Method of reducing fear memory

Abstract

A method of mitigating fear memory in a mammal comprising the step of inhibiting or reducing cAMP signaling in the mammal. cAMP signaling may be mitigated with an adenyl cyclase 1 (AC1) inhibitor, or using a phosphodiesterase, such as a cAMP-specific phosphodiesterase such as PDE4.

Description

METHOD OF REDUCING FEAR MEMORYField of the Invention

[0001] The present invention generally relates to fear memory, and more particularly, to methods of reducing fear memory and / or treating fear-related psychiatric conditions such as post traumatic stress disorder (PTSD).Background

[0002] Posttraumatic stress disorder (PTSD) is a serious psychiatric condition that can develop 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 reexperiencing, avoidance, and hyperarousal. Reexperiencing refers to the involuntary retrieval of traumatic memories, such as flashbacks, nightmares, and intrusive thoughts. While avoidance and hyperarousal are common to many anxiety-related disorders, reexperiencing is largely unique to PTSD and is recognized widely as a core feature of this disorder. Since reexperiencing can be understood as a persistent conditioned response, i.e., a learned response to a previously neutral stimulus, the learning and memory processes involved in fear memory regulation are postulated to underlie reexperiencing symptoms.

[0003] The development of PTSD depends on environmental factors such as traumatic experiences. However, there is growing evidence that genetic factors also play an important role, indicating that PTSD is caused by a combination of environmental and genetic factors. Pituitary adenylate cyclase-activating polypeptide (PACAP) in peripheral blood is associated with PTSD symptoms in females, while a single nucleotide polymorphism in the PAC1 receptor gene (ADCYAP1R1) is associated with PTSD symptoms in females, and shows fear- and estrogen-induced expression. Interestingly, a genome-wide association study identified corticotropin-releasing hormone receptor 1 gene (CRHR1) as relevant to intrusive reexperiencing in PTSD.

[0004] Fear memory is generated through traumatic experiences. In experimental animals, Pavlovian fear conditioning and inhibitory avoidance (IA) tasks, both of which generate fear memory, have been used widely as models of PTSD. Fear memory is initially labile up to several hours after a fear experience and then stabilized through gene expression-dependent memory consolidation. Importantly, memory retrieval is not a passive process; consolidated memory becomes labile again when it is retrieved and then maintained or enhanced through gene expression-dependent reconsolidation (restabilization). Conditioned fear memory has been observed in many animal species from insects tohumans; therefore, the mechanisms for fear memory regulation is considered to be similar between humans and other animals.

[0005] Given the prevalence of PTSD, and it debilitating effects, it would be desirable to develop a method of treatment, or a method that reduces one or more undesirable symptoms thereof.Summary of the Invention

[0006] It has now been determined that increased cyclic adenosine monophosphate (cAMP) signaling results in pronounced fear memory and more severe reexperiencing symptoms in patients with certain psychiatric conditions in which pathological fear is a characteristic, and that inhibition of cAMP signaling mitigates these symptoms. Increased expression of cAMP degrading enzymes also have a role to play in mitigation of such conditions.

[0007] Thus, in one aspect of the invention, a method of mitigating fear memory in a mammal is provided comprising the step of inhibiting or reducing cAMP signaling in the mammal.

[0008] In embodiments, cAMP signaling is inhibited or reduced by administration to the mammal of an adenyl cyclase 1 inhibitor.

[0009] In other embodiments, cAMP signaling is inhibited or reduced by upregulating expression of a cAMP degrading enzyme, including a phosphodiesterase.

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

[0011] In another aspect, use of a cAMP inhibitor to mitigate fear memory and / or treat PTSD in a mammal is provided.

[0012] In a further aspect of the invention, a composition for use to mitigate fear memory in a mammal 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 in the description and examples that follow by reference to the following Figures.Brief Description of the Figures

[0014] Figure 1 - illustrates effects of the systemic injection of rolipram (ROL) (A) or NB001 (B) on contextual fear memory (A, Vehicle (VEH), n = 10; ROL, n = 11. B, VEH, n = 10; NB001, n =11); the effects of the systemic injection of ROL (C) or NBOOl (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, post hoc Bonferroni's test; #p < 0.05, paired Ltest. Error bars indicate SEM);

[0015] Figure 2 - illustrates A) a schematic of virus injection and mGFP expression in the dorsal hippocampus (upper images) and photostimulation of photoactivatable adenylyl cyclase (bPAC; lower image); B) effects of optogenetic increased cAMP levels in the dorsal hippocampus on contextual fear memory. GFP, n = 12; no stimulation (No Stim.), n = 9; optical stimulation (Optical Stim.), n = 11; C) a schematic illustration of virus injection and GFP expression in the dorsal hippocampus (upper images) and photostimulation of light-activated phosphodiesterase (LAPD; lower image); and D) effects of optogenetic decreased cAMP levels in the dorsal hippocampus on contextual fear memory. GFP, n = 11; No Stim., n = 11; Optical Stim., n = 11. *p < 0.05; post hoc Bonferroni's test. Error bars indicate SEM;

[0016] Figure 3 - illustrates changes of mRNA levels in the murine dorsal hippocampus and peripheral blood following IA memory retrieval, including A) a schematic of experimental design; B) graphical illustration of changes of mRNA levels in the dorsal hippocampus. Non-reactivated (NR), n = 6-10; 30 min after reactivation (React-30), n = 6-10; and C) graphical illustration of changes of mRNA levels in peripheral blood. NR, n = 8-10; React-30, n = 8-11; 90 min after reactivation (React-90), n = 8-10. *p < 0.05, post hoc Bonferroni's test; #p < 0.05, Student’s Ltest. Error bars indicate SEM.

[0017] Figure 4 - illustrates AC1 inhibitors;

[0018] Figure 5 - illustrates the amino acid sequences of phosphodiesterases (PDEs), and specifically, of A) a PDE4, B) a PDE7A, C) a PDE7B, and D) a PDE8; and

[0019] Figure 6 - illustrates the nucleic acid sequence of a PDE4A.Detailed Description

[0020] A method of mitigating fear memory in a mammal is provided comprising the step of inhibiting or reducing cAMP signaling in the mammal. Fear memory is linked to re-experiencing, a symptom of fear-related psychiatric conditions.

[0021] The term “fear memory” is used herein to refer to memory of a traumatic event and a reaction to it. Fear memory is formed in multiple brain regions including the hippocampus (contextual conditioning and inhibitory avoidance), the basolateral amygdala (inhibitory avoidance), the lateral amygdala (conditioning to a tone), the anterior cingulate cortex (inhibitory avoidance), and the medial prefrontal cortex (inhibitory avoidance). Fear memory can be intrusive, causing re-experiencing of thetraumatic event and increased arousal and stress response, resulting in a fear-related psychiatric condition.

[0022] A fear-related psychiatric condition is a psychiatric condition in which fear memory plays a role. Generally, pathological fear is a characteristic of such a condition, which includes phobias, anxiety and post-traumatic stress disorder (PTSD). Pathological fear refers to an abnormal or extreme fear, or feeling of danger, which evokes a powerful emotional state or reaction that cannot be controlled.

[0023] Post-traumatic stress disorder (PTSD) is a mental or behavioural disorder that develops in a mammal who has experienced a traumatic event, e.g. a shocking, frightening, or dangerous event. PTSD may be diagnosed using a number of established diagnostic tools such as Posttraumatic Diagnostic Scale (PDS) according to the DSM 5 (Diagnostic and Statistical Manual of Mental Disorders, 5th edition) criteria, Clinician-Administered PTSD Scale 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 reactivity symptoms; and iv) at least two cognition and mood symptoms, for at least 1 month. Re-experiencing symptoms include: experiencing flashbacks — reliving the traumatic event, including physical symptoms such as a racing heart or sweating; having recurring memories (fear memory) or dreams related to the event; having distressing thoughts; and / or experiencing physical signs of stress. Thoughts and feelings can trigger these symptoms, as can words, objects, or situations that are reminders of the event. Avoidance symptoms include: staying away from places, events, or objects that are reminders of the traumatic experience, and / or avoiding thoughts or feelings related to the traumatic event. Avoidance symptoms may cause people to change their routines. For example, some people may avoid driving or riding in a car after a serious car accident. Arousal and reactivity symptoms include: being easily startled; feeling tense, on guard, or on edge; having difficulty concentrating; having difficulty falling asleep or staying asleep; feeling irritable and having angry or aggressive outbursts; and / or engaging in risky, reckless, or destructive behavior. Arousal symptoms are often constant. They can lead to feelings of stress and anger and may interfere with parts of daily life, such as sleeping, eating, or concentrating. Cognition and mood symptoms include: having trouble remembering key features of the traumatic event; having negative thoughts about oneself or the world; having exaggerated feelings of blame directed toward oneself or others; having ongoing negative emotions, such as fear, anger, guilt, or shame; losing interest in enjoyable activities; having feelings of social isolation; and / or having difficulty feeling positive emotions, such as happiness or satisfaction.

[0024] The present method comprises the treatment of fear memory and / or PTSD in a mammal. The term “mammal” is used herein to refer to humans, and non-human mammals such as domestic animals such as cats, dogs, rodents, livestock such as cows, horses, goats, sheep, and the like. The term “treat” or “treatment” refers to the prevention, cure, amelioration, or the slowing of the progression of a fear memory or a fear-related psychiatric condition such as PTSD.

