HK1143038A

HK1143038A - Methods for treating dependence

Info

Publication number: HK1143038A
Application number: HK10109609.2A
Authority: HK
Inventors: Tom Woiwode; Mark Moran; Lesley Pickford
Original assignee: Biotie Therapies, Inc.
Priority date: 2007-08-06
Filing date: 2008-08-06
Publication date: 2010-12-24

Description

Method of treating dependency

Cross Reference to Related Applications

Claims rights and priority to the following U.S. provisional patent application serial nos. in accordance with 35u.s.c. § 119 (e): 60/935,323, "Methods for Treating dependency Using Nepicastat", 8.8.6.2007; 60/956,555, "Methods for Treating Dependence Using Nepicastat", 8.8.17.2007; and 60/960,591, "Methods for Treating Dependence using Nepicacast", application No. 10/4, 2007. All of which are incorporated herein by reference in their entirety.

Technical Field

The present invention provides certain methods of treating a patient suffering from or susceptible to at least one of the following symptoms with compound a: abuse, dependence or withdrawal of at least one substance. The invention also provides certain methods of treating at least one stage of substance dependence on at least one substance in a patient, and certain methods of treating at least one stage of cocaine dependence in a patient.

Background

Substance abuse and dependence are characterized by: craving, searching and using substances while the restriction of the intake of the substances is out of control. These actions occur regardless of significant substance use related issues and compromise other actions. In 2004, americans about two thousand two hundred and fifty thousand years of age 12 or over 12 needed treatment for substance (alcohol or illicit drug) abuse. Recent estimates have shown that the social cost of just illegal drug abuse is 1810 hundred million (2002).

The problem of cocaine abuse and dependence is a major medical, social and legal concern. According to the data of National Survey on Drug Use and Health in 2005, about 13.9% of americans aged 12 and older than 12 tried cocaine at least once in a lifetime, and 3.3% tried cocaine of high purity at least once in a lifetime. More troublesome is that in 2005, the current cocaine user had 240 million people, while the number in 2004 was 200 million people. Also, the users of high purity cocaine at that time increased from 467,000 people in 2004 to 682,000 people in 2005. In 2004, Drug Abuse burning Network estimated 940,953 Drug-related emergency calls nationwide, with most of these involving cocaine.

Clearly, there is a need for a broadly effective therapeutic approach and an approach involving pharmaceutical ingredients that is more effective than current behavioral therapies such as cognitive behavioral therapy alone or occasional management. Various treatments have been studied in clinical trials, but none have significant effects. In particular, a number of randomized clinical trials for antidepressants have been completed, including trials with desipramine, fluoxetine, bupropion and imipramine. Clinical trials of mood stabilizers (including carbamazepine and lithium) were also completed, as were phenytoin, direct or indirect dopamine agonists including bromocriptine, pergolide, amantadine, mazindole (mazindole) and ritalin. A number of other agents have also been investigated, including ritanserin (ritanserin), gepirone, nimodipine and naltrexone. None of these compounds is truly effective. Several drugs acting on the GABA system have also been evaluated for cocaine-dependent treatments, including tiagabine, baclofen and vigabatrin (vigabatrin). The effect of tiagabine is unreliable and the effect of baclofen, although not convincing, is slightly encouraging. Investigation of vigabatrin (vigabatrin) may be similarly encouraging, although it is primarily based on open tests. The results of these development programs are often discouraging.

The dopamine β -hydroxylase (DBH) inhibitor disulfiram is the currently most effective pharmacological therapy for cocaine dependence. Unfortunately, disulfiram non-specifically inhibits some enzymes, including aldehyde dehydrogenase and plasma esterase. Disulfiram and related compounds chelate with copper, an essential cofactor for a variety of enzymes, including aldehyde dehydrogenase, plasma esterase and DBH. By inhibiting aldehyde dehydrogenase, disulfiram can alter alcohol (ethanol) metabolism, resulting in a disulfiram-ethanol reaction. Such reactions include flushing, nausea and hypotension.

Inhibition of plasma esterases may slow the elimination of cocaine, which may lead to elevated plasma cocaine levels. In a laboratory study evaluating the effect of intranasal cocaine during treatment with disulfiram, disulfiram treatment significantly increased plasma cocaine levels. However, increased cocaine levels are not associated with changes in the physiological or subjective effects of cocaine. In a controlled study, a 6-fold increase in plasma cocaine levels was observed, which could be increased even more without controlled illicit use. Subsequent studies with IV cocaine administration demonstrated that disulfiram can slow cocaine elimination, possibly due to inhibition of plasma esterases. Slow absorption following intranasal administration accounts for the previously observed increase in plasma concentration.

Several studies have shown the initial effect of disulfiram as a cocaine-dependent therapy. In human laboratory studies, treatment with disulfiram reduced the positive subjective effects produced by cocaine. Results in patients with morbidity (comorbid) alcohol and cocaine dependence improved when treated with disulfiram up to 500 mg. Similarly, buprenorphine-sustained opiate and cocaine-dependent patients may have reduced cocaine use during treatment with disulfiram. Recently, the results of a number of clinical trials have demonstrated that 250mg disulfiram per day can reduce cocaine use compared to placebo, regardless of the alcohol use pattern or type of psychotherapy provided. In this study, 112 cocaine-dependent volunteers were randomized to either placebo or disulfiram and provided one of two psychotherapies. Disulfiram treatment can provide fewer cocaine positive urine samples than placebo treatment, demonstrating that it is associated with reduced cocaine use. The magnitude of this effect is modest and the results can remain reproducible.

Disulfiram can inhibit DBH, a single enzyme that mediates the synthesis of Norepinephrine (NE). DBH is expressed in noradrenaline neurons, is located within synaptic vesicles, and is released along with NE. DBH can be measured in plasma, the concentration of DBH is highly inherited, and changes in activity are mainly accounted for by changes in the DBH site. The T variant (-1021C → T) is associated with an attenuated DBH gene transcription process and reduced DBH activity. Such alleles are quite common. The T allele is reported to occur in 20% of African-Americans, 22% of northern Europe-Americans and 16% of Japanese. For these populations, the corresponding haplotype frequencies were 0.32, 0.34, and 0.09, respectively.

Some reports indicate that disulfiram is more effective in patients with low DBH activity. It has been shown that in patients with low DBH activity the proportion of cocaine-positive urine decreases over time during treatment with 250 mg/day disulfiram (relative to placebo), but that this proportion increases significantly over time during treatment with 62.5mg and 125mg disulfiram/day (p's < 0.04). In patients with high DBH activity, the proportion of cocaine-positive urine treated with disulfiram (62.5 mg/day) increased over time relative to placebo (p ═ 0.001). Thus, the effect of 250 mg/day disulfiram treatment was limited to patients with low DBH activity (corresponding to C → T genotype). Disulfiram doses below 250 mg/day appear to increase cocaine use, probably due to a decrease in cocaine clearance rate through inhibition of plasma esterases, thereby increasing the psychotropic effects associated with cocaine abuse.

Disulfiram is more effective in reducing cocaine use in patients with DBH C → T genotype associated with low DBH activity. In patients with low DBH activity, it is likely that disulfiram can inhibit DBH more completely, making disulfiram more effective in patients with a reduced activity C → T genotype. The observation that disulfiram is more effective in patients with a low activity of DBH C → T genotype demonstrates that inhibition of DBH is a key mechanism of action of disulfiram as a cocaine-dependent therapy.

Although disulfiram provides evidence that DBH inhibitors can treat cocaine dependence, the use of disulfiram itself as a cocaine-dependent therapy is severely limited by its interaction with alcohol and cocaine.

Summary of the invention

The present invention provides methods of treating a patient suffering from or susceptible to at least one of the following symptoms: abuse, dependence or withdrawal of at least one substance. The method comprises the following steps: administering to the patient a therapeutically effective amount of compound a.

The invention also provides a method of treating at least one phase of substance dependence on at least one substance in a patient, wherein the at least one phase is selected from the group consisting of the acquisition, maintenance, regression and relapse phases. The method comprises the following steps: administering to the patient a therapeutically effective amount of compound a.

The invention also provides a method of treating at least one stage of cocaine dependence in a patient, wherein the at least one stage is selected from the group consisting of acquisition, maintenance, regression, and relapse stages. The method comprises the following steps: administering to the patient a therapeutically effective amount of compound a.

Drawings

Figure 1 illustrates the magnitude of the effect obtained from sample sizes from 5 to 15.

FIG. 2 illustrates the details of the independent enzymatic assay.

FIG. 3 shows the affinity (IC) of nepicastat (nepicastat) for DBH and a number of selected enzymes and receptors ₅₀Value or PKi) graph.

Figure 4 shows the% inhibitory effect of nepicastat (nepicastat) on enzyme activity.

Figure 5 shows urodoparn levels in normal volunteers after 24 hours of treatment with nepicastat (nepicastat).

Figure 6 shows norepinephrine levels in the cortex of SHRs following vehicle or different doses of nepicastat (nepicastat).

Fig. 7 shows dopamine levels in the cortex of SHRs after vehicle or different doses of nepicastat (nepicastat) were administered.

Figure 8 shows the dopamine/norepinephrine ratio in the cortex of SHRs following vehicle or different doses of nepicastat (nepicastat).

Figure 9 shows norepinephrine levels in mesenteric arteries of SHRs following vehicle or different doses of nepicastat (nepicastat).

Fig. 10 shows dopamine levels in mesenteric arteries of SHRs after vehicle or different doses of nepicastat (nepicastat) were administered.

Figure 11 shows the dopamine/norepinephrine ratio in mesenteric arteries of SHRs following vehicle or different doses of nepicastat (nepicastat).

Detailed Description

The following words and phrases used in this specification generally have the meanings as set forth below, except where the context indicates otherwise.

As used herein, "compound a" includes (S) -5-aminomethyl-1- (5, 7-difluoro-1, 2, 3, 4-tetrahydronaphthalen-2-yl) -2, 3-dihydro-2-thioxo-1H-imidazole, (R) -5-aminomethyl-1- (5, 7-difluoro-1, 2, 3, 4-tetrahydronaphthalen-2-yl) -2, 3-dihydro-2-thioxo-1H-imidazole and mixtures thereof and pharmaceutically acceptable salts, e.g., hydrochloride salts, thereof. In some embodiments, nepicastat (nepicastat) ((S) -5-aminomethyl-1- (5, 7-difluoro-1, 2, 3, 4-tetrahydronaphthalen-2-yl) -2, 3-dihydro-2-thioxo-1H-imidazole hydrochloride) is used.

As used herein, "compound B" refers to (R) -5-aminomethyl-1- (5, 7-difluoro-1, 2, 3, 4-tetrahydronaphthalen-2-yl) -2, 3-dihydro-2-thioxo-1H-imidazole and pharmaceutically acceptable salts thereof, e.g., the hydrochloride salt.

"pharmaceutically acceptable salts" include, but are not limited to: salts with inorganic acids, such as hydrochloride, phosphate, hydrogen phosphate, hydrobromide, sulfate, sulfinate, nitrate and the like; and salts with organic acids, e.g. malate, maleate, fumarate, tartrate, succinate, citrate, acetate, lactate, methanesulfonate, p-toluenesulfonate, 2-hydroxyethanesulfonate, benzoate, salicylate, stearate and alkanoates, e.g. acetate, HOOC- (CH) ₂)_n-COOH, wherein n is 0-4, and similar salts.

Alternatively, if the compound is obtained as an acid addition salt, the free base may be obtained by basification of an acid salt solution. Conversely, if the product is the free base, the addition salts (especially the pharmaceutically acceptable addition salts) may be prepared as follows: according to the conventional method for preparing an acid addition salt from a base compound, a free base is dissolved in a suitable organic solvent, and the solution is treated with an acid. Those skilled in the art will be aware of the various synthetic methods which may be used to prepare non-toxic pharmaceutically acceptable addition salts.

The term "patient" as used herein refers to a mammal. In certain embodiments, the term "patient" refers to a human.

The terms "administering," "administration," or "administering" as used herein refer to the direct administration of compound a or a composition thereof to a patient.

The terms "treat," "treating," or "treating" as used herein refer to the partial or complete alleviation, inhibition, prevention, amelioration, and/or alleviation of the disorder or at least one symptom thereof.

The term "suffering from" as used herein means that a patient has been diagnosed with or is estimated to have one or more conditions.

The term "sensitive" as used herein means having a likelihood of being affected by the symptoms of at least one disorder.

As will be appreciated by those of ordinary skill in the art, "substance abuse" is often associated with symptoms of physical and/or psychological "dependence". In addition, when a dependent individual is abstinent from substance abuse, the individual often exhibits certain symptoms, including sleep and mood disturbances and intense craving for substance abuse, referred to as "withdrawal symptoms". The methods described herein include the treatment of substance abuse itself, dependence, and withdrawal symptoms.

The term "substance abuse" as used herein may be defined with reference to the standards listed in the Diagnostic and statistical Manual of Mental Disorders, 4th Ed. text review (2000) ("DSM-IVTR") (which is formulated by Task Force on DSM-IV of the American psychiatric Association). Substance abuse is characterized by an ill-adapted pattern of substance use, manifested as periodic and significant adverse consequences associated with substance reuse. As listed in DSM-IV TR, substance abuse is defined as: an ill-adapted pattern of substance abuse leading to clinically significant damage or distress, manifested as: at least one of the following symptoms occurs within a 12 month period: (1) periodic substance use, resulting in failure to fulfill major obligations at work, school, or home; (2) periodic substance use in physically hazardous situations; (3) periodic material-related legal issues; and (4) continue to use substances despite having social or interpersonal problems caused or exacerbated by the effects of the substances, which are persistent or periodic. Additionally, DSM-IV TR requires that the symptoms of substance abuse not meet the criteria for substance dependence at all. In some embodiments, treating substance abuse with nepicastat (nepicastat) can reduce the amount or frequency of substance use by the patient. In some embodiments, treatment of substance abuse in a patient with compound a can alleviate at least one DSM-IV TR symptom of substance abuse. In some embodiments, treatment of a patient with compound a can alleviate at least one symptom of substance abuse, including, for example and without limitation, at least one of the following: intoxication, apathy, dysphoria, reckless, poor judgment, obsessive-compulsive disorder, aggression, anger, craving for abused substances and mood disorders. In some embodiments, treatment with compound a can reduce stress-induced substance craving in a patient.

The phrase "alleviating a symptom" as used herein refers to alleviating at least one of the occurrence and magnitude of symptoms of a disorder in a patient. In certain embodiments, the patient's symptoms are alleviated and no longer experienced.

As used herein, the phrase "increasing symptoms" refers to an increase in at least one of the incidence and magnitude of symptoms of a disorder in a patient.

The term "substance-dependent" as used herein may be defined with reference to the criteria set forth in DSM-IV TR. The substance-dependent symptoms listed in DSM-IV TR are modes of substance use that cause clinically significant damage or distress, manifested as: at any time within the same 12 month period, at least three symptoms selected from the group consisting of: (1) a tolerance defined by one of: (a) the amount of the substance needs to be significantly increased to obtain the desired effect; or (b) continuing to use the same amount of substance(s) results in a significant reduction in efficacy; (2) withdrawal symptoms are illustrated by one of the following: (a) characteristic withdrawal syndrome of a particular substance; or (b) ingesting the same or closely related substances in order to reduce or avoid withdrawal symptoms; (3) often, large quantities of the substance are ingested, or the time taken is longer than a predetermined time; (4) there is a constant desire or unsuccessful effort to curtail or control substance use; (5) much time is spent in activities to obtain a substance, use a substance, or restore its effect; (6) abandoning or reducing important social, occupational or recreational activities due to the use of substances; and (7) continuing to use the substance despite knowledge of persistent or periodic physical or psychological problems (which may be caused or exacerbated by the substance). Substance dependence can be physiological dependence, where there is evidence of tolerance or withdrawal, or non-physiological dependence, where there is no evidence of tolerance or withdrawal. In some embodiments, treating substance dependence with compound a can reduce the amount or frequency of substance use by the patient. In some embodiments, treating substance dependence in a patient with compound a can reduce at least one DSM-IV TR symptom of substance dependence in the patient. In some embodiments, treatment of a patient with compound a can alleviate at least one substance-dependent symptom, including, for example and without limitation, at least one of the following: intoxication, apathy, dysphoria, reckless, poor judgment, obsessive-compulsive disorder, aggression, anger, craving for substances and mood disorders to which it depends. In some embodiments, treatment with compound a can reduce stress-induced substance craving in a patient.

As used herein, "symptom relief" refers to a condition during which at least one symptom of substance abuse or dependence has been reduced in appearance. In some embodiments, the term symptom relief does not apply if the patient is using agonist therapy or is in a controlled environment where the substance of interest is restricted. In some embodiments, symptom relief refers to a condition during which at least one symptom of substance abuse or dependence does not occur. In some embodiments, symptom relief refers to a state of a patient during which all symptoms of substance abuse or dependence have been alleviated. In some embodiments, symptom relief refers to a state during which symptoms without substance abuse or dependence occur. In some embodiments, symptom relief refers to a condition during which use of the substance does not occur.

In some embodiments, the symptom relief is characterized by at least one of the following: early complete remission, early partial remission, sustained complete remission, and sustained partial remission, and is applicable only after at least one month in the absence of any symptoms of substance abuse and dependence. The definition of these four types of symptom relief is based on the time interval elapsed since cessation of dependency (early symptom relief relative to sustained symptom relief), and whether there is sustained presence of at least one symptom of substance dependency or abuse (partial symptom relief relative to complete symptom relief).

The qualifier "early complete symptom relief" is used when symptoms of substance dependence or substance abuse do not occur for at least one month (but less than 12 months).

The qualifier "early partial symptom relief" is used when at least one symptom of substance dependence or substance abuse occurs for at least one month (but less than 12 months) but the criterion for substance dependence or substance abuse is not met.

The term "sustained complete symptom relief" is used when there is no symptom of substance dependence or substance abuse at any time during at least 12 months.

The term "sustained partial symptom relief" is used when at least one symptom of substance dependence or substance abuse occurs for at least 12 months but does not meet the criteria for substance dependence or substance abuse.

In some embodiments, compound a treatment may promote remission in a patient. In some embodiments, compound a treatment can prolong the time to remission in a patient.

The phrase "extending the time to symptom relief" refers to increasing the time interval during which a patient is in symptom relief. In some embodiments, the stress condition may result in the patient ending remission. In some embodiments, the recurrence occurs at the end of remission. In some embodiments, treatment with compound a may reduce the likelihood of the patient ending remission after a stressful condition. In some embodiments, treatment with compound a can promote at least one of the following conditions: early partial symptom relief, sustained complete symptom relief, sustained partial symptom relief, and sustained complete symptom relief.

"withdrawal" refers to the collective symptoms that occur when administration of the relevant substance is reduced, delayed or discontinued. Substance-specific symptoms of withdrawal can result in, for example, clinically significant distress or impairment of social, occupational, or other important areas of function. These symptoms cannot be attributed to a conventional medical condition and cannot be well documented with another mental disorder. Withdrawal symptoms are often (but not necessarily) associated with substance dependence. In some embodiments, treatment with compound a may reduce at least one symptom of withdrawal in the patient. In some embodiments, withdrawal symptoms include, for example, but are not limited to: apathy, dysphoria, reckless, poor judgment, obsessive-compulsive disorder, aggression, anger, craving for substances, mood disorders and sleep disorders. In some embodiments, treatment with compound a can reduce stress-induced substance craving in a patient.

The term "substance-dependent" is characterized by: there is at least one condition identified by the following DSM-IV TR: alcohol abuse; alcohol dependence; alcohol poisoning; alcohol intoxication delirium; alcohol withdrawal symptoms; alcohol withdrawal confusion; alcohol-induced anxiety disorder; alcohol-induced mood disorders; alcohol-induced persistent amnesia; alcohol-induced persisting dementia; alcohol-induced psychotic disorder (with hallucinations); alcohol-induced psychotic disorder (hallucinogenic); alcohol-induced sexual dysfunction; alcohol-induced sleep disorders; alcohol related other non-referenced (NOS) disorders; amphetamine abuse; amphetamine dependence; amphetamine intoxication; intoxication and confusion with amphetamines; amphetamine withdrawal symptoms; amphetamine-induced anxiety disorder; amphetamine-induced mood disorders; amphetamine-induced psychotic disorder (with hallucinations); amphetamine-induced psychotic disorder (hallucinogenic); amphetamine-induced sexual dysfunction; amphetamine-induced sleep disorder; amphetamine-related NOS disorder; cannabis abuse; cannabis dependence; cannabis intoxication; cannabis-toxic delirium; cannabis-induced anxiety disorder; cannabis-induced psychotic disorder (with hallucinations); cannabis-induced psychotic disorder (hallucinogenic); cannabis-related NOS disorders; cocaine abuse; cocaine dependence; cocaine poisoning; cocaine toxic delirium; cocaine withdrawal symptoms; cocaine-induced anxiety disorder; cocaine-induced mood disorders; cocaine-induced psychotic disorder (with hallucinations); cocaine-induced psychotic disorder (hallucinogenic); cocaine-induced sexual dysfunction; cocaine-induced sleep disorders; cocaine-related NOS disorders; inhalant abuse; (ii) inhalant dependence; (ii) inhalation toxicosis; toxic delirium in inhalants; inhalant-induced anxiety disorder; inhalant-induced mood disorders; inhalant-induced persisting dementia; inhalant-induced psychotic disorder (with hallucinations); (ii) an inhalant-induced psychotic disorder (hallucinogenic); inhalant-related NOS disorders; opioid abuse; opioid dependence; opioid intoxication; opioid intoxication delirium; opioid withdrawal symptoms; opioid-induced mood disorders; opioid-induced psychotic disorder (with hallucinations); opioid-induced psychotic disorder (hallucinogenic); opioid-induced sexual dysfunction; opioid-induced sleep disorders; opioid-related NOS disorder; benzxedine abuse; benzoxetine dependence; phenicol poisoning; phencyclidine intoxication is delirium; phencyclidine-induced anxiety disorder; phencyclidine-induced mood disorders; phencyclidine-induced psychotic disorder (with hallucinations); phencyclidine-induced psychotic disorder (hallucinogenic); and phencyclidine-related NOS disorders.

The terms "stop" and "abstinence" may (but need not) be with respect to the following conditions identified by DSM-IV TR: nicotine withdrawal symptoms; other non-annotated nicotine-related disorders; nicotine dependence with physiological dependence; nicotine dependence without physiological dependence; nicotine dependence with early complete symptom relief; early partial remission of nicotine dependence; nicotine dependence that provides sustained complete symptomatic relief; sustained partial relief of nicotine dependence; nicotine dependence for agonist therapy; opioid withdrawal symptoms; other non-annotated opioid-related disorders; opioid dependence with physiological dependence; opioid dependence without physiological dependence; opioid dependence with early complete symptom relief; early partial remission of opioid dependence; opioid dependence with sustained complete symptom relief; sustained partial relief of opioid dependence; opioid dependence of agonist treatment; and opioid dependence in a controlled environment; ethanol withdrawal symptoms; ethanol dependence with physiological dependence; ethanol withdrawal symptoms without physiological dependence; early complete symptom relief of ethanol withdrawal symptoms; early partial relief of ethanol withdrawal symptoms; ethanol withdrawal symptoms that continue to provide complete symptom relief; sustained partial relief of ethanol withdrawal symptoms; agonist-treated ethanol withdrawal symptoms; ethanol withdrawal symptoms in a controlled environment; amphetamine withdrawal symptoms; and cocaine withdrawal symptoms.

As used herein, "agonist treatment" refers to the treatment of substance abuse, dependence or withdrawal symptoms with an agonist. The term "agonist" refers to an element, including but not limited to a compound, such as a small molecule or complex organic compound or protein, that elicits a response in a patient that is at least one response or partial response to a substance abused, dependent or abstained by the patient. For example, in some embodiments, "agonist treatment of opioid dependence" refers to treatment of opioid dependence with methadone.

Withdrawal symptoms may occur when any kind of substance is reduced. For example, discontinuation of use of smoking articles (all containing nicotine) typically causes nicotine withdrawal symptoms. Due to discontinuation of use of any form of tobacco (including but not limited to smoking cigarettes, cigars or pipe tobacco, or oral or intranasal ingestion of tobacco or chewing tobacco), individuals often experience nicotine withdrawal symptoms. Such oral or intranasal tobacco includes, but is not limited to, snuff and chewing tobacco. Termination of nicotine use or reduction of nicotine usage, often with symptoms occurring within 24 hours, include: a dysphoric depressed mood; light headedness; insomnia; irritability, frustration or anger; anxiety; tension tremor; difficulty in concentration; restlessness; a decrease in heart rate; appetite increase or weight gain; and the desire to obtain tobacco or nicotine. These symptoms often result in clinically significant distress or impairment of social, occupational, or other important areas of function. When nicotine withdrawal symptoms cannot be attributed to a conventional medical condition and cannot be well explained by another medical disorder, the methods described herein can be used to alleviate one or more symptoms resulting from nicotine withdrawal. The method is also helpful for those patients who have used tobacco instead of or in part instead of nicotine replacement therapy. Thus, such patients may be helped to reduce and even completely eliminate their dependence on all forms of nicotine.

Discontinuation or reduction of opioid administration (typically by injection or oral administration, from self-administration by inhalation or intranasal ingestion) is often characteristic of opioid withdrawal symptoms. This withdrawal symptom can also be promoted by administering an opioid antagonist such as naloxone or naltrexone after the opioid is administered (preconditioned). Opioid withdrawal symptoms are characterized by: symptoms that are generally opposite to opioid agonistic effects. These withdrawal symptoms may include: anxiety; restlessness; muscular soreness often occurs in the back and legs; desire to obtain opioids; dysphoria and increased sensitivity to pain; a restless mood; nausea or vomiting; tearing; rhinorrhea; the nipple expands; erecting wool; sweating; diarrhea; yawning; generating heat; and insomnia. When there is dependence on short-acting opioids, such as heroin, withdrawal symptoms usually appear within 6-24 hours after the last dose, whereas for long-acting opioids, such as methadone, symptoms may take 2-4 days to develop. These symptoms often result in clinically significant distress or impairment of social, occupational, or other important areas of function. When such symptoms are not attributable to a conventional medical condition and are not well documented in another medical condition, the methods described herein may be used to alleviate one or more symptoms resulting from opioid withdrawal.

Discontinuing or reducing the use of ethanol (e.g., a beverage containing ethanol) can cause ethanol withdrawal symptoms. Ethanol withdrawal symptoms are characterized in that: symptoms appear when the blood concentration of ethanol rapidly decreases within 4 to 12 hours after cessation or decrease of ethanol use. These ethanol withdrawal symptoms include: ethanol is desired; autonomic nervous system hyperactivity (e.g., sweating or pulse rate greater than 100); shaking hands; insomnia; nausea; vomiting; temporary hallucinations or illusions of vision, touch or hearing; psychomotor agitation; anxiety; and grand mal seizures. These symptoms often result in clinically significant distress or impairment of social, occupational, or other important areas of function. The methods described herein may be used to alleviate one or more symptoms of ethanol withdrawal when the symptoms of ethanol withdrawal cannot be attributed to a conventional medical condition and cannot be well interpreted with another medical condition.

Cocaine abuse and dependence can lead to cognitive, behavioral, and physiological symptoms. Symptoms of cocaine abuse and dependence may include: (ii) attention deficit hyperactivity disorder and intoxication at different levels; increased vitality, excitement and sociability; no hunger and fatigue; a clear sense of physical and psychological strength; dysphoria; a reduction in pain sensation; and the desire to obtain cocaine. Respiratory effects include the following symptoms: such as bronchitis, shortness of breath and chest pain, cardiovascular effects include the following symptoms: such as palpitations, arrhythmias, cardiomyopathy and heart failure. Symptoms also include dilated pupils, nausea, vomiting, headache, dizziness, anxiety, vertigo, mental disorders and confusion. Cocaine administration by inhalation or snuff can cause ear, nose, and throat effects including nasal inflammation, nasal crusting, periodic nasal bleeding, nasal stuffiness, and facial pain. In some embodiments, treatment with compound a can reduce at least one symptom of cocaine abuse and dependence in a patient. In some embodiments, the nepicstat treatment may increase at least one negative subjective symptom of cocaine abuse and dependence.

Cocaine withdrawal symptoms may include: fatigue, lack of pleasure, depression, irritability, sleep disorders, increased appetite, psychomotor depression, agitation, extreme suspicion and desire for cocaine. In some embodiments, treatment with compound a may reduce at least one symptom of cocaine withdrawal.

The substance dependence is characterized by the following stages: acquisition, maintenance, regression and relapse. The term "obtaining" as used herein refers to the stage at which a patient initiates and obtains substance-dependent substance dependence. In some embodiments, compound a treatment may inhibit the development of the acquisition phase in the patient. In some embodiments, treatment of the acquisition phase with compound a can reduce at least one of the amount or frequency of substance use by the patient. In some embodiments, treatment of the acquisition phase with compound a can alleviate at least one DSM-IV symptom of substance abuse and dependence in a patient. In some embodiments, treatment of the acquisition stage with compound a can alleviate at least one symptom of substance abuse and dependence, including, for example and without limitation, at least one of the following: intoxication, apathy, dysphoria, reckless, poor judgment, obsessive-compulsive disorder, aggression, anger, craving for abused or dependent substances and mood disorders. In some embodiments, treatment with compound a can reduce substance craving in a patient induced by stress during the acquisition phase.

By "maintenance" is meant a substance-dependent period during which a substance is stably administered to a patient or a substance is stably administered to a patient. In some embodiments, a change of 10% in at least one of the quantity and frequency of substance use by the patient is considered stable behavior. In some embodiments, treatment of the maintenance phase with compound a can reduce at least one of the amount and frequency of substance use by the patient. In some embodiments, treatment of the maintenance phase with compound a in a patient may alleviate at least one DSM-IV symptom of substance abuse and dependence in the patient. In some embodiments, treatment of the maintenance phase with compound a can alleviate at least one symptom of substance abuse and dependence, including, for example, but not limited to, at least one of the following: intoxication, apathy, dysphoria, reckless, poor judgment, obsessive-compulsive disorder, aggression, anger, craving for abused or dependent substances and mood disorders. In some embodiments, treatment with compound a can reduce substance craving induced by stress conditions during the maintenance phase in a patient.

By "resolution" is meant a substance-dependent stage in which the patient is not provided with a substance or is abstained from substance use. In some embodiments, the substance dependence at the stage of regression is diminished or reduced. In some embodiments, at least one withdrawal symptom is present during the resolution stage. In some embodiments, compound a treatment may promote the development of the remission stage in the patient. In some embodiments, treatment of the remission stage of a patient with compound a may alleviate at least one DSM-IV symptom of substance abuse and dependence in the patient. In some embodiments, treatment with compound a during the remission stage may alleviate at least one symptom of substance abuse and dependence, including, for example and without limitation, at least one of the following: intoxication, apathy, dysphoria, reckless, poor judgment, obsessive-compulsive disorder, aggression, anger, craving for abused or dependent substances and mood disorders. In some embodiments, compound a treatment may alleviate withdrawal symptoms in a patient during the remission stage. In some embodiments, treatment with compound a can alleviate substance craving in a patient induced by stress during the remission stage.

By "relapse" is meant that the patient reappears with at least one symptom of substance abuse or dependence after a period of withdrawal. In some embodiments, the recurrence occurs at the end of remission. In some embodiments, the patient is trained to regress prior to relapse. In some embodiments, relapse occurs after a drug triggers, stresses, or contacts a cued or irritated environment associated with prior substance use. In some embodiments, compound a treatment may reduce the frequency of relapse in a patient. In some embodiments, treatment of the relapsing phase with compound a may alleviate at least one DSM-IV symptom of substance abuse and dependence in the patient. In some embodiments, treatment of the relapsing phase with compound a may alleviate at least one symptom of substance abuse and dependence, including, for example, but not limited to, at least one of the following: intoxication, apathy, dysphoria, reckless, poor judgment, obsessive-compulsive disorder, aggression, anger, craving for abused or dependent substances and mood disorders. In some embodiments, compound a treatment may alleviate withdrawal symptoms of a patient during the relapsing phase. In some embodiments, treatment with compound a can alleviate substance craving in a patient induced by stress conditions during the relapse phase.

Treatment of substance abuse, dependence and withdrawal symptoms can be staged. In some embodiments, the initial period of withdrawal substance use is preferably prior to induction by treatment of the patient with compound a. In some embodiments, the patient is administered an initial low dose of compound a. In some embodiments, the amount of compound a administered to the patient is escalated until a targeted therapeutic response is observed. In some embodiments, the amount of compound a is escalated in order to determine an optimal amount for treating the condition while minimizing the patient's symptoms, side effects, and substance craving.

In some embodiments, compound a treatment may promote remission. In some embodiments, the dose of compound a is unchanged, or gradually decreases after the patient achieves remission.

