Methods of reducing fear memories

Abstract

A method of reducing fear memory in a mammal comprising the step of inhibiting or reducing cAMP signaling in the mammal. The cAMP signaling can be reduced using an adenylyl cyclase 1 (AC1) inhibitor or using a phosphodiesterase, such as a cAMP-specific phosphodiesterase, such as PDE4.

Description

Technical Field

[0001] This invention generally relates to fear memories, and more particularly, to methods for reducing fear memories and / or treating fear-related mental illnesses such as post-traumatic stress disorder (PTSD). Background Technology

[0002] Post-traumatic stress disorder (PTSD) is a serious mental illness that can occur after a major traumatic event, with an estimated lifetime prevalence of approximately 3.9% worldwide. Patients with PTSD exhibit a variety of psychological and behavioral symptoms, including re-experiencing, avoidance, and hypervigilance. Re-experiencing refers to the involuntary retrieval of traumatic memories, such as flashbacks, nightmares, and intrusive thoughts. While avoidance and hypervigilance are common in many anxiety-related disorders, re-experiencing is largely specific to PTSD and is widely considered a central feature of the disorder. Because re-experiencing can be understood as a persistent conditioned reflex—a learned response to a previously neutral stimulus—the learning and memory processes involved in the regulation of fear memories are presumed to underlie the symptoms of re-experiencing.

[0003] The development of PTSD depends on environmental factors such as traumatic experiences. However, mounting evidence suggests that genetic factors also play a significant role, indicating that PTSD is caused by a combination of environmental and genetic factors. The pituitary adenylate cyclase-activating polypeptide (PACAP) in peripheral blood is associated with PTSD symptoms in women, while a single nucleotide polymorphism in the PAC1 receptor gene (ADCYAP1R1) is associated with PTSD symptoms in women and exhibits fear- and estrogen-induced expression. Interestingly, a genome-wide association study found that the corticotropin-releasing hormone receptor 1 gene (CRHR1) is associated with invasive re-experiencing in PTSD.

[0004] Fear memories are generated through traumatic experiences. In laboratory animals, Pavlovian fear conditioning and inhibitory avoidance (IA) tasks (both of which can generate fear memories) have been widely used as models of PTSD. Fear memories are unstable in the first few hours after a fearful experience, subsequently stabilizing through gene expression-dependent memory consolidation. Importantly, memory retrieval is not a passive process; consolidated memories become unstable again upon retrieval, and are then maintained or enhanced through gene expression-dependent reconsolidation (restabilization). Conditioned fear memories have been observed in many animal species, from insects to humans; therefore, the mechanisms regulating fear memories in humans are considered similar to those in other animals.

[0005] Given the prevalence of PTSD and its allergic effects, there is a desire to develop a treatment or a method to alleviate one or more of its unpleasant symptoms. Summary of the Invention

[0006] It has now been established that increased cyclic adenosine monophosphate (cAMP) signaling leads to more pronounced fear memories and more severe re-experiencing in patients with certain mental illnesses characterized by pathological fears, and that inhibiting cAMP signaling can alleviate these symptoms. Increased expression of cAMP-degrading enzymes also plays a role in alleviating this condition.

[0007] Therefore, in one aspect of the present invention, a method for alleviating fear memories in mammals is provided, comprising the step of inhibiting or reducing cAMP signaling in mammals.

[0008] In one implementation, cAMP signaling is inhibited or reduced by administering a cAMP inhibitor to a mammal.

[0009] In other embodiments, cAMP signaling is inhibited or reduced by upregulating the expression of cAMP-degrading enzymes, including phosphodiesterases.

[0010] In another aspect of the invention, a method for treating PTSD in mammals is provided, comprising the step of inhibiting or reducing cAMP signaling in mammals.

[0011] On the other hand, cAMP inhibitors are provided for use in alleviating fear memories in mammals and / or treating PTSD in mammals.

[0012] In a further aspect of the invention, a composition for alleviating fear memories in mammals is provided, comprising a cAMP inhibitor and a pharmaceutically acceptable excipient or carrier.

[0013] These and other aspects of the invention are described in detail below with reference to the accompanying drawings in the description and embodiments. Attached Figure Description

[0014] Figure 1 - This study illustrates the effects of systemic injection of rolipram (ROL) (A) or NB001 (B) on situational fear memories (A, mediator (VEH)). n = 10; ROL, n = 11. B, VEH, n = 10; NB001, n = 11); Effects of systemic injection of ROL (C) or NB001 (D) on inhibitory avoidance memory. (C, VEH, n = 10; ROL, n = 10. D, VEH, n = 10; NB001, n = 10. p <0.05, p <0.01, Bonferroni test performed afterward; # p <0.05, paired t-test. Error bars indicate SEM).

[0015] Figure 2 - This illustrates A) viral injection and mGFP expression in the dorsal hippocampus (top image) and light stimulation of photoactivated adenylate cyclase (bPAC; bottom image); B) the effect of optogenetic increases in cAMP levels in the dorsal hippocampus on situational fear memories. GFP, n = 12; No Stimulation (No Stimulation) n = 9; Optical stimulation (Optical Stim.) n = 11; C) Schematic diagram of viral injection and GFP expression in the dorsal hippocampus (top) and light stimulation of photoactivated phosphodiesterase (LAPD; bottom); and D) Effect of optogenetic reduction of cAMP levels in the dorsal hippocampus on situational fear memory. GFP, n = 11; No stimulation, n = 11; light stimulation, n = 11. p <0.05; Bonferroni test performed afterward. Error bar indication SEM; Figure 3 - This illustrates the changes in mRNA levels in the dorsal hippocampus and peripheral blood of mice after IA memory retrieval, including A) a schematic diagram of the experimental design; and B) a graphical illustration of the changes in mRNA levels in the dorsal hippocampus. (Not reactivated (NR)). n = 6-10; 30 min after reactivation (reactivation -30). n = 6-10; and C) Graphical illustration of changes in peripheral blood mRNA levels. NR, n = 8-10; Reactivate -30, n = 8-11; 90 min after reactivation (reactivation -90). n = 8-10. p <0.05, Bonferroni test performed afterward; # p <0.05, Student's t-test. Error bar indicator SEM.

[0016] Figure 4 - This explains the AC1 inhibitor; Figure 5- The amino acid sequences of phosphodiesterases (PDEs) are described, specifically the amino acid sequences of A) PDE4, B) PDE7A, C) PDE7B, and D) PDE8; and Figure 6 - This describes the nucleic acid sequence of PDE4A. Detailed Implementation

[0017] A method for mitigating fear memories in mammals is provided, comprising steps of inhibiting or reducing cAMP signaling in mammals. Fear memories are associated with re-experiencing, a symptom of fear-related mental illnesses.

[0018] The term "fear memory" as used in this article refers to the memory of a traumatic event and the reaction to that event. Fear memories are formed in multiple brain regions, including the hippocampus (situational conditioning and inhibitory avoidance), the basolateral amygdala (inhibitory avoidance), the lateral amygdala (tonality conditioning), the anterior cingulate cortex (inhibitory avoidance), and the medial prefrontal cortex (inhibitory avoidance). Fear memories can be intrusive, leading to re-experiencing of the traumatic event and increased arousal and stress responses, resulting in fear-related mental illnesses.

[0019] Fear-related mental illnesses are those in which fear memories play a role. Typically, pathological fears are characteristic of these illnesses, including phobias, anxiety disorders, and post-traumatic stress disorder (PTSD). Pathological fear refers to an abnormal or extreme fear or sense of danger that triggers an uncontrollable and intense emotional state or reaction.

[0020] Post-traumatic stress disorder (PTSD) is a mental or behavioral disorder that occurs in mammals who have experienced a traumatic event, such as a shocking, frightening, or dangerous event. PTSD can be diagnosed using many established diagnostic tools, such as the Diagnostic Post-Traumatic Stress Scale (PDS) based on the DSM-5 (Diagnostic and Statistical Manual of Mental Disorders, 5th Edition) criteria, physician-managed PTSD scales, and the Mini-International Neuropsychiatric Interview (MINI). To be diagnosed with PTSD, an adult must have: i) at least one re-experiencing symptom; ii) at least one avoidance symptom; iii) at least two arousal and reactive symptoms; and iv) at least two cognitive and emotional symptoms, lasting for at least one month. Re-experiencing symptoms include: experiencing flashbacks—reliving the traumatic event, including physiological symptoms such as rapid heartbeat or sweating; recurring memories (fear memories) or dreams associated with the event; distressing thoughts; and / or physical signs of stress. Thoughts and feelings can trigger these symptoms, such as verbal, object, or situational reminders of the event. Avoidance symptoms include: avoiding places, events, or objects that remind of the traumatic experience, and / or avoiding thoughts or feelings associated with the traumatic event. Avoidance symptoms may cause people to change their daily lives. For example, some people may avoid driving or riding in a car after a serious car accident. Arousal and reactive symptoms include: being easily startled; feeling tense, alert, or anxious; difficulty concentrating; difficulty falling asleep or staying asleep; feeling irritable and having outbursts of anger or aggression; and / or engaging in dangerous, reckless, or destructive behavior. Arousal symptoms are usually persistent. They can cause feelings of stress and anger and may interfere with certain aspects of daily life, such as sleep, eating, or concentration. Cognitive and emotional symptoms include: difficulty recalling key features of the traumatic event; having negative thoughts about oneself or the world; feeling excessively guilty about oneself or others; persistent negative emotions such as fear, anger, guilt, or shame; loss of interest in favorite activities; feelings of social isolation; and / or difficulty experiencing positive emotions such as happiness or satisfaction.

