US 20170020892A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2017/0020892 A1 Thompson et al. (43) Pub. Date: Jan. 26, 2017
(54) USE OF NEGATIVE MODULATORS OF Related U.S. Application Data GABA RECEPTORS CONTAINING ALPHAS SUBUNITS AS FAST ACTING (60) Provisional application No. 61/972,446, filed on Mar. ANTDEPRESSANTS 31, 2014. (71) Applicant: University of Maryland, Baltimore, Publication Classification Baltimore, MD (US) (51) Int. Cl. A 6LX 3/557 (2006.01) (72) Inventors: Scott Thompson, Baltimore, MD (US); A6II 3/53 (2006.01) Mark D. Kvarta, Ellicott City, MD A6II 45/06 (2006.01) (US); Adam Van Dyke, Baltimore, MD (52) U.S. Cl. (US) CPC ...... A61 K3I/55.17 (2013.01); A61K 45/06 (2013.01); A61 K3I/53 (2013.01) (73) Assignee: University of Maryland, Baltimore, Baltimore, MD (US) (57) ABSTRACT Embodiments of the disclosure include methods and com (21) Appl. No.: 15/300,984 positions related to treatment of one or more medical conditions with one or more negative modulators of GABA (22) PCT Filed: Mar. 31, 2015 receptors. In specific embodiments, depression and/or Sui cidability is treated or ameliorated or prevented with one or (86) PCT No.: PCT/US2O15/023667 more negative modulators of GABA receptors, such as a S 371 (c)(1), partial inverse agonist of a GABA receptor comprising an (2) Date: Sep. 30, 2016 alpha5 subunit. Patent Application Publication Jan. 26, 2017. Sheet 1 of 12 US 2017/002O892 A1
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USE OF NEGATIVE MODULATORS OF conditions. In particular embodiments, the one or more GABA RECEPTORS CONTAINING ALPHAS medical conditions includes depression and similar condi SUBUNITS AS FAST ACTING tions. The condition may be of any kind, but in specific ANTIDEPRESSANTS embodiments the condition being treated is major depression and/or Suicidality. In specific embodiments, treatment of the CROSS-REFERENCE TO RELATED medical condition occurs at a more rapid rate than currently APPLICATIONS known treatments and has fewer deleterious side effects. In specific embodiments, the medical condition is a depression 0001. This application claims priority to U.S. Provisional related disorder; an anxiety-related disorder, an attention Patent Application No. 61/972,446, filed on Mar. 31, 2014, related disorder; a psychosis-related disorder; an eating which is incorporated by reference herein in its entirety. disorder; a personality disorder, cognitive impairment, including that follows traumatic brain injury (TBI) or that is STATEMENT REGARDING FEDERALLY non-TBI cognitive impairment; neuropathic pain; chronic SPONSORED RESEARCH OR DEVELOPMENT muscle or bone pain; diabetic complications resulting in 0002 This invention was made with government support nerve injury; generalized attack of muscular weakness; under Grant Number MH086828 awarded by the National recurring sleep episodes during the day; migraine; addiction; Institutes of Health. The government has certain rights in the or a combination thereof. invention. 0008 Embodiments of the present disclosure relate to a method for treating or ameliorating depression and/or Sui TECHNICAL FIELD cidality in a human Subject comprising administering to the 0003 Embodiments of the disclosure are related at least subject a therapeutically effective amount of a modulator of to the fields of cell biology, molecular biology, biochemistry, a GABA receptor. Although any modulator of a GABA neurology, psychiatry, pharmacology, psychology, and receptor that ameliorates a medical condition as described medicine. In specific embodiments, the disclosure relates to herein, in a specific embodiment the modulator is a negative methods for treating or ameliorating depression and/or Sui modulator of the receptor. In specific embodiments, the cidality in a Subject by administering one or more modula modulator is a negative allosteric modulator of GABA tors of the GABA receptor that can produce a rapid anti receptors containing an alpha5 subunit. depressant action. 0009 Specific embodiments of the disclosure concern the use of negative allosteric modulators of alpha5 subunit BACKGROUND containing GABA receptors as fast-acting antidepressants to address depression and/or reduce Suicidality. In specific 0004. There is a need to identify new drugs that can be embodiments, fast-acting as used herein is defined as the used to treat depressive disorders and reduce the incidence onset of therapeutic effects being within hours to days of suicide on a worldwide basis. Selective serotonin compared to current therapies that take days to weeks to reuptake inhibitors (SSRIs) are often the first line of treat months. ment, but many patients do not respond to these medications 0010. In particular embodiments, any and all drugs that and prolonged treatment (several weeks or months) is often are negative allosteric modulators of alpha5 subunit-con required to achieve therapeutic improvement. taining GABA receptors (including inverse agonists at the 0005. The GABA receptor is an ionotropic receptor and benzodiazepine binding site of GABA receptors containing ligand gated ion channel. Its endogenous ligand is gamma alpha5 Subunits) will produce a rapid antidepressant action aminobutyric acid (GABA), the major inhibitory neurotrans and will produce a rapid decrease in depression and/or mitter in the central nervous system. The primary activation Suicidality in patients Suffering from major depression or in site of the GABA receptor is the binding site for GABA and patients suffering from depression that are not suicidal. several drugs, such as muscimol, gaboXadol, and bicucul Because alpha5-containing GABA receptors are narrowly line. A second binding site is the so-called “benzodiazepine expressed in the hippocampus and cortex, and because in at receptor’ site. Drugs binding at this site can promote or least some embodiments the Substances are weak inverse impair the ability of GABA to activate the GABA receptor. agonists, in certain embodiments they have a significantly GABA receptors occur in all organisms with a central greater therapeutic potential than current treatments. nervous system. Because of their wide distribution within 0011. In one embodiment the present disclosure relates to the nervous system of mammals, they play a role in virtually a method for treating or ameliorating depression and/or all brain functions. Suicidality in a human Subject comprising administering to 0006 Ketamine, a recognized antagonist of the NMDA the Subject a therapeutically effective amount of a negative type glutamate receptor, has demonstrated fast-acting anti allosteric modulator of the GABA receptor, and in some depressant properties. However, ketamine is recognized to embodiments the modulator is administered orally, intrad have potent psychotomimetic effects and the mechanism of ermally, intramuscularly, intraperitonealy, intravenously, via action of ketamine remains uncertain. Thus, there is a clear insufflation, or in a dermal patch. clinical need for the identification of other fast-acting anti 0012. The present disclosure relates to a method for depressants for depression and Suicidality that have treating or ameliorating depression and/or Suicidality in a enhanced pharmacokinetic properties and that can yield human Subject comprising administering to the Subject a more robust responses with less negative side effects. therapeutically effective amount of a negative allosteric BRIEF SUMMARY modulator of the alpha5 subunit of the GABA receptor and in some embodiments the modulator is administered orally 0007 Embodiments of the disclosure concern methods intradermally, intramuscularly, intraperitonealy, intrave and compositions for the treatment of one or more medical nously, via insufllation, or in a dermal patch. US 2017/002O892 A1 Jan. 26, 2017