The present invention provides methods of treating a patient suffering from or susceptible to at least one of the following symptoms: abuse, dependence or withdrawal symptoms of at least one substance. The method comprises the following steps: administering to the patient a therapeutically effective amount of compound a. In some embodiments, the at least one substance is selected from drugs of abuse and drug therapy. In some embodiments, the drug of abuse is selected from psychostimulants, opioids, hallucinogens, inhalants, sedatives, tranquilizers Agents, hypnotics, anxiolytics and illicit substances. In some embodiments, the psychostimulant is a β -phenylisopropylamine derivative. In some embodiments, the β -phenylisopropylamine derivative is selected from amphetamine, dextroamphetamine, and methamphetamine. In some embodiments, the psychostimulant agent is selected from the group consisting of synthetic hallucinogens, phenmetrazine, ritaline, bupropion, pemoline, mazindol, (-) norpseudoephedrine cathinone, and fenfluramine. In some embodiments, the opioid is selected from the group consisting of Lortab, tramadol, heroin, methadone, hydrocodone, and oxycodone. In some embodiments, the hallucinogen is selected from the group consisting of nudeomushroom, hallucinogen, lysergic acid diethylamide (LSD), phencyclidine (PCP), and ketamine. In some embodiments, the inhalant is selected from the group consisting of benzene, toluene, o-xylene, m-xylene, p-xylene, ethylbenzene, fluorobenzene, o-difluorobenzene, 1, 3, 5-trifluorobenzene, 1, 2, 4-trifluorobenzene, pentafluorotoluene, pentafluorobenzene and perfluorobenzene. In some embodiments, the drug therapy is selected from the group consisting of anesthetics, analgesics, anticholinergics, antihistamines, muscle relaxants, non-steroidal anti-inflammatory drugs, over-the-counter drugs, and antidepressants. In some embodiments, the drug of abuse is cocaine, alcohol, caffeine, opiates, cannabinoids, cannabis, benzodiazepines (benzodiazapine) myo-inositol (carisoprodol), tobacco, nicotine, paracetamol (Vicodin), hydrocodone, oxycodone hydrochloride, oxycodone, and tylosin (Tylox). In some embodiments, the drug of abuse is cocaine, and compound a can reduce at least one symptom of cocaine abuse and dependence in the patient selected from the group consisting of: attention deficit hyperactivity disorder; the feeling of drunkenness; increased vitality, excitement and sociability; does not feel hunger and fatigue; a clear sense of physical and psychological strength; a reduction in pain sensation; bronchitis; short gas; chest pain; palpitations; cardiac arrhythmia; cardiomyopathy; heart failure; the pupil is dilated; nausea; vomiting; headache; dizziness; vertigo; anxiety; a mental disorder; confusion of consciousness; nasal inflammation; nasal crusts; periodic nasal bleeding; binein (nasal obstruction)Ventilating; facial pain; dysphoria; and cocaine craving. In some embodiments, the drug of abuse is cocaine and compound a can increase at least one negative subjective symptom of cocaine abuse and dependence. In some embodiments, the drug of abuse is cocaine, and compound a reduces at least one symptom of cocaine withdrawal selected from fatigue, lack of pleasure, depression, dysphoria, sleep disorders, increased appetite, mental retardation, agitation, extreme suspicion, and craving for cocaine. In some embodiments, treatment with compound a may increase the score of a patient on at least one of the following scales: attention deficit hyperactivity disorder evaluation scale IV (scale) (ADHD-IV), hamilton depression scale (HAM-D), hamilton anxiety scale (HAM-a), Beck depression scale (inventory) (BDI), apathy scale from neuropsychiatric scales, and cognitive function scale. In some embodiments, the cognitive function rating scale is selected from the group consisting of the wife-behcet's mental scale-revision (WAIS-R), the gomper's revised wecker memory scale (WMS-R), the heler auditory word learning test (RAVLT, Trials I-VII), the Rey complex graph test (RCFT), and the tracking wiring test (TMT, parts a and B). In some embodiments, compound a may reduce at least one of the amount and frequency of substance use by the patient in the patient. In some embodiments, compound a may alleviate at least one symptom of abuse, dependence, or withdrawal of at least one substance in a patient. In some embodiments, compound a can alleviate at least one symptom of substance abuse in a patient selected from the group consisting of: periodic material usage resulting in failure to fulfill major obligations at work, school, or home; periodic substance use in case the body is at risk; periodic substance use involving legal issues; and sustained substance use, although such substance use has persistent or periodic social or interpersonal problems (caused or exacerbated by the consequences of the substance). In some embodiments, compound a can reduce at least one symptom of substance dependence in a patient selected from the group consisting of: tolerance; withdrawal symptoms; often ingest large amounts of substances or are expected to last longer; with continued desire and/or unsuccessful efforts to reduce or control the use of substances (ii) a Spending a lot of time in at least one activity in order to obtain the substance, use the substance and restore its effect; abandoning and/or reducing at least one important social, occupational and recreational activity due to the use of the substance; and continuing to use the substance despite knowledge of persistent and/or periodic physical and/or psychological problems caused or exacerbated by the substance. In some embodiments, compound a may promote remission in a patient. In some embodiments, the symptom relief is characterized by: at least one of early complete symptom relief, early partial symptom relief, sustained complete symptom relief, and sustained partial symptom relief. In some embodiments, compound a can prolong the time to remission in a patient. In some embodiments, the method further comprises treating with at least one of strain (containment) management and cognitive behavioral therapy. In some embodiments, the method further comprises co-administering a therapeutically effective amount of at least one additional agent selected from the group consisting of: selective Serotonin Reuptake Inhibitors (SSRI), serotonin-norepinephrine reuptake inhibitors (SNRI), Norepinephrine Reuptake Inhibitors (NRI), norepinephrine-dopamine reuptake inhibitors (NDRI), serotonin 5-hydroxytryptamine 1A (5HT1A) antagonists, dopamine beta-hydroxylase inhibitors, adenylate receptor antagonists, adenosine A2A receptor antagonists, monoamine oxidase inhibitors (MAOI), monoamine oxidase B inhibitors, sodium channel blockers, calcium channel blockers, central and peripheral alpha adrenergic receptor antagonists, central alpha adrenergic agonists, central or peripheral beta adrenergic receptor antagonists, NK-1 receptor antagonists, Corticotropin Releasing Factor (CRF) antagonists, atypical antidepressants/antipsychotics, tricyclic drugs, anticonvulsants, glutamate antagonists, gamma-aminobutyric acid (GABA) agonists, GABA metabolic enzyme inhibitors, GABA synthesis activators, partial dopamine D2 agonists, dopamine metabolic enzyme inhibitors, catechol-O-methyl-transferase inhibitors, opioid receptor antagonists, mood stabilizers, direct or indirect dopamine agonists, partial 5HT1 agonists, serotonin 5HT2 antagonists, opioids, carboxylase inhibitors, partial opioid agonists, partial nicotinic acid agonists and inhalations And (3) preparing. In some embodiments, the at least one other agent is an SSRI selected from paroxetine, sertraline, citalopram, escitalopram and fluoxetine. In some embodiments, the at least one other agent is an SNRI selected from duloxetine, mirtazapine (mirtazapine), and venlafaxine. In some embodiments, the at least one other agent is NRI selected from bupropion and atomoxetine (atomoxetine). In some embodiments, the at least one other agent is NDRI BUPROPION (BUPROPION). In some embodiments, the at least one other agent is the dopamine β -hydroxylase inhibitor disulfiram. In some embodiments, the at least one other agent is an adenosine A2A receptor antagonist istradefylline. In some embodiments, the at least one additional agent is a sodium channel blocker selected from the group consisting of lenoda, carbamazepine, oxcarbazepine, and valproate. In some embodiments, the at least one additional agent is a calcium channel blocker selected from nimodipine (nimoopine), lenedane, and carbamazepine. In some embodiments, the at least one other agent is the central and peripheral alpha adrenergic receptor antagonist prazosin. In some embodiments, the at least one other agent is the central alpha adrenergic agonist clonidine. In some embodiments, the at least one other agent is the central and peripheral beta adrenergic receptor antagonist propranolol. In some embodiments, the at least one other agent is an atypical antidepressant/antipsychotic selected from the group consisting of bupropion, olanzapine (olanzapine), risperidone, and quetiapine. In some embodiments, the at least one other agent is a tricyclic agent selected from amitriptyline, amoxapine, desipramine, doxepin, imipramine, nortriptyline, prottyline, and trimipramine. In some embodiments, the at least one other agent is an anticonvulsant agent selected from the group consisting of phenytoin, lenoda, carbamazepine, oxcarbazepine, valproate, topiramate, tiagabine, vigabatrin (vigabatrin), and levetiracetam. In some embodiments, the at least one other agent is the glutamate antagonist topiramate. In some embodiments, the at least one other agent is a GABA agonist selected from baclofen, valproate and topiramate. At one end In some embodiments, the at least one additional agent is carbidopa, an inhibitor of dopamine-metabolizing enzymes. In some embodiments, the at least one other agent is the partial dopamine D2 agonist aripiprazole. In some embodiments, the at least one additional agent is an opioid receptor antagonist selected from naltrexone and naloxone. In some embodiments, the at least one additional agent is a mood stabilizer selected from carbamazepine and lithium. In some embodiments, the at least one other agent is a direct or indirect dopamine agonist selected from dopamine, bromocriptine, pergolide, amantadine, mazindole, and ritalin. In some embodiments, the at least one additional agent is gepirone, a partial 5HT1 agonist. In some embodiments, the at least one other agent is the serotonin 5HT2 antagonist ritanserin (ritanserin). In some embodiments, the at least one other pharmaceutical agent is the opioid methadone. In some embodiments, the at least one other agent is the partial opioid agonist buprenorphine. In some embodiments, the at least one additional agent is the partial nicotinic acid agonist champix. In some embodiments, the at least one additional pharmaceutical agent is an inhalant selected from the group consisting of benzene, toluene, o-xylene, m-xylene, p-xylene, ethylbenzene, fluorobenzene, o-difluorobenzene, 1, 3, 5-trifluorobenzene, 1, 2, 4-trifluorobenzene, pentafluorotoluene, pentafluorobenzene, and perfluorobenzene. In some embodiments, the method further comprises co-administering a therapeutically effective amount of at least one additional agent selected from the group consisting of benzodiazepines Levodopa, carnisprodol, modafinil, acamprosate, gamma-butyrolactone, gamma-hydroxybutyrate, opioids, dimethyl-4-hydroxytryptamine phosphate (psilopabin), tricholoma, tobacco and nicotine. In some embodiments, compound a is administered to the patient after the patient has abstinent from substance use for a period of time. In some embodiments, the therapeutically effective amount of compound a in the patient is determined by gradually increasing the amount of compound a administered to the patient until a targeted therapeutic response is observed. In some implementationsIn the regimen, the amount of compound a is gradually reduced after the patient achieves remission. In some embodiments, the amount of compound a is unchanged after the patient achieves remission.

The invention also provides a method of treating at least one phase of substance dependence of at least one substance in a patient. In some embodiments, the at least one phase of substance dependence is selected from the group consisting of an acquisition, maintenance, regression, and relapse phase. The method comprises the following steps: administering to the patient a therapeutically effective amount of compound a. In some embodiments, compound a may inhibit acquired stage progression in a patient. In some embodiments, compound a may promote the development of the remission stage in the patient. In some embodiments, compound a may reduce the frequency of relapse in a patient. In some embodiments, the at least one substance is selected from drugs of abuse and drug therapy. In some embodiments, the drug of abuse is selected from psychostimulants, opioids, hallucinogens, inhalants, sedatives, tranquilizers, hypnotics, anxiolytics, and illicit substances. In some embodiments, the psychostimulant is a β -phenylisopropylamine derivative. In some embodiments, the β -phenylisopropylamine derivative is selected from amphetamine, dextroamphetamine, and methamphetamine. In some embodiments, the psychostimulant agent is selected from the group consisting of synthetic hallucinogens, phenmetrazine, ritaline, bupropion, pemoline, mazindol, (-) norpseudoephedrine, and fenfluramine. In some embodiments, the opioid is selected from the group consisting of Lortab, tramadol, heroin, methadone, hydrocodone, and oxycodone. In some embodiments, the hallucinogen is selected from the group consisting of nudeomushroom, hallucinogen, lysergic acid diethylamide (LSD), phencyclidine (PCP), and ketamine. In some embodiments, the inhalant is selected from the group consisting of benzene, toluene, o-xylene, m-xylene, p-xylene, ethylbenzene, fluorobenzene, o-difluorobenzene, 1, 3, 5-trifluorobenzene, 1, 2, 4-trifluorobenzene, pentafluorotoluene, pentafluorobenzene and perfluorobenzene. In some embodiments, the drug therapy is selected from the group consisting of anesthetics, analgesics, anticholinergics, antihistamines, muscle relaxants, non-steroidal anti-inflammatory drugs, over-the-counter drugs, and antidepressants. In some embodiments, the drug of abuse is an alcohol Caffeine, opium, cannabinoid, cannabis, benzodiazepines(benzodiazapine), myo-quinine (carisoprodol), tobacco, nicotine, paracetamol (Vicodin), hydrocodone, oxycodone hydrochloride, oxycodone and tylosin (Tylox). In some embodiments, treatment with compound a may increase the score of a patient on at least one of the following scales: ADHD-IV, HAM-D, HAM-A, BDI, apathy and cognitive function scales from neuropsychiatric scales. In some embodiments, the cognitive function rating scale is selected from the group consisting of WAIS-R, WMS-R, RAVLT, Trials I-VII, RCFT, and TMT (parts A and B). In some embodiments, compound a may reduce at least one of the amount and frequency of use of the at least one substance by the patient in the patient. In some embodiments, compound a may alleviate at least one symptom of abuse, dependence, or withdrawal of at least one substance in a patient. In some embodiments, compound a can alleviate at least one symptom of substance abuse in a patient selected from the group consisting of: periodic material usage resulting in failure to fulfill major obligations at work, school, or home; periodic substance use in case the body is at risk; periodic substance use involving legal issues; and sustained substance use, although such substance use has persistent or periodic social or interpersonal problems (caused or exacerbated by the consequences of the substance). In some embodiments, compound a can reduce at least one symptom of substance dependence in a patient selected from the group consisting of: tolerance; withdrawal symptoms; often ingest large amounts of substances or then last for a longer time; there is a continuing goal and/or unsuccessful effort to curtail or control substance use; spending a lot of time in at least one activity in order to obtain the substance, use the substance and restore its effect; abandoning and/or reducing at least one important social, occupational and recreational activity due to the use of the substance; and continuing to use the substance despite knowledge of persistent and/or periodic physical and/or psychological problems caused or exacerbated by the substance. In some embodiments, compound a may contribute to the symptoms of a patient And (4) relieving. In some embodiments, the symptom relief is characterized by: at least one of early complete symptom relief, early partial symptom relief, sustained complete symptom relief, and sustained partial symptom relief. In some embodiments, compound a can prolong the time to remission in a patient. In some embodiments, the method further comprises treating with at least one of strain management and cognitive behavioral therapy. In some embodiments, the method further comprises co-administering a therapeutically effective amount of at least one additional agent selected from the group consisting of: selective Serotonin Reuptake Inhibitors (SSRI), serotonin-norepinephrine reuptake inhibitors (SNRI), Norepinephrine Reuptake Inhibitors (NRI), norepinephrine-dopamine reuptake inhibitors (NDRI), serotonin 5-hydroxytryptamine 1A (5HT1A) antagonists, dopamine beta-hydroxylase inhibitors, adenylate receptor antagonists, adenosine A2A receptor antagonists, monoamine oxidase inhibitors (MAOI), monoamine oxidase B inhibitors, sodium channel blockers, calcium channel blockers, central and peripheral alpha adrenergic receptor antagonists, central alpha adrenergic agonists, central or peripheral beta adrenergic receptor antagonists, NK-1 receptor antagonists, Corticotropin Releasing Factor (CRF) antagonists, atypical antidepressants/antipsychotics, tricyclic drugs, anticonvulsants, glutamate antagonists, gamma-aminobutyric acid (GABA) agonists, GABA metabolic enzyme inhibitors, GABA synthesis activators, partial dopamine D2 agonists, dopamine metabolic enzyme inhibitors, catechol-O-methyl-transferase inhibitors, opioid receptor antagonists, mood stabilizers, direct or indirect dopamine agonists, partial 5HT1 agonists, serotonin 5HT2 antagonists, opioids, carboxylase inhibitors, partial opioid agonists, partial nicotinic acid agonists, and inhalants. In some embodiments, the at least one other agent is an SSRI selected from paroxetine, sertraline, citalopram, escitalopram and fluoxetine. In some embodiments, the at least one other agent is an SNRI selected from duloxetine, mirtazapine (mirtazapine), and venlafaxine. In some embodiments, the at least one other agent is NRI selected from bupropion and atomoxetine (atomoxetine). At one end In some embodiments, the at least one other agent is NDRI BUPROPION (BUPROPION). In some embodiments, the at least one other agent is the dopamine β -hydroxylase inhibitor disulfiram. In some embodiments, the at least one other agent is an adenosine A2A receptor antagonist istradefylline. In some embodiments, the at least one additional agent is a sodium channel blocker selected from the group consisting of lenoda, carbamazepine, oxcarbazepine, and valproate. In some embodiments, the at least one additional agent is a calcium channel blocker selected from nimodipine (nimoopine), lenedane, and carbamazepine. In some embodiments, the at least one other agent is the central and peripheral alpha adrenergic receptor antagonist prazosin. In some embodiments, the at least one other agent is the central alpha adrenergic agonist clonidine. In some embodiments, the at least one other agent is the central and peripheral beta adrenergic receptor antagonist propranolol. In some embodiments, the at least one other agent is an atypical antidepressant/antipsychotic selected from the group consisting of bupropion, olanzapine (olanzapine), risperidone, and quetiapine. In some embodiments, the at least one other agent is a tricyclic agent selected from amitriptyline, amoxapine, desipramine, doxepin, imipramine, nortriptyline, prottyline, and trimipramine. In some embodiments, the at least one other agent is an anticonvulsant agent selected from the group consisting of phenytoin, lenoda, carbamazepine, oxcarbazepine, valproate, topiramate, tiagabine, vigabatrin (vigabatrin), and levetiracetam. In some embodiments, the at least one other agent is the glutamate antagonist topiramate. In some embodiments, the at least one other agent is a GABA agonist selected from baclofen, valproate and topiramate. In some embodiments, the at least one other agent is carbidopa, a dopamine-metabolizing enzyme inhibitor. In some embodiments, the at least one other agent is the partial dopamine D2 agonist aripiprazole. In some embodiments, the at least one additional agent is an opioid receptor antagonist selected from naltrexone and naloxone. In some embodiments, the at least one additional agent is mood stabilizer selected from carbamazepine and lithium And (4) dosing. In some embodiments, the at least one other agent is a direct or indirect dopamine agonist selected from dopamine, bromocriptine, pergolide, amantadine, mazindole, and ritalin. In some embodiments, the at least one additional agent is gepirone, a partial 5HT1 agonist. In some embodiments, the at least one other agent is the serotonin 5HT2 antagonist ritanserin (ritanserin). In some embodiments, the at least one other pharmaceutical agent is the opioid methadone. In some embodiments, the at least one other agent is the partial opioid agonist buprenorphine. In some embodiments, the at least one additional agent is the partial nicotinic acid agonist champix. In some embodiments, the at least one additional pharmaceutical agent is an inhalant selected from the group consisting of benzene, toluene, o-xylene, m-xylene, p-xylene, ethylbenzene, fluorobenzene, o-difluorobenzene, 1, 3, 5-trifluorobenzene, 1, 2, 4-trifluorobenzene, pentafluorotoluene, pentafluorobenzene, and perfluorobenzene. In some embodiments, the method further comprises co-administering a therapeutically effective amount of at least one additional agent selected from the group consisting of benzodiazepines Levodopa, carnisprodol, modafinil, acamprosate, gamma-butyrolactone, gamma-hydroxybutyrate, opioids, psilopubin, hallucinogenic mushroom, tobacco and nicotine. In some embodiments, compound a is administered to the patient after the patient has abstinent from substance use for a period of time. In some embodiments, the therapeutically effective amount of compound a in the patient is determined by gradually increasing the amount of compound a administered to the patient until a targeted therapeutic response is observed. In some embodiments, the amount of compound a is gradually reduced after the patient achieves remission. In some embodiments, the amount of compound a is unchanged after the patient achieves remission.

The invention also provides methods of treating at least one stage of cocaine dependence in a patient. In some embodiments, the at least one phase is selected from the group consisting of an acquisition, maintenance, regression, and relapse phase. The method comprises the following steps:administering to the patient a therapeutically effective amount of compound a. In some embodiments, compound a may inhibit acquired stage progression in a patient. In some embodiments, compound a may promote the development of the remission stage in the patient. In some embodiments, compound a may reduce the frequency of relapse in a patient. In some embodiments, compound a may alleviate at least one symptom of abuse, dependence, or withdrawal of cocaine in a patient. In some embodiments, compound a can alleviate at least one symptom of cocaine abuse in a patient selected from the group consisting of: periodic cocaine use resulting in failure to fulfill major obligations at work, school, or home; periodic cocaine use in hazardous situations of the body; periodic cocaine use involving legal issues; and sustained cocaine use, although such substance use has persistent or periodic social or interpersonal problems (caused or exacerbated by the consequences of cocaine). In some embodiments, compound a can reduce at least one symptom of cocaine dependence in a patient selected from the group consisting of: tolerance; withdrawal symptoms; often ingesting large amounts of cocaine or then desirably for longer periods of time; continued desire and/or unsuccessful efforts to curtail or control cocaine use; spending a lot of time in at least one activity in order to obtain cocaine, use cocaine and restore its effect; abandonment and/or reduction of at least one important social, occupational, and recreational activity due to cocaine use; and continued use of cocaine despite knowledge of persistent and/or periodic physical and/or psychological problems caused or exacerbated by cocaine. In some embodiments, compound a can reduce at least one symptom of cocaine abuse and dependence in a patient selected from the group consisting of: attention deficit hyperactivity disorder; the feeling of drunkenness; increased vitality, excitement and sociability; does not feel hunger and fatigue; a clear sense of physical and psychological strength; a reduction in pain sensation; bronchitis; short gas; chest pain; palpitations; cardiac arrhythmia; cardiomyopathy; heart failure; the pupil is dilated; nausea; vomiting; headache; dizziness; vertigo; anxiety; a mental disorder; confusion of consciousness; nasal inflammation; nasal crusts; periodic nasal bleeding; stuffy nose; facial pain; dysphoria; and cocaine thirst And (6) obtaining. In some embodiments, compound a can increase at least one negative subjective symptom of cocaine abuse and dependence. In some embodiments, compound a can alleviate at least one symptom of cocaine withdrawal selected from fatigue, lack of pleasure, depression, irritability, sleep disorders, increased appetite, mental retardation, agitation, extreme suspicion, and craving for cocaine. In some embodiments, compound a may increase the patient's score on at least one of the following scales: ADHD-IV, HAM-D, HAM-A, BDI, apathy and cognitive function scales from neuropsychiatric scales. In some embodiments, the cognitive function rating scale is selected from the group consisting of WAIS-R, WMS-R, RAVLT, Trials I-VII, RCFT, and TMT (parts A and B). In some embodiments, compound a may reduce at least one of the amount and frequency of cocaine use by the patient in the patient. In some embodiments, compound a may promote remission in a patient. In some embodiments, the symptom relief is characterized by: at least one of early complete symptom relief, early partial symptom relief, sustained complete symptom relief, and sustained partial symptom relief. In some embodiments, compound a can prolong the time to remission in a patient. In some embodiments, the method further comprises treating with at least one of strain management and cognitive behavioral therapy. In some embodiments, the method further comprises co-administering a therapeutically effective amount of at least one additional agent selected from the group consisting of: selective Serotonin Reuptake Inhibitors (SSRIs), serotonin-norepinephrine reuptake inhibitors (SNRIs), Norepinephrine Reuptake Inhibitors (NRIs), norepinephrine-dopamine reuptake inhibitors (NDRIs), serotonin 5-hydroxytryptamine 1A (5HT1A) antagonists, dopamine β -hydroxylase inhibitors, adenylate receptor antagonists, adenylate A2A receptor antagonists, monoamine oxidase inhibitors (MAOI), monoamine oxidase B inhibitors, sodium channel blockers, calcium channel blockers, central and peripheral alpha adrenergic receptor antagonists, central alpha adrenergic agonists, central or peripheral beta adrenergic receptor antagonists, NK-1 receptor antagonists, Corticotropin Releasing Factor (CRF) antagonists, atypical antidepressants/antipsychotics. Tricyclic drugs, anticonvulsants, glutamate antagonists, gamma-aminobutyric acid (GABA) agonists, GABA metabolic enzyme inhibitors, GABA synthesis activators, partial dopamine D2 agonists, dopamine metabolic enzyme inhibitors, catechol-O-methyl-transferase inhibitors, opioid receptor antagonists, mood stabilizers, direct or indirect dopamine agonists, partial 5HT1 agonists, serotonin 5HT2 antagonists, opioids, carboxylase inhibitors, partial opioid agonists, partial nicotinic acid agonists, and inhalants. In some embodiments, the at least one other agent is an SSRI selected from paroxetine, sertraline, citalopram, escitalopram and fluoxetine. In some embodiments, the at least one other agent is an SNRI selected from duloxetine, mirtazapine (mirtazapine), and venlafaxine. In some embodiments, the at least one other agent is NRI selected from bupropion and atomoxetine (atomoxetine). In some embodiments, the at least one other agent is NDRI BUPROPION (BUPROPION). In some embodiments, the at least one other agent is the dopamine β -hydroxylase inhibitor disulfiram. In some embodiments, the at least one other agent is an adenosine A2A receptor antagonist istradefylline. In some embodiments, the at least one additional agent is a sodium channel blocker selected from the group consisting of lenoda, carbamazepine, oxcarbazepine, and valproate. In some embodiments, the at least one additional agent is a calcium channel blocker selected from nimodipine (nimoopine), lenedane, and carbamazepine. In some embodiments, the at least one other agent is the central and peripheral alpha adrenergic receptor antagonist prazosin. In some embodiments, the at least one other agent is the central alpha adrenergic agonist clonidine. In some embodiments, the at least one other agent is the central and peripheral beta adrenergic receptor antagonist propranolol. In some embodiments, the at least one other agent is an atypical antidepressant/antipsychotic selected from the group consisting of bupropion, olanzapine (olanzapine), risperidone, and quetiapine. In some embodiments, the at least one additional agent is selected from amitriptyline, amoxapine, desipramine, doxepin, imipramine, nortriptyline Tricyclic agents of prottyline and trimipramine. In some embodiments, the at least one other agent is an anticonvulsant agent selected from the group consisting of phenytoin, lenoda, carbamazepine, oxcarbazepine, valproate, topiramate, tiagabine, vigabatrin (vigabatrin), and levetiracetam. In some embodiments, the at least one other agent is the glutamate antagonist topiramate. In some embodiments, the at least one other agent is a GABA agonist selected from baclofen, valproate and topiramate. In some embodiments, the at least one other agent is carbidopa, a dopamine-metabolizing enzyme inhibitor. In some embodiments, the at least one other agent is the partial dopamine D2 agonist aripiprazole. In some embodiments, the at least one additional agent is an opioid receptor antagonist selected from naltrexone and naloxone. In some embodiments, the at least one additional agent is a mood stabilizer selected from carbamazepine and lithium. In some embodiments, the at least one other agent is a direct or indirect dopamine agonist selected from dopamine, bromocriptine, pergolide, amantadine, mazindole, and ritalin. In some embodiments, the at least one additional agent is gepirone, a partial 5HT1 agonist. In some embodiments, the at least one other agent is the serotonin 5HT2 antagonist ritanserin (ritanserin). In some embodiments, the at least one other pharmaceutical agent is the opioid methadone. In some embodiments, the at least one other agent is the partial opioid agonist buprenorphine. In some embodiments, the at least one additional agent is the partial nicotinic acid agonist champix. In some embodiments, the at least one additional pharmaceutical agent is an inhalant selected from the group consisting of benzene, toluene, o-xylene, m-xylene, p-xylene, ethylbenzene, fluorobenzene, o-difluorobenzene, 1, 3, 5-trifluorobenzene, 1, 2, 4-trifluorobenzene, pentafluorotoluene, pentafluorobenzene, and perfluorobenzene. In some embodiments, the method further comprises co-administering a therapeutically effective amount of at least one additional agent selected from the group consisting of benzodiazepines Levodopa, carnisprodol, modafinil, acamprosate, gamma-butyrolactone, gamma-hydroxybutyrate, opioids, psilopubin, hallucinogenic mushroom, tobacco and nicotine. In some embodiments, compound a is administered to the patient after the patient has abstained from cocaine for a period of time. In some embodiments, the therapeutically effective amount of compound a in the patient is determined by gradually increasing the amount of compound a administered to the patient until a targeted therapeutic response is observed. In some embodiments, the amount of compound a is gradually reduced after the patient achieves cocaine-dependent remission. In some embodiments, the amount of compound a is unchanged after the patient achieves cocaine-dependent remission. In some embodiments, compound a can treat at least one symptom of abuse, dependence, or withdrawal of at least one secondary substance in a patient. In some embodiments, the at least one secondary substance is selected from drugs of abuse and drug therapy. In some embodiments, the drug of abuse is selected from psychostimulants, opioids, hallucinogens, inhalants, sedatives, tranquilizers, hypnotics, anxiolytics, and illicit substances. In some embodiments, the psychostimulant is a β -phenylisopropylamine derivative. In some embodiments, the β -phenylisopropylamine derivative is selected from amphetamine, dextroamphetamine, and methamphetamine. In some embodiments, the psychostimulant agent is selected from the group consisting of synthetic hallucinogens, phenmetrazine, ritaline, bupropion, pemoline, mazindol, (-) norpseudoephedrine, and fenfluramine. In some embodiments, the opioid is selected from the group consisting of Lortab, tramadol, heroin, methadone, hydrocodone, and oxycodone. In some embodiments, the hallucinogen is selected from the group consisting of nudeomushroom, hallucinogen, lysergic acid diethylamide (LSD), phencyclidine (PCP), and ketamine. In some embodiments, the inhalant is selected from the group consisting of benzene, toluene, o-xylene, m-xylene, p-xylene, ethylbenzene, fluorobenzene, o-difluorobenzene, 1, 3, 5-trifluorobenzene, 1, 2, 4-trifluorobenzene, pentafluorotoluene, pentafluorobenzene and perfluorobenzene. In some embodiments, the drug therapy is selected from the group consisting of anesthetics, analgesics, anticholinergics, antihistamines, muscle relaxants, Non-steroidal anti-inflammatory drug therapy, over-the-counter drug therapy, and antidepressant drug therapy. In some embodiments, the drug of abuse is an alcohol, caffeine, opioid, cannabinoid, cannabis, benzodiazepineMyo-inositol (carisprol), tobacco, nicotine, paracetamol (Vicodin), hydrocodone, oxycodone hydrochloride, oxycodone, and tylosin (Tylox).

Pharmaceutically acceptable derivatives include acid, base, enol ethers and ester, hydrate, solvate and prodrug forms. The derivative is selected such that its pharmacokinetic (pharmokinetic) properties are superior to at least one characteristic of the corresponding neutral agent. Compound a may be derivatized prior to processing of the formulation.

The therapeutically effective amount of compound a or a pharmaceutically acceptable derivative may vary widely depending on the severity of the addiction or dependency, the age and relative health of the patient, the potency of the compound used and other factors. In certain embodiments, a therapeutically effective amount is from about 0.1 milligrams per kilogram of body weight (mg/kg) per day to about 50mg/kg of body weight per day. In other embodiments, the amount is about 1.0 to about 10 mg/kg/day. Thus, in certain embodiments, a therapeutically effective amount is from about 7.0 to about 3500 mg/day for a 70kg human, while in other embodiments it is from about 70 to about 700 mg/day.

Without undue experimentation, and relying on self knowledge and the disclosure of this application, one of ordinary skill in the art of treating such diseases will be able to determine a therapeutically effective amount of compound a for treating or preventing addiction or dependence. Compound a may be administered in the form of a pharmaceutical composition, typically (for example, but not limited to) by one of the following routes: oral, systemic (e.g., transdermal, intranasal, or suppository), or parenteral (e.g., intramuscular, intravenous, or subcutaneous). The composition may take the form of (for example, but not limited to): tablets, pills, capsules, semisolids, powders, sustained release formulations, solutions, suspensions, elixirs, aerosols or any other suitable composition and generally comprise compound a in combination with at least one pharmaceutically acceptable excipient. Acceptable excipients are, for example and without limitation, non-toxic agents that facilitate administration, and do not adversely affect the therapeutic benefit of the compound. Such excipients may be, for example, any solid, liquid, semi-solid, or, in the case of aerosol compositions, gaseous excipients commonly available to those skilled in the art.

Solid pharmaceutical excipients include, for example but are not limited to, starch, cellulose, talc, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride, dried skim milk, and the like. Liquid and semi-solid vehicles may be selected from, for example, but not limited to, water, ethanol, glycerol, propylene glycol, and various oils, including those of petroleum products, animal, vegetable, or synthetic origin (e.g., peanut oil, soybean oil, mineral oil, sesame oil, and the like). Liquid carriers, particularly injection solutions, including, for example and without limitation, water, saline, aqueous dextrose, and glycols are preferred. The compressed gas may disperse the compound in the form of an aerosol. Inert gases suitable for this purpose are, for example but not limited to, nitrogen, carbon dioxide, nitrous oxide, and the like.

In addition, the pharmaceutical preparations may, for example and without limitation, contain preservatives, solubilizers, stabilizers, wetting agents, emulsifiers, sweeteners, pigments, flavorants, salts for varying the osmotic pressure, buffers, masking agents or antioxidants. In certain embodiments, they may also comprise other therapeutically valuable substances. Other suitable Pharmaceutical carriers and their formulations are described in a.r. alfonso Remington's Pharmaceutical Sciences 1985, 17th ed. easton, Pa.: mack Publishing Company.

The amount of compound a in the composition may vary widely, depending on, for example, the type of formulation, the size of the unit dose, the type of excipient, and other factors known to those skilled in the art of pharmaceutical science. Typically, the final composition comprises from 10% w to 90% w of the compound, preferably from 25% w to 75% w, with the remainder being excipients. Preferably, for continuous treatment, the pharmaceutical composition is administered in a single unit dosage form, or when relief of symptoms is specifically desired, in an unlimited single unit dosage form.

Examples

Example 1

Oral administration [ 2 ]¹⁴C]After nepicastat (nepicastat), the majority of the radioactivity in the plasma was associated with nepicastat (nepicastat), which is the N-linked glucuronide of nepicastat (nepicastat) (metabolite 2, M2), and the unidentified polar fragment (M1). With multiple doses, nepicastat (nepicastat) did not accumulate significantly, and after single and multiple doses, T _1/2Are similar. T is_1/2Is 10-14 hours. It is noted that for nepicastat (nepicastat), between patients with a fast acetylation phenotype and those with a slow acetylation phenotype, C_maxOr AUC, although C of N-acetyl metabolite in slow acetylation_maxAnd AUC vs C of N-acetyl metabolite in Rapid acetylation_maxAnd AUC is much lower (as expected). In a study comparing the pharmacokinetics of 40mg tablets (with fasting or after a meal), there was no significant difference in plasma concentrations. T is_axFrom 1.4 hours in the fasted state to 3.5 hours after the meal.

The pharmacokinetics of nepicastat (nepicastat) after a single administration of 40mg were compared in both men and women. In women, the AUC is approximately 43% greater than that in men, and in women, C is greater than that in men_maxApproximately 23% greater. T in female versus male_1/2Long. Comparing the pharmacokinetics of nepicastat (nepicastat) after 10 days of administration with a 40mg dose, the AUC in healthy subjects was higher than the AUC for subjects with CHEF, T_1/2There is no difference. In any population, no significant accumulation occurred with multiple doses.

In humans, the radioactivity associated with the compound can be eliminated rapidly. At the first 72 hours, an average of 87.4% of the administered radioisotope was recovered, 82.4% in urine and 5.01% in feces. After 10 days, the average total recovery of radioisotope was 93.8%. In plasma, radioactive T _axIs 1-2 hours (similar to the case of nepicastat). The N-linked glucuronide of nepicastat (nepicastat) showed the greatest percentage of total radioactivity in plasma (26.8%) and urine (57.9%) over 0 to 48 hours during the rapid and slow acetylation. Final T finding Total radioactivity in plasma_1/2Very long (-100 hours), probably due to the slow elimination of polar moieties present at low concentrations.

Example 2

In studies designed to evaluate the effect of nepicastat on cognitive function, treatment of subjects with 5 or 40mg nepicastat did not significantly impair mood, sleep or cognitive ability. In that¹²³I thyroid uptake studies, nepicastat was not differentiated from placebo at 5, 40 and 100mg doses. The decrease in absorption after administration of a single 200mg dose of nepicastat was significantly greater than placebo but significantly less than the value after a 10mg dose of methimazole. In a single dose of a first-phase study, nepicastat is generally better tolerated at a dose of 5 to 800mg (calculated dose based on the hydrochloride salt) in healthy men.

Example 3

In an internal piroxostat multi-dose first-phase study, doses of 5 and 40mg were generally better tolerated in healthy men.

Treatment with 200mg for 5 out of 6 patients for 8 days (or longer) resulted in the formation of a rash that spontaneously resolved.

One patient developed atrial arrhythmia and periodic right bundle branch block 6 days after administration of 200mg nepicastat.

Example 4

Within the subject design, sixteen non-therapeutic cocaine-dependent volunteers (in an inpatient fashion) were studied using double-blind, placebo-controlled studies. After informed consent, potential volunteers were subjected to outpatient psychosis and medical screening. Qualified volunteers were confirmed and physical examination, EKG, pregnancy test and mental test were completed. The study used a escalating dose schedule in which participants (n-12) received escalating doses of cocaine (0mg, 10mg, 20mg and 40mg) during daily treatment with escalating doses of nepicastat (0mg, 80mg, 160 mg). To maintain blindness, a parallel group of participants (n-4) received placebo treatment only daily throughout the study period. Treatment was performed daily at each dose level for 4 days, or for more than 4 half-lives (10 to 14 hours) of nepicastat. On day 4 of each dose level treatment, participants were allowed to receive cocaine in the order of 0mg, 10mg, 20mg and 40 mg. Cocaine is given at hourly intervals to provide sufficient time for cardiovascular and subjective effects to return to baseline. During all procedures associated with cocaine administration, continuous EKG and frequent blood pressure are used to carefully monitor cardiovascular indicators, and if the cardiovascular indicators exceed predetermined limits, parameter monitoring is discontinued and cocaine is no longer administered. Previous studies have shown that 6 doses of 32mg cocaine given at 14 minute intervals are safe, and extending the inter-dose intervals to 1 hour can further increase safety. Blood samples were collected and analyzed for pharmacokinetics of 10mg of cocaine (administered on the third day of treatment with 0mg of nepicastat, and re-administered on the third day of treatment with 80mg and 160mg of nepicastat). The pharmacokinetic effect of nepicastat on cocaine was studied. Based on existing information, there is no expected interaction.

Each participant took approximately 12 days to complete the study. Within a year, 16 participants could complete the study.

To participate in the study, the participants must:

1. that is, volunteers in english who did not seek treatment at the time of the study;

2. between 18-55 years of age;

3. compliance with the cocaine-dependent DSM-IV TR criteria;

4. having a self-recorded history of cocaine use (by IV route) and, prior to admission, providing at least one cocaine-positive urine;

5. has the following vital signs: resting pulse is between 50 and 95bpm, blood pressure is between 85-150mm Hg systolic pressure and 45-96mm Hg diastolic pressure; must meet the standard within 2 days of allowance

6. With hematological and chemical laboratory tests within the normal (+/-10%) range, with the following exceptions: a) liver function test (total bilirubin, ALT, AST and alkaline phosphatase) is less than or equal to 3x upper limit of normal, and b) kidney function test (creatinine and BUN) is less than or equal to 2x upper limit of normal;

7. having a baseline EKG showing clinically normal sinus rhythm, clinically normal conduction, and no clinically significant arrhythmia;

8. with a medical history and brief physical examination, showed no clinically significant contraindications for study participation, as judged by the hospitalized physician and the primary investigator.

Potential participants were excluded from the participants in the study if:

1. have any history or evidence that accounts for an epileptic disorder or brain injury;

2. any previous medical adverse reactions to cocaine including loss of consciousness, chest pain or seizures;

3. having a neurological or psychiatric disorder, for example:

● psychotic disorder, bipolar disease or major melancholia (assessed by SCID);

● organic brain disease or dementia, assessed by clinical interviews;

● history of any psychiatric condition that requires treatment or makes study compliance difficult;

● have a history of suicide attempts (as assessed by SCID) over the past three months and/or currently have a suicide concept/program (as assessed by SCID);

4. has clinically significant evidence of heart disease or hypertension, as determined by PI, even though the participants can take antihypertensive drugs;

5. family history in first-degree relatives with early cardiovascular morbidity or mortality, as determined by PI;

6. evidence of an untreated or unstable medical condition, including: neuroendocrine, autoimmune, renal, hepatic, or active infectious diseases;

7. has HIV, and is currently presenting with symptoms, has a diagnosis of AIDS, or is receiving antiretroviral drugs;

8. During pregnancy or lactation. Other women must not be pregnant (i.e., by surgical sterilization, anogenital or post-menopause) or use a reliable form of contraception (e.g., abstinence, contraceptive pill, contraceptive ring, condom or spermicide.) before entry into the study, when permitted by the hospital, and at the end of study participation, all women must provide a negative pregnancy urine test;

9. asthma or current use of alpha or beta agonists, theophylline or other sympathomimetic agents;

10. suffering from any other disease, condition, or using psychotropic drugs, which would prevent the safe and/or successful completion of the study from the point of view of the PI and/or the hospitalized physician.

Abort criteria after Start

11. Positive urine drug screening or breath testing indicates illegal use of cocaine, alcohol, opiates or other drugs of abuse, which are not delivered as part of the regimen;

12. failure to follow the study procedure;

13. due to the exaggerated response to cocaine, abort criteria were met as described below.

Abort criterion

Participants must continue to meet inclusion criteria in order to remain in the program. If there is a clinically significant arrhythmia, or if the vital signs are outside the acceptable range (resting pulse < 130bpm, blood pressure below 165mm Hg systolic and 100mm Hg diastolic), cocaine administration cannot begin. These values are higher than those of the inclusion/exclusion criteria, since a temporary increase in vital signs may occur in the expected case of cocaine acceptance. In addition, if there is a behavioral phenomenon of cocaine toxicity (agitation, mental abnormalities, failure to coordinate with the course of the study), repeated doses of cocaine cannot be administered (investigator discontinues sustained cocaine delivery).

Abort criteria for further participation

Subject participation is terminated if any of the following conditions occur:

1. the systole BP > 180mm Hg for 5 minutes or more than 5 minutes;

2. diastolic BP > 120mm Hg for 5 minutes or more than 5 minutes;

3. heart rate > (220-age x0.85) bpm for 5 minutes or more.