[0021] This approach includes treating fear memories and / or PTSD in mammals. As used herein, the term "mammal" refers to humans, as well as non-human mammals such as domestic animals (e.g., cats, dogs, rodents) and livestock (e.g., cattle, horses, goats, sheep, etc.). The term "treat" or "treatment" refers to preventing, curing, improving, or slowing the progression of fear memories or fear-related mental illnesses such as PTSD.

[0022] The method includes the step of inhibiting or reducing cAMP signaling. cAMP signaling can be inhibited or reduced by using a small molecule inhibitor that inhibits the activity of adenylate cyclase 1 (AC1).

[0023] In one implementation, cAMP signaling is inhibited or reduced by administering an AC1 inhibitor, as described in PCT / CA2006 / 001687, to a mammal, the contents of which are incorporated herein by reference. The AC1 inhibitor has the following general formula (1): (1) in: A is selected from the group consisting of H, OH, halogen, C1-C6 alkyl, C1-C6 alkyl halide, C2-C6 alkenyl, C2-C6 alkynyl and C1-C6 alkoxy; B is selected from the group consisting of: hydroxyl, thio, -OR 1 -NH2, -NO2, -NHR 1 -NR 1 R 2 -SR 1 -C1-C6 saturated or unsaturated alkyl groups, optionally substituted with hydroxyl, halogen, thio, OR 1 NH2, NO2, NHR 1 NR 1 R 2 SR 1 C3-C 10 One or more substituents in an aromatic or non-aromatic ring structure or a C3-C9 aromatic or non-aromatic heterocyclic structure, optionally substituted with OH, halogen, thio, NH2, C1-C6 alkyl, C1-C6 alkanol or C1-C6 alkoxy, wherein R 1 and R 2 Independently selected from the group consisting of C1-C6 alkyl, C1-C6 alkyl halides, C1-C6 alkenyl, C1-C6 alkynyl, C1-C6 alkanol, C1-C6 alkoxy and C1-C6 carboxylalkyl, or B is NR 1 R 2 It forms a C3-C6 aromatic or non-aromatic heterocycle, optionally substituted with OH, halogen, thio, NH2, NO2, C1-C6 alkyl, C1-C6 alkanol, C1-C6 alkoxy, or C1-C6 carboxylalkyl. D is selected from the group consisting of: H, halogen, hydroxyl, NH2, thio, NHR 1 NR 1 R 3 SR 1 C1-C6 alkyl, C1-C6 alkoxy, wherein R 1 As defined above and R 3 For example, regarding R 1 Definition; E is H or OH, or E represents C1-C6 alkyl, C1-C6 alkoxy, C 3-10 -Aryl-C 1-6 -alkyl or C 3-10 -Aryloxy-C 1-6 -alkyl, optionally substituted with C 1-6 -alkyl, amino, NHR 1 NR 1 R 2 thiolated, SR 1 Unsubstituted C3-C7 cycloalkyl, phenyl, or C4-C6 heterocycles, or substituted C3-C7 cycloalkyl, phenyl, or C4-C6 heterocycles, wherein the substituted C3-C7 cycloalkyl, phenyl, or C4-C6 heterocycle has one or more substituents selected from the group consisting of C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkanoyl, C1-C6 carboxyl, halogen, or OH. E is an unsubstituted C3-C7 cycloalkyl, phenyl, or C4-C6 heterocycle, or a substituted C3-C7 cycloalkyl, phenyl, or C4-C6 heterocycle, wherein the substituted C3-C7 cycloalkyl, phenyl, or C4-C6 heterocycle has one or more substituents selected from the group consisting of C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylacyl, C1-C6 carboxyl, halogen, or OH. Where R 1 and R 2 As defined above; and G, H, J, and M are all N, or H and J are both C, and G and M are both N, S, or O, or H, J, and M are all C, and G is N, S, or O.

[0024] The basic heterocyclic system in the compound of formula (1) can be such that each variable G, H, J, and M is nitrogen (N), i.e., a purine ring system. Alternatively, H and J can both be carbon, and G and M can be selected from N, S, or O, for example, benzothiazole. In another alternative, H, J, and M can each be carbon (C), and G can be N, S, or O, for example, forming indole, benzothiphene, or benzofuran ring systems, respectively.

[0025] Variable A can be H; OH; halogens, such as F, Cl, Br, and I; C1-C6 alkyl groups, including branched alkyl groups, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, 3-methylpentyl, hexyl, and isohexyl; C1-C6 alkyl halides, such as chloromethyl, fluoromethyl, bromoethyl, bromovinyl, propylfluorine, isopropyliodine, chlorobutyl, 1,1-dichloro-2,3-butyl, 2-bromopentyl, 3-chlorohexyl, 1-fluoro-3-methylhexyl, and 1,1-difluorohexyl; C2-C6 alkenyl groups, including branched alkenyl groups. Alkenyl groups, such as vinyl, propenyl, butenyl, isobutenyl, 2-butenyl, pentenyl, isopentenyl, 2-pentenyl, and hexenyl; C2-C6 alkynyl groups, such as ethynyl, propynyl, and butynyl, including branched alkynyl groups; C1-C6 alkoxy groups, including branched alkoxy groups, such as methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, methoxymethyl, ethoxymethyl, ethoxyethyl, methoxyethyl, methoxypropyl, ethoxypropyl, propoxymethyl, propoxyethyl, propoxypropyl, pentoyl, isopentenyl, hexoxy, and isohexoxy. Preferably, A is hydrogen.

[0026] Variable B can be hydroxyl, halogen, thio, or -OR. 1 -NH2, -NO2, -NHR 1 -NR 1 R 2 SR 1 -C1-C6 saturated or unsaturated alkyl groups (e.g., alkyl, alkenyl, or ynyl groups as exemplified above), optionally substituted with a group selected from hydroxyl, halogen, thio, -OR 1 -NH2, -NO2, -NHR 1 -NR 1 R 2 or -SR 1 or C3-C 10 One or more substituents in an aromatic or non-aromatic ring structure or a C3-C9 aromatic or non-aromatic heterocyclic structure are optionally substituted with OH, halogen, thio, NH2, C1-C6 alkyl, C1-C6 alkanol, or C1-C6 alkoxy. The term "ring structure" as used herein refers to structures consisting of a single ring as well as polycyclic structures, such as bicyclic structures. The term "heterocyclic" or "heterocyclic structure" is intended to include 3- to 9-membered ring structures that include at least one heteroatom selected from O, S, and N in the core ring structure. Examples of suitable ring structures include benzene, naphthalene, tetrahydronaphthalene, decahydronaphthalene, piperidine, pyrrolidine, furan, piperazine, tetrahydrothiophene, morpholine, imidazole, benzothiophene, quinoline, isoquinoline, indole, benzofuran, and purine. Preferably, B is -NH2, -NHR. 1 or -NR 1 R 2 , where R1 and R 2 It is independently selected from the group consisting of C1-C6 alkyl, C1-C6 alkanol or C1-C6 alkoxy groups.

[0027] variable R 1 and R 2 Independently selected from the group consisting of C1-C6 alkyl, C1-C6 alkyl halide, C1-C6 alkenyl, C1-C6 alkynyl, C1-C6 alkanol, C1-C6 alkoxy, or C1-C6 carboxylalkyl. Therefore, OR 1 It can be, for example, oxymethyl, oxydimethyl, oxyethyl, oxy-3-chlorobutyl, oxypropenyl, or oxypropanol. NHR 1 It can be, for example, alkylamines such as methylamine, as well as 2-chloropropylamine, NH-ethanol, NH-propanol, NH-ethylmethyl ether, or N-butyric acid. Similarly, NR 1 R 2 It can be a dialkylamine, such as diethylamine, or it can be, for example, N-chloro-N-propyl, N-methyl-N-butyric acid, or N-methyl-N-propanol.

[0028] NR 1 R 2 It can also form C3-C as exemplified above. 10 An aromatic or non-aromatic heterocyclic structure, which may optionally be substituted with OH, halogen, thio, NH2, NO2, C1-C6 alkyl, C1-C6 alkanol, C1-C6 alkoxy or C1-C6 carboxylalkyl.

[0029] Variable D can be H, halogen, hydroxyl, NH2, thio, or -NHR. 1 -NR 1 R 3 -SR 1 -C1-C6 alkyl or -C1-C6 alkoxy. R 1 As defined above and R 3 For example, regarding R 1 The definition is as follows. Therefore, D can be, for example, an unsubstituted group, such as H, halogen, hydroxyl, -NH2, thio (SH), -C1-C6 alkyl, or -C1-C6 alkoxy. D can also be a substituted group; for example, D can be -NHR. 1 , where R 1 It is a C1-C6 alkyl group, such as methyl, ethyl, isopropyl, butyl, isobutyl, pentyl, 2-methylbutyl; D can be -NR 1 R 3 Such as methyl ethylamine or N-propyl-N-bromoamine; or D can be -SR 1Examples include thioethyl, thiopentyl, and thioacetic acid. As mentioned earlier, C1-C6 alkyl and C1-C6 alkoxy groups may also optionally be substituted with halogens, hydroxyl groups, NH2, thio, or NHR. 1 NR 1 R 3 and SR 1 D is preferably hydrogen.