[0025] The method comprises the step of inhibiting or reducing cAMP signaling. cAMP signaling may be inhibited or reduced by the use of a small molecule inhibitor that inhibits adenylcyclase 1 (AC1) activity.

[0026] In one embodiment, cAMP signaling is inhibited or reduced by administration to the mammal of an AC1 inhibitor as described in PCT / CA2006 / 001687, the contents of which are incorporated herein by reference. The AC1 inhibitor has the following general formula (1):wherein:A is selected from the group consisting of H, OH, halogen, Ci-Ce alkyl, Ci-Ce alkyl halide, C2- Ce alkenyl, C2-C6 alkynyl and Ci-Ce alkoxy;B is selected from the group consisting of: hydroxy, thio, -OR1, -NH2, -NO2, -NHR1, -NR1R2, - SR1or -Ci-Ce saturated or unsaturated alkyl group optionally substituted with one or more substituents selected from hydroxy, halogen, thio, OR1, NH2, NO2, NHR1, NR1R2, SR1, a C3-C10 aromatic or nonaromatic ring structure or a C3-C9 aromatic or non-aromatic heterocyclic ring structure optionally substituted with OH, halogen, thio, NH2, Ci-Ce alkyl, Ci-Ce alkanol or Ci-Ce alkoxy, wherein R1and R2are independently selected from the group consisting of Ci-Ce alkyl, Ci-Ce alkyl halide, Ci-Ce alkenyl, Ci-Ce alkynyl, Ci-Ce alkanol, Ci-Ce alkoxy and Ci-Ce carboxyalkyl, orB is NR.1R.2forms a C3-C6 aromatic or non-aromatic heterocyclic ring optionally substituted with OH, halogen, thio, NH2, NO2, Ci-Ce alkyl, Ci-Ce alkanol, Ci-Ce alkoxy or Ci-Ce carboxyalkyl,D is selected from the group consisting of: H, halogen, hydroxy, NH2, thio, NHR1, NR1R3, SR1, Ci-Ce alkyl, Ci-Ce alkoxy, wherein R1is as defined above and R3is as defined for R1;E is H or OH, orE is Ci-Ce alkyl, Ci-Ce alkoxy, C3-io-aryl-Ci-6-alkyl or C3-io-aryloxy-Ci-6-alkyl optionally substituted with Ci-6-alkyl, amino, NHR1, NR.1R.2, thio, SR1, an unsubstituted C3-C7 cycloalkyl, phenyl or C4-C6 heterocyclic ring, or a substituted C3-C7 cycloalkyl, phenyl or C4-C6 heterocyclic ring having one or more substituents selected from the group consisting of Ci-Ce alkyl, Ci-Ce alkoxy, Ci-Ce alkanoyl, Ci-Ce carboxyalkyl, halogen or OH, orE is an unsubstituted C3-C7 cycloalkyl, phenyl or C4-C6 heterocyclic ring, or substituted C3-C7 cycloalkyl, phenyl or C4-C6 heterocyclic ring having one or more substituents selected from the groups consisting of Ci-Ce alkyl, Ci-Ce alkoxy, Ci-Ce alkanoyl, Ci-Ce carboxyalkyl, halogen or OH, wherein R1and R2are as defined above; andG, H, J and M are each N, orH and J are each C, and G and M are each N, S or O, orH, J and M are each C and G is N, S or O.

[0027] The base heterocyclic ring system in the compound of formula (1) may be such that each of variables G, H, J and M are nitrogen (N), i.e. a purine ring system. Alternatively, H and J may each be carbon, and G and M may be selected from N, S or O, for example, benzothiazole. In another alternative, H, J and M may each be carbon (C) and G may be either N, S or O, e.g. to yield an indole, benzothiophene or benzofuran ring system, respectively.

[0028] The variable A may be H; OH; halogen such as F, Cl, Br and I; Ci-Ce alkyl, including branched alkyl groups, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, 3 -methylpentyl, hexyl and isohexyl; Ci-Ce alkyl halide such as chloromethyl, fluoromethyl, bromoethyl, bromoethylenyl, propylfluoro, isopropyliodo, chlorobutyl, l,l-dichloro-2,3- butyl, 2-bromopentyl, 3 -chlorohexyl, l-fluoro-3 -methylhexyl and 1,1-difluorohexyl; C2-C6 alkenyl, including branched alkenyl groups, for example, ethylenyl, propylenyl, butenyl, isobutenyl, 2-butenyl, pentenyl, isopentenyl, 2-pentenyl and hexenyl; C2-C6 alkynyl such as ethynyl, propynyl and butynyl, including branched alkynyl groups; Ci-Ce alkoxy, including branched alkoxy groups, such as methoxy, ethoxy, propyloxy, isopropyloxy, butyloxy, isobutyloxy, methoxymethyl, ethoxymethyl, ethoxyethyl, methoxyethyl, methoxypropyl, ethoxypropyl, propyloxymethyl, propyl oxy ethyl, propyloxypropyl, pentyloxy, isopentyloxy, hexyloxy and isohexyloxy. Preferably A is hydrogen.

[0029] The variable B may be hydroxy, halogen, thio, -OR1, -NH2, -NO2, -NHR1, -NR1R2, SR1, or -Ci-Ce saturated or unsaturated alkyl group, for example, alkyl, alkenyl or alkynyl groups as exemplified above, optionally substituted with one or more substituents selected from hydroxy, halogen, thio, -OR1, -NH2, -NO2, -NHR1, -NR1R2or -SR1, or a C3-C10 aromatic or non-aromatic ring structure or a C3-C9 aromatic or non-aromatic heterocyclic ring structure optionally substituted with OH, halogen, thio, NH2, Ci-Ce alkyl, Ci-Ce alkanol or Ci-Ce alkoxy. The term “ring structure” is used herein to refer to structures comprised of a single ring as well as multi-cyclic structures, such as bicyclic structures. The term “heterocyclic ring” or “heterocyclic ring structure” is meant to include 3-9-membered ring structures that include at least one hetero atom selected from O, S and N within the core ring structure. Examples of suitable ring structures include benzene, naphthalene, tetralin, decalin, piperidine, pyrrolidine, furan, piperazine, tetrahydrothiphene, morpholine, imidazole, benzothiophene, quinoline, isoquinoline, indole, benzofuran and purine. Preferably, B is -NH2, -NHR1or -NR1R2, wherein R1and R2are independently selected from the group consisting of Ci-Ce alkyl, Ci-Ce alkanol or Ci-Ce alkoxy.

[0030] The variables R1and R2are independently selected from the group consisting of Ci-Ce alkyl, Ci-Ce alkyl halide, Ci-Ce alkenyl, Ci-Ce alkynyl, Ci-Ce alkanol, Ci-Ce alkoxy, or Ci-Ce carboxyalkyl. Thus, OR1may be, for example, oxymethyl, oxy -dimethyl, oxyethyl, oxy-3 -chlorobutyl, oxypropylenyl, or oxypropanol. NHR1may be, for example, alkylamine such as methylamine, as well as 2-chloro-propylamine, NH-ethanol, NH-propanol, NH-ethylmethyl ether or N-butyric acid. Similarly, NR.1R.2may be a dialkylamine such as di-ethylamine, or may be, for example, N-chloro-N-propyl, N- methyl-N-butyric acid, N-methyl-N-propanol.

[0031] NR' R2may also form a C3-C10 aromatic or non-aromatic heterocyclic ring structure, as exemplified above, that may optionally be substituted with OH, halogen, thio, NH2, NO2, Ci-Ce alkyl, Ci-Ce alkanol, Ci-Ce alkoxy or Ci-Ce carboxyalkyl.

[0032] The variable D may be H, halogen, hydroxy, NH2, thio, -NHR1, -NR' R3, -SR1, -Ci-Ce alkyl or -Ci-Ce alkoxy. R1is as defined above and R3is as defined for R1. Thus, D may be, for example, an unsubstituted group such as H, halogen, hydroxy, -NH2, thio (SH), -Ci-Ce alkyl or -Ci-Ce alkoxy. D may also be a substituted group, for example, D may be -NHR1in which R1is a Ci-Ce alkyl such as methyl, ethyl, isopropyl, butyl, isobutyl, pentyl, 2-methyl-butyl; D may be -NR1R3such as methylethyl amine or N-propyl-N-bromoamine; or D may be -SR1such as thioethyl, thiopentyl, thio-ethanoic acid. Ci-Ce alkyl and Ci-Ce alkoxy may also optionally be substituted with halogen, hydroxy, NH2, thio, NHR1, NR1R3and SR1as previously described. Preferably D is hydrogen.

[0033] The variable E may be H or OH. E may also be Ci-Ce alkyl, Ci-Ce alkoxy, Cs-io-aryl-Ci- 6-alkyl or C3-io-aryloxy-Ci-6-alkyl optionally substituted with Ci-6-alkyl, amino, -NHR1, -NR1R2, thio, - SR1, an unsubstituted C3-C7 cycloalkyl, phenyl or C4-C6 heterocyclic ring, or a substituted C3-C7 cycloalkyl, phenyl or C4-C6 heterocyclic ring having one or more substituents selected from the group consisting of Ci-Ce alkyl, Ci-Ce alkoxy, Ci-Ce alkanoyl, Ci-Ce carboxyalkyl, halogen and OH. E may also be an unsubstituted C3-C7 cycloalkyl, benzyl or C4-C6 heterocyclic ring, or a substituted C3-C7 cycloalkyl, benzyl or C4-C6 heterocyclic ring having one or more substituents selected from the group consisting of Ci-Ce alkyl, Ci-Ce alkoxy, Ci-Ce alkanoyl, Ci-Ce carboxyalkyl, halogen and OH. R1and R2are as previously defined.