Rationale for subject selection criteria

Participants were asked to have used cocaine by the IV route to avoid exposure to the route of administration which produces a more potent internal sensory effect. Age criteria were first selected to avoid enrolled participants with undiagnosed cardiovascular disease. Excluding participants with active HIV disease from possible exacerbation of their primary disease; participants with asymptomatic HIV may be included because the group is at high risk for cocaine dependence. Participants with asthma (or those taking asthma medication) were excluded due to possible adverse interactions between the beta agonist drug and cocaine.

Research medicine

Cocaine can produce a prototypic excitatory effect by inhibiting the uptake of DA, NE and serotonin into presynaptic storage particles. Cocaine has a short elimination half-life of about 90 minutes. The main clinical effects of cocaine are psychomotor stimulation and increased sympathetic tone, with significant increases in heart rate and blood pressure.

Up to 40mg of a single dose of cocaine is administered, and up to 200mg is administered in a self-administration phase, including 10 doses of 20mg administered at 13 minute intervals. These doses were moderate compared to the daily number of participants that had been reported in these studies; typical daily dosage patterns are on average 250mg to 500mg or more.

Higher doses than those recommended herein are associated with seizures and with severe cardiovascular toxicity and death. These potential toxicities were ameliorated by using relatively low doses, careful screening of potential volunteers, careful monitoring after cocaine administration to the participating humans, and rapid medical intervention in adverse circumstances.

The use of cocaine given IV can be made complete. Cocaine is metabolized primarily to benzoylecgonine by plasma esterase, which is not known to be affected by nepicastat. Benzoylecgonine and other minor metabolites are excreted via the kidney (renally).

In humans, cocaine for IV use is obtained from NIDA contractors, obtained with an authority to allow us to refer to the NIDA's IND of cocaine, and submitted to the FDA.

Increasing doses of nepicastat (0mg, 80mg, and 160mg) were administered at 7 AM. Treatment at each dose level lasted 4 days.

Starting with a lower dose and increasing the dose after completion of the first series of the study procedure, the risk of combination of nepicastat and cocaine can be minimized. This approach also reduced the risk of skin rash, which has occurred in 7% to 20% of volunteers to date. The incidence of rash is related to the dose and duration of treatment. Doses above 160mg create a greater risk of rash.

No pharmacokinetic interaction occurred because nepicastat is not an enzyme inhibitor, as evidenced by the pharmacokinetic evaluation of the 10mg dose of cocaine given on the third day of treatment at each dose level of nepicastat. Since nepicastat reduces NE synthesis, the rewarding effect of cocaine can be reduced during treatment with nepicastat. Because nepicastat increases plasma and brain concentrations of DA, DA-mediated side effects such as paranoia may occur. These symptoms were not observed during the trial for CHF, but no stimulant was administered in those studies.

After allowing, the participants were asked to submit a cocaine positive urine sample for recording ongoing drug use. Some participants (limited by the number of devices available) are also required to wear telemetry devices during the entire study to screen and record heart rate and motion. Based on the variation of these parameters, the data from such devices may identify the drug use scenario.

To control nicotine exposure, smoking was prohibited within 2 hours of the study procedure associated with cocaine administration or exposure cues. During the study, participants were asked to abstain from illegal and prescribed drug use and confirmed with daily urine and breath alcohol index tests.

For a given participant, the experimental period was performed at approximately the same time each day. Cocaine was administered in the laboratory. Cocaine was administered using a syringe pump that could administer the correct dose of cocaine or saline placebo within 2 minutes. Heart rate and blood pressure were monitored during and 1 hour after the drug administration period.

The participant was subjected to historical and physical examination of the target. Blood was drawn for standard laboratory tests including CBC, electrolytes, LFT and creatinine. Participants were screened for HIV as a service and counseling and treatment opinions were made on those participants who tested positive.

In some volunteers, an actifat MiniMitter was used to measure heart rate and exercise before admission (the number of volunteers was limited by the number of devices available). Minimist was mounted on the skin of the participants using glue and EKG and movements were recorded non-invasively (up to two weeks). The data may be downloaded to a PC for subsequent analysis.

The participants must meet the cocaine and nicotine dependent DSM-IV-TR criteria (as determined by Mini International Neuropsychiatric Interview (Mini)), and are defined by inclusion/exclusion criteria. MINI is a short, objective standard diagnostic interview developed by psychiatrists and clinicians in the United states and Europe in 1990 for DSM-IV TR and ICD-10 psychotic disorders. MINI is an objective standard interview for psychiatric evaluation selection and is a documented result of clinical psychopharmacological trials and epidemiological studies, the most widely used diagnostic interview tool for psychiatric disorders worldwide. This approach can be used to determine whether a patient meets the DSM-IV TR criteria for drug dependence and to rule out any severe psychiatric disorders (e.g., affective disorders, schizophrenia).

By training the investigator during the screening, a form of addiction severity index-Lite clinical coefficient (ASI-Lite CF) was performed. ASI-Lite is the estimated severity of a participant's state in seven areas (medical, occupational, drug use, alcohol use, legal, family/social and psychological) by interviewers. The Lite form is a shorter form of ASI that also preserves all the issues for computing ASI composite records. As with the ASI-Lite format, minimal family history information is collected, and the family history portion of the ASI is retained.

There is a third edition of the Beck depression scale (BDI) revised in 1996. The tool book retained its original 21 questionnaire structure, which took approximately 10 minutes to complete. BDI-II has been validated against BDI-IA and is still a superior index of depression/distress. This indicator is used to monitor participants who become clinically depressed during the trial, making it a measure of participant safety.

Current symptoms of Attention Deficit Hyperactivity Disorder (ADHD) are evaluated weekly using the ADHD-IV rating scale.

Apathy scales from neuropsychiatric scales were collected in baseline form.

DNA was collected with a buccal swab applied to a Whatman FTA card. These cards can provide secure and steady-state storage of biological samples for DNA extraction. The predicted yield of genomic DNA is 50-100. mu.g, which is suitable for testing over 500 genotypes (using current methods).

Genotype was determined using a 5' exonuclease (Taqman) based genotyping assay. The test was developed by Applied Biosystems (ABI; Assays by Design). Allele discrimination was performed using an ABI 3730 real-time PCR amplification apparatus (cycler).

Blood samples for analysis of cocaine pharmacokinetics were collected during treatment with 0mg nepicastat (study day 1) and during treatment with 80mg and 160mg nepicastat (study days 4 and 8). On the third day of treatment with nepicastat at each dose level, blood samples were collected-15, 20, 30, 40, 50, 60, 90, 120, 180, 240, 300, 360, 420 and 480 minutes after administration of 10mg cocaine. Note that: on day 4 of treatment with nepicastat at each dose level, an additional dose of cocaine (0-40mg) was administered, so the pharmacokinetic evaluation did not interfere with the other evaluations. Blood was collected, plasma was separated and frozen at-70 ℃ until analysis. Cocaine and BE were analyzed using liquid chromatography/tandem mass spectrometry (LC/MS/MS). For these analyses, the reference laboratory had a quantitative limit of 2.5 ng/ml. Pharmacokinetic analysis elucidated the pharmacokinetic effect of nepicastat on cocaine.

DBH is stored in NE depots and released along with NE. Thus, plasma DBH gives a good index of enzyme activity within the CNS. Blood samples will be taken at 10AM (prior to cocaine/placebo administration) daily and stored for subsequent analysis. DBH activity was determined using the tyramine-phenolethanolamine method using the high performance liquid chromatography-fluorescence system described previously. This allows testing of the DBH changes over time, providing insight into the efficacy of nepicastat in inhibiting DBH. Throughout the protocol, BDI was given repeatedly in order to monitor mood changes.

Subjective effects were measured using a computerized Visual Analog Scale (VAS) consisting of continuous 10cm digitized lines, with the aim of scoring from 0 to 100. The participant is asked to move the cursor away from the left hand end and onto the line by pressing the left or right mouse button for left and right movement on the line. VAS is designed to provide a rapidly acquired ranking of intoxication, irritability and craving for cocaine. These include the following ratings: "any drug effect", "high", "good effect", "motivation" and "bad effect", "paranoia feeling", "suspicion feeling" and "if possible cocaine is used", "craving cocaine", "cocaine can now be rejected" and "cocaine is needed". VAS assay results were collected prior to cocaine administration, and at 5, 10, 15, 20, 30, and 45 minutes after drug administration.

15 minutes after cocaine administration, participants were asked how much they paid for the dose of drug, based on $ 50/gram (based on current costs if purchased from an illegal channel). Such fixed numbers are provided to standardize the response (assuming that the price of cocaine varies over time and place).

On day 13 (the last day of treatment with study drug), all patients participated in the "experimental phase" where patients could make a series of choices between money and either placebo (saline) or double-blind infusion of 20mg cocaine. In one phase, only placebo (saline) is available. In another phase, only 20mg of cocaine was available. The participants chose to self-administer placebo, or to receive money and 20mg cocaine (relative to money). This phenomenon occurs in the morning (am) and afternoon (pm), the sequence being randomized and balanced, so that placebo or nepicastat is administered first to an equal number of patients.

Experimental (selection) phase:

during each phase, the patient is asked to make a series of choices between infusions and money commensurate with the color ("blue" or "green"). The color corresponds to the dose (0mg or 20mg of cocaine) given to the patient during the sampling period. For each of the 2 selection phases, the participant may make 10 selections of infusion (0mg IV cocaine in one phase, 20mg cocaine in another phase) or money. Participants made a series of choices between increasing value monetary schemes ($0.05, $0.05, $0.05, $1, $4, $7, $10, $13 and $16) or cocaine (0mg or 20 mg/IV/infusion) (using Patient Controlled Analgesia (PCA) pumps).

Using the PCA button, the participant made an infusion selection, and the money selection was verbally notified to the investigator. Infusion was carried out for more than 2 minutes followed by a rest period of 3 minutes. Thus, selection was performed at 5 minute intervals.

After indicating their selection, participants received cocaine doses immediately, provided vital signs were maintained within predetermined limits up to a maximum of 200mg cocaine (10X20 mg). Immediately after selection, the patient is given a choice of money directly, but the money must be spent before it is released.

The chart shows the experimental selection phase for a total of 16 participants.

	Selecting	8 participants	8 participants
	Selecting	8 participants	8 participants	am	Option 1	0mg cocaine IV or $0.05	20mg cocaine IV or $0.05
am	Option 2	0mg cocaine IV or $0.05	20mg cocaine IV or $0.05	am	Option 1	0mg cocaine IV or $0.05	20mg cocaine IV or $0.05
am	Option 2	0mg cocaine IV or $0.05	20mg cocaine IV or $0.05	am	Selection 3	0mg cocaine IV or $0.05	20mg cocaine IV or $0.05
am	Option 4	0mg cocaine IV or $0.05	20mg cocaine IV or $0.05	am	Selection 3	0mg cocaine IV or $0.05	20mg cocaine IV or $0.05
am	Option 4	0mg cocaine IV or $0.05	20mg cocaine IV or $0.05	am	Option 5	0mg cocaine IV or $1.00	20mg cocaine IV or $1.00
am	Selection 6	0mg cocaine IV or $4.00	20mg cocaine IV or $4.00	am	Option 5	0mg cocaine IV or $1.00	20mg cocaine IV or $1.00
am	Selection 6	0mg cocaine IV or $4.00	20mg cocaine IV or $4.00	am	Option 7	0mg cocaine IV or $7.00	20mg cocaine IV or $7.00
am	Selection 8	0mg cocaine IV or $10.00	20mg cocaine IV or $10.00	am	Option 7	0mg cocaine IV or $7.00	20mg cocaine IV or $7.00
am	Selection 8	0mg cocaine IV or $10.00	20mg cocaine IV or $10.00	am	Selection 9	0mg cocaine IV or $13.00	20mg cocaine IV or $13.00
am	Selection 10	0mg cocaine IV or $16.00	20mg cocaine IV or $16.00	am	Selection 9	0mg cocaine IV or $13.00	20mg cocaine IV or $13.00
am	Selection 10	0mg cocaine IV or $16.00	20mg cocaine IV or $16.00
pm	Option 1	20mg cocaine IV or $0.05	0mg cocaine IV or $0.05
pm	Option 1	20mg cocaine IV or $0.05	0mg cocaine IV or $0.05	pm	Option 2	20mg cocaine IV or $0.05	0mg cocaine IV or $0.05
pm	Selection 3	20mg cocaine IV or $0.05	0mg cocaine IV or $0.05	pm	Option 2	20mg cocaine IV or $0.05	0mg cocaine IV or $0.05
pm	Selection 3	20mg cocaine IV or $0.05	0mg cocaine IV or $0.05	pm	Option 4	20mg cocaine IV or $0.05	0mg cocaine IV or $0.05
pm	Option 5	20mg cocaine IV or $1.00	0mg cocaine IV or $1.00	pm	Option 4	20mg cocaine IV or $0.05	0mg cocaine IV or $0.05
pm	Option 5	20mg cocaine IV or $1.00	0mg cocaine IV or $1.00	pm	Selection 6	20mg cocaine IV or $4.00	0mg cocaine IV or $4.00
pm	Option 7	20mg cocaine IV or $7.00	0mg cocaine IV or $7.00	pm	Selection 6	20mg cocaine IV or $4.00	0mg cocaine IV or $4.00
pm	Option 7	20mg cocaine IV or $7.00	0mg cocaine IV or $7.00	pm	Selection 8	20mg cocaine IV or $10.00	0mg cocaine IV or $10.00
pm	Selection 9	20mg cocaine IV or $13.00	0mg cocaine IV or $13.00	pm	Selection 8	20mg cocaine IV or $10.00	0mg cocaine IV or $10.00
pm	Selection 9	20mg cocaine IV or $13.00	0mg cocaine IV or $13.00	pm	Selection 10	20mg cocaine IV or $16.00	0mg cocaine IV or $16.00

In the nepicastat treatment group, a sample size of 12 could detect moderate to large effects, which is suitable for initial evaluation. The figure (fig. 1) illustrates the magnitude of the effect obtained with sample sizes from 5 to 15. Increasing the sample size (above 12) increases the efficiency of the assay to detect differences between treatments, but increases costs. The placebo treated group was included only to maintain blindness and not as a comparative group.

The analysis was mainly focused on the effect of nepicastat in the nepicastat treatment group. The placebo-treated group was included primarily to maintain blindness. Side effects and adverse conditions (AEs) were tabulated and compared between the entire treatment condition using ANOVA or chi square. The subjective and cardiovascular effects produced by cocaine IV and placebo during treatment with nepicastat were compared to those produced during treatment with placebo using repeated assays (time bearing the repeat measure), analysis of variance (ANOVA), peak effect one-way ANOVA, and, if indicated, the area under the ANOVA curve.

Example 5

Bovine and human dopamine β -hydroxylase activity was analyzed by measuring the conversion of tyramine to ethanolamine phenolate. Bovine kidney dopamine β -hydroxylase was obtained from Sigma Chemicals (St Louis, MO, USA) and human dopamine β -hydroxylase was purified from the culture medium of neuroblastoma cell line SK-N-SH. The assay was performed at pH5.2 and 32 ℃ in a medium containing 0.125M NaAc, 10mM fumarate, 0.5-2. mu.M CuSO₄、0.1mg.ml^-1Catalytic enzyme, 0.1mM tyramine and 4mM ascorbate. In a typical assay, 0.5-1 milliunits of enzyme is added to the reaction mixture, followed by the addition of a substrate mixture comprising the catalytic enzyme, tyramine and ascorbate to initiate the reaction (final volume of 200 μ l). The samples were incubated at 37 ℃ for 30 to 40 minutes with or without appropriate concentrations of nepicastat or compound B. The reaction was quenched by a stop solution containing 25mM EDTA and 240. mu.M 3-hydroxytyrosamine (internal standard). The samples were analyzed for ethanolamine by reverse phase High Pressure Liquid Chromatography (HPLC) using uv detection (at 280 nM). HPLC chromatographic operation was performed as follows: flow rate 1ml.min ^-1Elution was carried out with 10mM acid (acid), 10mM 1-heptanesulfonic acid, 12mM tetrabutylammonium phosphate and 10% methanol in the absence of a gradient (isocratic) using a LiChrocarT 125-4RP-18 column. Percent activity retained was calculated based on controls, corrected using internal standards, and fitted to a non-linear four-parameter concentration response curve.

Nepicastat (S-enantiomer) and compound B (R-enantiomer) produced concentration-dependent inhibition of the activity of bovine and human dopamine β -hydroxylase. Calculated IC of nepicastat for bovine and human enzymes₅₀Values were 8.5. + -. 0.8nM and 9.0. + -. 0.8nM, respectively. Compound B was slightly less effective than nepicastat (for cattle and humans)Enzyme, IC₅₀Values were 25.1. + -. 0.6nM and 18.3. + -. 0.6nM), respectively). Nepicastat was demonstrated to be a potent inhibitor of human and bovine dopamine β -hydroxylase in vitro. The inhibitory effect of the compounds is stereospecific, since the S-enantiomer (nepicastat) is to some extent (marginally) (but significantly) more potent than the R-enantiomer (compound B).

Using established assays, nepicastat activity was determined for 12 selected enzymes and receptors. A brief description of the principle constituting each enzymatic assay is given in FIG. 2. The binding data were analyzed by iterative curves (fitted with a four parameter logistic equation). Using Cheng-Prusoff equation, from IC ₅₀Values Ki values were calculated. The enzyme inhibitory activity may be expressed as IC₅₀Value (concentration required to produce 50% inhibition of enzyme activity).

Nepicastat against a large number of other enzymes (tyrosine hydroxylase, acetyl-CoA synthetase, acyl-CoA-cholesterol transacylase, Ca2 +/calmodulin protein kinase II, cyclooxygenase-I, HMG-CoA reductase, neutral endopeptidase, nitric oxide synthase, phosphodiesterase III, phospholipase A2, and protein kinase C) and neurotransmission mediator receptor (alpha.) (II, III_1A，α_1B，α_2A，α_2B，β₁And beta₂Adrenergic receptors, M1 muscarinic receptors, D1 and D2 dopamine receptors, mu opioid receptors, 5-HT_1A，5-HT_2AAnd 5-HT_2CSerotonin receptor) has negligible affinity (IC50 value or Kis > 10 μ M). Because the compounds have negligible affinity for 12 other enzymes and 13 neurotransmitter receptors, nepicastat has been shown to be highly selective for dopamine β -hydroxylase.

In a study related to SHRs, the drug nepicastat ((S) -5-aminomethyl-1- (5, 7-difluoro-1, 2, 3, 4-tetrahydronaphthalen-2-yl) -1, 3-dihydroimidazole-2-thione hydrochloride) and the corresponding R-enantiomer (compound B) were dissolved in distilled water and administered orally with a gavage needle. In a dog study, the drug was filled in capsules and administered orally. All doses are expressed as free base equivalents.

Male SHRs (15-16 weeks old, Charles River, Wilmington, MA, USA) were used for in vivo studies. On the day of the study, animals were weighed and randomized three times with vehicle (control) or appropriate dose of nepicastat (3, 10, 30 or 100mg^-1Po) or Compound B (30 mg.kg)^-1Po), 12 hour intervals. 6 hours after the third dose, rats were anesthetized with halothane (halothame), decapitated, tissues (cerebral cortex, mesenteric artery and left ventricle) were rapidly harvested, weighed, placed in frozen perchloric acid (0.4M), frozen in liquid nitrogen, and stored at-70 ℃ until later analysis. To quantify the noradrenaline and dopamine concentrations, the tissues were homogenized by brief sonication and centrifuged (13,000rpm) for 30 minutes at 4 ℃. The supernatants were tested for norepinephrine and dopamine (added to the supernatant) by HPLC using electrochemical detection with 3, 4-dihydroxybenzylamine (internal standard).

Catecholamine content of basal tissue in control animals (μ g.g)^-1Wet weight) as follows: mesenteric artery (noradrenaline, 10.40 + -1.03; dopamine, 0.25 + -0.02), left ventricle (noradrenaline, 1.30 + -0.06; dopamine, 0.02 + -0.00) and cerebral cortex (noradrenaline, 0.76 + -0.03; dopamine, 0.14 + -0.01). Nepicastat resulted in a dose-dependent reduction in noradrenaline content and an increase in dopamine to dopamine/noradrenaline ratio in three tissues studied.

In mesenteric artery and left ventricle, at 3mg.kg or more^-1At doses of (2), these changes were statistically significant (p < 0.05), but in the cerebral cortex, only at 30 and 100mg.kg^-1Statistical significance was obtained at the dose of (c). At the highest dose studied (100 mg.kg)^-1Po), norepinephrine decreased by 47%, 35%, 42%, and dopamine increased by 820%, 800%, and 86%, respectively, in the mesenteric artery, left ventricle, and cerebral cortex. When the concentration is 30mg.kg^-1(po) Nepirstat caused a higher catecholamine content in mesenteric arteries and left ventricle than the R-enantiomer (Compound B) when testedA significant change.

Male beagles (10-16kg, Marshall farm USA Inc, North Rose, NY, USA) were also used for in vivo testing. On the day of the study, dogs were weighed and randomly dosed orally with empty capsules (control) or appropriate doses of nepicastat (0.05, 0.5, 1.5 or 5 mg.kg)^-1(ii) a po, b.i.d), for 5 days. Dogs were euthanized with pentobarbital and tissues (cerebral cortex, renal artery, left ventricle) were rapidly harvested 6 hours after the first dose on day 5. The tissue was subsequently processed and analyzed for norepinephrine and dopamine.

Data are expressed as mean (mean) ± standard error of the mean (SEM). Tissue and plasma catecholamine data were analyzed using nonparametric one-way analysis of variance (ANOVA) or two-way ANOVA, respectively, followed by pairwise comparisons using the Fisher LSD test. P < 0.05 was considered statistically significant.

Catecholamine content of basal tissue in control animals (μ g.g)^-1Wet weight) as follows: renal arteries (noradrenaline, 10.7 + -1.05; dopamine, 0.22 + -0.01), left ventricles (noradrenaline, 2.11 + -0.18; dopamine, 0.07 + -0.03) and cerebral cortex (noradrenaline, 0.26 + -0.02; dopamine, 0.03 + -0.00). Nepicastat resulted in a dose-dependent reduction in noradrenaline content and an increase in dopamine to dopamine/noradrenaline ratio in the three tissues studied when compared to control animals.

In three tissues, the weight is more than or equal to 0.1mg.kg^-1Sky^-1These changes were statistically significant (p < 0.05). At the highest dose studied (5 mg.kg)^-1Po), norepinephrine decreased 88%, 91% and 96% and dopamine increased 627%, 700% and 166% respectively in the renal arteries, left ventricle and cerebral cortex.

Male beagle dogs were randomized to oral administration of empty capsules (control) or nepicastat (2mg^-1(ii) a po, b.i.d), for 15 days. Venous blood samples were taken 6 hours after the first dose each day and assayedPlasma concentrations of dopamine and norepinephrine. Samples were collected in tubes containing heparin and glutathione, centrifuged at-4 ℃ and the separated plasma stored at-70 ℃ until analysis. The baseline concentrations of catecholamines in the two groups of animals did not differ significantly from each other: in the control group, plasma norepinephrine and dopamine concentrations were 460.3 + -59.6 and 34.4 + -11.9 pg/ml, respectively^-1In the nepicastat treatment group, the concentrations are 401.9 +/-25.5 and 41.1 +/-8.8 pg^- ¹. Nepicastat (2 mg.kg) when compared to control group^-1B.i.d, po) caused a significant decrease in plasma concentration of noradrenaline, resulting in an increase in plasma concentration of dopamine and dopamine/noradrenaline ratio.

For the control of congestive heart failure, the inhibition and modulation of sympathetic function is an attractive therapeutic strategy by pharmacological means, due to the increased activity of this system and the progressive worsening of the disease. The aim of this study was to pharmacologically identify the effect of nepicastat, a compound that modulates norepinephrine synthesis in sympathetic nerves by inhibiting the enzyme dopamine β -hydroxylase.

Inhibition of dopamine β -hydroxylase in vivo is expected to result in increased levels of substrate (dopamine) and decreased levels of product (norepinephrine) in tissue innervated by norepinephrine. This expectation was confirmed in experiments investigating the effect of nepicastat on the production of catecholamine levels in central and peripheral tissues in vivo. In SHRs and beagle dogs, nepicastat causes a dose-dependent decrease in noradrenaline levels and an increase in dopamine levels in peripheral (mesenteric or renal arteries, left ventricle) and central (cerebral cortex) tissues. In this respect, compound B was less effective than nepicastat, which had a lower IC for the enzyme than the former enantiomer did₅₀The values are consistent. Although the dopamine/norepinephrine ratio also increased, it does not appear that dopamine is used to stoichiometrically replace norepinephrine. For this finding, the most likely explanation is: tissue levels of dopamine are underestimated due to their intra-neural metabolism.

In the cerebral cortex, the ability of nepicastat to alter catecholamine levels indicates that the drug substance has penetrated the blood brain barrier. In dogs, the degree of catecholamine changes in the cerebral cortex is comparable to that in the surrounding tissues. However, in SHRs, the dosage is low (≦ 10 mg.kg) ^-1) Nepicastat (b) causes significant changes in noradrenaline and dopamine levels in the surrounding tissues, without affecting catecholamines in the cerebral cortex. This indicates that, at least in SHRs, the drug has moderate peripheral selectivity.

Plasma norepinephrine concentrations provide an effective measure of overall sympathetic activity, although such parameters may be affected by changes in neuronal uptake and metabolic clearance rates of catecholamines. In dogs, the baseline concentration of norepinephrine in plasma is unexpectedly elevated, perhaps as a reflection of the initial stress induced by the phlebotomy blood sampling procedure. Nevertheless, nepicastat caused a significant decrease in plasma norepinephrine concentration compared to the control, consistent with a decrease in mediator synthesis and release, although indirect effects (secondary to promotion of neuronal uptake or metabolic clearance) could not be seen. Since released noradrenaline represents a small fraction of the total neuronal noradrenaline reserve, inhibitors of noradrenaline biosynthesis affect noradrenaline release only after sufficient depletion of the existing reserve of catecholamines has occurred. Accordingly, a statistically significant decrease in plasma norepinephrine concentration (indicative of a gradual regulation of the sympathetic nervous system) was not achieved until 4 days of nepicastat administration.

There is increasing evidence that long-term activation of the sympathetic nervous system is a maladaptive response in congestive heart failure. This argument is supported by clinical trials showing the beneficial effect of carvedilol on long-term morbidity and mortality in congestive heart failure patients. It should be noted, however, that most patients require a certain level of sympathetic induction (drive) in order to support cardiovascular homeostasis. Indeed, the therapeutic value of beta-blockers (including carvedilol) is limited by their propensity to cause hemodynamic degeneration, particularly during the initiation of treatment. This undesirable effect resulting from acute withdrawal of sympathetic vectors requires careful dose determination. Inhibitors of dopamine β -hydroxylase, such as nepicastat, may not have this undesirable effect for the following reasons. Firstly, such drugs can attenuate, rather than eliminate, the release of norepinephrine and secondly, they produce gradual modulation of the system, thereby avoiding the need for dose determination. Another advantage of nepicastat over β -blockers is that dopamine levels can be increased by agonism of dopamine receptors, which can have beneficial effects on renal function such as renal vasodilation, diuresis and natriuresis.

Nepicastat is a potent, selective and orally active inhibitor of dopamine β -hydroxylase that can treat cardiovascular disorders associated with hyperactivation of the sympathetic nervous system.

Example 6

Nepicastat was prepared based on the following: reacting tetralone 3 (from AlCl) under the conditions described by Terashima (LAH, (-) -1R, 2S-N-methylephedrine, 2-ethylaminopyridine)₃Catalyzed reaction of 3, 5-difluorophenylacetyl chloride with ethylene (in CH)₂Cl₂Friedel-crafts reaction at-65 ℃) to obtain R- (+) -tetrahydronaphthol 4a (92-95% ee) which is converted to R- (+) -methanesulfonate and then reacted with sodium azide to obtain a mixture of azide and dihydronaphthalene 7 (9: 1). The azide was hydrogenated and the product was treated with anhydrous HCl to give S- (-) -amine hydrochloride which was converted to S- (-) -aminonitrile by Strecker reaction (formaldehyde bisulphite complex and KCN). The formation of the heterocycle is achieved by successive diformylation of the aminonitrile and subsequent treatment with thiocyanic acid. Competing hydrolysis of the nitrile gives the corresponding amount of primary amide. Reduction of the nitrile to the amine was accomplished using LAH in THF (93-96% ee). By following the same route as described above, (+) -1S, 2R-N-methylephedrine is used as a chiral auxiliary in the Terashima reduction of ketones The enantiomer (91.6% ee) was obtained. The absolute configuration of the chiral center is based on the previously described S- (-) -2-tetrahydronaphthol preferred in the literature.

Melting points were determined on a Uni-Melt Thomas Hoover capillary melting point apparatus or Metttler FB 81HT cell (with Mettler FP90 processor) and were not corrected. Mass spectra were obtained using a Finnigan MAT 8230 (electron-bombardment or chemical ionization) or Finnigan MAT TSQ70(LSIMS) spectrometer. Recording on Bruker ACF300, AM300, AMX300 or EM390 spectrometer¹The H NMR spectrum gives the chemical shifts in ppm (. delta.) (tetramethylsilane as internal standard). The IR spectra were recorded on a Nicolet SPC FT-IR spectrometer. The UV spectra were recorded on a Varian Cary 3 UV-visible spectrometer (Leeman Labs Inc). Optical rotations were measured with a Perkin-Elmer Model 141 polarimeter. Chiral HPLC assay on a region Chiral AGP column (4.6X100mM) with 2% acetonitrile-98% 20mM KH₂PO₄(pH4.7) elution (1mL/min at 20 ℃ C.).

5, 7-difluoro-2-tetralone. Adding SOCl₂(100ml) were added in one portion to 3, 5-difluorophenylacetic acid (100g, 0.58mol), stirred for 15 h, after which the volatiles were evaporated under reduced pressure. Dissolving the obtained oily acid chloride in CH₂Cl₂(200mL) and added dropwise to mechanically stirred AlCl ₃(154g，1.16mol)/CH₂Cl₂(1.0L) in suspension. The stirred suspension was cooled to an internal temperature of-65 ℃ in a dry ice/acetone bath and the acid chloride solution was added at the rate described below so as to maintain an internal temperature < -60 ℃. After the addition was complete, ethylene gas was rapidly bubbled through the reaction mixture for 10 minutes at-65 ℃. The reaction mixture was allowed to warm to 0 ℃ over 2 hours with stirring, then cooled to-10 ℃, initially treated dropwise with water (500mL) and then added rapidly. The organic layer was separated, washed with brine (100mL), MgSO₄And (5) drying. Evaporation under reduced pressure gave a black oily residue which was vacuum distilled on a Kugelrohr collecting material boiling between 90-110 deg.C (1.0 to 0.7mm Hg). The distillate was redistilled at 100 ℃ and 105 ℃ (0.3mm Hg) to give the product as a white solid (73.6g, 0.40 mol; 70%): mp 46 ℃; IR (KBr)1705cm^-1；¹H NMR(CDCl₃)δ2.55(t，J＝7.5Hz，2H)，3.10(t，J＝7.5Hz，2H)，3.58(s，2H)，6.70(m，2H)；MS m/z 182(M⁺)。C₁₀H₈F₂Analytical calculation of O: c, 65.93; h, 4.42. Measured value: c, 65.54; h, 4.42.

(R) - (+) -2-hydroxy-5, 7-difluoro-1, 2, 3, 4-tetrahydronaphthalene. (-) -1R, 2S-N-Methylephedrine (81.3g, 0.454mol) in anhydrous Et₂O (1.1 liters) solution was added dropwise (45 minutes) to 1.0M lithium aluminum hydride (416mL, 0.416mol)/Et at a rate sufficient to maintain a gentle reflux ₂And (4) in O. After the addition was complete, the reaction mixture was heated at reflux for 1 hour and then cooled to room temperature. 2-Ethanaminopyridine (111g, 0.98 mol) in anhydrous Et was added at the following rate₂O (100ml) solution (45 min) to maintain a gentle reflux. The reaction mixture was heated at reflux for a further 1 hour, during which a pale yellow-green suspension appeared. The mixture was cooled to an internal temperature of-65 ℃ using a dry ice-acetone bath, and 5, 7-difluoro-2-tetralone (23.0g, 126mmol) of Et was added dropwise at a rate to maintain the internal temperature below-60 ℃₂O (125mL) solution. After the addition was complete, the mixture was stirred at-65 ℃ to-68 ℃ for 3 hours and quenched by addition of MeOH (100mL), keeping the internal temperature below-60 ℃. The reaction was stirred for a further 10 minutes at-65 ℃ and warmed to about-20 ℃. Then 3N HCl solution (2L) was added at a rate limiting the temperature < 35 ℃. After stirring at high speed to achieve complete dissolution, the layers were separated, the ether layer was washed with brine (200mL), dried (MgSO)₄). The ethereal solution was evaporated under reduced pressure and the residue was dissolved in hot Et₂O (20mL), followed by hexane (200 mL). The resulting precipitated crystals were collected by cooling the seeded solution in an ice bath and held at 0 ℃ for 1 hour, and dried under vacuum to give the alcohol (10.9g, 47%): mp 85 ℃; [ alpha ] to ]²⁵ _D+38.1°(c＝1.83，CHCl₃) (ii) a 93.4% ee, by chiral HPLC:¹H NMR(CDCl₃)δ1.70(br s，1H)，1.76-1.88(m，2H)，1.99-2.06(m，2H)，2.63-3.08(m，3H)，4.15(m，1H)，6.60(m，2H)。C₁₀H₁₀F₂analytical calculation of O: c, 65.21; h, 5.47. Measured value: c, 65.38: h, 5.42. The spectrum of (S) -enantiomer 4b is identical: mp84-85 deg.C; [ alpha ] to]²⁵ _D-37.8°(c＝1.24，CHCl₃) (ii) a 92.4% ee by chiral HPLC. C₁₀H₁₀F₂Analytical calculation of O: c, 65.21; h, 5.47. Measured value: c, 65.47; h, 5.39.

(R) - (+) -2-methanesulfonyloxy-5, 7-difluoro-1, 2, 3, 4-tetrahydronaphthalene. R- (+) -5, 7-difluoro-2-tetrahydronaphthol (59.0g, 320mmol) and Et using an ice-MeOH bath₃N (74.2mL, 53.9g, 530mmol) in anhydrous Et₂The O (1.78 l) solution was cooled (-15 ℃ C.) and treated with MsCl (37.2ml, 55.3g, 480mmol) under argon (stirring) for 5-10 min. After 5 hours, the reaction was complete (as determined by TLC), water was added to dissolve the solid. A small amount of EtOAc was added to help complete the dissolution of the solid. The organic phase was separated and washed with 1N HCl, NaHCO₃The aqueous solution and brine were washed sequentially with MgSO₄And (5) drying. Evaporation of the solvent gave an off-white solid (87.1g, 332mmol), which was used directly in the next step. By i-Pr₂O grinding a small sample to obtain an analytical sample: mp is 78.8-80.5 ℃; [ alpha ] to]²⁵ _D+16.8°(c＝1.86，CHCl₃)；¹H NMRδ2.13-2.28(m，2H)，2.78-2.96(m，2H)，3.07(s，3H)，3.09(dd，J＝17.1Hz，4.7，1H)，3.20(dd，J＝17.2，4.7Hz，1H)，5.20(m，1H)，6.67(m，2H)。C₁₁H₁₂F₂O₃Analytical calculation of S: c, 50.37; h, 4.61. Measured value: c, 50.41; h, 4.64. The spectrum of (S) -enantiomer 5b was identical: mp79.9-80.9 ℃; [ alpha ] to ]²⁵ _D-16.6°(c＝2.23，CHCl₃)。C₁₁H₁₂F₂O₃Analytical calculation of S: c, 50.37; h, 4.61. Measured value: c, 50.41; h, 4.65.

(S) - (-) -2-amino-5, 7-difluoro-1, 2, 3, 4-tetrahydronaphthalene hydrochloride. Sodium azide (40.0g, 0.62mol) was added to DMSO (1L) with stirring until a clear solution was obtained. The mesylate (138g, 0.53mol) was added in one portion, andin N₂The mixture was heated at 50 ℃ for 16 hours under an atmosphere. The reaction mixture was diluted with water (1.8L), extracted with pentane (4X250mL), and the combined pentane extracts were washed sequentially with water (2X100mL), brine (100mL), and MgSO₄And (5) drying. The solvent was evaporated under reduced pressure to give a volatile oil which was subjected to flash silica gel chromatography using pentane as eluent to give a dihydronaphthalene (8.50g, 51.2mmol) volatile oil. With pentane/CH₂Cl₂(9: 1) further elution gave azide (101g, 483mmol) as a colorless oil: IR (CHCl)₃)2103cm^-1；m/z 171(M⁺). The azide 6a was dissolved in EtOAc (1200mL) and hydrogenated with 10% Pd/C (6g) in a 2.5L Parr bottle (60psi) for 6 hours. After each hour, the bottle was vented and refilled with hydrogen to remove evolved N₂. The resulting mixture was filtered through celite, stirred with etherified HCl (1N, 500mL), and the fine precipitate filtered off, washed with EtOAc, then with anhydrous ether. (about 4 hours was required for filtration). The wet solid was transferred to a round bottom flask and the remaining solvent was removed in vacuo to give a white solid (90.4g, 412 mmol; 77.9%): mp is more than 280 ℃; [ alpha ] to ]²⁵ _D-60.2°(c＝2.68，MeOH)；¹H NMR(d₆-DMSO)δ1.79(m，1H)，2.33(m，1H)，2.63(m，1H)，2.83-2.92(m..2H)，3.14(dd，J＝16.7，5.0Hz，1H)，3.46(m，1H)，6.93(d，J＝9.4Hz，1H)，7.00(dt，J＝9.4，2.5Hz，1H)。C₁₀H₁₂ClF₂Analytical calculation of N: c, 54.68; h, 5.51; n, 6.37. Measured value: c, 54.31; h, 5.52; n, 6.44. The spectrum of (R) -enantiomer 8b is identical: mp is more than 280 ℃; [ alpha ] to]²⁵ _D+58.5°(c＝1.63，MeOH)。C₁₀H₁₂ClF₂Analytical calculation of N: c, 54.68; h, 5.51; n, 6.37. Measured value: c, 54.64; h, 5.51; and N, 6.40.

