HK1173372A - Use of brimonidine for preventing and reducing the severity of stress-associated conditions - Google Patents

Description

Methods for preventing and reducing the severity of stress-related conditions

The present application is a divisional application of an invention patent application having an application date of 2004, 6/22, application No. 200480017842.2, entitled "method for preventing and reducing the severity of stress-related disorders".

Background

Technical Field

The present invention relates generally to the sympathetic nervous system and various stress-related conditions, and in particular to the alpha-2 adrenergic agonist brimonidine.

Background

Stress-related or exacerbating disorders are mediated at least in part by the sympathetic nervous system. Such stress-related disorders include, but are not limited to, gastrointestinal disorders; irritable bowel syndrome; dyspepsia; tachycardia is treated; a panic attack; insulin resistance; type II diabetes; skin diseases; disorders of muscle contraction such as tension-type headache; sensory hypersensitivity associated with migraine, such as nausea, photophobia, and phonophobia; and stress-related behavioral abnormalities such as binge eating and drug dependence.

Unfortunately, treatment of the stress-related conditions described above is not effective or satisfactory due to, for example, deleterious side effects, such as sedation. Therefore, there is a need for a new method to prevent or reduce the severity of stress-related disorders. The present invention satisfies the above-identified needs and provides related advantages.

Disclosure of Invention

The present invention provides a method for preventing or reducing the severity of a stress-related disorder in a subject by systemically administering to the subject an effective amount of brimonidine or a pharmaceutically acceptable salt, ester, amide, stereoisomer or racemic mixture thereof, wherein the stress-related disorder is one of: gastrointestinal disorders; irritable bowel syndrome; dyspepsia; tachycardia is treated; a panic attack; insulin resistance; type II diabetes; non-inflammatory skin diseases; disorders of muscle contraction; hyperesthesia with migraine; or stress-related behavioral abnormalities.

In one embodiment, the methods of the invention prevent or reduce the severity of gastrointestinal disorders. In another embodiment, the methods of the invention prevent or reduce the severity of irritable bowel syndrome or dyspepsia. In another embodiment, the methods of the invention prevent or reduce tachycardia other than tachycardia associated with myocardial ischemia, for example tachycardia associated with a pulmonary disorder. In yet another embodiment, the method of the invention prevents or reduces the severity of panic attacks. In another embodiment, the methods of the invention prevent or reduce the severity of insulin resistance, or prevent or reduce the severity of type II diabetes. In another embodiment, the methods of the invention prevent or reduce the severity of non-inflammatory skin disorders. In another embodiment, the methods of the invention prevent or reduce the severity of a muscle contraction disorder, e.g., a skeletal muscle contraction disorder or a smooth muscle contraction disorder, such as a smooth muscle contraction disorder with cystitis or non-bacterial prostatitis, or a muscle contraction disorder with tension-type headache. In another embodiment, the methods of the invention prevent or reduce the severity of hyperesthesia associated with migraine. In another embodiment, the methods of the invention prevent or reduce the severity of hyperesthesia associated with stress-related behavioral abnormalities. In the methods of the invention, an effective amount of brimonidine may be administered by any of a variety of methods including, but not limited to, oral administration, topical administration, intravenous administration, or administration via a patch (patch).

Drawings

Figure 1 shows tactile hypersensitivity observed with several different chemical models. Each experimental group included 5-6 wild-type mice. The evaluation of tactile hypersensitivity is as follows; sensitization scores measured every 5 minutes over a 35 minute measurement period were summed and the mean +/-SEM calculated. Unpaired two-tailed t-test (unpaired two-tailed t-test) was used for each group (^*p＜0.1，^**p < 0.01) compared to vehicle controls. (a) Spinal injection of the alpha-1 agonist phenylephrine induces dose-dependent tactile hypersensitivity. Various doses of phenylephrine (filled circles) were injected. Alpha-1 antagonist 5-MU (30ug/kg i.p.; filled squares) was administered 15 minutes prior to intrathecal administration of 30ng phenylephrine. (b) Systemic administration of phenylephrine induces dose-dependent tactile hypersensitivity. Different doses of phenylephrine (filled circles) were injected intraperitoneally. Alpha-1 antagonist 5-MU (30ug/kg i.p.; filled squares) was administered 15 minutes prior to the administration of 30ng/kg phenylephrine. (c) Selective administration of EP to the spine₁/EP₃The agonist sulprostone induces dose-dependent chemical tactile hypersensitivity. The dose of sulprostone (filled circles) injected intrathecally was increased. An EP injection 15 minutes before administration of 200ng sulprostone₁Antagonist (100ng/kg i.t.; filled squares). (d) Spinal NMDA induces dose-responsive tactile hypersensitivity. Different doses of NMDA (filled circles) were injected intrathecally. The NMDA antagonist, memantine (1ug i.t.; filled squares), was administered 15 minutes prior to 100ng of NMDA.

FIG. 2 shows that increased sympathetic tone in α -2A and α -2C knockout mice enhances exposure to alpha-1 receptor activationAnd (3) allergy induction. Wild type (filled circles), α -2A knockout (filled squares) and α -2C knockout (filled triangles) mice were injected intraperitoneally with increasing doses of phenylephrine and tested for tactile hypersensitivity. alpha-2A knockout mice were pretreated with 50mg/kg i.p. guanethidine to produce temporary chemical sympathetic nerve blockade 24-30 hours prior to i.p. phenylephrine (open squares). Each group of mice consisted of 5-6 animals. Calculate mean sensitization score and SEM and use unpaired two-tailed t-test (^*p＜.01，^**p <. 001) was compared to the vehicle control group.

Figure 3 shows that the sympathetic nervous system increases sulprostone-induced tactile hypersensitivity. Wild-type (filled circles), α -2A knockout (filled squares), and α -2C knockout (filled triangles) mice were intrathecally injected with increased doses of sulprostone and tested for tactile hypersensitivity. alpha-2A knockout mice were pretreated with guanethidine (50mg/kgi. p.) to create a temporary chemical sympathetic nerve block 24 hours prior to intrathecal sulprostone injection (open squares). Each group of mice consisted of 5-6 animals. Calculate mean sensitization score and SEM and use unpaired two-tailed t-test (^*p＜.01，^**p <. 001) was compared to the vehicle control group.

FIG. 4 shows that α -2 knockout mice do not exhibit NMDA-alteration induced tactile hypersensitivity. Wild type (filled circles), α -2A knockout (filled squares) and α -2C knockout (filled triangles) mice were injected intrathecally with increasing doses of NMDA. Tactile hypersensitivity was scored for 5-6 mice in each group. Calculate the average reaction and SEM and use unpaired two-tailed t-test (^*p＜.01，^**p <. 001) was compared to the vehicle control group.

Figure 5 shows that the alpha adrenergic agonists differ in the relief of sympathetically enhanced hyperesthesia. Scoring the responses of 5-6 mice in each group; and the average reaction and SEM calculated as described above. Unpaired two-tailed t-test was used for each drug treatment group (^*p＜.01，^**p <. 001) was compared to the vehicle control group. (a)Spinal administration of brimonidine and clonidine alleviated NMDA-induced tactile hypersensitivity in wild-type mice. Mice were injected intrathecally with DMSO vehicle or intrathecally co-injected with 100ng NMDA and saline, 0.4 μ g brimonidine (UK14304) or 1 μ g clonidine ("Clon"). (b) Spinal administration of brimonidine and clonidine alleviated sulprostone-induced tactile hypersensitivity in wild-type mice. Mice were injected intrathecally with DMSO vehicle or intrathecally co-injected with 200ng sulprostone with saline, 0.4 μ g brimonidine (UK14304) or 0.4 μ g clonidine. (c) Spinal administration of brimonidine and clonidine alleviated NMDA-induced tactile hypersensitivity in α -2C knockout mice, but did not alleviate α -2A knockout mice. Mice were injected intrathecally with DMSO vehicle or intrathecally co-injected with 100ng NMDA and saline, 0.4 μ g brimonidine (UK14304) or 1 μ g clonidine. (d) Brimonidine and clonidine injected spinally in α -2C knockout mice differ in their ability to alleviate sulprostone-induced tactile hypersensitivity. Mice were injected intrathecally with DMSO vehicle or intrathecally co-injected with 200ng (. alpha. -2C knockout) or 30ng (. alpha. -2A knockout) sulprostone and saline, 0.4. mu.g brimonidine (UK14304) or 0.4. mu.g clonidine. In alpha-2A knockout mice, there is no alpha-2 agonist analgesic effect; clonidine analgesia was also lost in α -2C knockout mice.

Figure 6 shows that brimonidine can alleviate sulprostone-induced tactile hypersensitivity without producing sedation, but clonidine or tizanidine does not. The dose response antiallergic and sedative effects of three alpha-2 agonists (tizanidine, triangle; clonidine, square; brimonidine, circle) were compared in a sulprostone-induced tactile hypersensitivity model and a motor activity model, respectively. The average total score of the sensitivity and the standard deviation of the average were calculated and represented as a solid line (left axis). The locomotor activity of the treated animals relative to vehicle is expressed as a percentage, and percent sedation is calculated by subtracting the percent locomotor activity from 100% and is shown as a dashed line (right axis).

