women often complain of discomfort with intercourse, dryness of the vagina and diminished vaginal arousal.
Studies comparing sexual dysfunction in couples have revealed 40% of the men had erectile or ejaculatory dysfunction, whereas 63% of the women had arousal or orgasmic dysfunctions. Similar to male sexual dysfunction, the prevalence of female sexual dysfunction has been shown to increase with age and be associated with the presence of vascular risk factors and the development of the menopause.
The clitoris is the homologue of the penis. It is a cylindrical, erectile organ composed of the glans, corporal body and the crura. The corporal body is surrounded by a fibrous sheath, tunica albuginea, which encases cavernosal tissue consisting of sinusoids and surrounding smooth muscle. The clitoris responds to sexual excitement by tumescence and erection, although this does not occur with the degree of pressure elevation as found during penile erection. The characteristics of the clitoral blood flow, however, approximately parallel those of the male. See K. Park et al., "Vasculogenic female sexual dysfunction: The hemodynamic basis for vaginal engorgement insufficiency and clitoral erectile insufficiency," Irat. J.
Impotence Res.
9:27 (1997).
Post-menopausal women and women with a history of vascular risk factors have been shovsm to have significantly more complaints of self reported female vaginal and clitoral dysfunctions than pre-menopausal women or women without vascular risk factors. Such problems include, but are not limited to, atherosclerosis-induced vaginal engorgement insufficiency and clitoral erectile insufficiency syndromes.
Determination whether a human female is suffering from poor blood flow or supply is readily made by a person skilled in the art using a number of readily available diagnostic procedures. The human vagina receives arterial blood supply from the vaginal artery, the vaginal branch of the uterine artery, the internal pudendal artery, and the vaginal branches of the middle rectal artery. Blood flow in these areas can readily be assessed by a number of techniques. Arterial blood can be obtained and the blood levels of cholesterol and triglycerides can be analyzed as a first step.
However, the preferred method is imaging.
While relatively non-invasive imaging is preferred, more invasive techniques can be used. For example, vaginal wall blood flow can be measured by laser Doppler flow probes placed into the vaginal muscularis layer within the spongy region of blood-filled spaces and vascular smooth muscle. Clitoral intracavernosal erectile tissue blood flow can be measured with a similar laser Doppler flow probe placed into the corporal bodies. The flow probes are connected to a laser Doppler flowmeter (Transonic Systems, Inc.) which is calibrated against an internal standard reading flow in units of ml/min/100 gm of tissue.
The laser Doppler probe uses the Doppler shift of a projected beam of laser light that registers on a photodetector. Static tissues will produce no Doppler shift in wavelength but moving red blood cells will produce a shift proportional to the red cell velocity.
4. Treatment of Male and Female Erectile Dysfunction It is not intended that the present invention be limited by the particular nature of the therapeutic preparation. In one embodiment, the (+) enantiomer of flosequinan is provided together with physiologically tolerable liquid, gel or solid carriers, diluents, adjuvants and excipients. In addition, enantiomers of flosequinan may be used together with other chemotherapeutic agents. On the other hand, formulations may also contain such normally employed additives as binders, fillers, Garners, preservatives, stabilizing agents, emulsifiers, buffers and excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. These compositions typically contain 1%-95% of active ingredient, preferably 2%-70%.
The present invention is not limited by the method of introduction of the therapeutic compound to the body. Among other methods, the present invention contemplates administering cutaneously, orally, intracavernosally, transurethrally, by standard injection (e.g. intravenous or intramuscular), intranasally or through respiratory inhalation.
It is believed that oral administration of the (+) flosequinan enantiomer and pharmaceutical compositions comprising the (+) enantiomer of flosequinan is an effective mode of administration. Peak plasma concentrations of flosequinan (+) enantiomer are observed 1-2 hours following oral administration, while peak metabolite plasma levels are observed about seven hours following oral dosage.
In one 15' embodiment, the enantiomer of flosequinan is introduced into the male or female orally. It is believed that an oral dosage up to approximately 200 milligrams of a racemic mixture of flosequinan is an effective oral dosage. It is also believed that the oral administration of a purified of enantiomer of flosequinan is effective at even lower dosages (e.g. less than 200 mg). While the present invention is not limited to a specific dosage level, in one embodiment, the male or female is an adult human and the oral dosage of a purified enantiomer of flosequinan is in a single dose per day of up to approximately two hundred milligrams, more preferably, between approximately fifty to approximately seventy-five milligrams. In an even more preferred embodiment, a purified enantiomer of flosequinan is administered in a single oral dose per day of between approximately twenty and approximately fifty, and even more preferably, between approximately ten and approximately twenty milligrams per day.
The enantiomers of flosequinan are water soluble and are soluble in many organic solvents. Thus, while the present invention is not limited by the form of oral administration, aqueous and organic solutions of flosequinan enantiomer for oral administration are contemplated. Likewise, flosequinan enantiomers can be associated with a solid pharmaceutical carrier for solid oral administration (i.e., in pill form). , One skilled in the art is able to readily prepare such solid formulations, and in one embodiment, the inactive ingredients include croscarmellose sodium, hydroxypropyl methylcellulose, lactose, magnesium stearate, methocel E5, microcrystalline cellulose, povidine, propylene glycol and titanium dioxide.
Flosequinan enantiomers and pharmaceutical compositions comprising flosequinan enantiomers may also be administered cutaneously in a carrier adapted for topical administration. Such carriers include creams, ointments, lotions, pastes, jellies, sprays, aerosols, bath oils, or other pharmaceutical carriers which accomplish direct contact between flosequinan and the pore of the skin. In general pharmaceutical preparations may comprise from about 0.001 % to about 10%, and preferably from about 0.01 to 5% by w/w of the active compound (e.g., flosequinan) in a suitable carrier. In some cases it may be necessary to dissolve the flosequinan enantiomer or pharmaceutical compositions comprising a flosequinan enantiomer in an appropriate solvent such as ethanol or DMSO (dimethylsulfoxide), and the like, to facilitate incorporation into a pharmaceutical preparation. Likewise, the present invention can be incorporated in other products associated with sexual activity. For example, a coated, erection inducing condom as disclosed in U.S. Pat. No. 4,29,991, hereby incorporated by reference, can be utilized with flosequinan enantiomers or pharmaceutical compositions comprising flosequinan enantiomers.
While the present invention is not limited by a specific method of introducing flosequinan enantiomers, and pharmaceutical compositions thereof, injection of flosequinan enantiomers, or a pharmaceutical composition thereof can be carried out by any conventional injection means (e.g., employing an hypodermic syringe and needle or a similar device such as the NovolinPen. sold by Squibb-Novo, Inc., Princeton, N.J., USA). This injection may be by subject injecting himself or by another person (such as a partner during sexual relations or a physician prior to sexual relations) injecting the male whose erection is to be induced. In one embodiment, flosequinan enantiomers are introduced intracavernously as described in U.S.
Patent No. 5,447,912 to Gerstenberg et al., hereby incorporated by reference.