0013. In another embodiment the present disclosure stimulants, anti-glucocorticoids, antagonists of NMDA-type relates to a method for treating or ameliorating depression glutamate receptors, tricylic antidepressants (TCAS), and and/or Suicidality in a human Subject comprising adminis combinations thereof. tering to the subject a therapeutically effective amount of 0019. In another embodiment the present disclosure L655,708 or a derivative or analog thereof and wherein relates to a method for treating or ameliorating unipolar and L655,708 is (but not limited to) administered orally, intra bipolar forms of depression, Suicidality, and other depressive dermally, intramuscularly, intraperitoneally, intravenously, symptoms of unipolar and bipolar depression, posttraumatic via insufflation, or in a dermal patch, in specific embodi stress disorder, and/or postpartum depression in a human mentS. Subject comprising administering to the Subject a therapeu 0014. In another embodiment the present disclosure tically effective amount of one or more negative modulators relates to a method for treating or ameliorating depression of a GABA receptor, including an alpha5 subunit-contain and/or Suicidality in a human Subject comprising adminis ing GABA receptor and, in some embodiments, the modu tering to the subject a therapeutically effective amount of lator is a partial inverse agonist of an alpha5-containing MRK-016 or a derivative or analog thereof and wherein GABA receptor. MRK-016 is (but not limited to) administered orally, intra 0020. In another embodiment the present disclosure dermally, intramuscularly, intraperitoneally, intravenously, relates to a method for treating or ameliorating unipolar and via insufflation, or in a dermal patch, in specific embodi bipolar forms of depression, Suicidality, and other depressive mentS. symptoms of unipolar and bipolar depression, posttraumatic 0015. In another embodiment the present disclosure stress disorder, and/or postpartum depression in a human relates to a method for treating or ameliorating depression Subject comprising administering to the Subject a therapeu and/or Suicidality in a human Subject comprising adminis tically effective dose of one or more negative modulators of tering to the subject a therapeutically effective amount of a GABA receptors, such as an inverse agonist of the alpha5 CP-457,920 or a derivative or analog thereof and wherein subunit-containing GABA receptor, for example, either CP-457,920 is (but not limited to) administered orally, alone or in combination with one or more other antidepres intradermally, intramuscularly, intraperitoneally, intrave sant drugs selected from the group consisting of monoamine oxidase inhibitors (MAOis), selective serotonin reuptake nously, via insufflation, or in a dermal patch, in specific inhibitors(SSRis), serotonin-norepinephrine reuptake embodiments. inhibitors (SNRis), norepinephrine reuptake inhibitors 0016. In another embodiment the present disclosure (NRIs), triple reuptake inhibitors, modulators of CNS ace relates to a method for treating or ameliorating depression tylcholine function, stimulants, anti-glucocorticoids, antago and/or Suicidality in a human Subject comprising adminis nists of NMDA-type glutamate receptors, tricylic antide tering to the subject a therapeutically effective amount of pressants (TCAS), and a combination thereof. RG-1662 or a derivative or analog thereof and wherein 0021. In another embodiment, the present disclosure RG-1662 is (but not limited to) administered orally, intrad relates to a method for boosting excitatory synapse function ermally, intramuscularly, intraperitoneally, intravenously, in multiple brain regions of a human Subject comprising via insufflation, or in a dermal patch, in specific embodi administering to the subject a therapeutically effective mentS. amount of a negative modulator of the alpha5 subunit 0017. In another embodiment the present disclosure containing GABA receptor. relates to a method for treating or ameliorating depression 0022. In another embodiment, the present disclosure and/or Suicidality in a human Subject comprising adminis relates to a method for restoring Sucrose preference in a tering to the subject a therapeutically effective dose of a rodent animal model with stress-induced anhedonia wherein negative allosteric modulator of the alpha5 subunit-contain the Sucrose preference increases following administration of ing GABA receptor either alone or in combination with an a therapeutically effective amount of a negative modulators another antidepressant drug including, for example, one or of a GABA receptors, such as a negative modulator of the more selected from the group consisting of monoamine alpha5 subunit-containing GABA receptor, such as L-655, oxidase inhibitors (MAOis), selective serotonin reuptake 708; MRK-016; CP-457,920; RG-1662; a derivative or inhibitors(SSRis), serotonin-norepinephrine reuptake analog thereof, or a combination thereof. inhibitors (SNRis), norepinephrine reuptake inhibitors 0023. In another embodiment, the present disclosure (NRIs), triple reuptake inhibitors, modulators of CNS ace relates to a method for treating or ameliorating depression tylcholine function, stimulants, anti-glucocorticoids, antago and/or Suicidality in a human Subject comprising adminis nists of NMDA-type glutamate receptors, tricylic antide tering to the subject a therapeutically effective amount of pressants (TCAS), and a combination thereof. compound L-655, 708 (ethyl (13aS)-7-methoxy-9-oxo-11, 0018. In another embodiment the present disclosure 12,13,13 a-terahydro-9H-imidazo 1.5-alpyrrolo2,1-c. 14 relates to a method for treating or ameliorating depression benzodiazepine-1-carboxylate; MRK-016; CP-457,920; and/or Suicidality in a human Subject comprising adminis RG-1662; a derivative or analog thereof; or a combination tering to the subject a therapeutically effective dose of one thereof. or more of L655,708; MRK-016; CP-457,920; RG-1662; or 0024. In another embodiment, the present disclosure a derivative or analog thereof, either alone or in combination relates to a method for treating or ameliorating depression with one or more other antidepressant drugs selected from and/or Suicidality in a human Subject comprising adminis the group consisting of monoamine oxidase inhibitors tering to the subject a therapeutically effective amount of (MAOis), selective serotonin reuptake inhibitors(SSRis), compound L-655,708; MRK-016; CP-457,920; RG-1662; a serotonin-norepinephrine reuptake inhibitors (SNRis), nor derivative or analog thereof; or a combination thereof. epinephrine reuptake inhibitors (NRIs), triple reuptake 0025. In another embodiment, the present disclosure inhibitors, modulators of CNS acetylcholine function, relates to a method for treating or ameliorating depression US 2017/002O892 A1 Jan. 26, 2017