(S) - (-) - (5, 7-difluoro-1, 2, 3, 4-tetrahydronaphthalen-2-yl) (cyanomethyl) amine. Amine hydrochloride 8a (50.27g, 229mmol) was treated with a solution of NaOH (10.0g, 250mmol) in water (150ml) followed by several additional NaOH pellets sufficient to obtain a solution. Further water (300ml) was added to the reaction solution, and the mixture was placed in a jarInto a 50 ℃ bath, treated with sodium formaldehyde bisulfite complex (30.8g, 230 mmol). After stirring the mixture for 30 min, KCN (15.0g, 230mmol) was added. The reaction mixture was stirred at 80 ℃ for a further 1 h, cooled to room temperature and extracted with EtOAc to give an oil (51.3g) which solidified. TLC (5% MeOH-CH)₂Cl₂) Indicating that about 10-15% of the starting amine remained. Chromatography on silica gel gave the nitrile product (39.4g) and the starting free amine (7.12g), which rapidly formed the carbonate in air. The amine was recycled to give an additional 5.35g of product. The combined yields (44.8g, 202 mmol; 87.5%): mp73.1-76.5 ℃; [ alpha ] to ]²⁵ _D-58.0°(c＝1.63，CHCl₃)；¹H NMR(CDCl₃)δ1.50(brs，1H)，1.70(m，1H)，2.05(m，1H)，2.55-3.04(m，4H)，3.22(m，1H)，3.70(s，2H)，6.62(m，2H)；MS m/z 222(M⁺)。C₁₂H₁₂F₂N₂Analytical calculation of (a): c, 64.85; h, 5.44; and N, 12.60. Measured value: c, 65.07; h, 5.47; n, 12.44. The spectrum of (R) -enantiomer 9b is identical: mp 64.4-73.6 ℃; [ alpha ] to]²⁵ _D+52.3°(c＝2.12，CHCl₃)。C₁₂H₁₂F₂N₂Analytical calculation of (a): c, 64.85; h, 5.44; and N, 12.60. Measured value: c, 65.14; h, 5.54; n, 12.53.

(S) - (-) -1- (5, 7-difluoro-1, 2, 3, 4-tetrahydronaphthalen-2-yl) -5-cyano-2, 3-dihydro-2-thioxo-1H-imidazole. In N₂Nitrile (44.7g, 201mmol) was heated under reflux (120 ℃ bath) in butyl formate (240ml) for 19 hours under ambient conditions, and the solvent was removed under reduced pressure. Toluene was added, evaporated, the last traces of solvent removed and the residue dried under high vacuum to give an oil (53.2 g). The resulting formamide and ethyl formate (48.7ml, 44.7g, 604mmol) were cooled in dry THF (935ml) in ice/MeOH (-15 deg.C) and stirred while t-BuOK (1M in THF, 302ml, 302mmol) was added over 20 min. After stirring the reaction for 18 h, the solvent was evaporated and the residue was dissolved in 1N HCl (990ml) and ethanol (497ml) and treated with KSCN (78.1g, 804 mmol). The mixture was stirred at 85 ℃ for 135 minutes and then placed in an ice bath to give a precipitate. Filtering the solid to obtain a filtrate10％MeOH/CH₂Cl₂The slurry in (b) was loaded onto a silica gel (1kg) packed column (with hexane). With 10% acetone/CH ₂Cl₂Elution afforded the product (18.05g, 62.1 mmol; 30.8%): m.p.240.7-249.2 deg.c;-69.1°(c＝1.18，DMSO)；¹H NMR(d₆-DMSO)δ2.18(br m，1H)，2.47(m，1H)，2.75(m，1H)，3.03-3.35(m，3H)，5.19(m，1H)，6.94(d，J＝9.3Hz，1H)，7.03(dt，J＝9.3，2.4Hz，1H)，8.29(s，1H)，13.3(br s，1H)；MS m/z 291(M⁺)。C₁₄H₁₁F₂N₃analytical calculation of S: c, 57.72; h, 3.80; n, 14.42. Measured value: c, 57.82; h, 3.92; n, 14.37. (with 1: 1 MeOH/CH)₂Cl₂The column was further eluted to give the primary amide 11 a: mp 261.9-262.7 deg.C;-90.5°(c＝0.398)；IR(KBr)1593，1630cm^-1；¹H NMR(d₆-DMSO)δ2.14(m，1H)，2.15-2.28(m，1H)，2.74-3.05(m，4H)，5.64(m，1H)，6.90(d，J＝9.2Hz，1H)，7.05(dt..J＝9.5，2.4Hz，1H)，8.73(s，1H)，9.70(br s，1H)，13.7(br s，1H)；MS m/z 309(M⁺)。C₁₄H₁₃F₂N₃OS 0.25H₂analytical calculation of O: c, 53.57; h, 4.33; n, 13.39. Measured value: c, 53.32; h, 3.96: and N, 13.24. The spectra of the (R) -enantiomers are identical: mp 243.1-244.7 ℃;+74.9°(c＝2.14，DMSO)。C₁₄H₁₁F₂N₃analytical calculation of S: c, 57.72; h, 3.80; n, 14.42. Measured value: c, 57.85; h, 3.85; n, 14.45.

(S) -1- (5, 7-difluoro-1, 2, 3, 4-tetrahydronaphthalen-2-yl) -5-aminomethyl-2, 3-dihydro-2-thio-1H-imidazole. The above nitrile (5.00g, 17.2mmol) was stirred in THF (75ml) in an ice bath under argon atmosphere until a homogeneous solution was obtained. LAH/THF solution (1M, 34.3ml, 34.3mmol) was added dropwise over 10 minutes, and then the solution was stirred at 0 ℃ for 30 minutes and allowed to reach room temperature for 1.5 hours. The reaction was cooled again to 0 ℃ and treated with saturated sodium potassium tartrate solution until the mixture became freely stirrable. A further tartrate solution (30mL) was added followed by 10% MeOH/CH ₂Cl₂(200mL), the mixture was stirred for 15 minutes and treated with water (100-150 mL). The organic layer was separated with 10% MeOH/CH₂Cl₂(2x125mL) the aqueous phase was extracted. The combined extracts were washed and dried (MgSO)₄) And (4) evaporating. The residue (5.2g) was chromatographed on silica gel with 5% MeOH/CH₂Cl₂Elution afforded the free amine (2.92g, 9.89 mmol; 58%): mp 170 ℃;-11.0°(c＝1.59，DMSO)。C₁₄H₁₅F₂N₃S 0.25H₂analytical calculation of O: c, 56.07; h, 5.21; and N, 14.01. Measured value: c, 56.11; h, 5.10; n, 14.14.

(S) -1- (5, 7-difluoro-1, 2, 3, 4-tetrahydronaphthalen-2-yl) -5-aminomethyl-2, 3-dihydro-2-thioxo-1H-imidazole hydrochloride (nepicastat). The hydrochloride salt was prepared as follows: etherified HCl (1M, 20mL, 20mmol) is added to the free amine 2a (3.12g, 10.6mmol), which has been dissolved in MeOH (250mL) by heating. Part of the solvent was removed under reduced pressure and then the solvent was replaced by co-evaporation several times with EtOAc without evaporation to dryness. The resulting precipitate was treated with EtOAc (150mL) and ether (150mL), filtered off, washed with ether, dried under nitrogen and then dried at 78 ℃ under high vacuum for 20 hours to give the hydrochloride salt (3.87 g): mp 245 ℃ (dec); [ a ] A]²⁵ _D+9.65 ° (c ═ 1.70, DMSO); (93% ee by chiral HPLC); ¹H NMR(T＝320°K，DMSO)δ2.07(m，1H)，2.68-3.08(m，4H)，4.09(m，3H)，4.77(m，1H)，6.84(m，2H)，7.05(s，1H)，8.57(br s，3H)，12.4(br s，1H)。C₁₄H₁₆ClF₂N₃S 0.5H₂Analytical calculation of O: c, 49.33; h, 5.03; n, 12.33. Measured value: c, 49.44; h, 4.96; and N, 12.18. The spectrum of (R) -enantiomer (R) -1- (5, 7-difluoro-1, 2, 3, 4-tetrahydronaphthalen-2-yl) -5-aminomethyl-2, 3-dihydro-is identical; mp261-263 deg.C; [ alpha ] to]²⁵ _D10.8 ° (c ═ 1.43, DMSO), 91.6% ee, by chiral HPLC. C₁₄H₁₆ClF₂N₃S 0.35H₂Analytical calculation of O: c, 49.73; h, 4.98; n, 12.42. Measured value: c, 49.80; h, 4.93; n, 12.39.

Nepirstat was demonstrated to be bovine (IC)₅₀8.5 ± 0.8nM) and human (IC)₅₀9.0 ± 0.8nM) competitive inhibitors of DBH. R-enantiomer (R) -1- (5, 7-difluoro-1, 2, 3, 4-tetrahydronaphthalen-2-yl) -5-aminomethyl-2, 3-dihydro- (IC)₅₀25.1 ± 0.6 nM; 18.3. + -. 0.6nM) and SKF102698 (IC)₅₀67.0 ± 4.2 nM; 85.0 ± 3.7nM) are less effective inhibitors of the bovine and human enzymes, respectively. DBH activity was tested by determining the conversion of tyramine to ethanolamine. Bovine DBH derived from adrenal glands was obtained from Sigma Chemical Co (St Louis, Mo.). Human secreted DBH was purified from the culture medium of the neuroblastoma cell line SK-N-SH. The assay was performed at pH5.2 and 32 ℃ at 0.125M NaOAc, 10mM fumarate, 0.5-2. mu.M CuSO ₄0.1mg/mL of catalytic enzyme, 0.1mM of tyramine and 4mM of ascorbate. In a typical experiment, 0.5-1 milliunits of enzyme is added to the reaction mixture, followed by the addition of a substrate mixture comprising the catalytic enzyme, tyramine and ascorbate to initiate the reaction (final volume of 200 μ L). The samples were incubated at 37 ℃ for 30-40 minutes with or without the appropriate concentration of inhibitor. The reaction was quenched by a stop solution containing 25mM EDTA and 240. mu.M 3-hydroxytyrosamine (internal standard). The samples were analyzed for ethanolamine by reverse phase HPLC using UV detection (at 280 nM). Percent activity retained was calculated based on control (no inhibitor), corrected using internal standard, and fitted to a non-linear four-parameter concentration response curve to obtain IC₅₀The value is obtained.

Using established assays, the activity of nepicastat was determined for 11 different enzymes. The affinity of nepicastat for the 13 selected receptors was determined by radioligand binding assay using standard filtration techniques and membrane preparations. The binding data were analyzed by iterative curves (fitted with a four parameter logistic equation). K is calculated using the Cheng-Prusoff equation_iThe value is obtained. Figure 3 shows a graph depicting the interaction of nepicastat with DBH and a number of selected enzymes and receptors. Nepicastat shows weak affinity for a large number of other enzymes and neurotransmitters receptors. These data indicate that nepicastat is a potent and highly selective inhibitor of DBH in vitro. Furthermore, the S-enantiomer nepicastat is more potent than the R-enantiomer, approximately 2-3 fold, indicating stereoselectivity.

Oral administration of nepicastat to Spontaneously Hypertensive Rats (SHRs) and normal dogs produced an effective and dose-dependent increase in the tissue Dopamine (DA)/Norepinephrine (NE) ratio in the peripheral arteries (renal or mesenteric), left ventricle, and cerebral cortex. Nepitastat administered orally to normal dogs for a long period of time also consistently increases the DA/NE ratio in the plasma. In mentally conscious SHRs, oral urgent nepicastat produces a dose-dependent and durable (> 4 hours) antihypertensive effect, and may also attenuate the hypertensive (pressor) response to preganglionic sympathetic nerve stimulation. Serum T₃And T₄The levels were not affected by the dose (6.2mg/kg, po, b.i.d., 10 days) that could elevate the dopamine/norepinephrine ratio in the mesenteric artery. Nepicastat is clinically evaluated for the treatment of congestive heart failure based on its ability to effectively modulate sympathetically driven cardiovascular tissue.

Congestive Heart Failure (CHF) is the leading cause of death in the united states. CHF is characterized by: significant activation of the Sympathetic Nervous System (SNS) and the renin-angiotensin system (RAS). The simultaneous activation of these two neurohormonal systems is increasingly implicated in the maintenance and progression of CHF. Therapeutic interventions that block the effects of these neurohormonal systems are likely to favorably alter the natural course of CHF. Indeed, angiotensin-converting enzyme (ACE) inhibitors, which block the formation of angiotensin II, have been shown to reduce morbidity and mortality in CHF patients. However, ACE inhibitors have limited indirect capacity to attenuate SNS. Inhibition of SNS with beta-adrenergic receptor antagonists is a promising approach currently evaluated clinically. An alternative strategy to directly modulate the SNS is to inhibit the biosynthesis of Norepinephrine (NE) by inhibiting dopamine β -hydroxylase (DBH), the enzyme responsible for the conversion of NE to Dopamine (DA). Inhibition of DBH is expected to reduce the tissue level of NE and increase the tissue level of DA, thereby increasing the DA/NE ratio of tissue. This approach has potential advantages over β -adrenergic receptor antagonists, for example, reduced stimulation of α -adrenergic receptors, and increased levels of DA, which can cause renal vasodilation, natriuresis, and impaired aldosterone release. Previous DBH inhibitors, such as fusaric acid and SKF-102698, have drawbacks, e.g., low potency and specificity, which may hinder their clinical development in heart failure.

Nepicastat was used in vivo biochemical studies to study the effects in Spontaneously Hypertensive Rats (SHRs) and normal beagle dogs. On the day of the study, animals were weighed and randomized to receive either placebo (vehicle) or the appropriate dose of nepicastat. Each mouse was dosed orally three times, 12 hour intervals, starting in the morning. At 6 hours after the third dose, rats were anesthetized with halothane, decapitated, tissues (cerebral cortex, mesenteric artery and left ventricle) were rapidly harvested, weighed, placed in frozen perchloric acid (0.4M), frozen in liquid nitrogen, and stored at-70 ℃ until later analysis. The NE and DA concentrations of the tissues were determined by HPLC using electrochemical detection. In this study, male puppies (10-16kg, Marshall farm USA Inc, NorthRose, NY) were used. On the day of the study, dogs were randomized to receive either placebo (empty capsules) or an appropriate dose of nepicastat. Each dog was dosed twice daily for 4.5 days. Dogs were euthanized with pentobarbital 6 hours after the first dose on day 5, tissues (cerebral cortex, renal artery and left ventricle) were collected, weighed, placed in frozen perchloric acid (0.4M), frozen in liquid nitrogen, and stored at-70 ℃ until analysis. The NE and DA concentrations of the tissues were determined by HPLC using electrochemical detection.

Oral nepicastat caused a dose-dependent increase in the DA/NE ratio in SHRs and in the arteries (mesentery or kidney), left ventricle and cerebral cortex of dogs.

In the highest dose trials (100 mg/kg in SHRs, 5mg/kg in dogs), the greatest increases in DA/NE ratios in arteries, left ventricle and cerebral cortex were 14, 11 and 3.2 fold (in SHRs) and 95, 151 and 80 fold (in dogs), respectively. When tested at 30mg/kg in SHRs, SKF-102698(1) increased the DA/NE ratio by 5.5-fold, 3.5-fold and 2.7-fold, respectively, in the mesenteric artery, left ventricle and cerebral cortex, while nepicastat at the same dose increased the ratio by 8.3, 7.5 and 1.5-fold, respectively. In SHRs, 30mg/kg of Compound B only caused 2.6, 3.5 and 1.1 fold increases in the DA/NE ratio in the mesenteric artery, left ventricle and cerebral cortex, respectively. These data indicate that nepicastat elicits the desired biochemical effects in SHRs and dogs, but is more effective in later species. Furthermore, nepicastat was more potent than its compound B and SKF-102698(1) in SHRs.

In normal dogs, the long-term effect of nepicastat (14.5 days treatment) on the plasma DA/NE ratio was studied. Animals were randomized to receive either oral placebo (empty capsules) or nepicastat (2mg/kg, b.i.d.) for 14.5 days. Blood samples were drawn daily (6 hours after the first dose) for determination of the plasma concentrations of DA and NE. Samples were collected in tubes containing heparin and glutathione, centrifuged at-4 ℃ and stored at-70 ℃ until analysis.

Oral nepicastat (2 mg/kg; b.i.d) resulted in a significant increase in the DA/NE ratio, with a peak effect obtained at approximately 6-7 days, followed by a plateau to a new steady state between 7-14 days.

Nepicastat was further evaluated for its in vivo hemodynamic activity in conscious, restricted SHRs (a model with high sympathetic drive to cardiovascular tissue). Hemodynamics were studied in SHRs. In this study, male SHRs (15-16 weeks old) were used. The animals were lightly anesthetized with ether and the left femoral artery and vein were catheterized for blood pressure determination and drug administration, respectively. The animals were placed in the restraint and allowed to recover for 30-40 minutes. After obtaining a baseline determination, animals were orally treated with vehicle or appropriate dose of nepicastat and hemodynamic parameters were continuously recorded for 4 hours. The animals were then anesthetized with pentobarbital, placed on a hot plate (37 ℃) and ventilated with a Harvard rodent (rodent) ventilator. After administration of atropine (1mg/kg, iv) and tubocurarine (1mg/kg, iv), the animals were punctured with stainless steel strips through the orbit to destroy the cerebrospinal fluid. The cerebrospinal-piercing rods were electrically stimulated (at different frequencies (0.15, 0.45, 1.5, 5, 15Hz)) with a 1ms pulse of 80V to obtain frequency-blood pressure response curves.

Oral administration of nepicastat resulted in a dose-dependent antihypertensive effect. The animals were placed in the restraint and allowed to recover for 30-40 minutes. After obtaining a baseline determination, animals were orally treated with vehicle or appropriate dose of nepicastat and hemodynamic parameters were continuously recorded for 4 hours. Nepicastat significantly (p < 0.05) reduced mean arterial pressure at all doses and time points except 0.3mg/kg (180, 210, and 240 minutes) and 1mg/kg (30, 210, and 240 minutes).

With the 10mg/kg dose, a maximum reduction in mean blood pressure of 53 + -4 mmHg (33% reduction relative to vehicle control) was observed. The initial response is slow and reaches its plateau over 3-4 hours the exact reason for the loss of the antihypertensive effect at the highest dose (30mg/kg) is not known at present. There was no significant effect on heart rate, but there was still a slightly significant decrease (9.8 and 10.5% decrease, respectively) at 10 and 30 mg/kg. Following this study, rats were punctured of the cerebrospinal fluid and 5 hours after dosing, the effect of nepicastat on the increased blood pressure response of Preganglionic Nerve Stimulation (PNS) of the spinal cord was evaluated. The frequency-blood pressure response curve shifts significantly (p < 0.05) to the right (maximum shift-5 times in the frequency response curve) in a dose-dependent manner. The heart rate response to PNS was not significantly affected. These data suggest that nepicastat can inhibit sympathetic drive to the vascular system in SHRs and is a possible mechanism for its antihypertensive effect.

Since the heterocyclic moiety of nepicastat is structurally similar to methimazole, a known potent inhibitor of mammalian thyroid function, the effect of nepicastat on thyroid function was evaluated in iodine deficient Sprague Dawley rats (n ═ 9-12) at doses of 2.0 and 6.2mg/kg (po, b.i.d, 10 days). Methimazole (1mg/kg, po, b.i.d.) used as a positive control resulted in T4 hours after administration₃Significantly reduced serum levels of (31% on days 3, p < 0.05; 7 and 9, 42% and 44%, p < 0.01) and T4 (46% and 58%, p < 0.01) while nepicastat showed no significant effect throughout the study (days 3, 7 and 9). Two doses of nepicastat significantly increased the DA/NE ratio in the mesenteric artery (p < 0.01, relative to vehicle control) 4 hours after the last dose on day 10, but not in the cortex.

The findings of this study indicate that nepicastat is a potent, selective and orally active inhibitor of DBH. This compound also lacks significant behavioral effects in animal models, and these findings will be the subject of future disclosures. Because the compound nepicastat is effective in modulating sympathetic drive to cardiovascular tissue, it has been tested for the treatment of CHF.

Example 7

Concentrations of dopamine and norepinephrine were determined in 942 plasma samples collected from Congestive Heart Failure (CHF) patients. The research aims are as follows:

1. the effect of various doses of nepicastat on myocardial blood transport (arterial coronary sinus) and coronary sinus catecholamine levels after four weeks was evaluated, and the safety and tolerability of nepicastat over 12 weeks was evaluated.

2. The effect of nepicastat on changes from baseline was evaluated as follows:

a) plasma (intravenous) catecholamine levels after 4 and 12 weeks

b) Quality of life (QoL), CHF symptoms, Global (Global) assessment and NYHA categories after 4 and 12 weeks

c) Hemodynamic parameters including cardiac output, systemic vascular resistance, MVO after 4 weeks₂Pulmonary artery pressure and pulmonary artery wedge pressure

d) Hospitalization and drug dose variation over 12 weeks of CHF treatment

e) Blood pressure and heart rate at 4 and 12 weeks

f) 6 minute walk test after 4 and 12 weeks

g) Left ventricular ejection fraction, left ventricular end-systole and left ventricular end-diastole volumes of 12 weeks.

2 hours after dosing between 4 and 12 cycles, the patient was supine and blood samples were collected from the patient's peripheral veins. On day 0 (i.e. the day before the initial dosing), further samples were collected from supine patients at a time equivalent to 2 hours post-dosing. In addition, during week 4, at 2 hours after dosing and on day 0 (i.e., the day before the initial dosing) (at a time equivalent to 2 hours after dosing), right heart and coronary sinus catheterization was performed on one group of patients. Three samples of blood were collected from the arteriovenous and coronary sinuses of these patients.

The free base concentrations of dopamine and norepinephrine were determined by the radioactive zymogen method. The method comprises the following steps: plasma samples were incubated with catechol-O-methyltransferase and tritiated S-adenosylmethionine. After the culture was completed, the O-methylated catecholamine was extracted from the plasma by liquid/liquid extraction, and then separated by thin layer chromatography. The relevant band for each catecholamine was labeled and then scraped into scintillation vials for counting. The quantitative limit of this method is 1pg dopamine or norepinephrine per mL plasma. The linear range is 1 to 333000pg dopamine or norepinephrine per mL of plasma (0.045 mL to 1mL aliquots are used).

Human plasma samples collected were used as quality control samples (QC) and single sample (single) analysis was performed daily during routine use of the method to monitor the efficacy of the method.

Example 8

Pre-clinical in vitro and in vivo pharmacological studies were performed on nepicastat. In vitro studies evaluate the ability of compounds to inhibit DBH activity and its binding affinity to selected receptors. In vivo assays are subdivided into four categories: 1) biochemical effects (i.e., the ability to reduce tissue noradrenaline levels and increase dopamine levels), 2) effects on thyroid function, 3) cardiovascular effects, and 4) behavioral effects.

Nepicastat is a potent inhibitor of DBH in cattle and humans. IC of nepicastat on DBH of human₅₀The value was 9nM (CL 6960), IC vs. DBH inhibitor SKF-102698₅₀The value (85nM) decreased significantly. Nepicastat, the S enantiomer, was more potent than the R enantiomer (18nM), expressed as compound B.

Nepicastat was screened for binding affinity to the selected receptor. For M1, D1 and D2 and 5HT_1A，2AAnd 2_CNepicastat showed a binding affinity of less than 5.0. The N-acetyl metabolites of nepicastat in rats and monkeys show a similar lack of binding affinity for these receptors. Thus, nepicastat and its major metabolites are not potent inhibitors of the above receptors.

In Spontaneously Hypertensive Rats (SHRs), the in vitro aortic systolic response to phenylephrine was impaired relative to normotensive Wistar-Kyoto rats. Treatment with nepicastat (10mg/kg, p.o.) daily for 21 days in SHRs restored phenylephrine responses to values similar to those of Wistar-Kyoto rats.

Generally, nepicastat is a potent inhibitor of DBH in rats and dogs. In both species, oral or intravenous administration can result in a significant (p < 0.05) reduction in tissue noradrenaline, an increase in dopamine and an increase in dopamine/noradrenaline levels in the heart, mesentery or renal arteries and cerebral cortex.

In a study on male Spontaneously Hypertensive Rats (SHRs), nepicastat significantly reduced norepinephrine and increased dopamine and dopamine/norepinephrine ratios in the mesenteric artery 0.5 to 4 hours after oral or i.v. administration (6.2 mg/kg). Significant changes in these parameters were also observed in the left ventricle of male Sprague-Dawley rats 6 hours after the second of two i.v injections (15mg/kg) (12 hour intervals). In male SHRs, the 24-hour time course of tissue catecholamines was studied after 10 or 30mg/kg oral administration, respectively. At 1 hour, the dopamine/norepinephrine ratio increased significantly and was persistent (10mg/kg for 12 hours, mesenteric artery, and 30mg/kg for 24 hours, left ventricle). After 10 days of administration of 2.0 and 6.2mg/kg (p.o.b.i.d.) in male Sprague-Dawley rats, significant changes in mesenteric arterial dopamine and norepinephrine levels were observed, with no significant effect observed in the cerebral cortex. Given SHRs at a dose of 1 or 10mg/kg/d (p.o.) for 7 or 25 days, the dopamine and dopamine/norepinephrine ratios are significantly increased in the mesenteric artery and cerebral cortex. Taken together, nepicastat caused a significant decrease in noradrenaline and an increase in dopamine and dopamine/noradrenaline ratio in the mesenteric artery in rats given either acutely or chronically (up to 25 days).

The effect of nepicastat was found to be dose-responsive when evaluated 6 hours after single oral doses of 0.3, 1, 3, 10, 30 and 100mg/kg in male SHRs and Sprague-Dawley rats. In SHRs, there were significant changes in the dopamine/norepinephrine ratio in the mesenteric artery (0.3mg/kg dose), in the left ventricle (3.0mg/kg dose), and in the cerebral cortex (10mg/kg dose). In Sprague-Dawley rats, there was a significant increase in the dopamine/norepinephrine ratio in the mesenteric artery (3.0mg/kg dose), in the left ventricle (1.0mg/kg dose), and in the cerebral cortex (only 100mg/kg dose). In a second dose-response study in SHRs, three doses were given at 3.0, 10, 30 or 100mg/kg (12 hour interval), with tissue harvested 6 hours after the third dose. Nepicastat resulted in a significant dose-dependent decrease in noradrenaline (10mg/kg) and an increase in dopamine (3.0mg/kg) and dopamine/noradrenaline ratio (3.0mg/kg) in the left ventricle and mesenteric artery. In the cerebral cortex, the effect of nepicastat on dopamine and norepinephrine concentrations and dopamine/norepinephrine ratios was only significant at 30 and 100 mg/kg. In the left ventricle, similar significant dose-response effect effects were observed in female Wistar rats dosed with nepicastat (7 days by drinking water, 0.3, 0.6 and 1.0 mg/ml). In conclusion, nepicastat has a poorer DBH inhibitory effect in the cerebral cortex of rats (60-100mg/kg/d) than in the left ventricle and mesenteric artery (1-6 mg/kg/d).

In SHRs, nepicastat (S enantiomer) was significantly more potent than the R enantiomer in the left ventricle and mesenteric arteries after three doses (30mg/kg p.o.) given at 12 hour intervals. Nepicastat was significantly more effective than the DBH inhibitor SKF-102698 in terms of norepinephrine reduction and an increase in dopamine to dopamine/norepinephrine ratio in the left ventricle and mesenteric arteries following a single administration or three doses at a dose of 30mg/kg in SHRs. The efficacy relationships resulting from these in vivo tests strongly paralleled those obtained with in vitro tests using purified DBH (see above) in the left ventricle and mesenteric artery. However, in the cerebral cortex, nepicastat was significantly less effective than SKF-102698 in reducing norepinephrine levels and increasing dopamine levels. Norepinephrine has been shown to stimulate renin release and increase plasma renin activity. Therefore, it is of interest to evaluate whether lowering norepinephrine levels with nepicastat leads to a decrease in plasma renin activity. However, nepicastat (30 and 100mg/kg/d, p.o., 5 days) did not alter plasma renin activity in male SHRs. Thus, nepicastat does not alter plasma renin activity in SHRs when administered at doses that reduce tissue norepinephrine levels.

Nepicastat resulted in a significant reduction in noradrenaline levels and an increase in the dopamine/noradrenaline ratio in the mesenteric artery of male beagle dogs 5 hours after intraduodenal administration of 30mg/kg, but did not alter dopamine levels. When nepicastat (5, 15 and 30mg/kg b.i.d., or 10, 30 and 60mg/kg/d) was administered to male beagle dogs for 4.5 days, norepinephrine was significantly reduced, dopamine and dopamine/norepinephrine ratios increased, and response plateaus started at 10mg/kg/d and extended to 60mg/kg/d in the renal arteries, renal cortex and renal medulla. Similar results were observed in the left ventricle, except that there was no significant increase in dopamine. In the cerebral cortex, norepinephrine was significantly reduced at doses of 30 and 60mg/kg/d, and dopamine/norepinephrine ratios were significantly increased at all doses. In conclusion, nepicastat is a potent orally active inhibitor of DBH in dogs at a dose of at least 10 mg/kg/d.

Nepicastat has structural similarity to methimazole, an in vivo potent inhibitor of thyroid peroxidase. In male Sprague-Dawley rats fed a low-iodine diet and administered for 10 days, nepicastat at a dose of 4 or 12.4mg/kg/d (p.o) had no effect on the serum level of triiodothyronine (triiodothyronine) or thyroxine, while methimazole (2mg/kg/d) significantly reduced the serum level of triiodothyronine (triiodothyronine) or thyroxine. Thus, unlike methimazole, nepicastat does not affect the serum levels of triiodothyronine (triiodothyronine) or thyroxine.

Nepicastat elicited significant antihypertensive effects for up to 4 hours and significantly reduced heart rate (10 and 30mg/kg) in conscious, restricted SHRs (1.0-30mg/kg, p.o.). In conscious, restricted SHRs (10mg/kg, p.o.), the antihypertensive effect of nepicastat was not diminished by prior treatment with the dopamine receptor (DA-1) antagonist SCH-23390. Nepicastat (10mg/kg) also reduced blood pressure for 4 hours after administration in conscious, restricted normotensive Wistar-Kyoto rats; however, the decrease in pressure (-13mmHg) was less than the decrease in pressure (-46mmHg) with SHRs. Taken together, nepicastat can cause a decrease in blood pressure in SHRs and normotensive rats, but the antihypertensive effect in SHRs is more pronounced. The antihypertensive effects in SHRs are not as mediated through DA-1 receptors.

Nepicastat also significantly attenuated hypertension and tachycardia response to preganglionic nerve stimulation 5 hours after administration (3mg/kg p.o.) in cerebrospinal SHRs. Thus, nepicastat reduces the increase in blood pressure in response to sympathetic nerve stimulation.

SHRs under emergency intravenous therapy anesthesia with nepicastat (3.0mg/kg, i.v) reduced mean arterial pressure over a 3 hour period, but did not reduce renal blood flow or alter urine production, or sodium or potassium urinary excretion. After administration, the calculated renal vascular resistance decreased. Attempts were made to evaluate whether the renal vasodilatory effect of nepicastat is mediated by DA-1 receptors using the DA-1 antagonist SCH-23390. However, when given alone, such compounds can lower blood pressure, thereby producing unexplained results. Overall, although nepicastat causes a decrease in arterial blood pressure, it does not impair renal function of anesthetized SHRs and does not reduce renal blood flow.

In SHRs, daily treatment with nepicastat (1 and 10mg/kg, p.o.) for 21 days did not change heart rate or systolic blood pressure (as measured by the tail-cover method). However, nepicastat (10mg/kg, p.o.) elicits significant antihypertensive effects when rats were confined and their blood pressure was measured directly through the arterial cannula.

At doses of 30 and 100mg/kg/d (30 days) nepicastat significantly reduced blood pressure in SHRs (with meter equipped with radio telemetric blood pressure sensor), but no significant effect was observed at doses of 3 and 10 mg/kg/d. After a single administration, the effect lasted for a period of 24 hours at 30 and 100mg/kg/d, and was not lost over 30 days. The heart rate did not increase and the motor activity was unaffected. The combination of the angiotensin converting enzyme inhibitor enalapril (1mg/kg, p.o.) which does not lower blood pressure, and nepicastat (30mg/kg) at doses enhanced the antihypertensive effect over the 30 day dosing period and resulted in a significant reduction in left ventricular mass (mass). For enalapril alone, no reduction in left ventricular mass occurred. Thus, treatment of the SHRs with nepicastat (30 and 100mg/kg/d) for 30 days resulted in a lower blood pressure drop and, when combined with enalapril, an additional blood pressure drop with a concomitant reduction in left ventricular mass.

At doses of 30 and 100mg/kg/d (7 days of administration), the blood pressure lowering effect of nepicastat was less than that observed in SHRs in normotensive Wistar rats (with a meter fitted with a radio telemetric blood pressure transducer). At a dose of 30mg/kg/d, the highest reduction in blood pressure compared to-20 mmHg in SHRs is-10 mmHg. At a dose of 100mg/kg/d, the highest reduction in blood pressure compared to-42 mmHg in SHRs was-17 mmHg. Thus, nepicastat had a greater blood pressure lowering effect in SHRs than in normotensive rats.

Studies in normally anesthetized dogs showed that nepicastat had no effect on the cardiovascular system following acute intravenous administration (1-10mg/kg i.v.), with no change in arterial blood pressure, left ventricular pressure (including peak dp/dt), heart rate, cardiac output, or renal blood flow for up to five hours following administration. In a study of long-term instrumented, conscious dogs, a similar lack of effect was observed 12 hours after a single dose (3-30mg/kg i.v.).

Nepicastat (30mg/kg, administered intraduodenally) did not significantly inhibit a decrease in renal blood flow in response to direct renal nerve stimulation, or an increase in arterial blood pressure in response to carotid artery occlusion, up to 5 hours after administration in anesthetized male beagle dogs. However, nepicastat resulted in a significant decrease in noradrenaline levels and an increase in the dopamine/noradrenaline ratio (rather than dopamine levels) in the mesenteric artery 5 hours after administration. Thus, although tissue norepinephrine levels are significantly reduced, sympathetic-induced functional responses are not significantly inhibited.

When nepicastat was administered to male beagle dogs (4.5 days, 10mg/kg/d), there was no statistically significant decrease in blood pressure and increase in heart rate in anesthetized animals in response to carotid artery occlusion. Nepicastat treatment significantly reduced the increase in heart rate during response to i.v. tyramine stimulation, but resulted in only slight and insignificant inhibition of blood pressure increase. Thus, the chronic administration of nepicastat at a dose that results in the maximal reduction of tissue norepinephrine levels does not have a significant inhibitory effect on sympathetic-induced functional responses.

In mice given 1.0-30mg/kg (p.o.) urgently, nepicastat did not cause a significant effect on gross (gross) motor behavior, and it did not affect the motor (loco motor) activity of mice (10-100mg/kg i.p.). Acute dosing of rats did not affect locomotor (lococotor) activity or acoustic startle response (3-100mg/kg i.p.).

No behavioral effects were observed in rats 10 days after administration of 10, 30 and 100mg/kg/d (p.o.). Rectal temperature has not been affected. Motor activity and auditory startle reflex were significantly reduced by treatment with the DBH inhibitor SKF-102698(100mg/kg/d, p.o.) and the centrally acting a-adrenergic agonist clonidine (20mg/kg, b.i.d., p.o.). Locomotor (motor) activity was also unaffected by administration in SHRs over 30 days (3-100mg/kg/d, p.o.). Thus, nepicastat does not cause detectable changes in central nervous system-mediated behavioral effects in rats.

Nepicastat is a potent competitive inhibitor of DBH in vitro in humans and in vivo in rats and dogs. Oral treatment with nepicastat at a dose of 6mg/kg/d in rats produced evidence of significant DBH inhibition in the heart and mesenteric arteries. In contrast to another DBH inhibitor, SKF-102698, nepicastat showed some selectivity for the left ventricle and mesenteric artery over the cerebral cortex. In rats, no effect of nepicastat on behavior was observed. In dogs, at a dose of 10mg/kg/d, a plateau effect of DBH inhibition occurs in the heart, renal arteries and kidneys. Nepicastat significantly reduced the hypertensive response to sympathetic stimulation in rats (3mg/kg p.o.), and significantly reduced blood pressure throughout the day when administered once a day (30mg/kg/d p.o.) in SHRs (30 days of administration). In conclusion, nepicastat is a potent DBH inhibitor that modulates the action of the sympathetic nervous system.

Example 9

The study described herein was designed to evaluate the pharmacokinetics of higher oral doses of nepicastat, to compare pharmacokinetics in male and female rats, and to determine the permeation of nepicastat into the CNS by determining nepicastat levels in the brain.

Male rats weighing 180-220g (Crl: CD BR Vaf +) were fasted overnight prior to dosing until 4 hours after dosing. The dose was formulated in water containing 2% 1-hydroxypropyl methylcellulose (50 centipoise viscosity), 1% benzyl alcohol, and 0.6% Tween 80 (all available from Sigma Chemical Company). For doses of 10, 30 and 100mg/kg, the drug concentrations in the dosing solutions were 5, 15 and 50mg/ml, respectively, and the dose volumes were 2.0 ml/kg. at various times after dosing by liquid chromatography (LC confirmed.5 mg/ml dose is a clear solution, higher concentration dose is a translucent suspension. blood samples were obtained by puncturing the heart with a heparinized syringe and plasma was prepared by centrifugation.