FIG. 7 shows the changes in alpha-2/alpha-1 agonist selectivity for the alpha adrenergic agonists clonidine and brimonidine. Detection using in vitro cell-based functional assaysIncreasing concentrations of phenylephrine (filled squares), clonidine (filled diamonds), tizanidine (filled circles), dexmedetomidine (filled triangles), and brimonidine (filled inverted triangles) for alpha-1 and alpha-2 agonist activity. (a, B) alpha 1-1A agonist activity and alpha 2-1B agonist activity of an alpha 0 adrenergic agonist. The increase in intracellular calcium was determined by measuring changes in the fluorescence of calcium-sensitive dyes in HEK cells stably expressing the bovine alpha 4-1A receptor (a) or the hamster alpha 5-1B receptor (B) after addition of various concentrations of alpha 3 adrenergic agonists. Each agonist was tested 6-15 times, repeated 3 times each, and the mean fluorescence and SEM at each concentration were calculated. The results of a typical experiment are shown in the figure. (C, d) alpha-2A and alpha-2C agonist activity of alpha adrenergic agonists. Inhibition of cAMP accumulation induced by forskolin (forskolin) was determined in PC12 cells stably expressing either human α -2A receptor (C) or human α -2C receptor (d) after addition of various concentrations of α adrenergic agonist. Each agonist was tested 3-5 times, repeated 3 times each, and the average% inhibition and SEM at each concentration were calculated. The results of a typical experiment are shown in the figure. (e) Co-administration of prazosin and clonidine restored clonidine-mediated analgesia in α -2C knockout mice. Wild-type mice ("WT", open bars) and α -2C knockout mice ("2 CKO", hatched bars) were injected with vehicle, prazosin ("Praz", 100ng/kg i.p.), sulprostone ("Sulp", 200ng i.t.), clonidine ("Clon", 400ng i.t.), or various combinations as shown. Tactile hypersensitivity was scored for 5-6 mice in each group and the mean response and SEM calculated. Unpaired two-tailed t-test was used for each drug treatment group (^*p＜.01，^**p <. 001) was compared to the vehicle control group.

Detailed Description

Adrenergic receptors mediate physiological responses to catecholamines, norepinephrine, and epinephrine, and are members of the G protein-coupled receptor superfamily that have seven transmembrane domains. These receptors, which are pharmacologically classified into α -1, α -2 and β adrenergic receptor types, are involved in a variety of physiological functions, including cardiovascular and central nervous system functions. The α adrenergic receptors mediate most agonistic functions: alpha-1 adrenergic receptors generally mediate responses in effector organs, while alpha-2 adrenergic receptors are present both postsynaptic and presynaptic and regulate the release of neurotransmitters therein. At present, agonists of alpha-2 adrenergic receptors have been clinically used for the treatment of hypertension, glaucoma, spasticity and attention deficit disorder and for the suppression of drug withdrawal (opiate with dry) and as an adjuvant drug for general anesthesia.

At present, the α -2 adrenergic receptor is divided into three subtypes according to its pharmacological and molecular characterization: α -2A/D (α -2A in humans and α -2D in rats); alpha-2B; and alpha-2C (Bylund et al,Pharmacol.Rev.46: 121-136 (1994); and Hein and Kobilka,Neuropharmacol.34: 357 to 366 (1995)). The α -2A and α -2B subtypes modulate arterial constriction in some vascular beds, while the α -2A and α -2C subtypes mediate feedback inhibition of norepinephrine release from sympathetic nerve endings. The alpha-2A subtype also mediates the various central effects of alpha-2 adrenergic agonists (Calzada and artiano,Pharmacol.Res.44: 195-208 (2001); hein and the like,Ann.NY Acad.Science881: 265 to 271 (1999); and Ruffolo (editorial),α-Adrenoreceptors：Molecular Biology， Biochemistry and Pharmacology S.Karger Publisher’s Inc.Farmington，CT(1991))。

previous studies have shown the affinity (K) of noradrenaline for the alpha-2C receptor_i650nM) is higher than the affinity (K) for the alpha-2A receptor_i5800 nM; link, and the like, in a computer system,Mol.Pharm.42: 16-27 (1992)). Thus, in the case of low concentrations of noradrenaline, the autoamtic inhibition of noradrenaline release is mediated through the alpha-2C receptor, whereas in the case of high concentrations of noradrenaline, it is mediated through the alpha-2A receptor (Altman et al,MOL.Pharm.56: 154 to 161 (1999). As a result, feedback inhibition of the norepinephrine-based release is mediated by the α -2C receptor, whereas α -2A receptor mediates release under high frequency stimulationFeedback inhibition (Hein et al,Ann.N.Y.Acad.Sci.881: 256 to 271 (1999)). As disclosed in example II of the present invention, α -2C knockout mice are less inhibited presynaptically by sympathetic efferent under basal (or low frequency stimulation) conditions and are therefore more sensitive to increased α -1 receptor activity following phenylephrine treatment (see FIG. 2). Furthermore, as shown in FIG. 3, α -2A knockout mice are more sensitive to sulprostone-induced tactile hypersensitivity, whereas in α -2C knockout mice, sulprostone sensitivity is the same as that of wild-type mice. These results indicate that sulprostone treatment produces high frequency sympathetic stimulation, as evidenced by the fact that only α -2A knockout mice lacking presynaptic inhibition of high frequency sympathetic efferents exhibit a decrease in the threshold for sulprostone-induced tactile hypersensitivity.

As further disclosed in example III of the present invention, brimonidine has analgesic effects in sulprostone-induced tactile hypersensitivity wild-type mice and alpha-2C knockout mice. In contrast, clonidine had an analgesic effect in wild-type mice, but not in α -2C knockout mice (compare FIGS. 5b and 5 d). As expected, neither clonidine nor brimonidine was analgesic in α -2A knockout mice, which lack the spinal α -2A adrenergic receptor that mediates analgesic activity. Thus, α -2C knockout mice treated with sulprostone, as a model in sympathetically enhanced conditions, where the activity of the pan agonists brimonidine and clonidine were significantly different. Other results disclosed herein indicate that in wild type mice, brimonidine, but not other pan agonists such as tizanidine or clonidine, had analgesic activity without concomitant sedation (see figure 6). Furthermore, brimonidine was more selective (more than 1000-fold) for the α -2 adrenergic receptor than the α -1 receptor in functional assays than other pan agonists, such as clonidine and tizanidine, which exhibited less than 10-fold selectivity (see figure 7 and table 2). These results indicate that the pan agonists brimonidine and clonidine differ in functional activity and that alpha-2/alpha-1 functional selectivity can advantageously treat sympathetically-enhanced conditions, such as stress-related conditions, without concomitant sedation.

Dyspepsia has been called biopsychological disorder and is often characterized in part by upper abdominal discomfort after meals. In addition to upper abdominal discomfort or pain after meals, dyspepsia can also be characterized by: i.e. early satiety, nausea, vomiting, abdominal distension, bloating or anorexia in the absence of disease in the organ (thumbshirn,Gut51 supplement 1: i 63-66 (2002; Anderson,Dorland’s Illustrated Medical Dictionary28 th edition, w.b. saunder's Company, philadelphia (1994))).

The methods of the invention can be used to prevent or reduce the severity of dyspepsia, which is a term used to denote digestive damage. Any of a variety of dyspepsia can be treated by the methods of the present invention. The term dyspepsia includes, but is not limited to, acidic dyspepsia, which is associated with gastric hyperacidity; appendiceal dyspepsia, also known as appendiceal dyspepsia, in which the symptoms of dyspepsia are accompanied by chronic appendicitis; catarrhal dyspepsia, which is accompanied by gastritis; rice flour dyspepsia, a condition of starch malnutrition found in malnourished infants; gallstone dyspepsia, which involves the onset of sudden dyspepsia associated with gallbladder disturbances; colonic dyspepsia, which is related to large bowel dysfunction; fermentative dyspepsia, which is characterized by fermentation of ingested food; flatulent dyspepsia, which is associated with gas produced in the stomach and usually involves upper abdominal discomfort with frequent hiccups; gastric dyspepsia, which is caused by the stomach; intestinal dyspepsia, which is caused by the intestine. It will be appreciated that the above and other mild or acute symptomatic forms of the condition are included in the definition of "dyspepsia" as described herein. In one embodiment, the method of the invention is used to prevent or reduce the severity of dyspepsia other than dyspepsia associated with gastritis.

In another embodiment, the invention relates to the treatment of gastrointestinal disorders. Inflammatory Bowel Disease (IBD) or Irritable Bowel Syndrome (IBS) is a gastrointestinal disease that affects half of the americans throughout their lifetime, with IBD causing losses of more than $ 26 billion and IBS causing losses of more than $ 80 billion. For visceral hypersensitivity associated with IBD, IBS and other gastrointestinal disorders including inflammatory gastrointestinal disorders, the frequency or severity is exacerbated by stress. As disclosed herein, the methods of the invention can be used to prevent or reduce the severity of visceral hypersensitivity related to stress-related gastrointestinal disorders such as, but not limited to, Ulcerative Colitis (UC), Crohn's Disease (CD), or Irritable Bowel Syndrome (IBS). Accordingly, the present invention provides a method for preventing or reducing the severity of visceral hypersensitivity associated with stress-related gastrointestinal disorders in a subject by systemically administering to said subject an effective amount of brimonidine or a pharmaceutically acceptable salt, ester, amide, stereoisomer or racemic mixture thereof.