[0034] Specific examples of groups that E may be include -methylphenyl, -ethylphenyl, -propyl -phenyl, -methylamine-phenyl, -methylamine-propanol, -ethylamine-pentanol, -l-methyl-2,6- dichlorophenyl,-l-2,3-dihydroxy-4-methan-ol-tetrahydrofuran and -p-ethoxy-tolyl.

[0035] In embodiments, the AC1 inhibitor is a compound of formula (1) in which G, H, J and M are each N; A is hydrogen; B is -NH2, -NHR1or -NR.1R.2, wherein R1and R2are independently selected from the group consisting of Ci-Ce alkyl, Ci-Ce alkanol or Ci-Ce alkoxy; D is NH2, SH, -NHR1, -NR1R3or -SR1, wherein R1and R2are independently selected from the group consisting of Ci-Ce alkyl, Ci-Ce alkanol, Ci-Ce carboxyalkyl or Ci-Ce alkoxy; and E is one of: -methylphenyl, -ethylphenyl, propyl-phenyl, -methylamine-phenyl, -methylamine-propanol, -ethylamine-pentanol, -l-methyl-2,6- dichlorophenyl,-l-2,3-dihydroxy-4-methan-ol-tetrahydrofuran and -p-ethoxy-tolyl.

[0036] In embodiments, the AC1 inhibitor is a compound as shown in Fig. 4.

[0037] In a particular embodiment, the AC1 inhibitor is a compound of formula (1) in which: A is H; B is NH2: D is H: E is CH2CH2-NH(CH2)5OH; and G, H, J and M are each N.

[0038] To determine whether a compound of formula (1) inhibits AC1, well-established assays may be used such as the cAMP assay. Since AC1 catalyzes the conversion of ATP to cAMP, production of cAMP by AC 1 -expressing cells in the presence of a test compound can be monitored to determine the AC1 inhibitory activity of the test compound. Briefly, non-ACl -expressing cells transfected with DNA encoding AC1 are incubated with varying concentrations of a potential AC 1 -inhibiting compound. Following a suitable reaction time, AC1 activity is measured by determining the amount of cAMP in the reaction mixture. Little or no cAMP is indicative of inhibitory activity.

[0039] A dual luciferase reporter system may also be used to determine AC1 inhibitory activity, as described in more detail in the specific examples that follow. In this assay, changes in intracellular cAMP concentration are detected as changes in expression level of firefly luciferase, the transcription of which is regulated by the transcription factor cAMP response element binding protein (CREB) binding to upstream cAMP response element (CRE). Cells, such as HEK293 cells, are transfected with luciferase-encoding constructs and incubated with a test compound. Following a suitable incubation period, luciferase activity is determined. Inhibition of luciferase activity is indicative of an AC1 inhibitor.

[0040] Examples of compounds in accordance with formula (1) include those compounds illustrated in Figure 4. Although these compounds can readily be synthesized using standard chemical synthesis protocols, as one of skill in the art would appreciate, they may also be commercially available.

[0041] The AC1 inhibitor may be in the form of a pharmaceutically acceptable salt. A "pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects (see e.g., Berge, S. M. et a (1977) J. Pharm. Set. 66: 1-19). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from nontoxic inorganic acids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous and the like, as well as from nontoxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like. Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium and the like, as well as from nontoxic organic amines, such as N,N'-dibenzylethylenediamine, N- methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine and the like.

[0042] In another embodiment, cAMP signaling is inhibited or reduced by upregulating expression of a cAMP degrading enzyme. cAMP degrading enzymes include cyclic nucleotide phosphodiesterases (PDEs) such as PDE1, 2, 3, 4, 7, 8, 10 and 11. Preferably, cAMPl signaling is inhibited by upregulation of a PDE which is cAMP selective, e.g. cAMP-specific 3',5'-cyclic phosphodiesterase, such as PDE4, PDE7 and PDE8.

[0043] PDE4 refers herein to mammalian PDE4, including human and non-human PDE4, and functionally equivalent forms thereof such as isoforms and variants thereof. The sequence of human PDE4A isoform 1 is illustrated in Fig. 5 and corresponds with NCBI Ref. Seq. NP 001104777.1. Other isoforms correspond with NCBI Ref. Seq. NP_001104778, NP_001104779, NP_006193 andNP 001230050, including non-human sequences which correspond with NCBI Ref Seq. NP_001297679, NP_062772 and NP_899668.

[0044] PDE7 refers herein to mammalian PDE7, including human and non-human PDE7A and PDE7B, and functionally equivalent forms thereof such as isoforms and variants thereof. The sequence of human PDE7A isoform c and PDE7B are illustrated in Fig. 5 and correspond respectively with NCBI ref. Seq. NP_001229247.1 and NP_061818.1. Another isoform corresponds with NCBI Ref. Seq. NP_002594.1, and non-human sequences correspond with NCBI Ref. Seq. NP_001116231 and NP_032828.

[0045] PDE8 refers herein to mammalian PDE8B, including human and non-human PDE8B, and functionally equivalent forms thereof such as isoforms and variants thereof. The sequence of human PDE8B isoform 3 is illustrated in Fig. 5 and corresponds with NCBI ref. Seq. NP 001025022. Other isoforms correspond with NCBI Ref. Seq. NP_001025023, NP_001025024, NP_001025025 and NP_003710, and non-human sequences correspond with NCBI Ref. Seq. NP_001164140, NP_001333711, NP_758467, NP_001360932 and NP_001360933.

[0046] The term "functional equivalent variants" as they relate to a phosphodiesterase include naturally or non-naturally occurring variants of an endogenous phosphodiesterase that retain the biological activity of a phosphodiesterase, e.g. to regulate the intracellular levels of cyclic adenosine monophosphate. The variant need not exhibit identical activity to an endogenous phosphodiesterase, but will exhibit sufficient activity to render it useful to downregulate cAMP, e.g. by at least about 10% of the biological activity of a phosphodiesterase, and preferably at least about 20-50% or greater of the biological activity of a phosphodiesterase. The term “about” is used herein to refer to a variance from the listed value by about 10%, either more or less than the listed value. Such functionally equivalent variants may result naturally from alternative splicing during transcription or from genetic coding differences and may retain significant sequence homology with a wild-type phosphodiesterase, e.g. at least about 70% sequence homology, preferably at least about 80% sequence homology, and more preferably at least about 90% or greater sequence homology. Such variants can readily be identified using established cloning techniques employing primers derived from the native phosphodiesterase. Additionally, such modifications may result from non-naturally occurring synthetic alterations made to the phosphodiesterase to render functionally equivalent variants which may have more desirable characteristics for use in a therapeutic sense, for example, increased activity or stability. Non-naturally occurring variants of a phosphodiesterase include analogues, fragments and derivatives thereof.

[0047] A functionally equivalent analogue of a phosphodiesterase in accordance with the present invention may incorporate one or more amino acid substitutions, additions or deletions. Amino acid additions or deletions include both terminal and internal additions or deletions to yield a functionally equivalent peptide. Examples of suitable amino acid additions or deletions include those incurred at positions within the protein that are not closely linked to activity. Amino acid substitutions within a phosphodiesterase, particularly conservative amino acid substitutions, may also generate functionally equivalent analogues thereof. Examples of conservative substitutions include the substitution of a nonpolar (hydrophobic) residue such as alanine, isoleucine, valine, leucine or methionine with another nonpolar (hydrophobic) residue; the substitution of a polar (hydrophilic) residue with another such as between arginine and lysine, between glutamine and asparagine, between glutamine and glutamic acid, between asparagine and aspartic acid, and between glycine and serine; the substitution of a basic residue such as lysine, arginine or histidine with another basic residue; or the substitution of an acidic residue, such as aspartic acid or glutamic acid with another acidic residue.

[0048] A functionally equivalent fragment in accordance with the present invention comprises a portion of a phosphodiesterase sequence which maintains the function of an intact phosphodiesterase, e.g. with respect to downregulating cAMP. Such biologically active fragments of a phosphodiesterase can readily be identified using assays useful to evaluate the activity of selected phosphodiesterase fragments.

[0049] A functionally equivalent derivative of a phosphodiesterase in accordance with the present invention is a phosphodiesterase, or an analogue or fragment thereof, in which one or more of the amino acid residues therein is chemically derivatized. The amino acids may be derivatized at the amino or carboxy groups, or alternatively, at the side “R” groups thereof. Derivatization of amino acids within the peptide may render a peptide having more desirable characteristics such as increased stability or activity. Such derivatized molecules include for example, those molecules in which free amino groups have been derivatized to form, for example, amine hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups, t-butyl oxy carbonyl groups, chloroacetyl groups or formyl groups. Free carboxyl groups may be derivatized to form, for example, salts, methyl and ethyl esters or other types of esters or hydrazides. Free hydroxyl groups may be derivatized to form, for example, O-acyl or O-alkyl derivatives. The imidazole nitrogen of histidine may be derivatized to form N-im-benzylhistidine. Also included as derivatives are those peptides which contain one or more naturally occurring amino acid derivatives of the twenty standard amino acids, for example: 4-hydroxyproline may be substituted for proline; 5 -hydroxylysine may be substituted for lysine; 3-methylhistidine may be substituted forhistidine; homoserine may be substituted for serine; and ornithine may be substituted for lysine. Terminal derivatization of the protein to protect against chemical or enzymatic degradation is also encompassed including acetylation at the N-terminus and amidation at the C-terminus of the peptide.