Aliquots of plasma (0.05 or 0.5ml) were mixed with an internal standard (50 μ l methanol containing 5 μ g/ml of the monofluoro analogue of nepicastat and 5mg/ml dithiothreitol). The sample was mixed with 200mM sodium phosphate buffer (pH7.0, 0.5ml) and extracted with 3ml ethyl acetate/hexane (1/1, v/v). Analyte-containing solution was back-extracted with 250. mu.l of 250mM acetic acid The organic phase and 100 μ l aliquots of the aqueous phase were tested by LC. LC System Using Keystone Hypersil BDS 15cm C₈Column, at ambient temperature. Mobile phase a was 12.5mM potassium phosphate (ph3.0, containing 5mM dodecanesulfonic acid) and mobile phase B was acetonitrile. The solvent composition was 40% B and was pumped at a flow rate of 1 ml/min. Detection was by UV absorption (261 nm). The concentration of the analyte was determined using a standard curve generated from the analysis of plasma (from untreated rats) to which known concentrations of analyte were added. Plasma concentration data are expressed in μ g (free base)/ml.

The brains were briefly rinsed with saline, blotted dry on a paper towel, and then weighed (1.5-2.0 g). Internal standard (50. mu.l methanol, containing the monofluoro analogue of nepicastat at 20. mu.g/ml) was added and the brain was homogenized in 5ml 200mM sodium phosphate (pH7.0) containing 0.5mg/ml dithiothreitol an aliquot of the tissue homogenate (2ml) was extracted with 10ml ethyl acetate/hexane (1/1, v/v). The organic phase was gently back-extracted with 150. mu.l of 250mM acetic acid.

After adding 100. mu.l of methanol to the aqueous phase (dispersing any emulsion), 100. mu.l aliquots were analyzed by LC as described for plasma. Levels in the brain are expressed in μ g (free base)/g brain tissue.

Pharmacokinetic parameters were calculated from the mean plasma concentrations. Plasma half-life (T)_1/2) The calculation is as follows: 0.693/β, where β is the elimination rate constant determined by linear regression of log plasma concentration versus time data (within the terminal linear portion of the data). The area under the plasma concentration versus time curve (time from zero to the last at which the plasma concentration can be measured) is calculated using the trapezoidal rule (AUC). AUC (AUC) from zero to infinity_{All are}) The calculation is as follows:

AUC_{all are}＝AUC(0-C_{Finally, the})+C_{Finally, the}B is, wherein C_{Finally, the}Is the final quantifiable plasma concentration.

Concentrations of nepicastat were obtained in plasma of male rats given a single oral dose of 10, 30 or 100 mg/kg. Concentration of nepicastat in plasmaThe degree increases with increasing dose and is in AUC_{All are}And dose is linear. At higher doses (1.70, 2.09 and 3.88 hours after oral dosing of 10, 30 and 100mg/kg, respectively, in male rats), the elimination half-life appeared to be slightly increased. Plasma AUC of nepicastat in female rats after oral administration of nepicastat dose of 30mg/kg to female rats_{All are}Plasma AUC in male rats over administration of equal doses of nepicastat_{All are}The height is 77%. Initially, nepicastat was present at lower levels in the brain (expressed as μ g/g) than in plasma (expressed as μ g/ml). However, from 2 hours after administration, the concentration of nepicastat in the brain exceeded the concentration in plasma.

In male rats, based on AUC_{All are}Values, between 10 and 100mg/kg, plasma levels of nepicastat increased linearly with increasing dose.

After an oral dose of 30mg/kg, plasma levels of nepicastat were higher in female rats than in male rats.

After administration of an oral dose of nepicastat of 10mg/kg to male rats, nepicastat levels in the brain were initially lower than in the plasma, but from 2 hours onwards, nepicastat levels in the brain were greater than in the plasma.

Example 10

The aim of this study was to determine the effect of nepicastat (10mg/kg) on dopamine and norepinephrine levels in the mesenteric artery over a 24 hour period following a single oral administration in spontaneously hypertensive rats. In a single oral administration of nepicastat (10mg/kg) or vehicle (dH)₂O; 10ml/kg) were followed by 1, 2, 4, 6, 8, 12, 16 and 24 hours, catecholamine levels were determined.

Male Spontaneous Hypertensive Rats (SHRs) 16-17 weeks old weighing 300-. In the afternoon prior to the study, animals were weighed and randomly assigned to one of the following test groups (each group n-9): the animals were orally administered a single dose of 10mg/kg nepicastat, or a single oral vehicle (10ml/kg), 1, 2, 4, 6, 8, 12, 16, or 24 hours prior to killing.

Nepirstat was synthesized as the hydrochloride salt and dissolved in the excipient (dH)₂O) to give an oral dose that can be administered in a repeated volume of 10 ml/kg. All doses of nepicastat were administered as free base equivalents and were prepared in the morning of the administration.

Animals were dosed at each measured time of the morning (minute) when the animals were sacrificed. At 1, 2, 4, 6, 8, 12, 16 and 24 hours after dosing, 9 treated and 9 vehicle animals were anesthetized with halothane, decapitated, the left ventricle and mesenteric artery were rapidly harvested and weighed. The mesenteric artery was filled into 0.5ml of 0.4M perchloric acid (in a centrifuge tube) and the left ventricle was placed in an empty cryostraw. Both tissues were immediately frozen in liquid nitrogen and stored at-70 ℃. The catecholamine levels in the mesenteric arteries were determined using HPLC (with electrochemical detection). At the time of decapitation, plasma samples were obtained by draining blood from the carcass into tubes containing heparin and centrifuged at 4 ℃.

At each time point, each treatment group was compared to the vehicle group. Two-way variance (ANOVA) analysis with effects TRT, HARVEST and their interactions was performed. For each acquisition time, a one-way ANOVA with a factor TRT was performed. At each time point, pairwise analysis was performed between treated and vehicle animals using Fisher's LSD strategy in order to control experimental-wise error rates. Except at the 4 hour time point, the norepinephrine value was significantly lower (p < 0.05) than for the vehicle group. At the 6 hour point, the levels decreased to some extent (0.05 < p < 0.1). At 2 and 6 hour collection times, dopamine levels were significantly (p < 0.05) higher than those of the vehicle group. At the 1, 2, 4, 6 and 12 hour time points, the dopamine/norepinephrine ratio was significantly (p < 0.05) greater than the dopamine/norepinephrine ratio of vehicle-treated animals.

Typically, nepicastat has little statistically significant effect on mesenteric artery norepinephrine or dopamine levels 1, 2, 4, 6, 8, 12, 16, or 24 hours after administration following a single oral dose of 10mg/kg in spontaneously hypertensive rats. However, a steady increase in the dopamine/norepinephrine ratio was observed during the majority of the first 12 hours of treatment. At 16 and 24 acquisition times, no change in all three parameters was observed.

Example 11

The purpose of this study was: the effect of intravenous administration of nepicastat (hereinafter nepicastat) on dopamine and norepinephrine levels in the left ventricle was determined in Sprague-Dawley rats. Animals received two intravenous (iv) administrations (12 hour interval): excipient (75% propylene glycol + 25% DMSO; 1.0ml/kg) or nepicastat 15 mg/kg. Tissue norepinephrine and dopamine levels were measured 6 hours after the final compound administration.

Male Sprague-Dawley rats 16 to 17 weeks old weighing 300-. In the afternoon prior to the study, animals were weighed and randomly assigned to one of the following test groups (each group n 10): excipient (1.0ml/kg) or nepicastat 15 mg/kg.

Nepicastat was synthesized and dissolved in the appropriate amount of vehicle (75% propylene glycol + 25% DMSO) to give a dosing volume of 1.0 ml/kg. Nepicastat was administered as a free base equivalent and prepared in the afternoon prior to the first dose.

In the afternoon prior to sampling, each mouse was dosed iv in the tail vein. The following morning, 12 hours later, dosing was repeated. 6 hours after the last dose, rats were anesthetized with halothane, decapitated, and the left ventricle was rapidly harvested and weighed. The ventricles were placed in 1.0ml of frozen 0.4M perchloric acid. The tissue was immediately frozen in liquid nitrogen and stored at-70 ℃. Tissue dopamine and norepinephrine concentrations were determined by high performance liquid chromatography using electrochemical detection.

For norepinephrine, one-way analysis of variance (ANOVA) with the main effects of treatment was performed. The Kruskal Wallis test was performed on dopamine and their ratios, mainly due to the variance of heterogeneity among the test groups. A pairwise comparison was then made between nepicastat-treated rats and vehicle using the Fisher's LSD assay. Bonferroni correction was performed for all p-values to ensure a total experimental type 1 error rate of 5%.

Compared to vehicle treated animals, nepicastat given at 15mg/kg significantly (p < 0.01) reduced noradrenaline levels by 51%, significantly (p < 0.01) increased dopamine levels by 472%, and significantly (p < 0.01) increased the dopamine/noradrenaline ratio by 1117%.

In conclusion, intravenous administration of nepicastat significantly inhibited DBH in the left ventricle of Sprague-Dawley rats.

Example 12

This study evaluated the effect of nepicastat on altering dopamine and norepinephrine levels in the cortex, left ventricle, and mesenteric arteries of male Spontaneously Hypertensive Rats (SHRs). Animals were given three doses (12 hour intervals) (3, 10, 30 or 100mg/kg p.o.).

The study also compared the effect of the S enantiomer (nepicastat) with the R enantiomer (compound B) after three doses (30 mg/kg). This study also compared the effect of nepicastat with SKF-102698 (a DBH inhibitor), whose oral activity was previously demonstrated in rats.

The compounds are prepared and administered as free base equivalents. Nepicastat is dissolved in a suitable amount of excipient (dH)₂O (for nepicastat), PEG 400: dH₂O, 50: 50 vol: vol (for SKF-102698)). Medicaments of 3, 10, 30 and 100mg/kg nepicastat and 30mg/kg SKF-102698 were prepared in a dosing volume of 10.0 ml/kg.

Spontaneously hypertensive 15-16 week old males (SHRs) (Charl)es river labs) use food and water ad libitum. Animals were weighed and randomly assigned to one of the following test groups: 1) distilled water excipient (dH)₂O), or 3, 10, 30 and 100mg/kg nepicastat, 2)30mg/kg compound B (in distilled water), or 3) PEG 400: dH₂Excipient O or SKF-102698(30 mg/kg). Each mouse was dosed orally three times (p.o., using a gavage needle), 12 hour intervals, beginning in the morning. 6 hours after the third dose, rats were anesthetized with halothane, decapitated, the cortex, mesenteric artery and left ventricle were collected rapidly, weighed, placed in frozen perchloric acid (0.4M), frozen in liquid nitrogen, and stored at-70 ℃. The tissue dopamine and norepinephrine concentrations are determined by high performance liquid chromatography and electrochemical detection.

Four series of statistical analyses were performed. The first series compares rats treated with various doses of nepicastat and 30mg/kg compound B with vehicle control animals. Non-parametric one-way analysis of variance (ANOVA) was performed for factor Dose (Dose) and blocking factor Day (Day) for each tissue and strain (strain), respectively. The overall results are reported. Pairwise analysis was performed between treatment and control groups at each dose using Dunnett's test in order to control the experimental error rate. Second statistical test for SKF-102698 and PEG-dH ₂The O vehicle treated groups were compared (using a non-parametric t-test). A third statistical test compares the 30mg/kg dose of compound B with nepicastat (using a non-parametric t-test). A fourth statistical analysis compared a 30mg/kg dose of nepicastat to SKF-102698. Due to the use of two different excipients, a linear comparison (contrast) was performed, which calculates the difference in difference as follows:

change ═ 30 mg/kg-vehicle)_Nepitastat- (30 mg/kg-excipient)_SKF-102698

Zero equivalence (for equality to zero) of this new variable was tested by the SAS procedure General Linear Models.

At 30 and 100mg/kg doses of nepicastat, the dopamine concentration in the cerebral cortex was significantly (p < 0.05) greater than vehicle, the norepinephrine concentration was significantly (p < 0.05) lower than vehicle, and the dopamine/norepinephrine ratio was significantly (p < 0.05) greater than vehicle.

At doses of 3, 10, 30 and 100mg/kg, dopamine concentrations in the left ventricle were significantly (p < 0.05) greater than vehicle. At doses of 10, 30 and 100mg/kg, the noradrenaline concentration was significantly (p < 0.05) lower than that of the vehicle. At doses of 3, 10, 30 and 100mg/kg nepicastat, the dopamine/norepinephrine ratio in the left ventricle was significantly (p < 0.05) greater than in the vehicle group.

At doses of 3, 10, 30 and 100mg/kg, the dopamine concentration in the mesenteric arteries of SHRs was significantly (p < 0.05) greater than in the vehicle group. At doses of 10, 30 and 100mg/kg, the noradrenaline concentration was not significantly (p < 0.05) less than in the vehicle group. At all doses of nepicastat, the dopamine/norepinephrine ratio in the mesenteric artery was significantly (p < 0.05) greater than in the vehicle group.

In the cerebral cortex, compound B resulted in a significant (p < 0.01) increase in dopamine and norepinephrine relative to vehicle treatment, and had no effect on the dopamine/norepinephrine ratio. With nepicastat norepinephrine levels were significantly lower than compound B (p < 0.01).

In the left ventricle, compound B resulted in a significant increase in dopamine and dopamine/norepinephrine ratio (p < 0.01) relative to vehicle treatment, but did not significantly reduce norepinephrine levels. Nepicastat was significantly more potent than compound B in reducing norepinephrine levels and increasing dopamine to dopamine/norepinephrine ratio (p < 0.01).

In the mesenteric artery, compound B resulted in a significant increase in dopamine and dopamine/norepinephrine ratio (p < 0.01) relative to vehicle treatment, but did not significantly reduce norepinephrine levels. Nepicastat was significantly more potent than compound B in reducing norepinephrine levels and increasing dopamine to dopamine/norepinephrine ratio (p < 0.01).

In the cerebral cortex, comparing nepicastat with SKF-102698(30mg/kg), at a dose of 30mg/kg SKF-102698, the dopamine concentration in the cortex was significantly greater than the vehicle (p < 0.01). SKF-102698 is higher than nepicastat (p < 0.01) relative to the increase of excipient. For SKF-102698, its norepinephrine concentration was significantly lower than the vehicle, and for this decrease, SKF-102698 was reduced more than nepicastat (p < 0.01). For SKF-102698, the dopamine/norepinephrine ratio in the cortex was significantly greater (p < 0.01) than for vehicle, and for vehicle enhancement, SKF-102698 was more enhanced than nepicastat (p < 0.01).

For SKF-F102698, dopamine concentration in the left ventricle was significantly greater than vehicle (p < 0.01), and for vehicle enhancement, nepicastat was more enhanced than SKF-F102698 (p < 0.01). There was no difference in noradrenaline concentration between treatment with SKF-102698 and vehicle, however, treatment with nepicastat significantly reduced noradrenaline relative to vehicle, more than SKF-102698 (p < 0.01). For SKF-102698, the dopamine/norepinephrine ratio in the left ventricle was significantly greater than vehicle (p < 0.05), and nepicastat was increased more than SKF-102698 (p < 0.05) relative to vehicle increase.

For SKF-102698, dopamine concentrations were significantly greater in the mesenteric artery than for vehicle, and for vehicle enhancement, nepicastat was increased more than SKF-102698. The noradrenaline concentration for treatment with SKF-102698 was significantly lower than the vehicle, and treatment with nepicastat significantly reduced noradrenaline relative to the vehicle, which was more reduced than SKF-102698. For SKF-102698, the dopamine/norepinephrine ratio in the left ventricle was significantly greater than the vehicle, and for the vehicle increase, nepicastat increased more than SKF-102698.

In summary, the data indicate that nepicastat is an effective inhibitor of DBH in vivo in the mesenteric artery, left ventricle and cerebral cortex of SHRs 6 hours after the third of the three oral doses (12 hours between doses). At a dose of 30mg/kg, the S enantiomer (nepicastat) was more potent than the R enantiomer (compound B) in all three tissues. Furthermore, nepicastat was more effective than SKF-102698 in the mesenteric arteries and left ventricle but less effective in the cerebral cortex after three doses (30mg/kg) given over 24 hours.

Example 13

Nepicastat was prepared and administered as a free base equivalent. Nepicastat and methimazole were dissolved in vehicle (66.7% propylene glycol: 33.3% dH) ₂O), a suitable concentration of the dosing solution is obtained so that all doses can be administered in a dose of 1.0ml/kg volume.

Male Sprague-Dawley rats (weight 180-. Animals were weighed and randomly assigned to one of the following test groups (each group n-12): nepicastat 2.0mg/kg, nepicastat 6.2mg/kg, methimazole 1mg/kg or excipient 1 ml/kg. Rats in each group were dosed orally for 10 consecutive days in the evening and the next morning (approximately 12 hour interval).

Four hours after the second dose (on day 10), rats were anesthetized with halothane, decapitated, and the cortical, striatal, and mesenteric arteries were harvested and weighed. Tissue samples were not taken from the methimazole group as they served only as positive controls for thyroid function assays. Mesenteric arteries, cortex and striatum were immediately placed in 0.4M frozen perchloric acid and analyzed for norepinephrine and dopamine levels using HPLC on the same day.

Orbital blood samples were taken on days-3, 0, 3, 7 and 9 (day 0 being the first day of administration). Analysis of T in serum samples Using radioimmunoassay ₃And T₄And (4) horizontal.

To statistically evaluate T₃And T₄Changes in levels were calculated from day-3 time points from baseline. Non-parametric two-way analysis of variance was performed within the scope of the target analysis of variance (ANOVA). One-way ANOVA was also performed to test whether significant differences occurred compared to the control. Pairwise analysis was performed between control and each treatment group using Fisher's LSD strategy to control experimental error rates. For statistical analysis of catecholamine levels, one-way ANOVA (with factor DOSE) was performed. Pairwise analysis was performed between treatment and control at each dose using the Fisher's LSD strategy in order to control experimental error rates.

At doses of 2.0 and 6.2mg/kg, norepinephrine levels in the cortex were not significantly (p > 0.05) different compared to vehicle control in nepicastat-treated animals. Norepinephrine levels in the mesenteric artery were significantly (p < 0.05) reduced in the 2.0 and 6.2mg/kg dose groups and to some extent (p < 0.10) in the striatum in the 2.0 and 6.2mg/kg dose groups compared to the vehicle control.

In the dose groups of either 2.0 or 6.2mg/kg of endostatin, there was no significant (p > 0.05) difference in dopamine levels in all three tissues compared to the vehicle control.

At doses of 2.0 and 6.2mg/kg nepicastat, there was no significant (p > 0.05) difference in dopamine/norepinephrine ratios in the cortex and striatum compared to the vehicle control, whereas at doses of 2.0 and 6.2mg/kg nepicastat, the ratio in the mesenteric artery was higher than that in the vehicle control (p < 0.05).

By altering free T in mouse serum₃Or all of T₄At the level, neither 2.0 nor 6.2mg/kg nepicastat affected thyroid function. The dose of 1.0mg/kg methimazole (positive control) significantly (p < 0.05) reduced T compared to vehicle control₃Level (on all treatment days) and T₄Levels (on days 3 and 7). Methimazole for treating animalsT₄The levels only decreased to some extent (p < 0.10) on day 9.

Nepicastat (2.0 or 6.2mg/kg) did not cause any significant (p > 0.05) changes in dopamine or norepinephrine levels or dopamine/norepinephrine ratios when compared to vehicle. In the striatum, in the 6.2mg/kg dose group, a somewhat significant (p < 0.10) decrease in noradrenaline levels was observed, but no other significant changes were observed. In mesenteric arteries, 2.0 and 6.2mg/kg nepicastat caused a significant (p < 0.05) decrease in noradrenaline levels and a significant (p < 0.05) increase in the dopamine/noradrenaline ratio compared to vehicle, with no significant change in dopamine levels observed. Thus, nepicastat appears to be an in vivo potent inhibitor of dopamine β -hydroxylase with greater efficacy in the mesenteric artery than in the cerebral cortex or striatum 10 days after administration in Sprague-Dawley rats.

Example 14

This study was conducted to determine dopamine and norepinephrine concentrations in the renal medulla and renal cortex of dogs taking nepicastat. Adult male beagle dogs were randomly assigned to four groups of 8 dogs each and were orally administered nepicastat. Nepicastat was delivered at doses of 5, 15 and 30mg/kg (in a single capsule). The excipient is an empty capsule. Each dog was given 2 doses per day, morning and afternoon (8-10 hour interval), for 4 days. On the fifth day, each dog received a single dose in the morning, and the dogs were euthanized 6 hours after the last dose. Samples of kidney medulla and kidney cortex were collected rapidly, weighed, placed in cold 0.4M perchloric acid, frozen in liquid nitrogen, and stored at-70 ℃.

To determine the concentration of Noradrenaline (NE) and dopamine (D), each tissue was homogenized by brief sonication in 0.4M perchloric acid. After sonication, the tissue homogenate was centrifuged (13,000rpm) in a microtube for 30 minutes at 4 ℃. An aliquot of each supernatant was removed and 3, 4-Dihydroxybenzylamine (DHBA) was added as an internal standard. The extract from each sample was subjected to HPLC separation using electrochemical detection. The method has a quantitative limit of 2.0ng/mL, with a linear range of 2.0ng/mL to 400ng/mL per analyte.

Each analyte assay data was normalized to the weight of the tissue sample and expressed as: μ g analyte per gram of tissue. The concentration of dopamine, norepinephrine, and the ratio of dopamine to norepinephrine concentration (D/NE) were obtained for each dog. In addition, for each treatment group, the mean and standard deviation and D/NE ratio calculated for each analyte were provided.

Example 15

In this study, male puppies (Marshall farms, North Rose, NY) weighing between 9-16kg were used. Animals were given unlimited access to water and the food was administered once a day at-10.00 AM. Animals were randomly assigned to one of the following treatment groups (n-8/group): placebo (empty capsules), or nepicastat 2mg/kg b.i.d. (4 mg/kg/day). Each animal received 2 doses per day, morning and afternoon (8-10 hour interval). 6 hours after AM administration, a daily blood sample (10ml) was drawn for the determination of the plasma levels of nepicastat and catecholamine. Blood was collected in tubes containing heparin and glutathione, centrifuged at-4 ℃ and collected within 1 hour. The plasma was separated and divided into two samples, one for the determination of plasma catecholamine and the other for the analysis of nepicastat.

If tissue catecholamines need to be analyzed at a later point, tissue samples are also taken from the dogs at the end of the study. On day 15, 6 hours after AM administration, a final blood sample (10ml) was taken. Dogs were anesthetized with sodium pentobarbital (40mg/kg, iv), placed on autopsy tables, and euthanized with a second injection of pentobarbital (80mg/kg, iv). A rapid two-way thoracotomy and laparotomy was performed. Biopsies were taken from the renal arteries and left ventricle. The skull is opened and frontal lobe of the cerebral cortex is exposed to obtain a biopsy. Tissue samples were weighed, placed in ice-cold 0.4M perchloric acid, frozen in liquid nitrogen, and stored at-70 ℃ until analyzed.

Plasma Norepinephrine (NE), Dopamine (DA), and Epinephrine (EPI) were analyzed by HPLC using electrochemical detection. Plasma concentrations of nepicastat were determined by HPLC using electrochemical detection.

The Box-Cox transform indicates that the logarithm is a suitable variable for the stable transform; all analyses were thus performed with log-values. The BQL (below the quantitation limit) in the DA concentration for dog 1 on day 10 was set to 0; 1n (0) is set to none. Analysis was performed using a MIXED model (using PROC MIXED), day and treatment category variables were fixed, and dogs within the treatment range were randomized factors. For a fixed effect, the interplay between days and treatment was included, as the difference between the drug and placebo groups changed daily. The CONTRAST instruction is used to calculate the CONTRAST (CONTRAST) which correctly takes into account the error term for each particular CONTRAST. In particular, the comparison of the treatment groups to the drug groups uses mean square error terms for the dogs, while the comparisons used to establish steady state are all within dog comparisons and mean square error terms are required.

The steady state time period is calculated using the Helmert transform (cf. SAS PROC GLM manual). These transformations compare the mean of the treatment averages for the time points following each treatment average (mean). The steady state period is defined to start at the first point in time after the maximum time when the Helmert contrast is statistically significant. However, since this method cannot determine a smoothly changing process, as may be the case herein, it is also necessary to calculate the slope of the analyte concentration during the steady state period. For each individual dog, the slope during the steady state period was calculated, one slope for each animal. A univariate statistic over the slope is then calculated, the average slope is used to establish the normal theoretical confidence interval, and its normal theoretical p-value is calculated, testing the assumption that the slope equals zero. This slope analysis serves as the basis for the determination whether or not the steady state period is the period of changing concentration.

Nepicastat (2mg/kg, b.i.d) caused a significant decrease in plasma NE (2.1-fold) and EPI (1.91-fold) and a significant increase in plasma DA (7.5-fold) and DA/NE ratio (13.6-fold) when compared to placebo.

A peak-like decrease in plasma NE and EPI was observed on days 6 and 8, respectively, while a peak-like increase in plasma DA and DA/NE ratio was observed on days 7 and 6, respectively. At approximately 4, 8 and 6 days post-administration, the effects on plasma NE, DA and EPI, respectively, reached steady state. The changes in plasma DA and DA/NE ratios were significantly different from placebo on all days post-dose. The changes in plasma NE were significantly different from placebo on days 4-9 and 11-13 post-dose. The changes in plasma EPI were significantly different from placebo on days 7-9 and 12 post-dose.

On all days, administration of nepicastat (2mg/kg, bid) resulted in significant plasma drug levels. Peak levels were observed 2 days after dosing. On any day, no significant levels of the N-acetyl metabolite of nepicastat were determined.

Long-term (14.5 days) administration of nepicastat (2mg/kg, bid, po) resulted in a significant decrease in plasma NE and EPI, and a significant increase in the ratio of plasma DA to DA/NE. These changes reflect inhibition of the sympathetic adrenal system by inhibition of the enzyme dopamine β -hydroxylase.

Example 16

Nepicastat was weighed and placed into capsules (13-Torpac size; East Hanover, NJ) to give doses of 5, 15 and 30mg/kg per capsule (b.i.d. administration to give doses of 10, 30 and 60 mg/kg/day). Initial dog weights were used to determine the dose for each animal. Dogs receiving 0 mg/kg/day received an empty capsule (placebo). All doses of nepicastat were given as free base equivalents.

32 male beagle dogs (weighing 10-12kg) were randomly assigned to one of the following 4 treatment groups (each group n-8): nepicastat 0 mg/kg/day (placebo), 10 mg/kg/day (5mg/kg, b.i.d.), 30mg/kg (15mg/kg, b.i.d.), or 60 mg/kg/day (30mg/kg, b.i.d.). Dogs numbered 1-16 were designated as dose group a and dogs numbered 17-32 were designated as dose group B. To harvest tissue, lethal surgery was performed on 16 animals studied each day for a period of 2 days. Each dog was weighed 2 or 3 days prior to administration of the first compound and the area of skin covering the head, saphenous vein and jugular vein was trimmed. Administration includes orally administering one capsule, the second administration being 8-10 hours later. Dogs were dosed on days 1-3 as scheduled. On day 4, 3ml of blood was obtained from the jugular vein prior to AM administration, and baseline plasma compound levels were determined. Dogs were then given an AM dose and 1, 2, 4 and 8 hours after dosing, additional 3ml blood samples were collected and plasma compound levels were determined. Blood samples were placed into tubes containing heparin, centrifuged at 4 ℃ and stored at-20 ℃ until analysis. The PM dose is then administered as planned. AM doses were given as scheduled on the day of surgery. Approximately 6 hours after the AM dose, a final 3ml blood sample was taken from the jugular vein and plasma compound levels were determined. The dogs were then anesthetized with sodium pentobarbital (-40 mg/kg) (i.v. administration in the head or saphenous vein), transported to an autopsy room, and given additional doses of sodium pentobarbital (-80 mg/kg, iv). The left ventricle, renal artery, kidney, renal medulla, renal cortex and cerebral cortex were then rapidly harvested, weighed, placed in 2ml of frozen 0.4M perchloric acid, frozen in liquid nitrogen, and stored at-70 ℃ until the catecholamines were analyzed by HPLC using electrochemical detection. All tissue samples were divided into 2 aliquots, the second aliquot was immediately frozen in liquid nitrogen and stored at-70 ℃ for determination of tissue compound levels. The third transmural sample (taken from the left ventricle) was immediately frozen in liquid nitrogen and stored at-70 ℃ for receptor binding studies.

Ventricles were disrupted in 50mM Tris-HCl, 5mM Na using a Polytron P-10 tissue disruptor (set at 10, 2X15 seconds)₂EDTA buffer (pH7.4, at 4 ℃) was homogenized. The tissue homogenate was centrifuged at 500Xg for 10 minutes and the supernatant was stored on ice. The pellet was washed by resuspension and centrifuged at 500Xg, and the supernatants were combined. The combined supernatants were centrifuged at 48,000Xg for 20 minutes. The pellets were washed by resuspension and centrifuged in homogenization bufferOnce, it was centrifuged twice in 50mM Tris-HCl, 0.5mM EDTA buffer (pH7.4, at 4 ℃). The membrane was stored at-70 ℃ until needed. In a buffer solution (pH7.4 at 32 ℃) containing 50mM Tris-HCl, 0.5mM EDTA, the use of [ 2 ], [ solution ]³H]CGP-12177 was subjected to a saturation experiment. Nonspecific binding was defined by 10 μ M isoproterenol. For eight concentrations of [ 2 ]³H]CGP-12177 (from 0.016nM to 2nM), tubes for total binding, non-specific binding and total counts were prepared. The samples were incubated at 32 ℃ for 60 minutes. Samples were filtered using a Brandel cell harvester with 0.1% PEI (pre-treated GF/B glass fiber filter pad). The sample was washed three times with room temperature water (3 seconds). A hydrosol scintillation fluid was added to each tube and radioactivity was determined by liquid scintillation counting. After the first conversion of total ligand concentration to free ligand concentration (total-bound vs. free), the saturation binding isotherm was analyzed. An independent saturation isotherm for each tissue was completed. The membrane was tested for proteins using the Bio-Rad protein binding method and gamma globulin as a standard. Receptor density per mg protein is expressed as the mean value for each treatment group. The nepicastat-treated group was compared with the placebo (control) -treated group and tissue catecholamine levels were analyzed. For each tissue and each catecholamine assay, non-parametric one-way analysis of variance (ANOVA) was performed with factor DOSE, respectively. Pairwise analysis was performed between each dose of treatment and control using Dunnett's test to control the experimental error rate. Student-Neuman-Kuels and Fisher's LSD tests were performed as validation. Analysis of tissue and plasma compound levels was performed in two ways. First, an independent t-test is performed to compare each dose level for each parameter to the factor level for its partner dose. For example, in a particular tissue or plasma, there would be three times the level of compound at 10mg/kg, which should be comparable to that observed in the 30mg/kg group. In addition, linear orthogonal contrasts were calculated for all three doses over the range of one-way ANOVA. The paired t-test was used to determine any differences in binding between the vehicle treated group and the 10 mg/kg/day nepicastat group.

Dogs were orally administered 0, 5, 15 or 30mg/kg nepicastat capsules (b.i.d.) to give doses of 10, 30 and 60 mg/kg/day for 4.5 days, and tissues were harvested 6 hours after the last dose. Nepicastat was administered at doses of 10, 30 and 60 mg/kg/day in the renal artery, significantly (p < 0.01) reducing noradrenaline levels by 86%, 81% and 85%, respectively. At doses of 10, 30 and 60 mg/kg/day, dopamine levels were significantly (p < 0.01) increased by 180%, 273% and 268%, respectively. The doses of nepicastat at 10, 30 and 60 mg/kg/day significantly (p < 0.01) increased the dopamine/norepinephrine ratio by 1711%, 1767% and 1944%, respectively, compared to placebo. After administration of 10 and 60 mg/kg/day nepicastat, dopamine levels were significantly (p < 0.01) increased 632% and 411% in the cerebral cortex, respectively. The dopamine/norepinephrine ratio increased significantly (p < 0.01) by 531% after administration of 10 mg/kg/day nepicastat and by 612% after administration of 60 mg/kg/day nepicastat. At these 2 doses, norepinephrine levels were not significantly (p > 0.01) affected. At a dose of 30 mg/kg/day, noradrenaline was significantly (p < 0.01) reduced by 63%, proportionally significantly (p < 0.01) increased by 86%, and dopamine levels were increased to some extent (0.05 < p < 0.10) by 174% compared to placebo. After administration of 10, 30 and 60 mg/kg/day nepicastat, norepinephrine levels were significantly (p < 0.01) reduced by 85%, 58% and 79%, respectively, in the left ventricle. The dopamine/norepinephrine ratio was significantly (p < 0.01) increased 852%, 279%, and 607%, respectively, compared to placebo animals. No significant changes in dopamine levels were observed at doses of nepicastat at 10, 30 and 60 mg/kg/day.

In the renal cortex, norepinephrine levels were significantly reduced (p < 0.01) by 86%, 66% and 85%, respectively, after doses of 10, 30 and 60 mg/kg/day nepicastat compared to placebo. At this dose, dopamine levels were significantly (p < 0.01) increased by 156%, 502% and 208%, respectively. At doses of 10, 30 and 60 mg/kg/day, the dopamine/norepinephrine ratio increased significantly (p < 0.01) 1653%, 1440% and 1693%, respectively. In the renal medulla, at doses of 10, 30 and 60 mg/kg/day nepicastat, the dopamine/norepinephrine ratio was significantly (p < 0.01) increased by 555%, 636% and 677%, respectively, compared to placebo. At a dose of 30 mg/kg/day, dopamine levels were significantly (p < 0.01) increased 522%, and at doses of 10 and 60 mg/kg/day, dopamine levels were increased to some extent (0.05 < p < 0.10) by 150% and 156%, respectively. After administration of nepicastat at 10 mg/kg/day, norepinephrine levels were significantly (p < 0.01) reduced by 72% compared to placebo, and after administration of 60 mg/kg/day, norepinephrine levels were reduced to some extent (0.05 < p < 0.10) by 69%.

Statistical analysis showed that the nepicastat concentration in plasma obtained on day 4 and in tissue and plasma obtained on day 5 was dose-proportional between each dose level and the factor (factor) level of its partner (partner) dose. Thus, the dose points determined were linear, except for the following (significant results indicate that the data is not linear):

Renal medulla: 3X10 < 30(p < 0.05)

Renal medulla: 6X10 < 60(p ═ 0.077)

Plasma (day 4): 2X30 > 60(p ═ 0.076)

At day 5, nepicastat levels were higher in all tissues tested than in plasma.

The results show that there was no difference between the left ventricular samples from the 10 mg/kg/day nepicastat-treated group and the vehicle-treated group.

Example 17

Evaluation of Nepitastat Activity against a number of enzymes including tyrosine hydroxylase, NO synthase, phosphodiesterase III, phospholipase A₂Neutral endopeptidase, Ca²⁺Calponin protein kinase II, acetyl CoA synthetase, acyl CoA-cholesterol transacylase, HMG-CoA reductase, protein kinase (non-selective) and cyclooxygenase-I, Nepirstat has an IC > 10. mu.M for all 12 enzymes studied, as shown in FIG. 4₅₀The value of the one or more of,it is therefore a highly selective (> 1000 fold) inhibitor of dopamine-beta-hydroxylase.

Example 18

Bovine DBH derived from adrenal glands was obtained from Sigma Chemicals (St Louis, MO). Human secreted DBH was purified from the culture medium of the neuroblastoma cell line SK-N-SH and used to obtain inhibition data. A column of lentil lectin containing 25ml of gel was prepared and applied with 50mM KH ₂PO₄(pH6.5), 0.5M NaCl balance. 35ml of 10% methyl alpha, D-mannoside (at 50mM KH) was used₂PO₄(pH6.5) in 0.5M NaCl) at a rate of 0.5 ml/min. The fraction containing most of the enzymatic activity was collected and concentrated using YM30 membrane with an Amicon stirred cell (cell). By heating at 50mM KH₂PO₄(pH6.5) and 0.1M NaCl, and methyl α, D-mannoside was removed. The concentrated enzyme solution was aliquoted and stored at-25 ℃.

DBH activity was determined by HPLC assay using tyramine and ascorbate as substrates. The method is based on the separation and quantification of tyramine and ethanolamine by reverse phase HPLC chromatography (Feilchenfeld, n.b., Richter, H.&Waddlell, W.H, (1982) anal. biochem: a time-delayed assay of subpamine β -hydroxyase activity analyzing high-pressure chromatography.122: 124-128.). The test is carried out at pH5.2 and 37 deg.C under conditions of 0.125M NaAc, 10mM fumarate, 0.5-2.0 μ MCuSO₄0.1mg/ml of catalytic enzyme (6,500u, Boeriger Mannheim, Indianapolis, IN), 0.1mM tyramine and 4mM ascorbate. In a typical experiment, 0.5-1.0 milliunits of enzyme is added to the reaction mixture, followed by the addition of a substrate mixture comprising the catalytic enzyme, tyramine and ascorbate to start the reaction (final volume of 200 μ l). The samples were incubated at 37 ℃ for 30-40 minutes. The reaction was quenched by a stop solution containing 25mM EDTA and 240. mu.M 3-hydroxytyrosamine (internal standard). Samples (150 μ l) were loaded into a Gilson autosampler and analyzed by HPLC using UV detection (at 280 nM). PC-1000 software (Thermo Separations products, Fremont, Calif.) for integration and data analysis. HPLC chromatographic operation was performed as follows: the flow rate was 1ml/min, and elution without gradient was performed using a LiChroCART 125-4 RP-18 column with 10mM of acid, 10mM of 1-heptanesulfonic acid, 12mM of tetrabutylammonium phosphate and 10% of methanol. Percent activity retained was calculated based on control (no inhibitor), corrected using internal standard, and fitted to a non-linear four-parameter dose response curve to obtain IC₅₀The value is obtained.

[¹⁴C]-purification of tyramine. Purification by means of a C18 lightly loaded column (two columns combined into one)¹⁴C]Tyramine hydrochloride, column applied with 2ml MeOH, 2ml 50mM KH₂PO₄(pH2.3), 30% acetonitrile, then 4ml 50mM KH₂PO₄(pH2.3) washing. A vacuum manifold (Speed Mate 30, available from Applied Separations) was used to vacuum wash and elute the column. Filling [ 2 ]¹⁴C]After tyramine, 6ml of 50mM KH was used₂PO₄(pH2.3) the column was washed with 2ml of 50mM KH₂PO₄(containing 30% acetonitrile). The eluate was freeze-dried to remove acetonitrile, resuspended in water and stored at-20 ℃.