The methods of the invention may also be used to prevent or reduce the severity of tachycardia that is not accompanied by myocardial ischemia. The term "tachycardia" as used herein means tachycardia and includes tachyarrhythmia. In adults, the term tachycardia generally refers to heart rates greater than 100 beats/minute. The term tachycardia encompasses tachycardias associated with a variety of diseases (other than myocardial ischemia) including, but not limited to: paroxysmal tachycardia, wherein the tachycardia has a sudden onset and cessation and is ventricular or supraventricular; and non-paroxysmal tachycardia, which is a slow-onset tachycardia, the heart rate is usually 70-130 times/min. In one embodiment, the methods of the invention prevent or reduce the severity of autonomic tachycardia that is not accompanied by myocardial ischemia. In another embodiment, the methods of the invention prevent or reduce the severity of central tachycardia in an adult individual. In yet another embodiment, the methods of the invention prevent or reduce the severity of central tachycardia in a pediatric individual.

Tachycardias to be treated according to the methods of the invention include tachycardias produced by any part of the heart, such as ventricular tachycardias and supraventricular tachycardias, which may be classified, for example, as atrial tachycardias and junctional (nodal) tachycardias. Thus, the methods of the invention can be used to prevent or reduce the severity of, for example, ventricular tachycardia, an abnormally rapid ventricular rhythm with abnormal ventricular stimulation that occurs in the ventricles, typically in excess of 150 beats/minute, sometimes accompanied by atrioventricular separation. The methods of the invention may also be used to prevent or reduce the severity of supraventricular tachycardia (SVT), which is a regular tachycardia in which the stimulation site is located above the bundle branch, such as the sinoatrial node, the atrial junction, or the atrioventricular junction, or in which the SVT is generated in the great reentrant circuit (reentrant circuit) including the atrial site and the ventricular site. In one embodiment, the method of the invention is used to prevent or reduce the severity of atrial tachycardia characterized by: the heart rate is fast, typically between 160 beats/min to 190 beats/min, and is produced at the atrial site; such tachycardias include, but are not limited to, paroxysmal atrial tachycardias. In another embodiment, the method of the invention is used to prevent or reduce the severity of a junctional tachycardia that occurs in response to a pulse generated at the atrioventricular junction and is characterized by a heart rate greater than 75 beats/minute. Junctional tachycardias include nonparoxysmal junctional tachycardias and paroxysmal junctional tachycardias, such as junctional tachycardias due to reentry or increased autonomy. It is to be understood that the present method may also be used to prevent or reduce the severity of tachycardia, which is not limited to: dual tachycardias, which include two types of ectopic tachycardias; sinus tachycardia, which arises from the sinoatrial node and can be accompanied by shock, hypotension, congestive heart failure, or fever; upright tachycardia, which is characterized by disproportionately accelerated heart rates when rising from a reclined position to an upright position; and a turbulent atrial tachycardia characterized by an atrial rate of 100-130 beats/minute, a significantly variable P-waveform, and an irregular P-P interval.

The cardiac acceleration to be treated according to the methods of the invention can be accompanied by one or more diseases, such as pulmonary disease, diabetes, or trauma, and can occur, for example, in the elderly. For example, a turbulent atrial tachycardia (a polytropic atrial tachycardia) can exist, for example, in patients with chronic obstructive pulmonary disease, in patients with diabetes, and in the elderly. By way of further example, non-paroxysmal junctures tachycardia can be accompanied by, for example, trauma. It will be appreciated that many of the above-described and well-known autonomic tachycardias and other tachycardias not accompanied by myocardial ischemia may be prevented or reduced in severity according to the methods of the invention. In another embodiment, the invention provides a method of preventing or reducing the severity of all types of tachycardia, including tachycardia associated with myocardial ischemia.

The method of the invention can also be used to prevent or reduce the severity of panic attacks, a common disease with an incidence of about 3% in the general population (Potokar and Nutt,Int.J.Clin.Pract.54: 110 to 114 (2000)). Panic attacks involving recurrent episodes of panic are common in young adults, with an average age of onset of 24 years, and are more common in women than in men. The term "panic attack" as used herein means a discontinuous period of intense fear or discomfort accompanied by one or more of the following symptoms: heart rate or palpitations are accelerated; chest pain; chills or hot flashes; physical disintegration or personality disintegration; fear of death; fear of losing control or going crazy; dizziness or asthenia; feeling choking; nausea or abdominal distress; paresthesia; feeling shortness of breath or choking; sweating; or chatter or vibration. Panic attacks usually start with a strong apprehension or sudden onset of fear and usually last about 5-20 minutes. The term panic attack encompasses both full-blown attacks and attacks with limited symptoms. Full-scale attacks involve four or more of the above symptoms, while limited-symptom attacks involve less than four symptoms. The methods of the invention may completely prevent panic attacks, or prevent or reduce the severity of one or any combination of the above attendant symptoms.

Some patients with panic attacks develop a "panic disorder," the severity of which can also be prevented or reduced using brimonidine according to the methods of the present invention. The term panic attack as used herein encompasses panic disorders defined as: panic attacks are repeated and are accompanied by persistent anxiety for at least one month after one or more panic attacks with additional attacks or altered outcome or behavior of the attack.

The central sympathetic nervous system may play a key role in the development of the features of type II diabetes, namely insulin resistance and hypertension (Rocchini et al,Hypertension33[ part II]: 548 to 553 (1999). The present invention also provides a method of preventing or reducing the severity of type II diabetes mellitus which is characterized by hypertension, hyperlipidemia and insulin resistance and which is exacerbated by stress effects. As disclosed herein, brimonidine or a pharmaceutically acceptable salt, ester, amide, stereoisomer, or racemic mixture thereof can be administered systemically to a subject to prevent or reduce the severity of type II diabetes in the subject.

The methods of the invention may also be used to prevent or reduce the severity of non-inflammatory skin conditions. The method may be used, for example, to prevent or reduce the severity of one or more symptoms such as itching or other discomfort associated with non-inflammatory skin disorders. The term "non-inflammatory skin disease" as used herein means any skin disease or other skin disease or skin condition that is not caused by or accompanied by inflammation. Non-inflammatory skin conditions to be treated according to the methods of the present invention may develop or worsen under stress conditions. Non-inflammatory skin diseases encompass, but are not limited to, non-inflammatory skin diseases including non-inflammatory blistering diseases such as epidermolysis bullosa and porphyria; ichthyosis; keratosis of hair; juvenile Plantar Dermatoses (JPD); lichen planus dermatosis; and xeroderma. It will be appreciated by those skilled in the art that the above and other non-inflammatory skin conditions known in the art can be treated by the methods disclosed herein.

In a separate embodiment, the present invention provides a method of preventing or alleviating a stress-related inflammatory skin disorder in a subject, comprising systemically administering to the subject an effective amount of brimonidine or a pharmaceutically acceptable salt, ester, amide, stereoisomer, or racemic mixture thereof. The method may be used, for example, to prevent or reduce the severity of one or more symptoms such as itching or other discomfort associated with inflammatory skin diseases. Any of a variety of inflammatory dermatoses encompassed by the methods of the present invention include, but are not limited to, any of a variety of forms of acute or chronic dermatitis, such as psoriasis, allergic dermatitis such as allergic contact dermatitis, atopic dermatitis, heat shock dermatitis, contact dermatitis, cosmetic dermatitis, eczema, exfoliative dermatitis, human dermatitis, irritant dermatitis, lichen simplex chronicus, marine dermatitis, neurodermatitis, perioral dermatitis, phototoxic dermatitis, seborrheic dermatitis, stasis dermatitis, and proliferative dermatitis.

The methods of the invention are useful for preventing or reducing the severity of various disorders of muscle contraction that result, at least in part, from inappropriate muscle contraction. Muscle contraction disorders to be treated according to the methods of the present invention include, but are not limited to, disorders of skeletal muscle contraction, disorders of smooth muscle contraction, disorders of gland-associated muscle contraction, disorders of cardiac muscle contraction such as congestive heart failure; the above and other disorders whose severity is to be prevented or reduced according to the methods of the present invention include disorders of muscle contraction in which muscle cells are innervated or not innervated. By way of non-limiting example, the methods of the invention can be used to prevent or reduce the severity of disorders of muscle contraction such as, for example, back muscle or other muscle spasms; muscle contraction disorders associated with cystitis; muscle contraction disorders associated with non-bacterial cystitis; muscle contraction disorders with bruxism; muscle contraction disorder with tension-type headache; and muscle contraction disorders with congestive heart failure.

The methods of the invention can be used, for example, to prevent or reduce the severity of muscle spasms, such as back spasms. Muscle spasms are well known in the art. The term "spasm" as used herein means a sudden involuntary contraction of a muscle or group of muscles, accompanied by pain and dysfunction. Spasticity may result, for example, involuntary movement or deformation. In one embodiment, the methods of the invention prevent or reduce the severity of back spasms.