Flosequinan enantiomers and pharmaceutical compositions comprising flosequinan enantiomers can be introduced intracavernosally in a physiologically acceptable composition. Such compositions are aqueous solutions that are physiologically acceptable for administration by intracavernosal injection into the penis. The physiologically acceptable carrier is selected such that it is not painful or irritating upon intracavernosal injection. The physiologically acceptable compositions will preferably be sterile at the time of administration by intracavernosal injection.
Among the physiologically acceptable compositions for use in the methods is physiological saline or phosphate buffered saline, in which flosequinan enantiomers or a pharmaceutical composition comprising enantiomers of flosequinan, is dissolved or suspended, such that the resulting composition is suitable for intracavernosal injection.
Such a physiologically acceptable composition can also include a non-irritant preservative, such as, e.g., benzalkonium chloride at 0.05% (w/v) to'0./2%
(w/v). As the skilled artisan will understand, there are numerous non-toxic salts of VIP, PHM
and a-adrenergic blockers that can be employed in a physiologically acceptable composition for use in the methods herein, including, among others, the chloride, bromide, acetate, sulfate, and mesylate salts.
In carrying out the methods, it is preferred that, for a period of dine between about 1 minute and about 15 minutes (preferably about 5 minutes-10 minutes), the penis is constricted near the base thereof and between the base and the point at which the injection into a corpus cavernosum occurs, in order to limit loss of injected fluid from the corpus cavernosum before the ingredients in the fluid, that are active in inducing erection, have been able to have erection-inducing effects. The constriction can be effected by any means known in the art, such as with a tourniquet, cuff, rubber band or the like, or even manually, in order to slow the release of the injected fluid and the pharmacologically active substances) therein into the general circulation.
Likewise, the present invention is not limited by a particular method for introducing flosequinan enantiomers or a pharmaceutical composition comprising enantiomers of flosequinan transurethrally. In one embodiment, the (+) flosequinan enantiomer, or a pharmaceutical composition thereof, is introduced to the urethra in a carrier as described for cutaneous administration. Devices and methods for transurethral introduction of pharmaceutical compositions is described in U.S.
Patent No. 5,474,535 to Place et al.; Voss, U.S. Pat. No. 4,801,587 and Kock, EPA
0357581, all hereby incorporated by reference.
Additional methods of the transurethral introduction of flosequinan enantiomers, or pharmaceutical compositions thereof, include the use of medicated catheters, such as those used to prevent or treat localized infections and irritation of the urethra and bladder (See U.S. Pat. No. 4,640,912, hereby incorporated by reference).
Alternatively, transurethral administration of pharmaceutical compositions is presented a in U.S. Pat. Nos. 4,478,822, 4,610,868, 4,640,912 and 4,746,508, all hereby incorporated by reference, and medicated urethral suppositories, inserts or plugs, typically containing anti-infective agents or spermicide are disclosed in U.S.
Pat. Nos.
1,897,423, 2,584,166, 2,696,209 and 3,373,746, all incorporated by reference.
While the present invention is not limited to the method of injecting enantiomers of flosequinan, or pharmaceutical compositions thereof, in the preferred embodiment, flosequinan or a pharmaceutical composition comprising flosequinan is , injected with a standard syringe. One skilled in the art would be capable of injecting flosequinan or a pharmaceutical composition comprising flosequinan with a carrier as described for intracavernosal injection.
Flosequinan enantiomers and pharmaceutical compositions comprising flosequinan enantiomers may also be administered intranasally. Formulations suitable for intranasal administration include ointments, creams, lotions, pastes, gels, sprays, aerosols, oils and other pharmaceutical carriers which accomplish direct contact between flosequinan or a pharmaceutical composition comprising flosequinan and the nasal cavity. Examples of pharmaceutical compositions administered intranasally are described in U.S. Patents Nos. 5,393,773 and 5,554,639 to Craig et al.; and 5,801,161 to Merkus, all hereby incorporated by reference.
Flosequinan enantiomers and pharmaceutical compositions comprising flosequinan enantiomers may also be administered through respiratory inhalation.
Formulations suitable for respiratory inhalation include ointments, creams, lotions, pastes, gels, sprays, aerosols, oils and other pharmaceutical carriers which accomplish direct contact between flosequinan enantiomers or a pharmaceutical composition comprising flosequinan enantiomers and the respiratory tract. Examples of pharmaceutical compositions administered through respiratory inhalation are described in U.S. Patent 4,552,891 to Hu et al.; 5,869,479 to I~reutner et al., and 5,864,037 to Chasis et al.
In some embodiments, intranasal administration and respiratory inhalation are the preferred modes of administration due to the ease of administration and faster onset of therapeutic activity. It is contemplated that intranasal administration and respiratory inhalation are advantageous as they may allow a smaller effective dosage to be administered than would be possible with the oral route of administration. A
preferred mode of administration comprises administration to the lung. Intrapulmonary delivery of pharmacologic agents to patients can be accomplished via aerosolization.
Alternatively, the agent may be administered to the lung through a bronchoscope. Of course, the therapeutic agents may be investigated for their efficacy via other routes of administration, including parenteral administration.
In one embodiment, the administration of the compositions of the present invention is accompanied by sexual stimulation to induce an erection. The sexual stimulation can,begin before or after the introduction of flosequinan or a pharmaceutical composition comprising flosequinan. If the stimulation begins after the injection, it is preferably begun within 5 to 10 minutes to insure that there is significant overlap of the pharmacological effects of the pharmaceutical composition administered and the stimulative effects of the sexual stimulation. Whether the stimulation begins before or after the injection, it will continue preferably at least until an erection sufficient for vaginal penetration is achieved.
Sexual stimulation as prescribed by these methods, includes any form of sexual stimulation that would induce an erection in a normal male who is not suffering from erectile insufficiency. The sexual stimulation can be that which occurs in the course of sexual relations between the subject and another person or can be outside sexual relations with another person. Examples of methods of sexual stimulation include, alone or in combination, touching or erotically manipulating erogenous areas of the genital organs or other erogenous parts of the body; providing visual stimulation, as with a sexually explicit media (e.g., pornographic film) or other form of sexually stimulative show or display. Additionally, providing vibratory stimulation to the penis, at between about 30 Hz and about 100 Hz with an amplitude of about 1 mm to about 5 ram, as can be provided, for example, by resting the penis on the table of a vibrating apparatus such as that of a Vibrector system (Multicept, Genofte, Denmark).
In inducing an erection in an impotent male outside of sexual relations, as, for example, when a physician induces an erection in a patient suffering from psychogenic impotence, a preferred method of sexual stimulation includes providing visual stimulation, as with a pornographic film, simultaneously with vibratory stimulation of the penis, as with a Vibrector system set to between about 30 Hz and about 60 Hz (usually about 50 Hz)in frequency and between about 1 mm and about 2.5 mm (usually about 2.2 mm) in amplitude.
From the above, it should be clear that the present invention provides methods of treatment.of sexual dysfunction (e.g. erectile dysfunction) with pharmaceutical agents. In particular, flosequinan enantiomers, or pharmaceutical compositions comprising flosequinan enantiomers, are administered therapeutically to patients having such dysfunction.
B. The Treatment of Cardiovascular Disease The present invention relates to methods for the treatment of cardiovascular disease. In one embodiment, the present invention contemplates compositions and methods for the treatment of CHF and hypertension. These compositions comprise quinolinones, including derivatives and enantiomers thereof. A variety of quinolinone derivatives are shown in Figure 2. Methods for producing antibiotic derivatives of a particular quinolone carboxylic acid skeleton are provided in U.S. Patent No.
4,623,650 to Gilligan et al., hereby incorporated by reference.
While the present invention is not limited by the nature of the derivatives, in one embodiment, the present invention encompasses the use of a variety of quinolinone derivatives (e.g., 5-bromoquinoline, 5-nitroisoquinoline, 8-nitroisoquinoline and 1-methylisoquinoline). One skilled in the art can readily produce such derivatives as set forth in McMurry, Organic Chemistry, 2nd Ed., Brooks/Cole Publishing, Belinont, CA
(1988), pages 1044-1045 and 1076.
In another embodiment, the present invention contemplates the use of methylthio and methylsulphinyl derivatives of quinolinone. In a preferred embodiment, the methylsulphinyl derivative is flosequinan (whether as a racemic mixture or as a purified enantiomer).
In another embodiment, the present invention contemplates compositions and methods for the treatment of CHF and hypertension in subjects who are not concurrently being treated with nitrites or nitrates. In a preferred embodiment, the present invention contemplates the administration of the enantiomers of flosequinan as a methods of treating CHF and hypertension. In another embodiment, the optically active derivatives and metabolites of flosequinan are also contemplated.
In one embodiment, a purified enantiomer.preparation of flosequinan is administered. It is contemplated that an enantiomer of flosequinan be administered cutaneously, by standard injection, intranasally, or through respiratory inhalation. It is not intended that the methods of the present invention be limited to the mode of administration of an enantiomer of flosequinan.
1. Evaluation Of Compounds For Efficacy As Anti-Hypertensives The antihypertensive effects exerted by the enantiomers of flosequinan is readily demonstrated by means of tests on standard laboratory animals. It is not intended that the present invention be limited by the species of animal used as a test platform. Suitable animal models include, but are not limited to, species including rodents, non-human primates, ovines, bovines, ruminants, lagomorphs, porcines, caprines, equines, canines, felines, and aves.
In one embodiment, female rats (weight range 180-240 g) of the Aoki-Okamoto strain of spontaneously hypertensive can be used. The rats axe divided into groups of four and will be fasted overnight before administration of either the (+) or (-) enantiomer of flosequinan. Blood pressure will be determined in the following way.
The rats are placed in a restraining cage, maintained at 38°C, with their tails protruding through holes in the cage. After 30 minutes in the cage, blood pressure will be measured using an inflatable cuff placed round the base of the tail and arterial pulsations will be monitored with a pneumatic pulse transducer. In other animal models, however, a limb or a phalanx as a site to monitor blood pressure. A
pressure, greater than the expected blood pressure, will be applied to the cuff and this pressure will be slowly reduced. The pressure in the cuff at which arterial pulsations reappears will be recorded as the blood pressure. The rats will be removed from the cage and each group will be orally administered a given dose of either the (+) or (-) enantiomer of flosequinan. In addition to the pre-dose reading, blood pressure will be measured at 1.5 and 5.0 hours after dosing. In one embodiment, a effective dosage will be defined as follows.
Either the (+) or (-) enantiomer of flosequinan will be tested initially at a given dose level (for example 90 mg/kg). If the compound is considered sufficiently active (giving a reduction of blood pressure equal to or greater than of approximately 15%
after correction) it will be retested at a lower dose level, for example 30 mg/kg. By testing at successively lower dose levels, a threshold antihypertensive dose (dose giving a reduction of blood pressure of between S and 15% after correction) will be determined. Compounds which are inactive at a particular dose level and which produce a reduction of blood pressure equal to or greater than 15% after correction at the next highest dose level will be designated as having a threshold antihypertensive dose within the range covered by the two dose levels.
The above described protocol is suited it a variety of hypertensive and normotensive animal models. In a preferred embodiment; a normotensive marmoset monkey will be used.

