阅读说明：本技术 脂肪酸酰胺及其在治疗成瘾紊乱和成瘾相关状况中的用途 (Fatty acid amides and their use in treating addictive disorders and addiction-related conditions ) 是由 R·梅克霍拉姆 温琴佐·迪马索 菲比安娜·皮希泰利 阿伦·H·里奇曼 伊马德·M·达马杰 于 2018-05-22 设计创作，主要内容包括：本发明涉及氨基酸的脂肪酸酰胺,包括其立体异构体和盐,用于治疗罹患任何类型的成瘾紊乱、物质滥用紊乱的患者,包括罹患与所述成瘾紊乱、物质滥用紊乱相关的任何状况和症状的患者,并且包括在所述患者的康复治疗期间和之后的戒断综合征和成瘾复发。(The present invention relates to fatty acid amides of amino acids, including stereoisomers and salts thereof, for use in the treatment of patients suffering from any type of addictive disorder, substance abuse disorder, including patients suffering from any condition and symptom associated with said addictive disorder, substance abuse disorder, and including withdrawal syndrome and relapse of addiction during and after rehabilitation therapy of said patients.)

1. A fatty acid amide of an amino acid, including stereoisomers and salts thereof, for use in treating a patient suffering from an addictive disorder, including any conditions and symptoms associated with said addictive disorder.

2. A fatty acid amide of an amino acid, including stereoisomers and salts thereof, for use in the treatment of a substance abuse disorder, including conditions and symptoms associated with the substance abuse disorder.

3. A fatty acid amide of an amino acid, including stereoisomers and salts thereof, for use in treating a patient suffering from substance addiction, including any disorders, conditions, and symptoms associated with said substance addiction.

4. A fatty acid amide of an amino acid, including stereoisomers and salts thereof, for use in the treatment of a patient suffering from withdrawal syndrome during rehabilitation or detoxification from addiction to a substance of abuse.

5. A fatty acid amide of an amino acid, including stereoisomers and salts thereof, for use in the treatment of a patient suffering from relapse to addiction during or after rehabilitation or detoxification from addiction to substances of abuse.

6. The fatty acid amide according to any of claims 1 to 5, wherein the fatty acid moiety is selected from the group consisting of a saturated fatty acid moiety, a monounsaturated fatty acid moiety, and a polyunsaturated fatty acid moiety.

7. The fatty acid amide according to any of claims 1 to 6, wherein the amino acid is selected from glycine, dimethylglycine, alanine, serine, cysteine, tyrosine and phenylalanine.

8. The fatty acid amide according to any of claims 1 to 7, wherein the amino acid is substituted with at least one group selected from: straight or branched chain-C₁-C₆Alkyl, straight or branched-C₂-C₆Alkenyl, straight-chain or branched-C₂-C₆Alkynyl, -OH and-O (C)₁-C₁₀Alkyl groups).

9. Fatty acid amide according to any of the preceding claims, wherein the amino acid is substituted with at least one straight or branched chain-C₁-C₆Alkyl substitution.

10. Fatty acid amide according to any of the preceding claims, wherein the amino acid is substituted with at least two straight or branched-C chains₁-C₆Alkyl substitution.

11. The fatty acid amide of claim 9 or 10 wherein said-C₁-C₆Alkyl is methyl.

12. The fatty acid amide according to any of claims 9-11 wherein the substitution is at position α -of the amino acid moiety.

13. A fatty acid amide according to any of the preceding claims, wherein said fatty acid moiety is substituted with at least one group selected from: -C₁-C₆Alkyl, -OH, -O (C)₁-C₁₀Alkyl), -SH and-S (C)₁-C₁₀Alkyl groups).

14. The fatty acid amide of claim 13 wherein the fatty acid moiety is substituted with at least one-C₁-C₆Alkyl substitution.

15. The fatty acid amide of claim 13 wherein at least one C₁-C₆Alkyl is methyl.

16. The fatty acid amide of any of claims 13-15 wherein the at least one substitution is at least one of the α -position or β -position of the fatty acid moiety.

17. The fatty acid amide according to any of claims 1 to 5, which is a compound of general formula (I), including stereoisomers and salts thereof:

wherein

R₁Selected from straight or branched chain-C₁₃-C₂₂Alkyl, straight or branched-C₁₃-C₂₂Alkenyl and straight-chain or branched-C₁₃-C₂₂An alkynyl group; optionally substituted with at least one group selected from: straight or branched chain-C₁-C₆Alkyl, -OH, -O (C)₁-C₁₀Alkyl), -SH and-S (C)₁-C₁₀Alkyl groups);

R₂and R₃Independently selected from H, straight or branched-C₁-C₆Alkyl, straight or branched-C₂-C₆Alkenyl, straight-chain or branched-C₂-C₆An alkynyl group; each optionally substituted by at least one-OH, -SH, -O (C)₁-C₆Alkyl), phenyl and phenol;

provided that R is₂And R₃At least one of which is differentAt H.

18. The fatty acid amide of claim 17 wherein R₂is-C₁-C₆An alkyl group.

19. The fatty acid amide of claim 17 or 18 wherein R₃is-C₁-C₆An alkyl group.

20. The fatty acid amide of claim 17 wherein R₂And R₃Each independently is-C₁-C₆An alkyl group.

21. The fatty acid amide according to any of claims 17 to 20, wherein the-C₁-C₆Alkyl is methyl.

22. The fatty acid amide according to any of claims 17-21, wherein R₁Is straight-chain or branched-C₁₃-C₂₂An alkenyl group.

23. The fatty acid amide of claim 22 wherein the linear or branched-C₁₃-C₂₂Alkenyl groups contain between 1 and 6 double bonds.

24. The fatty acid amide of claim 1 wherein said addiction is a drug addiction, a cigarette addiction, an alcohol addiction, a food addiction, a behavioral addiction, and any combination thereof.

25. The fatty acid amide according to claim 1, wherein said addiction is nicotine addiction.

26. The fatty acid amide of claim 1 wherein the addiction is an opioid addiction.

27. The fatty acid amide of claim 1 wherein the addiction is a drug addiction.

28. The fatty acid amide of claim 1 wherein said addiction is an analgesic drug addiction.

29. The fatty acid amide of claim 1 wherein the addiction is ***e addiction.

30. The fatty acid amide of claim 1 wherein the addiction is a behavioral addiction.

31. The fatty acid amide according to claims 2 to 5, wherein said substance is a medicament, a cigarette, an alcoholic beverage, a food and any combination thereof.

32. A fatty acid amide according to claims 2 to 5 wherein the substance is nicotine.

33. A fatty acid amide according to claims 2 to 5 wherein the substance is an opioid.

34. The fatty acid amide of claims 2-5 wherein the substance is ***e.

35. The fatty acid amide according to claims 2 to 5 wherein said substance is an alcohol.

36. The fatty acid amide according to claims 2 to 5 wherein the substance is a food.

37. A fatty acid amide according to claims 2 to 5 wherein the substance is an analgesic drug.

38. A method of treating an addictive disorder, including any conditions and symptoms associated with said addictive disorder, in a patient suffering from said addictive disorder, including any conditions and symptoms associated with said addictive disorder, said method comprising administering to said patient a fatty acid amide of an amino acid, including stereoisomers and salts thereof.

39. A method of treating substance abuse disorders, including conditions and symptoms associated with said substance abuse disorders, in a patient suffering from, including conditions and symptoms associated with, said substance abuse disorders, comprising administering to said patient a fatty acid amide of an amino acid, including stereoisomers and salts thereof.

40. A method of treating substance addiction, including any disorders, conditions, and symptoms associated with said substance addiction, in a patient suffering from said substance addiction, including any disorders, conditions, and symptoms associated with said substance addiction, said method comprising administering to said patient a fatty acid amide of an amino acid, including stereoisomers and salts thereof.

41. A method of treating a patient suffering from withdrawal syndrome during rehabilitation or detoxification from addiction to substances of abuse comprising administering to the patient a fatty acid amide of an amino acid, including stereoisomers and salts thereof.

42. A method of treating a patient suffering from relapse from addiction during or after rehabilitation or detoxification from addiction treatment for substances of abuse comprising administering to the patient a fatty acid amide of an amino acid, including stereoisomers and salts thereof.

Background

Cigarette smokers with Traumatic Brain Injury (TBI) -induced damage to islet leaf cortex display showed cessation of nicotine addiction (Naqvi et al, 2007; Naqvi et al, 2014). Donvito et al subject anesthetized mice to a TBI weight drop model (weight drop model), and harvested the islet cortex, hippocampus, and hypothalamus after 24 hours. Using targeted lipidomics techniques, they demonstrated a significant increase in OlGly in the islet cortex of brain-injured mice, but not in the hippocampus or hypothalamus, and not in sham (sham) mice. OlGly by itself produces neither location preference nor location aversion (aversion), but it interferes with nicotine-induced location preference and with reduced promised withdrawal response and withdrawal-induced location aversion in nicotine-dependent mice.