Enzyme assay using radioactive method. Use of¹⁴C]Tyramine was used as a substrate and the product was isolated using a C18 column to test for enzyme activity. The assay was performed in a 200ml volume containing 100mM NaAc (pH5.2), 10mM fumaric acid, 0.5. mu.M CuSO ₄4mM ascorbic acid, 0.1mg/ml catalytic enzyme and tyramine at various concentrations. The total count for each reaction was-150,000 cpm. Bovine DBH (0.18 ng per reaction) was mixed with tyramine and inhibitors in reaction buffer at 37 ℃. The reaction was initiated by adding the ascorbate/catalase mixture and incubated at 37 ℃ for 30 minutes. By adding 100ml of 25mM EDTA, 50mM KH₂PO₄(pH2.3) to terminate the reaction. The entire mixture was loaded to a pre-wash with MeOH and 50mM KH₂PO₄(pH2.3) balanced C18 lightly loaded column (two combined into one). With 1ml KH₂PO₄(pH2.3) elution was performed twice with the same buffer, and then eluted into a scintillation vial with 2ml of the same buffer. ReadySafe scintillation fluid (16ml) was added to the scintillation vial and the samples counted¹⁴C, radioactivity.

Nepicastat concentrations of 0, 1, 2, 4, 8nM were used to study inhibition kinetics at the following tyramine concentrations: 0.5, 1, 2, 3, 4 mM. In each of the reactions carried out as described above,¹⁴the C count is the same. A blank without enzyme was used to obtain background. Data were corrected for background, converted to activity (nmol/min) and plotted (1/V vs 1/S). Km' is calculated from the slope and Y-intercept, and linear regression is used to obtain Ki values.

IC of SKF-102698, nepicastat and Compound B against human and bovine DBH₅₀Values were obtained using HPLC assays with substrate concentrations of 0.1mM tyramine, 4mM ascorbate, pH5.2 and 37 ℃. All three compounds lead to dose-dependent inhibition of DBH activity for both bovine and human enzymes.

IC calculated for nepicastat, Compound B and SKF-102698₅₀The values show that the S enantiomer (nepicastat) is more potent than the R enantiomer (compound B), 3 times more potent against bovine DBH and 2 times more potent against human enzymes. Nepicastat is more effective than SKF-102698, is 8 times more effective against bovine enzyme, and is 9 times more effective against human DBH.

Km was determined from the Lineweaver-Burk plot at 0.6 mM. Nepicastat (1-8nM) caused a large change in Km, as expected for competitive inhibitors. Nepicastat appears to compete with tyramine for inhibition of bovine DBH. Ki was calculated by linear regression at 4.7. + -. 0.4 nM.

Nepicastat is a potent inhibitor of DBH in both humans and cattle. It is 8-9 times more effective than SKF-102698. Nepicastat (S enantiomer) was 2-3 fold more potent than compound B (R enantiomer). Nepicastat appears to compete with tyramine for inhibition of bovine DBH with a Ki of 4.7 ± 0.4 nM.

Example 19

In the binding assay described using standard radioligands, the affinity of nepicastat was determined using the filter binding method.

Competition binding data were analyzed by iterative curves (fitted with a four parameter logistic equation). Direct acquisition of Hill coefficient and IC₅₀The value is obtained. Using Cheng-Prusoff equation, from IC₅₀Values calculate the pKi (log of Ki) of the competing ligands.

Nepirstat against alpha₁The receptor has a moderate affinity (pKi of 6.9-6.7). Its affinity for all other receptors tested was relatively low (pKi < 6.2).

Example 20

At the time of administration, a 60mg/ml nepicastat preparation was prepared by mixing the excipient with nepicastat powder and then shaking. Nepitastat formulations at 6 and 20mg/ml were prepared by diluting the 60mg/ml formulation with the excipient. The recombinant nepicastat formulation retained potency during use. The aqueous vehicle solution and the nepicastat formulation comprised hydroxypropyl methylcellulose, benzyl alcohol, and polysorbate 80.

The dose was selected based on acute toxicity studies in which mice were given a single oral dose of 250, 1000 or 2500mg/kg nepicastat. At 1000 and 2500mg/kg, clinical signs of toxicity and death occurred.

A single oral dose of vehicle or nepicastat formulation was administered by gavage to each mouse using a rodent introducer. The oral route was chosen because it is the recommended clinical route of administration. Dose volumes were calculated based on individual body weights recorded prior to dosing (body weight data not included in the report). 2.5 to 3.5 hours prior to dosing (not 1.5 hours specifically given in the protocol), mice were given no food and no water. Such deviations do not affect the completeness of the study.

Clinical observations were recorded prior to dosing. Mice in each treatment group (in up to 3 groups, approximately 10 minute intervals) were evaluated, beginning 60 minutes after dosing, each time for clinical observation and protocol-specific behavior trials. After administration, one mouse in the 30mg/kg group and one mouse in the 100mg/kg group died and they were removed from the study. Surviving mice were euthanized and at the end of the observation/testing period, they were removed from the study.

Mice in 6 male groups were given a single oral dose of nepicastat at 0 (vehicle), 30, 100 or 300mg/kg by gavage. Clinical observations and behavioral tests were initiated 60 minutes after administration of the test formulations. At the end of the observation period, all surviving mice were euthanized and removed from the study.

In the 30, 100 and 300mg/kg groups, a decrease in body temperature occurred, compared to the vehicle control group. No treatment-related clinical or macroscopic behavioral changes occurred. Rectal temperature data and observation and behavioral testing data were obtained. No treatment-related clinical or macroscopic behavioral changes occurred. Among the mice in the 100mg/kg group, an abnormal social group (listed as other reactions) appeared, but this phenomenon was not observed in the 300mg/kg group; this finding was considered to be incidental. Clinical/behavioral changes in 1 mouse in the 100mg/kg group included inactivity, gait and posture abnormalities, decreased induced activity, abnormal negativity, and soft/continuous vocalization; these changes were not the result of nepicastat. After dosing, one mouse died in each of the 30 and 100mg/kg groups; the death was considered to be incidental and the mice were removed from the study.

Example 21

The objective of this study was to determine whether the DBH inhibitors SKF-102698 and nepicastat can cause changes in locomotor activity or acoustic startle responsiveness. These behavioral changes may therefore reflect the activity of these compounds in the central nervous system.

Adult male Sprague Dawley rats (250-350 g on the day of the study) were obtained from Charles river Labs. The rats were placed in a normal light/dark cycle environment and were lit between 0900hrs and 2100 Hrs. Animals were placed in pairs in standard metal cages with unlimited use of food and water.

The motion activity box comprisesBox (size 18 "x 18" x12 "high). Around the surface of the steel pipeThe box is an Omnitech Digiscan monitor (model # RXCM 16) consisting of one inch of ban of a photon beam and 32 photosensors per box. The number of photon beam interruptions was analyzed by an Omnitech Digiscan analyzer (model # DCM-8). Animals were tested in a sealed room while a white (white) noise generator was run to shield against extraneous noise.

Sound startle reactivity tests were performed in eight SR-Lab (San Diego Instruments, San Diego, Calif.) automated testing stations. Rats were individually placed inCylinder (10cm diameter), the cylinder was placed in a ventilated sound-attenuating housing. Burst noise (wide range noise, rise time and decay time 1msec) was provided through a speaker mounted 30cm above the animal. Piezoelectric accelerometers convert target movement into random (arbitrary) voltages on a scale of 0 to 4095.

Each of the 72 rats was placed in a startle device prior to drug administration, and after 5 minutes of acclimation, they were provided with a sudden noise every 20 seconds for 15 minutes (45 startles total). The average startle for each rat was calculated by taking the average startle number from 11 to 45 (the first ten startles were negligible). 64 of these rats were then placed into one of eight treatment groups so that each group had a similar average startle value. Eight treatment groups were as follows: SKF-102698(100mg/kg) and its excipients (50% water/50% polyethylene glycol), clonidine (40. mu.g/kg), nepicastat (3, 10, 30 and 100mg/kg) and their excipients, dH ₂And O. The previous work has shown that it is possible to,this matching method is most suitable for startle, since there is significant variability in startle response between rats, but there is a high degree of consistency in rats over the course of one day.

Each day following the test procedure, eight rats (from each of the eight treatment groups) were injected with their assigned drug treatments and immediately placed individually in a sports activity box. The rats were monitored for 4 hours of motor activity. Next, the rats were placed in transfer cages for 15 minutes. At the beginning of this 15 minute period, rats for which clonidine treatment has been prescribed receive an additional injection of 40. mu.g/kg. Next, the rats were placed in a startle device and after 5 minutes of acclimation period, they were provided with a 90dB burst of noise every minute for 4 hours.

To evaluate the movement activity, the transverse activity (number of photon beam interruptions), the number of movements were determinedThe therapeutic effect of the retardation of the day. Pairwise comparisons of the treated groups with vehicle control groups were also performed using the Dunnett's t test.

To evaluate startle responsiveness, the average and maximum forces generated by each startle rat over the entire 200 milliseconds were measured within 200 milliseconds immediately after each successive startle. The mean maximum and mean voltage per treatment (TREAT) per Trial (TRIALN) were calculated (MAXMEAN and AVGMEAN) and these values were then plotted against the number of trials per treatment. The figures are attached in the report. Runs 1-60 were set to: time 1, trials 61-120 were set to: time 2, trial 121-: time 3, trial 181-. The average maximal and average startle responses over the respective time period and per treatment were calculated. This average is then used in statistical analysis. Startle responses were analyzed using analysis of co-variance. The investigators were interested in treatment contrast over time, rather than time effects within treatment. Therefore, startle response was analyzed by time. This model includes the day of the rat trial (date), baseline startle response and terms of treatment. Date is the chunking factor and baseline startle response is a covariate. For each of the above purposes, there are three independent models. Varying doses of nepicastat were compared to vehicle using Dunnett's method to control multiple comparisons.

When the 4 nepicastat-treated groups were compared to the vehicle-treated control group, there was no gross and pairwise significant difference in any of the 3 parameters at any of the test times.

The clonidine-treated group had significantly more lateral mobility at 2 and 2.5 hours, significantly more mobility at 2 hours, and significantly less resting time at 2 hours (all p < 0.05) when compared to vehicle-treated controls. Note that at 1 hour, the clonidine-treated group had significantly more resting time (p < 0.05) than the vehicle-treated control group.

The SKF-102698 treated group had significantly less lateral movement at 2.5 hours and significantly less movement at 2.5 hours (two p < 0.05) when compared to vehicle treated controls. Note that at 1.5 and 4 hours, SKF-102698 treated groups had significantly more movement than vehicle treated controls (two p < 0.05). There was no significant difference in resting time between SKF-102698 and the vehicle at any test time.

Typically, the amount of lateral movement and mobility decreases during the first 2 hours, and remains low during the last 2 hours. Similarly, the first 2 hours of rest time increased, and the last 2 hours remained increased.

Nepicastat had no significant effect on the locomotor activity of the rats. Animals treated with 3, 10, 30 or 100mg/kg nepicastat did not differ significantly from vehicle-treated controls at any of the test times in terms of lateral movement, number of movements or resting time.

In startle response, the overall therapeutic effect of nepicastat and vehicle was not significant for either response at any time (p > 0.05). At time 2, the overall therapeutic effect of the mean startle response was somewhat significant (p 0.0703) and the Dunnett's test showed that nepicastat 30mg/kg had a significantly higher mean startle response (p < 0.05) than the vehicle group. At times 3 and 4 of both responses, the baseline average response was statistically significant (p ≦ 0.05), and to some extent was significant (p ≦ 0.10) at times 1 and 2 of the maximum startle response and at time 2 of the average startle response.

SKF-102698(100mg/kg) was not statistically significantly different from the vehicle at any time during the startle response assay.

At time 1, clonidine statistically significantly reduced the maximal and average startle responses over vehicle, (p < 0.01), while at time 2, only the average startle was (p ═ 0.0352). The maximum startle response for time 2 and the average startle response for time 3 were somewhat significantly lower in the clonidine group than in the water group.

Administration of 3, 10, 30 or 100mg/kg nepicastat does not appear to affect the maximal or average startle response at any time in rats when compared to vehicle. For both startle responses, SKF-102698 behaved similarly to vehicle (PEG) at all times. Clonidine successfully reduced the maximal and average startle responses during the earlier time period, and during the later time period, it behaved similarly to the vehicle.

Example 22

The effect of long-term administration of nepicastat was tested in rats. Between 3 and 13 days prior to the first dosing day, rats were placed inside a startle device and after a 5 minute acclimation period, they were provided with 118dB of sudden noise, one minute on average (using a variable test interval in the middle of the 30 and 90 second range), for 20 minutes. Startle responses were measured and the average of the last 20 startle responses for each rat was calculated. Rats were randomly placed in one of eight experimental groups (nepicastat, 5, 15 or 50mg/kg,bid; SKF-102698, 50mg/kg, bid; clonidine, 20. mu.g/kg, bid: d-amphetamine, 2mg/kg, bid; dH₂O or cyclodextrin (excipient of SKF-102698)). Rats were given a 10ml/kg dose volume by oral gavage. Rats were dosed daily in the morning and in the evening for 10 days. The administration was performed at a time between morning and evening, between 6 and 10 hours. Previous work has shown that this matching method is best suited for acoustic startle responses, due to the significant variability in startle response among rats, but rats have a high degree of consistency over the day-by-day period.

Since it was not possible to test all 96 rats on the same day (8 test groups, n-12), the dosing schedule was staggered and only 8 rats were operated per day. Of these 12 groups (eight rats per group), each group of rats included one rat from each of the eight treatment groups, such that the treatment groups were balanced throughout the day. Furthermore, all treatment groups could be balanced in eight sports activity cabins, however, treatment groups could not be balanced in startle cabins.

During and after the long-term administration, the following behavioral tests were performed: body internal temperature, athletic activity, acoustic startle responsiveness, and pre-pulse suppression of acoustic startle.

Animals were tested in a sealed room while running a white noise generator. The locomotor activity test was performed immediately after taking an internal body temperature reading on day 10 of administration (approximately 3 hours 35 minutes after the 10 th day of administration, the daily dose of nepicastat and SKF-102698, 20 minutes before the daily administration of clonidine and d-amphetamine). The exercise activity test was performed for one hour. Before each test phase, a diagnostic procedure is performed on each locomotion pod to ensure that the light beam and the light sensitive element are functioning properly. It has been shown that motor activity is sensitive to changes in central dopamine levels (diet and Kuschinsky, 1994), making this behavioural test a potential sensitivity test for the effects of DBH inhibitors in vivo. D-amphetamine was used as a positive control for this assay.

By probing the rectumThe needle was inserted 2cm into the colon of each rat to obtain the internal body temperature of the rat. The internal body temperature of each rat was measured three times, and the average of the three readings was calculated. Internal body temperature readings were obtained immediately prior to ten days of chronic dosing (to obtain baseline), three and a half hours after the morning daily dose of nepicastat and SKF-102698 on days 1, 5, and 10 of dosing, and 15 minutes prior to daily administration of clonidine and d-amphetamine. It has been demonstrated that body internal temperature is sensitive to dopamine and norepinephrine levels, making this behavioral test a potential sensitivity test for the effects of DBH inhibitors in vivo. Clonidine (alpha)₂Agonist) and d-amphetamine (dopamine releaser) were used as positive controls for this assay.

In eight SR-Lab (San Diego Instruments, San Diego, CA) test stations, acoustic startle responsiveness (a series of muscle contractions with rapid onset caused by intense burst disturbances) and pre-pulse inhibition (sensorimotor gating was determined by analyzing any decrease in startle responsiveness (occurring when startle stimulation was performed immediately prior to non-startle stimulation)). Rats were individually placed in Plexiglas cylinders (10cm diameter) which were placed in a ventilated sound-attenuating housing. Burst noise (wide range noise, rise time and decay time 1msec) was provided through a speaker mounted 30cm above the animal. Furthermore, these speakers produced background noise at a 68dB level during all experimental phases. A piezoelectric accelerometer attached under the plexiglas cylinder converts the target movement into a voltage, which is then modified and digitized (on a scale of 0 to 4095) by a PC computer (equipped with SR-Lab software and interface assembly). In each of the eight test stations, a decibel meter was used to calibrate the speakers to ± 1% of the mean. In addition, the SR-Lab calibration meter was used to calibrate each of the eight startle detection meters to. + -. 2% of the mean. Startle responsiveness and pre-pulse suppression tests were performed simultaneously immediately after the exercise activity test (approximately 4 hours 40 minutes after the daily morning injection of nepicastat and SKF-102698 on day 10 of dosing, and 10 minutes after the adjunctive administration of clonidine and d-amphetamine). Startle responsiveness and pre-pulse inhibition tests included: each rat was placed individually in the SR-Lab testing station and after a five minute acclimation period, the rats were provided with one of three different types of burst noise (startle response determination), one minute on average (using variable test intervals ranging between 30 and 90 seconds), and one hour (60 total burst noise and startle response). Three different types of burst noise include: loud burst noise (118dB) and relatively mild burst noise (77dB), providing mild burst noise 100msec (pre-pulse suppression test) before loud burst noise. These experiments are provided in a pseudo-random order. Prepulse inhibition has been shown to be sensitive to changes in the level of mesolimbic dopamine. Furthermore, it has also been demonstrated that acoustic startle responsiveness is sensitive to changes in dopamine and norepinephrine levels, making these behavioral tests a potentially sensitive test for the effects of DBH inhibitors in vivo. Clonidine and d amphetamine serve as positive controls for acoustic startle responsiveness and pre-pulse inhibition of the acoustic startle test.

The daily behavioral test is planned as follows. When t is 0, a DBH inhibitor is injected. At 3.5 hours, the internal body temperature was measured. At 3 hours and 35 minutes, locomotor activity was evaluated. Startle responsiveness and pre-pulse inhibition were evaluated at 4 hours and 40 minutes.

Three temperature readings were taken from each target at each time of the experiment. The average of these three readings is then calculated.

The spontaneous motility of each rat was obtained by counting the total number of photon beams interrupted by the target during the experimental phase.

After providing the stimuli, the response of the target during 40msec of each experiment was determined. Each startle response was calculated by taking the average of 40 readings (one per millisecond) starting immediately after each sudden noise burst. Acoustic startle responsiveness was calculated by measuring the average response of each target startle caused by 118dB burst sounds. The pre-pulse inhibition values were calculated as follows: for each rat, the average startle response elicited by the 77dB pulse-118 dB pulse paired test (the pre-pulse suppression test described above) was subtracted from the average startle response elicited by the 118dB individual test and then divided by the 118dB individual test value, i.e.: ([118dB test value-pre-pulse inhibition test value ] ÷ 118dB test value).

For the primary effect of treatment on baseline changes per animal at each time, a total one-way ANOVA was performed. Subsequently, for each comparison in the study, a t-test was performed.

Spontaneous locomotor activity was measured every 15 minutes for each animal for 1 hour. Each time group was analyzed separately (every 15 minutes). Kruskal-Wallis test (non-parametric technique) was performed to test the differences between treatment groups. If no overall significant difference was determined, Bonferroni's correction for multiple comparisons was then performed.

In each Trial (TRIALN), the mean voltage (AVGMEAN) and the mean percent pre-pulse inhibition (RATIO) were calculated for each treatment (TREAT) and trial type (TRIALT). The pre-pulse inhibition values were calculated as follows: for each rat, the average startle response elicited by the 77dB pulse-118 dB pulse paired test (the pre-pulse suppression test described above) was subtracted from the average startle response elicited by the 118dB individual test and then divided by the 118dB individual test value, i.e.: ([118dB test value-pre-pulse inhibition test value ] ÷ 118dB test value).

For each treatment and trial type, the values were plotted against the trial number, which is attached to the report. Note the y-axis variation on the graph. Runs 1-15 correspond to time 1, runs 16-30 correspond to time 2, runs 31-45 correspond to time 3, and runs 46-60 correspond to time 4. Also attached are graphs showing the average percent pre-pulse inhibition for each treatment and the average startle of the animals over time.

Mean startle response and percent pre-pulse inhibition were analyzed using analysis of variance. The model includes the terms treatment, animal within treatment, time and interaction of treatment with time. The treatment effect was tested using error terms for animals under treatment. The overall therapeutic effect and the effect of the treatment over time were studied. Fisher's least significant difference method was used for the adjustment of multiple comparisons. If the overall therapeutic effect or the effect of the treatment over time is not significant (p-value > 0.05), a Bonferroni correction is performed. If the overall therapeutic effect is not significant, then the correction is used for a specific pair-by-pair comparison. Further, if the specific pair-wise therapeutic effect is not significant (p-value > 0.05), then within the time frame, a correction is also applied to the therapeutic effect. If the overall therapeutic effect and the effect of the treatment over time are not significant (p-value > 0.05), Bonferroni correction is performed for the independent comparisons over time and averaged over time.

For each animal analyzed, the change in body weight from the previous dose was calculated. Two-way ANOVA with repeated measures was used to test the overall effect of treatment, time and treatment-time interaction. One-way ANOVAS was then performed to test the daily treatment effect.

On the first day of chronic dosing, the positive controls (d-amphetamine and clonidine) significantly increased the body internal temperature, but the other compounds did not have any significant effect on the body internal temperature at any time.

The d-amphetamine group had significantly higher locomotor activity than the vehicle control group at all test times. However, at any test time, the clonidine group did not differ significantly from the vehicle control group. Compared to vehicle control group, SKF-102698(50mg/kg, b.i.d.) group significantly reduced locomotor activity in the first 45 min (i.e. samples 1-3), but not significantly after 45 min.

At any test time, nepicastat had no overall significant therapeutic effect. The comparison shows that none of the nepicastat-treated groups differed significantly from the vehicle control group at any of the test times. In addition, at any test time, in both vehicle control groups (dH)₂Excipients for O and SKF) were not significantly different.

None of the treatment groups produced any significant change in pre-pulse inhibition. For SKF-102698 group and RingThe overall time effect was statistically significant for the dextrin group (p ═ 0.0001). For cyclodextrins and dH ₂O, the interaction of treatment with time was statistically significant (p ═ 0.0283), but others did not. The therapeutic effect was not significant for any comparison in the study. However, the SKF group had a somewhat higher percentage of pre-pulse inhibition (p-0.0782) than the cyclodextrin group.

During times 1 and 2, the clonidine group had a significantly higher percentage of pre-pulse inhibition compared to the vehicle control group, but during times 3 and 4 it was not significantly different from the vehicle. At any one time, neither d-amphetamine nor SKF-102698 are significantly different from their own excipients. At any time, none of the nepicastat dose groups differed significantly from dH₂O。

Only the SKF-102698 treated group caused a significant change in acoustic startle responsiveness. The overall temporal effect was statistically significant for all comparisons in the study (all p ═ 0.0001). Mixing amphetamine with dH₂O, clonidine and dH₂O and Cyclodextrin with dH₂In comparison, the interaction of treatment with time was statistically significant (all p < 0.05), but not others. The therapeutic effect of SKF-102698(50mg/kg, b.i.d.) was significant relative to cyclodextrin (p 0.0007) and nepicastat (50mg/kg, b.i.d.) relative to SKF-102698(50mg/kg, b.i.d.) (p 0.0047), but not others. The SKF-102698(50mg/kg, b.i.d.) group significantly reduced startle response compared to cyclodextrin, and also significantly reduced startle response compared to nepicastat (50mg/kg, b.i.d.) group.

At all times, the SKF-102698(50mg/kg b.i.d.) group significantly reduced startle response compared to the cyclodextrin group. The nepicastat (50mg/kg, b.i.d.) group significantly improved startle response compared to the SKF-102698(50mg/kg, b.i.d.) group during times 1 and 3. No other significant differences were measured.

There was no gross or pairwise significant difference in body weight between groups at the pre-dose baseline.

The d-amphetamine group had significantly less change in body weight from the previous dose (p < 0.01) compared to the vehicle control group. When analyzed daily, the vehicle control group gained significantly more body weight (from the previous dose) than the amphetamine group on days 4-10 of treatment. However, at any test time, the clonidine group did not differ significantly from the vehicle control group. The group of SKF-102698(50mg/kg, b.i.d.) showed significantly less increase in body weight (p < 0.01) from the pre-dose baseline compared to the vehicle control (SKF-vehicle). When analyzed daily, the SKF-vehicle control significantly increased body weight compared to the SKF-102698 group starting from the previous dose on days 2-10 of treatment (except days 3 and 6). Importantly, there was no difference in body weight change between SKF-vehicle and vehicle control groups on any day.

At any test time, any dose of nepicastat had no overall significant therapeutic effect on body weight. The comparison shows that none of the nepicastat-treated groups differed significantly from the vehicle control group at any of the test times. Interestingly, there was a significant (p < 0.05) overall difference in weight change between the SKF-102698(50mg/kg, b.i.d.) and nepicastat (50mg/kg, b.i.d.) groups. When analyzed daily, on days 7-9, SKF-102698(50mg/kg, b.i.d.) group significantly reduced body weight compared to nepicastat (50mg/kg, b.i.d.) group.

Example 23

1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) is purchased from RBI, Inc, (Natick, Mass.). For administration, MPTP was suspended in water at a concentration of 2mg/ml (free base) and injected subcutaneously in a volume equal to the weight (kg) of each animal. For example, 950 gram animals receive an injection of 0.95ml MPTP (2mg/ml), resulting in a final injection of 2.0mg/kg per injection.

The monkeys were kept in a 13h/11h light-dark cycle and food and water were available ad libitum. All methods used in this study were as instructed by NIH and approved by the Institutional Animal Care and Use Committee (IACUC). Animals were placed individually and allowed to acclimate to the population for a minimum of one month prior to starting the behavioral study.

Six squirrel monkeys (three non-lesioned, three lesioned) (receiving 2mg/kgMPTP 3 months earlier) were used to study the optimal route of administration of nepicastat. Three different approaches were examined, including: (i) embedded therapy, (ii) oral injection and (ii) oral feeding. Testing solutions of nepicastat embedded in hollyhock sugar (5mg/ml) in 3 non-diseased monkeys proved to be a poor route of administration, as animals were not willing to swallow due to adverse taste. Oral injection of nepicastat injections (0.5, 2 and 5mg/kg) into the mouths of three non-lesioned and three lesioned monkeys is also an unacceptable route, as animals tend to spit out the solution at the highest drug concentration. Oral feeding was performed at the highest dose (5mg/kg) in 3 MPTP-affected monkeys, and administration was well received.

Six squirrel monkeys (three non-lesioned, three lesioned) (received 2mg/kgMPTP 3 months earlier) were used to study the safety and tolerability of nepicastat. Animals received nepicastat at a concentration of 0.5, 2.0, or 5.0mg/kg twice daily (10am and 2pm), for 5 days with two-day clearance between different dose levels. Nepicastat (0.5, 2.0 and 5.0mg/kg doses) was administered by oral syringe, and when fed orally, the dose was 5.0 mg/kg. At the two lower doses, the drug was better tolerated. On the last two days of administration, a non-diseased monkey receiving 5.0mg/kg developed a light beige thin stool and administration was stopped for one day, which was eliminated.

24 squirrel monkeys (14 females, 10 males) were used in the Parkinsonian model. Twenty-four animals were randomly assigned to one of four treatment groups of 6 animals each. Each group includes the following: group a (6 animals), receiving placebo (water) treatment; group B (5 animals) receiving the drug nepicastat at 1 mg/kg/day (0.5mg/kg, twice daily); group C (6 animals) received 4 mg/kg/day (2mg/kg, twice daily); and group D (6 animals), receiving 10 mg/kg/day (5mg/kg, twice daily). In group B, one animal died acutely after MPTP-induced dysfunction without replacement.

Animals were subjected to quantitative assessment of spontaneous locomotor activity prior to lesions using infrared activity monitoring (IRAM) cages. All recording phases were 60 minutes, with 10 phases performed over a 2 week period. Animals were evaluated for behavior once per day (12 pm to 1pm at noon) and 3 to 5 consecutive days by 1 to 3 clinical evaluators using the parkinson's disease-like Clinical Rating Scale (CRS). Normal animals generally score no more than 3 on CRS. Activity Monitoring (IRAM) and clinical rating assessment established an average baseline activity per animal.

Animals were lesioned (by subcutaneous injection) to achieve the state of Parkinson's disease by administering MPTP (free base) at a concentration of 2.0 mg/kg. Finally, behavioral assessment after MPTP-lesions was performed 2 to 4 weeks after MPTP-lesions. Locomotor activity was monitored over a 60 minute period by IRAM for 3 to 5 days. Clinical behavior (CAS) was evaluated by one to three independent ratings over a period of 3 to 5 days.

In some cases, the animals required an additional dose of MPTP (2mg/kg) in order to obtain a sufficient degree of pathology showing symptoms of Parkinson's disease (defined as an average overall clinical rating score greater than 3). Within three weeks of initiating the effect study, all animals received a final post-MPTP behavioral assessment (IRAM and CRS). This final post-MPTP evaluation was used to establish the baseline clinical parkinson's disease status and as a pre-treatment value for statistical analysis.

Animals were tested for their response to L-Dopa and for their effect on nepicastat drug. After the final MPTP dose, the experiment was performed for 4 to 12 weeks. L-Dopa (at concentrations of 2.5, 5 or 7.5mg/kg) was administered by oral feeding twice daily (at 10am and 2pm) for 7 consecutive days. Behavior was measured by IRAM and CRS. Clinical ratings of 60 to 90 minutes were performed after the 10am morning dose in the last 4 days of treatment. Evaluators (one to three persons) were not known for the different treatment groups. In the last 2 to 5 days of drug treatment, a 90 minute IRAM assessment was performed immediately after 2pm drug administration. Between each treatment dose, there was a minimum of 2 days of clearance cycles.

Following a minimum of 2 days of clearance after administration of L-Dopa, nepicastat or water (as placebo) was administered for 12 days. Drugs were administered twice daily by oral feeding at 10am and 2 pm. Behavior was assessed by IRAM and CRS. CRS was performed in the morning 60 to 90 minutes after the dose of 10am nepicastat on the last 5 days of drug treatment. Evaluators (one to three persons) were not known for the different treatment groups. In the last 5 days of drug treatment, a 90 minute IRAM assessment was performed immediately after 2pm drug administration.

For statistical analysis, athletic activity and clinical rating scores were monitored. For each animal, mean motor activity was calculated before and after MPTP lesions. Baseline pre-MPTP lesions were determined by averaging 10 1 hour monitoring sessions. Behavioral assessments were obtained after MPTP (pretreatment) within three weeks of initiating the effect study. Locomotor activity following MPTP lesions (IRAMS) was determined by averaging 3 to 5 1 hour monitoring sessions. Activity monitoring is reported as "movement/10 minutes". High scores are considered to be faster animals. For each group of animals (group a to group D), a Wilcoxon symbolic ranking test was used to compare activity before and after MPTP lesions.

After each drug level, IRAM motor activity was monitored every 10 minutes for at least 90 minutes. Higher ratings were considered faster animals (less parkinson's disease).

The statistical analysis comprises: statistics are described and mean values of data blocks per 10 minutes for placebo versus 1, 4 and 10mg/kg experimental drug are plotted. The curve was then examined to determine any trends. Since no differences were determined from the graphical analysis, no further statistical analysis was performed.

Due to inadequate IRAM data collection, statistical analyses comparing post-MPTP lesions (pretreatment) to 2.5, 5.0 and 7.5mg/kg L-Dopa and nepicastat (1, 4, 10 mg/kg/day or placebo) were not performed. Only a 60 min period was collected after MPTP relative to nepicastat (90 min).

In the clinical rating score assay, animals pre-MPTP lesions scored no more than 3 on CRS. Clinical grade scores after MPTP were determined within three weeks of the start of the efficacy study by averaging the total CRS of 1 to 3 independent evaluators (using data from 3 to 5 consecutive days).

In each animal, eight parkinson's disease features were assessed, and the total score was derived from the sum of these eight features. For each animal, a single clinical rating score for each drug dose was provided by averaging the clinical rating scores performed by all evaluators (one to three) over consecutive multiple dosing (same dose) days. This average CRS was used for statistical analysis. A low score is considered a parkinson's disease-free behavior state.

The statistical analysis comprises: (1) comparisons were made between placebo and the mean CRS of 1, 4 and 10 mg/kg/day nepicastat (using Kruskal-Wallis (non-parametric analysis of variance)). This comparison was repeated using the average CRS for each experimental drug concentration (corrected by final MPTP post-rating for each animal). The corrected clinical record is the ratio of the clinical record of the experimental drug at each concentration as the post-MPTP clinical record. (2) Using Friedman's analysis (non-parametric ANOVA, repeated measures), pairwise comparisons were made between post-MPTP lesion (pretreatment) and 2.5, 5.0 and 7.5mg/kgL-Dopa and placebo treatment. The same analysis was performed for nepicastat at concentrations of 1, 4 and 10 mg/kg. If necessary, Dunnett's post hoc analysis (for nonparametric data) was performed.

In each squirrel monkey, IRAM (activity monitoring) and CRS (clinical rating scale) were used to assess the extent of MPTP lesions.

There was no significant difference between the pre-and post-lesion IRAM groups due to group a: placebo treatment, high variability of movement per animal every 10 minutes. Wilcoxon signed rank (rank) test: w19, N6, P < 0.06, accepting a null hypothesis. The average CRS for group a was 8.9, ranging from 4.8 to 15.4. All animals showed significant improvement in clinical rating scores following MPTP lesions. Normal animals (non-diseased) generally have a score of less than 3.

There was no significant difference between the pre-and post-lesion IRAM groups due to the high variability of movement per 10 min for each animal in group B (1 mg/kg/day treatment group). Wilcoxon signed rank test: w9, N5, P < 0.06, and accepted a zero hypothesis Clinical Rating Score (CRS). The average CRS for group B was 10.32, ranging from 4.3 to 16.1. All animals showed significant improvement in clinical rating scores following MPTP lesions. Normal animals (non-diseased) generally have a score of less than 3.

There was no significant difference between the pre-and post-lesion IRAM groups due to the high variability of movement per 10 min for each animal in group C (4 mg/kg/day treatment group). Wilcoxon signed rank test: w17, N6, P > 0.06, accepting a null hypothesis. The average CRS for group C was 8.97, ranging from 6.5 to 17.3. All animals showed significant improvement in clinical rating scores following MPTP lesions. Normal animals (non-diseased) generally have a score of less than 3.

There was no significant difference between the pre-and post-lesion IRAM groups due to the high variability of movement per 10 minutes per animal in group D (10 mg/kg/day treatment group). Wilcoxon signed rank test: w is 21, N is 6, P > 0.06, and the null hypothesis is accepted. All animals showed significant improvement in clinical rating scores following MPTP lesions. The average CRS for group C was 8.02, ranging from 4.0 to 15.6. Normal animals (non-diseased) generally have a score of less than 3.

Due to the high variability of RAM results for each animal, there was no significant difference in overall in locomotor activity as determined by IRAM (within each group, as determined between baseline (pre-MPTP lesion) and post-MPTP lesion). CRS results show the difference between pre-MPTP and post-MPTP lesion status. The animal score before MPTP lesion was no greater than 3 in CRS. All animals post MPTP lesion had scores greater than 3. All groups (a-D groups) have an average CRS of 8 to 10, for (out of) a total possible CRS score of 24.

In MPTP-lesioned squirrel monkeys, there were no detectable differences between placebo treatment and three different concentrations of nepicastat (1, 4, 10 mg/kg/day). 4 and 10 mg/kg/day nepicastat with placebo showed significant improvement in post-MPTP (pre-treatment) status. All animal groups showed significant improvement for 5mg/kg and 7.6mg/kg L-Dopa, except for the 7.5mg/kg dose group C and the 5mg/kg dose group B, when compared to post MPTP (pretreatment). No animal groups showed significant improvement under 2.5mg/kg L-Dopa when compared to post MPTP.

At time points 10 to 90 minutes after dosing, comparisons of treatment groups and L-DOPA, Friedman test results, descriptive statistics and Dunnett's test post hoc analysis were made, and comparisons were made between the activity monitoring of placebo treatment and the activity monitoring of all other nepicastat concentrations. For each drug dose level, 10 minute intervals were plotted. There was no difference in the treatment with the drug (nepicastat) at the 4 and 10 mg/kg/day dose levels when compared to placebo. At 1 mg/kg/day, the animals were moving more slowly compared to placebo treatment. In a non-human primate model of MPTP lesions in PD, nepicastat did not produce significant effects on parkinson symptoms compared to placebo (water treatment) based on unpaired comparative analysis of 4 different treatment groups (1, 4 and 10mg/kg nepicastat and placebo). Nepicastat at concentrations of 4 and 10 mg/kg/day showed significant effects on parkinson's disease symptoms compared to post MPTP lesion (pre-treatment evaluation) based on pairwise comparative analysis of animals (same group of animals tested pre-and post-treatment). Placebo had an undefined significant effect. Using the same pairwise comparison, in all groups (except animals in group B (5mg/kg L-Dopa had no effect) and group C (7.5mg/kg L-Dopa had no effect), 5 and 7.5mg/kg L-Dopa showed significant effects when compared to the post-MPTP lesion state. However, 2.5mg/kg L-Dopa showed no significant effect.

Pharmacokinetic studies were performed in squirrel monkeys in order to determine the plasma concentration of nepicastat. This study was performed simultaneously with safety and tolerability studies. Three MPTP-lesioned squirrel monkeys (#353, 358, and 374) were used. One milliliter of blood (removed from the femoral vein of each animal) was collected for analysis. Nepicastat was administered at concentrations of 1, 4 and 10mg/kg for 5 days with 2 days of clearance between each drug concentration. Blood was collected for analysis 1 hour before the first dose to establish baseline, and 6 hours after the first drug dose at each different drug level.

A second pharmacokinetic study was performed to determine steady state plasma levels of nepicastat. This study was performed concomitantly with an efficacy study in which animals were tested at each of three different drug concentrations for 12 days. One milliliter of blood was removed from the femoral vein 6 hours after the first dose on day 1, then 6 hours after the first dose on day 7, and finally 6 hours after the first dose on day 12. One week prior to drug administration, baseline plasma levels of the collected samples were determined.