2. Evaluation Of Compounds For Efficacy In The Treatment of CHF
a. Surgical Preparation Mature M. fascicularis primates will be premeditated with ketamine hydrochloride (15 mg/kg im) and glycopyrrolate (0.01 mg/kg im). Following intubation, anesthesia will be maintained with 0.5% to 1% isoflurane. A left thoracotomy, including removal of the fourth or fifth rib, will be performed under sterile conditions and the pericardium will be opened and reflected to expose the heart.
. Silastic catheters attached to subcutaneous vascular access ports (VAPs;
Access Technology) will be inserted into the descending thoracic aorta and into the right atrium via the atrial appendage. To measure LV pressure, a solid-state micromanometer (Konigsberg Instruments) will be introduced via a stab wound in the ventricular apex and secured with a purse-string suture. A unipolar pacing lead will be sutured to the posterior wall of the LV and connected to a programmable pacemaker (Medtronic, Model Minix X340) that will be situated subcutaneously over the left chest wall. Internal electrocardiogram (ECG) leads will be sutured to the chest wall. All catheters and wires will be tunneled subcutaneously to the dorsal midline below the scapula. Transdermal titanium skin buttons (Konigsberg Instruments) attached to the wires from the LV pressure transducer and ECG leads will be secured along the midline, while the catheter VAPs will be situated in subcutaneous pockets. The chest will be closed in layers and negative intrapleural pressure restored via a temporary chest tube. Antibiotics (Cefazolin, 30 mg/kg) will be administered subcutaneously prior to surgery and postoperatively for 10 days (30 mg/kg bid). Buprenorphine (0.01 mg/kg s~ will be administered for analgesia immediately after surgery and as needed the first postoperative week. All monkeys will be allowed to recover for a minimum of 2 weeks after surgery and were conditioned to sit in upright/recliung primate restraint chairs prior to commencement of experimental investigations.
b. Hemodynamic Measurements Arterial blood pressure will be measured by inserting into the aortic VAP a saline-filled needle-tipped catheter, the other end of which was connected to a Statham P23 Xh pressure transducer. Intravenous agents will be delivered via a similar catheter extension set inserted into the right atrial VAP. Left ventricular pressure (LVP) will be measured via the Konigsberg micromanometer and will be cross-calibrated with the aortic pressure signal, and the transthoracic ECG will be measured using a Gould ECGBiotach amplifier (Gould Instrument Systems). All physiological signals will be sampled at 500 Hz using a computerized digital data acquisition system (Po-Ne-Mah, Gould Instrument Systems), while being simultaneously recorded on digital audio tape (Model RD 111T, Teac) and displayed on an MT 95000 eight-channel thermal-array recorder (Astro-Med). Measurements of heart rate (HR), mean arterial blood pressure (MAP), left ventricular systolic and end-diastolic blood pressures (LVSP and LVEDP, respectively), and the peak positive rate of change in LVP (+dP/dt",~) will derived using algorithm-based analyses of the digitized waveforms.
c. Myocardial Function Measurements LV dimensions will be recorded with the primates under ketamine sedation using M-mode echocardiographic imaging (Hewlett Packard Sons 100CF) of the LV
via a left transthoracic approach. Images will be recorded on VHS tape for subsequent calculation of LV fractional shortening and wall thickening percentage (WT) from the short axis and wall thickness dimensions. In addition, ejection fraction (EF) will be calculated by applying the Teicholz formula to the respective short axis dimensions measured at end-diastole and end-systole (EDV=[7/(2.4+LVDd)](LVDd3) and ESV=[7/(2.4+LVDS)](LVDS3) for end diastolic and end systolic volumes, respectively).
Utilizing simultaneous ECG recordings on the videotape, end-diastolic dimensions will be defined as occurring at the peak of the R-wave, while end-systole will be associated with the peak of the T-wave. Systolic WT will be then calculated as the difference between end-systolic and end-diastolic dimensions and is expressed as percent change from end-diastolic thickness [(EST - EDT)/EDT] x 100.
It is not intended, however, that the present invention be limited by the species of the animal model used to evaluate the efficacy of a given compound as potential CHF therapeutic. The above described protocol is suited it a variety of hypertensive and normotensive animal models. In a preferred embodiment, a normotensive marmoset monkey will be used.
The compositions utilized in the methods of the present invention comprise purified enantiomers of flosequinan, including derivatives thereof. Although the present invention is not limited by a specific means of producing enantiomers of flosequinan, methods of producing a racemic mixture of flosequinan (and methylsuphinyl /
methylthio derivatives of quinolinone) are set forth in U.S. Patent Nos.
5,079,264 and 5,011,931 to MacLean et al., hereby incorporated by reference. Moreover, a means of resolving the enantiomers of flosequinan is set forth in Morita et al., "Synthesis and Absolute Configuration of the Enantiomers of 7-Fluoro-1-methyl-3-(methylsulfmyl)-4(lI~-quinolinone (Flosequinan)," Chem. Pharm. Bull., 42(10): 2157-2160 (1994), hereby incorporated by reference.
3. Resolution of the (+) and (-) Enantiomers of Flosequinan In a preferred embodiment, the present invention contemplates the administration of a purified enantiomer of flosequinan. Many organic compounds, including flosequinan, exist in optically active forms (i.e., they have the ability to rotate the plane of plane-polarized light). In describing an optically active compound, the prefixes D and L or R and S are used to denote the absolute configuration of the molecule about its chiral center(s). The prefixes d and 1 or (+) and (-) are employed to designate the sign of rotation of plane-polarized light by the compound, with (-) or 1 meaning that the compound is levorotatory (rotates to the left). A compound prefixed with (+) or d is dextrorotatory (rotates to the right).
The present invention is not limited by any specific means of resolving the (+) and (-) enantiomers of flosequinan to obtain a substantially purified enantiomer of flosequinan. In one embodiment, the enantiomers were resolved according to the methods in Example 2.
It is not intended that the present invention be limited to any specific mechanism. However, the enantiomers of flosequinan can function as a vasodilator.