Another substance abuse disorder that carries a great expense to society and individuals is opiate addiction. In 2014, 190 ten thousand of the us people aged 2150 ten thousand 12 years or 12 years old or older who suffered from substance use disorders had substance use disorders related to the prescribed analgesic Drug (pain reliever), and 586,000 had substance use disorders related to heroin (National Institute of Drug Abuse, Abuse 2015). Withdrawal from opiates is the driving force for maintenance of opiate addiction (e.g., Koob, 2009a, b). Morphine Withdrawal (MWD) can be produced by terminating chronic exposure to morphine or by administering an opioid antagonist to morphine-pretreated animals. Even after a single exposure to high doses of morphine, naloxone (naloxone) administered after several hours produces withdrawal symptoms in humans (Heishman et al, 1990; June et al, 1995) and other animals (Eisenberg, 1982; Martin and Eades, 1964). Withdrawal is evident not only in the behavioral symptoms of withdrawal (abstinence), but also in the ability of such withdrawal to serve as an aversive motivation stimulus. Parker et al (Parker & Joshi, 1998; Parker et al, 2002) demonstrated that the aversive nature of naloxone-promoted MWD in the Conditional Positional Aversion (CPA) paradigm is evident up to 48 hours after a single injection of morphine instead of saline.

Summary of The Invention

Accordingly, the present invention provides fatty acid amides of amino acids, including stereoisomers and salts thereof, for use in treating patients suffering from addictive disorders, including any conditions and symptoms associated with such addictive disorders.

Accordingly, the present invention provides fatty acid amides of amino acids, including stereoisomers and salts thereof, for use in the treatment of substance abuse disorders, including conditions and symptoms associated with said substance abuse disorders.

In a further aspect, the invention provides fatty acid amides of amino acids, including stereoisomers and salts thereof, for use in treating patients suffering from substance addiction, including any disorders, conditions and symptoms associated with said substance addiction.

The term "addiction" or "addictive disorder" is to be understood as a primary chronic disease that includes brain reward, motivation, memory, and associated circuits. The term refers to both irritant/compulsive seeking behavior and substance abuse dependence. The terms "substance addiction" and/or "substance abuse disorder" and/or "substance dependence" are to be taken as generic addictive disorders and in particular to the dependence of a subject on a specific substance or substances, which corresponds to an addictive disorder as defined above.

Compulsive pursuit includes, but is not limited to gambling, sex addiction, shopping addiction, compulsive addiction (such as over-cleaning and other compulsions that are typically within the OCD spectrum), and any combination thereof.

Substance abuse addiction includes, but is not limited to, drug addiction (including, but not limited to, opioids, such as heroin or other morphine derivatives, ***e, amphetamines (amphetamines), cannabis, any type of addictive drug, including, but not limited to, sleep inducers, analgesics, antihistamines, and the like), smoking, drinking, food consumption, and any combination thereof.

Without being bound by theory, addiction affects neurotransmission and interactions within brain reward structures, including nucleus accumbens (nucleus accumbens), cortices cingulate cortices (cortices), basal forebrain, and amygdala, such that the motor hierarchy is altered and addictive behaviors (which may or may not include alcohol and other drug use) replace healthy self-care related behaviors. Addiction also affects neurotransmission and interaction between cortical and hippocampal circuits and brain reward structures, such that memory previously exposed to rewards (such as food, sex, alcohol and other drugs) leads to biological and behavioral responses to external causes (external waters), which in turn trigger thirst and/or participation in addictive behaviors.

Addiction is characterized by a failure to continue withdrawal of substances or behavioral patterns, impaired behavioral control, craving substance or reward experience/behavior, cognitive impairment of subject behavior and major problems of interpersonal relationship; and emotional reactions of dysfunction. External triggers trigger thirst and drug use, as well as the strength to increase the frequency of participation in other potentially addictive behaviors, where the hippocampus is important in memory of previous euphoric or dysphoric experiences, and the amygdala is important in concentrating the motor on selecting behaviors associated with these past experiences, are also features of addiction.

The continued risk of relapse and/or reoccurrence after the withdrawal period is another essential feature of addiction. This can be triggered by exposure to reward substances and behaviors, by exposure to environmental cues for use, and by exposure to emotional stressors (emotional stressors) that trigger increased activity in the brain stress circuit.

Some of the symptoms associated with addiction include, for example, problems with impaired executive function, perception, learning, impulse control, compulsions and judgment, a low willingness to alter their dysfunctional behavior, showing a clear lack of understanding of the severity of cumulative problems and complications. Additional symptoms include the behavioral, cognitive, emotional, and interactive aspects of an individual, including the ability of an individual to associate with its family members, its community members, its own mental state, and things beyond its daily experience.

Performance and complications associated with addiction, primarily due to impaired control, may include: overuse and/or participation in addictive behaviors at a higher frequency and/or amount than expected by an individual, often associated with a sustained craving and unsuccessful attempts at behavior control; the loss of too long a time in substance use or recovery from the effects of substance use and/or participation in addictive behaviors has a major adverse effect on social and occupational functioning (e.g., development of interpersonal problems or neglect of liability in the home, school, or work); continued use and/or participation in addictive behaviors despite the existence of persistent or recurring physical or psychological problems that may be caused or exacerbated by substance use and/or related addictive behaviors; the behavioral set (behaviorerperoire) is narrowed down, focusing on rewards that are part of the addiction; and even if a problem is recognized, the ability and/or willingness to take consistent improvement action is clearly lacking.

Cognitive symptoms associated with addiction may include: addiction to substance use (precoccupation); evaluation of changes in relative pros and cons associated with drug or reward behavior; and to mistakenly believe that a problem experienced in one's life is due to other causes, rather than the predictable consequences of addiction.

Emotional symptoms associated with addiction include: increased anxiety, irritability and emotional distress; increased sensitivity to stressors associated with the recruitment of brain stress systems, making "things seem more stressful" a consequence; and difficulty in recognizing emotions, physical perception to distinguish emotions from emotional arousal, and describing emotions to others (sometimes referred to as dysemotions).

Since addiction is a chronic disease, a relapse phase that may interrupt the remission phase is a common feature of addiction. It is also important to recognize that the return of medication use or the pursuit of a reward for a disease state is not inevitable.

The qualitative way in which the brain and behavior respond to drug exposure and participate in addictive behavior differs in the later stages of addiction from the early stages, indicating a progression that may not be overtly evident.

The invention also provides a method of treating an addictive disorder, including any conditions and symptoms associated with said addictive disorder, in a patient suffering from said addictive disorder, including any conditions and symptoms associated with said addictive disorder, said method comprising administering to said patient a fatty acid amide of an amino acid, including stereoisomers and salts thereof.

In a further aspect, the present invention provides a method of treating substance abuse disorders, including conditions and symptoms associated with said substance abuse disorders, in a patient suffering from, said substance abuse disorders, including conditions and symptoms associated with said substance abuse disorders, said method comprising administering to said patient a fatty acid amide of an amino acid, including stereoisomers and salts thereof.

In yet another aspect, the invention provides methods of treating substance addiction, including any disorders, conditions, and symptoms associated with said substance addiction, in a patient suffering from said substance addiction, including any disorders, conditions, and symptoms associated with said substance addiction, comprising administering to said patient a fatty acid amide of an amino acid, including stereoisomers and salts thereof.

In some embodiments, the substance is a drug (including stimulants such as ***e and heroin, barbiturates, nicotine, analgesic drugs, sleep inducing drugs), cigarette, alcoholic beverages, food, and any combination thereof.

In some embodiments, the addiction is a drug addiction (including analgesic drugs, opioids, sleep-inducing drugs, etc.), cigarette addiction (also nicotine addiction), alcohol addiction, food addiction, behavioral addiction (including any type of OCD behavior, sexual addiction, narcolepsy, etc.), and any combination thereof.

In some embodiments, the addiction is nicotine addiction. In other embodiments, the addiction is an opioid addiction (a substance that acts on opioid receptors to produce a morphine-like effect).

In some embodiments, the substance is a drug, a cigarette, an alcoholic beverage, a food, and any combination thereof. In further embodiments, the substance is nicotine. In other embodiments, the substance is an opioid.