[0030] Variable E can be H or OH. E can also be C1-C6 alkyl, C1-C6 alkoxy, C... 3-10 -Aryl-C 1-6 -alkyl or C 3-10 -Aryloxy-C 1-6 -alkyl, which may optionally be substituted with C 1-6 -alkyl, amino, -NHR 1 -NR 1 R 2 Thio, -SR 1 E can be an unsubstituted C3-C7 cycloalkyl, phenyl, or C4-C6 heterocycle, or a substituted C3-C7 cycloalkyl, phenyl, or C4-C6 heterocycle, wherein the substituted C3-C7 cycloalkyl, phenyl, or C4-C6 heterocycle has one or more substituents selected from the group consisting of C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylacyl, C1-C6 carboxyl, halogen, and OH. E can also be an unsubstituted C3-C7 cycloalkyl, benzyl, or C4-C6 heterocycle, or a substituted C3-C7 cycloalkyl, benzyl, or C4-C6 heterocycle, wherein the substituted C3-C7 cycloalkyl, benzyl, or C4-C6 heterocycle has one or more substituents selected from the group consisting of C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylacyl, C1-C6 carboxyl, halogen, and OH. 1 and R 2 As previously defined.

[0031] Specific examples of groups that E can represent include -methylphenyl, -ethylphenyl, -propylphenyl, -methylamine-phenyl, -methylamine-propanol, -ethylamine-pentanol, -1-methyl-2,6-dichlorophenyl, -1-2,3-dihydroxy-4-methanol-tetrahydrofuran, and -p-ethoxy-tolyl.

[0032] In the embodiments, the AC1 inhibitor is a compound of formula (1), wherein G, H, J and M are all N; A is hydrogen; and B is -NH2 or -NHR. 1 or -NR 1 R 2 , where R 1 and R 2 Independently selected from the group consisting of C1-C6 alkyl, C1-C6 alkanol or C1-C6 alkoxy groups; D is NH2, SH, -NHR1 -NR 1 R 3 or -SR 1 , where R 1 and R 2 Independently selected from the group consisting of C1-C6 alkyl, C1-C6 alkanol, C1-C6 carboxyl or C1-C6 alkoxy; E is one of the following: -methylphenyl, -ethylphenyl, -propylphenyl, -methylaminophenyl, -methylaminopropanol, -ethylaminopentanol, -1-methyl-2,6-dichlorophenyl, -1-2,3-dihydroxy-4-methanol-tetrahydrofuran and -p-ethoxy-tolyl.

[0033] In several implementations, AC1 inhibitors are as follows: Figure 4 The compound shown.

[0034] In one particular embodiment, the AC1 inhibitor is a compound of formula (1), wherein: A is H; B is NH2; D is H; E is CH2CH2-NH(CH2)5OH; and G, H, J and M are all N.

[0035] To determine whether a compound of formula (1) inhibits AC1, established assays, such as cAMP assays, can be used. Since AC1 catalyzes the conversion of ATP to cAMP, the cAMP produced by AC1-expressing cells in the presence of the test compound can be monitored to determine the AC1 inhibitory activity of the test compound. In short, non-AC1-expressing cells transfected with DNA encoding AC1 are incubated with different concentrations of the potential AC1 inhibitory compound. After an appropriate reaction time, AC1 activity is measured by determining the amount of cAMP in the reaction mixture. Very low or no cAMP indicates inhibitory activity.

[0036] A dual-luciferase reporter system can also be used to determine AC1 inhibitory activity, as described in more detail in the specific examples below. In this assay, changes in intracellular cAMP concentration were detected as changes in firefly luciferase expression levels, the transcription of which is regulated by the transcription factor cAMP response element-binding protein (CREB) and the upstream cAMP response element (CRE). Cells such as HEK293 cells were transfected with the luciferase-encoded construct and incubated with the test compound. After an appropriate incubation period, luciferase activity was determined. Inhibition of luciferase activity indicated an AC1 inhibitor.

[0037] Examples of compounds according to formula (1) include Figure 4 The compounds shown are examples of those compounds. While these compounds can be readily synthesized using standard chemical synthesis protocols, those skilled in the art will understand that they are also commercially available.

[0038] AC1 inhibitors can be in the form of pharmaceutically acceptable salts. A "pharmaceutically acceptable salt" is one that retains the desired biological activity of the parent compound without producing any adverse toxicological effects (see, for example, Berge, SM, et al.). ， (1977) J. Pharm. Sci . 66 Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from non-toxic inorganic acids (such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphorus, etc.) and non-toxic organic acids (such as aliphatic monocarboxylic acids and dicarboxylic acids, phenyl-substituted alkyl acids, hydroxyalkyl acids, aromatic acids, aliphatic and aromatic sulfonic acids, etc.). Base addition salts include those derived from alkaline earth metals (such as sodium, potassium, magnesium, calcium, etc.) and non-toxic organic amines (such as N,N'-dibenzylethylenediamine, N-methylglucosamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine, etc.).

[0039] In another embodiment, cAMP signaling is inhibited or reduced by upregulating the expression of cAMP-degrading enzymes. cAMP-degrading enzymes include cyclic nucleotide phosphodiesterases (PDEs), such as PDE1, 2, 3, 4, 7, 8, 10, and 11. Preferably, cAMP1 signaling is inhibited by upregulating cAMP-selective PDEs, such as cAMP-specific 3',5'-cyclic phosphodiesterases, such as PDE4, PDE7, and PDE8.

[0040] PDE4 in this paper refers to mammalian PDE4, including human and non-human PDE4, as well as its functionally equivalent forms, such as its isotypes and variants. The sequence of human PDE4A isotype 1 is as follows: Figure 5 The sequence shown corresponds to the NCBI reference sequence NP_001104777.1. Other identical sequences correspond to NCBI reference sequences NP_001104778, NP_001104779, NP_006193, and NP_001230050, including non-human sequences corresponding to NCBI reference sequences NP_001297679, NP_062772, and NP_899668.

[0041] PDE7 in this paper refers to mammalian PDE7, including human and non-human PDE7A and PDE7B, as well as their functionally equivalent forms, such as their isotypes and variants. The sequences of human PDE7A isotype c and PDE7B are as follows: Figure 5 The sequences shown correspond to NCBI reference sequences NP_001229247.1 and NP_061818.1, respectively. Another isotype corresponds to NCBI reference sequence NP_002594.1, and the non-human sequences correspond to NCBI reference sequences NP_001116231 and NP_032828.

[0042] PDE8 in this paper refers to mammalian PDE8B, including human and non-human PDE8B, as well as its functionally equivalent forms, such as its isotypes and variants. The sequence of human PDE8B isotype 3 is as follows: Figure 5 The sequence shown corresponds to the NCBI reference sequence NP_001025022. Other similar sequences correspond to NCBI reference sequences NP_001025023, NP_001025024, NP_001025025, and NP_003710, while non-human sequences correspond to NCBI reference sequences NP_001164140, NP_001333711, NP_758467, NP_001360932, and NP_001360933.

[0043] The term "functionally equivalent variant" in relation to phosphodiesterases includes natural or non-natural variants of endogenous phosphodiesterases that retain the biological activity of the phosphodiesterase, such as regulating intracellular levels of cyclic adenosine monophosphate (cAMP). These variants need not exhibit the same activity as the endogenous phosphodiesterase, but should exhibit sufficient activity to enable them to downregulate cAMP, for example, at least about 10% of the phosphodiesterase biological activity, and preferably at least about 20-50% or more. The term "about" as used herein means a difference of about 10%, more or less than the listed value. Such functionally equivalent variants may arise naturally from alternative splicing during transcription or genetic coding differences and may maintain significant sequence homology with wild-type phosphodiesterases, for example, at least about 70% sequence homology, preferably at least about 80% sequence homology, and more preferably at least about 90% or more sequence homology. These variants can be readily identified using primers derived from native phosphodiesterases through well-established cloning techniques. Furthermore, such variants can arise from non-naturally occurring synthetic alterations to phosphodiesterases, resulting in functionally equivalent variants that may possess more desirable properties for therapeutic use, such as enhanced activity or stability. Non-naturally occurring variants of phosphodiesterases include their analogues, fragments, and derivatives.

[0044] According to the present invention, functional analogues of phosphodiesterases can be incorporated by substitution, addition, or deletion of one or more amino acids. Amino acid additions or deletions include both terminal and internal additions or deletions to produce functionally equivalent peptides. Examples of suitable amino acid additions or deletions include those occurring at positions in the protein that are not closely related to activity. Amino acid substitutions in phosphodiesterases, particularly conserved amino acid substitutions, can also produce their functional analogues. Examples of conserved substitutions include: substituting a nonpolar (hydrophobic) residue such as alanine, isoleucine, valine, leucine, or methionine with another nonpolar (hydrophobic) residue; substituting a polar (hydrophilic) residue such as between arginine and lysine, between glutamine and asparagine, between glutamine and glutamate, between asparagine and aspartic acid, and between glycine and serine with another polar (hydrophilic) residue; substituting a basic residue such as lysine, arginine, or histidine with another basic residue; or substituting an acidic residue such as aspartic acid or glutamate with another acidic residue.

[0045] The functionally equivalent fragment according to the invention comprises a portion of a phosphodiesterase sequence that retains the complete function of the phosphodiesterase, such as in relation to the downregulation of cAMP. Such bioactive fragments of phosphodiesterases can be readily identified using assays for assessing the activity of selected phosphodiesterase fragments.