This vasodilation decreases the total peripheral resistance offered by the vasculature, thereby, decreasing blood pressure. Flosequinan has also been reported to be a selective inhibitor of phosphodiesterase III (PDE3). (See Gristwood et al., BY. J.
Pharmacol., 105: 985 (1992); Frodsham et al., Euf~. J. Plzarmacol. 211: 383 (1992)).
Inhibitors of PDE3 cause vasodilation leading to a concomitant reduction in arterial blood pressure. See Shiraishi et al., Br. J. Pharmacol., 123: 869-878 (1998).
It is not intended that the present invention be limited to any particular mechanism to reduce symptoms of cardiovascular disease. In one embodiment, the (+) and (-) enantiomers of flosequinan were subjected to biochemical assays to determine their respective percent inhibition of PDE3. In one embodiment, it was shown that the (+) enantiomer of flosequinan has an eight-fold increase in PDE3 inhibition as compared to that of the (-) enantiomer of flosequinan at the same molar concentration.
Moreover, in the same embodiment, the (+) enantiomer of flosequinan exhibited greater inhibition of PDE3 than a racemic mixture of flosequinan at the same molar concentration. Further data concerning this is provided in Example 3.
Moreover, it is not intended that the present invention be limited to any particular mechanism to reduce the side effects associated with various pharmacological treatments for cardiovascular disease. In one embodiment, a 100 ~,M
mixture of the (+) and (-) enantiomers of flosequinan were subjected to biochemical assays to determine their respective percent inhibition of PDE6 as compared to that of sildenafil citrate at a concentration of 1 ~M. In one embodiment, it was shown that the mixture of the (+) and (-) enantiomers of flosequinan exhibited approximately half as much inhibition of PDE6 as compared to that of a 100-fold lower molar concentration of sildenafil citrate. Further data concerning this is provided in Example 1.
4. Diagnosis of Cardiovascular Disease Determination whether an adult human is suffering from cardiovascular disease is readily made by a person skilled in the art using a number of readily available diagnostic procedures. Thus, an adult human suffering from cardiovascular disease can first be given a physical examination with particular attention to possible blood pressure or heart rhythm pathology, whereby many anatomical electrophysiological cardiovascular abnormalities can be detected.
Tests to determine hypertension may be performed by doppler investigation of the venous blood return system sufficiency. Blood pressures can be determined by a number of standard techniques.
Tests to determine myocardial infarction include detecting aberrations in blood enzymes. Following a myocardial infarction the damaged heart cells release of lactate dehydrogenase (LDH) subtypes result in circulating ratios that are not normally observed in a healthy person.
Test to determine angina pectoris include testing procedures as with myocardial infarction, only with the appearance of normal LDH subtype ratios.
Test to determine congestive heart failure include comparisons of blood pressures, cardiac output and stroke volume measurement. Congestive heart failure is a debilitating progressive condition that is fatal if immediate action is not taken.
5. Treatment of Cardiovascular Disease It is not intended that the present invention be limited by the particular nature of a preparation. In one embodiment, the (+) enantiomer of flosequinan is provided together with physiologically tolerable liquid, gel or solid carriers, diluents, adjutants and excipients. In addition, enantiomers of flosequinan may be used together with other chemotherapeutic agents. On the other hand, formulations may also contain such normally employed additives as binders, fillers, carriers, preservatives, stabilizing agents, emulsifiers, buffers and excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. These compositions typically contain 1%-95% of active ingredient, preferably 2%-70%.
The present invention is not limited by the method of introduction of the compound to the body. Among other methods, the 'present invention contemplates administering cutaneously, orally, or by standard injection (e.g.
intravenous).