The term "addictive treatment" as used herein refers to the administration of a therapeutic amount of a compound and/or composition disclosed herein effective to ameliorate an addictive disorder, including its undesirable symptoms and conditions associated therewith, so as to prevent the addictive disorder, including the manifestations of its symptoms and conditions, prior to the occurrence of the addictive disorder, including its symptoms and conditions (e.g., in a subject in need of a treatment regimen with a drug having addictive potential, such as prior to or during treatment with an opioid), so as to slow the progression of addiction, slow the worsening of addiction and its symptoms, so as to promote the onset of a remission period, slow the irreversible damage caused in the progressive chronic phase of addiction, so as to delay the onset of the progressive phase, so as to reduce the severity of addiction and addictive behavior or to cure addiction and addictive behavior, so as to promote recovery, or so as to prevent the onset of addictive forms, so as to reduce the frequency and intensity of relapse of addiction, so as to maintain a remission period of addiction and addictive behavior, so as to optimize the level of function of the subject during the remission period; and any combination of the above.

The invention also provides fatty acid amides of amino acids, including stereoisomers and salts thereof, for use in treating patients suffering from withdrawal syndrome during rehabilitation or detoxification from addiction to substances of abuse therapy.

When referring to "withdrawal syndrome during rehabilitation or detoxification from addiction to a substance of abuse" it is to be understood as referring to any symptom occurring in a patient undergoing rehabilitation or detoxification treatment during which there is a complete or partial cessation of use of said substance of abuse or a reduction in the dose of said substance of abuse.

In another aspect, the invention provides fatty acid amides of amino acids, including stereoisomers and salts thereof, for use in treating a patient suffering from relapse to addiction during or after rehabilitation or detoxification from treatment for addiction to abused substances.

When referring to "relapse of addiction during or after rehabilitation or detoxification from addiction to a substance of abuse" is understood to refer to the result of a violation during or after rehabilitation or detoxification from addiction to a substance of abuse.

In some embodiments, the addiction is nicotine addiction. In other embodiments, the addiction is an opioid addiction. In other embodiments, the addiction is a drug addiction. In additional embodiments, the addiction is an analgesic drug (pain killer drug) addiction (including addiction to analgesic drugs (also known as analgesic drug addiction), addiction to drugs used to alleviate pain. In additional embodiments, the addiction is an analgesic drug addiction. In other embodiments, the addiction is a ***e addiction. In additional embodiments, the addiction is a behavioral addiction (including, but not limited to, eating addiction, drinking addiction, vomiting addiction, sex addiction, shopping addiction, gaming addiction, compulsive behavior addiction, gambling addiction, and the like).

In some embodiments, the substance is selected from the group consisting of a drug, a cigarette, an alcoholic beverage, a food, and any combination thereof. In some embodiments, the substance is nicotine. In other embodiments, the substance is an opioid. In further embodiments, the agent is ***e. In further embodiments, the substance is an alcohol. In a further embodiment, the substance is a food. In another embodiment, the substance is an analgesic drug.

The invention also provides a method of treating a patient suffering from withdrawal syndrome during rehabilitation or detoxification from addiction to substances of abuse comprising administering to the patient a fatty acid amide of an amino acid, including stereoisomers and salts thereof.

The invention also includes a method of treating a patient suffering from relapse from addiction during or after rehabilitation or detoxification from addiction treatment for substances of abuse comprising administering to the patient a fatty acid amide of an amino acid, including stereoisomers and salts thereof.

The term "fatty acid amide of an amino acid" as used herein is intended to include through the nitrogen atom in the amino acid moiety-NHCR₂R₃C (═ O) OH) and a carbonyl atom of a fatty acid moiety (-C (═ O) R₁) Forming amide bond between them, and making fatty acid portion (having general formula-C (═ O) R₁Wherein R is₁As defined herein) and an amino acid moiety (having the general formula-NHCR₂R₃C (═ O) OH, wherein R₂And R₃As defined herein) to the conjugate. It is to be understood that while the compounds of the invention are generally referred to as conjugates of fatty acid moieties and amino acid moieties, the conjugates of the invention can be formed from a variety of precursors using single-step or multi-step synthetic methods.

When reference is made to a "fatty acid moiety", it is to be understood as including an acyl moiety derivable from a fatty acid, i.e. typically R₁A form of C (═ O) -, wherein R₁Represents the aliphatic chain (saturated or unsaturated) of the corresponding fatty acid, and wherein the point of attachment of the fatty acid moiety to the amino acid moiety of the fatty acid amide is through the carbonyl carbon atom of the fatty acid moiety.

The term "fatty acid" as used herein is intended to include monocarboxylic acids having an aliphatic chain ("tail"), wherein the aliphatic chain can be saturated, monounsaturated (having one unsaturated bond anywhere on the aliphatic chain), or polyunsaturated (having at least two unsaturated bonds anywhere on the aliphatic chain). The unsaturated bonds on the aliphatic chain may be double bonds (in cis and/or trans configuration) or triple bonds. The length of the aliphatic chain (saturated, monounsaturated, or polyunsaturated) of the fatty acid may vary between 10 and 30 carbon atoms, or in some embodiments between 13 and 22 carbon atoms. The fatty acids may be derived from natural sources (animal or vegetable), synthetic or semisynthetic.

Non-limiting examples of saturated fatty acids are lauric, myristic, palmitic and stearic acids, non-limiting examples of monounsaturated fatty acids are myristoleic, palmitoleic and oleic acids, non-limiting examples of polyunsaturated fatty acids are linoleic, α -linolenic, arachidonic, eicosapentaenoic, erucic and docosahexaenoic acids.

In some embodiments, the fatty acid moiety of a fatty acid amide is selected from saturated fatty acid moieties (i.e., R)₁Is a hydrocarbon consisting of only a single saturated bond), monounsaturated fatty acid moiety (i.e., R)₁Is a hydrocarbon containing one unsaturated bond (double or triple bond)) and a polyunsaturated fatty acid moiety (i.e., R₁Is a hydrocarbon containing at least two unsaturated bonds (each independently a double or triple bond). In other embodiments of the invention, the fatty acid moiety is an oleoyl fatty acid moiety (CH)₃(CH₂)₇CH＝CH(CH₂)₇C (═ O) -), i.e. derived from the corresponding oleic acid.

In some further embodiments, the fatty acid moiety is substituted with at least one group selected from: -C₁-C₆Alkyl, -OH, -OR ', -SH and-SR', wherein R 'and R' are each independently straight OR branched-C₁-C₆An alkyl group. In other embodiments, the fatty acid moiety is substituted with at least one straight chain orBranched chain-C₁-C₆Alkyl substitution. In other embodiments, the fatty acid moiety is substituted with at least two straight or branched chains-C₁-C₆Alkyl substitution. In still other embodiments, the at least one C₁-C₆Alkyl is methyl.

In additional embodiments, the at least one substitution is at least one of the α -position or the β -position of the fatty acid moiety As is known in the art, "the α -position of the fatty acid moiety" is the carbon atom on the aliphatic chain of the fatty acid moiety that is directly adjacent to the carbonyl carbon atom of the fatty acid moiety, "the β -position of the fatty acid moiety" is the carbon atom on the aliphatic chain of the fatty acid moiety that is adjacent to the second carbon atom of the carbonyl carbon atom of the fatty acid moiety.

In some embodiments, the fatty acid amides of the present invention are substituted at the α -position of the fatty acid moiety.

When reference is made to an "amino acid moiety", it is to be understood as including radicals derivable from amino acids, i.e. generally of the formula-NHCR₂R₃COOH, wherein the point of attachment of the amino acid moiety to the fatty acid moiety as defined herein is through an amine of the amino acid moiety as set forth above.

An "amino acid" is an amino acid as known in the art (i.e., α -amino acid or β -amino acid)₂NCR₂R₃Amino acid of COOH, wherein R₂And R₃As defined above. Non-limiting examples of amino acids corresponding to the amino acid portion of the compounds defined herein are alanine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, dimethylglycine, proline, serine, tyrosine, arginine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine. As used hereinThe amino acids used may be derived from natural, synthetic or semisynthetic sources. Amino acids as used herein may also be in the D-configuration or the L-configuration. In some embodiments, the amino acid is an L-amino acid.

In some embodiments, the amino acid moiety is selected from the group consisting of serine, glycine, dimethylglycine, alanine, cysteine, tyrosine, and phenylalanine. In other embodiments, the amino acid moiety is serine.

In some embodiments of the invention, the fatty acid moiety is optionally substituted with one group selected from: -C₁-C₆Alkyl, -OH, -O (C)₁-C₁₀Alkyl), -SH and-S (C)₁-C₁₀Alkyl groups); and the amino acid moiety is optionally substituted with one group selected from: -C₁-C₆Alkyl, -OH and-O (C)₁-C₁₀Alkyl), phenyl, and phenol.

In further embodiments, the amino acid moiety is unsubstituted.