The study also shows that when a small number of animals are used, the paired analysis (by comparing the same animals before and after treatment, reducing animal-to-animal variability) is best suited for determining significant efficacy compared to a non-paired study design.

Example 24

Male spontaneously hypertensive rats (280-345 g; Charles River Labs, Kingston, NY) were fasted overnight and then anesthetized with ether. The femoral artery and vein were cannulated with PE50 for recording blood pressure and administration of compounds, respectively. The animals were then placed in the MAYO restrainer and their feet were loosely tied to the restrainer. Heparinized saline (50 units heparin sodium/ml) was used to keep each tube unclosed throughout the experiment. Using Modular Instruments MI²BioReport^TMThe software (installed on an IBM personal computer) continuously records the following parameters: mean Arterial Pressure (MAP), Heart Rate (HR) and baseline changes for each parameter at specific time points in the experiment.

On the day of use, all compounds were dissolved. Nepicastat was dissolved in deionized water (vehicle) to give a free base concentration of 1 mg/ml. The oral dosage volume of nepicastat or vehicle was 10 ml/kg. SCH-23390 was dissolved in saline (vehicle) to give a free base concentration of 0.2 mg/ml. Nepicastat or saline was administered intravenously in a bolus form at a volume of 1.0ml/kg, followed directly by 0.2ml of isotonic saline.

After surgical preparation, each animal was allowed to recover for a minimum of one hour. Animals were randomly assigned to four treatment groups: excipient (iv)/excipient (po); excipient (iv)/nepicastat (po); SCH-23390 (iv)/excipient (po); or SCH-23390 (iv)/nepicastat (po). Once the animals were stable (one hour minimum), baseline blood pressure and heart rate were determined by averaging each parameter over a 15 minute period. Once baseline blood pressure and heart rate were determined, animals were administered SCH-23390 (200. mu.g/kg) or vehicle (saline, 1ml/kg) intravenously. After 15 minutes, nepicastat (10mg/kg) or vehicle (deionized water, 10ml/kg) was orally administered to the animals.

The recorded parameters were measured 15 minutes prior to intravenous administration in order to establish baseline blood pressure and heart rate. The recorded parameters were then determined 5, 10 and 15 minutes after intravenous administration of either SCH-23390 or vehicle. After oral administration of nepicastat or vehicle, the recorded parameters were determined at 15, 30, 60, 90, 120, 150, 180, 210 and 240 minutes.

At the end of the experiment, each animal was anesthetized with halothane and euthanized by decapitation. The cortex, left ventricle (apex) and mesenteric artery were dissected out, weighed and placed in 0.4M perchloric acid. The tissue was then frozen in liquid nitrogen and stored at-70 ℃. At a later stage, these tissues were subjected to biochemical analysis to determine catecholamine levels, including dopamine and norepinephrine levels. Blood pressure and heart rate were analyzed separately. Baseline changes in blood pressure and heart rate were analyzed by analysis of variance (ANOVA), along with treatment effects, time, and their interactions. This analysis was performed after the iv time period and after the oral time period. The main effect on time at each time was further analyzed by ANOVA. When the overall therapeutic effect was not significant, a comparative comparison was performed after each ANOVA using the Fisher's LSD strategy (with Bonferroni correction).

Additional analysis was performed by ANOVA for the primary effect of treatment to compare baseline averages for each treatment group, followed by comparison-by-comparison. The following comparisons were made: SCH-23390 (iv)/excipient (po) with excipient (iv)/excipient (po), excipient (iv)/nepicastat (po) with excipient (iv)/excipient (po), and SCH-23390 (iv)/nepicastat (po) with excipient (iv)/nepicastat (po).

There was no significant difference in baseline heart rate or mean arterial pressure between treatment groups.

Intravenous treatment with SCH-23390 resulted in a significant reduction in heart rate (p < 0.05) compared to vehicle controls at 120 and 240 minutes after oral administration. Nepicastat did not reduce heart rate as much as was observed in vehicle treated animals. This was statistically significant (p < 0.05) at 150 and 180 minutes post-dose. It should be noted that over the time of the experiment, a large change in heart rate was observed.

Intravenous administration of SCH-23390 still resulted in a small (5 + -1 mmHg) significant decrease in mean arterial pressure (p < 0.05) during the 15 min period following the iv cycle, compared to the vehicle-receiving animals. Oral treatment with nepicastat resulted in a significant decrease in mean arterial pressure (p < 0.05) 30 minutes after dosing, which continued to persist for the duration of the experiment. Pretreatment with SCH-23390 did not significantly reduce the antihypertensive effect, which was observed with nepicastat alone.

Example 25

Male Crl 15 weeks old was used: COBS (WI) BR rats. 24 rats were chronically implanted with a telemetric implant (TA11PA-C40, Data Sciences, Inc., St. Paul, MN) for measurement of arterial blood pressure, heart rate and locomotor activity. The rats were anesthetized with sodium pentobarbital (60mg/kg, ip) and their abdomens were trimmed. Under sterile conditions, incisions were made on the midline. The abdominal aorta is exposed and the catheter of the telemetry transmitter is inserted. After the transmitter was attached to the abdominal musculature, the skin was sutured closed. Each rat was allowed to recover for at least 2 weeks prior to drug administration. Three days before the start of the experiment, rats were randomly divided into 4 treatment groups: excipient (p.o.), hydralazine (10mg/kg, p.o.), nepicastat (30mg/kg, p.o.), nepicastat (100mg/kg, p.o.).

Systolic Blood Pressure (SBP), Diastolic Blood Pressure (DBP), Mean Blood Pressure (MBP), Heart Rate (HR) and locomotor activity (MA) were monitored. Nepicastat and hydralazine were prepared in water with traces of Tween 80. All doses of 10ml/kg were given orally to rats and are expressed as free base equivalents.

A computerized data collection system is used to continuously collect SBP, DBP, MBP, HR, and MA data. Data was collected for each rat every 5 minutes for 10 sec. These data were then averaged hourly and the standard mean error (SEM) was calculated. All values are expressed as mean ± SEM. Statistical significance was defined as P levels less than 0.05. Data on MBP, HR and MA were analyzed separately. Each analysis was performed at 26 time points measured daily. Two-way ANOVA was used for the main effects and time of treatment and their interactions. If the overall therapeutic effect or significant interaction is determined, a series of one-way ANOVA is performed at each time point. Using the Dunn's method, a phase-by-phase comparison was performed at each time point. If no overall therapeutic effect is detected, analysis of pairwise differences from controls can be performed by adjusting the cut-off values using the Bonferroni correction.

After establishing the pre-dose values of these parameters, the corresponding groups of rats received daily treatment with vehicle, nepicastat or hydralazine for 7 days.

Oral nepicastat (30mg/kg) (all doses indicated below are po doses) tended to slowly lower blood pressure, but did not elicit a stable hypotensive effect on day 1. As the effect progressed, on day 2, at 13 hours, a peak hypotensive effect of-10 mmHg was observed. A similar degree of antihypertensive effect was elicited throughout the study. At 100mg/kg, the compound elicited a peak antihypertensive response of-11 mmHg (p < 0.01) 22 hours after dosing on day 1. MBP continued to decrease, reaching its lowest point of about-17 mmHg on day 3 (p < 0.01). MBP remained reduced throughout the study.

10mg/kg hydralazine resulted in a rapid hypotensive effect that was attenuated at 10 hours and a maximum decrease of-24 mmHg (p < 0.01) in MBP was observed within 1 hour after the administration on day 1. Throughout the study, a similar temporary hypotensive effect was observed.

On day 1, 30 and 100mg/kg nepicastat did not continuously affect HR. However, on day 2, 3 hours after dosing, 100mg/kg nepicastat resulted in a slowed heart rate response of-100 b/mm. On days 3-7, a meaningful but less pronounced response slowing down the heart rate was observed. In the course of the comparison, 10mg/kg hydralazine caused varying degrees of tachycardia throughout the study.

None of the drug treatments showed consistent effects on MA throughout the study.

Body weight was recorded daily. For body weight, two-way ANOVA with respect to change before dosing was used to analyze treatment overall effect, day, and treatment-day interaction. One-way ANOVA was then performed daily and pairwise comparisons of drug treated groups with vehicle controls were performed using Dunn's method and Fisher's LSD strategy to correct multiple comparisons. None of the drug treatments had any effect on body weight (p < 0.05) compared to vehicle treatment. Although treatment with nepicastat at 100mg/kg caused weight loss on day 3, it was not statistically significant.

Example 26

Nepicastat can reduce the conversion of dopamine to norepinephrine. A basic assay for nepicastat activity can determine plasma or urodopa levels or dopamine to norepinephrine ratios. Nepicastat treatment can increase the plasma or urine levels of dopamine or increase the dopamine/norepinephrine ratio in the plasma or urine.

The urodoparn levels in normal volunteers after 24 hours of treatment with nepicastat are shown in figure 5.

Using a repeated measures analysis of variance model, a significant increase in the mean supine position (pinnate) plasma dopamine/norepinephrine ratio (p < 0.05) was measured in subjects receiving 200mg nepicastat when compared to those receiving placebo. Urodoparn levels increased 10 days after administration of 40mg and 200mg nepicastat.

Example 27

Daily doses of 40, 80 and 120mg nepicastat given over 10 days are generally better tolerated in Chronic Heart Failure (CHF) patients. The dose with increased frequency causing significant adverse conditions was 160 mg.

Of the 8 patients treated with 160mg for 8 days or longer, four developed rashes. Two of the rash patients were accompanied by itching. One patient also had tachypnea with a rash.

One patient treated with the 80mg dose dropped out of the study because symptoms of orthostatic hypotension appeared. Concomitant medications include hydralazine and three diuretics. In patients using all doses (including placebo), orthostatic hypotension was occasionally reported. Of the 8 patients who received 160mg, 6 of them developed symptoms of orthostatic hypotension.

In an ongoing study, there have been 2 deaths in patients with CHF: one was death due to CHF worsening (patients received 80mg, qd) and one patient who received placebo suddenly died.

One serious adverse condition reported to be potentially drug related is elevated creatinine levels, which requires hospitalization. Medically important conditions not considered relevant for the study of drugs include: CHF exacerbations (one of which followed by acute MI and cardiac arrest) occurred in 2 patients, unstable angina in 1 patient, atypical chest pain in 1 patient, and renal mass in 1 patient with a history of breast cancer.

In a study of congestive heart failure patients, changes in plasma dopamine levels, norepinephrine levels and dopamine/norepinephrine ratio were determined after 10 days of treatment with nepicastat. Nepicastat treatment increased dopamine/norepinephrine levels in the 10-day study.

In a study of congestive heart failure patients, plasma dopamine levels, norepinephrine levels and dopamine/norepinephrine ratios and changes in such levels and ratios were determined after 30 days of treatment with nepicastat. Nepicastat treatment increased dopamine/norepinephrine levels in the 30-day study.

Example 28

The dopamine/norepinephrine ratio in the brain of rodents treated with nepicastat was determined. In rodent brains treated with nepicastat or disulfiram, the dopamine/norepinephrine ratio is increased.

It will be apparent to those of ordinary skill in the relevant art that other suitable improvements and modifications to the methods and applications described herein are suitable and may be made without departing from the scope of the invention or any embodiment thereof. While the invention has been described in connection with certain embodiments, it is intended to cover alternatives, modifications, and equivalents as may be included within the spirit and scope of the invention as defined by the following claims.

Example 29

In rats, a delayed position matching (DMTP) assay was used to examine the potential effect of drugs on short-term or working memory.

In the delayed position matching study, prior to starting the test nepicastat, a pilot study was conducted in order to evaluate the behavioral/physiological effects of urgent and repeated administrations of the highest recommended dose of nepicastat (100mg/kg, p.o.) and to determine the maximum tolerated oral dose of repeatedly administered physostigmine.

In experimental studies male Sprague Dawley rats (n-8) were given nepicastat (30 and 100mg/kg, p.o.) or vehicle acutely, within the same weight range of the training animals (400-480 g). Animals were observed 1, 3 and 24 hours after drug or vehicle administration by observers blinded to the treatment status of each animal. Similarly, in a separate study, 8 rats were acutely administered either histosostigmine (1, 3, 10 or 30mg/kg, p.o.) or vehicle. Observations were made 1, 3 and 24 hours after drug or vehicle administration.

In a repeated dose trial study, 8 rats were administered intralipistat (100mg/kg, p.o.) or vehicle twice daily (06:00 and 18:00h) for 10 days (once on day 11). Throughout the study, the weight of the animals was monitored and on day 5, the animals were observed by "blinded" independent observers in order to evaluate the apparent behavioral/physiological effects after repeated dosing. In a separate study, groups of 8 rats received either vehicle or physostigmine (0.3, 1, 3 or 10mg/kg, p.o.) using the same dosing schedule. Throughout the study, the weight of the animals was monitored.

Nepicastat (30 or 100mg/kg, p.o., acute) did not cause any significant behavioral physiological changes. Similarly, repeated administration of nepicastat at a dose of 100mg/kg (p.o) had no significant effect. However, in the latter study, after 11 days, drug treated animals showed an average body weight loss of 28g, while the control had an average body weight gain of 1 g. Drug-treated animals also became more irritable than control-treated animals when operated during the 11-day study.

Acute dosing of physostigmine (3mg/kg or higher) resulted in significant behavioral effects (chewing and salivation in the mouth). At a dose of 30mg/kg (p.o), signs of toxicity (cyanosis, tremor, head spasm, dyskinesia) were observed. Toxicity occurred with repeated administration of 3 and 10mg/kg (p.o.) physostigmine (3 of 8 animals were found dead on day 2 in the 10mg/kg group and 2 of 8 animals had cramps on day 5 in the 3mg/kg group). Repeated administrations of 0.3 or 1mg/kg physostigmine were not effective.

As a result of these studies, the highest nepicastat dose for the DMTP study was reduced to 30mg/kg (p.o.), and a physostigmine dose of 1mg/kg (p.o.) was selected for repeat administration.

In the current DMTP study, rats were trained to remember the position of the lever for food reward during the 0, 8, 16 or 32 second delay of the entire schedule. After training, the effect of repeated administration of nepicastat (1, 3, 10 and 30mg/kg, p.o, b.i.d.) or physostigmine (1.0mg/kg, p.o., b.i.d.) was examined over 10 consecutive days tested in the DMTP task. On day 11 of the experiment, scopolamine Hbr (0.1mg/kg, s.c, 30mm pretreatment time) was administered to animals treated with nepicastat and physostigmine together. The dosage of scopolamine was selected based on data in the experimental DMTP study, where it was found that 0.1mg/kg scopolamine caused a significant compromise in selection accuracy. In addition to animals treated with nepicastat or physostigmine, a group of animals previously treated with vehicle was treated with scopolamine. Throughout the experiment, rats in the other group received vehicle treatment only. The objective of the final scopolamine test was: it was determined whether long-term administration of nepicastat or physostigmine reversed the impairment of selection accuracy in the DMTP task caused by scopolamine.

In the current study, dependency assays include: the percentage of correct selection, the waiting time to generate the selection response, and the number of trials that the animal is able to complete during the 70 minute trial period. The former measured changes may indicate changes in memory and/or attention function, while the latter two measured changes may indicate changes in other non-cognitive effects of the drug.

56 male Sprague Dawley rats were used weighing between 200 and 290g at the start of training. They were placed in groups of four per cage and each rat was fed approximately 12-15g of food per day. This amount of food allowed the rats to maintain approximately 85% of their free-feeding weight. Any animal starting below this weight is given additional food. Water is freely available. Animals were maintained under a 12: 12 hour light/dark cycle with a light period starting at 6 a.m.

12 Camden Instruments workpods (with two retractable levers and a centrally located food hopper) were used for behavioral testing. A baffle (which the rat may make back to enable it to pick up food pellets) is provided in front of the food hopper. The box is modified so that partitions can be installed at both sides of the food hopper. The partition is transparent perspex, reaching from the floor of the cabin grid to the ceiling of the cabin and extending 105mm to the central area. The work box is housed in a sound and illumination attenuating housing. Paul Fray Control System interfaces and Acorn A5000 computer programmed with Arachnid software were used to Control the work equipment.

Throughout the period, the room was illuminated and the rats were initially trained to retrieve Noyes 45mg Formula 'A' food pellets from behind the hopper baffle. Rats were then trained to squeeze the left and right levers in order to obtain a food reward. During the 30mm phase, either left or right side levers were randomly provided. The reaction to the inserted lever causes the lever to retract, the delivery of the food pellets, and the illumination of the hopper light. The hopper light is kept on until the pellet is retrieved.

Position matching training is then started. This training and all subsequent training is performed with the spacer placed in the work box. This phase is initially 50 minutes long. Rats were placed in a work box and the session was initiated when the room light was on. After a 30s test interval (ITI), one of the two levers (sample lever) was inserted into the capsule. The lever remains inserted into the compartment until a lever squeeze reaction occurs. The reaction to the lever causes the lever to retract and the hopper light to illuminate (but no pellets to be delivered). Once the hopper shutter is squeezed, the hopper light is extinguished and the two levers are inserted. The reaction to the sample lever (i.e., the same lever as previously provided) results in retraction of both levers, delivery of the food pellets, and illumination of the hopper lamp. The hopper light is kept on until the shutter is squeezed. No food pellets were available TO the improper lever (the lever opposite the sample lever provided), and a 10s pause (TO) cycle was initiated during which the room lights were extinguished. Before starting a new experiment, 30s of ITI were started. The levers inserted as sample levers are semi-randomly determined so that in a unit of 16 trials, 8 times for the right and left levers are provided as sample levers.

In this training and all subsequent training, a correction method is used. For uncorrected trials (i.e., the first trial at this stage and the trial immediately after the correctly selected trial occurred), the lever (left or right lever) inserted as a sample lever was randomly determined by the computer. Each time an inappropriate reaction occurs, the unselected levers (i.e. 'correct' levers) are provided as samples in subsequent 'calibration' tests. These calibration tests prevent position habituation (i.e. always responding to left or right levers and achieving 50% accuracy). The number of correction trials was recorded, but only the data collected in the non-correction trials was used to evaluate the percentage of correct selection.

After 24 stages, the animals were subjected to position matching work with a high degree of accuracy. In stage 25, a variable delay interval is inserted between the reduction of the sample lever and the provision of the lever in the selection test. After a 0s (immediate), 4, 8 or 16s delay after responding to the sample lever, a selection lever is inserted with the occurrence of the first baffle squeeze. The order of the four types of trials (0, 4, 8 or 16s delay) was determined semi-randomly, with the constraints: each delay occurred 4 times in 16 test units; two for the left lever test and two for the right lever test. A limited control (hold) is used so that if the rat does not produce a selective response within 30s of the end of the planned delay period, the test can be terminated and the test interval started. Such tests are considered incomplete and do not contribute to data analysis. After the end of ITI, the same experiment was resumed. Starting from stage 25, the timeout period after improper selection is ignored, and the stage length is increased to 70 minutes.

After 26 stages with delays of 0-16s (stages 25-50), the trial interval is reduced to 10s, and in the next 8 stages (stages 51-58) delays of up to 64s are used. However, since the performance at 64s delay is poor, such delay is not used in the further stages. At stage 59, delays of 0, 8, 16 and 32s are used. These delays are used at all subsequent stages. During phase 59, only 51 out of 56 rats completed more than 24 trials (corrected plus uncorrected). These rats were selected and semi-randomly assigned to the following 7 groups to match the groups in terms of performance (percent correct, response latency and number of trials completed): excipient/excipient (n ═ 7), excipient/scopolamine (n ═ 7), nepicastat 1.0 mg/kg/scopolamine (n ═ 7), nepicastat 3.0 mg/kg/scopolamine (n ═ 7), nepicastat 10.0 mg/kg/scopolamine (n ═ 7), nepicastat 30 mg/kg/scopolamine and Phys/scopolamine (n ═ 8).

At 6:00am and 6:00pm on consecutive days, during which phases 60-69, rats are given oral vehicle, physostigmine (Phys) or nepicastat (1, 3, 10 or 30 mg/kg). Due to the highly accurate selection shown by vehicle treated animals during stages 68 and 69, 0, 8, 16 and 32s delays were also used during the final test stage (stage 70) where almost all vehicle/vehicle treated groups received 0.1mg/kg scopolamine HBr (given 30 minutes s.c. prior to the test). Animals in the vehicle/vehicle group received s.c. injections of saline 30 minutes prior to the final experimental period. Thus, during the 11 consecutive days of the experiment provided, the drug treatments given to the seven groups were:

Group of	Stages 60-69(0, 8, 16 and 32s delay)	Stage 70(0, 4, 8 and 32s delay)
Group of	Stages 60-69(0, 8, 16 and 32s delay)	Stage 70(0, 4, 8 and 32s delay)	1	Excipient	Excipients/excipients
2	Excipient	Scopolamine excipient/scopolamine	1	Excipient	Excipients/excipients
2	Excipient	Scopolamine excipient/scopolamine	3	Nepitastat 1.0mg/kg	Nepitastat 1.0 mg/kg/scopolamine
4	Nepitastat 3.0mg/kg	Nepitastat 3.0 mg/kg/scopolamine	3	Nepitastat 1.0mg/kg	Nepitastat 1.0 mg/kg/scopolamine
4	Nepitastat 3.0mg/kg	Nepitastat 3.0 mg/kg/scopolamine	5	Nepitastat 10mg/kg	Nepitastat 10 mg/kg/scopolamine

Group of	Stages 60-69(0, 8, 16 and 32s delay)	Stage 70(0, 4, 8 and 32s delay)
Group of	Stages 60-69(0, 8, 16 and 32s delay)	Stage 70(0, 4, 8 and 32s delay)	6	Nepitastat 30mg/kg	Nepitastat 30 mg/kg/scopolamine
7	Physostigmine	Physostigmine/scopolamine	6	Nepitastat 30mg/kg	Nepitastat 30 mg/kg/scopolamine

Data collected and analyzed in the DMTP study provided included: 1) the percentage of correct reaction; 2) a waiting time between performance of the lever reaction on the sample and performance of the selection reaction; and 3) the total number of calibration and non-calibration trials completed. Only the first two dependency measurements were collected for non-calibration experiments.

Although the figures include data collected on days 1-10 of the study, to improve the efficiency and sensitivity of the statistical analysis, during the first 10 drug treatment phases (phases 60-69), the data was divided into two phase units (cells 1-5). Also, for the first 10 days of the trial, since the animals in the vehicle/vehicle and vehicle/scopolamine groups received the same treatment, the animals in both groups were pooled for statistical analysis during units 1-5.

Two-way analysis of variance (ANOVA) for drug treatment as an inter-subject factor and delay (0, 8, 16 or 32s) as an intra-subject factor were used to analyze the percentage of correct selection and response latency. These analyses were performed separately for each set of data. One-way ANOVA (performed at each delay stage) was performed after significant interaction. After significant primary effect from one-way ANOVA, a two-tailed Dunnett's t test was performed. One-way ANOVA was followed by a subsequent Dunnett's test, when appropriate, to analyze the average number of completed tests.

All statistical tests were performed with a Macintosh computer (using SuperAnova software). Throughout this period, α was set to 0.05. Animals that failed to complete the trial in each of the four delay periods were excluded from the analysis of the percentage of correct selection and reaction latency. The number of animals included in the analysis of the percentage of correct selection and latency to response was recorded for each of the 5 unit drug trials and the scopolamine (scop) trial day. To evaluate the overall effect of the drug on the ability to perform delayed matching of sample work, all animals were included in the analysis of the number of completed trials.

Physostigmine sulfate (1.0mg/kg, provided by RBI) and nepicastat (1, 3, 10 and 30mg/kg, provided by Roche) were administered (p.o. twice daily) starting at 6:00am and 6:00 pm. Scopolamine HBr (0.1mg/kg, supplied by Sigma) was administered (s.c.) 30 minutes prior to the final experimental period. Physostigmine and nepicastat were dissolved or suspended in distilled water and injected in a volume of 2.5 ml/kg. Scopolamine HBr was dissolved in saline and injected at a volume of 1.0 ml/kg. All drug doses are expressed as base weights.

During the first test unit, the drug had no significant effect on the percentage of correct selection or the waiting time for the selection reaction to proceed in DMTP operation. Drug treatment also failed to affect the number of completed trials, F (5, 45) ═ 0.319, and p ═ 0.899.

In unit 2, nepicastat and physostigmine had no significant effect on any dependency determination during the unit trial. Although the effect was not statistically significant (F (5, 45) ═ 1.717, p ═ 0.150), there was a tendency to reduce the number of experiments completed. The significant effect was slightly more pronounced in the group treated with nepicastat at 3 or 30mg/kg and in the group treated with physostigmine. In fact, only 4 of 7 animals treated with 30.0mg/kg nepicastat during this training unit were able to complete the trial in all four delays.

During unit 3 of the experiment, animals treated with physostigmine showed an independent delay defect in terms of selection accuracy. ANOVA showed significant primary effects for drug treatment for the correctly selected percentage of ANOVA, but the interplay of drug treatment X delay failed to reach statistical significance. The latter Dunnett's test for the main effects of drug treatment showed that only the physostigmine-treated group was significantly different from the vehicle-treated group. During this test unit, drug treatment did not significantly affect response latency or number of completed tests, F (5, 45) ═ 0.701, and p ═ 0.625.

In unit 4, drug treatment achieved an effect on the correctly selected percentage but failed to achieve statistical significance (p ═ 0.056). However, drug treatment significantly reduced response latency, while ANOVA showed significant interplay of drug treatment X delay. Subsequent one-way ANOVAs performed at delays of 0, 8, 16 and 32s found that only the 32s delay had a statistically significant effect; 0. the results of the 8, 16 and 32s delay analyses are as follows: f (5, 40) ═ 2.115, p ═ 0.084; f (5, 40) ═ 1.403, p ═ 0.244; f (5, 40) ═ 2.259, p ═ 0.067; f (5, 40) ═ 3.325, p ═ 0.013. At a delay of 32s, the latter Dunnett's test found that there was a longer waiting time for the selective reaction in the group treated with only nepicastat at 10.0mg/kg compared to the vehicle-treated group. During unit 4 of the trial, drug treatment did not significantly affect the number of completed trials, F (5, 45) ═ 1.533, and p ═ 0.199.

During unit 5 of the experiment, nepicastat induced significant dose-and delay-dependent defects in terms of selection accuracy. Two-way ANOVA showed significant interplay of drug treatment X delay, and subsequent one-way ANOVAs (0, 8, 16 and 32s delay) were found to have significant group differences only in the percentage correctly selected at 32s delay, with the results of the 0, 8, 16 and 32s delays analysis being as follows: f (5, 39) ═ 0.327, p ═ 0.894; f (5, 39) ═ 0.825, p ═ 0.539; f (5, 39) ═ 1.188, p ═ 0.333; f (5, 39) ═ 3.018, p ═ 0.021. At a delay of 32s, the latter Dunnett's trial found that the groups treated with 10 and 30mg/kg nepicastat showed a defect in the correct selection percentage relative to vehicle treated animals.

During unit 5 of the experiment, nepicastat and physostigmine did not significantly affect the reaction waiting time or number of completed experiments, F (5, 45) 1.692, p 0.156.

Many animals did not complete the delay match to the test sample after the administration of scopolamine HBr. In each of the four delays, only 1 rat treated with 10.0mg/kg nepicastat plus scopolamine and only 2 rats treated with 30.0mg/kg nepicastat plus scopolamine were able to complete the trial. In fact, in all groups, n < 4, except for the vehicle/vehicle and vehicle/scopolamine groups, seven and four rats, respectively, completed the test in each of the four delays.

In all scopolamine treated groups, the number of trials completed by animals was significantly reduced, F (6, 16) ═ 8.801, and p ═ 0.001.

In the scopolamine treated group, no ANOVA was performed on the selection accuracy and response latency data due to the low number of subjects. In addition, the average percentage of correct selections dropped sharply in the four delays. In a t-test comparing the accuracy of selection of the vehicle/scopolamine group versus the vehicle/vehicle group, it was found that scopolamine significantly reduced the percentage of correct selections, t (9) ═ 4.15, and p ═ 0.003. Since there were fewer than 4 subjects in the other groups, no further statistical analysis was performed. However, interestingly, two animals in the group given 30.0mg/kg nepicastat plus scopolamine performed better than the scopolamine treated group alone: two animals treated with 30.0mg/kg nepicastat plus scopolamine yielded more correct selections than either the vehicle/scopolamine treated group or any of the other scopolamine treated groups.

In the DMTP trial, nepicastat alone did not appear to cause a memory-improving effect. Notably, the delay-induced memory deficits observed in the five unit trial appeared to have disappeared in vehicle-treated control animals. However, vehicle treated control animals also showed delay-dependent memory deficits for the fifth test unit with 100% selection accuracy at 0s delay and 80% selection accuracy at 32s delay. Thus, in vehicle-treated animal performance, no upper-limit effect was observed at 32s delay.

In particular, nepicastat has a selective memory-destroying effect for the fifth training unit. Physostigmine did not improve performance on any treatment day, and actually produced a delayed independence defect in selection accuracy during unit 3 of the trial (days 5 and 6). On day 11, the results from the scopolamine stimulation test, in which scopolamine HBr (0.1mg/kg) and nepicastat or physostigmine were co-administered to the animals, could not be analyzed, since the number of subjects in the nepicastat and physostigmine treatment groups who were able to perform DMTP work was small. However, two rats (capable of DMTP work) receiving 30.0mg/kg nepicastat and scopolamine showed higher selection accuracy compared to any other scopolamine treated animals. Nepicastat may reverse some cognitive disorders caused by scopolamine, an effect which may be masked by other "non-cognitive" effects of the compound.

Nepicastat caused significant dose-and delay-dependent deficiencies in selection accuracy. Animals treated with 10.0mg/kg nepicastat absolutely showed no deficiency in selection accuracy at 0, 8 and 16s delay. In contrast, at the 32s delay, animals in the nepicastat group at 10.0mg/kg were weakened relative to the vehicle treated group. At 0s delay, the group treated with the highest dose of nepicastat 30.0mg/kg showed no defect in selection accuracy, at 8 and 16s delays there was a tendency for impaired selection accuracy, and at 32s delay, selection accuracy appeared to be significantly deficient relative to the vehicle treated group. The delay-dependent nature of the defects caused by these drugs in terms of accuracy of selection suggests that the compounds may directly contribute to short-term or working memory. At short delays, the animals can be fully stimulated and can perform DMTP work accurately, showing defects only when the retention intervals are long. In this model, a few compounds have been tested that show this property. It has been claimed that many compounds that can impair memory generally lead to a deficiency in selection accuracy, which has been observed in all delays (e.g., MK-801, scopolamine). Nepicastat had little effect on the latency to complete the trial, as is evident in the fourth unit of training, during which time animals treated with 10.0mg/kg nepicastat took longer to complete the 32s delay trial compared to vehicle treated animals. This effect was not dose dependent and was not observed in the group treated with 30.0 mg/kg. There is also a trend towards fewer trials being completed for animals treated with nepicastat compared to vehicle treated animals: during the last two training units, a trend was observed in which the number of completed trials decreased. However, due to the variability of the data, this trend did not reach statistical significance. This variability of data is unexpected. It appears that the initial stress caused by long-term oral dosing may interfere with the performance of these food deprived animals, especially during the first few experimental units. Between the first and second training units, a reduced number of completed trials occurred for all groups. Animals recovered from this initial reduction and showed more stable performance in the next three training units.

We found that some animals began a weight loss in this study, in some cases more than 5% of total body weight. Animals showing weight loss were divided and given additional food at the end of their daily training period. This additional feeding may contribute to the variability in the number of trials completed. Although no systematic recording was made, random observations suggest that more animals in the 30mg/kg group must be given additional food than the other groups. This observation is consistent with the results of an exploratory study in which administration of nepicastat at 100mg/kg (p.o.) per day resulted in significant weight loss.

Physostigmine did not improve rat performance in the DMTP assay. In fact, the animals treated with physostigmine during unit 3 of training showed a significant drawback in terms of percentage of correct selection. Contrary to the effect obtained with nepicastat, the drawback of selection accuracy caused by physostigmine is the delayed independence: the interacting terms derived from the analysis of variance are not close to statistical significance. Thus, the effect of physostigmine on the accuracy of the response is likely to be secondary to the behavioral toxic effects of the drug when administered at this dose. In the last two training units, during which the defect in selection accuracy caused by physostigmine no longer reaches statistical significance, the animals appear to develop tolerance to these effects.

Finally, physostigmine did not appear to reverse the effects of scopolamine during the scopolamine test. It is possible that different doses of physostigmine are effective against scopolamine. We have not previously attempted to reverse scopolamine with physostigmine (using the administration regime provided) and therefore there is no historical data to compare with the results provided. This lack of efficacy of physostigmine compared to acute administration may be due to the low dose of physostigmine which must be administered over a long period of time. Animals cannot tolerate the repeated high doses of physostigmine required to reverse the effects of scopolamine (see results of exploratory studies). In addition, nepicastat did not reverse the effect of scopolamine, although it was of interest that two animals treated with 30.0mg/kg nepicastat (which could be completed during the scopolamine trial) showed higher selection accuracy than any of the other scopolamine treated groups. In this trial, further studies must be made to determine unambiguously whether acute or chronic treatment with nepicastat can reverse the effect of scopolamine.

Nepicastat appears to have a specific memory-interfering effect, which is evident 8 days after administration. Physostigmine did not improve performance on any treatment day, and actually produced a delayed independence defect in selection accuracy during unit 3 of the trial (days 5 and 6). On day 11, the results from the scopolamine stimulation test, in which scopolamine HBr (0.1mg/kg) and nepicastat or physostigmine were co-administered to the animals, could not be analyzed, since the number of subjects in the nepicastat and physostigmine treatment groups who were able to perform DMTP work was small. However, two rats (capable of DMTP work) receiving 30.0mg/kg nepicastat and scopolamine showed higher selection accuracy compared to any other scopolamine treated animals. Nepicastat is able to reverse some cognitive disorders caused by scopolamine, an effect which may be masked by other "non-cognitive" effects of the compound. In the last training unit, nepicastat caused significant dose-and delay-dependent deficiencies in selection accuracy. This was an unexpected finding because considering that many other memory-interfering drugs, such as scopolamine and MK-801, could lead to delayed independence deficits in selection accuracy (which may be due to deficits in attention and/or motor/motivation factors). In contrast, it is unlikely that the changes in attention or motor/stimulation performance would explain the results provided by nepicastat. If the drug is selective for a new receptor or pharmacological mechanism, then these results demonstrate an important role for this substrate in working memory.

Example 30

Recently, we have demonstrated that nepicastat (a selective dopamine β -hydroxylase inhibitor) shows potent antihypertensive activity in acute studies in SHRs. In rats of the same strain, nepicastat was examined for its antihypertensive effect for a long time. In addition, we have also explored the potentiating effect of co-administration of the compound with the angiotensin converting enzyme inhibitor enalapril. In SHRs, the therapeutic effect on cardiac hypertrophy was also examined.

Male SHRs/NCr1BR rats (22-28 weeks old at the start of dosing) and weight-matched WKY/NCrI BR rats were used. Four series of experiments were performed in sequence:

series I

Excipient

Enalapril 10mg/kg

Nepitastat 3mg/kg

Nepitastat 10mg/kg

Series II

Excipient

Enalapril 10mg/kg

Nepitastat 30mg/kg

Nepitastat 100mg/kg

Series III

Excipient

Enalapril 1mg/kg

Nepitastat 30mg/kg

Nepitastat 30mg/kg + enalapril 1mg/kg

Series IV

Enalapril 1mg/kg (E1)

Nepitastat 15mg/kg + E1

Nepitastat 30mg/kg + E1

Nepitastat 60mg/kg + E1

In each series, 24 SHRs were chronically implanted with telemetric implants for measuring arterial blood pressure, heart rate and locomotor activity. The rats were anesthetized with sodium pentobarbital (60mg/kg, i.p) and their abdomens were trimmed. Under sterile conditions, incisions were made on the midline. The abdominal aorta is exposed and the catheter of the telemetry transmitter is inserted. After the transmitter was attached to the abdominal musculature, the skin was sutured closed. Each rat was allowed to recover for at least 2 weeks prior to drug administration. Rats were placed individually in a quiet room with inverted light/dark cycles (08:00-20:00 lights off).

Three days before the start of the experiment, rats were randomly divided into 4 groups and their Systolic Blood Pressure (SBP), Diastolic Blood Pressure (DBP), Mean Blood Pressure (MBP), Heart Rate (HR) and Motor Activity (MA) were monitored. The pre-dose values of these parameters were determined and the corresponding rat groups received a 30 day (daily) treatment with nepicastat and/or enalapril (see below).

Twenty-four hours after the final treatment, rats were sacrificed, the left ventricle was collected, weighed (wet weight), and freeze-dried for at least 24 hours to obtain dry weight.

At the beginning of each experiment, the number of rats in each group that were telemetrically monitored was always 6. However, in series I, 7 Wistar Kyoto (WKY) rats were similarly placed and vehicle (water) was given, while in series III and IV an additional 2 rats in each group were similarly treated (increasing the number of animals for statistical analysis of the effects of excessive increase in SHRs). No telemetry equipment installation or monitoring was performed on these rats.

Nepicastat and enalapril were prepared in water. All doses of 10ml/kg were given orally to rats and are expressed as free base equivalents. EnalaprilCommercially available from local drug stores.

A computerized data collection system is used to continuously collect SBP, DBP, MBP, HR, and MA data. Data was collected for each rat every 5 minutes for 10 sec. These data were then averaged hourly and the Standard Error (SEM) of the mean was calculated. At the end of the treatment, left ventricular mass (dry and wet weight) was obtained. Body weight was recorded daily.

All values are expressed as mean ± SEM. Statistical significance was defined as P levels less than 0.05.

Data on MBP, HR and MA were analyzed separately. Each analysis was performed at 26 time points measured daily. Two-way ANOVA was used for the primary effect and duration of treatment and their interplay. If the overall therapeutic effect or significant interplay is determined, a series of one-way ANOVA is performed at each time point. Using the Dunn's method, a phase-by-phase comparison was performed at each time point. If the overall therapeutic effect is not determined, pair-wise differential analysis is performed starting from the control by adjusting the cut-off values using the Bonferroni correction.