In one embodiment, the methods of the invention are used to prevent or reduce the severity of muscle contractions associated with cystitis. The term "cystitis" as used herein refers to inflammation of the bladder. The term cystitis encompasses, but is not limited to, allergic cystitis, bacterial cystitis, acute catarrhal cystitis, cystoceles, diphtheria (guru's) cystitis, eosinophilic cystitis, exfoliative cystitis, follicular cystitis, glandular cystitis, crusted cystitis, chronic interstitial (submucosal) cystitis, mechanical irritative cystitis, papillomic cystitis, and senile female cystitis (cystitis senilis feminium). Reference is made, for example, to Anderson, supra, 1994. Cystitis may be associated with one or more of the following clinical symptoms: frequent urination, burning, suprapubic discomfort, lassitude, turbid or bloody urine and in some cases low fever (Bennett and Plum (ed),Cecil Textbook of medicinesixth edition, w.b. saunders Company, philadelphia, 1996). It will be appreciated by those skilled in the art that muscle contractions associated with any of the above or other forms of mild, severe, acute or chronic cystitis can be treated according to the methods of the present invention.

The method of the invention may also be used to prevent or reduce muscle contraction associated with non-bacterial prostatitis, as disclosed herein. About 50% of men have symptoms of prostatitis in adulthood; of these, about 95% are due to factors other than bacterial infection. The term "abacterial prostatitis" as used herein is synonymous with "abacterial prostatitis" and means inflammation of the prostate gland not caused by bacterial infection. Non-bacterial prostatitis encompasses, but is not limited to, chronic non-bacterial prostatitis, allergic or eosinophilic prostatitis, and non-specific granulomatous prostatitis. It is to be understood that the term non-bacterial prostatitis includes, but is not limited to, prostatitis of unknown etiology characterized by abnormally Expressed Prostatic Secretion (EPS) and normal bacterial culture. In some cases, non-bacterial prostatitis can be effectively treated with antibiotics or by stress management (Bennett and Plum, supra, 1996). It will be appreciated that muscle contractions associated with mild, severe, acute or chronic non-bacterial prostatitis in the above or other forms may be treated according to the method of the invention.

In another embodiment, the methods of the invention are used to prevent or reduce muscle contraction associated with Tension Type Headache (TTH), a common form of headache affecting up to 90% of american adults. The term "tension type headache" as used herein means a headache caused at least in part by muscle contraction, which may be initiated by, for example, stress or exertion. The term "tension type headache" encompasses both episodic headache and chronic headache and includes, but is not limited to, ordinary tension headache. Tension-type headache usually involves the posterior part of the head and neck, although it may also occur at the top or anterior part of the skull, and is also often characterized by symmetry and non-disability (non-disability). Although not necessarily all diagnostic features, diagnostic features of tension-type headache include bilateral pain; mild to moderate severity; has a press-like characteristic and is stable in state; progressively worsen over time; the frequency may be higher, such as daily or continuous episodes; and exacerbation by less common migraine features such as nausea, light sensitivity, sound sensitivity, and physical activity such as head movement.

Tension-type headaches are caused by tightening of muscles of the face, neck and scalp, such as from stress, strain, eyestrain or poor posture. The headache can last for days or weeks and can cause pain of varying intensity. Tension-type headaches that occur over extended periods of time, such as weeks or months, are known as chronic tension headaches and are encompassed by the term tension-type headache as used herein.

Tension-type headache is distinguished from migraine headache by the absence of vascular features and symptoms, such as nausea, vomiting and sensitivity to light and the absence of aura (Spira,Austr.Family Phys.27: 597 to 599 (1988)). The term tension-type headache refers to a headache without a significant vascular component, and this term is used to distinguish tension-type vascular headaches, cluster headaches, migraine headaches, and other headaches with a major vascular component. However, the methods of the invention may also be used to prevent or reduce the severity of hyperesthesia with other headaches including, but not limited to, cervicogenic headache, post-injury headache, cluster headache, and temporomandibular joint disorder (TMJ).

The methods of the invention are useful for preventing or reducing the severity of hyperesthesia associated with migraine, a headache afflicting more than 10% of the population and which may be associated with vascular composition. In one embodiment, the methods of the invention prevent or reduce the severity of ocular allergies with migraine headaches, e.g., photophobia. The methods of the present invention are useful for preventing or reducing the severity of hyperesthesia associated with any of a variety of forms of migraine, including, but not limited to, migraine without aura ("MO"), migraine with aura ("MA"), and migraine disorders. The hyperesthesia to be prevented or reduced in severity according to the method of the invention may also be accompanied by, for example, abdominal migraine, acute psychosocial migraine, basilar (basilar) migraine, hemiplegic or hereditary hemiplegic migraine, lightning-like migraine, ocular (eye-related) migraine, ophthalmoplegic migraine or retinal migraine. In addition, the methods of the present invention are useful for preventing or reducing the severity of hyperesthesia associated with migraine equivalent disorders, wherein migraine is aurated but does not include headache. Migraine aura is accompanied by abnormal visual, motor, mental, sensory or other neurological abnormalities. With reference to the Elrington, the following description,J.Neurol.Neurosurq. Psychiatry72 supplement II: ii 10-ii 15 (2002); anderson, supra, 1994; bennett and Plum, supra, 1996.

The methods of the invention are useful for preventing or reducing the severity of one or more of a variety of types of hyperesthesia associated with migraine. The sensory hypersensitivity includes, but is not limited to, nausea; vomiting; diarrhea; photophobia (intolerance of light); and phonophobia (phonophobia). The hyperesthesia also includes visual abnormalities such as bright light (bright flashing light) visual abnormalities (glistening or accentuating scotomas) or monocular (retinal) visual abnormalities or loss of partial blindness; paresthesia (abnormal tactile sensation), such as unilateral paresthesia; aphasia (loss of language or comprehension); hemiparesis (muscle weakness or incomplete paralysis on one side of the body); unilateral sensory deficit; or vertigo, ataxia (loss of muscle coordination) or diplopia. It will be appreciated that the method of the invention may be used to prevent or reduce the severity of one of the above or other types of hyperesthesia that occurs before, simultaneously with or after migraine headache, or that occurs as part of a migraine equivalent in the absence of headache.

The methods of the invention can be used to prevent or reduce the severity of one or more of a variety of types of hyperesthesia with other disorders. Such other disorders are, for example, fibromyalgia, also known as fibrositis. Fibromyalgia is a disorder involving widespread chronic musculoskeletal pain and tenderness at multiple sites in the absence of connective tissue or other musculoskeletal disease signals. Specifically, according to the definition of the american college of rheumatology, fibromyalgia is defined as pain or tenderness occurring in 11 or more of 18 sites. Fibromyalgia is often accompanied by sleep disturbances, chronic fatigue, headache, and irritable bowel syndrome.

Various types of hypersensitivity, including but not limited to, light, noise, touch or smell hypersensitivity, intolerance to cold heat, nausea or development of allergic-like symptoms such as rhinitis, pruritus or rash without a true allergic response, may be associated with fibromyalgia and may be prevented or reduced in severity according to the methods of the present invention. It will be appreciated by those skilled in the art that the methods of the invention may be used to prevent or reduce the severity of any of the above or other types of hyperesthesia associated with fibromyalgia.

The methods of the invention may also be used to prevent or reduce the severity of stress-related behavioral abnormalities, which are any behavioral abnormalities that are induced or exacerbated by stress. By way of non-limiting example, the stress-related behavioral abnormality may be stress-induced or exacerbated compulsive behavior or repetitive adverse behavior, such as, but not limited to, binge eating or obesity, Obsessive Compulsive Disorder (OCD), tics, Tourette's Syndrome (TS), alcohol abuse, drug abuse, gambling, self-disabling behavior such as scratching or pulling hair (self-inflicted injurious behavor), or impotence or sexual arousal. In one embodiment, the stress-related behavioral abnormality is an abnormality other than substance abuse. In another embodiment, the stress-related behavioral abnormality is an abnormality other than drug abuse or alcohol abuse.

The methods of the invention may also be used to prevent or alleviate stress-related psychiatric disorders, which are any psychiatric disorders that are induced or exacerbated by stress. By way of non-limiting example, the methods of the invention may be used to prevent or reduce the severity of a psychotic disorder such as schizophrenia.

The present invention also provides a method of preventing or reducing the severity of an ocular condition in a subject comprising systemically administering to the subject an effective amount of brimonidine or a pharmaceutically acceptable salt, ester, amide, stereoisomer or racemic mixture thereof. Brimonidine, as disclosed herein, is useful as a neuroprotective agent, i.e., in a variety of ocular diseases affecting the sensory neuroretina, e.g., to prevent retinal damage. Eye diseases in which brimonidine may be used to prevent or reduce its severity according to the methods of the present invention include, but are not limited to, diabetic retinopathy; macular edema, such as macular edema associated with diabetes or other conditions; retinal degeneration such as age-related macular degeneration or retinitis pigmentosa; inflammation of the retina; retinal vascular occlusion disorders, such as retinal vein occlusion or retinal artery branch occlusion or central retinal artery occlusion; retinopathy of prematurity; retinopathy with dyscrasia of blood such as sickle cell anemia; damage after retinal detachment; damage or injury caused by vitrectomy surgery or retinal surgery; and other retinal injuries, including those caused by, for example, retinal excitationTherapeutic damage caused by phototherapy, such as pan-retinal photocoagulation for diabetic retinopathy or retinal photodynamic therapy for, for example, age-related macular degeneration and other eye diseases such as scrapie. Ocular diseases whose severity can be prevented or reduced according to the methods of the present invention also include, but are not limited to, hereditary optic neuropathies and acquired optic neuropathies, such as those characterized primarily by loss of central vision, e.g., Leber's Hereditary Optic Neuropathy (LHON), autosomal dominant optic atrophy (Kjer disease), and other optic neuropathies, such as those involving mitochondrial defects, abnormal motility-related proteins, or inappropriate apoptosis. Reference is made, for example, to Carelli et al,Neurochem.Intl.40: 573-584 (2002); and an acid addition reaction of the compounds of the formula Olichon and the like,J.Biol.Chem.278：7743～7746(2003)。