In still further embodiments, the amino acid moiety is substituted with at least one group selected from: -C₁-C₆Alkyl, -OH and-O (C)₁-C₁₀Alkyl) in which R is₃is-C₁-C₆An alkyl group. In other embodiments, the amino acid is substituted with at least one-C₁-C₆Alkyl substitution. In other embodiments, the amino acid is substituted with at least two-C₁-C₆Alkyl substitution. In a further embodiment, said-C₁-C₆In yet another embodiment, the substitution is at the α -position of the amino acid moiety.

"position α of said amino acid moiety" is the carbon atom on the amino acid moiety directly adjacent to the carbonyl carbon atom of the amino acid moiety.

In some further embodiments, the amino acid moiety is selected from the group consisting of serine, cysteine, glycine, dimethylglycine, alanine, tyrosine, and phenylalanine moieties. In some embodiments, the amino acid isThe moiety is substituted with at least one group selected from: straight or branched chain-C₁-C₆Alkyl, straight or branched-C₂-C₆Alkenyl, straight-chain or branched-C₂-C₆Alkynyl, -OH and-O (C)₁-C₁₀Alkyl groups).

In some other embodiments, the amino acid is substituted with at least one-C₁-C₆Alkyl substitution. In other embodiments, the amino acid is substituted with at least two-C₁-C₆Alkyl substitution. In still further embodiments, the-C₁-C₆In some embodiments, the substitution is at position α -of the amino acid moiety.

In some embodiments, the fatty acid moiety is substituted with at least one group selected from: -C₁-C₆Alkyl, -OH, -O (C)₁-C₁₀Alkyl), -SH and-S (C)₁-C₁₀Alkyl groups). In a further embodiment, the fatty acid moiety is substituted with at least one-C₁-C₆Alkyl substitution. In some embodiments, at least one C₁-C₆In other embodiments, the at least one substitution is at least one of the α -position or the β -position of the fatty acid moiety.

In some embodiments, the fatty acid amides of the present invention are compounds of general formula (I), including stereoisomers and salts thereof:

wherein R is₁Selected from straight or branched chain-C₁₃-C₂₂Alkyl, straight or branched-C₁₃-C₂₂Alkenyl and straight-chain or branched-C₁₃-C₂₂An alkynyl group; optionally substituted with at least one group selected from: -C₁-C₆Alkyl, -OH, -O (C)₁-C₁₀Alkyl), -SH and-S (C)₁-C₁₀Alkyl groups); r₂And R₃Independently selected from H, straight or branched-C₁-C₆Alkyl, straight or branched-C₂-C₆Alkenyl, straight-chain or branched-C₂-C₆An alkynyl group; each optionally substituted by at least one-OH, -SH, -O (C)₁-C₆Alkyl), phenyl and phenol; provided that R is₂And R₃Is different from H.

In some embodiments, R₂Is straight-chain or branched-C₁-C₆An alkyl group. In other embodiments, R₃Is straight-chain or branched-C₁-C₆An alkyl group. In other embodiments, R₂And R₃Each independently is-C₁-C₆An alkyl group. In still other embodiments, the-C₁-C₆Alkyl is methyl. In some embodiments, R₁Is straight-chain or branched-C₁₃-C₂₂An alkenyl group. In some embodiments, the straight or branched chain-C₁₃-C₂₂Alkenyl groups contain between 1 and 6 double bonds.

The term "stereoisomer" as used herein is intended to include isomers which have the same composition as the corresponding stereoisomer, but whose atoms are arranged in space other than the corresponding stereoisomer. For example, stereoisomers may be enantiomers, diastereomers and/or cis-trans (E/Z) isomers. It is to be understood that compositions comprising the fatty acid amides of the present invention may comprise a single enantiomer, a single diastereomer, and mixtures thereof in any ratio (e.g., racemic mixture, non-racemic mixture, mixture of at least two diastereomers, etc.). Furthermore, the present invention includes any stereoisomer of the fatty acid amide of the present invention obtained by in vivo or in vitro metabolism or by any type of synthetic route.

The term "salt" as used herein is intended to include any salt obtained by acid addition or base addition. In some embodiments, the salts are acid addition salts obtained by protonation of the fatty acid amides of the invention (e.g., at the amide moiety). In other embodiments, the salts are base addition salts obtained by deprotonation of a proton derived from a fatty acid amide of the invention (e.g., from the acidic moiety, i.e., -COOH of the fatty acid amide). The counter-ions forming the salts of fatty acid amides of the present invention may include, in a non-limiting manner, inorganic or organic cations, which in some embodiments are pharmaceutically acceptable, such as alkali metal cations, e.g., potassium or sodium cations, alkaline earth metal cations, such as magnesium or calcium or ammonium cations, including, for example, cations derived from organic nitrogen-containing bases, such as trialkylamine-derived cations, e.g., triethylammonium ions.

The term "alkyl" is intended to include monovalent linear (unbranched), branched, or cyclic saturated hydrocarbon radicals. When referring to "C₁-C₆Alkyl "is to be understood to include any linear or branched alkyl group having 1,2, 3, 4, 5 or 6 carbon atoms. C₁-C₆Non-limiting examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, 2-butyl, 3-butyl, n-isobutyl, 2-isobutyl, n-pentyl, 2-pentyl, 3-pentyl, 2-methylbutyl, 2-dimethylpropyl, n-hexyl, 2-hexyl, 3-hexyl, 2-methylpentyl, 3-methylpentyl, 2, 3-dimethylbutyl, 2-dimethylbutyl, 2-methyl-2-ethylpropyl, cyclobutyl, 1-methylcyclobutyl, 2-methyl-cyclobutyl, 1-dimethylcyclobutyl, 1, 2-dimethylcyclobutyl, 2-dimethylcyclobutyl, methyl-1-cyclobutyl, 1-cyclobutylethyl, tert-butyl, tert-butyl, 2-methyl-1-cyclobutyl, 1-ethyl, tert-butyl, 2-cyclobutylethyl, cyclopentyl, 1-methylcyclopentyl, 2-methylcyclopentyl. Similarly, when referring to "-C₁₀-C₃₀Alkyl "is to be understood to include any linear or branched alkyl radical having 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 carbon atoms. Similarly, when referring to "-C₁₁-C₂₀Alkyl "is to be understood to include any linear or branched alkyl radical having 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 carbon atoms. Similarly, when referring to "-C₁₃-C₂₂When "alkyl" it is to be understood to include alkyl groups having 13, 14, 15, 16, 17,Any linear or branched alkyl radical of 18, 19, 20, 21, 22 carbon atoms.

The term "alkenyl" is intended to include linear (unbranched) or branched hydrocarbon chains having at least one double bond. The double bond may be between any two carbon atoms of the alkenyl chain and may be in either the cis or trans (or E or Z) configuration. The double bond of the alkenyl group may be unconjugated or conjugated to another unsaturated group. When mentioning "-C₁₃-C₂₂When alkenyl "it is to be understood as including any linear or branched alkenyl radical having 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 carbon atoms. Similarly, when referring to "-C₁₁-C₂₀Alkenyl "is to be understood to include any linear or branched alkenyl radical having 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 carbon atoms. Similarly, when referring to "-C₁₀-C₃₀When alkenyl "it is to be understood as including any linear or branched alkenyl radical having 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 carbon atoms.

The term "alkynyl" is intended to include linear (unbranched) or branched hydrocarbon chains having at least one triple bond. The triple bond may be between any two carbon atoms of the alkynyl chain. The triple bond of the alkynyl group may be unconjugated or conjugated to another unsaturated group. When mentioning "-C₁₃-C₂₂Alkynyl "is to be understood to include any linear or branched alkynyl radical having 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 carbon atoms. Similarly, when referring to "-C₁₁-C₂₀Alkynyl "is to be understood to include any linear or branched alkynyl radical having 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 carbon atoms. Similarly, when referring to "-C₁₀-C₃₀Alkynyl "is to be understood to include groups having 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22Any linear or branched alkynyl radical of 23, 24, 25, 26, 27, 28, 29, 30 carbon atoms.

The term "phenyl" is understood to mean a compound having the formula C₆H₅The aromatic cyclic group of (1). The term "phenol" is understood to mean a compound having the formula C₆H₄An aromatic group of OH, wherein the-OH group at any point on the cyclic ring may be substituted.

Certain of the above-defined terms may occur more than once in a structural formula, and upon such occurrence, each term should be defined independently of the other terms.

The term "optionally substituted" as used herein means that the group in question is unsubstituted or substituted with one or more of the substituents specified. When a group in question is substituted with more than one substituent, the substituents may be the same or different.