and/or Suicidality in a human Subject comprising adminis when one or more other therapies are utilized with the tering to the subject a therapeutically effective amount of modulator, the other therapies may be provided in repetitive compound L-655,708; MRK-016; CP-457,920; RG-1662; a dosing regimens. derivative or analog thereof; or a combination thereof that is 0030. In another embodiment the present disclosure administered to the Subject orally, intradermally, intramus relates to a method for treating or ameliorating depression cularly, intravenously, intraperitoneally, via insufflation, or and/or Suicidality in a human Subject comprising adminis in a dermal patch, in certain embodiments. tering to the subject a therapeutically effective amount of 0026. In another embodiment, the present disclosure compound L-655,708; MRK-016; CP-457,920; RG-1662; a relates to a method for treating or ameliorating depression derivative or analog thereof, or a combination thereof, in and/or Suicidality in a human Subject comprising adminis combination with another benzodiazepine binding site tering to the subject a therapeutically effective dose of a inverse agonist such as RO4882224, RO4938581 or other negative allosteric modulator of GABA receptor, including negative allosteric modulators of GABA receptor function. one that comprises an alpha5 subunit, and in some cases is 0031. In another embodiment the present disclosure an inverse agonist of the alpha5 subunit-containing GABA relates to a method for treating or ameliorating depression receptor, wherein the modulator is one or more benzodiaz and/or Suicidality in a human Subject comprising adminis epine binding site inverse agonists or other binding sites that tering to the subject a therapeutically effective amount of produce negative allosteric modulation of GABA receptors compound L-655,708; MRK-016; CP-457,920; RG-1662; a containing alpha5 subunits. derivative or analog thereof, or a combination thereof, in combination with a 3-phenyl-5-arylpyridazine class of com 0027. In another embodiment the present disclosure relates to a method for treating or ameliorating depression in pounds such as RO 15-4513, RY-010, RY-023, RY-080, or a human Subject comprising administering to the Subject a RG-1662. therapeutically effective dose of a negative allosteric modu 0032 Embodiments of the disclosure include methods to lator of GABA receptor (including a negative modulator of reduce the risk of suicide in an individual prone to suicide a GABA receptor that has an alpha5 subunit, such as an and/or depression. inverse agonist of the alpha5 subunit-containing GABA 0033. In one embodiment, there is a method of treating or receptor) such as L-655,708; MRK-016; CP-457,920; preventing or ameliorating at least one symptom of a medi RG-1662; a derivative or analog thereof; or a combination cal condition in an individual, comprising the step of pro thereof in combination with one or more antidepressant viding to the individual atherapeutically effective amount of drugs selected from the group consisting of monoamine one or more negative modulators of GABA receptors, oxidase inhibitors (MAOis), selective serotonin reuptake wherein the medical condition is selected from the group inhibitors (SSRis), serotonin-norepinephrine reuptake consisting of a depression-related disorder, an anxiety inhibitors (SNRis), norepinephrine reuptake inhibitors related disorder; an attention-related disorder, a psychosis (NRIs), modulators of CNS acetylcholine function, stimu related disorder; an eating disorder, a personality disorder; lants, antagonists of NMDA-type glutamate receptors, tri cognitive impairment following traumatic brain injury; neu ropathic pain; chronic muscle or bone pain; diabetic com cylic antidepressants, or a combination thereof. plications resulting in nerve injury; generalized attack of 0028. In another embodiment the present disclosure muscular weakness; recurring sleep episodes during the day; relates to a method for treating or ameliorating depression in migraine; addiction; Suicidality; and a combination thereof. a human Subject comprising administering to the Subject a In specific embodiments, the onset of amelioration of one or therapeutically effective dose of an inverse agonist of the more depression-related symptoms occurs within hours, alpha5 subunit-containing GABA receptor, Such as L-655, days, or weeks. In particular embodiments, the GABA 708; MRK-016; CP-457,920; RG-1662; a derivative or receptor comprises an alpha5 subunit. The negative modu analog thereof; or a combination thereof, wherein the effec lator may be selected from the group consisting of a negative tive dose results in the presence of the inverse agonist in the allosteric modulators acting at the benzodiazepine binding cerebrospinal fluid at a concentration sufficient to bind 10 to site; negative allosteric modulators acting at the barbiturate 75% of all GABA receptors and thereby reduce GABA steroid binding site; negative allosteric modulators acting at receptor function by 10 to 75%. the neuroactive steroid binding site; competitive antagonists 0029. In another embodiment the present disclosure of the GABAR: negative modulators acting in the pore of relates to a method for treating or ameliorating depression in the GABAR channel; nicotinamide and related com a subject comprising administering to the Subject a thera pounds; inverse agonists and antagonists of the propofol peutically effective dose of one or more negative allosteric binding site of the GABAR; and a combination thereof. modulators of GABA receptor (including a negative allos 0034. In certain embodiments, the depression-related dis teric modulator of an alpha5 subunit-comprising GABA order is major depressive disorder (MDD); dysthymia; receptor, Such as an inverse agonist of the alpha5 Subunit cyclothymic disorder; seasonal affective disorder/seasonal containing GABA receptor), such as L-655,708 or deriva depression; depression after traumatic brain injury; postpar tives or analogs thereof (or MRK-16 or analogs or deriva tum depression; premenstrual dysphoric disorder; depres tives thereof; or CP-457,920, or analogs or derivatives sive symptoms associated with menopause; depression fol thereof; or RG-1662 or analogs or derivatives thereof), or a lowing substance abuse? withdrawal; bipolar disorder; combination thereof. In particular embodiments, the effec bipolar disorder in remission; or depressive episodes of tive dose of the negative allosteric modulator of GABA bipolar disorder. In specific embodiments, the anxiety-re receptor is administered to the subject every 0.5, 1, 2, 3, or lated disorder is general anxiety disorder, obsessive com more days or in repetitive dosing regimens. Repetitive pulsive disorder, Impulse control disorder; anxiousness dosing may occur when the modulator is provided alone or associated with depression; repeated episodes of anxiety, when it is provided with one or more other therapies, and extreme apprehension or fear of Social interaction (social US 2017/002O892 A1 Jan. 26, 2017