[0050] A phosphodiesterase for use in the present methods, and functionally equivalent variants thereof, may be made using standard, well-established solid-phase peptide synthesis methods (SPPS). Two methods of solid phase peptide synthesis include the BOC and FMOC methods. The phosphodiesterase and variants thereof may also be made using any one of a number of suitable techniques based on recombinant technology. It will be appreciated that such techniques are well- established by those skilled in the art, and involve the expression of a phosphodiesterase-encoding nucleic acid in a genetically engineered host cell. DNA encoding the phosphodiesterase may be synthesized de novo by automated techniques well-known in the art given that the protein and nucleic acid sequences are known.

[0051] Phosphodiesterase-encoding nucleic acid molecules or oligonucleotides may also be used to increase plasma phosphodiesterase levels. In this regard, the expression “phosphodiesterase-encoding nucleic acid” is used herein to encompass mammalian phosphodiesterase-encoding nucleic acid, including human and non-human forms, and functionally equivalent forms thereof (e.g. that encode a functionally equivalent phosphodiesterase, or nucleic acids which differ due to degeneracy of the genetic code). The sequence of such genes may be based on the PDE amino acid sequences disclosed herein. The sequence of a human phosphodiesterase-encoding gene encoding PDE4A, transcript variant 1, is shown in Figure 6, and examples of genes encoding other PDE4 transcripts, and transcripts of PDE7 and PDE8, functionally equivalent forms thereof, genes encoding other isoforms and / or non-human forms may be readily accessed, for example, at the NCBI sequence databank, 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.

[0052] The term “oligonucleotide” refers to an oligomer or polymer of nucleotide or nucleoside monomers consisting of naturally occurring bases, sugars, and intersugar (backbone) linkages. The term also includes modified or substituted oligonucleotides comprising non-naturally occurring monomers or portions thereof, which function similarly. Such modified or substituted oligonucleotides may be preferred over naturally occurring forms because of properties such as enhanced cellular uptake, or increased stability in the presence of nucleases. The term also includes chimeric oligonucleotides which contain two or more chemically distinct regions. For example, chimeric oligonucleotides may containat least one region of modified nucleotides that confer beneficial properties (e.g. increased nuclease resistance, increased uptake into cells), or two or more oligonucleotides of the invention may be joined to form a chimeric oligonucleotide. Other oligonucleotides of the invention may contain modified phosphorous, oxygen heteroatoms in the phosphate backbone, short chain alkyl or cycloalkyl intersugar linages or short chain heteroatomic or heterocyclic intersugar linkages. For example, oligonucleotides may contain phosphorothioates, phosphotriesters, methyl phosphonates, and phophorodithioates. Oligonucleotides of the invention may also comprise nucleotide analogs such as peptide nucleic acid (PNA) in which the deoxribose (or ribose) phosphate backbone in the DNA (or RNA), is replaced with a polymide backbone similar to that found in peptides. Other oligonucleotide analogues may contain nucleotides containing polymer backbones, cyclic backbones, or acyclic backbones, e.g. morpholino backbone structures.

[0053] Such oligonucleotide molecules are readily synthesized using procedures known in the art based on the available sequence information. For example, oligonucleotides may be chemically synthesized using naturally occurring nucleotides or modified nucleotides as described above designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed with mRNA or the native gene, e.g. phosphorothioate derivatives and acridine substituted nucleotides. Selected oligonucleotides may also be produced biologically using recombinant technology in which an expression vector, e.g. plasmid, phagemid or attenuated virus, is introduced into cells in which the oligonucleotide is produced under the control of a regulatory region.

[0054] Once prepared and suitably purified, a phosphodiesterase, phosphodiesterase-encoding oligonucleotides, or functionally equivalent variants thereof, may be utilized in accordance with the invention to downregulate cAMP signaling in the treatment of fear memory and / or PTSD. In this regard, increasing the expression of a phosphodiesterase in a mammal, by administration of a phosphodiesterase or by administration of phosphodiesterase-encoding nucleic acid, results in phosphodiesterase expression or over-expression in the mammal. While not wishing to be bound by any particular mode of action, upregulation of a phosphodiesterase in accordance with the invention results in downregulation of cAMP signaling.

[0055] The AC1 inhibitor, phosphodiesterase or nucleic acid encoding a phosphodiesterase such as PDE4, may be administered either alone or as a composition in combination with at least one pharmaceutically acceptable adjuvant, for use in treatments in accordance with embodiments of the invention. The expression "pharmaceutically acceptable" means acceptable for use in the pharmaceuticaland veterinary arts, i.e. not being unacceptably toxic or otherwise unsuitable. Examples of pharmaceutically acceptable adjuvants are those used conventionally with small molecule, peptide- or nucleic acid- based drugs, such as diluents, excipients and the like. Reference may be made to "Remington's: The Science and Practice of Pharmacy", 21st Ed., Lippincott Williams & Wilkins, 2005, for guidance on drug formulations generally. The selection of adjuvant depends on the intended mode of administration of the composition. In one embodiment of the invention, the compounds are formulated for administration by infusion, or by injection either subcutaneously or intravenously, and are accordingly utilized as aqueous solutions in sterile and pyrogen-free form and optionally buffered or made isotonic. Thus, the compounds may be administered in distilled water or, more desirably, in saline, phosphate-buffered saline or 5% dextrose solution. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents that delay absorption such as aluminum monostearate and gelatin. Compositions for oral administration via tablet, capsule or suspension are prepared using adjuvants including sugars, such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and derivatives thereof, including sodium carboxymethylcellulose, ethylcellulose and cellulose acetates; powdered tragancanth; malt; gelatin; talc; stearic acids; magnesium stearate; calcium sulfate; vegetable oils, such as peanut oils, cotton seed oil, sesame oil, olive oil and corn oil; polyols such as propylene glycol, glycerine, sorbital, mannitol and polyethylene glycol; agar; alginic acids; water; isotonic saline and phosphate buffer solutions. Wetting agents, lubricants such as sodium lauryl sulfate, stabilizers, tableting agents, anti-oxidants, preservatives, colouring agents and flavouring agents may also be present. Creams, lotions and ointments may be prepared for topical application using an appropriate base such as a triglyceride base. Such creams, lotions and ointments may also contain a surface active agent. Aerosol formulations may also be prepared in which suitable propellant adjuvants are used. Other adjuvants may also be added to the composition regardless of how it is to be administered, for example, anti-microbial agents may be added to the composition to prevent microbial growth over prolonged storage periods, for example, paraben, chlorobutanol, phenol sorbic acid, and the like.

[0056] 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 and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxy anisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

[0057] Examples of suitable aqueous and nonaqueous carriers that may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin and by the use of surfactants.

[0058] A phosphodiesterase-encoding oligonucleotide may be introduced into tissues or cells using techniques in the art including vectors (retroviral vectors, adenoviral vectors and DNA virus vectors) or physical techniques such as microinjection. Therapeutic oligonucleotides may be directly administered in vivo or may be used to transfect cells in vitro which are then administered in vivo. Administration of such cells may be achieved, for example, by encapsulated cell biodelivery.

[0059] To downregulate cAMP in the treatment of fear memory and / or PTSD, a therapeutically effective amount of AC1 inhibitor, a phosphodiesterase or nucleic acid encoding a phosphodiesterase is administered to a mammal. The term "therapeutically effective amount" is an amount of the AC1 inhibitor, phosphodiesterase or nucleic acid encoding the phosphodiesterase required to sufficiently downregulate cAMP signaling, while not exceeding an amount which may cause significant adverse effects, e.g. an amount which treats fear memory by alleviating one or more symptoms thereof such as a re-experiencing symptom, an avoidance symptom, an arousal and / or reactivity symptom or a cognition and / or mood symptom. Dosages that are therapeutically effective will vary on many factors including the nature of the condition to be treated as well as the particular individual being treated. An appropriate dosage may be an amount of an AC1 inhibitor that downregulates cAMP signaling by about 1% in a patient, for example, an amount that results in a 5-10% decrease in cAMP signaling, or more. Appropriate dosages of AC1 inhibitor for use include dosages sufficient to result in plasma levels of about 100-1500 ng / ml, preferably 120-1200 ng / ml, or 150-1000 ng / ml.

[0060] Similarly, appropriate dosages of a phosphodiesterase such as PDE4 for use include dosages sufficient to result in an increase in the plasma levels of the PDE in a patient with a fear-related psychiatric condition such as PTSD such that it alleviates one or more symptoms thereof, such as a reexperiencing symptom, an avoidance symptom, an arousal and / or reactivity symptom or a cognition and / or mood symptom. An appropriate dosage may be an amount of PDE which results in an increase of at least about 1% in the plasma PDE levels, for example, an amount that results in a 5-10% PDE plasma increase, or more, while not exceeding an amount which may cause significant adverse effects.