[0046] According to the present invention, the functionally equivalent derivative of a phosphodiesterase is a phosphodiesterase or an analogue or fragment thereof, wherein one or more amino acid residues are chemically derivatized. The amino acid may be derivatized at an amino or carboxyl group, or alternatively, at a side "R" group. Derivatization of amino acids in a peptide can endow the peptide with more desired properties, such as increased stability or activity. For example, such derivatized molecules include those in which the free amino group has been derivatized to form amine hydrochloride, p-toluenesulfonyl, benzyloxycarbonyl, tert-butoxycarbonyl, chloroacetyl, or formyl, etc. The free carboxyl group may be derivatized to form, for example, salts, methyl esters and ethyl esters, or other types of esters or hydrazides. The free hydroxyl group may be derivatized to form, for example, an O-acyl or O-alkyl derivative. The imidazole nitrogen of histidine may be derivatized to form N-imidazolium benzylhistidine. In addition, the derivatives included are peptides containing one or more naturally occurring amino acid derivatives of the twenty standard amino acids, such as: 4-hydroxyproline replacing proline; 5-hydroxylysine replacing lysine; 3-methylhistidine replacing histidine; homoserine replacing serine; and ornithine replacing lysine. Terminal derivatization of proteins is also covered to prevent chemical or enzymatic degradation, including N-terminal acetylation and C-terminal amidation of peptides.

[0047] Phosphodiesterases and their functionally equivalent variants for this method can be prepared using standard, well-established solid-phase peptide synthesis (SPPS) methods. Two methods for solid-phase peptide synthesis include the BOC method and the FMOC method. Phosphodiesterases and their variants can also be prepared using any of a variety of suitable techniques based on recombinant technology. It is understood that such techniques are well-established by those skilled in the art and involve the expression of phosphodiesterase-encoded nucleic acids in genetically engineered host cells. Given the known protein and nucleic acid sequences, DNA encoding phosphodiesterases can be synthesized de novo using automated techniques well known in the art.

[0048] Plasma phosphodiesterase levels can also be increased using phosphodiesterase-encoded nucleic acid molecules or oligonucleotides. In this context, the expression "phosphodiesterase-encoded nucleic acid" as used herein encompasses mammalian phosphodiesterase-encoded nucleic acids, including human and non-human forms, as well as their functionally equivalent forms (e.g., nucleic acids encoding functionally equivalent phosphodiesterases, or nucleic acids that differ due to the degeneracy of the genetic code). The sequences of such genes can be based on the PDE amino acid sequences disclosed herein. Figure 6 The sequence of the human phosphodiesterase-encoding gene encoding PDE4A transcript variant 1 is shown, along with genes encoding other PDE4 transcripts, as well as transcripts of PDE7 and PDE8, their functionally equivalent forms, and examples of genes encoding other isotypes and / or non-human forms, readily available in the NCBI sequence database, including NM_001111308, NM_001111309, NM_001243121, NM_006202, NM_001242318, NM_002603, NM_002604, NM_001029851, NM_001029852, NM_001029853, NM_001029854, and NM_003719.

[0049] The term "oligonucleotide" refers to an oligomer or polymer of nucleotide or nucleoside monomers composed of naturally occurring bases, sugars, and inter-sugar (backbone) linkages. The term also includes modified or substituted oligonucleotides containing non-naturally occurring monomers or portions thereof, which function similarly. Such modified or substituted oligonucleotides may be preferred over their naturally occurring forms due to properties such as enhanced cellular uptake or increased stability in the presence of nucleases. The term also includes chimeric oligonucleotides containing two or more chemically distinct regions. For example, a chimeric oligonucleotide may contain at least one modified nucleotide region that imparts beneficial properties (e.g., increased nuclease resistance, increased cellular uptake), or two or more oligonucleotides of the present invention may be linked together to form a chimeric oligonucleotide. Other oligonucleotides of the present invention may contain modified phosphoric or phosphate backbones with oxygen heteroatoms, short-chain alkyl or cycloalkyl sugar linkages, or short-chain heteroatoms or heterocyclic sugar linkages. For example, oligonucleotides may contain thiophosphates, triphosphates, methyl phosphonates, and dithiophosphates. The oligonucleotides of the present invention may also include nucleotide analogs, such as peptide nucleic acids (PNAs), wherein the deoxyribose (or ribose) phosphate backbone of DNA (or RNA) is replaced by a polyamide backbone similar to that in peptides. Other oligonucleotide analogs may contain nucleotides with polymeric, cyclic, or acyclic backbones (e.g., morpholine backbone structures).

[0050] Such oligonucleotide molecules can be readily synthesized using procedures known in the art, based on available sequence information. For example, oligonucleotides can be chemically synthesized using naturally occurring nucleotides or modified nucleotides as described above, said modified oligonucleotides being designed to increase the biological stability of the molecule or the physical stability of the double strand formed with mRNA or a natural gene, such as phosphate thioester derivatives and acridine-substituted nucleotides. Selected oligonucleotides can also be produced biologically using recombinant technologies, wherein an expression vector (e.g., plasmid, bacteriophage, or attenuated virus) is introduced into a cell, where oligonucleotides are produced under the control of regulatory regions.

[0051] Once prepared and properly purified, phosphodiesterase, phosphodiesterase-encoded oligonucleotides, or functionally equivalent variants thereof can be used according to the invention to downregulate cAMP signaling in the treatment of fear memories and / or PTSD. In this respect, increasing the expression of phosphodiesterase in mammals by administering the phosphodiesterase or by administering the phosphodiesterase-encoded nucleic acid can lead to the expression or overexpression of the phosphodiesterase in mammals. While not wishing to be limited to any particular mode of action, according to the invention, upregulation of phosphodiesterase leads to downregulation of cAMP signaling.

[0052] AC1 inhibitors, phosphodiesterases, or nucleic acids encoding phosphodiesterases (such as PDE4) may be administered alone or in combination with at least one pharmaceutically acceptable adjuvant for use in treatment according to embodiments of the invention. The expression "pharmaceutically acceptable" means acceptable for use in the pharmaceutical and veterinary fields, i.e., without unacceptable toxicity or other unsuitability. Examples of pharmaceutically acceptable adjuvants are those commonly used with small molecule, peptide, or nucleic acid-based drugs, such as diluents, excipients, etc. General guidance on drug formulation can be found in "Remington's: The Science and Practice of Pharmacy", 21st Ed., Lippincott Williams & Wilkins, 2005. The choice of adjuvant depends on the intended route of administration of the composition. In one embodiment of the invention, the compound is formulated for administration by infusion or by subcutaneous or intravenous injection, and thus as a sterile, pyrogen-free aqueous solution, optionally buffered or prepared as an isotonic solution. Therefore, the compound may be administered in distilled water, or more preferably in saline, phosphate-buffered saline, or a 5% glucose solution. In addition, agents that delay absorption, such as aluminum monostearate and gelatin, may be included to provide prolonged absorption in injectable drug form. Compositions for oral administration via tablets, capsules, or suspensions are prepared using adjuvants including: sugars, such as lactose, glucose, and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, including sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; stearic acid; magnesium stearate; calcium sulfate; vegetable oils, such as peanut oil, cottonseed oil, sesame oil, olive oil, and corn oil; polyols, such as propylene glycol, glycerin, sorbitol, mannitol, and polyethylene glycol; agar; alginic acid; water; isotonic saline and phosphate buffer solutions. Wetting agents, lubricants (such as sodium lauryl sulfate), stabilizers, tableting agents, antioxidants, preservatives, colorants, and flavoring agents may also be present. Creams, lotions, and ointments for topical application can be prepared using suitable matrices (such as triglyceride matrices). These creams, lotions, and ointments may also contain surfactants. Aerosol formulations can also be prepared, using suitable propellant adjuvants. Other adjuvants can also be added to the composition, regardless of the route of administration; for example, antimicrobial agents such as parabens, chlorobutanol, and phenolic sorbic acid can be added to the composition to prevent microbial growth during long-term storage.

[0053] Examples of pharmaceutically acceptable antioxidants that may be included in the composition include, but are not limited to: (1) water-soluble antioxidants, such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, etc.; and (3) metal chelating agents, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0054] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the present invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters (such as ethyl oleate). For example, appropriate flowability can be maintained by using coating materials such as lecithin and by using surfactants.

[0055] Using techniques in the art, including vectors (retroviral vectors, adenoviral vectors, and DNA viral vectors) or physical techniques (such as microinjection), phosphodiesterase-encoded oligonucleotides can be introduced into tissues or cells. Therapeutic oligonucleotides can be administered directly in vivo or can be used to transfect cells in vitro and then administered in vivo. For example, such cell delivery can be achieved via encapsulated cell biodelivery.

[0056] To downregulate cAMP in the treatment of fear memories and / or PTSD, a therapeutically effective amount of an AC1 inhibitor, phosphodiesterase, or nucleic acid encoding a phosphodiesterase is administered to a mammal. The term "therapeuticly effective amount" is the amount of an AC1 inhibitor, phosphodiesterase, or nucleic acid encoding a phosphodiesterase required to adequately downregulate cAMP signaling, while not exceeding an amount that could cause significant adverse effects, such as by alleviating one or more symptoms of fear memories (e.g., re-experiencing symptoms, avoidance symptoms, arousal and / or reactive symptoms, or cognitive and / or emotional symptoms). The therapeutically effective dose will vary depending on many factors, including the nature of the disease being treated and the specific individual receiving treatment. A suitable dose may be an amount of AC1 inhibitor that downregulates cAMP signaling by about 1% in a patient, for example, a dose that results in a 5-10% or greater reduction in cAMP signaling. Suitable doses of AC1 inhibitors used include doses sufficient to result in plasma levels of about 100-1500 ng / ml, preferably 120-1200 ng / ml, or 150-1000 ng / ml.