phobia); panic disorders; posttraumatic stress syndrome or description when considered in connection with the accom posttraumatic stress disorder, or separation anxiety disorder. panying figures. It is to be expressly understood, however, In some embodiments, the attention-related disorder is atten that each of the figures is provided for the purpose of tion deficit hyperactive disorder; or adult attention deficit illustration and description only and is not intended as a hyperactive disorder. The psychosis-related disorder may be definition of the limits of the present invention. Schizophrenia, Schizophrenia-spectrum disorder or psy chotic depressive illness, in some embodiments. When the BRIEF DESCRIPTION OF THE DRAWINGS individual has an eating disorder, it may be anorexia ner 0039 For a more complete understanding of the present Vosa; bulimia; or obesity. In some embodiments, the indi invention, reference is now made to the following descrip vidual has avoidant personality disorder; antisocial person tions taken in conjunction with the accompanying drawing, ality disorder; borderline personality disorder; conduct in which: disorder, dependent personality disorder, depressive person 0040 FIG. 1 illustrates an example of ketamine's fast ality disorder; histrionic personality disorder, narcissistic acting therapeutic action and the involvement of excitatory personality disorder; negativistic personality disorder; synapses in depression. obsessive-compulsive personality disorder, paranoid per 004.1 FIG. 2. Injection of L-655,708 produced a rapid Sonality disorder, Schizoid personality disorder; or schizo increase in oscillatory activity Instr. pyramidale of area CA1 typal personality disorder. (upper row) and the NAc shell (lower row) in vivo, particu 0035. An individual treated with embodiments of the larly in the theta and beta frequency bands, that persisted for disclosure includes an individual that has been or has not more than one hour. been diagnosed with the medical condition. 0042 FIG. 3A. In control slices (black), anpirtoline pro 0036. In particular embodiments, the negative modulator duced a fully reversible potentiation of TA-CA1 EP8P slope of GABA receptor is a negative allosteric modulators of-175%. After CUS (gray), the potentiation was larger and acting at the benzodiazepine binding site; the negative persisted for >60 min after washout. FIG. 3B. In two slices modulator of GABA receptor is a negative allosteric modu from CUS rats given an injection of L-655,708 24 hours lator acting at the barbiturate steroid binding site; the earlier, the potentiation was similar to controls. negative modulator of GABA receptor is a negative allos 0043 FIG. 4. 24 hours after injection of L-655,708, but teric modulator acting at the neuroactive steroid binding site; not vehicle, phosphorylation of S831 and total GluA1 the negative modulator of GABA receptor is a competitive expression were increased in SLM of hippocampal tissue antagonist of the GABAR; negative modulator acting in the from CRS rats to levels comparable to unstressed controls. pore of the GABAR channel; and/or the negative modula In contrast, L-655,708 had no effect in unstressed animals. tor of GABA receptor is a partial inverse agonist of a 0044 FIG. 5. One hour after injection of L-655,708 there GABA receptor comprising an C5 subunit. In specific was an increase in expression of Foss and AFosB in embodiments, the partial inverse agonist of a GABA recep unstressed animals in the NAc but not in the hippocampus. tor comprising an O.5 subunit is L-655,708, RO4938581, No changes were apparent 24 hrs after injection. CP-457,920, MRK-016, or a combination thereof. 004.5 FIGS. 6A and 6B. L-655,708 rapidly reverses loss 0037. In particular embodiments, the method further of Sucrose preference and Social interaction behaviors after comprises the step of providing to the individual a thera chronic restraint stress. Quantification of results from the peutically effective amount of another therapy, such as one sucrose preference test (SPT, FIG. 6A) and the social selected from the group consisting of monoamine oxidase interaction tests (SIT, FIG. 6B) at two time points: before inhibitors (MAOis), selective serotonin reuptake inhibitors CRS and after 10 days of CRS. 24 hrs prior to the second (SSRis), serotonin-norepinephrine reuptake inhibitors (SN round of tests, rats received an injection of either vehicle Ris), norepinephrine reuptake inhibitors (NRIs), triple (black, left bar in pairs of bars; vehicle) or L-655,708 (white, reuptake inhibitors, modulators of CNS acetylcholine func right bar in pairs of bars; L-655,708). Mean sucrose pref tion, stimulants, anti-glucocorticoids, antagonists of erence differed significantly following vehicle injection NMDA-type glutamate receptors, tricylic antidepressants compared with all other groups (2x2 repeated-measures (TCAs), and a combination thereof. The other therapy may ANOVA group-time interaction F(1,11)=7.514, p=0.019; *, be an anti-depressant. p-0.05 compared to pre-CUS baseline, Bonferroni post-hoc 0038. The foregoing has outlined rather broadly the fea test; n=7 CRS+vehicle rats, 6 CRS+L-655,708 rats). Mean tures and technical advantages of the present invention in Social interaction ratios differed significantly following order that the detailed description of the invention that vehicle injection compared with all other groups (F(1,12) follows may be better understood. Additional features and =2.85, p=0.115; n=7 CRS+vehicle rats, 7 CRS+L-655,708 advantages of the invention will be described hereinafter rats). which form the subject of the claims of the invention. It 0046 FIGS. 7A, 7B, 7C, and 7D. L-655,708 rapidly should be appreciated by those skilled in the art that the reverses loss of social interaction behaviors after chronic conception and specific embodiment disclosed may be read unpredictable stress but does not affect hedonic behavior in ily utilized as a basis for modifying or designing other unstressed rats. Quantification of results from the Social structures for carrying out the same purposes of the present interaction test (SIT) at three time points: before CUS, after invention. It should also be realized by those skilled in the 4 weeks of CUS and a vehicle injection given 24 hrs earlier, art that Such equivalent constructions do not depart from the and after an additional week of CUS and an injection of spirit and scope of the invention as set forth in the appended L-655,708 given 24 hrs earlier. FIG. 7A, Mean social claims. The novel features which are believed to be char interaction ratio differed significantly in the CUS+vehicle acteristic of the invention, both as to its organization and group compared to all other groups (3x2 repeated-measures method of operation, together with further objects and ANOVA group-time interaction F(2.50)=6.538, p=0.005; *, advantages will be better understood from the following p-0.05 vs all other groups, Bonferroni test, n=18 CUS rats, US 2017/002O892 A1 Jan. 26, 2017