The present invention also contemplates administering the enantiomers of flosequinan to the subject intranasally or through respiratory inhalation.
Formulations suitable for intranasal administration include ointments, creams, lotions, pastes, gels, sprays, aerosols, oils and other pharmaceutical carriers which accomplish direct contact between an enantiomer of flosequinan or a pharmaceutical composition comprising an enantiomer of flosequinan and the nasal cavity. Examples of pharmaceutical compositions administered intranasally are described in U.S. Patents 5,393,773 and 5,554,639 to Craig et al.; and 5,801,161 to Merkus, all hereby incorporated by reference. Formulations suitable for respiratory inhalation include ointments, creams, lotions, pastes, gels, sprays, aerosols, oils and other pharmaceutical carriers which accomplish direct contact between an enantiomer of flosequinan or a pharmaceutical composition comprising an enantiomer of flosequinan and the respiratory tract.
Examples of pharmaceutical compositions administered through respiratory inhalation are described in U.S. Patent 4,552,891 to Hu et al.; 5,869,479 to Kreutner et al., and 5,864,037 to Chasis et al., all hereby incorporated by reference.
In some embodiments, intranasal administration and respiratory inhalation are the preferred modes of administration due to the ease of administration and faster onset of therapeutic activity. It is contemplated that intranasal administration and respiratory inhalation are advantageous as they may allow a smaller effective dosage to be administered than would be possible with the oral route of administration. A
preferred mode of administration comprises administration to the lung. Intrapulmonary delivery of pharmacologic agents to subjects can be accomplished via aerosolization.
Alternatively, the agent may be administered to the lung through a bronchoscope. Of course, the therapeutic agents may be investigated for their efficacy via other routes of administration, including parenteral administration.
Oral administration of an enantiomer of flosequinan is an effective mode of administration, with a mean absolute bioavailability of 72% following a single dose of fifty milligrams. Peak plasma concentrations of an enantiomer of flosequinan are observed 1-2 hours following oral administration, while peak metabolite plasma levels are observed about seven hours following oral dosage. While the present invention is not limited to a specific dosage level, fox adult humans, in one preferred embodiment the dosage is a single dosage per day of 10 milligrams, while in another preferred embodiment the dosage is a single dosage per day of 25 milligrams, while in another preferred embodiment the dosage is a single dosage per day of 50 milligrams, while in yet another preferred embodiment the dosage is a single dosage per day of 75 milligrams. In another preferred embodiment, the dosage is a single dosage per day of approximately 125 milligrams and in another preferred embodiment, the dosage is a single dosage per day of approximately 150 milligrams. In another preferred embodiment, the dosage is a single dosage per day of approximately 200 milligrams.
Multiple dosage is also contemplated.
Flosequinan and its enantiomers are water soluble and soluble in many organic solvents. Thus, while the present invention is not limited by the form of oral administration, aqueous and organic solution of an enantiomer of flosequinan for oral administration is contemplated. Likewise, an enantiomer of flosequinan can be associated with a solid pharmaceutical carrier for solid oral administration (i.e., in pill form). One skilled in the art is able to readily prepare such solid formulations, and in one embodiment, the inactive ingredients include croscarmellose sodium, hydroxypropyl methylcellulose, lactose, magnesium stearate, methocel E5, microcrystalline cellulose, povidine, propylene glycol and titanium dioxide.
The enantiomers of flosequinan may also be administered cutaneously in a carrier adapted for topical administration. Such carriers include creams, ointments, lotions, pastes, jellies, sprays, aerosols, bath oils, or other pharmaceutical carriers which accomplish direct contact between an enantiomer of flosequinan and the pore of the skin. In general pharmaceutical preparations may comprise from about 0.001% to about 10%, and preferably from about 0.01 to 5% by w/w of the active compound (e.g., an enantiomer of flosequinan) in a suitable carrier. In some cases it may be necessary to dissolve the active compound in an appropriate solvent such as ethanol or DMSO (dimethylsulfoxide), and the like, to facilitate incorporation into a pharmaceutical preparation.

While the present invention is not limited by a specific method of introducing an enantiomer of flosequinan by injection, injection of an enantiomer of flosequinan (or a formulation comprising an enantiomer of flosequinan) can be carried out by any conventional injection means (e.g., employing an hypodermic syringe and needle or a similar device such as the NovolinPen. sold by Squibb-Novo, Inc., Princeton, N.J., USA). This injection may be by the subject injecting him or herself or by another person injecting the subject.
Flosequinan and its enantiomers can be introduced by injection in a physiologically acceptable composition. Such compositions are aqueous solutions that are physiologically acceptable for administration by injection. The physiologically acceptable carrier is selected such that it is not painful or irritating upon injection.
The physiologically acceptable compositions will preferably be sterile at the time of administration by injection.
Among the physiologically acceptable compositions for use in the methods is physiological saline or phosphate buffered saline, in which the enantiomers of flosequinan are dissolved or suspended, such that the resulting composition is suitable for injection. Such a physiologically acceptable composition can also include a non-irritant preservative, such as, e.g., benzalkonium chloride at 0.05% (w1v) to 0.l2%
(w/v). As the skilled artisan will understand, there are numerous non-toxic salts of VIP, PHM and oc-adrenergic blockers that can be employed in a physiologically acceptable composition for use in the methods herein, including, among others, the chloride, bromide, acetate, sulfate, and mesylate salts.
While the present invention is not limited to the method of injecting the enantiomers of flosequinan, in the preferred embodiment, it is injected with a standard syringe. One skilled in the art would be capable of injecting the enantiomers of flosequinan with .a carrier as described above.
In view of the above, the present invention provides methods for the treatment human cardiovascular disease with the enantiomers of flosequinan (or a formulation comprising the enantiomers of flosequinan).

6. Other Compounds Useful in the Treatment of Cardiovascular Disease The invention is further ,directed to pyridinone compounds with therapeutic activity, and to compositions containing such comp~iunds, wherein the compounds have the general formula I:
O

RI is hydrogen, lower alkyl optionally substituted by hydroxy or Cl_4 alkoxycarbonyl, allyl, propynyl or phenyl-lower alkyl in which the phenyl ring is optionally substituted by 1 or 2 Cl_4 alkoxy groups; RZ is hydrogen or lower alkyl; R3 is (X)m S(O)"R4, CORS, SR6, or S(OH) (O)NR~, wherein m is 0 or l, n is 0, 1, or 2, X is oxygen or lower alkylene, R~ is C,_ø alkyl, RS is hydroxyl, lower alkyl carbonyl, amino, or lower alkyl amino, and R6 and R7 are lower alkyl; and ring A represents an optionally substituted phenyl ring of the formula:

Riot wherein Rg, Rg and Rlo, which may be the same or different, are hydrogen, lower alkyl, lower allcoxy, lower alkanoyl, halo, tri-fluoromethyl, lower alkylthio, lower alkylsulphinyl, lower alkylsulphonyl, halogenated lower alkyl, halogenated lower alkoxy, cyano, phenyl, or phenyl substituted by 1 to 3 groups independently selected from lower alkyl, lower alkoxy and trifluoromethyl; or ring A represents an optionally substituted thiophene ring of the formula:

S
R~ i wherein Rll is hydrogen, lower alkyl, lower alkoxy, lower alkanoyl, halo, trifluoromethyl, lower alkylthio, phenyl, or phenyl substituted by halogen, or a pharmaceutically acceptable salt thereof.
The compounds of the general formula I have been found to have antihypertensive activity and cardiac activity in warm-blooded animals. The compounds, methods of making the compounds, antihypertensive and cardiac therapeutic compositions of the compounds, and methods for treating hypertension and heart failure using the compounds are described in US Patent 4,302,460, US
Patent 4,522,884, US Patent 4,855,291, US Patent 4,877,793, US Patent 4,710,506, US
Patent 4,772,614 and US Patent 4,997,840, the.disclosures of which patents are expressly incorporated herein, in their entirety, by reference.
In a preferred embodiment, the invention provides pharmaceutical compositions comprising compounds of the general formula I, and methods of using the compositions to treat subjects with symptoms of cardiovascular disease, such that said symptoms are reduced. As described above for the enantiomers of flosequinan, the invention is not limited by the particular nature of the pharmaceutical composition, or by the method of introduction of the active or therapeutic compound to the body. All of the treatment methods and compositions contemplated above axe also contemplated here for compounds of formula I. The active ingredient in the compositions is preferably administered in unit dosage form. In one embodiment, tablets and capsules may conveniently contain a unit dosage of the active compound of 1-500 rng/kg, more preferably 5-100 mg/kg and still more preferably 5-50 mglkg.