[0057] Similarly, appropriate doses of phosphodiesterases (such as PDE4) used include doses sufficient to increase plasma PDE levels in patients with fear-related mental disorders (such as PTSD) to alleviate one or more of their symptoms (such as re-experiencing symptoms, avoidance symptoms, arousal and / or reactive symptoms, or cognitive and / or emotional symptoms). An appropriate dose may be a PDE amount that results in an increase in plasma PDE levels of at least about 1%, for example, an amount that results in a 5-10% or greater increase in plasma PDE, while not exceeding an amount that could cause significant adverse reactions.

[0058] In this treatment, AC1 inhibitors, phosphodiesterases, or nucleic acids may be administered via any route suitable for increasing their plasma levels. Suitable routes of administration include, but are not limited to, parenteral routes, including intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal, intraarticular, intrathecal, intracapsular, intraorbital, intracardiac, tracheal, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions. Alternatively, other preferred routes of administration include non-parenteral routes, including local, epidermal, or mucosal routes, such as intranasal, oral, vaginal, rectal, sublingual, or local administration.

[0059] The embodiments of the present invention are described with reference to the following specific examples, but these examples should not be construed as limiting.

[0060] Example The following research was conducted to determine what influences the re-experiencing of symptoms in conditions such as PTSD.

[0061] Mouse studies Male C57BL / 6N mice were obtained from Charles River (Yokohama, Japan). Mice were housed in 5 or 6 cages under a 12-hour light / dark cycle with free access to food and water. Mice were at least 8 weeks old at the time of testing. Testing was conducted during the cyclical light period. All experiments were performed without knowledge of the mice's treatment conditions (total n = 208). All animal experiments were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals (Japanese Society for Neuroscience and Tokyo University of Agriculture) and approved by the Animal Care and Use Committee of Tokyo University of Agriculture (Authorization No. 2021031). All surgeries were performed under pentobarbital anesthesia.

[0062] drug - The phosphodiesterase 4 (PDE4) inhibitor rolipran (ROL; Tocris Bioscience, UK) was dissolved in dimethyl sulfoxide (DMSO; Wako, Osaka, Japan) and then diluted with distilled water for systemic injection experiments. Figure 1A, C). Mice were given a systemic injection of ROL (0.1 mg / kg body weight) or a mediator (VEH) 30 min before re-exposure. The adenylate cyclase 1 (AC1) inhibitor NB001 (HTS 09836), an adenylate cyclase 1 inhibitor [Wang et al., SciTransl Med. 2011;3:65ra3], was dissolved in saline for the systemic injection experiment. Figure 1 (B, D). Two hours before re-exposure, mice were given two systemic injections of NB001 (30 mg / kg body weight) or VEH every hour.

[0063] Situational fear conditioning task Mice were trained and tested in a conditioned reflex chamber (17.5 × 17.5 × 15 cm) (O'Hara&Co., Ltd., Tokyo, Japan), which had a stainless steel mesh floor through which plantar electric shocks could be delivered. Training consisted of placing mice in the chamber and delivering a plantar electric shock after 148 s [duration 2 s; 0.2 mA ( Figure 1 A and Figure 2 B) or 0.4 mA ( Figure 1 B and Figure 2 D)], and the mice were returned to their cages 30 seconds after receiving a foot shock (training).

[0064] To examine the effects of increased or decreased cAMP levels on the retrieval and maintenance of situational fear memories ( Figure 1 A, B and Figure 2 Mice were trained as described above, and after 24 h, they were returned to the conditioned reflex chamber for 3 min (re-exposure). 24 h after re-exposure, the mice were again placed in the conditioned reflex chamber for 5 min, and their freeze (test) was assessed. Memory was assessed as the percentage of time spent frozen in the training environment. Freeze behavior (defined as complete lack of movement other than respiration) was automatically measured via video (O'Hara&Co., Ltd., Tokyo, Japan).

[0065] Inhibitory avoidance (IA) task - The walk-in IA device (O'Hara&Co., Ltd., Tokyo, Japan) consists of a box containing separate light and dark compartments (each 15.5 × 12.5 × 11.5 cm). The light compartment is illuminated by a fluorescent lamp (2500 lux). During training, each mouse was allowed acclimatization in the light compartment for 30 s, after which the gate was raised to allow entry into the dark compartment. The latency to enter the dark compartment was considered a measure of learning. The gate was closed as soon as the mouse entered the dark compartment. Five seconds later, a plantar electric shock [0.1 mA ( Figure 1 C) or 0.2 mA ( Figure 1 D)] 2 s (training).

[0066] To examine the effects of increased or decreased cAMP levels on IA memory retrieval and maintenance ( Figure 1 (C, D) Mice were trained as described above, and after 24 h, they were returned to the light compartment until they entered the dark compartment without being subjected to foot shocks (re-exposure). Memory was assessed twice at 48 h after re-exposure, as a cross-latency when the mouse was returned to the light compartment and entered the dark compartment, as in the reactivation (test).

[0067] Virus – As previously reported (Stierl et al., J Biol Chem. 2011;286(2):1181-8), a photoactivated adenylate cyclase (bPAC) was constructed by PCR using the S27A mutant. A photoactivated phosphodiesterase (LAPD) (Gasser et al., Proc Natl Acad Sci US A. 2014;111(24):8803-8) with the catalytic domain of human phosphodiesterase 4 (NM_002600) (GenScript, NJ, USA) was synthesized. AAV9-CaMKII-mGFP-bPAC (titer: 1.21 × 10⁻⁶) was packaged and purified by SignaGenlaboratories (SL100863 and SL100864, ​​Rockville, MD, USA). 13 VG / mL), AAV9-CK0.4-LAPD-GFP (titer: 3.16 × 10⁻⁶) 13 VG / mL), AAV9-CaMKII-GFP (titer: 2.98 × 10⁻⁶) 13 VG / mL).

[0068] Virus injection and fiber optic implantation- For viral injection, stereotactic injection of adeno-associated virus (AAV) vector was performed in a biosafety cabinet. Mice were anesthetized with a combination of metoprimidine-midazolam-butorphanol and placed in a stereotactic frame. The skull was exposed, and a small portion of the skull above the dorsal hippocampus was removed bilaterally using a drill. AAV (0.3 μL / site, 0.1 μL / min) was injected into the mouse brain using a glass capillary pipette drawn using a micropipettes retractor (P-87, Sutter Instruments, Novato, California, USA) [anteroposterior (A / P) -1.6 mm, mid-lateral (M / L) ±1.6 mm, dorsoventral (D / V) (measured from the dura mater) -1.6 mm]. After injection, the glass pipette was held in place for 5 min, then slowly lifted and removed. The mice were sutured, and an antibiotic ointment was applied. The mice were then placed on a warm heater for recovery. One week after viral injection, mice underwent a standard stereotactic procedure using metoprimidine-midazolam-butorphanol combined anesthesia to implant a stainless steel guide cannula (size 22) into the dorsal hippocampus (-1.6 mm, ±1.6 mm, -1.6 mm). The procedure was performed as previously described (Fukushima et al., J Neurosci. 2021;41:1288-1300). Mice were allowed one week of postoperative recovery before behavioral analysis. Stereotactic coordinates for dorsal hippocampal localization were based on brain atlases (Franklin and Paxinos, The mouse brain in stereotaxic coordinates. Elsevier Academic: San Diego; 1997).

[0069] Optogenetic manipulation of cAMP levels in the dorsal hippocampus - To examine the effects of increased or decreased cAMP levels on fear memory, mice were micro-injected into the dorsal hippocampus with AAV vectors (AAV9-CaMKII-mGFP-bPAC or AAV9-CaMKII-GFP) expressing photoactivated adenylate cyclase (bPAC) under the control of the CaMKII promoter. Figure 2 A, B) or AAV vectors expressing light-activated phosphodiesterase (LAPD) (AAV9-CK0.4-LAPD-GFP or AAV9-CaMKII-GFP); Figure 2(C, D) (Luyben et al., Front Neural Circuits. 2020;14:24). Starting 40 min before re-exposure, mice were stimulated with blue light for 30 min using a 473-nm laser at 4 Hz (pulse width 15 ms, ~1.0 mW, Lucir Inc., Tsukuba, Japan). Successful transduction in the hippocampus was histologically confirmed by native GFP fluorescence. Subsequent data analysis only included mice exhibiting bilateral GFP expression in the hippocampus.

[0070] Measurement of phosphorylated CREB levels To examine the effects of increased or decreased cAMP levels on CREB phosphorylation, mice were microinjected into the dorsal hippocampus with AAV vectors expressing bPAC, LAPD, or GFP under the control of the CaMKII promoter, and then a stainless steel guide cannula was implanted into the dorsal hippocampus as described above. Blue light stimulation was performed for 30 min as described above, and after 30 min, the mice were perfused for immunohistochemical staining of phosphorylated CREB-positive cells.

[0071] Immunohistochemistry Immunohistochemistry was performed as previously described (Ishikawa et al., Hippocampus. 2014;24:784–793; Fukushima et al., J Neurosci. 2021;41:1288-1300). After anesthesia, all mice were perfused with 4% paraformaldehyde. Brains were removed, fixed overnight, transferred to 30% sucrose, and stored at 4°C. Coronal sections (30 µm) were excised in a cryostat. Free-floating sections were treated with 1% H2O2 and incubated overnight in blocking solution (phosphate-buffered saline with 1% goat serum albumin and 1 mg / ml bovine serum albumin) with rabbit polyclonal antiphospho-CREB (serine 133; S133) antibody (1:2000; #06-519, Millipore). Sections were washed with phosphate-buffered saline and incubated at room temperature for 1 h with horseradish peroxidase-conjugated donkey anti-rabbit IgG (1:500; Jackson ImmunoResearch). The pCREB signal was amplified by biotinylate and visualized using Alexa Fluor-conjugated streptavidin (Invitrogen). Sections were mounted on glass slides and sealed with a DAPI-containing mounting medium (VECTASHIELD).