9 unstressed rats). FIG. 7B, Analysis of individual animals mediated responses (1-Way ANOVA, F(2,17)=0.549, p=0. revealed that 14 of 18 animals displayed robust responses to 588). FIG. 10C, AMPA:NMDA ratios were computed from CUS and L-655,708 (black). The 4 rats that failed to respond the initial slopes of the responses in each slice before and to L-655,708 appeared largely resilient to the effects of CUS after application of DNQX (shown by dotted lines in A). (gray symbols). Unstressed controls were also given vehicle AMPA:NMDA ratios were decreased significantly in slices and L-655,708 injections at the same time points. Neither from vehicle-treated CRS rats compared to slices from either injection produced a significant change in Social interaction unstressed or L-655,708-treated CRS rats (1-Way ANOVA (FIG. 7C) or sucrose preference (FIG. 7D). F(2,17)=4.345 p=0.03; n=5 unstressed, 8 CRS+vehicle, 7 0047 FIG. 8. Mean sucrose preference following 4 CRS+L-655,708; *, p<0.05 compared to unstressed and weeks of CUS+vehicle injection differed significantly from CRS+L-655,708, LSD post-hoc test). Bar notations in FIG. all other groups, (3x2 repeated-measures ANOVA group 10C are the same as in FIG. 10B. time interaction F(2,18)=12.309, p=0.006: *p-0.05 vs all 0050 FIGS. 11A and 11B. L-655,708 rapidly reverses other groups, Bonferroni test) N=5 CUS rats and N=7 stress-induced decreases in GluA1 expression at TA-CA1 unstressed rats. The left bar of the pairs (black) reflects CUS synapses of the rathippocampus. FIG. 11A, Representative rats and the right bar of the pairs (white) reflects unstressed Western blots of GluA1 and B-actin protein levels in SLM ratS. samples from unstressed rats (left) and rats subjected to 10 0048 FIGS. 9A, 9B, and 9C. MRK-016 rapidly reverses days of CRS then given an injection 24 hrs earlier of either loss of Sucrose preference and social interaction behaviors vehicle solution (middle) or L-655,708 (right). FIG. 11B, after chronic restraint stress. FIG. 9A, Quantification of GluA1 expression, normalized to B-actin levels, was results from one group of rats in the Sucrose preference test decreased significantly in tissue from vehicle-treated CRS at five time points: baseline, after 2 weeks of CRS, 24 hrs rats (gray, middle bar of triplet of bars) compared to tissue after an additional 3 days of CRS and 24 hrs after injection from unstressed (black, left bar of triplet of bars) or L-655, of vehicle, after one week of CRS and 24 hrs after an 708-treated CRS rats (white, right bar of triplet of bars) injection of MRK-016 (3 mg/kg), and in the same animals (Kruskal-Wallis H test: 202)=10.62, p=0.0049; n=6 after an additional week of CRS and 7 days after the unstressed, 6 CRS+vehicle, 8 CRS+L-655,708; *, p<0.05 injection of MRK-016. Mean sucrose preference differed compared to unstressed and CRS+L-655,708, post-hoc significantly following vehicle injection compared with all Mann-Whitney U test). other groups (F(3.33)—20.63, p<0.0001, n=12 rats). *, p<0. 0051 FIGS. 12A and 12B. Neither stress nor L-655,708 05 compared to pre-CUS baseline, Tukey's post-hoc test). injection affect time in the interaction Zone in the target FIG. 9B. Results in individual animals for the full experi absent condition. FIG. 12A, Percent time spent in the ment. FIG. 9C, Quantification of results from one group of interaction Zone is quantified for rats during the baseline rats in the Social interaction test at four time points: baseline, period and 24 hrs after injection of either vehicle (white, after 2 weeks of CRS and 24 hrs after injection of vehicle, right bar of pair of bars) or L-655,708 (black, left bar of pair after one week of CRS and 24 hrs after an injection of of bars) for the experiments illustrated in FIG. 12A. FIG. MRK-016 (3 mg/kg), and in the same animals after an 12B. Percent time spent in the interaction Zone is quantified additional week of CRS and 7 days after the injection of for rats subjected to CUS (black, left bar of pair of bars) or MRK-016. Mean social interaction ratios differed signifi unstressed rats (white, right bar of pair of bars) during the cantly following vehicle injection compared with all other baseline period, 24 hrs after injection of vehicle, and 24 hrs groups (F*2,14* 11.84, p=0.0009, n=8 rats). *, p<0.05 after injection of L-655,708 for the experiments illustrated compared to pre-CUS baseline, Tukey's post-hoc test). in FIG. 6B. 0049 FIGS. 10A, 10B, and 10C. L-655,708 rapidly reverses stress-induced weakening of AMPAR-mediated DETAILED DESCRIPTION synaptic transmission in TA-CA1 synapses of the rat hip pocampus. FIG. 10A, representative traces showing the I. Exemplary Definitions AMPAR- and NMDAR-mediated components of the fBPSP. 0052. As used herein, the words “a” and “an when used recorded extracellularly in SLM of area CA1 in response to in the present specification in concert with the word com simulation of TA afferents in Saline lacking added Mg2+. prising, including the claims, denote "one or more.” Some Traces are shown before (black) and after (gray) addition of embodiments of the invention may consist of or consist 50M DNQX to block AMPARs. Traces were recorded from essentially of one or more elements, method steps, and/or slices taken from unstressed controls (left) and rats subjected methods of the invention. It is contemplated that any method to 10 days of CRS then given an injection 24 hrs earlier of or composition described herein can be implemented with either vehicle solution (middle) or L-655,708 (right). FIG. respect to any other method or composition described 10B, the mean slope of the relationship between AMPAR herein. (left) and NMDAR-mediated response (right) and FV ampli 0053. The terms “therapeutic level:” “therapeutically tude over a range of Stimulation intensities from the three effective dose.” “therapeutically effective amount,” and the groups of animals. The slope of the AMPAR-mediated like, as used herein, refer to an amount or concentration of responses was decreased significantly in slices from vehicle a compound or agent, e.g., a GABAR antagonist, that treated CRS rats (gray, middle bar of triplet of bars) com achieves a therapeutic effect in a subject, wherein the pared to slices from unstressed (black, left bar of triplet of therapeutic effect can be an amelioration, or alleviation, if bars) or L-655,708-treated CRS rats (white, right bar of not complete cessation, of one or more symptoms of clinical triplet of bars) across the range of stimulation intensities depression and other similar conditions, including Suicidal (1-Way ANOVA, F(2,17), 3.675, p=0.047; n=5 unstressed, 8 ity as defined elsewhere herein. In particular embodiments, CRS+vehicle, 7 CRS+L-655,708; *, p<0.05, LSD post hoc an effective dose is the dose of the compound that results in test). There were no significant differences in NMDAR the presence of the compound in the cerebrospinal fluid at a US 2017/002O892 A1 Jan. 26, 2017