The compounds of formula I may contain one or more asymmetric centers and, therefore, can exist as enantiomers or diastereoisomers. Furthermore, certain compounds of formula I containing alkenyl groups may exist as cis-isomers or trans-isomers. In each case, the invention includes both mixtures and separate individual isomers. The compounds of formula I may also exist in tautomeric forms and the invention includes both mixtures and separate individual tautomers.
Preferred compounds of the general formula I for use in the pharmaceutical compositions and methods of the invention are compounds having the general fo~xnulas II and III:
R O

JI cin R~o N
R~
O

Ri ~ ~ (III) N
R~
wherein Ri, R3, R8, R~, Rlo, and R11 are defined as above.
- 49~ -With regard to compounds of formula II, still more preferred compounds have the formula IIA:
O
R~
Rio ~ N
R, wherein R3 is (X)m S(O)nR4, CORS, SR$, or S(OH) (O)NR~; and (a) Rlo is hydrogen and R~ is 6-lower alkoxy, 8-lower alkoxy, S-halo or 6-halo;
(b) R9 is hydrogen and Rlo is halo; lower alkyl, lower alkoxy, trifluoromethyl or lower alkyl-thio;
(e) Rlo is halo, lower alkoxy or lower alkyl and R9 is 6-lower alkyl, 6-lower alkoxy or 6-halo of a different value from Rlo; or (d) R9 and Rlo are hydrogen.
Preferred embodiments include compounds of formula IIA wherein Rl and RZ
are methyl, R9 is hydrogen and Rlo is halo, lower alkyl or trifluoromethyl.
More preferably, Rlo is halo or CI-C4 alkyl. In yet another preferred embodiment, R9 is G-lower alkoxy and Rlo is halo or lower alkoxy. In a further preferred embodiment, R9 is 6-halo and Rlo is lower alkoxy. In another preferred embodiment, Rlo is C,-C4 alkyl.
Thus, preferred embodiments include compounds of formula IIB, IIC, IID, IIE, IIF, IIIA and IIIB as follows:

U O
Can-S(O)nR4 R CORS
I (IIBj I
Rte Rt Rto \ N
Rt O O
(O)nRa R9 Rto ' I NJ II ~ (IIE) I ( ~ R \ N
R to 1 t R
t O S(OI~(O)R~
O
I S CORS
(IIIB) Rya \ N (I~ Rig I J
Rt N

s s(o)~~a Rt t I (IIIA) N
Rt Preferred compounds of formula IIB are those in which m is 1, n is 2, and X is oxygen. Preferred compounds of formula IIC include those in which m is 0, n is 1 or 2, and R4 is methyl. Preferred compounds of formula IID include those in which RS is amino or lower alkyl amino. Preferred compounds of formula IIE include those in which R6 is methyl. Preferred compounds of formula IIF include those in which R~ is methyl. Preferred compounds of formula IIIA include those in which n is 1 and R4 is methyl. Preferred compounds of formula IIIB include those in which RS is amino or lower alkyl amino.
Specific preferred compounds of these formulae include:

1-methyl-3-methylsulphinyl-4-quinolone, 7-fluoro-1-methyl-4-oxo-1,4-dihydro-quinolone-3-carboxamide, 4-methyl-7-oxo-4,7-dihydrothieno[3,2-b]pyridine-6-carboxamide, 4-methyl-6-methylsulphinyl-7(4H)-thieno[3,2-b]pyridinone, 7-chloro-1-methyl-3-methylsulphamoyl-4-quinolone, 1-methyl-4-oxo-1,4-dihydroquinol-3-yl methanesulphonate, 7-chloro-1-methyl-4-oxo-1,4-dihydroquinoline-3-carboxamide, fluoro-1-methyl-3-methylsulphonyl-4-quinolone, or 7-fluoro-1-methyl-3-methylthio-4-quinolone, or pharmaceutically acceptable salts thereof.
EXPERIMENTAL
The following examples serve to illustrate certain preferred embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof.
In the experimental disclosure which follows, the following abbreviations apply: eq (equivalents); M (Molar); ~,M (micromolar); N (Normal); mol (moles);
mmol (millimoles); ~mol (inicromoles); nmol (nanomoles); g (grams); mg (milligrams); ~,g (micrograms); L (liters); ml (milliliters); ~1 (microliters); cm (centimeters); rmn (millimeters); ~,m (micrometers); nm (nanometers);
°C (degrees Centigrade).
Example 1 In this example, a biochemical assay was performed to test the percentage of phosphodiesterase 6 (PDE6) inhibition of various molar concentrations of sildenafil citrate (Viagra) as compared to that of a 100 ~,M concentration of a racemic mixture of flosequinan as follows.
PDE6 partially purified from bovine retinal rod and activated by trypsin was used. In four separate reactions, Viagra at molar concentrations of 0.3 ~.M, 1.0 ~.M, and 3.0 ~M, and a 100 ~M racemic mixture of flosequinan were incubated with 0.2 ~,g/ml active PDE6 and 100 ~,M cGMP containing 0.1 ~,M [3H]cGMP in Tris buffer pH 7.5 for 20 minutes at 30°C. Each reaction was terminated by raising the temperature to 100°C for 2 minutes. The resulting GMP was converted to guanosine by addition of 10 mg/ml snake venom nucleotidase and further incubated at 30°C for minutes. Unhydrolyzed cGMP was bound to AGI-X2 resin, and [3HJguanosine remaining in the aqueous phase was quantitated by scintillation counting.
The results of the assays, as noted in the table below, indicate that although Viagra inhibits PDE6 around 50% at concentrations as low as 0.3 ~,M, such levels of 5 inhibition of PDE6 require greater than 100 ~,M amounts of flosequinan (i.
e. more than 300 times more compound on a molar basis). These empirical results could not be predicted.
Compound Concentration % Inhibition of Viagra 3.0 ~M ~7 10 Viagra 1.0 ~,M 62 Viagra 0.3 p,M 57 Flosequinan 100 ~.M 36 Example 2 In this example, the enantiomers of flosequinan were resolved by high-performance liquid chromatography (HPLC) as follows. A 5.0 g sample of a racemic mixture of flosequinan was resolved over a 10 cm ID x 50 cm L CHIRALCEL OD
HPLC column (Chiral Technologies, Exton, PA) at 25°C and with a flow rate of 1.0 rnUminute such that the column pressure was 37 bar. The mobile phase employed was 100% methanol and the detection of the mixture was performed at 270 nm. The (-) enantiomer had a retention time of 3.13 minutes, while the (+) enantiomer had a retention time of 4.40 minutes. A total of 2.1 g of the (-) enantiomer having an optical purity greater than 99% was produced. A total of 2.3 g of the (+) enantiomer having an optical purity greater than 99% was produced. (See Figure 1).