[0072] Quantitative– Quantification was performed as previously described (Ishikawa et al., Hippocampus. 2014;24:784–793; Fukushima et al., J Neurosci. 2021;41:1288–1300). Structures were anatomically defined according to the atlas of Franklin and Paxinos (1997). All immunoreactive neurons were counted by experimental personnel unaware of the treatment conditions. Fluorescence images were acquired using a TCS SP8 (Leica) confocal microscope. Confocal 2-μm z stacked images were acquired using LAS AF software (Leica). The same cutoff threshold was applied to all sections. We used 40× objectives for pCREB. + GFP + and DAPI + Cell counts were quantified. To quantify pCREB in hippocampal fields (290 × 290 mm; anterior fontanelle between -1.46 and -1.82 mm) in at least two sections... + Cells were analyzed using WinROOF version 5.6 software (Mitani Corporation, Fukui, Japan).

[0073] RNA analysis – Mouse hippocampal RNA analysis was performed as previously described (Tsujii et al., Neuropsychopharmacol Rep. 2021;41(2):230-36). Mice were euthanized by cervical dislocation. The dorsal hippocampus (anterior fontanelle between -1.46 and -2.18 mm) was dissected using a rodent brain matrix (MUROMACHI KIKAI Co., Ltd., Tokyo, Japan) and flash-frozen in liquid nitrogen. Total RNA was prepared from the dorsal hippocampal region of mice using the RNeasy mini kit (Qiagen, Valencia, California, USA). For peripheral blood mRNA analysis, mice were anesthetized, and blood was collected via cardiac puncture. Total RNA was isolated from peripheral blood using RNA-protected animal blood tubes (Qiagen) and the RNeasy protected animal blood kit (Qiagen), following the manufacturer's instructions.

[0074] Quantitative reverse transcription PCR (qRT-PCR) was performed as previously described (Tsujii et al., 2021). Total RNA (500 ng) was reverse transcribed using Superscript III reverse transcriptase (Invitrogen) and oligo-dT primers. qRT-PCR was performed using a SYBR Green PCR Master mix (Thermo Fisher Scientific), according to the manufacturer's protocol, and an ABIPRISM 7000 (Applied Biosystems, California, USA). The reaction was first incubated at 50 °C for 2 min, then at 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. Amplification of individual PCR products was confirmed by monitoring the dissociation curves. The amplification curves were visually examined to establish appropriate baseline ranges and threshold levels. Target molecules were quantified using a relative quantification method according to the manufacturer's protocol, where the ratio of each target molecule to a reference molecule in the same sample was calculated. All measurements were performed triplicate. Gapdh The mRNA level was used to normalize the relative expression level of the target mRNA. The primer sequences used for qRT-PCR analysis are listed in Table 1.

[0075] Table 1 - List of all primer sequences used in mouse qRT-PCR assays

[0076] RNA sequencing- RNA sequencing was performed as previously described (Tsujii et al., 2021). Total RNA was isolated from the dorsal hippocampus of mice using the RNeasy mini kit (Qiagen). RNA-seq analysis was performed for each experimental group (n = 5 animals per group). After RNA quality checks [all RNA integrity digital (RIN) values ​​> 8.0] were performed on an Agilent Bioanalyzer using the RNA Nanokit (Agilent Technologies), 1 μg of total RNA was used to prepare cDNA libraries using the TruSeq RNA Sample Preparation Kit v2 (Illumina, San Diego, CA, USA). The derived cDNA libraries were analyzed using the DNA 1000 kit on an Agilent Bioanalyzer and quantified by qPCR using the KAPA Library Quantification Kit (KAPA Biosystems, Wilmington, MA, USA). cDNA libraries were pooled in lanes and clustered on cBot (Illumina) to obtain 100-bp single-end reads on a HiSeq 2500 sequencer (Illumina). Demultiplexed fastq files were generated using bcl2fastq version 2.18 (Illumina). Filtering, mapping, and differential expression analysis were performed using CLC Genomics Workbench software version 9.5 (Qiagen). Raw sequence reads were filtered to exclude adapter sequences, ambiguous nucleotides, and low-quality sequences, and the retained sequences were aligned to the mouse genome (mm10). RNA sequencing data have been deposited into the DDBJ Sequence Read Archive (DRA) and are available under accession number DRA013665.

[0077] Statistical analysis – The effects of drug, time, and group were analyzed using one-way ANOVA and post-hoc Newman-Keuls test, as well as two-way ANOVA and post-hoc Bonferroni comparison. Paired t-tests were used to analyze differences in cross-latency between groups between two tests (re-exposure comparison test). Student's t-tests were used to analyze differences in mRNA expression levels.

[0078] All references mentioned in this article are incorporated by way of citation.

[0079] result The retrieval and maintenance of fear memories are related to cAMP levels.– The effects of cAMP levels on the retrieval and subsequent fate of hippocampus-dependent situational fear memories were determined using pharmacology. To examine the effect of increased cAMP levels on situational fear memories, mice were given systemic injections of ROL, a PDE4 inhibitor, or a mediator (VEH / solvent) prior to memory retrieval. Mice were trained with a single foot shock (0.2 mA, 2 s; training) and then re-exposed to the training environment for 3 min (re-exposure) and 5 min (test) every 24 h. Mice were given a systemic injection of ROL (0.1 mg / kg body weight) or VEH 30 min before re-exposure. Two-way ANOVA showed significant effects of the drugs (ROL vs. VEH: F(1,38) = 10.191, p < 0.05), but not significant effects of time (re-exposure vs. test: F(1,38) = 1.775, p > 0.05) or time × drug interaction (F(1,38) = 0.000, p > 0.05). Figure 1 A). In re-exposure and testing, the freeze rate in the ROL group was significantly higher than that in the VEH group (ps < 0.05; post-hoc Bonferroni test). These results indicate that ROL injection enhances the retrieval and subsequent maintenance of situational fear memories. Therefore, these observations suggest that upregulation of cAMP levels enhances situational fear memories.

[0080] To examine the effect of reduced cAMP levels, the adenylate cyclase 1 inhibitor NB001 was used. Similar experiments to those described above were performed, except that mice were trained with plantar electric shocks (0.4 mA, 2 s) and were administered NB001 (30 mg / kg body weight) or VEH twice, every hour starting 2 h before re-exposure. In both re-exposure and testing, the freezing point in the NB001 group was significantly lower than in the VEH group (two-way ANOVA: drug, F(1,38) = 13.317, p < 0.05; time, F(1,38) = 0.43, p = 0.516; and time × drug interaction, F(1,38) = 0.305, p = 0.584; post-hoc Bonferroni test, p < 0.05). Figure 1 B). These results indicate that adenylate cyclase 1 inhibitors (such as NB001) impair the retrieval and subsequent expression of situational fear memories, suggesting that downregulation of cAMP levels disrupts situational fear memories.

[0081] The effects of drug activation (ROL) and inactivation (NB001) on IA memory (another type of hippocampal-dependent fear memory) were also investigated. Mice were first placed in a light-lit compartment. A brief plantar electric shock (0.1 mA, 2 s) was delivered 5 s after they entered the dark compartment (training). 24 h after training, the mice were re-exposed to the light compartment, and their cross-latency to the dark compartment was assessed (re-exposure). Mice were immediately returned to their housing cages after entering the dark compartment from the light compartment. Prior to re-exposure, the mice received a systemic injection of either VEH or ROL. Cross-latency was assessed after 48 h (test). Compared with the VEH group, the ROL group showed significantly longer cross-latency at re-exposure and testing (two-way ANOVA: drug, F(1,36) = 30.872, p<0.05; time, F(1,36) = 24.355, p<0.05; and drug × time interaction, F(1,36) = 10.239, p<0.05; post-hoc Bonferroni test, ps<0.05). Figure 1 C). With Figure 1 Consistent with the results in A, these observations suggest that ROL enhances the retrieval and maintenance of IA memories. Importantly, both the VEH and ROL groups exhibited significantly increased cross-latency in the test compared to re-exposure (paired t-test, ps < 0.05); Figure 1 C).

[0082] In contrast, systemic injection of NB001 weakened IA memory retrieval and maintenance (two-factor ANOVA: drug, F(1,36) = 30.692, p < 0.05; time, F(1,36) = 15.02, p < 0.05; and drug × time interaction, F(1,36) = 17.531, p < 0.05). Figure 1 D). Compared with the VEH group, the NB001 group showed a significantly shorter cross latency at both re-exposure and testing (post-hoc Bonferroni test, p < 0.05), although the VEH group (but not the NB001 group) showed a significantly increased cross latency at testing compared to re-exposure (paired t-test, p < 0.05). Figure 1 D).

[0083] It is important to note that previous studies have demonstrated that memory reconsolidation enhances IA memories rather than situational fear memories after retrieval (Fukushima et al., 2021a, 2021b). The results of the two-factor ANOVA reflect the difference in the "reconsolidation effect" between IA memories and situational fear memories (a significant effect of time and time-time × drug interaction was observed in IA memories, but not in situational fear memories).