concentration sufficient to bind to GABA receptors, includ nism via which rapid therapeutic AD actions are exerted. ing those containing alpha 5 subunits. In specific embodi Therefore, other means were sought to produce Such ments, an effective dose is the dose of the compound that changes. In one embodiment, it was considered to produce results in the presence of the compound in the cerebrospinal a direct decrease in the strength of inhibition mediated by fluid at a concentration sufficient to bind to 10-75% of all y-aminobutyric acid type-A receptors (GABARS). Disin GABARs and thereby reduce GABAR function by hibition promotes anxiety and epileptiform discharge, how 10-75%, although in specific embodiments the percentages ever. In another embodiment, related to efforts to develop are 10-100%, 10-90%, 10-80%, 10-70%, 10-60%, 10-50%, memory-enhancing drugs, an alternative to ketamine as a 10-40%, 10-30%, 10-20%, 20-100%, 20-90%, 20-80%, rapidly acting AD includes partial inverse agonists of 20-70%, 20-60%, 20-50%, 20-40%. 20-30%, 30-100%, C.5-containing GABARS. 30-90%, 30-80%, 30-70%, 30-60%, 30-50% 30-40%, 0057. GABARs are heteropentameric, ligand-gated ion 40-100%, 40-90%, 40-80%, 40-70%, 40-60%, 40-50%, channels. Nineteen GABA receptor subunits have been 50-100%, 50-90%, 50-80%, 50-70%, 50-60% 60-100%, identified to date. Most GABA receptors contain C, B, and 60-90%, 60-80%, 60-70%, 70-100%, 70-80%, 70-90%, Y2 subunits in a 2:2:1 stoichiometry. B subunits direct 70-80%, 80-100%, 80-90%, 90-100, and so forth. In specific membrane insertion whereas adjacent y2 and C. Subunits embodiments, the percentage of receptors that are bound is form the benzodiazepine site, an allosteric modulator of greater than 10, 20, 30, 40, 50, 60, 70, 80, 90, or 95%. channel gating. The identity of the C. Subunit determines the pharmacological profile of this site. Receptors containing II. General Embodiments C1, C2, or C3 Subunits are potentiated by Zolpidem, a 0054 Major depressive disorder (MDD) afflicts nearly 20 benzodiazepine type 1 agonist, whereas receptors containing percent of the world population, with Suicide as a too C5 subunits are essentially insensitive to Zolpidem. Recent common and tragic end. Monoaminergic antidepressants studies have shown that C1 subunits mediate the sedative, (ADs), such as selective serotonin reuptake inhibitors (SS anticonvulsant and amnestic effects of benzodiazepines RIs), are the first line treatment for MDD, but many patients preferentially, whereas the C.2 and C3 subunits mediate their do not respond to these medications and clinical improve anxiolytic effects. In addition to the anxiolytic agonists of ment generally requires prolonged treatment (weeks to benzodiazepines, there is a class of drugs, exemplified by the months) in patients that do. The delay in efficacy, as well as B-carbolines, that act as full or partial inverse agonists at the the high non-response rate, have motivated the search for benzodiazepine site to decrease GABA receptor function. better AD drugs. Ketamine, a noncompetitive NMDA recep Partial inverse agonists offer the advantage of a wider tor (NMDAR) antagonist, has demonstrated efficacy as a therapeutic concentration range and a lower likelihood of rapidly acting AD in treatment refractory patients. Low producing anxiety or epileptiform discharge, in specific doses of ketamine result in a rapid and Sustained improve embodiments of the disclosure. ment in core depressive symptoms and a decrease in Suicidal 0.058 C.5-subunit mRNA is highly expressed in the pyra ideation in individuals with treatment-resistant MDD (Ber midal cells of the hippocampus and cortex (Allen Brain man et al., 2000; Zarate et al., 2006). Similarly, systemic Atlas) and C.5-containing GABA receptors are localized in ketamine administration at sub-anesthetic doses (3-50 the dendrites of hippocampal CA1 pyramidal cells at Syn mg/kg) results in AD-like behavior in several rodent models aptic and extrasynaptic sites. Because disinhibition pro of depression (Maeng et al., 2008: Li et al., 2011). Although motes induction of long-term potentiation (LTP), and ketamine exerts a rapid AD action in humans, its potential because of their selective forebrain localization, drugs that for use outside of the research setting is limited because it is selectively inhibit C.5-containing receptors are being devel addictive and because it produces dissociative, psychotomi oped as cognitive enhancers. It has been shown, for metic effects even at low doses (e.g. Machado-Vieira et al., example, that partial inverse agonists of C.5-containing 2009). Thus, even though ketamine itself has limited thera GABA receptors enhance associative memory acquisition peutic potential, its rapid action has kindled great enthusi in hippocampal-dependent learning tasks. asm for the possibility of developing rapidly acting ADS. 0059. Thus, in particular embodiments of the disclosure Ketamine has also provided a critical mechanistic insight one or more partial inverse agonists of the benzodiazepine into how rapid AD actions may be produced (e.g. Li et al., binding site of GABA receptors, particularly those con 2011), raising hopes that new drugs can be developed that taining an C5 subunit, produce a rapid antidepressant action lack its disadvantages and risks. in unipolar and bipolar forms of depression and reduce 0055. The mechanism of ketamine's fast-acting therapeu Suicidal ideation by restoring the normal function of excit tic action remains uncertain. It has been postulated that atory synapses. Such considerations may be characterized, ketamine is somewhat selective for NMDARs on inhibitory by example, using a rodent model of depression with face, interneurons, so that their excitation is diminished by ket construct, and predictive validity, chronic unpredictable amine, resulting in a mild disinhibition of the neuronal stress (CUS) to compare the actions of acute administration population and overall increase in its activity. Ketamine (24 hrs) of the C.5-selective partial inverse agonists induced increases in activity then cause stimulation of (RO4938581, Ballard et al., 2009; MRK-016, Atack et al., activity-dependent processes such as long-term potentiation 2009) and the non-selective inverse agonist (FG7142; Shan and activation of the mammalian target of the rapamycin sky et al., 2004) with the actions of acutely administered (mTOR) signaling pathway leading to the rapid induction of ketamine. Antidepressant efficacy of partial inverse agonists synaptogenesis via increased synthesis of synaptic proteins of C5 subunit-containing GABA receptors are then tested (Li et al., 2010) and restoration of normal activity. in vivo using the Sucrose preference and Social exploration 0056. In the present disclosure, in certain embodiments it tests (Amat et al., 2010), for example. One could also was considered that it is ultimately the mild increase in determine the ability of partial inverse agonists of C.5 global activity in forebrain neural circuits that is a mecha Subunit-containing GABA receptors to reverse the electro US 2017/002O892 A1 Jan. 26, 2017 physiological correlates of stress-induced depression using order; adult attention deficit hyperactive disorder); psycho electrophysiological and biochemical analyses in vitro. sis-related disorders (such as Schizophrenia, Schizophrenia 0060. In embodiments of the disclosure, partial inverse spectrum disorder, psychotic depressive illness); eating agonists of C5 subunit-containing GABA receptors exert disorders (such as anorexia nervosa; bulimia: obesity); per an antidepressant action comparable to ketamine's (Ibrahim Sonality disorders (such as avoidant personality disorder; et al., 2012). In certain embodiments, patients may undergo antisocial personality disorder; borderline personality disor standard psychiatric screening for DSM-IV criteria for der; conduct disorder, dependent personality disorder; major depressive episodes. In specific embodiments, a saline depressive personality disorder; histrionic personality dis Solution comprising one or more of the agonists are provided order; narcissistic personality disorder; negativistic person to the individual, such as infused slowly (ca. 30-60 min), for ality disorder, obsessive-compulsive personality disorder; example. Depression-related and other rating tests, such as paranoid personality disorder, Schizoid personality disorder; the