Example 3 In this example, a racemic mixture of flosequinan and the (+) and (-) enantiomers of flosequinan were subjected to biochemical enzyme assays to determine them respective percent inhibition of a variety of phosphodiesterases (PDE1-PDE6).
The reaction conditions for each PDE assay were as follows.
PDEl : PDE 1 partially purif ed from bovine heart was used. A racemic mixture of flosequinan, and each enantiomer of flosequinan, all at a molar concentration of 100 ~,M, were independently incubated with 13 ~Cg PDE1 enzyme, 1.0 ACM [3H]CAMP
and CaCl2/calmodulin in Tris buffer pH 7.5 for 20 minutes at 30°C. The reaction was terminated by boiling for 2 minutes, and the resulting AMP was converted to adenosine by addition of 10 mg/ml snake venom nucleotidase and further incubation at 30°C for 10 minutes. Unhydrolyzed cAMP was bound to AGI-X2 resin, and the remaining [3H]adenosine in the aqueous phase was quantitated by scintillation counting.
PDE2: PDE2 partially purified from human platelets was used. A racemic mixture of flosequinan, and each enantiomer of flosequinan, all at a molar concentration of 100 ~,M, were independently incubated with 23 ~.g PDE2 enzyme, 25 p,M cAMP containing 0.05 ~.M [3H]CAMP in Tris buffer pH 7.5 for 20 minutes at 30°C. The reaction was terminated by boiling for 2 minutes, and the resulting AMP
was converted to adenosine by addition of 10 mg/ml snake venom nucleotidase and further incubation at 30°C for 10 minutes. Unhydrolyzed cAMP was bound to AGI-X2 resin, and the remaining [3H]adenosine in the aqueous phase was quantitated by scintillation counting.
PDE3: PDE3 partially purified from human platelets was used. A racemic mixture of flosequinan, and each enantiomer of flosequinan, all at a molar concentration of 100 p,M, were independently incubated with 13 p.g PDE3 enzyme and 1 ~.M cAMP containing 0.01 ~M [3H]CAMP in Tris buffer pH 7.5 for 20 minutes at 30°C. The reaction was terminated by boiling for 2 minutes, and the resulting AMP
was converted to adenosine by addition of 10 mg/ml snake venom nucleotidase and further incubation at 30°C for 10 minutes. Unhydrolyzed CAMP was bound to AGI-X2 resin, and the remaining [3H]adenosine in the aqueous phase was quantitated by scintillation counting.
PDE4: PDE4 partially purified from human U-937 pronocytic cells was used.
A racemic mixture of flosequinan, and each enantiomer of flosequinan, all at a molar concentration of 100 ~,M, were independently incubated with 20 ~.g PDE4 enzyme and 1 p.M cAMP containing 0.01 ~.M [3H]CAMP in Tris buffer pH 7.5 for 20 minutes at 30°C. The reaction was terminated by boiling for 2 minutes, and the resulting AMP
was converted to adenosine by addition of 10 mg/ml snake venom nucleotidase and further incubation at 30°C for 10 minutes. Unhydrolyzed cAMP was bound to AGI-X2 resin, and the remaining [3H]adenosine in the aqueous phase was quantitated by scintillation counting.
PDES: PDES partially purified from human platelets was used. A racemic mixture of flosequinan, and each enantiomer of flosequinan, all at a molar concentration of 100 ~,M, were independently incubated with 120 pg PDES enzyme and 1 p.M cGMP containing 0.01 p,M [3H]cGMP in Tris buffer pH 7.5 for 20 minutes at 30°C. The reaction was terminated by boiling for 2 minutes, and the resulting GMP
was converted to guanosine by addition of 10 mg/ml snake venom nucleotidase and further incubation at 30°C for 10 minutes. Unhydrolyzed cGMP was bound to AGI-X2 resin, and the remaining [3H]guanosine in the aqueous phase was quantitated by scintillation counting.
PDE6: PDE6 partially purified from bovine retinal rod and activated by trypsin was used. A racemic mixture of flosequinan, and each enantiomer of flosequinan, all at a molar concentration of 100 p.M, were independently incubated with 0.2 ~.g/ml active PDE6 and 100 ~M cGMP containing 0.1 ~,M [3H]cGMP in Tris buffer pH 7.5 for 20 minutes at 30°C. Each reaction was terminated by boiling for 2 minutes. The resulting GMP was converted to guanosine by addition of 10 mg/ml snake venom nucleotidase, and further incubated at 30°C for 10 minutes.
Unhydrolyzed cGMP was bound to AGI-X2 resin, and [3H]guanosine remaining in the aqueous phase was quantitated by scintillation counting.

The results of the above PDE assays are presented in the following table. The results indicate that the (+) enantiomer of flosequinan demonstrated more PDE1 and PDE3 inhibitory activity when compared with the (-) enantiomer of flosequinan.
These empirical results could not be predicted.
Target % Inhibition % Inhibition % Inhibition Phosphodiesterasew/ w/ w/
100 ~.M racemic100 ~M (+)- 100 p,M (-)-mixture of flosequinan flosequinan flosequinan PDEl 31 28 11 Example 4 This example describes how certain preferred compounds of general formula (I) can be prepared.
a. 7-fluoro-1-methyl-4-oxo-1,4-dihydroquinolone-3-carboxamide can be prepared as described in Example 1 of US Patent 4,855,291.
(i) A mixture of ethyl 7-fluoro-4-hydroxyquinoline-3-carboxylate (4.7 g), anhydrous potassium carbonate (3.0 g), dimethyl sulphate (2.52 g) and butanone (200 ml) can be boiled under reflux for 14 hours. The solvent can be evaporated and the residue can be triturated with dichloromethane (150 ml).
The mixture can be filtered and the filtrate evaporated to a small volume.