[0061] In the present treatment, the AC1 inhibitor, phosphodiesterase or nucleic acid may be administered by any route suitable to increase the plasma levels thereof. Examples of suitable administrable routes include, but are not limited to, parental routes, including intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, transtracheal, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion. Alternatively, other preferred routes of administration include non-parenteral routes, including topical, epidermal or mucosal routes of administration, for example, intranasally, orally, vaginally, rectally, sublingually or topically.

[0062] Embodiments of the invention are described by reference to the following specific examples which are not to be construed as limiting.Example

[0063] The following study was conducted to determine what impacts reexperiencing symptoms in conditions such as PTSD.Mouse Study

[0064] Male C57BL / 6N mice were obtained from Charles River (Yokohama, Japan). The mice were housed in cages of 5 or 6, maintained on a 12-h light / dark cycle, and allowed access to food and water ad libitum. The mice were at least 8 weeks of age when tested. Testing was performed during the light phase of the cycle. All experiments were conducted blind to the treatment condition of the mice (n = 208 in total). All animal experiments were conducted according to the Guide for the Care and Use of Laboratory Animals (Japan Neuroscience Society and Tokyo University of Agriculture) and were approved by the Animal Care and Use Committee of Tokyo University of Agriculture (authorization #2021031). All surgical procedures were performed under Nembutal anesthesia.

[0065] Drugs - The phosphodiesterase 4 (PDE4) inhibitor rolipram (ROL; Tocris Bioscience, UK) was dissolved in dimethyl sulfoxide (DMSO; Wako, Osaka, Japan) and then diluted with distilled water for systemic injection experiments (Fig. 1A, C). Mice were systemically injected with ROL (0.1 mg / kg b.w.) or vehicle (VEH) 30 min before re-exposure. The adenylyl cyclase 1 (AC1) inhibitor NB001 (HTS 09836), an adenylyl cyclase 1 inhibitor [Wang et al. Sci Transl Med. 201 l;3:65ra3], was dissolved in saline for systemic injection experiments (Fig. IB, D). Mice were systemically injected with NB001 (30 mg / kg b.w.) or VEH twice every hour from 2 h before re-exposure.

[0066] Contextual fear conditioning task - The mice were trained and tested in conditioning chambers (17.5 x 17.5 x 15 cm) (O’Hara & Co., Ltd., Tokyo, Japan) that had a stainless-steel grid floor through which a footshock could be delivered. Training consisted of placing the mice in the chamber and delivering a single electric footshock [2 s duration; 0.2 mA (Fig. 1A and Fig. 2B) or 0.4 mA (Fig. IB and Fig. 2D)], at 148 s later, and the mice were returned to their home cage at 30 s after the footshock (training).

[0067] To examine the effects of increased or decreased cAMP levels on retrieval and maintenance of contextual fear memory (Fig. 1A, B and Fig. 2), the mice were trained as described above, and at 24 h later, the mice were placed back in the conditioning chamber for 3 min (re-exposure). At 24 h after re-exposure, the mice were once again placed back in the conditioning chamber for 5 min and freezing was assessed (test). Memory was assessed as the percentage of time spent freezing in the training context. Freezing behavior (defined as a complete lack of movement, except for respiration) was measured automatically by video (O’Hara & Co., Ltd., Tokyo, Japan).

[0068] Inhibitory avoidance (IA) task - The step-through IA apparatus (O’Hara & Co., Ltd., Tokyo, Japan) consisted of a box with separate light and dark compartments (both 15.5 x 12.5 x 11.5 cm). The light compartment was illuminated by a fluorescent light (2500 lux). During the training sessions, each mouse was allowed to habituate to the light compartment for 30 s, and the guillotine door was raised to allow access to the dark compartment. Latency to enter the dark compartment was considered as a measure of acquisition. As soon as the mouse had entered the dark compartment, the guillotine door was closed. After 5 s, a footshock [0.1 mA (Fig. 1C) or 0.2 mA (Fig. ID)] was delivered for 2 s (training).

[0069] To examine the effects of increased or decreased cAMP levels on retrieval and maintenance of IA memory (Fig. 1C, D), the mice were trained as described above, and at 24 h later, the mouse was placed back in the light compartment until it entered the dark compartment without a footshock (re-exposure). Memory was assessed twice at 48 h after the re-exposure as the crossover latency for the mouse to enter the dark compartment when replaced in the light compartment, as in reactivation (test).

[0070] Viruses - A photoactivatable adenylyl cyclase (bPAC) was constructed as previously reported with S27A mutation by PCR (Stierl et al. J Biol Chem. 2011;286(2): 1181-8). A light-activated phosphodiesterase (LAPD) (Gasser et al. Proc Natl Acad Sci U S A. 2014;l l l(24):8803-8) with a catalytic domain of Homo sapiens phosphodiesterase 4 (NM_002600) was synthesized (GenScript, New Jersey, USA). AAV9-CaMKII-mGFP-bPAC (titer: 1.21 x 1013VG / mL), AAV9-CK0.4-LAPD-GFP(titer: 3.16 x 1013VG / mL), AAV9-CaMKII-GFP (titer: 2.98 x 1013VG / mL) were packaged and purified by SignaGen laboratories (SL100863 and SL100864, Rockville, MD, USA).

[0071] Virus injection and optical fiber implantation - For virus injections, the stereotaxic injection of adeno-associated virus (AAV) vectors was performed in a biological safety cabinet. Mice were anesthetized with a combination of medetomidine-midazolam-butorphanol anesthesia and placed in a stereotaxic frame. The skull was exposed and a small portion of the skull over dorsal hippocampus was removed bilaterally with a drill. AAVs (0.3 pL / site at a speed of 0.1 pL / min) were injected into the mouse brains using glass capillary pipettes [The anteroposterior (A / P) -1.6 mm, mediolateral (M / L) ±1.6 mm, dorsoventral (D / V) (from Dura) -1.6 mm] pulled with a micropipette puller (P-87, Sutter Instruments, Novato, CA, USA). After the injection, the glass pipettes were left in place for another 5 min before being slowly lifted up and removed. The mice were sutured, and antibiotic ointment was applied. Mice were then kept on a warm heater for recovery. At 1 week after the virus injections, a stainless-steel guide cannula (22 gauge) was implanted into the dorsal hippocampus (-1.6 mm, ±1.6 mm, -1.6 mm), under a combination of medetomidine-midazolam-butorphanol anesthesia, using standard stereotaxic procedures. Surgery was performed as described previously (Fukushima et al. J Neurosci. 2021;41 : 1288- 1300). The mice were allowed to recover for 1 week after surgery and then subjected to behavioral analyses. Stereotaxic coordinates for the dorsal hippocampus placement were based on the brain atlas (Franklin and Paxinos, The mouse brain in stereotaxic coordinates. Elsevier Academic: San Diego; 1997).

[0072] Optogenetic manipulation of cAMP levels in the dorsal hippocampus - To examine the effects of increased or decreased cAMP levels on fear memory, mice received a micro-infusion of an AAV vector expressing a photoactivatable adenylyl cyclase (bPAC) (AAV9-CaMKII-mGFP-bPAC or AAV9-CaMKII-GFP; Fig. 2A, B) or a light-activated phosphodiesterase (LAPD) (AAV9-CK0.4-LAPD- GFP or AAV9-CaMKII-GFP; Fig. 2C, D) under the control of the CaMKII promoter into the dorsal hippocampus, respectively (Luyben et al. Front Neural Circuits. 2020; 14:24). The blue light was stimulated at 4 Hz (pulse width, 15 ms, ~1.0 mW, Lucir Inc., Tsukuba, Japan) using a 473-nm laser for 30 min from 40 min before the re-exposure. Successful transduction of the hippocampus region was confirmed histologically by native GFP fluorescence. Only mice showing bilateral GFP expression in the hippocampus were included in subsequent data analyses.

[0073] Measurement of phosphorylated CREB levels - To examine the effects of increased or decreased cAMP levels on CREB phosphorylation, mice received a micro-infusion of an AAV vector expressing bPAC, LAPD or GFP under the control of the CaMKII promoter into the dorsal hippocampus,respectively, and then a stainless-steel guide cannula was implanted into the dorsal hippocampus as described above. The blue light stimulation was performed for 30 min as described above, and at 30 min later, mice were perfused and used for immunohistochemical staining of phosphorylated CREB positive cells.

[0074] Immunohistochemistry - Immunohistochemistry was performed as described previously (Ishikawa et al., Hippocampus. 2014;24:784-793; Fukushima et al. J Neurosci. 2021;41 : 1288-1300). After anesthetization, all mice were perfused with 4% paraformaldehyde. The brains were removed, fixed overnight, transferred to 30% sucrose, and stored at 4°C. Coronal sections (30 pm) were cut in a cryostat. Free-floating sections were treated with 1% H2O2 and incubated overnight with a rabbit polyclonal antiphospho-CREB (serine 133; S133) antibody (1 :2000; #06-519, Millipore) in blocking solution (phosphate-buffered saline plus 1% goat serum albumin, 1 mg / ml bovine serum albumin). The sections were washed with phosphate-buffered saline and incubated with horseradish peroxidase-conjugated donkey anti-rabbit IgG (1 :500; Jackson ImmunoResearch) for 1 h at room temperature. pCREB signals were amplified by biotin tyramide and visualized using Alexa Fluor-conjugated streptavidin (Invitrogen). The sections were mounted on slides and coverslipped using mounting medium with DAPI (VECTASHIELD).