In another aspect, the invention includes a pharmaceutical composition comprising a fatty acid amide as disclosed herein, including any stereoisomers and salts thereof. The present invention also provides a pharmaceutical composition comprising at least one fatty acid amide as disclosed herein, including any stereoisomers and salts thereof, in combination with at least one other therapeutic agent. The invention also provides the use of the fatty acid amides disclosed herein for the preparation of a pharmaceutical composition.

The present invention also relates to a pharmaceutical composition comprising a fatty acid amide disclosed herein in combination (e.g., admixture) with a pharmaceutically acceptable adjuvant and optionally at least one additional therapeutic agent. Adjuvants are necessarily "acceptable" in the sense that they are compatible with the other ingredients of the composition and not deleterious to the recipient thereof.

Pharmaceutical compositions include those suitable for oral, rectal, intranasal, topical (including transdermal, buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration or administration via an implant.

In some embodiments, the pharmaceutical compositions disclosed herein are transdermal compositions. In some other embodiments, the fatty acid amides disclosed herein are administered to a patient using a transdermal formulation. In some embodiments, the transdermal formulation/composition employs a skin patch.

In some embodiments, the pharmaceutical compositions disclosed herein are intranasal compositions. In some other embodiments, the fatty acid amides disclosed herein are administered to a patient using an intranasal formulation. In some embodiments, the intranasal formulation/composition employs a delivery device (e.g., a nebulizer).

The compositions may be prepared by any method well known in the pharmaceutical arts. Such methods include the step of associating the fatty acid amides of the present invention or combinations thereof with any adjuvant. Adjuvants, as auxiliary ingredients, are generally selected from those conventional in the art, such as carriers, fillers, binders, diluents, disintegrants, lubricants, colorants, flavoring agents, antioxidants and wetting agents.

Pharmaceutical compositions suitable for oral administration may be presented as discrete dosage units, such as pills, tablets, dragees or capsules, or as powders or granules, or as solutions or suspensions. The active ingredient may also be presented as a bolus or paste. The compositions may also be formulated as suppositories or enemas (enema) for rectal administration.

The invention also includes a pharmaceutical composition as described above in combination with a packaging material comprising instructions for use of the composition for use as described above.

For parenteral administration, suitable compositions include aqueous and non-aqueous sterile injections. The compositions may be presented in unit-dose or multi-dose containers, for example, sealed vials and ampoules, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water, prior to use. For transdermal administration, for example, gels, patches or sprays are contemplated. Compositions or formulations suitable for pulmonary administration, for example by nasal inhalation, include fine dusts or mists which may be generated by means of a metered dose pressurised aerosol, nebuliser or insufflator.

The precise dosage and regimen of administration of the composition will necessarily depend on the effect to be achieved and may vary with the particular formulation, route of administration, and age and condition of the individual subject to which the composition is to be administered.

The invention also provides a kit comprising at least one compound of the invention as described above or a pharmaceutical composition comprising a compound of the invention and instructions for its use.

The invention also provides methods of treating patients suffering from addictive disorders, including any conditions and symptoms associated with such addictive disorders; the method comprises administering to the patient a fatty acid amide of at least one amino acid, including stereoisomers and salts thereof.

The present invention provides methods of treating substance abuse disorders, including conditions and symptoms associated with said substance abuse disorders, comprising administering to a patient a fatty acid amide of at least one amino acid, including stereoisomers and salts thereof.

In a further aspect, the invention provides methods of treating a patient suffering from substance addiction, including any disorders, conditions, and symptoms associated with such substance addiction; the method comprises administering to the patient a fatty acid amide of at least one amino acid, including stereoisomers and salts thereof.

In the following detailed description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. However, it will be understood by those skilled in the art that the present invention may be practiced without these specific details. In other instances, well-known methods, procedures, and components have not been described in detail so as not to obscure the present invention.

Part I: oleoyl glycine interferes with morphine withdrawal but does not interfere with morphine reward

Test subject

Male Sprague-Dawley rats (200g to 250g) were used as subjects. Animals were housed in pairs in opaque shoe box cages while receiving food and water ad libitum. They were exposed to 12/12h of reverse light/dark cycle with the lights turned on at night 7 h. All experiments were performed during the dark cycle of rats. The living room (colony) of all rats housed was maintained at 21 ℃. All Animal procedures were approved by the Animal Care Committee of Guilph, University of Guilfu, and followed the guidelines of the Canadian Council of Animal Care.

Medicine

Morphine and naloxone were prepared with saline at concentrations of 20mg/ml and 1mg/ml, respectively, and then injected subcutaneously (sc) in an amount of 1ml/kg (volume). OlGly and AM251 were dissolved in a vehicle mixture of ethanol, Tween 80 and saline in a ratio of 1:1: 18. Both oleoyl glycine and AM251 were first dissolved in ethanol, then Tween 80 was added to the solution and the ethanol was evaporated off with a stream of nitrogen; after that, brine was added. The final Vehicle (VEH) consisted of 1:9 (Tween/saline). Oleoyl glycine was prepared at a concentration of 5mg/ml or 30mg/ml and injected intraperitoneally.

Device

A position training device with a movable floor is used. The training device is a rectangular box (60X 25cm) made of black Plexiglas and a wire mesh cover. During training, a removable metal floor characterized by a perforated surface (1 cm diameter, 1cm apart from each other) or a grid surface (1/2cm horizontal bars, 1cm apart) was placed on a black rubber mat on top of the black Plexiglas surface. The different backplanes serve as situational cues to differentiate between the processing backplane and the VEH backplane. During the test and pre-test trials, ferrous metal floor plates (half perforated surface and half grid surface) divided into two equal halves were placed in a training box. The tactile stimulation characteristics of the two floor halves are identical to their matching floor counterparts used in training. Ethovision software was used to define the box and floor type perimeters, as well as to define the neutral zone.

Procedure for measuring the movement of a moving object

All rats received a 10min drug-free pre-test to measure baseline floor preference. Ethovision tracked the movement of the rats throughout the experiment to determine how much time was spent on each floor. Each rat is then assigned to a specific drug group and drug floor (well floor or grid floor) in a balanced manner. Rats with a bias of over 200s for either floor were removed. Between each test, the floor and training box were cleaned.

Experiment 1: potential of OlGly in generating CPP or CPA

Rats (n-12) received two training trials with oleoyl glycine. For each test they received 5mg/kg of oleoyl glycine or VEH as intraperitoneal (ip) injections (24 hours apart; in order of equilibration) and after 20 minutes were placed in a training chamber lined with grid or well plates (equilibrated) for 20 minutes. Three days after the last training day, rats received a 10min drug-free test using a separate grid/well plate.

Experiment 2: effect of systemic OlGly on naloxone-promoted establishment of MWD-CPA

Rats (n-22) received two 3-day training cycles to obtain naloxone-induced site avoidance. On day 1, the floor opposite the designated drug floor was paired with a subcutaneous saline injection. After 10 minutes of saline injection, rats were placed in a training chamber with a designated saline paired floor for 20 minutes while their movements were tracked with Ethovision. On day 2, rats received a high dose of morphine (20mg/kg) subcutaneously 24h after the saline training trial of the previous day. After injection, they were placed in empty shoe box cages and monitored for signs of respiratory distress and stimulated as necessary until they recovered and were returned to the containment cage. On day 3, rats were injected with VEH (n-12) or OlGly (n-12) 24h after morphine injection, and received subcutaneous injections of naloxone 10min later. After 10min, they were placed in a training box with a prescribed naloxone pairing floor for 20 minutes while their movements were tracked using Ethovision. Four days later, all rats were subjected to a second 3-day training cycle. Five days after the last naloxone test, a 10min drug free test was performed. The test trial consisted of the same procedure as the pre-test trial, but rats were given subcutaneous saline injections 10min prior to the test. Ethovision tracked the amount of time rats spent on each floor surface during the test trials.

Experiment 3: effect of systemic OlGly on establishment of Morphorphine-induced CPP

Rats received four 2-day training trials to generate morphine-induced conditioned place preference. During each training trial, all rats received a subcutaneous injection of morphine (10mg/kg) on one day and saline (in balanced order) on another day, and were placed 10min later in a training room with morphine-paired or saline-paired floors, respectively, for a duration of 30 min. For morphine training experiments rats were administered an intraperitoneal injection of VEH (n-11), 5mg/kg OlGly (n-11) or 30mg/kg OlGly (n-10), followed by a morphine injection 10min later. For saline training experiments, all rats were injected with VEH and saline injection was performed 10min later. Three days after the last training day, rats received a 10min drug-free test using a separate grid/well plate. All rats received subcutaneous administration of saline, 10 minutes later for each test experiment.