the methods of the invention are useful for preventing or reducing the severity of stress-related conditions without producing sedation. Sedation according to the present invention is a term that indicates a decrease in locomotor activity. The phrase "does not produce sedation" as used herein means that there is a concomitant reduction in locomotor activity at the same time as the severity of one or more symptoms of the stress-related disorder is reduced at one or more drug doses. A drug is generally referred to as "without concomitant sedation" if it is administered peripherally at least 3-fold greater than the dose required to produce a 20% decrease in locomotor activity than the dose required to produce a significant decrease in one or more symptoms of a stress-related disorder. As shown in figure 6, brimonidine was administered at a dose that resulted in a decrease in sensitization score (solid line, left axis) and an increase in sedation (dashed line, right axis) of less than 20%, but not tizanidine or cola. By way of non-limiting example, the dose required to reduce 20% of locomotor activity may be at least 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 25-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1000-fold, 2000-fold, or 5000-fold higher than the dose required to significantly reduce one or more symptoms of a stress-related disorder. Methods for determining the degree of reduction in the severity of symptoms of stress-related conditions, as well as methods for determining the degree of sedation, are well known in the art.

The term "brimonidine" as used herein denotes a compound having the formula

Or a pharmaceutically acceptable derivative thereof, such as a salt, ester, amide, stereoisomer, racemic mixture, polymorph, hydrate or solvate. The pharmaceutically acceptable derivatives can have substantially the activity of D-5-bromo-6- (2-imidazolin-2-ylamino) quinoxaline tartrate (1: 1) in terms of reducing tactile hypersensitivity in sulprostone-treated mice without producing sedation. The term brimonidine encompasses, but is not limited to, Alphagan^TMAnd UK 14304. Brimonidine and pharmaceutically acceptable salts, esters, amides, stereoisomers and racemic mixtures thereof are commercially available, e.g., Alphagan^TM(allergy). In addition, brimonidine and its pharmaceutically acceptable salts, esters, amides, stereoisomers and racemic mixtures thereof can be prepared by conventional methods as described in example I below. Reference may also be made to U.S. Pat. No. 6,323,204.

Thus, it is to be understood that the methods of the present invention encompass the use of pharmaceutically acceptable salts, esters and amides derived from brimonidine of the formula shown above. Suitable pharmaceutically acceptable salts of brimonidine include, but are not limited to, acid addition salts, which may be formed, for example, by mixing a solution of brimonidine with a suitable acid solution, such as hydrochloric acid, sulfuric acid, fumaric acid, maleic acid, succinic acid, acetic acid, benzoic acid, citric acid, tartaric acid, carbonic acid, or phosphoric acid. Pharmaceutically acceptable salts further include, but are not limited to, acid phosphates, acetates, benzenesulfonates, benzoates, bicarbonates, bisulfates, bitartrates, borates, bromides, calcium ethylenediaminetetraacetate, dextrocamphorsulfonates, carbonates, chlorides, clavulanates, citrates, dihydrochloride, ethylenediaminetetraacetate, edisylates, propionates lauryl sulfate, ethanesulfonates, fumarates, glucoheptonates, gluconates, glutamates, p- α -hydroxyacetaminophenylarsonates, hexylresorcinol (hexyresoranate), hydrabamine (hydrabamine), bromates, hydrochlorides, iodates, hydroxynaphthalates, iodides, isothiocyanates, lactates, lactobionates, laurates, malates, maleates, mandelates, methanesulfonates, methane bromides, methyl nitrates, methyl sulfates, hydrogen tartrates, borates, bromides, salts of p-hydroxy-acetamides, n-ethyl-citrates, n-ethyl-methyl propionates, n-propionates, n, Mucate (mucate), naphthalenesulfonate, nitrate, N-methylglucamine, oleate, oxalate, pamoate, palmitate, pantothenate, phosphate/biphosphate, polygalacturonate, saccharate, salicylate, stearate, sulfate, subacetate, succinate, tannate, tartrate, 8-chlorotheyl salt, p-toluenesulfonate, tosylate, triethyliodide and valerate. In one embodiment, the method of the invention is practiced using brimonidine tartrate.

It is also understood that the functional groups of brimonidine may be modified, for example, to enhance the pharmacological utility of the compound. Such modifications are within the knowledge of the skilled chemist and include, but are not limited to, esters, amides, ethers, N-oxides and prodrugs of brimonidine, which are encompassed within the term "brimonidine" as used herein. Examples of modifications which may increase activity include, for example, esterification, such as C formation₁To C₆Alkyl esters, preferably C₁To C₄Alkyl esters, wherein the alkyl group is linear or branched. Other useful esters include, for example, C₅To C₇Cycloalkyl esters and aromatic alkyl esters such as benzyl esters. The esters may be prepared from the compounds of the present invention by conventional methods well known in the art of organic chemistry.

Other pharmaceutically acceptable modifications include amide formation. Useful amide modifications include, for example, amines derived from ammonia; c₁To C₆A primary dialkyl amine of an alkyl group, wherein the alkyl group is linear or branched; and aromatic amines having various substituents. When a secondary amine, the amine may also be in the form of a five or six membered ring. Processes for preparing the above and other amides have beenAre well known in the art.

It is also to be understood that chemically differentiated enantiomers and tautomers of brimonidine are also encompassed within the term "brimonidine" and can be used in the methods of the invention. Furthermore, when the compound is in a crystalline form, it may exist as a polymorph; when present, the compounds may form solvates with, for example, water or common organic solvents. This polymorph, hydrate, and other solvates are also encompassed within the term "brimonidine" and may be used in the disclosed methods.

It is also understood that pharmaceutically acceptable compositions containing brimonidine may be used in the methods of the present invention. The pharmaceutically acceptable composition comprises brimonidine and may optionally include an excipient, such as a pharmaceutically acceptable carrier or diluent, i.e., any carrier or diluent that is substantially free of long-term or permanent deleterious effects when administered to a subject. The excipients are usually mixed with the active compound or can be used to dilute or encapsulate the active compound. The carrier may be a solid, semi-solid, or liquid agent that acts as an excipient or carrier for the active compound. Examples of solid carriers include, but are not limited to, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, polyalkylene glycols, talcum, cellulose, glucose, sucrose, and magnesium carbonate. Suppository formulations may include, for example, propylene glycol as a carrier. Examples of pharmaceutically acceptable carriers and diluents include, but are not limited to, water, such as distilled or deionized water; brine; aqueous glucose, glycerol, ethanol, and the like. It will be understood that the active ingredient may be soluble or may be delivered as a suspension in the desired carrier or diluent.

The pharmaceutical composition may also optionally include one or more agents such as, but not limited to, emulsifying agents, wetting agents, sweetening or flavoring agents, tonicity adjusting agents (tonicity adjust), preserving agents, buffering agents or antioxidants. Tonicity adjusting agents which may be used in the pharmaceutical composition include, but are not limited to, salts such as sodium acetate, sodium chloride, potassium chloride, mannitol or glycerol, and other pharmaceutically acceptable tonicity adjusting agents. Useful in pharmaceutical compositionsPreservatives include, but are not limited to, benzalkonium chloride, chlorobutanol, thimerosal, phenylmercury acetate, and phenylmercury nitrate. A variety of pH-adjusting buffers and methods may be used in preparing the pharmaceutical compositions, including, but not limited to, acetate buffers, citrate buffers, phosphate buffers, and borate buffers. Similarly, antioxidants useful in pharmaceutical compositions are well known in the art and include, for example, sodium metabisulfite, sodium thiosulfate, acetylcysteine, butylated hydroxyanisole, and butylated hydroxytoluene. It is to be understood that the above and other materials known in the art of pharmacology may be included in the pharmaceutical compositions used in the methods of the present invention. Reference, for example, Remington' sPharmaceutical SciencesMack Publishing Company, Easton, Pa, 16 th edition, 1980. In addition, the brimonidine-containing compositions may be administered by the same or different route of administration, in the same or different pharmaceutical composition, in combination with one or more other therapeutic substances.

Brimonidine or a pharmaceutically acceptable salt, ester, amide, stereoisomer, or racemic mixture thereof is administered in an effective amount. The effective amount is generally the minimum dose required to achieve the desired prophylactic or palliative effect on the severity of one or more symptoms of the stress-related disorder, e.g., an amount generally required to reduce the discomfort caused by the stress-related disorder to a tolerable level. The above dose is usually in the range of 0.1 to 1000 mg/day and may be, for example, in the range of 0.1 to 500 mg/day, 0.5 to 100 mg/day, 0.5 to 50 mg/day, 0.5 to 20 mg/day, 0.5 to 10 mg/day or 0.5 to 5 mg/day, the actual dose being determined by a physician in consideration of the relevant circumstances, including the severity of the stress-related disorder, the age and weight of the patient, the general physical condition of the patient, and the pharmaceutical preparation and route of administration. Suppositories and sustained release agents may also be used in the methods of the invention, including, for example, skin patches, formulations applied to or under the skin, and formulations for intramuscular injection.