[0075] Quantification - Quantification was performed as described previously (Ishikawa et al., Hippocampus. 2014;24:784-793; Fukushima et al. J Neurosci. 2021;41 : 1288-1300). Structures were defined anatomically according to the atlas of Franklin and Paxinos (1997). All immunoreactive neurons were counted by an experimenter blind to the treatment condition. Fluorescence images were acquired using TCS SP8 (Leica) confocal microscope. Confocal 2-pm z-stack images were obtained using LAS AF software (Leica). Equal cutoff thresholds were applied to all slices. We quantified the number of pCREB+, GFP+and DAPI+cells using a 40* objective. For quantification of pCREB+cells in the field of view within the hippocampus (290x290 mm; bregma between -1.46 and -1.82 mm) across at least two sections, computerized image analyses were performed using WinROOF version 5.6 software (Mitani Corporation, Fukui, Japan).

[0076] RNA analysis - Mouse hippocampal RNA analyses were performed as described previously (Tsujii et al. Neuropsychopharmacol Rep. 2021;41(2):230-36). Mice were sacrificed by cervical dislocation. The dorsal hippocampus (bregma between -1.46 and -2.18 mm) was dissected by Rodent Brain Matrices (MUROMACHI KIKAI Co., Ltd, Tokyo, Japan) and snap-frozen in liquid nitrogen. Total RNA was prepared from mouse dorsal hippocampus region using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA). To analyze the peripheral blood mRNA, the mice were anesthetizedbefore blood collection by cardiac puncture. Total RNA from peripheral blood was isolated using the RNAprotect Animal Blood Tubes (Qiagen) and RNeasy Protect Animal Blood Kit (Qiagen) according to the manufacturer’s instructions.

[0077] Quantitative reverse transcription PCR (qRT-PCR) was performed as described previously (Tsujii et al. 2021). Total RNA (500 ng) was reverse transcribed using Superscript III reverse transcriptase (Invitrogen) and an oligo dT primer. qRT-PCR was performed with the ABI PRISM 7000 (Applied Biosystems, CA, USA) using SYBR Green PCR Master Mix (Thermo Fisher Scientific) according to the manufacturer's protocol. 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 a single PCR product was confirmed by monitoring the dissociation curve. Amplification curves were visually inspected to set a suitable baseline range and threshold level. The relative quantification method was employed for the quantification of target molecules according to the manufacturer’s protocol, where the ratio between the amount of each target molecule and a reference molecule within the same sample was calculated. All measurements were performed in triplicate. The levels of Gapdh mRNA were used to normalize the relative expression levels of target mRNA. The primer sequences for qRT-PCR analyses are listed in Table 1.Table 1 - List of all primer sequences used in mouse qRT-PCR assay

[0078] RNA-sequencing - RNA-sequencing was performed as described previously (Tsujii et al. 2021). Total RNA from mouse dorsal hippocampus was isolated using the RNeasy Mini Kit (Qiagen). They were subjected to RNA-seq analysis for each experimental group (n = 5 animals per pool). After RNA quality check [all RNA integrity number (RIN) values > 8.0] with the RNA nano kit (Agilent Technologies) on Agilent Bioanalyzer (Agilent Technologies), 1 pg of total RNA was used for preparing cDNA libraries with the TruSeq RNA Sample Preparation Kit v2 (Illumina, San Diego, CA, USA). Thederived cDNA libraries were analyzed on an Agilent Bioanalyzer with DNA 1000 Kit and quantified by qPCR using the KAPA Library Quantification Kit (KAPA Bio systems, Wilmington, MA, USA). cDNA libraries were pooled in lanes and clusters were generated on a cBot (Illumina) to obtain 100-bp single reads in a HiSeq 2500 sequencer (Illumina). Demultiplexed fastq files were generated using bcl2fastq ver. 2.18 (Illumina). Filtering, mapping, and differential expression analysis were performed using the CLC Genomics Workbench software ver. 9.5 (Qiagen). The raw sequence reads were filtered to exclude adapter sequences, ambiguous nucleotides, and low-quality sequences and the retained sequences were aligned against the mouse genome (mm 10). RNA sequencing data have been deposited to the DDB J Sequence Read Archive (DRA) and are available at the accession number DRA013665.

[0079] Statistical analysis - One-way analysis of variance (ANOVA) followed by post hoc Newman-Keuls test and 2-way ANOVA followed by post hoc Bonferroni’s comparisons were used to analyze the effects of drug, time, and group. A paired Ltest was used to analyze the differences in crossover latency within each group between two sessions (re-exposure vs. test). A Student’s Ltest was used to analyze differences in mRNA expression levels.

[0080] References referred to herein are incorporated by reference.Results

[0081] Retrieval and maintenance of fear memory correlate with cAMP levels - The effect of cAMP levels on the retrieval and subsequent fate of hippocampus-dependent contextual fear memory was determined using pharmacology. To examine the effects of increased cAMP levels on contextual fear memory, the mice received a systemic injection ofROL, aPDE4 inhibitor, or vehicle (VEH / solvent). before memory retrieval. The mice were trained with a single footshock (0.2 mA, 2 s; training) and then re-exposed to the training context for 3 (re-exposure) and 5 (test) min every 24 h. The mice were systemically injected with ROL (0.1 mg / kg b.w.) or VEH at 30 min before re-exposure. Two-way ANOVA revealed significant effects of drug (ROL vs. VEH: F(l,38) = 10.191, p < 0.05), but not time (re-exposure vs. test: F(l,38) = 1.775, p > 0.05) or time x drug interaction (F(l,38) = 0.000, p > 0.05) (Fig. 1A). The ROL group froze significantly more than the VEH group at re-exposure and test (ps < 0.05; post hoc Bonferroni’s test). These results suggest that ROL injection enhances the retrieval and subsequent maintenance of contextual fear memory. Thus, these observations suggest that the upregulation of cAMP levels strengthens contextual fear memory.

[0082] To examine the effects of decreased cAMP levels, the adenylyl cyclase 1 inhibitor, NB001 was used. A similar experiment to that described above was used, except that the mice were trained witha footshock (0.4 mA, 2 s) and systemically injected with NB001 (30 mg / kg b.w.) or VEH twice every hour from 2 h before re-exposure. The NB001 group froze significantly less than the VEH group at reexposure and test (two-way ANOVA: drug, F(l,38) = 13.317, p < 0.05; time, F(l,38) = 0.43, p = 0.516; and time x drug interaction, F(1 ,38) = 0.305, p = 0.584; post hoc Bonferroni’s test, ps < 0.05; Fig. IB). These results indicate that an adenyl yl cyclase 1 inhibitor such as NB001 impairs the retrieval and subsequent expression of contextual fear memory, indicating that the downregulation of cAMP levels disrupts contextual fear memory.

[0083] The effects of pharmacological activation (ROL) and inactivation (NB001) on IA memory, another type of hippocampus-dependent fear memory, was also investigated. The mice were first placed in the light compartment. At 5 s after they entered the dark compartment, a brief electrical footshock (0.1 mA, 2 s) was delivered (training). The mice were re-exposed to the light compartment at 24 h after training and their crossover latency to enter the dark compartment was assessed (re-exposure). The mice were returned to their home cages immediately after they entered the dark compartment from the light compartment. Prior to re-exposure, the mice received a systemic injection of VEH or ROL. At 48 h later, crossover latency was assessed (test). The ROL group showed significantly longer crossover latency at re-exposure and test than the VEH group (two-way ANOVA: drug, F(1 ,36) = 30.872, p < 0.05; time, F(l,36) = 24.355, p < 0.05; and drug x time interaction, F(l,36) = 10.239, p < 0.05; post hoc Bonferroni’s test, ps < 0.05; Fig. 1C). Consistent with the results of Figure 1A, these observations indicate that ROL enhances the retrieval of IA memory and its maintenance. Importantly, the VEH and ROL groups displayed significantly increased crossover latency at test compared with re-exposure (paired t-test, ps < 0.05; Fig. 1C).

[0084] In contrast, systemic injection of NB001 impaired IA memory retrieval and its maintenance (two-way ANOVA: drug, F(l,36) = 30.692, p < 0.05; time, F(l,36) = 15.02, p < 0.05; and drug x time interaction, F(l,36) = 17.531, p < 0.05; Fig. ID). The NB001 group showed significantly shorter crossover latency at re-exposure and test than the VEH group (post hoc Bonferroni’s test, ps < 0.05), although the VEH group, but not the NB001 group, displayed significantly increased crossover latency at test compared with re-exposure (paired t-test, p < 0.05; Fig. ID).

[0085] It is important to note that previous studies have shown IA memory, but not contextual fear memory, is enhanced following memory retrieval through memory reconsolidation (Fukushima et al., 2021a, 2021b). The differences in “reconsolidation effect” between IA memory and contextual fear memory are reflected by the results of two-way ANOVA (a significant effect of time and time x drug interaction were observed in the IA memory, but not contextual fear memory).