Hamilton Depression Rating Scale, Beck Depression Schizotypal personality disorder) and other conditions such Inventory, Visual Analog Scales score for intoxication as cognitive impairment following traumatic brain injury; “high”, and the Brief Psychiatric Rating Scale, may be given neuropathic pain; chronic muscle or bone pain; diabetic repeatedly; as an example only, they may be given over a complications resulting in nerve injury; generalized attack of four hour period after drug administration, and daily over the muscular weakness; recurring sleep episodes during the day; following seven days. Antidepressant efficacy, as well as migraine treatment and prevention; and nicotine addiction. psychotomimetic or anxiogenic responses, is determined Also, treatment-related issues may be addressed. Such as upon changes in the test scores, in specific embodiments to lack of compliance with current treatment(s) and intolerance show efficacy with a particular compound. of side-effects from current treatments(s). Embodiments of 0061 The studies encompassed herein are the first to the disclosure provide for an improvement over other thera characterize the antidepressant-like efficacy of partial pies in the art in cases wherein the treatment of the disclo inverse agonists of C5 subunit-containing GABA recep sure is effective with fewer doses than other therapies in the tors. Rapid onset of antidepressant action, shown using art established preclinical models, for example, is highly desir 0064. In particular embodiments, the individual is known able from a clinical perspective and has never been to have or is suspected of having MDD (which is also known attempted with these compounds. The studies allow one to as clinical depression, major depression, unipolar depres demonstrate that partial inverse agonists of C5 subunit Sion, or unipolar disorder, or as recurrent depression in the containing GABA receptors are useful at least as a treat case of repeated episodes), which is a mental disorder ment for depression. characterized by a pervasive and persistent low mood that is accompanied by low self-esteem and by a loss of interest or III. Medical Conditions of the Disclosure pleasure in normally enjoyable activities. In specific 0062. There are a variety of medical conditions in which embodiments, the individual with MDD (or with another treatment as encompassed in this disclosure produces a medical condition) is Suicidal, although in specific embodi beneficial therapeutic response. The medical condition may ments the Suicidal person does not have depression. As an be diagnosed prior to treatment. In specific embodiments, example, an individual may be suicidal who has one or more the individual is suspected of having the medical condition, of the following attributes: depression; previous suicide is at risk for having the medical condition, has shown attempts; preoccupation with death; making troubling or behavior indicative of the medical condition, has a personal morose statements; talking openly about wanting to kill or family history of the medical condition, and so forth. In oneself having developed a Suicide plan; having acquired certain embodiments, symptoms of the medical condition the means to carry out a suicide plan: “rehearsal' behavior; may overlap with symptoms form other types of related setting a time for a Suicidal attempt; self-inflicted injuries, medical conditions. Such as cuts, burns, and/or headbanging; engaging in risky 0063. In specific embodiments, there is clinical efficacy behavior, such as driving recklessly, using drugs and/or in any condition in which antidepressant medication(s) are high-risk sexual behavior, making out a will or giving away currently being used, either with regulatory approval or favorite possessions; inappropriately saying goodbye, and so through off label use. Specific examples of medical con forth. In cases where the individual has depression, it may be ditions in which the methods and compositions of the unipolar or bipolar. The individual may have a propensity disclosure are useful include but are not limited to depres for depression and/or Suicidal thoughts because of a genetic sion-related disorders (such as Major depressive disorder or familial predisposition, for example, but not limited to, (MDD); dysthymia; cyclothymic disorder; seasonal affec having the brain-derived neurotrophic factor (BDNF) tive disorder/seasonal depression; depression after traumatic Va166Met polymorphism and/or the transporter gene brain injury; postpartum depression; premenstrual dysphoric 5HTLPPR promoter region polymorphism. disorder, depressive symptoms associated with menopause: 0065. In specific embodiments, the condition being depression following substance abuse? withdrawal; bipolar treated with methods and compositions encompassed by the disorder; bipolar disorder in remission; and depressive epi disclosure is considered resistant to treatment with previ sodes of bipolar disorder); anxiety-related disorders(such as ously existing treatments for depression (including SSRIs, general anxiety disorder, obsessive compulsive disorder; SNRIs and/or TCAS for example), such as treatment-resis Impulse control disorder, anxiousness associated with tant MDD, for example. depression; repeated episodes of anxiety, extreme apprehen 0.066 Although in particular embodiments the medical sion or fear of social interaction (Social phobia); panic condition is diagnosed prior to treatment, in other embodi disorders; posttraumatic stress syndrome or posttraumatic ments the individual is treated without formal diagnosis of stress disorder, and separation anxiety disorder); attention the condition, Such as in cases where a first responder, related disorders (such as attention deficit hyperactive dis medical responder, family member, hospital worker, or so US 2017/002O892 A1 Jan. 26, 2017
forth considers the treatment to be warranted. The individual and/or by blocking or reducing binding of an agonist to the may or may not be treated in a medical facility. In specific GABAR (i.e. GABAR antagonists) and/or blocking or embodiments, the individual suffers from depression, has reducing an effect of an agonist on the GABAR (i.e. had one or more concussions or other brain injury, is being negative allosteric modulators). An exemplary GABAR treated with a therapy (Such as a drug) that has suicide and/or channel blocker is picrotoxin. Exemplary GABA Rantago depression as a side effect, abuses alcohol or other prescrip nists include bicuculline and pentylenetetrazole. An exem tion or recreational drugs, is a victim of mental and/or sexual plary GABAR channel blocker is picrotoxin. Exemplary abuse, a combination thereof, and so forth. The individual GABAR negative allosteric modulators include non-selec may be a professional athlete that has had one or more tive and selective benzodiazepine receptor inverse agonists. concussions, as an example. The individual may currently be in the military or may have been in the military. The 0070. Examples of GABA receptor negative modulators individual may be of any race or gender or age. are as follows: 0067. In specific embodiments, the treatment for the 0071 1. Negative allosteric modulators acting at the individual is considered to be effective in a shorter period of benzodiazepine binding site. Beta-subunits direct