Diethyl ether can be added, causing a solid to precipitate. The solid can be collected, washed with ether, dried and recrystallized from industrial methylated spirit to give the compound ethyl 7- fluoro-1-methyl-4-oxo-1,4-dihydroquinoline-3-carboxylate, m.p. 164°C-166°C.
(ii) A mixture of 19.0 g of the above carboxylate ester, aqueous ammonia (specific gravity 0.88, 750 ml) and caproyl alcohol (2 drops) can be stirred on a steam bath for 1.5 hours, then cooled to room temperature.
The solid product can be collected by filtration and recrystallized from industrial methylated spirit/water 3:2 to give the compound 7-fluoro-1-methyl-4-oxo-1,4-dihydroquinoline3-carboxamide, m.p.
317°C-318°C.
b. 4-methyl-7-oxo-4,7-dihydrothieno[3,2-b]pyridine-6-carboxamide can be prepared as described in Example 1 of US Patent 4,877,793.
(i) Dimethyl sulphate (3.9 ml) can be added to a stirred solution of ethyl 1S 7-hydroxythieno[3,2-b]-pyridine-6-carboxylate (4.63 g) and potassium hydroxide (3.5 g) in water (SO ml) at 0°C-5°C. More water (20 ml) can be added and the mixture can be stirred at ambient temperature for 24 hours. The solid product can be collected by filtration, washed with water and dried to give the compound ethyl 4-methyl-7-oxo-4,7-dihydrothieno[3,2-b]pyridine-6-caxboxylate, m.p.
122°C-128°C.
(ii) A mixture of 3.0 g of the product from (i) above and aqueous ammonia (specific gravity 0.880, 60 ml) can be stirred and heated on a steam bath. Effervescence will occur and ocean-1-of (2 ml) and more aqueous 2S ammonia (specific gravity 0.880, 20 ml) can be added and heating on the steam bath can be continued overnight. The mixture can then be cooled to ambient temperature and the solid product collected by filtration, dried and crystallized from industrial methylated spirit to give the compound 4-methyl-7-oxo-4,7-dihydrothieno[3,2-b]pyridine-6-carboxamide, m.p.
255°C-258°C.
c. 4-methyl-6-methylsulphinyl-7(4H)-thieno[3,2-b]pyridinone can be prepared as described in Example 2 of US Patent 4,710,506.
A solution of 3-chloroperbenzoic acid (85%; 1.63 g) in dichloromethane (60 ml) can be added dropwise during 20 minutes to a stirred solution of 4-methyl-6-methylthiothieno[3,2-b]pyrid-7(4H)-one (2.0 g) in dichloromethane (60 ml) at 0°C-5°C. After 4 hours, more 3-chloroperbenzoic acid (0.15 g) in dichloromethane (10 ml) can be added and the mixture stirred overnight at ambient temperature.
More 3-chloroperbenzoic acid (0.15 g) in dichloromethane (10 ml) can be added and the mixture can again be stirred overnight at ambient temperature. The resulting solution can be extracted with saturated aqueous sodium bicarbonate solution (5 x 150 ml) and saturated aqueous sodium chloride solution (1 x 150 ml) and the organic phase can be discarded. The aqueous extracts are combined and extracted with dichloromethane (S x 100 ml). The organic extracts are combined, dried over anhydrous magnesium sulphate and evaporated to give a solid product. This product can be crystallized from industrial methylated spirit to give the compound 4-methyl-6-methylsulphinylthieno[3,2-b]pyrid-7(4H)-one, m.p. 174°C-176°C.
d. 7-chloro-1-methyl-3-methylsulphamoyl-4-quinolone can be prepared as described in Example 4 of US Patent 4,772,614.
(i) 7-Chloro-1-methyl-4-quinolone (6.9 g) and chlorosulphonic acid (14 ml) can be stirred and heated at 140°C for 2 hours. The reaction mixture can be cooled to room temperature and carefully added dropwise to ice water (200 ml). The solid which forms can be collected, washed with water and dried in air to give the compound 7-chloro-1-methyl-4-oxo-1,4-dihydroquinoline-3-sulphoi~yl chloride, m.p. >300 °C.
(ii) 6.5 g of the above sulphonyl chloride and aqueous methylamine (30% w/v;

ml) can be stirred at room temperature for 3 hours. The resulting solid can be collected, washed with water and crystallized from dichloromethane/industrial methylated spirit 1:1. The product can be collected and partitioned between water (200 ml) and dichloromethane (200 ml). The organic layer can be separated, dried over anhydrous sodium sulphate and evaporated to dryness.
The residue can be crystallized from industrial methylated spirit to give the compound 7-chloro-1,N-dimethyl-4-oxo-1,4-dihydroquinoline-3-sulphonamide, m.p. 220 °C-223 °C.
e. 7-chloro-1-methyl-4-oxo-1,4-dihydroquinoline-3-carboxamide can be prepared as described in Example 4 of US Patent 4,855,291.
A mixture of ethyl 7-chloro-1-methyl-4-oxo-1,4-dihydroquinoline-3-carboxylate (S.0 g) and aqueous ammonia (specific gravity 0.88, 100 ml) can be stirred and heated on a steam bath for 3.5 hours. More aqueous ammonia (100 ml) can be added and heating continued for a further 21 hours. The mixture can be cooled in ice.
The solid product can be collected by filtration and dried to give the compound 7-chloro-1-methyl-4-oxo-1,4-dihydroquinoline-3-carboxamide, m.p.
>240°C.
From the above, it should be clear that the present invention provides methods of treatment of symptoms of cardiovascular disease with pharmaceutical agents.
In particular, quinolinone enantiomers are administered therapeutically to subjects having such symptoms.
All publications and patents mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those sleilled in the art are intended to be within the scope of the following claims.

Claims (21)

We claim:

1. A method, comprising:
a) providing:
i) a patient suffering from one or more symptoms of sexual dysfunction, and ii) a pharmaceutical composition comprising a purified enantiomer of flosequinan, or a pharmaceutically acceptable salt thereof; and b) administering said pharmaceutical composition to said patient such that at least one of said one or more symptoms are reduced.

2. The method of Claim 1, wherein said purified enantiomer of flosequinan is a (+) enantiomer.

3. The method of Claim 1, wherein said composition is substantially free of the (-) enantiomer of flosequinan.

4. The method of Claim 1, wherein said patient is a male.

5. The method of Claim 1, wherein said patient is a female.

6. The method of Claim 1, wherein said patient is free from cardiac disease.

7. The method of Claim 1, wherein said administering step is selected from the group consisting of intranasal administration and respiratory inhalation.

8. A method, comprising:
a) providing:

i) a patient suffering from symptoms of sexual dysfunction who is not being treated with a drug that causes hypotensive effects, and ii) a pharmaceutical composition comprising a purified enantiomer of flosequinan, or a pharmaceutically acceptable salt thereof; and b) administering said pharmaceutical composition to said patient such that such symptoms are reduced.

9. The method of Claim 8, wherein said administering step is selected from the group consisting of intranasal administration and respiratory inhalation.

10. The method of Claim 8 wherein, said patient has not been treated in the past with a drug that causes hypotensive effects.

11. The method of Claim 8, wherein said purified enantiomer of flosequinan is a (+) enantiomer.

12. The method of Claim 8, wherein said composition is substantially free of the (-) enantiomer of flosequinan.

13. The method of Claim 8, wherein said patient is a male.

14. The method of Claim 8, wherein said patient is a female.

15. A method, comprising:
a) providing:
i) a patient suffering from symptoms of sexual dysfunction who is not being treated with a nitrite or nitrate, and ii) a pharmaceutical composition comprising a purified enantiomer of flosequinan, or a pharmaceutically acceptable salt thereof; and b) administering said pharmaceutical composition to said patient such that such symptoms are reduced.

16. The method of Claim 15, wherein said administering step is selected from the group consisting of intranasal administration and respiratory inhalation.

17. The method of Claim 15, wherein said nitrate is selected from the group consisting of glyceryl trinitrate, isosorbide dinitrate, isosorbide-5'-mononitrate and erythrityl tetranitrate.

18. The method of Claim 15, wherein said purified enantiomer of flosequinan is a (+) enantiomer.

19. The method of Claim 15, wherein said composition is substantially free of the (-) enantiomer of flosequinan.

20. The method of Claim 15, wherein said patient is a male.

21. The method of Claim 15, wherein said patient is a female.