For left ventricular mass, covariance analysis with final body weight was used to analyze tissue wet weight and tissue dry weight, while Kruskal-Wallis test was used to analyze the ratio of tissue wet weight/body weight and tissue dry weight/body weight. If the overall therapeutic effect was not determined among all groups, Bonferron's correction for multiple comparisons was then performed.

For body weight, two-way ANOVA with respect to change before dosing was used to analyze the overall effect of treatment, day, and the interplay of treatment with day. One-way ANOVA was then performed daily and pairwise comparisons of drug treated groups with vehicle controls were performed using Dunn's method and Fisher's LSD strategy to correct multiple comparisons.

For series I and II, oral nepicastat (3 and 10mg/kg) (all doses indicated below are po administered) did not significantly affect blood pressure on any of the 30 days of treatment (data not shown). At 30mg/kg, nepicastat gradually reduced MBP on day 1, and continued to reduce MBP to a maximum of-20 mmHg (p < 0.01) on day 3, with a slight rebound within 24 hours. Throughout the study, a similar antihypertensive effect was elicited. At 100mg/kg, the compound elicited a peak antihypertensive response of-29 mmHg (p < 0.01) 21 hours after dosing on day 1. MBP continued to decrease, reaching its lowest point of about-42 mmHg on day 3 (p < 0.01). MBP remained reduced throughout the study.

Throughout the study, 10mg/kg enalapril consistently reduced MBP. On day 5, the greatest decrease in MBP-29mmHg was observed within 1 hour after dosing (p < 0.01).

In series III, although single administration of enalapril (1mg/kg (n-6)) or nepicastat (30mg/kg (n-6)) resulted in only a small antihypertensive effect, co-administration of both compounds (n-6) resulted in a greater antihypertensive response (at 16 h on day 1, reaching-21 mmHg, p < 0.01). The initiation of the reaction is slow and gradual. With the second dose on day 2, a greater antihypertensive response of the combination treatment was observed for most of the day (p < 0.01 at 13 hours, -25 mmHg). Throughout the study, an enhancement was observed.

In series IV, the effect of nepicastat was further investigated to enhance enalapril at non-antihypertensive doses. Although nepicastat (60mg/kg) produced a greater and longer antihypertensive effect initially in the presence of enalapril (1mg/kg) than that caused by 15 or 30mg/kg of the compound, no greater effect was observed on days 8 to 30. Thus, the potentiating effect was independent of the nepicastat doses tested (15, 30 and 60 mg/kg). The group receiving nepicastat (15mg/kg) and E1 showed reduced mean blood pressure with a large standard error (two rats showed a greater antihypertensive response than the remaining rats).

Nepicastat (3 and 10mg/kg) did not consistently affect Heart Rate (HR) in the 30-day study. However, at least the first few hours after administration, the group treated with nepicastat (100mg/kg) had a tendency to lower HR compared to the vehicle control group. Although enalapril (1mg/kg) did not affect HR, compound (10mg/kg) tended to cause transient mild tachycardia within 2 hours after administration. In series III, none of the treatments (i.e., enalapril (1mg/kg), nepicastat (30mg/kg), or a combination thereof) consistently affected HR. In series IV, the nepicastat (15, 30 and 60mg/kg) and enalapril (1mg/kg) treated groups had a tendency to lower HR as compared to the enalapril alone (1mg/kg) treated group.

None of the drug treatments showed significant effects on Motor Activity (MA) throughout the study.

Nepitastat (3-100mg/kg) did not affect cardiac hypertrophy observed in SHRs (p > 0.05). In series II (but not in series I), enalapril (10mg/kg) significantly reduced the mass of the left ventricle. In series III, enalapril (1mg/kg) did not provide excessive enlargement regression, but the co-administration of enalapril (1mg/kg) and nepicastat (30mg/kg) significantly reduced the left ventricular mass of SHRs (p < 0.01). However, in series IV, the effect of co-administration of enalapril (1mg/kg) and nepicastat (15, 30, and 60mg/kg) on the quality of the left ventricle was not different from that of enalapril alone (p > 0.05).

Treatment of SHRs with nepicastat (3 and 10mg/kg) did not have any effect on the body weight of SHRs compared to vehicle treatment (p > 0.05). However, treatment with compound (30 and 100mg/kg) resulted in more weight gain (p < 0.05).

By comparison, enalapril (10mg/kg) significantly reduced (p < 0.05) or had no effect on the body weight of rats. Although treatment with enalapril (1mg/kg) slightly reduced body weight, co-administration of enalapril (1mg/kg) and nepicastat (30 and 60mg/kg) slightly increased body weight in rats.

The pre-dose dimer of rats treated with vehicle, enalapril and nepicastat (3 and 10mg/kg) was: 387. + -. 11, 415. + -. 12, 407. + -. 4 and 415. + -.12 g.

The pre-dose dimer of rats treated with vehicle, enalapril and nepicastat (30 and 100mg/kg) was: 399 + -10, 389 + -6, 389 + -9 and 401 + -10 g.

The pre-dose dimer of rats treated with vehicle, enalapril and nepicastat (30mg/kg) (with or without enalapril) was: 365 +/-9, 371 +/-8, 361 +/-7 and 369 +/-7 g.

The pre-dose (pre-dose) body weights of rats treated with enalapril alone and with nepicastat (15, 30 and 60mg/kg) were respectively: 357 + -6, 363 + -6, 347 + -8 and 346 + -8 g.

In 4 series of 30 days treatment, four dead rats were observed. The cause of these deaths has not been determined, but it appears that these deaths are unlikely to be associated with nepicastat treatment.

Four series of experiments were performed in Spontaneously Hypertensive Rats (SHRs) with radio telemetry implants to evaluate the effect of 30 days of long-term oral nepicastat on blood pressure, heart rate, locomotor activity and left ventricular mass. Daily treatment with nepicastat (3 and 10mg/kg) (n-6) did not affect blood pressure. On day 3, nepicastat (30mg/kg) (n ═ 6) resulted in a peak antihypertensive effect of-20 mmHg (p < 0.01). The antihypertensive effect was moderate throughout the study, but could be measured. Nepicastat (100mg/kg) (n-5) resulted in greater antihypertensive effect. The effect was gradually increasing and reached a peak of-42 mmHg on day 3 (p < 0.01). In the rest of the studies, antihypertensive effects of similar magnitude were observed. By comparison, the angiotensin converting enzyme inhibitor enalapril (10mg/kg, n-6) resulted in antihypertensive effects of-20 to-30 mmHg throughout the study. Although enalapril (1mg/kg) administered alone did not result in a significant antihypertensive effect, co-administration with nepicastat (30 mg/kg; n ═ 6) resulted in a greater and sustained antihypertensive effect (p < 0.01). Throughout the 30 day study, an enhancement was observed. The enhanced antihypertensive effects of nepicastat due to enalapril (1mg/kg) can also be seen at doses of 15, 30 and 60mg/kg, although these effects are not dose-dependent.

No significant effect on heart rate was observed in the group treated with nepicastat (3-10mg/kg) or enalapril (1 mg/kg). However, the group receiving nepicastat (30 or 100mg/kg) showed a slight bradycardia during the waking hours in the rats. In contrast, enalapril (10mg/kg) caused transient tachycardia. The coadministration of nepicastat (15, 30 and 60mg/kg) and enalapril (1mg/kg) tended to reduce heart rate compared to enalapril alone (1 mg/kg). In any treatment group, no significant effect on motor activity was measured.

In SHRs, treatment with nepicastat (30 (n-6) and 100 (n-5) mg/kg) did not have a significant effect on left ventricular hypertrophy. Although enalapril (1mg/kg, n-8) or nepicastat alone (30mg/kg, n-7) did not provide a regression of the exaggerated increase, co-administration of both compounds (n-8) significantly reduced the left ventricular mass of the SHRs. However, the effect of co-administration on left ventricular mass was independent of nepicastat doses (15, 30 and 60mg/kg) and not statistically different from that of enalapril alone (1 mg/kg).

In four series of experiments, four dead rats appeared during the 30 day treatment period. Three rats were found in the group treated with nepicastat, one in the vehicle group. The cause of these deaths has not been established, but appears to be unrelated to nepicastat treatment.

In SHRs, nepicastat (30 and 100mg/kg) significantly reduced blood pressure over a 30 day period and did not cause any reflex tachycardia. In SHRs, nepicastat (30mg/kg) was co-administered with a non-antihypertensive dose of enalapril (1mg/kg) with greater antihypertensive effects and greater effects on excessively increased decline compared to nepicastat alone. However, these effects were independent of nepicastat doses (15, 30 and 60 mg/kg).

Example 31

A study was conducted in anesthetized (instrumented) dogs to evaluate the effect of nepicastat response to an autonomic nervous system agent (auto nomic agent).

Beagle dogs were given a single intraduodenal dose of 0 (vehicle) or 60mg/kg nepicastat via the duodenal tube. The vehicle control group consisted of 1 male and 1 female, and the nepicastat-treated group consisted of 2 males and 2 females. When anesthetized with isoflurane gas, each animal was surgically instrumented. Prior to administration of the test formulations, mean blood pressure responses to intravenous doses of autonomic nervous system agents, norepinephrine (3 μ g/kg), isoproterenol (0.3 μ g/kg), and acetylcholine (10 μ g/kg) were evaluated. A single bolus (bolus) dose of the test formulation was then administered to each animal, and the response of blood pressure to the autonomic nervous system agent was evaluated approximately 1, 2, and 3 hours after administration. After completion of the experiment, each dog was euthanized and removed from the study.

Dogs were selected because of the general utility in evaluating the effect of test compounds on hemodynamic parameters. Beagles were obtained from Marshall Farms, inc., North Rose, New York. Each dog was uniquely identified by a harsh number applied by the supplier. Animals were allowed to acclimate to laboratory conditions for at least 3 weeks prior to dosing. During the acclimation period, the general conditions of each animal were evaluated, and healthy ones were used. Dogs were randomized to treatment groups; males are designated single and females are designated double.

After assignment to the study, dogs were individually placed in stainless steel cages, indicating study number, animal number and harshness designation. The environment of the room in which the dog is placed can be controlled. The cages were cleaned daily and the animals were transferred to a sterile cage every other week. Providing a Purina Certified Canine once a dayWater is provided indefinitely.

On the day of treatment, dogs are approximately 14 to 16 months old. The male weighs 10.3-12.9 kg, and the female weighs 8.5-11.2 kg.

At the time of administration, a 60mg/ml suspension was prepared by mixing nepicastat powder with excipients. The recombinant 60mg/ml nepicastat formulation retained potency during use. At the time of daily administration, aqueous solutions of noradrenaline (60. mu.g/ml), isoproterenol (6. mu.g/ml) and acetylcholine (200. mu.g/ml) were prepared in sterile water.

Vehicle control groups (1 male and 1 female) were given 1ml/kg vehicle and nepicastat treatment groups (2 male and 2 female) were given 1ml/kg of 60mg/ml nepicastat solution. The total dose of nepicastat administered to each animal was 60 mg/kg.

Doses were selected based on data from two studies of nepicastat. In acute toxicity studies, a single oral dose of 400mg/kg resulted in temporary clinical signs of toxicity in dogs. Dogs were given doses of 5, 20 or 80mg/kg once daily orally in a 1 month study. At 80 mg/kg/day, clinical signs of toxicity appeared.

The single intraduodenal dose of vehicle or nepicastat formulation was administered directly into the duodenum via the duodenal tube. The intraduodenal route was chosen because the oral route is the recommended clinical route of administration of nepicastat. Dose volumes were calculated based on individual body weights recorded prior to dosing (body weight data not included in the report). At the end of each experiment, the dogs evaluated were euthanized by an excess of sodium pentobarbital (300mg/kg, IV) and removed from the study.

The dogs were surgically instrumented according to the method described in the protocol. Animals were deprived of food overnight prior to surgical installation of the meter. Anesthesia of each animal evaluated was initiated by injection of a mixture of (IV) ketamine (10mg/kg) and diazepam (0.5 mg/kg). Each animal was placed on an operating table with a circulating heated water cushion to maintain body temperature, maintaining mechanical ventilation throughout the experiment. The operative anesthesia plane (plane) is maintained with isoflurane gas (1.5% to 2% tidal volume delivered in oxygen, approximately 1.5L/min flow rate). Rectal temperature was monitored and used only to determine blood gas levels, and data are not provided in this report. External needle electrodes were placed subcutaneously in order to monitor a standard limb lead (limb) II Electrocardiogram (ECG) for evaluation of anesthesia.

Inserting a catheter into the left femoral vein, advancing the tip of a polyethylene tube into the vena cava for administration to the autonomic nervous systemAn agent. The catheter was inserted into the left femoral artery using a polyethylene tube filled with 50U/ml heparin-salt solution. The tip of the arterial cannula is advanced into the thoracic aorta and coupled to an external pressure transducer to record systolic and diastolic aortic blood pressure. Arterial blood samples were drawn from arterial cannulae for blood pH, PCO₂And PO₂And (6) analyzing.

A midline laparotomy was performed and the duodenum was separated just caudally to the pyloric sphincter. A needle is inserted into the duodenum and the tip of the saline filled tube is advanced through the needle into the lumen for administration of the test formulation. The needle is withdrawn from the incision site, the cannula is secured in place, the stopcock of the tube is removed from the abdomen, and the laparotomy skin is re-apposed.

After the instrument is surgically installed, ventilation (ventilatory) adjustments are made, if necessary, to adjust arterial blood pH and PCO₂The levels were adjusted to be within approximately the normal physiological range (pH 7.43 to 7.50, PCO)₂22 to 27 mmHg).

Using a femoral vein tube, with approximately 10 minute intervals between each dose, the autonomic nervous system agent, norepinephrine (3 μ g/kg), isoproterenol (0.3 μ g/kg), and acetylcholine (10 μ g/kg) were administered intravenously by bolus injection (over approximately 15 seconds). After each dose administration, the tube was rinsed with 3ml of water. Approximately 20 minutes after the first administration of acetylcholine, the dose is repeated.

Approximately 30 minutes after the second acetylcholine administration, vehicle or nepicastat is administered to each animal. The dose volume was 1ml/kg and was administered directly into the duodenum in a bolus using an intraduodenal tube. Immediately after administration, the intraduodenal tube was rinsed with 3ml of vehicle solution. Administration of the autonomic nervous system agent was repeated approximately 50, 110, and 170 minutes after administration (with approximately 10 minute intervals between administration of each agent).

Aortic blood pressure, heart rate and ECG parameters were continuously recorded directly on a polygraph. At about the time of drawingAt the time point of blood sampling, the pH, PCO of blood obtained from the blood gas analyzer₂And PO₂Values were manually recorded on a polygraph. Heart rate, ECG and blood gas parameters were used only to assess the level of anesthesia and stability of the animal preparation; these data are not provided in the report.

Systolic, diastolic, and mean aortic blood pressures were evaluated just prior to dosing (baseline) and at the time of peak response (maximum change from baseline) for each agent. Systolic, diastolic and mean aortic blood pressure and blood pH, PCO were evaluated prior to dosing and approximately 50, 110 and 170 minutes after dosing with the test formulations ₂And PO₂。

The method of characterizing the response to norepinephrine is: for each norepinephrine administration, the mean aortic blood pressure was evaluated just prior to and at the time of peak pressure increase. The reaction of isoproterenol and acetylcholine was characterized by: aortic blood pressure at diastole was assessed just before the peak pressure drop and at this drop for each administration of isoproterenol and acetylcholine.

At the end of the experiment, the dogs evaluated were euthanized by an excess of sodium pentobarbital (approximately 300mg/kg, IV) and removed from the study.

There was no treatment-related difference between the pre-dose and post-dose responses to norepinephrine. In vehicle control dogs, the magnitude of the post-dose response to norepinephrine was smaller compared to the pre-dose response; this was considered to be incidental. There was no treatment-related difference between pre-dose and post-dose responses to isoproterenol. There was no treatment-related difference between pre-dose and post-dose responses to acetylcholine.

After the instrumentation was surgically installed, a single intraduodenal dose of 60mg/kg of nepicastat was administered to the anesthetized beagle dog. The response of blood pressure to intravenous doses of autonomic nervous system agents (norepinephrine, isoproterenol, and acetylcholine) was evaluated before and approximately 1, 2, and 3 hours after administration. There was no treatment-related difference between pre-dose and post-dose responses to autonomic nervous system agents.

Example 32

Effect of acute intraperitoneal administration of nepicastat, DBH inhibitor (DBHI) on locomotor (lococotor) activity in mice. It is demonstrated that such compounds have an effect on locomotor (lococotor) activity.

Adult male CD-1(ICR) mice (30-40 g on the day of the study) were placed in groups of eight and lit at between 0900hr and 2100hr under normal light/dark cycles. Allowing the animal to use food and water ad libitum. All animals were first (naive) used for drug treatment and behavioral testing. Each animal was used only once.

The motion activity was monitored with an automated 14 workstation activity monitoring system (San Diego Instrument Co.). Each workstation comprises: a transparent plexiglas cage (25cmx45cmx20 cm; wx1xh) placed inside the metal frame, containing 3 light emitters and 3 light detectors (arranged evenly along the length of the wall). The bottom of each cage was slightly covered with a layer of clean cedar.

Prior to testing, mice were placed in the laboratory for at least 1 hour. Mice were individually placed in a mobility cage and allowed to explore (explore) for 30 minutes. After this habituation period, nepicastat (10, 30 and 100mg/kg), SKF-102698(30 and 100mg/kg), cocaine (30mg/kg) or vehicle was administered intraperitoneally to mice and returned immediately to the same cages. After a 60 minute pretreatment period, motor activity was monitored for 180 minutes. The activity statistics and movements of each animal were recorded every 30 minutes (defined as: 2 consecutive photon beams were interrupted).

Two-way analysis of variance (ANOVA) with repeated measures was performed using overall grading data (non-parametric techniques) to test the overall effect of treatment, time intervals and the interplay of treatment with time intervals. At each interval, one-way ANOVA was performed in order to see the therapeutic effect present at any interval. Comparison-by-comparison was then performed at each interval using the Dunn's method and Fisher's LSD strategy to adjust for the multiple comparison problem.

For nepicastat, the dosage range is 3-100mg/kg, and is dissolved in dH₂And O, ultrasonic treatment. For SKF-102698, the dosage range is 30-100 mg/kg. For cocaine hydrochloride, the dosage range is 30 mg/kg. The compound was administered in a volume of 1ml/100 g. All reported doses are expressed as free base, except cocaine (where salt weight is used).

Treatment and time had significant effects throughout the model (both p < 0.01), while treatment-time interactions were not significant. Analysis of each time point showed significant overall therapeutic effect at time intervals 1-4 (i.e., the first 120 minutes of the trial; all p < 0.01), whereas no overall significant therapeutic effect was measured at time intervals 5 and 6 (i.e., the last 60 minutes of the trial).

When cocaine was compared to vehicle groups, there was a significant overall effect of treatment and time (two p < 0.01) in terms of activity statistics and movement, while the interplay of treatment and time was not significant. Analysis of each time interval showed that at time intervals 1-4 (instead of 5 and 6), the cocaine group had significantly larger total activity statistics and significantly larger number of movements (all p < 0.05).

In contrast, there was no significant difference in total activity statistics or movements at any time interval for any nepicastat-or SKF-102698-treated group compared to vehicle controls.

It is effectively demonstrated that cocaine acts as a sports (lococotor) stimulant at a dose of 30 mg/kg. In contrast, acute administration of nepicastat (3, 10, 30 or 100mg/kg dose) did not result in any significant change in total activity or movement at any time interval compared to vehicle controls. Similarly, at any time interval tested, 30 and 100mg/kg doses of SKF-102698 had no significant effect on overall activity or movement. These data indicate that these DBHIs have no muscle motor (motoric) effect in mice.

Example 33

It has been shown that acute administration of the dopamine- β -hydroxylase inhibitor nepicastat can inhibit the enzyme in the mesenteric arteries and left ventricle of spontaneously hypertensive rats. Changes in norepinephrine and dopamine levels after 7 and 25 days of oral administration of 1mg/kg or 10mg/kg nepicastat were examined in the cerebral cortex and mesenteric artery of spontaneously hypertensive rats.

Nepirstat was prepared at 1 and 10mg/kg based on the free base. The weighed material was dissolved in the excipient (dH)₂O) to give an oral dose that can be administered in a volume of 10.0 ml/kg.

At the beginning of the study, 16-17 week old male Spontaneously Hypertensive Rats (SHRs) were used. Animals receive food and water ad libitum. Animals were randomly assigned to one of the following test groups: nepirstat 10mg/kg, 1mg/kg, or 10ml/kg of deionized water excipient groups were orally administered. Rats (once a day, given 7 or 25 days) were given oral vehicle, 1mg/kg or 10mg/kg nepicastat (n-8), except 25, where n-9. On day 7, four hours after compound administration, animals were anesthetized with halothane, decapitated, cortical and mesenteric arteries harvested, weighed, and analyzed in 24 rats (n-8/treatment group). On the next 18 days, the remaining 31 rats were allowed to continue to receive one of three treatments given orally. Mesenteric arteries and cortex were harvested from this group 4 hours after the last treatment, weighed, and analyzed for catecholamine levels.

Animals sacrificed on day 25 were also used for blood pressure measurements. The last blood pressure measurement was taken on day 22.

Statistically, three treatments were compared at each time period (7 or 25 days) using non-parametric one-way analysis of variance (ANOVA). Adjusting the difference in sample size using Fisher's LSD strategy for the mean values, for each Treatment was compared pair-by-pair with controls in order to control experimental error rates. Each variable was analyzed separately. With respect to the figures 6-11,^*p < 0.05 and^**，p＜0.01。

in the cerebral cortex, the 10mg/kg dose group had significantly lower norepinephrine levels (p < 0.1) and significantly higher dopamine/norepinephrine ratios (p < 0.05) compared to the vehicle group seven days after treatment. Seven days after treatment, there was no significant difference in dopamine levels (p > 0.05) or in norepinephrine levels or dopamine/norepinephrine ratios in the 1mg/kg nepicastat dose groups compared to vehicle in either of the two treatment groups (1 or 10mg/kg nepicastat) (FIGS. 6-8). There was a slightly significant (p < 0.05) increase in the cortical dopamine/norepinephrine ratio for day 7 of the 10mg/kg nepicastat dose.

After 25 days of treatment, the cortical level of dopamine was significantly (p < 0.05) increased compared to the vehicle group in the 1mg/kg nepicastat dose group. In this group, the cortical dopamine/norepinephrine ratio was also significantly (p < 0.01) greater than that of the vehicle group. This ratio was significantly greater for the group of nepicastat doses at 10mg/kg (p < 0.05) compared to vehicle. Norepinephrine levels were not significantly (p > 0.05) different from controls in any of the dose groups, as were dopamine levels in the 10mg/kg dose groups (FIGS. 6-8).

In the mesenteric artery, after 7 days (p < 0.05) and 25 days (p < 0.01) of administration, the 10mg/kg dose group had significantly higher dopamine levels and dopamine/norepinephrine ratios than the vehicle group, but no difference in norepinephrine levels. In the 1mg/kg nepicastat dose group, none of the parameters measured differed significantly (p < 0.05) from the control (FIGS. 9-11).

Nepitastat given orally for 7 and 25 days can significantly (p < 0.05) inhibit dopamine-beta-hydroxylase in the cortex and mesenteric arteries of Spontaneously Hypertensive Rats (SHRs). The administration of nepicastat at 10mg/kg resulted in greater inhibition than 1mg/kg, and the observed effect was therefore dose-dependent.

It will be apparent to those of ordinary skill in the relevant art that other suitable improvements and modifications to the methods and applications described herein are suitable and may be made without departing from the scope of the invention or any embodiment thereof. While the invention has been described in connection with certain embodiments, it is not intended to be limited to the specific form set forth, but on the contrary, it is intended to cover such alternatives, modifications, and equivalents as may be included within the spirit and scope of the invention as defined by the appended claims.

Claims

1. A method of treating a patient suffering from or susceptible to at least one of the following symptoms: abuse, dependence or withdrawal of at least one substance, the method comprising: administering to the patient a therapeutically effective amount of compound a.

2. The method of claim 1, wherein the at least one substance is selected from the group consisting of drugs of abuse and drug therapy.

3. The method of claim 2, wherein the drug of abuse is selected from the group consisting of psychostimulants, opioids, hallucinogens, inhalants, sedatives, tranquilizers, hypnotics, anxiolytics, and illicit substances.

4. The method of claim 3 wherein the psychostimulant is a β -phenylisopropylamine derivative

5. The method of claim 4 wherein the β -phenylisopropylamine derivative is selected from amphetamine, dextroamphetamine, and methamphetamine.

6. The method of claim 3 wherein the psychostimulant agent is selected from the group consisting of synthetic hallucinogens, phenmetrazine, ritaline, bupropion, pemoline, mazindol, (-) norpseudoephedrine, and fenfluramine.

7. The method of claim 3, wherein the opioid is selected from Lortab, tramadol, heroin, methadone, hydrocodone, and oxycodone.

8. The method of claim 3, wherein the hallucinogen is selected from the group consisting of nudeomushroom, hallucinogen, lysergic acid diethylamide (LSD), phencyclidine (PCP), and ketamine.

9. The method of claim 3 wherein the inhalant is selected from the group consisting of benzene, toluene, o-xylene, m-xylene, p-xylene, ethylbenzene, fluorobenzene, o-difluorobenzene, 1, 3, 5-trifluorobenzene, 1, 2, 4-trifluorobenzene, pentafluorotoluene, pentafluorobenzene and perfluorobenzene.

10. The method of claim 2, wherein the drug therapy is selected from the group consisting of anesthetics, analgesics, anticholinergics, antihistamines, muscle relaxants, nonsteroidal anti-inflammatory drugs, over-the-counter drugs, and antidepressants.

11. The method of claim 2, wherein the drug of abuse is cocaCaffeine, alcohol, caffeine, opiates, cannabinoids, cannabis, benzodiazepinesMyo-inositol (carisprol), tobacco, nicotine, paracetamol (Vicodin), hydrocodone, oxycodone hydrochloride, oxycodone, and tylosin (Tylox).

12. The method of claim 11, wherein the drug of abuse is cocaine and the compound a reduces at least one symptom of cocaine abuse and dependence in the subject selected from the group consisting of: attention deficit hyperactivity disorder; the feeling of drunkenness; increased vitality, excitement and sociability; does not feel hunger and fatigue; a clear sense of physical and psychological strength; a reduction in pain sensation; bronchitis; short gas; chest pain; palpitations; cardiac arrhythmia; cardiomyopathy; heart failure; the pupil is dilated; nausea; vomiting; headache; dizziness; vertigo; anxiety; a mental disorder; confusion of consciousness; nasal inflammation; nasal crusts; periodic nasal bleeding; stuffy nose; facial pain; dysphoria; and cocaine craving.

13. The method of claim 11, wherein the drug of abuse is cocaine and compound a increases at least one negative subjective symptom of cocaine abuse and dependence.

14. The method of claim 11, wherein the drug of abuse is cocaine and compound a reduces at least one symptom of cocaine withdrawal selected from fatigue, lack of pleasure, depression, irritability, sleep disorders, increased appetite, mental retardation, agitation, extreme suspicion, and craving for cocaine.

15. The method of claim 1, wherein the method further comprises: co-administering a therapeutically effective amount of at least one other agent selected from the group consisting of: selective Serotonin Reuptake Inhibitors (SSRI), serotonin-norepinephrine reuptake inhibitors (SNRI), Norepinephrine Reuptake Inhibitors (NRI), norepinephrine-dopamine reuptake inhibitors (NDRI), serotonin 5-hydroxytryptamine 1A (5HT1A) antagonists, dopamine beta-hydroxylase inhibitors, adenylate receptor antagonists, adenosine A2A receptor antagonists, monoamine oxidase inhibitors (MAOI), monoamine oxidase B inhibitors, sodium channel blockers, calcium channel blockers, central and peripheral alpha adrenergic receptor antagonists, central alpha adrenergic agonists, central or peripheral beta adrenergic receptor antagonists, NK-1 receptor antagonists, Corticotropin Releasing Factor (CRF) antagonists, atypical antidepressants/antipsychotics, tricyclic drugs, anticonvulsants, glutamate antagonists, gamma-aminobutyric acid (GABA) agonists, GABA metabolic enzyme inhibitors, GABA synthesis activators, partial dopamine D2 agonists, dopamine metabolic enzyme inhibitors, catechol-O-methyl-transferase inhibitors, opioid receptor antagonists, mood stabilizers, direct or indirect dopamine agonists, partial 5HT1 agonists, serotonin 5HT2 antagonists, opioids, carboxylase inhibitors, partial opioid agonists, partial nicotinic acid agonists, and inhalants.

16. The method of claim 1, wherein the method further comprises: co-administering a therapeutically effective amount of at least one other agent selected from the group consisting of: benzodiazepineLevodopa, carnisprodol, modafinil, acamprosate, gamma-butyrolactone, gamma-hydroxybutyrate, opioids, dimethyl-4-hydroxytryptamine phosphate, maghemia, tobacco and nicotine.

17. The method of claim 1, wherein compound a is administered to the patient after a.

18. A method of treating at least one phase of substance dependence of a patient on at least one substance, wherein the at least one phase of substance dependence is selected from the group consisting of an acquisition, maintenance, regression, and relapse phase, the method comprising: administering to the patient a therapeutically effective amount of compound a.

19. The method of claim 18, wherein compound a inhibits progression to the acquisition stage in the patient.

20. The method of claim 18, wherein compound a promotes the progression through the remission stage of the patient.

21. The method of claim 18, wherein Compound A reduces the frequency of relapse in the patient.

22. The method of claim 18, wherein the at least one substance is selected from the group consisting of drugs of abuse and drug therapy.

23. The method of claim 22, wherein the drug of abuse is selected from the group consisting of psychostimulants, opioids, hallucinogens, inhalants, sedatives, tranquilizers, hypnotics, anxiolytics, and illicit substances.

24. The method of claim 23 wherein the psychostimulant is a β -phenylisopropylamine derivative

25. The method of claim 24 wherein the β -phenylisopropylamine derivative is selected from amphetamine, dextroamphetamine, and methamphetamine.

26. The method of claim 23 wherein the psychostimulant agent is selected from the group consisting of synthetic hallucinogens, phenmetrazine, ritaline, bupropion, pemoline, mazindol, (-) norpseudoephedrine, and fenfluramine.

27. The method of claim 23, wherein the opioid is selected from the group consisting of Lortab, tramadol, heroin, methadone, hydrocodone, and oxycodone.

28. The method of claim 23, wherein the hallucinogen is selected from the group consisting of nudeomushroom, hallucinogen, lysergic acid diethylamide (LSD), phencyclidine (PCP), and ketamine.

29. The method of claim 23 wherein the inhalant is selected from the group consisting of benzene, toluene, o-xylene, m-xylene, p-xylene, ethylbenzene, fluorobenzene, o-difluorobenzene, 1, 3, 5-trifluorobenzene, 1, 2, 4-trifluorobenzene, pentafluorotoluene, pentafluorobenzene and perfluorobenzene.

30. The method of claim 22, wherein the drug therapy is selected from the group consisting of anesthetics, analgesics, anticholinergics, antihistamines, muscle relaxants, nonsteroidal anti-inflammatory drugs, over-the-counter drugs, and antidepressants.

31. The method of claim 22, wherein the drug of abuse is alcohol, caffeine, opiates, cannabinoids, cannabis, benzodiazepinesMyo-inositol (carisprol), tobacco, nicotine, paracetamol (Vicodin), hydrocodone, oxycodone hydrochloride, oxycodone, and tylosin (Tylox).

32. The method of claim 18, wherein the method further comprises: co-administering a therapeutically effective amount of at least one other agent selected from the group consisting of: selective Serotonin Reuptake Inhibitors (SSRI), serotonin-norepinephrine reuptake inhibitors (SNRI), Norepinephrine Reuptake Inhibitors (NRI), norepinephrine-dopamine reuptake inhibitors (NDRI), serotonin 5-hydroxytryptamine 1A (5HT1A) antagonists, dopamine beta-hydroxylase inhibitors, adenylate receptor antagonists, adenosine A2A receptor antagonists, monoamine oxidase inhibitors (MAOI), monoamine oxidase B inhibitors, sodium channel blockers, calcium channel blockers, central and peripheral alpha adrenergic receptor antagonists, central alpha adrenergic agonists, central or peripheral beta adrenergic receptor antagonists, NK-1 receptor antagonists, Corticotropin Releasing Factor (CRF) antagonists, atypical antidepressants/antipsychotics, tricyclic drugs, anticonvulsants, glutamate antagonists, gamma-aminobutyric acid (GABA) agonists, GABA metabolic enzyme inhibitors, GABA synthesis activators, partial dopamine D2 agonists, dopamine metabolic enzyme inhibitors, catechol-O-methyl-transferase inhibitors, opioid receptor antagonists, mood stabilizers, direct or indirect dopamine agonists, partial 5HT1 agonists, serotonin 5HT2 antagonists, opioids, carboxylase inhibitors, partial opioid agonists, partial nicotinic acid agonists, and inhalants.

33. A method of treating at least one stage of cocaine dependence in a patient, wherein the at least one stage is selected from the group consisting of acquisition, maintenance, regression, and relapse stages, the method comprising: administering to the patient a therapeutically effective amount of compound a.

34. The method of claim 33, wherein compound a inhibits progression to the acquisition stage in the patient.

35. The method of claim 33, wherein compound a promotes the progression through the remission stage of the patient.

36. The method of claim 33, wherein compound a reduces the frequency of relapse in the patient.

37. The method of claim 33, wherein compound a reduces at least one symptom of cocaine abuse, dependence, or withdrawal in the patient.

38. The method of claim 37, wherein compound a reduces at least one symptom of cocaine abuse in the subject selected from the group consisting of: periodic cocaine use resulting in failure to fulfill major obligations at work, school, or home; periodic cocaine use in hazardous situations of the body; periodic cocaine use involving legal issues; and sustained cocaine use, although such substance use has persistent or periodic social or interpersonal problems caused or exacerbated by the consequences of cocaine.

39. The method of claim 37, wherein compound a reduces at least one symptom of cocaine dependence in the subject selected from the group consisting of: tolerance; withdrawal symptoms; often, large amounts of cocaine are ingested or then persist for longer periods; continued objective and/or unsuccessful efforts to curtail or control cocaine use; spending a lot of time in at least one activity in order to obtain, use and recover from cocaine; abandonment and/or reduction of at least one important social, occupational, and recreational activity due to cocaine use; and continued use of cocaine despite knowledge of persistent or periodic physical or psychological problems caused or exacerbated by cocaine.

40. The method of claim 37, wherein compound a reduces at least one symptom of cocaine abuse and dependence selected from the group consisting of: attention deficit hyperactivity disorder; the feeling of drunkenness; increased vitality, excitement and sociability; does not feel hunger and fatigue; a clear sense of physical and psychological strength; a reduction in pain sensation; bronchitis; short gas; chest pain; palpitations; cardiac arrhythmia; cardiomyopathy; heart failure; the pupil is dilated; nausea; vomiting; headache; dizziness; vertigo; anxiety; a mental disorder; confusion of consciousness; nasal inflammation; nasal crusts; periodic nasal bleeding; stuffy nose; facial pain; dysphoria; and cocaine craving.

41. The method of claim 37, wherein compound a increases at least one negative subjective symptom of cocaine abuse and dependence.

42. The method of claim 37 wherein compound a reduces at least one symptom of cocaine withdrawal selected from fatigue, lack of pleasure, depression, irritability, sleep disorders, increased appetite, mental retardation, agitation, extreme suspicion, and craving for cocaine.

43. The method of claim 33, wherein compound a increases the patient's score for at least one of: ADHD-IV, HAM-D, HAM-A, BDI, apathy and cognitive function scale from Neuropsychiatric inventory.

44. The method of claim 43, wherein the cognitive function rating scale is selected from the group consisting of WAIS-R, WMS-R, RAVLT, Trials I-VII, RCFT, and portions A and B of TMT.

45. The method of claim 33, wherein compound a reduces at least one of the amount and frequency of cocaine usage by the patient.

46. The method of claim 33, wherein compound a promotes remission in the patient.

47. The method of claim 46, wherein the symptom relief is characterized by: at least one of early complete symptom relief, early partial symptom relief, sustained complete symptom relief, and sustained partial symptom relief.

48. The method of claim 33, wherein compound a prolongs the period of remission in the patient.

49. The method of claim 33, wherein the method further comprises: treating with at least one of strain management and cognitive behavioral therapy.

50. The method of claim 33, wherein the method further comprises: co-administering a therapeutically effective amount of at least one other agent selected from the group consisting of: selective Serotonin Reuptake Inhibitors (SSRI), serotonin-norepinephrine reuptake inhibitors (SNRI), Norepinephrine Reuptake Inhibitors (NRI), norepinephrine-dopamine reuptake inhibitors (NDRI), serotonin 5-hydroxytryptamine 1A (5HT1A) antagonists, dopamine beta-hydroxylase inhibitors, adenylate receptor antagonists, adenosine A2A receptor antagonists, monoamine oxidase inhibitors (MAOI), monoamine oxidase B inhibitors, sodium channel blockers, calcium channel blockers, central and peripheral alpha adrenergic receptor antagonists, central alpha adrenergic agonists, central or peripheral beta adrenergic receptor antagonists, NK-1 receptor antagonists, Corticotropin Releasing Factor (CRF) antagonists, atypical antidepressants/antipsychotics, tricyclic drugs, anticonvulsants, glutamate antagonists, gamma-aminobutyric acid (GABA) agonists, GABA metabolic enzyme inhibitors, GABA synthesis activators, partial dopamine D2 agonists, dopamine metabolic enzyme inhibitors, catechol-O-methyl-transferase inhibitors, opioid receptor antagonists, mood stabilizers, direct or indirect dopamine agonists, partial 5HT1 agonists, serotonin 5HT2 antagonists, opioids, carboxylase inhibitors, partial opioid agonists, partial nicotinic acid agonists, and inhalants.