[0086] To investigate further the roles of cAMP levels in fear memory, the effects of optogenetic manipulation of cAMP levels in the hippocampus were examined. Newly developed optogenetic probes that enable the generation or degradation of cAMP in a blue light-dependent manner were used. To examine the effects of increased cAMP levels, the mice received a micro-infusion of an AAV vector expressing bPAC under the control of the CaMKII promoter (AAV9-CaMKII-mGFP-bPAC or AAV9- CaMKII-GFP) into the dorsal hippocampus (Fig. 2A). A similar experiment to that used to generate the results of Figure 1 A was used, except that blue light was administered at 4 Hz (pulse width, 15 ms) using a 473 -nm laser for 30 min from 40 min before re-exposure. The bPAC group froze significantly more on re-exposure and test compared to the GFP and No light control groups (two-way ANOVA: group, F(2, 58) = 12.566, p < 0.05; time, F(l, 60) = 0.362, p > 0.05; and group x time interaction, F(2, 60) = 0.393, p > 0.05; post hoc Bonferroni’s test, ps < 0.05; Fig. 2B). These observations indicate that an optogenetic increase of cAMP levels in the hippocampus facilitates the retrieval and maintenance of contextual fear memory.

[0087] The effects of an optogenetic decrease of cAMP levels was then investigated. The mice received a micro-infusion of AAV expressing LAPD under the control of the CaMKII promoter (AAV9- CK0.4-LAPD-GFP or AAV9-CaMKII-GFP) into the dorsal hippocampus (Fig. 2C). A similar experiment to that used to generate the results of Figure IB was used, except that blue light was administered at 4 Hz (pulse width, 15 ms) using a 473-nm laser for 30 min from 40 min before reexposure. The LAPD group froze significantly less in re-exposure and test compared to the GFP and No light control groups (two-way ANOVA: group, F(2,60) = 10.825, p < 0.05; time, F(l,60) = 2.498, p > 0.05; and group x time interaction, F(2,60) = 0.362, p > 0.05; post hoc Bonferroni’s test, ps < 0.05; Fig. 2D). These results indicate that an optogenetic decrease of cAMP levels in the hippocampus impairs the retrieval and maintenance of contextual fear memory.

[0088] It is important to note that the effects of optogenetic manipulations of bPAC and LAPD on cAMP-signal transduction in the hippocampus were examined. To do this, the phosphorylation levels of CREB at serine 133 (pCREB levels) which is a target of protein kinase A (PKA) activated by cAMP were examined. The mice expressing bPAC or LAPD showed significantly increased or decreased, respectively, pCREB levels in the CAI region of the dorsal hippocampus 30 min after blue light stimulation for 30 min compared to the control groups. These observations indicate that optogenetic manipulations of bPAC and LAPD are reflected by activation or inactivation, respectively, of cAMP- PKA signal transduction.

[0089] Collectively, these observations indicate that an increase or decrease of cAMP levels around retrieval enhances or impairs, respectively, the retrieval and subsequent maintenance of fear memory. Therefore, these observations indicate that the state of fear memory reflects cAMP levels.

[0090] Mouse transcriptomes - Transcriptome profiles associated with mouse fear memory retrieval and those associated with human reexperiencing symptoms were then integratively analyzed. For mouse transcriptome analysis, similar behavioral experiments as used to generate the results of Figure ID were used, followed by RNA-seq analysis of the mouse dorsal hippocampus at 30 min following IA memory retrieval (re-exposure). Significantly increased (3,997) and decreased (3,140) mRNAs were observed compared to the control group (non-reactivated; NR) that were trained, but not re-exposed to the light compartment during the re-exposure session. These observations indicate that IA memory retrieval rapidly induces gene expression changes in the dorsal hippocampus.

[0091] The integrative transcriptome analysis to identify key genes involved in fear memory was performed. For the mouse hippocampus transcriptome data, the expression levels of 4,908 genes (3,034 upregulated and 1,874 downregulated) were significantly changed after fear memory retrieval to induce reconsolidation compared to no-retrieval control using the FDR threshold of q < 0.001 and |FC| > 1.36. These analyses identified 97 genes of interest. Of the 97 genes, 15 showed significant differences between healthy mice and mice conditioned with fear memory using the t-test threshold of p < 0.05. The curated 15 genes included PDE4B. Specifically, PDE4B expression levels were significantly downregulated after fear memory retrieval in mice (FC = -1.4, p < 0.001).

[0092] Decreased Pde4b mRNA levels in the murine hippocampus and blood after fear memory retrieval - The mRNA expression changes in the mouse dorsal hippocampus following IA memory retrieval were validated using qRT-PCR (Fig. 3). To do this, similar experiments to those used to generate the data in Figure ID were conducted. Hippocampal mRNA levels were measured at 30 min after reactivation [React-30 and non-reactivated (NR) groups] (Fig. 3A). The mRNA levels of Pde4b, Rapla, and Nkap were measured since these genes showed marked downregulated in human blood and mouse hippocampus transcriptomes in relation to reexperiencing / fear memory retrieval. Similar to previous studies, significantly increased c-fos mRNA levels were observed in the hippocampus of the React-30 group compared to the NR group (t-test, p < 0.05; Fig. 3B). Importantly, Pde4b, Rapla, and Nkap mRNA levels were significantly decreased in the hippocampus of the React-30 group compared to the NR group (t-test, ps < 0.05; Fig. 3B). To examine the relationship of mRNA levels between the hippocampus and peripheral blood following IA memory retrieval in mice, blood mRNA levels were measured at 30 and 90 min after reactivation (Fig. 3C). Similar to the results obtained in the hippocampus, Pde4b, Rapla,and Nkap mRNA levels were significantly decreased in the peripheral blood of the React-30 and / or -90 groups compared to the NR group (one-way ANOVA followed by post hoc Newman-Keuls test: Pde4b, F(2,21) = 2.649, p < 0.05; Rapla, F(2,28) = 4.727, p < 0.05; Nkap, F(2,28) = 3.674, p < 0.05; ps < 0.05; Fig. 3C), indicating that Pde4b, Rapla, and Nkap mRNA expression levels in the hippocampus and peripheral blood were decreased following IA memory retrieval. Thus, the results suggest that the IA memory retrieval-induced changes in these mRNA levels were consistent between the hippocampus and peripheral blood in mice.Discussion

[0093] The effects of the gain- and loss-of-function of the cAMP signaling pathway on hippocampus-dependent contextual fear and IA memories were examined in mice. Pharmacological and optogenetic activation or inactivation of cAMP signaling facilitated or impaired, respectively, the retrieval and maintenance of these memories. These findings indicate that the state of the cAMP signaling pathway determines the state of fear memory. Importantly, integrative transcriptome analysis identified the downregulated mRNA expression of genes, including PDE4B, an enzyme that degrades cAMP, in the hippocampus of mice following fear memory retrieval. Furthermore, PDE4B mRNA expression was downregulated in the hippocampus and peripheral blood of mice following fear memory retrieval. Since the downregulation of PDE4B mRNA expression leads to the activation of the cAMP-signaling pathway via a decrease in cAMP degradation, the downregulation of PDE4B expression appears to be associated with the mechanisms for reexperiencing symptoms in PTSD through the activation of the cAMP signaling pathway.

[0094] The cAMP signaling pathway plays a role in learning and memory and neural plasticity from invertebrate to mammalian animal models. The present mouse behavioral studies surprisingly showed that the gain- or loss-of-function of the cAMP signaling pathway enhanced or blocked, respectively, fear memory retrieval.

[0095] In the present study, memory maintenance was facilitated or impaired by activated and inactivated cAMP signals, respectively, before memory retrieval. Additionally, optogenetic manipulations of bPAC and LAPD increased or decreased respectively, pCREB levels in the dorsal hippocampus. Therefore, this indicates that activation or inactivation of cAMP signaling enhances or impairs, respectively, the reconsolidation of fear memory through CREB-mediated transcription, thereby modulating memory maintenance.

[0096] It was further observed that IA memory retrieval decreased Pde4b mRNA levels in mouse hippocampus and peripheral blood, indicating synchronous gene expression regulation between brain and blood cells, indicating a correlation between reexperiencing symptoms of PTSD patients with decreased PDE4B mRNA levels in peripheral blood. Therefore, the observations indicate that PTSD patients exhibiting reexperiencing symptoms may have reduced PDE4B expression in the brain. More importantly, since cAMP levels are increased by decreased PDE4B levels, the downregulation of PDE4B contributes to the enhancement of fear memory retrieval via an increase of cAMP levels. Therefore, elevated cAMP levels mediated by the decreased expression of PDE4B are indicated to have an impact on the pathophysiology of PTSD, especially reexperiencing symptoms.

[0097] The cAMP signaling pathway is regulated by abundant signal transduction molecules. As shown herein, the present findings indicate that the underlying mechanism of PTSD is by the activation of this pathway.

[0098] The genes co-expressed with PDE4B were also examined. Gene Ontology analysis of the co-expressed genes identified ontology related to MAPK activation as the top signal, and among the MAPK family, RAP1A was strongly co-expressed with PDE4B as well as NKAP / Nkap. The present data also shows that RAPIA / Rapla expression was consistently downregulated in mouse fear memory retrieval. I.

[0099] In conclusion, this study shows that increased cAMP levels promote memory retrieval and that PDE4B expression is downregulated in the hippocampus and peripheral blood in mice after fear memory retrieval. These findings indicate that the increased cAMP levels caused by the downregulation of PDE4B mRNA expression enhance traumatic memory, thereby playing a key role in the reexperiencing symptoms of PTSD patients as a functional index of these symptoms.