Results

Experiment 1: potential of oleoyl glycine to generate CPP or CPA

OlGly did not produce a significant preference or aversion to drug paired bottom plates, t (11) ═ 0.09, ns. Rats spent equal time on VEH paired floors (M232.16 sec, +36.44) and their spent equal time on oleoyl glycine paired floors (M299.00 sec, + 36.44). Furthermore, activity measurements showed that oleoyl glycine had no motor effect during training (motoric effect) compared to VEH.

Experiment 2: effect of systemic oleoyl Glycine on establishment of MWD-CPA

OlGly significantly interfered with naloxone to promote the establishment of MWD-induced CPA. Figure 1 shows the average number of seconds (+ sem) spent by rats receiving VEH or oleoyl glycine on the saline paired baseplate and MWD paired baseplate in the no drug test trial during each MWD trial of experiment 2.2 x 2 mixed factor analysis of variance (ANOVA) with factors between groups of pre-treated drugs (VEH, 5mg/kg OlGly) and factors within groups of the plates (MWD, saline) showed significant drug plate interactions, F (1,20) ═ 6.80, p ═ 0.017. Subsequent paired t-tests showed that there was floor aversion in only the VEH group, t (11) ═ 4.59, and p < 0.001. The activity evaluation during the training trial showed a significant effect of the training drug, F (1,20) ═ 118.75; p <0.001, where rats apparently lack activity during the naloxone training trial compared to the saline training trial, but pretreatment with OlGly did not change activity.

Experiment 3: effect of systemic OlGly on establishment of Morphorphine-induced CPP

5mg/kg or 30mg/kg of OlGly did not alter the establishment of morphine-induced positional preference. Figure 2 presents the average (+ sem) seconds spent by rats receiving VEH, 5mg/kg or 30mg/kg OlGly on the saline paired board and MWD paired board during the no drug test trial during each MWD training trial. A 3 x 2 mixed factor anova with interclass factors (VEH, 5mg/kg OlGly, 30mg/kg OlGly) for the pre-treatment drug and intraclass factors (morphine, saline) for the floor showed significant effect only for the floor, with F (1,31) ═ 5.62, p ═ 0.025, with no significant drug floor interaction. Overall, all rats showed morphine-induced CPP, but systemic OlGly administration did not alter this preference. Furthermore, the activity evaluation during the training trial showed a significant experimental effect, F (1,31) ═ 26.40; p <0.001, wherein rats apparently lack activity during morphine training trials compared to saline training trials, but pretreatment with OlGly did not change activity.

Part II: oleoyl glycine produced by brain trauma in mice reduced nicotine reward and withdrawal

Animal(s) production

Male C57BL/6 mice (Charles River, Italy) weighing 18-20 g were used in a mild TBI Weight Drop (WD) model. Mice were housed for at least 1 week in three cages under controlled illumination (12h light/dark cycle; 6:00 light in the morning) and standard environmental conditions (ambient temperature 20 ℃ -22 ℃, humidity 55% -60%), before the experiment was started. Animal food and tap water are available ad libitum. Male ICR mice (6-8 weeks old; Harlan, Indianapolis, IN) with a body weight of 27 g-32 g were used as subjects for all IN vivo pharmacological experiments. Mice were housed in groups (four per cage) with 12/12 light/dark cycles (0600h light) and given food and water ad libitum. All Animal protocols were approved by The Virginia Commission on The university of Federal Animal Care and UseCommittee, following The guidelines for The Care and Use of The national institutes of Health (The national institutes of Health Guide for The Care and Use of Laboratory Animals), 2011, and approved by The Navie Second university Animal Ethics Committee (The Animal Ethics Committee of The Second university of Laboratory Resources), in Italy (D.L.116/92) and in European Committee (O.J.of E.C.L358/118/12/86) for The protection of Animals. All efforts were made to reduce the number of animals and the pain during the experiment.

Surgical preparation and injury (mouse WD model)

Experimental mild tbi (mtbi) was performed using a weight drop device developed by the neapolit laboratory. Mice were anesthetized by intraperitoneal injection of 250mg/kg Avertin (Avertin) followed by mTBI. After the midline longitudinal incision, the skull was exposed to locate the impact area and placed under a metal tube device with the opening directly over the animal's head. Damage was induced by dropping a cylindrical metal weight (50g) from a vertical metal tube of 20cm height. The point of impact is between the anterior coronal slot (bregma) and the posterior coronal slot (lambda). Immediately after injury, the skin was closed with surgical wound clips (surgical wound clip) and the mice were returned to their cages to allow recovery from anesthesia and mTBI. Sham operated mice received the same procedure as described for mTBI, but did not release the weight.

Medicine

[²H]₈AEA、[²H]₅2-AG、[²H]₄PEA、[²H]₄OEA、[²H]₈N-arachidonoyl dopamine (NADA), (A) and (B)²H]₈AraSier and [2 ]²H]₈AraGly is available from Cayman Chemicals (MI, USA). OlGly was synthesized in Mechoulam laboratories and CP55,940((-) -cis-3- [ 2-hydroxy-4- (1, 1-dimethylheptyl) phenyl)]Trans-4- (3-hydroxypropyl) cyclohexanol) and morphine sulfate were generously provided by NIDA (Rockville, MD). OlGly and CP55,940 were dissolved in a vehicle solution consisting of ethanol (5% of total volume), alkamuls-620(Sanofi-Aventis, Bridgewater, NJ) (5% of total volume) and saline (0.9% NaCl) (90% of total volume). Oleoyl glycine and CP55,940 were administered via the intraperitoneal (i.p.) route of administration. (-) -Nicotine bitartrate [ (-) -1-methyl-2- (3-pyridyl) pyrrolidine (+) -bitartrate]And mecamylamine HCl hydrochloride (mecamyamine HCl) was purchased from Sigma-aldrich inc. Morphine sulfate [ morphine hemisulfate pentahydrate]]Nicotine and mecamylamine (2mg/kg) were dissolved in physiological saline and given in amounts of 10ml/kg via the subcutaneous (s.c.) route of administration. For the nicotine CPP study, a 0.5mg/kg nicotine dose was used, as this dose reliably produced significant CPP in ICR mice (18). Morphine CPP was performed at 10mg/kg (subcutaneously) as recently described (19). For the nicotine withdrawal study, 24 mg/kg/day nicotine or saline was infused continuously for 14 days using a subcutaneous osmotic minipump (model 2000; Alzet Corporation, Cupertino, CA) implanted under isoflurane anesthesia. Among the three behavioral paradigms used herein, this long-term nicotine administration regimen reliably produced significant withdrawal syndrome.

Synthesis of oleoyl glycine

To a solution of oleic acid (1gm, 3.54mmol) and N, N-dimethylformamide (266 μ L,3.64mmol) in dry dichloromethane (10mL) under a nitrogen atmosphere was added oxalyl chloride (2.0M solution in dichloromethane, 3.5mL, 7mmol) dropwise. The reaction mixture was stirred for 1h, and then the solvent was evaporated under a flow of nitrogen. The crude material in dichloromethane (01mL) was added to a solution of glycine (800mg,01.01mmol) and 2N potassium hydroxide in an ice bath. The reaction mixture was then stirred for 1h, water (10mL) was added, and the mixture was acidified to pH 3 with 1N HCl. The product was extracted with ether (3X 01mL) and dried (MgSO)₄) And evaporating the solution under reduced pressureAnd (3) preparing. The crude material was chromatographed on silica gel (eluting with chloroform: methanol) to give a crystalline solid. Melting point 93-94 deg.C (degradation); LC-MS: (M-H)⁺＝339m/z；NMR(CD₃OH,ppm)：5.35-5.32(m,2H),4.45(s,2H),2.13-2.18(m,6H),1.58(m,2H),1.32-1.29(m,20H),0.88(t,3H)。

Extraction and quantification of endocannabinoids, N-acylethanolamines, N-acyldopamine, N-acylserines and N-acylglycines

Immediately after dissection brain tissue was frozen in liquid nitrogen, which occurred within 5min after sacrifice. The frozen tissue was then homogenized (dounce-homogenized) and extracted with chloroform/methanol/Tris-HCl 50mM pH 7.5(2:1:1, v/v) containing internal deuterated standards (10pmol for [ 10 ] pmol for [ 1 ]) by isotopic dilution for AEA, 2-AG, PEA, OEA, NADA, AraSer and AraGly quantitation²H]₈AEA; 50pmol for [ alpha ], [ alpha²H]₅2-AG、