[0084] To further investigate the role of cAMP levels in fear memory, the effects of optogenetic manipulation of cAMP levels in the hippocampus were examined. Newly developed optogenetic probes capable of generating or degrading cAMP in a blue light-dependent manner were used. To examine the effects of increased cAMP levels, mice were microinjected into the dorsal hippocampus with AAV vectors expressing bPAC under the control of the CaMKII promoter (AAV9-CaMKII-mGFP-bPAC or AAV9-CaMKII-GFP). Figure 2 A). Used to generate Figure 1 Experiment A yielded similar results, but differed in that blue light was applied for 30 minutes using a 473-nm laser at 4Hz (pulse width, 15 ms), starting 40 minutes before re-exposure. Compared to the GFP and dark control groups, the bPAC group showed significantly more freezing at re-exposure and testing (two-way ANOVA: group, F(2, 58) = 12.566, p < 0.05; time, F(1, 60) = 0.362, p > 0.05; and group × time interaction, F(2, 60) = 0.393, p > 0.05; post-hoc Bonferroni test, p < 0.05). Figure 2 B). These observations suggest that optogenetic increases in cAMP levels in the hippocampus contribute to the retrieval and maintenance of situational fear memories.

[0085] The effects of optogenetic reduction in cAMP levels were then investigated. Mice were microinjected into the dorsal hippocampus with AAV expressing LAPD under the control of the CaMKII promoter (AAV9-CK0.4-LAPD-GFP or AAV9-CaMKII-GFP). Figure 2 C). Used to generate Figure 1 Experiment B yielded similar results, except that blue light was applied for 30 minutes using a 473-nm laser at 4 Hz (pulse width, 15 ms) starting 40 minutes before re-exposure. Compared to the GFP and dark control groups, the LAPD group experienced significantly less freezing during re-exposure and testing (two-way ANOVA: group, F(2,60) = 10.825, p < 0.05; time, F(1,60) = 2.498, p > 0.05; and group × time interaction, F(2,60) = 0.362, p > 0.05; post-hoc Bonferroni test, p < 0.05). Figure 2 D). These results indicate that optogenetic reduction of cAMP levels in the hippocampus weakens the retrieval and maintenance of situational fear memories.

[0086] Notably, the effects of optogenetic manipulation of bPAC and LAPD on cAMP signaling in the hippocampus were examined. To this end, the phosphorylation level of CREB at serine 133 (pCREB level), a target of cAMP-activated protein kinase A (PKA), was examined. Compared to the control group, mice expressing bPAC or LAPD showed significantly increased or decreased pCREB levels in the CA1 region of the dorsal hippocampus, respectively, 30 min after blue light stimulation. These observations indicate that optogenetic manipulation of bPAC and LAPD reflects activation or inactivation of cAMP-PKA signaling, respectively.

[0087] In summary, these observations suggest that increases or decreases in cAMP levels before and after memory retrieval enhanced or weakened the retrieval and subsequent maintenance of fear memories, respectively. Therefore, these observations indicate that the state of fear memories reflects cAMP levels.

[0088] mouse transcriptome - A comprehensive analysis was performed on the transcriptomic profiles associated with the retrieval of fear memories in mice and those associated with symptom re-experiencing in humans. For the mouse transcriptomic analysis, [the text abruptly ends here, likely due to an incomplete sentence or missing information]. Figure 1 A similar behavioral experiment was conducted, followed by RNA-seq analysis of the dorsal hippocampus of mice 30 min after IA memory retrieval (re-exposure). Significantly increased (3,997) and decreased (3,140) mRNA levels were observed compared to a trained control group (not reactivated; NR) that was not re-exposed to the light compartment during re-exposure. These observations suggest that IA memory retrieval rapidly induces changes in gene expression in the dorsal hippocampus.

[0089] Integrated transcriptome analysis was performed to identify key genes associated with fear memory. For mouse hippocampal transcriptome data, using an FDR threshold of q<0.001 and |FC|>1.36, the expression levels of 4908 genes (3034 upregulated and 1874 downregulated) changed significantly after fear memory extraction to induce reconsolidation compared to the unextracted control. These analyses identified 97 target genes. Using a t-test threshold of p<0.05, 15 of these 97 genes showed significant differences between healthy mice and mice subjected to fear memory conditioning. The 15 selected genes included PDE4B. Specifically, PDE4B expression levels were significantly downregulated in mice after fear memory extraction (FC=-1.4, p<0.001).

[0090] Fear memory retrieval reduced Pde4b mRNA levels in the hippocampus and blood of mice. - The changes in mRNA expression in the dorsal hippocampus of mice after IA memory retrieval were verified using qRT-PCR. Figure 3Therefore, work was carried out with the method used to generate... Figure 1 The experiment with data in D was similar to that in the experiment. Hippocampal mRNA levels were measured 30 min after reactivation [reactivation-30 group and non-reactivation (NR) group] ( Figure 3 A). Since Pde4b, Rap1a, and Nkap showed significant downregulation in the transcriptomes of human blood and mouse hippocampus associated with re-experiencing / fear memory retrieval, we measured the mRNA levels of these genes. Similar to previous studies, significantly increased c-fos mRNA levels were observed in the hippocampus of the reactivation-30 group compared to the NR group (t-test, p < 0.05). Figure 3 B). Importantly, compared with the NR group, the levels of Pde4b, Rap1a, and Nkap mRNA in the hippocampus of the reactivation-30 group were significantly reduced (t-test, ps < 0.05); Figure 3 B). To examine the relationship between mRNA levels in the hippocampus and peripheral blood in mice after IA memory retrieval, blood mRNA levels were measured at 30 and 90 min after reactivation. Figure 3 C). Similar to the results obtained in the hippocampus, the peripheral blood levels of Pde4b, Rap1a, and Nkap mRNA were significantly lower in the reactivation -30 and / or -90 groups compared with the NR group (one-way ANOVA followed by post-hoc Newman-Keuls test: Pde4b, F(2,21) = 2.649, p < 0.05; Rap1a, F(2,28) = 4.727, p < 0.05; Nkap, F(2,28) = 3.674, p < 0.05; ps < 0.05; Figure 3 (C) indicates that IA memory retrieval resulted in decreased expression levels of Pde4b, Rap1a, and Nkap mRNA in the hippocampus and peripheral blood. Therefore, these results suggest that the changes in these mRNA levels induced by IA memory retrieval are consistent between the hippocampus and peripheral blood in mice.

[0091] discuss The effects of gain- and loss-of-function of the cAMP signaling pathway on hippocampal-dependent situational fear and IA memories were examined in mice. Pharmacological and optogenetic activation or inactivation of cAMP signaling promoted or weakened the retrieval and maintenance of these memories, respectively. These findings suggest that the state of the cAMP signaling pathway determines the state of fear memories. Importantly, integrated transcriptomic analysis revealed downregulation of the mRNA expression of genes, including PDE4B, an enzyme that degrades cAMP, in the mouse hippocampus after fear memory retrieval. Furthermore, PDE4B mRNA expression was downregulated in the mouse hippocampus and peripheral blood after fear memory retrieval. Since downregulation of PDE4B mRNA expression leads to activation of the cAMP signaling pathway by reducing cAMP degradation, downregulation of PDE4B expression appears to be related to the mechanism by which re-experiencing symptoms occur in PTSD through activation of the cAMP signaling pathway.

[0092] Animal models ranging from invertebrates to mammals show that the cAMP signaling pathway plays a role in learning and memory, as well as neuroplasticity. This behavioral study in mice unexpectedly revealed that gain or loss of function of the cAMP signaling pathway enhanced or blocked fear memory retrieval, respectively.

[0093] In this study, activated and inactivated cAMP signaling promoted or weakened memory maintenance prior to memory retrieval. Furthermore, optogenetic manipulation of bPAC and LAPD increased or decreased pCREB levels in the dorsal hippocampus, respectively. Therefore, this suggests that activation or inactivation of cAMP signaling enhances or weakens the reconsolidation of fear memories via CREB-mediated transcription, thereby modulating memory maintenance.

[0094] Further observation revealed that IA memory retrieval reduced Pde4b mRNA levels in the hippocampus and peripheral blood of mice, indicating synchronized gene expression regulation between brain and blood cells, and demonstrating a correlation between re-experiencing symptoms in PTSD patients and decreased PDE4B mRNA levels in peripheral blood. Therefore, the observations suggest that PTSD patients exhibiting re-experiencing symptoms may have reduced PDE4B expression in the brain. More importantly, since cAMP levels increase due to decreased PDE4B levels, downregulation of all PDE4B contributes to enhanced fear memory retrieval through increased cAMP levels. Therefore, the increased cAMP levels mediated by decreased PDE4B expression are considered to have implications for the pathophysiology of PTSD, particularly re-experiencing symptoms.

[0095] The cAMP signaling pathway is regulated by a large number of signal transduction molecules. As shown in this paper, our findings suggest that a potential mechanism of PTSD is through activation of this pathway.

[0096] Genes co-expressed with PDE4B were also examined. Gene ontology analysis of co-expressed genes identified the ontology associated with MAPK activation as the most important signal, and within the MAPK family, RAP1A was strongly co-expressed with PDE4B and NKAP / Nkap. These data also indicate that RAP1A / Rap1a expression was persistently downregulated in mouse fear memory retrieval.

[0097] In summary, this study demonstrates that increased cAMP levels promote memory retrieval, and that PDE4B expression is downregulated in the hippocampus and peripheral blood of mice after fear memory retrieval. These findings suggest that increased cAMP levels induced by downregulation of PDE4B mRNA enhance traumatic memory, thus playing a crucial role in using the re-experiencing of symptoms in PTSD patients as a functional indicator of these symptoms.