The pharmaceutical compositions used in the methods of the invention can be administered to a subject by a variety of means depending, for example, on the type of condition to be treated, the pharmaceutical formulation, and the subject's medical history, risk factors, and symptoms. Routes of administration suitable for use in the methods of the invention include systemic and topical administration. By way of non-limiting example, a pharmaceutical composition for preventing or reducing the severity of a stress-related disorder can be administered orally; parenteral administration; a subcutaneous pump; a skin patch; intravenous, intra-articular, subcutaneous, or intramuscular injection; topical drops, creams, gels or ointments; as long-acting release agents for implantation or injection; subcutaneous micropumps or other implanted devices; intrathecal pumps or intrathecal injections; or an epidural injection. Depending on the mode of administration, brimonidine may be incorporated into any pharmaceutically acceptable dosage form, such as, but not limited to, tablets, pills, capsules, suppositories, powders, liquids, suspensions, emulsions, aerosols, and the like, and may optionally be packaged in unit dosage forms suitable for single administration of precise dosages, or in sustained release dosage forms suitable for continuous controlled administration.

The methods of the invention may be practiced by peripheral administration of brimonidine or a pharmaceutically acceptable salt, ester, amide, stereoisomer, or racemic mixture thereof. The term "peripheral administration" or "administered peripherally" as used herein means the introduction of brimonidine or a pharmaceutically acceptable salt, ester, amide, stereoisomer or racemic mixture thereof, outside of the central nervous system of an individual. Peripheral administration encompasses any route of administration other than directly to the spine or brain.

Peripheral administration may be local or systemic. Topical administration results in significantly more of the pharmaceutical composition being delivered to or around the site of topical administration than to areas remote from the site of administration. Systemic administration results in the delivery of the pharmaceutical composition to substantially the entire peripheral system of the subject.

Peripheral routes of administration useful in the methods of the invention include, but are not limited to, oral administration, topical administration, intravenous or other injection, and implanted micropumps or other sustained release devices or agents. The pharmaceutical composition for use in the present invention may be administered peripherally, for example, orally in any acceptable form such as tablets, oral liquids, capsules, powders, and the like; by intravenous, intraperitoneal, intramuscular, subcutaneous or parenteral injection; diffusion through the skin or electrophoresis; topical administration in any acceptable form such as drops, cream, gel or ointment; and sustained release devices or agents by micro-pump or other implantation.

The following examples are intended to illustrate the invention without constituting a limitation thereto.

Example I

Preparation of brimonidine

This example describes the preparation of brimonidine (5-bromo-6- (2-imidazolin-2-ylamino) quinoxaline).

Preparation of 6-amino-5-bromoquinoxaline hydrobromide

6-aminoquinoxaline (2.08g, 14.4mmol) is dissolved in 11.5ml of glacial acetic acid. The solution was cooled in water and a solution of bromine (0.74ml, 2.3g, 14.4mmol) in 1.5ml glacial acetic acid was added slowly over 15 minutes. After stirring for an additional 30 minutes, the orange solid formed was filtered and washed thoroughly with dry ether. The solid was dried in vacuo overnight to give 4.44g of crude product (100% yield). The compound 6-amino-5-bromoquinoxaline hydrobromide has no defined melting point. A phase change from fine powder to red crystals was observed at about 220 ℃. Decomposition was observed at about 245 ℃. This material was used directly to prepare 6-amino-5-bromoquinoxaline, as described below.

6-amino-5-bromoquinoxaline

The crude 6-amino-5-bromoquinoxaline prepared above is dissolved in water and a saturated solution of sodium bisulfite is added until the resulting solution is negative as determined by starch-iodide paper. The solution was then basified with 2N sodium hydroxide and extracted thoroughly with ethyl acetate. The organic extract phase was dried over magnesium sulfate and concentrated under reduced pressure to give the free base. The crude product is recrystallized from boiling benzene to give yellow crystals with a melting point of 155-6 ℃. The yellow crystal was determined to be 6-amino-5-bromoquinoxaline using various analytical methods. The yield was 82%.

6-bromo-6-isothiocyanatoquinoxaline

The above crude hydrobromide salt (4.27g, 14.0mmol) was dissolved in 60ml of water; carbon dichloride sulfide (Aldrich, 1.28ml, 16.8mmol) was added in small portions with vigorous stirring. After 2 hours, the red solution was drained. The solid formed was filtered and washed thoroughly with water. Drying at 25 ℃ in vacuo gave 3.38g of brick-red crystals with a melting point of 157-8 ℃ in an indication of a yield of 90%. A portion of this material was further purified by column chromatography to give white crystals with a melting point of 157-8 ℃. The white crystal was determined to be 5-bromo-6-isothiocyanatoquinoxaline using various analytical methods.

5-bromo-6 (-N- (2-aminoethyl) thioureido) quinoxaline

A solution of isothiocyanate (3.25g, 12.2mmol) in 145ml of benzene was added to a solution of ethylenediamine (Aldrich, 5.43g, 90.0mmol) in 18ml of benzene at 25 ℃ over a period of two hours. After stirring for a further 30 minutes, the supernatant was decanted off. The remaining oil phase was washed three times by swirling with dry ether and used directly in the next step.

Subjecting a portion of the above product to column chromatography (SiO)₂，CHCl₃) Further purified for characterization. A white solid was recovered, which decomposed at 175 ℃ with gas evolution (sparging). The white solid was determined to be 5-bromo-6 (-N- (2-aminoethyl) thioureido) quinoxaline.

5-bromo-6- (2-imidazolin-2-ylamino) quinoxalines

The crude product obtained above was dissolved in 100ml of dry methanol and the brown solution was refluxed for 19 hours until no hydrogen sulfide gas escaped. The mixture was cooled to room temperature and concentrated to about 50 ml. The yellow solid was filtered and dried in vacuo; the solid weighed 2.52g (yield 70%) and had a melting point of 242-4 ℃.

Since the crude product is insoluble in most common organic solvents, primary purification is achieved by acid-base extraction. The crude product (23g) was dissolved in 100ml of 0.5N hydrochloric acid. The cloudy yellow solution was filtered to give a clear orange solution, which was extracted twice with ethyl acetate (10 ml for each extraction). The aqueous phase was cooled to 0 ℃ and basified with 6N sodium hydroxide, keeping the temperature of the solution below 15 ℃ all the time. The precipitated yellow solid was filtered and washed thoroughly with water until the wash was neutral with pH paper. The solid was dried under vacuum overnight to give 1.97g of a yellow solid with a melting point of 249-250 ℃. The recovery was about 88%.

Further purification was achieved by recrystallization. The partially purified product was dissolved in N, N-dimethylformamide (about 17ml/g) at 100 ℃ with vigorous stirring. The solution was filtered hot and left to cool overnight. The bright yellow crystals were collected by filtration with a melting point of 252-253 ℃. The recovery rate is 65-77%. The bright yellow solid was determined to be 5-bromo-6- (2-imidazolin-2-ylamino) quinoxaline using a variety of analytical methods.

Example II

Mouse model with different sensory sensitization mechanisms

This example shows that increasing sympathetic tone in α -2A and α -2C knockout mice promotes induction of tactile hypersensitivity by activating the α -1 receptor.

A. Sulprostone-induced tactile hypersensitivity is driven by the sympathetic nervous system, while phenylephrine is induced Tactile hypersensitivity independent of sympathetic nervous system afferent

To analyze the contribution of the sympathetic nervous system to sensory sensitization in detail, mouse models with different mechanisms of sensory sensitization were established. Following intrathecal or intraperitoneal injection of the inducer, the flank of the mice were tapped with a brush and the response was scored to measure tactile hypersensitivity in the mice. To simulate an increase in sympathetic tone, phenylephrine, an alpha-1 adrenergic receptor agonist, was injected. As shown in fig. 1a and 1b, intrathecal (i.t.) or intraperitoneal (i.p.) administration of phenylephrine produces tactile hypersensitivity and significant responses are initially observed at doses of 3ng i.t. and 3ng/kg i.p.. The induction of tactile hypersensitivity is dependent on the alpha-1 receptor, as evidenced by the fact that the alpha-1 receptor antagonist 5-methylurapidil (5-MU) blocks the allergic response upon intraperitoneal injection.

In addition, synthetic EP was examined₁/EP₃Activity of receptor-selective prostaglandin agonist sulprostone. As shown in fig. 1c, increasing the intrathecal dose of sulprostone injection found that tactile hypersensitivity was dose dependent; doses of 100ng and 200ng produced significant allergic reactions. Simultaneous administration of specific EP₁Receptor antagonists completely block sulprostone-induced tactile hypersensitivity, suggesting that sulprostone is activated by EP₁Receptors mediate tactile hypersensitivity.

In a third mouse model, where chemical sensitization is induced by intrathecal injection of increasing doses of NMDA, NMDA can activate NMDA channels on postsynaptic dorsal horn neurons (Woolf et al,Science288: 1765-1769 (2000)). Intrathecal administration of NMDA produces dose-dependent tactile hypersensitivity, the greatest effect of which occurs at the 100ng dose. The allergy was blocked with the NMDA antagonist memantine, as shown in figure 1 d.