Claims

CLAIMS1. A method of mitigating fear memory in a mammal comprising the step of inhibiting or reducing cAMP signaling in the mammal.

2. The method of claim 1, wherein cAMP signaling is inhibited or reduced by administration to the mammal of an adenyl cyclase 1 (AC1) inhibitor.3 The method of claim 2, wherein the AC1 inhibitor has the following general formula (1):wherein:A is selected from the group consisting of H, OH, halogen, Ci-Ce alkyl, Ci-Ce alkyl halide, C2- Ce alkenyl, C2-C6 alkynyl and Ci-Ce alkoxy;B is selected from the group consisting of: hydroxy, thio, -OR1, -NH2, -NO2, -NHR1, -NR1R2, - SR1or -Ci-Ce saturated or unsaturated alkyl group optionally substituted with one or more substituents selected from hydroxy, halogen, thio, OR1, NH2, NO2, NHR1, NR1R2, SR1, a C3-C10 aromatic or nonaromatic ring structure or a C3-C9 aromatic or non-aromatic heterocyclic ring structure optionally substituted with OH, halogen, thio, NH2, Ci-Ce alkyl, Ci-Ce alkanol or Ci-Ce alkoxy, wherein R1and R2are independently selected from the group consisting of Ci-Ce alkyl, Ci-Ce alkyl halide, Ci-Ce alkenyl, Ci-Ce alkynyl, Ci-Ce alkanol, Ci-Ce alkoxy and Ci-Ce carboxyalkyl, orB is NR.1R.2forms a C3-C6 aromatic or non-aromatic heterocyclic ring optionally substituted with OH, halogen, thio, NH2, NO2, Ci-Ce alkyl, Ci-Ce alkanol, Ci-Ce alkoxy or Ci-Ce carboxyalkyl,D is selected from the group consisting of: H, halogen, hydroxy, NH2, thio, NHR1, NR1R3, SR1, Ci-Ce alkyl, Ci-Ce alkoxy, wherein R1is as defined above and R3is as defined for R1;E is H or OH, orE is Ci-Ce alkyl, Ci-Ce alkoxy, C3-io-aryl-Ci-6-alkyl or C3-io-aryloxy-Ci-6-alkyl optionally substituted with Ci-6-alkyl, amino, NHR1, NR1R2, thio, SR1, an unsubstituted C3-C7 cycloalkyl, phenyl or C4-C6 heterocyclic ring, or a substituted C3-C7 cycloalkyl, phenyl or C4-C6 heterocyclic ring havingone or more substituents selected from the group consisting of Ci-Ce alkyl, Ci-Ce alkoxy, Ci-Ce alkanoyl, Ci-Ce carboxyalkyl, halogen or OH, orE is an unsubstituted C3-C7 cycloalkyl, phenyl or C4-C6 heterocyclic ring, or substituted C3-C7 cycloalkyl, phenyl or C4-C6 heterocyclic ring having one or more substituents selected from the groups consisting of Ci-Ce alkyl, Ci-Ce alkoxy, Ci-Ce alkanoyl, Ci-Ce carboxyalkyl, halogen or OH, wherein R1and R2are as defined above; andG, H, J and M are each N, orH and J are each C, and G and M are each N, S or O, orH, J and M are each C and G is N, S or O.4 The method of claim 3, wherein the AC1 inhibitor is a compound of formula (1) in which G, H,J and M are each N; A is hydrogen; B is -NH2, -NHR1or -NR1R2, wherein R1and R2are independently selected from the group consisting of Ci-Ce alkyl, Ci-Ce alkanol or Ci-Ce alkoxy; D is NH2, SH, - NHR1, -NR1R3or -SR1, wherein R1and R2are independently selected from the group consisting of Ci- Ce alkyl, Ci-Ce alkanol, Ci-Ce carboxyalkyl or Ci-Ce alkoxy; and E is one of: -methylphenyl, - ethylphenyl, -propyl -phenyl, -methylamine-phenyl, -methylamine-propanol, -ethylaminepentanol, -l-methyl-2,6-dichlorophenyl,-l-2,3-dihydroxy-4-methan-ol-tetrahydrofuran and -p-ethoxy- tolyl.5 The method of claim 3, wherein the AC1 inhibitor is a compound selected from a compound of Fig. 4.6 The method of claim 3, wherein, the AC1 inhibitor is a compound of formula (1) in which: A is H B is NH2: D is H: E is CH2CH2-NH(CH2)5OH; and G, H, J and M are each N.7 The method of claim 1, wherein cAMP signaling is inhibited or reduced by upregulating expression of a cAMP degrading enzyme.8 The method of claim 7, wherein the cAMP degrading enzyme is a phosphodiesterase.9 The method of claim 8, wherein the phosphodiesterase is PDE4, PDE7 or PDE8.10 The method of claim 9, wherein the phosphodiesterase is PDE4B.

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

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

13. The method of any one of claims 1-12, wherein the mammal has a fear-related psychiatric condition.

14. The method of claim 13, wherein the mammal has post-traumatic stress disorder (PTSD).

15. Use of a cAMP inhibitor to treat fear memory in a mammal.

16. Use of a phosphodiesterase to treat fear memory in a mammal.

17. The use of claim 15 or 16, wherein the mammal has a fear-related psychiatric disorder.

18. A composition for use to treat fear memory in a mammal comprising an AC1 inhibitor and a pharmaceutically effective carrier, wherein the AC1 inhibitor has the following general formula (1):wherein:A is selected from the group consisting of H, OH, halogen, Ci-Ce alkyl, Ci-Ce alkyl halide, C2- Ce alkenyl, C2-C6 alkynyl and Ci-Ce alkoxy;B is selected from the group consisting of: hydroxy, thio, -OR1, -NH2, -NO2, -NHR1, -NR1R2, - SR1or -Ci-Ce saturated or unsaturated alkyl group optionally substituted with one or more substituents selected from hydroxy, halogen, thio, OR1, NH2, NO2, NHR1, NR1R2, SR1, a C3-C10 aromatic or nonaromatic ring structure or a C3-C9 aromatic or non-aromatic heterocyclic ring structure optionally substituted with OH, halogen, thio, NH2, Ci-Ce alkyl, Ci-Ce alkanol or Ci-Ce alkoxy, wherein R1and R2are independently selected from the group consisting of Ci-Ce alkyl, Ci-Ce alkyl halide, Ci-Ce alkenyl, Ci-Ce alkynyl, Ci-Ce alkanol, Ci-Ce alkoxy and Ci-Ce carboxyalkyl, orB is NR.1R.2forms a C3-C6 aromatic or non-aromatic heterocyclic ring optionally substituted with OH, halogen, thio, NH2, NO2, Ci-Ce alkyl, Ci-Ce alkanol, Ci-Ce alkoxy or Ci-Ce carboxyalkyl,D is selected from the group consisting of: H, halogen, hydroxy, NH2, thio, NHR1, NR1R3, SR1, Ci-Ce alkyl, Ci-Ce alkoxy, wherein R1is as defined above and R3is as defined for R1;E is H or OH, orE is Ci-Ce alkyl, Ci-Ce alkoxy, Cs-io-aryl-Ci-e-alkyl or Cs-io-aryloxy-Ci-e-alkyl optionally substituted with Ci-6-alkyl, amino, NHR1, NR.1R.2, thio, SR1, an unsubstituted C3-C7 cycloalkyl, phenyl or C4-C6 heterocyclic ring, or a substituted C3-C7 cycloalkyl, phenyl or C4-C6 heterocyclic ring having one or more substituents selected from the group consisting of Ci-Ce alkyl, Ci-Ce alkoxy, Ci-Ce alkanoyl, Ci-Ce carboxyalkyl, halogen or OH, orE is an unsubstituted C3-C7 cycloalkyl, phenyl or C4-C6 heterocyclic ring, or substituted C3-C7 cycloalkyl, phenyl or C4-C6 heterocyclic ring having one or more substituents selected from the groups consisting of Ci-Ce alkyl, Ci-Ce alkoxy, Ci-Ce alkanoyl, Ci-Ce carboxyalkyl, halogen or OH, wherein R1and R2are as defined above; andG, H, J and M are each N, orH and J are each C, and G and M are each N, S or O, orH, J and M are each C and G is N, S or O.

19. The composition of claim 18, wherein the AC1 inhibitor is a compound of formula (1) in which G, H, J and M are each N; A is hydrogen; B is -NH2, -NHR1or -NR1R2, wherein R1and R2are independently selected from the group consisting of Ci-Ce alkyl, Ci-Ce alkanol or Ci-Ce alkoxy; D is NH2, SH, -NHR1, -NR1R3or -SR1, wherein R1and R2are independently selected from the group consisting of Ci-Ce alkyl, Ci-Ce alkanol, Ci-Ce carboxyalkyl or Ci-Ce alkoxy; and E is one of: -methylphenyl, -ethylphenyl, -propyl -phenyl, -methylamine-phenyl, -methylamine-propanol, -ethylamine-pentanol, - 1 -methyl-2,6-dichlorophenyl,- 1 -2,3 -dihydroxy-4-methan-ol -tetrahydrofuran and -p-ethoxy-tolyl.

20. The composition of claims 18 or 19, wherein the AC1 inhibitor is a compound selected from a compound of Fig. 4.

Images