[²H]₄PEA and [2 ]²H]₄OEA; 5pmol for [ alpha ], [ alpha²H]₈NADA、[²H]₈AraSier and [2 ]²H]₈AraGly). The lipid extract is then purified by open bed chromatography on silica. The fractions were eluted in CHCl as previously described (22, 23)₃In increments of CH₃In OH, and a portion of the 9:1(v/v) fraction was analyzed by liquid chromatography-atmospheric pressure chemical ionization-single quadrupole mass spectrometry for AEA, 2-AG, PEA and OEA levels. AEA, 2-AG, PEA and OEA levels were calculated based on their area ratios to the internal deuterated standard signal areas. A portion of the 9:1 fraction was used for N-acyl dopamine identification, while the 7:3 fraction was used for N-acyl glycine and N-acyl serine identification and quantification, by LC-MS-IT-TOF (Shimadzu Corporation, Kyoto, Japan) equipped with ESI interface, using Multiple Reaction Monitoring (MRM). The method for NADA is as described previously. By using molecular ions [ M + H ] corresponding to deuterated and non-deuterated AraGly]⁺370.3192 and 362.2692; or the molecular ions [ M + H ] corresponding to deuterated and non-deuterated AraGly]⁺400.3297 and 392.2795 by co-locationQuantification was performed by dilution of the extract. The recovery of AraGly and AraSer from rat brain tissue using the extraction and analysis procedure reported herein (see methods) was 49.1 ± 15.7% and 42.1 ± 15.9%, respectively (n ═ 7). The LC-ESI-IT-ToF method is specific and shows a detection limit of 50fmol (LOD, defined as a concentration with a signal-to-noise ratio greater than 3: 1) in MS mode and 1pmol in MS/MS mode for all compounds analyzed. Furthermore, the [ M + H ] of undeuterated (0.025pmol to 10pmol) versus deuterated (1pmol) AraGly and AraSar]⁺The ratio between peak areas varies linearly with the amount of the respective deuterated standard. The limit of quantitation of the compounds is 100fmol and the reproducibility of the method is 95% -99%. High resolution [ M + H]⁺Chromatograms of values are extracted and used for calibration and quantification. LC analysis in isocratic mode using a Kinetex C18 column (10 cm. times.2.1 mm, 5 μm) and CH₃OH/Water/acetic acid (85: 15:0.1 by volume) as mobile phase at a flow rate of 0.15 ml/min. The identification of N-acyl dopamine, N-acyl glycine and N-acyl serine was performed using ESI ionization in positive mode using an atomizing gas flow of 1.5ml/min and a curvilinear desolvation line temperature of 250 ℃.

Conditional Positional Preference (CPP) study

As previously described, an unbiased CPP paradigm was performed. In short, a CPP device consists of three chambers (MedAssociates, st. albans, VT, ENV3013) arranged linearly, with white and black chambers (20 × 20 × 20cm each) that are also different in floor texture (white mesh or black bar). These chambers were separated by a small grey chamber with a smooth PVC floor. Partitions (partitions) may be removed to allow access from the grey chamber to the black and white chambers. On day 1, animals were confined to the middle compartment for a habitual time of 5min, and then allowed to move freely in all three compartments for 15 min. The time spent in each chamber was recorded and no systematic bias was observed in the baseline chamber preference. Training was performed twice daily for 20min (days 2-4). During the training period, the mice were confined to one of the larger chambers. The control group received saline in one large room in the morning and in another large room in the afternoon. The nicotine group receives nicotine in one large chamber and saline in another large chamber. The treatments were equally balanced to ensure that some mice received nicotine in the morning while other mice received nicotine in the afternoon. The nicotine pairing chamber is randomized in the subject. Epoch intervals were 4h and were performed by the same investigator. On each day of the training day, mice were pretreated with OlGly (intraperitoneal) or vehicle and nicotine or morphine (subcutaneous) injections were performed 15min later. After 5min of nicotine administration, the subjects were given a training period of 20 min. In the morphine CPP comparison study, mice were given a 30min training period 15min after morphine pretreatment (10mg/kg subcutaneously) (19). On the test day (day 5), mice were allowed to enter all chambers without drug for 15 min. The preference score is calculated by determining the difference between the time spent on the drug-mating side during the test day relative to the time spent on the drug-mating side during the baseline day.

Research on nicotine prompting withdrawal

Mice were implanted with subcutaneous osmotic minipumps (model 2000; Alzet Corporation, Cupertino, CA) under isoflurane anesthesia. The pump delivers 24 mg/kg/day nicotine or saline for 14 days. The concentration of nicotine was adjusted based on animal weight and micropump flow rate. On day 15 morning, mice were given subcutaneous injections of the non-selective nicotinic acetylcholine receptor (nAChR) antagonist mecamylamine (2mg/kg, subcutaneous) and 15min later were administered vehicle or OlGly (10mg/kg, 30mg/kg and 60mg/kg, intraperitoneal). As previously described (24), signs of nicotine withdrawal from the emotion (anxiety-like behavior) and body (physical signs, hyperalgesia) were evaluated for 10min beginning after mecamylamine administration. The anxiety-related behavior of the mice was first evaluated in the plus maze test for 5 min. The duration of time spent in the open arm (openarm) of the plus maze was evaluated as a measure of anxiety-related responses. The number of arm crossings between the opened and closed arms (closed arms) is also counted as a measure of the motion activity. Immediately after the maze assessment, observations were made for 20min for the measured body signs including paw and body tremor, shaking head, receding, jumping, curl (curl) and ptosis (ptosis). During the observation time, the mice were placed in a transparent activity cage without bedding. The total number of body signs was recorded for each mouse and the mean number of body signs during the observation time was plotted for each test group. Immediately after the time of observation of the somatic signs, hyperalgesia was evaluated using the hot plate test. The mice were placed in a 10-cm wide glass cylinder on a hot plate (Thermojust Apparatus, Richmond, Va.) maintained at 52 ℃. The delay (latency) of the reaction time (jumping or licking of the paw) was recorded. The particular test sequence was selected based on our previous studies which showed that this test sequence reduced intra-group variability and produced the most consistent results (24). All studies were performed by observers blinded to experimental treatment.

Tetrad behavior assessment

Mice were acclimated to the test environment for at least 1h, and then the four body parts were tested: spontaneous activity, catalepsy (catalepsy), antinociceptive effects and hypothermia (7-9). In the exercise study, subjects were administered a vehicle or drug and placed 5min later in a clear acrylic box (approximately 44.5cm × 22.25cm × 20.0cm) located within a sound-deadening cabinet equipped with an LED light source and a fan for general air circulation and white noise generation. Use of Fire-i from Unibrain (San Ramon, Calif., USA)^TMDigital camera and ANY-maze from Stoelting Company (Wood Dale, IL, USA)^TMVideo tracking software, collecting and recording the distance traveled (cm) and immobility time spent(s) for each mouse for 10 min. The mice were evaluated for baseline tail-tailing delay and body temperature, given intraperitoneal (i.p.) injection of vehicle or drug (OlGly), and 30min later evaluated in the following order: catalepsy, tail-shortening test and body temperature. The stiffness was measured using a horizontal bar test in which the two forelimbs of the mouse were placed on a horizontal bar (approximately 1.25cm in diameter and 4.5cm parallel to the bench top) and the duration of a fixed and immobile posture (except for normal breathing) was recorded by a stopwatch at intervals of 60 s. The antinociceptive effect was determined in a warm water (52 ℃) tail dip test, in which the distal end of the tail (about 1cm) was dipped into a water bath and the small record was madeDelay for mice to retract their tail (to the nearest 0.1 s). A 10s cutoff was used to minimize tail damage. The antinociceptive effect data is converted to a percentage of maximum effect (% MPE) by the following equation: % MPE [ [ (test delay-pretreatment delay)/(10-pretreatment delay)]X 100. Body temperature measurements (recorded to the nearest 0.1 ℃) were collected by inserting a rectal probe to a depth of 2cm, lubricated with mineral oil, and attached to a remote thermometer (Yellow Springs industries inc., Yellow Springs, OH, USA).

Cumulative CP55,940 dose response study

Mice were pre-treated with OlGly (60mg/kg intraperitoneally) or vehicle, and after 10min they received a first dose of CP55,940, followed by each subsequent dose every 40 min. Measurements of catalepsy, tail flick, and rectal temperature were taken 30min after each CP55,940 administration and prior to any injections to determine baseline responses. Cumulative dosages of CP55,940 were 0.3mg/kg, 1mg/kg, and 3mg/kg i.p.. The motor activity was not evaluated due to habitual effects that occurred after repeated testing.