To assess whether the three stimuli sensitize sensory channels by different mechanisms, a panel of agents was tested for their ability to prevent or ameliorate tactile hypersensitivity. As shown in Table 1, each receptor antagonist (5-MU, EP)₁Receptor antagonists or memantine) blocks only tactile hypersensitivity caused by each respective receptor agonist (phenylephrine, sulprostone or NMDA, respectively). Additionally, the ability of gabapentin to block tactile hypersensitivity was tested, which clinically relieves neuropathic pain by reducing spinal sensitization. Gabapentin inhibits tactile hypersensitivity caused by sulprostone and NMDA, but not phenylephrine, further suggesting a difference between sensory channels involved in different stimuli.

Alpha-2 knockout mice were supplied by doctor Brian Kobilka (university of stanford; Link et al,Mol.Pharmacol.48: 48-55 (1995); an Altman, etc. and the like,Mol.Pharmacol.56: 154 to 161 (1999). The alpha-2 knockout mice have a C57BL/6 background and are bred from homozygous knockout mouse breeding pairs. Age and sex matched C57BL/6 wild type mice were used as controls.

Sulprostone (Cayman Chemical; Ann Arbor, Michigan) and NMDA (Sigma; St Louis, MO) were dissolved in dimethyl sulfoxide (DMSO). EP to be synthesized substantially in accordance with U.S. Pat. No. 5,843,942₁Receptor antagonists

And gabapentin (Victor Medical; Irvine, CA) in 50% DMSO, 50% saline. Substantially in accordance with us patent 5,061,703 (see also Schneider et al,Dtsch Med. Wochenschr.109: 987(1984)) Synthesis of memantine (1-amino-3, 5-dimethyladamantane hydrochloride), a well-known analog of the antiviral agent amantadine (1-amantadine hydrochloride). 5-Methylurapidil, brimonidine, phenylephrine, clonidine and guanethidine were purchased from Sigma and dissolved in saline. Prazosin (Sigma) and tizanidine (Biomol; Plymouth Meeting, Pa.) were dissolved in distilled water.

Spinal drug injections were performed as follows. According to the specifications of Hylden and Wilcox,Eur.J. Pharmacol.67: 313-316 (1980) the intrathecal injection is carried out on mice (20-30 g). Briefly, a 30 gauge 1/2 inch sterile needle attached to a micro-syringe was pushed between the L5 and L6 vertebrae. One hand grips the pelvic girdle of the mouse and the other hand gripsThe syringe was held at an angle of about 20 ° above the spinal column. The needle was pushed into the tissue on one side of the spinous process of L6, i.e. into the groove between the spinous process and the transverse process. The needle angle was reduced to about 10 deg., and the needle was then slowly pushed into the intervertebral space until the dehiscence sound was felt and the tail appeared to have a pronounced sinuous movement. A volume of 5. mu.l of the compound was slowly injected into the subarachnoid space. Various compounds were tested at various doses. The minimum effective dose was used throughout all subsequent experiments.

The flank of the mouse was tapped with a small brush (normally painless) and the sensitivity to light touch was quantified by scoring the response. Mice were scored every 5 minutes between 15 and 50 minutes post injection according to the following criteria: animals showing a strong escape response and having a sharp bite of the brush are scored "2"; animals exhibiting a soft scream and attempting to escape were scored "1"; animals that did not respond to the brush were scored "0". The total score of the score meter generates an accumulated score of 0-16, such as Minami and the like,Pain57: 217 to 223 (1994). Statistical calculations of significance were performed for the in vivo studies using the two-tailed student t-test.

Guanethidine sympathetic blockade proceeds essentially as follows. Animals were first injected intraperitoneally with 50mg/kg guanethidine (Malmberg and Basbaum,Pain76: 215-222 (1998)), and their baseline tactile sensitivity was evaluated after 24 hours. Animals exhibiting normal tactile sensitivity were tested for sensitivity to tactile hypersensitivity chemoinduction. After 6-8 days, the mice recovered from the sympathetic blockade, as shown by the response prior to the sympathetic blockade.

B. Increased sympathetic tone in alpha-2A and alpha-2C knockout mice will be increased by activation of the alpha-1 receptor Strong sensitivity to tactile hypersensitivity

To assess whether sympathetic tone can affect susceptibility to sensory sensitization, the sensitivity of α -2A and α -2C knockout mice to chemical induction of tactile hypersensitivity was compared to that of wild-type mice. alpha-2A and alpha-2C knockout mice did not exhibit baseline tactile hypersensitivity as compared to wild-type controls. First, the concentrations of phenylephrine causing tactile hypersensitivity were compared between knockout and wild type mice. As shown in FIG. 2, the phenylephrine dose response was significantly shifted to the left in both α -2A and α -2C knockout mice. The results indicate that phenylephrine has an improved ability to cause tactile hypersensitivity in the alpha-2 knockout mouse line, with a greater improvement in the alpha-2C knockout mice. Specifically, 0.1 and 0.3ng/kg phenylephrine have caused the greatest sensitization in α -2C and α -2A knockout mice, respectively, compared to a strong tactile sensitization inducing dose of 30ng/kg phenylephrine in wild type mouse lines. Figure 2 also demonstrates that the progressive biphasic dose response in wild type mice becomes a steep dose response in two knockout mouse lines.

Systemic administration of guanethidine produces a functional sympathetic blockade by consuming noradrenaline from sympathetic nerve endings. To examine whether the change of phenylephrine dose response curve is due to an increase in sympathetic tone in α -2 knockout mice, α -2A knockout mice were chemically sympathically denervated by guanethidine treatment (50mg/kg i.p.) and tested for phenylephrine-induced sensitivity after 24-30 hours. In guanethidine-treated α -2A mice, the increased sensitivity to phenylephrine was partially removed to allow a dose response similar to the two-phase dose response observed in wild-type mice (see figure 2). This result demonstrates that increasing sympathetic tone will enhance sensory sensitization in α -2A knockout mice.

C. Sympathetic nervous system enhancement of sulprostone-induced tactile hypersensitivity

Sulprostone at increasing concentrations was intrathecally injected into wild type mice and α -2 knockout mice to determine if the knockout mice are more sensitive to primary afferent sensitization. As shown in FIG. 3, the sulprostone dose response was the same in wild-type mice and α -2C knockout mice, but there was a left shift in α -2A knockout mice. Specifically, 30ng of the alpha-2A knockout mouse had reached the maximum effective dose as compared with 100ng of the partial allergy-inducing dose and 200ng of the maximum dose in the wild-type mouse and the alpha-2C knockout mouse. Guanethidine (50mg/kg i.p.) chemosympathetic blockade reduced the susceptibility of α -2A knockout mice to sulprostone. As shown in FIG. 3, the dose response of sulprostone-induced tactile hypersensitivity shifted to the right by about 10-fold compared to guanethidine-treated α -2A knockout mice. The results indicate that the sympathetic nervous system enhances sulprostone sensitization.

D. Sympathetic nervous system does not affect NMDA-induced tactile hypersensitivity

To assess whether α -2 knockout mice are susceptible to NMDA-induced dorsal horn sensitization, wild-type mice and α -2 knockout mice were injected with varying concentrations of NMDA. As shown in FIG. 4, α -2A and α -2C knockout mice are less sensitive to NMDA than wild type mice. This result indicates that the sympathetic nervous system appears to have no effect on NMDA-induced tactile hypersensitivity.

In summary, the above results indicate that α -2 knockout mice exhibit higher levels of sympathetic activity, and that the α -2 knockout mice also exhibit higher stimulation site and stimulation pattern specific sensitization.

Example III

Comparison of alpha-2 agonist brimonidine and clonidine Activity

This example demonstrates that alpha adrenergic agonists differ in their ability to alleviate sensory hypersensitivity, which can be enhanced by the sympathetic nervous system.

A. Brimonidine relieves sympathetically enhanced tactile hypersensitivity, while cola does not

Spinal administration of alpha-2 adrenergic agonists relieves neuropathic pain via spinal alpha-2A receptors. To determine whether increased sympathetic activity in α -2 knockout mice alters the analgesic activity of an α -2 agonist, assays are performedSeveral agonist activities were tested. The alpha-2 agonists brimonidine and clonidine were first tested in the NMDA model, where sensitization was not affected by the basal sympathetic tone of knockout mice. Intrathecal co-administration of NMDA with clonidine or brimonidine in wild type mice and α -2C knockout mice (fig. 5a and 5C, respectively) resulted in complete suppression of tactile hypersensitivity. As expected, neither clonidine nor brimonidine inhibited NMDA-induced tactile hypersensitivity in α -2A knockout mice (fig. 5c), consistent with previous studies, suggesting that spinal α -2A adrenergic receptor subtypes mediate the analgesic effects of α -2 adrenergic agonists (Lakhlani et al,Proc.Natl.Acad.Sci.USA94: 9950 to 9955 (1997); stone, etc. in the presence of a catalyst,J.Neurosci.17: 7157-1765 (1997); the Hunter et al, the number of the patents,Br.J. Pharmacol.122: 1339 to 1344 (1997). The same pattern of brimonidine analgesic activity was also observed in the sulprostone-induced tactile hypersensitivity model, which is sensitive to sympathetic tone (see fig. 5b and 5 d). In contrast, clonidine gives quite different results: clonidine was analgesic in wild-type mice, but not in alpha-2A or alpha-2C knockout mice (compare FIG. 5b and FIG. 5 d). The results indicate that alpha-2 pan agonists may have different activities under conditions of increased sympathology, e.g., brimonidine appears active and clonidine is inactive.