Claims

1. A method for mitigating fear memories in mammals, comprising steps of inhibiting or reducing cAMP signaling in mammals.

2. The method according to claim 1, wherein, cAMP signaling was inhibited or reduced by administering an adenylate cyclase 1 (AC1) inhibitor to the mammal.

3. The method according to claim 2, wherein, The AC1 inhibitor has the following general formula (1): （1） in: A is selected from H, OH, halogen, C1-C6 alkyl, C1-C6 alkyl halide, C2-C6 alkenyl, C2-C6 alkynyl and C1-C6 alkoxy; B is selected from hydroxyl, thio, -OR 1 -NH2, -NO2, -NHR 1 -NR 1 R 2 -SR 1 or -C1-C6 saturated or unsaturated alkyl groups, optionally substituted with one or more substituents selected from hydroxyl, halogen, thio, OR 1 NH2, NO2, NHR 1 NR 1 R 2 SR 1 C3-C 10 Aromatic or non-aromatic ring structure or C3-C9 aromatic or non-aromatic heterocyclic structure, optionally substituted with OH, halogen, thio, NH2, C1-C6 alkyl, C1-C6 alkanol or C1-C6 alkoxy, wherein R 1 and R 2 Independently selected from C1-C6 alkyl, C1-C6 alkyl halide, C1-C6 alkenyl, C1-C6 alkynyl, C1-C6 alkanol, C1-C6 alkoxy and C1-C6 carboxylalkyl, or B is NR 1 R 2 It forms a C3-C6 aromatic or non-aromatic heterocycle, optionally substituted with OH, halogen, thio, NH2, NO2, C1-C6 alkyl, C1-C6 alkanol, C1-C6 alkoxy, or C1-C6 carboxylalkyl. D is selected from H, halogen, hydroxyl, NH2, thio, NHR 1 NR 1 R 3 SR 1 C1-C6 alkyl, C1-C6 alkoxy, wherein R 1 As defined above and R 3 For example, regarding R 1 Definition; E is H or OH, or E represents C1-C6 alkyl, C1-C6 alkoxy, C 3-10 -Aryl-C 1-6 -alkyl or C 3-10 -Aryloxy-C 1-6 -alkyl, optionally substituted with C 1-6 -alkyl, amino, NHR 1 NR 1 R 2 thiolated, SR 1 Unsubstituted C3-C7 cycloalkyl, phenyl, or C4-C6 heterocycles, or substituted C3-C7 cycloalkyl, phenyl, or C4-C6 heterocycles, wherein the substituted C3-C7 cycloalkyl, phenyl, or C4-C6 heterocycle has one or more substituents selected from C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkanoyl, C1-C6 carboxyl, halogen, or OH. E is an unsubstituted C3-C7 cycloalkyl, phenyl, or C4-C6 heterocycle, or a substituted C3-C7 cycloalkyl, phenyl, or C4-C6 heterocycle, wherein the substituted C3-C7 cycloalkyl, phenyl, or C4-C6 heterocycle has one or more substituents selected from C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkanoyl, C1-C6 carboxyl, halogen, or OH. Among them, R 1 and R 2 As defined above; and G, H, J, and M are all N, or H and J are both C, and G and M are both N, S, or O, or H, J, and M are all C, and G is N, S, or O.

4. The method according to claim 3, wherein, The AC1 inhibitor is a compound of formula (1), wherein G, H, J and M are both N; A is hydrogen; B is -NH2, -NHR 1 or -NR 1 R 2 , where R 1 and R 2 Independently selected from C1-C6 alkyl, C1-C6 alkanol, or C1-C6 alkoxy groups; D is NH2, SH, or -NHR. 1 -NR 1 R 3 or -SR 1 , where R 1 and R 2 It is independently selected from C1-C6 alkyl, C1-C6 alkanol, C1-C6 carboxylalkyl or C1-C6 alkoxy; E is one of the following: -methylphenyl, -ethylphenyl, -propylphenyl, -methylaminophenyl, -methylaminopropanol, -ethylaminopentanol, -1-methyl-2,6-dichlorophenyl, -1-2,3-dihydroxy-4-methanol-tetrahydrofuran and -p-ethoxy-tolyl.

5. The method according to claim 3, wherein, The AC1 inhibitor is a compound selected from the compounds in Figure 4.

6. The method according to claim 3, wherein, The AC1 inhibitor is a compound of formula (1), wherein: A is H; B is NH2; D is H; E is CH2CH2-NH(CH2)5OH; and G, H, J and M are all N.

7. The method according to claim 1, wherein, cAMP signaling can be inhibited or reduced by upregulating the expression of cAMP-degrading enzymes.

8. The method according to claim 7, wherein, The cAMP-degrading enzyme is a phosphodiesterase.

9. The method according to claim 8, wherein, The phosphodiesterase is PDE4, PDE7, or PDE8.

10. The method according to claim 9, wherein, The phosphodiesterase is PDE4B.

11. The method according to any one of claims 7-10, wherein, The enzyme is administered to the mammal.

12. The method according to any one of claims 7-10, wherein, The nucleic acid encoding the enzyme is administered to the mammal.

13. The method according to any one of claims 1-12, wherein, The mammal suffers from fear-related mental illness.

14. The method according to claim 13, wherein, The mammal suffers from post-traumatic stress disorder (PTSD).

15. The use of cAMP inhibitors in the treatment of fear memories in mammals.

16. The use of phosphodiesterase in the treatment of fear memories in mammals.

17. The use according to claim 15 or 16, wherein, The mammal suffers from fear-related mental disorders.

18. A composition for treating fear memories in mammals, comprising an AC1 inhibitor and a pharmaceutically effective carrier, wherein, The AC1 inhibitor has the following general formula (1): （1） in: A is selected from H, OH, halogen, C1-C6 alkyl, C1-C6 alkyl halide, C2-C6 alkenyl, C2-C6 alkynyl and C1-C6 alkoxy; B is selected from: hydroxyl, thio, -OR 1 -NH2, -NO2, -NHR 1 -NR 1 R 2 -SR 1 -C1-C6 saturated or unsaturated alkyl groups, optionally substituted with hydroxyl, halogen, thio, OR 1 NH2, NO2, NHR 1 NR 1 R 2 SR 1 C3-C 10 One or more substituents in an aromatic or non-aromatic ring structure or a C3-C9 aromatic or non-aromatic heterocyclic structure, optionally substituted with OH, halogen, thio, NH2, C1-C6 alkyl, C1-C6 alkanol or C1-C6 alkoxy, wherein R 1 and R 2 Independently selected from C1-C6 alkyl, C1-C6 alkyl halide, C1-C6 alkenyl, C1-C6 alkynyl, C1-C6 alkanol, C1-C6 alkoxy and C1-C6 carboxylalkyl, or B is NR 1 R 2 It forms a C3-C6 aromatic or non-aromatic heterocycle, optionally substituted with OH, halogen, thio, NH2, NO2, C1-C6 alkyl, C1-C6 alkanol, C1-C6 alkoxy, or C1-C6 carboxylalkyl. D is selected from H, halogen, hydroxyl, NH2, thio, NHR 1 NR 1 R 3 SR 1 C1-C6 alkyl, C1-C6 alkoxy, wherein R 1 As defined above and R 3 For example, regarding R 1 Definition; E is H or OH, or E represents C1-C6 alkyl, C1-C6 alkoxy, C 3-10 -Aryl-C 1-6 -alkyl or C 3-10 -Aryloxy-C 1-6 -alkyl, optionally substituted with C 1-6 -alkyl, amino, NHR 1 NR 1 R 2 thiolated, SR 1 Unsubstituted C3-C7 cycloalkyl, phenyl, or C4-C6 heterocycles, or substituted C3-C7 cycloalkyl, phenyl, or C4-C6 heterocycles, wherein the substituted C3-C7 cycloalkyl, phenyl, or C4-C6 heterocycle has one or more substituents selected from C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkanoyl, C1-C6 carboxyl, halogen, or OH. E is an unsubstituted C3-C7 cycloalkyl, phenyl, or C4-C6 heterocycle, or a substituted C3-C7 cycloalkyl, phenyl, or C4-C6 heterocycle, wherein the substituted C3-C7 cycloalkyl, phenyl, or C4-C6 heterocycle has one or more substituents selected from C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkanoyl, C1-C6 carboxyl, halogen, or OH. Among them, R 1 and R 2 As defined above; and G, H, J, and M are all N, or H and J are both C, and G and M are both N, S, or O, or H, J, and M are all C, and G is N, S, or O.

19. The composition according to claim 18, wherein, The AC1 inhibitor is a compound of formula (1), wherein G, H, J and M are all N; A is hydrogen; and B is -NH2 or -NHR. 1 or -NR 1 R 2 , where R 1 and R 2 Independently selected from C1-C6 alkyl, C1-C6 alkanol, or C1-C6 alkoxy groups; D is NH2, SH, or -NHR. 1 -NR 1 R 3 or -SR 1 , where R 1 and R 2 It is independently selected from C1-C6 alkyl, C1-C6 alkanol, C1-C6 carboxyl or C1-C6 alkoxy; and E is one of the following: -methylphenyl, -ethylphenyl, -propylphenyl, -methylaminophenyl, -methylaminopropanol, -ethylaminopentanol, -1-methyl-2,6-dichlorophenyl, -1-2,3-dihydroxy-4-methanol-tetrahydrofuran and -p-ethoxy-tolyl.

20. The composition according to claim 18 or 19, wherein, The AC1 inhibitor is a compound selected from the compounds in Figure 4.

Images