Statistical analysis

Unless otherwise stated, lipid levels are expressed as the mean ± standard error (M ± SEM) of pmols/g wet tissue weight. One-way anova followed by a graph-based test (Tukey's test) was used to compare AEA, 2-AG, PEA, OEA and OlGly levels between the different groups. P values below 0.05 were considered significant. For conditional location studies, a preference score is calculated by subtracting: the time spent in the nicotine pairing chamber after training minus the time spent before training. Positive values indicate a preference for the nicotine (or morphine) counterpart compartment, while negative values indicate avoidance of the nicotine (or morphine) counterpart compartment. A value of zero or close to zero indicates no preference. Data were analyzed by one-way analysis of variance and further analyzed by student Neuman-Keuls post hoc tests. In the tetrad study and luciferase assay, data were analyzed by one-way anova followed by Dunnett's post-hoc test. In the cumulative dose response of CP55,940, data were analyzed by two-way anova followed by Sidak post hoc tests. In the luciferase assay, student's t-test with Welch correction was applied.<A P value of 0.05 was considered statistically significant. In binding studies, K_iValues were calculated by applying the Cheng-Prusoff equation to IC50 values for displacement of bound radioligand by increasing the concentration of test compound. The computer program GraphPad Prism version 6.0 (GraphPad Software inc., San Diego, CA) was used for all statistical analyses. All data are expressed as mean +/-SEM.

Results

Fig. 3A-3C show representative chromatograms depicting the presence of OlGly in island leaves of TBI mice, but not in island leaves of sham or naive mice. In FIG. 3A (1), the damaged island leaves showed the formation of OlGly as confirmed by MS and MS/MS spectra. Endogenous OlGly was not detectable at the retention time of synthetic OlGly shown by the arrow in sham operated mice (fig. 3B) as well as in mice used for the first time in the experiment (fig. 3C). The chromatogram trace in fig. 3A (2) represents Total Ion Current (TIC), and the chromatogram trace in fig. 3A (3) represents the extracted chromatogram m/z of about 340 amu.

Figure 4 shows that OlGly had no effect on morphine-CPP. Mice were trained with saline or morphine (10mg/kg, subcutaneously) for 3 days. Robust CPP was observed in mice trained with morphine pre-treated with vehicle. Expression of morphine CPP was not attenuated by OlGly (30mg/kg, intraperitoneally). P <0.05, vs vehicle/vehicle. Values represent mean ± SEM of 7-8 mice per group.

Figures 5A-5E show the evaluation of cannabis mimetic effects with cannabinoid tetrads after OlGly administration. OlGly produced no antinociceptive effect (5A), hypothermia (5B) or motor behaviour as reflected by the following measurements: travel distance (5C), speed (5D), and motionless time (5E). Furthermore, OlGly did not elicit catalepsy responses as assessed in the pole climbing test. Values represent mean ± SEM of 9 mice per group.

Part III: effect of OLGL-like molecules on the acquisition of ***e-induced behavior。

A preliminary sensitization protocol (Schumann et al, 2009) was performed to examine whether the endogenous defense system responded to drug damage (insult). Therefore, mice received repeated injections of ***e (20mg/kg) daily for 10 days, and their locomotor activity was measured. As shown in fig. 6, the animals developed a sensitization response (increasing locomotor activity) to chronic ***e treatment. On day 11, animals were sacrificed and NAc and hippocampus were dissected and analyzed for levels of various compounds. As clearly seen in fig. 7A-7L, levels of OlGl were significantly increased in NAC in ***e-treated mice. Similarly, in hippocampus, the level of endocannabinoid 2AG known to have neuroprotective effects (Panikashvily et al, 2001) is elevated. Taken together, these results indicate that this improvement is associated with a self-defense mechanism against ***e damage.

We have also shown that potentiation of the endogenous system by exogenous administration of a compound of the invention, such as oleoyl glycine or a compound with similar properties, is beneficial in preventing addictive states. Thus, in two different behavioral paradigms of addiction: psychomotor Sensitization (PS) and Conditioned Place Preference (CPP), the effect of exogenous administration of OIGI and OIGI-like molecules was tested. PS represents an increase in psychomotor response following repeated drug exposure, which is called a sensitization response, and resembles the behavioral response of human addicts to abused drugs. CPP represents a preference for a drug-related environment and resembles the fortification/reward profile of a drug in a human addict. The ability of OLGL-like molecules to influence the acquisition of PS and CPP was first tested. Sprague-Dawley rats (12 per group) were injected with saline on the first two days in order to habituate to an open field room and, after acclimation, they were injected intraperitoneally with 15mg/kg of ***e for 10 consecutive days (developmental stage of sensitization). During all periods of behavior, athletic activity is continuously monitored. The group of experimental animals consisted of: ***e-injected group, OLGL (0.5 mg/kg; 5.0mg/kg and 10.0mg/kg) or OLGL-like molecules before ***e, saline-injected group and OLGL-like molecules before saline injection. The detailed procedure for PS was performed as described in Schumann and Yaka, 2009. After the behavioral period, animals were sacrificed and the levels of endocannabinoids were determined.

To test the effect of OLGL-like molecules on reward, the CPP paradigm was used. The same group described above was trained on ***e in a CPP device as described in Beiser et al, 2017. Briefly, the same group of rats (described above) were injected intraperitoneally with ***e 15mg/kg or an OLGL-like molecule every other day in different chambers, followed by ***e or saline injection, after habituation to the CPP chamber. After 8 days of training, rats were tested for expression of CPP by allowing them to explore both chambers, and their preferences were calculated.

The effect of OLGL-like molecules after ***e withdrawal.

Given the high rate of relapse in drug addicts after long-term withdrawal, exogenous administration of OLGL-like compounds was tested to show the beneficial effect of preventing drug re-use (drug relapse) during withdrawal. Thus, the administration of OLGL-like molecules was tested to show that it is beneficial to attenuate the expression of addictive behaviour after withdrawal. The same group of rats described above was assigned to these experiments. Both PS and CPP were performed as described above, but during withdrawal OLGL-like molecules were administered. The effective dose found in the previous examples and the time course of treatment are used to determine the optimal dose and time to prevent expression of PS or CPP.

Sensitization in mental exercise (PS)

All animals were assigned to saline treatment and ***e treatment groups after one week of acclimation to their home cage environment. Two days before the first ***e or saline injection, animals were habituated to the behavioral testing procedure by being placed in photocell cages (MedAssociates, st. albans, VT) for 30min after the saline injection. On the first day of treatment (day 1), animals were habituated to the photocell cage for 20min, followed by injection of ***e (15mg/kg, i.p.) or saline (1ml/kg, i.f., in the membrane). The motion activity (total beam break) is measured for another 30 min. On the next 4 days (days 2-5), the same procedure was applied. For the ifenprodil (ifenprodil) experiment, the same procedure was applied, except that ifenprodil or vehicle was injected intra-abdominally after 20min habituation, and then ***e or saline was injected after 30 min. The athletic activity was measured for an additional 30 min. All rats were returned to their home cages for 21 days. On day 21, all rats were removed from their containment cages and sacrificed for biochemical analysis. As previously described (Boudreau and Wolf, 2005), sensitization criteria were based on the Coefficient of Variation (CV) of the beam break ratio on day 5/day 1 of the saline group (CV ═ SD/mean). CV provides a measure of variability within the saline group. A ***e-injected rat is considered sensitized if its activity during ***e treatment increases (day 5/day 1 beam interruption ratio) beyond the CV of saline. For this analysis, the day 5/day 1 beam interruption ratio was calculated based on the activity of the first 30min after injection.

Conditional Position Preference (CPP)

CPP devices (Med Associates) consist of two visually distinct training compartments. One containing white walls and a wire mesh floor (28cm x 21cm) and the other containing black walls and a steel rod floor (28cm x 21 cm). The compartments are connected by a smaller central compartment (12cm x 21 cm). An infrared beam at the bottom of the wall allows the preference of the animal for each compartment to be evaluated. CPP experiments were performed at predetermined times per day. After 3 days of acclimation, a biased CPP design was performed as follows: animals were placed in the central grey compartment for 5min and then allowed to freely explore all three compartments for 15 min. The time spent in each compartment was analyzed by automated software and the results used to determine initial preferences. The least preferred compartment of each subject was then designated as the drug-paired compartment. The training session begins the day after the habitual period. Cocaine injections or saline injections were given daily. Animals received four saline injections (1ml/kg, i.p.) and four ***e injections (15mg/kg, i.p.) on alternating days and were confined to the assigned compartment for a period of 15 min. Thus, training was performed for a total of 8 days. To evaluate the establishment of ***e-induced CPP, animals were tested one day after the last training day. Each animal was placed in the central compartment for 5min, followed by free access to all compartments for a period of 15 min. The CPP score is defined as a percentage determined by: 100 × (time spent in drug paired chamber-time spent in saline paired chamber)/(time spent in drug paired chamber + time spent in saline paired chamber).

When the drug was administered during withdrawal, a standard ***e CPP regimen was performed. One day after ***e training was complete, half of the animals from each treatment group received daily injections of drug for seven days, while the other half received daily injections of saline. On day 7, CPP testing was performed as described above.

While certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will now occur to those of ordinary skill in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the invention.