B. Brimonidine relieves sulprostone-induced hypersensitivity without producing sedation, but clonidine or telotide Zany rule of failure

Sedation limits the use of many drugs, including alpha-2 agonists. Thus, alpha-2 agonists are compared to test whether the dose that causes allelochemicals differs relative to the dose that causes sedation.

For three alpha-2 agonists (tizanidine, clonidine and brimonidine), their ability to calm and block tactile hypersensitivity was compared at different doses in a motor activity model and a sulprostone-induced tactile hypersensitivity model, respectively. Tactile hypersensitivity was scored for 5-6 mice in each group every 5 minutes for 15-50 minutes after intraperitoneal administration. Vehicle treated animals generally receive about 4 points. In addition, 30 minutes after intraperitoneal administration, locomotor activity of 5 to 6 mice in each group was measured over a period of 5 minutes. Locomotor activity of the treated animals relative to vehicle is expressed as a percentage; percent sedation was calculated by subtracting percent locomotor activity from 100%. As shown in fig. 6, of the three α -adrenergic agonists tested, only brimonidine produced analgesia that could be separated from sedation. This result demonstrates the ability of brimonidine to alleviate sympathetically-enhanced disorders, such as sulprostone-induced tactile hypersensitivity, without concomitant sedation, unlike other alpha-2 pan agonists, such as clonidine and tizanidine.

C. Alpha-2/alpha-1 functional selectivity changes for alpha adrenergic pan agonists

In the assay, brimonidine and clonidine were analyzed for their alpha adrenergic receptor drug profiles using cell lines stably expressing the alpha-2A, alpha-2C, alpha-1A and alpha-1B receptors.

Consistent with previous studies, in PC12 cells stably expressing either the alpha-2A receptor or the alpha-2C receptor (FIGS. 7a, b; Table 2), the order of potency for inhibition of forskolin-induced cAMP accumulation was dexmedetomidine ≧ brimonidine ≧ clonidine ≧ tizanidine ≧ phenylephrine (Jasper et al,Biochem.Pharmacol.55: 1035 to 1043 (1998); a combination of Pihlavisto et al,Eur.J.Pharmacol.385: 247 to 253 (1999). Brimonidine, clonidine and tizanidine are approximately 10-fold more potent at the α -2A receptor than at the α -2C receptor.

The ability of the same compound to stimulate alpha-1 mediated increases in intracellular calcium was tested functionally in HEK293 cells stably expressing both the alpha-1A receptor and the alpha-1B receptor (FIGS. 7c, d; Table 2). The order of potency at the α -1A and α -1B receptors is phenylephrine > clonidine > tizanidine ═ dexmedetomidine > brimonidine. The alpha-2 agonists clonidine, tizanidine and dexmedetomidine are partial agonists, whereas brimonidine exhibits weak activity at the alpha-1A receptor and no activity at the alpha-1B receptor. Thus, while clonidine and tizanidine have previously been classified as "alpha-2 selective" agonists in binding assays, the compounds exhibit 10-fold less selectivity between alpha-2 and alpha-1 receptors in functional assays. In contrast, dexmedetomidine was approximately 300-fold selective in the functional assay, and brimonidine, the most selective compound in the functional assay, was 1000-fold more selective for the α -2 receptor than for the α -1 receptor (see Table 2). This result indicates that brimonidine is a highly α -2/α -1 selective agonist, and that the difference in α -2/α -1 selectivity of brimonidine is in sharp contrast to the selectivity of other pan agonists such as clonidine.

The difference in alpha-2/alpha-1 selectivity between clonidine and brimonidine indicates that alpha-1 agonist activity of clonidine increases higher sympathetic tone in alpha-2C knockout mice and masks the analgesic activity of clonidine in the sulprostone model. This result is supported by the fact that co-administration of the α -1 antagonists prazosin with clonidine restored the analgesic activity of clonidine in α -2C knockout mice (fig. 7 e). In wild-type mice or alpha-2C knockout mice, prazosin itself has no analgesic activity.

Taken together, the results demonstrate that clonidine loses analgesic activity, while brimonidine is not lost, in α -2C knockout mice, probably due to clonidine α -1 agonist activity, and that the α -1 agonist activity of many "α -2 agonists" may limit its ability to treat stress-related disorders as well as other sympathetically-enhanced disorders.

Stable cell lines expressing adrenergic receptors were established as follows. The cDNA blunt ends of bovine alpha-1A, hamster alpha-1B, human alpha-2A and human alpha-2C receptors were subcloned into the NheI-EcoRI site in the retroviral vector pCL BABE Puro. The retroviral construct was verified by double-stranded DNA sequencing. High titer pseudotyped retroviral particles are produced by the following method: cotransfection of HEK2939GP (a stably expressed Mal) with appropriate retroviral vectors and PMD.G (an expression vector for vesicular stomatitis virus envelope protein, VSV-G)HEK293 cell line for oney leukemia virus Gag-Pol). 16 hours after transfection, medium was changed (DMEM, 10% FCS); high titer (-1X 10) was harvested after 48 hours⁶pfu/mL) medium. The supernatant was filtered through a 0.4uM filter.

Varying amounts of human alpha-2A and alpha-2C receptor supernatants were added to naive PC12 cells, followed by 48 hours of incubation. The transduced cell population was replated (replated) at a lower density and grown in medium containing 100. mu.g/ml puromycin. Untransduced cells were killed within three days and single foci grew within two months. Foci were picked and expanded and receptor density was measured by brimonidine radioligand binding. Functional alpha-2 receptor activity was demonstrated by inhibition of forskolin-induced cAMP accumulation.

Varying amounts of bovine alpha-1A receptor and hamster alpha-1B receptor supernatants were added to naive HEK293 cells and then incubated for 48 hours. The transduced cell population was replated at a lower density and grown in medium containing 0.25ug/ml puromycin. Significant cell death was evident within three days and single foci appeared within two weeks. After picking and expanding the foci, intracellular Ca induced by measurement of phenylephrine⁺²Accumulation A functional assay was performed on the expression of the amplified subcloned alpha-1 receptor. Receptor density is measured in a prazosin radioligand binding assay.

Measurement of intracellular Ca in HEK293 cells stably expressing bovine alpha-1A or hamster alpha-1B adrenergic receptors⁺²The response is as follows. 40,000-50,000 cells were coated in each well of a poly-D-lysine coated 96-well plate in 0.2ml DMEM containing 10% heat inactivated killed calf serum, 1% antibiotic-antifungal agent and 0.25. mu.g/ml puromycin the day before use. The cells were washed twice with HBSS supplemented with 10mM HEPES, 2.0mM CaCl₂And 2.5mM probenecid (probenicid), followed by incubation with 4. mu.M Fluo-4(Molecular Probes; Eugene, Oregon) for 60 minutes at 37 ℃. Extracellular dye on the plate was washed twice and then the plate was placed in a fluorometric imaging plate reader (FLIPR; molecular devices; Sunny)vale, California). The ligands were diluted in HBSS and aliquoted into 96-well microplates. Drugs were tested at concentrations ranging from 0.64nM to 10,000 nM. Ca⁺²Data for the response were obtained in arbitrary fluorescence units.

Measurement of intracellular cAMP was performed as follows. PC12 cells stably expressing human alpha-2A or human alpha-2C adrenergic receptors were plated at a density of 30,000 cells/well in poly-D-lysine coated 96-well plates in 100 μ l DMEM supplemented with 10% horse serum, 5% heat inactivated lethal bovine serum, 1% antibiotic-antifungal agents, and 100 μ g/ml puromycin. Cells were incubated at 37 ℃ and 5% CO₂And grown overnight. Cells were dosed by adding equal volumes of medium containing IBMX (to a final concentration of 1mM), forskolin (to a final concentration of 10. mu.M) and appropriate drug dilutions (to a final concentration of 10. mu.M)^-5M to 10^-12M). After 10 min of incubation, the medium was aspirated and the cells were lysed with 200. mu.l lysis buffer (Amersham biosciences; Piscataway, New Jersey). Before testing, the plates were stored at-20 ℃ for up to 24 hours. Intracellular cAMP was measured using a Biotrak cAMP enzyme immunoassay system (amersham biosciences) according to the manufacturer's instructions. The plate was read at 450nm with a plate reader.

Using Kaleidagraph (Synergy Software; Reading, PA) by applying the equation: response ═ maximal response + ((minimal response-maximal response)/(1 + (ligand concentration/EC)₅₀) ) was least squares fit to generate a dose response curve for the in vitro assay. Percent efficacy is determined by comparing the maximal effect of the compound to the effect of a standard full agonist, phenylephrine for the alpha-1 receptor and brimonidine for the alpha-2 receptor.

All journal articles, references and patent citations mentioned above, whether previously stated or not, in parentheses or elsewhere, are hereby incorporated by reference in their entirety.

While the invention has been described in conjunction with the embodiments described above, it will be understood that various modifications may be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the claims.

Claims (5)

1. Use of brimonidine or a pharmaceutically acceptable salt, tautomer, solvate, amide or N-oxide thereof in the manufacture of a medicament for preventing or reducing the severity of hyperesthesia associated with migraine by systemic administration of the compound.

2. The use of claim 1, wherein the compound is administered orally.

3. The use of claim 1, wherein the compound is administered topically.

4. The use of claim 1, wherein the compound is administered by patch.

5. The use of claim 1, wherein the compound is administered intravenously.