membrane time than known treatment(s) for the condition. For insertion whereas adjacent gamma2 and alpha Subunits form example, in the case of compositions of the disclosure, the the so-called benzodiazepine site, an allosteric modulator of individual may have a therapeutic effect within minutes, channel gating. The identity of the a subunit determines the hours, days or weeks of the first administration. In particular pharmacological profile of this site. GABARS containing embodiments, the individual is given the therapeutic com alpha1, alpha2 or alpha3 subunits are potentiated by Zolpi position(s) once or more than once. In specific embodi dem, a benzodiazepine type 1 agonist, whereas GABARs ments, there are one or more therapeutic effects with the containing alpha5 Subunits are essentially insensitive to composition of the disclosure no more than days, hours, or Zolpidem (Burgard et al., 1996). Recent studies have shown minutes after the first administration. For example, the that al Subunits mediate the sedative/anticonvulsant and therapeutic effect may take place no more than 14, 13, 12. amnestic effects of benzodiazepines preferentially, whereas 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 day after the first the alpha2 and alpha3 subunits mediate their anxiolytic administration. The therapeutic effect may take place no effects (Rudolph et al., 1999; Mohler et al., 2002). In more than 96, 72, 48, 24, 23, 22, 21, 5, 4, 3, 2, or 1 hour after addition to the anxiolytic agonists of benzodiazepines there the first administration. The therapeutic effect may take is a class of drugs, exemplified by the B-carbolines which act place no more than 60, 59,58, 57, 56, 55, 54, 53, 52, 51, 50, as full or partial inverse agonists at the benzodiazepine site 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, to decrease GABAR function. Partial inverse agonists offer 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, the advantage of a wider therapeutic concentration range and 17, 16, 15, 14, 13, 12, 11, 10,9,8,7,6, 5, 4, 3, 2, or 1 minute a lower likelihood of producing anxiety or epileptiform after the first administration. A therapeutic effect may com discharge, in specific embodiments. prise a situation where the individual no longer wants to 0072 alpha5-subunit mRNA is highly expressed in the commit Suicide, or has a reduced desire to commit Suicide, pyramidal cells of the hippocampus and cortex (Allen rain or is having second thoughts about committing Suicide, or is Atlas) and alpha5-containing GABARS are localized in the now uncertain about committing Suicide, for example. A dendrites of hippocampal CA1 pyramidal cells (Fritschy and therapeutic effect may comprise when an individual no Mohler, 199S; Wainwright et al., 2000) at synaptic and longer has depression or when the individual is less extrasynaptic sites (Serwanski et al. 2006). Because disin depressed or has fewer depression symptoms or has longer hibition promotes induction of long-term potentiation (LTP), periods of time in between depressive moods, for example. and because of their selective forebrain localization, drugs that selectively inhibit alpha5-containing receptors are being IV. Compositions of the Disclosure developed as cognitive enhancers (Ballard et al., 2009). It 0068. In specific embodiments of the disclosure, one may has been shown, for example, that partial inverse agonists of employ one or more compositions for the treatment of one C.5-containing GABARs enhance associative memory or more medical conditions in which modulation of the acquisition in hippocampal-dependent learning tasks (Col GABA receptor is therapeutically effective for the condi linson et al., 2002). tion(s). In specific embodiments, the GABA receptor com 0073 Representative examples of benzodiazepine bind prises an alpha5 Subunit. Although in particular embodi ing site inverse agonists are as follows: ments any such condition is treatable, in specific embodiments the condition has depression or Suicidality as 0074 General, non-subunit-selective: Sarmazenil, beta at least one symptom. In particular embodiments, adminis carbolines, S-8510, DMCM, FG 7142. tration of one or more compositions of the disclosure to the 0075 Alpha5 subunit-selective: the imidazo 1.5-a 1.2, individual provides treatment of the condition or prevention 4-triazolo 1.5-d1.4benzodiazepine class of compounds of the condition and/or reduces the onset of the condition (such as R04882224, R04938581, L-655,708), the triazolo and/or reduces the severity of one or more symptoms of the phthalazine class of compounds (such as C.5IA), the 7,8,9, condition. 10-tetrahydro-(7,10-ethano)-1,2,4-triazolo 3,4-alphthala 0069. In specific embodiments, the composition utilized Zine class of compounds, the pyrazolotriazine class of for methods of the disclosure includes one or more GABA compounds (such as MRK-016), the 6,7-Dihydro-2-benzo receptor negative modulators. The term GABA receptor thiophen-4(5H)-one class of compounds, 6,6-Dimethyl-3- negative modulators, as used in this disclosure, may refer to (2-hydroxyethyl)thio-1-(thiazol-2-yl)-6,7-dihydro-2-benzo compounds that decrease the activity of the GABA recep thiophen-4(5H)-one, the 3,4-Dihydronaphthalen-1 (2H)-one tor, e.g., by blocking or reducing the influx of chloride ions class of compounds, (3-tert-butyl-7-(5-methylisoxazol-3- through the pore of the GABAR (i.e. channel blockers), yl)-2-(1-methyl-1H-1,2,4-triazol-5-ylmethoxy)pyrazolo US 2017/002O892 A1 Jan. 26, 2017
1,5-d. 1.2.4 triazine, the 3-Phenyl-5-arylpyridazine class 0080 Representative examples of GABAR antagonists of compounds, Ro 15-4513, RY-010, RY-023, RY-024, are as follows: picrotoxin, bilobalide, pentylenetetrazol, RY-080, and RG1662. thujone, and ginkgolide B. 0076 2. Negative allosteric modulators acting at the I0081 5. Negative modulators acting in the pore of the barbiturate steroid binding site. Barbiturates are drugs that GABAR channel. Numerous compounds have been devel were originally developed for their sedative properties. They oped that act to block the flux of C1-ions through the pore of act as positive allosteric modulators of GABARs to the GABA.R. Because they do not compete with GABA at increase the efficacy of GABA through an increase in its receptor on the channel, they are referred to as noncom channel open time. Barbiturates are thought to bind at the petitive antagonists (NCA). The best known example is interface between an C. and B subunit of the GABA.R picrotoxinin, which is also highly epileptogenic. Drugs (Sieghart et al., 2012). Barbiturates at supra-clinical con active at this site are widely used as insecticides and act by centrations may also directly open the channel in the physically blocking the channel, although allosteric modu absence of GABA. Although negative allosteric modulators lation has not been ruled out. The B3 subunit may be acting at this site have not yet been described, in specific particularly relevant for NCA binding (Chen et al., 2006). embodiments they exert antidepressant actions and may or Low affinity blockers would be helpful for clinical utility. may not be selective for alpha5 subunit containing receptors. I0082 6. Other negative modulators. Nicotinamide and related compounds, as well as inverse agonists and antago 0077 3. Negative allosteric modulators acting at the nists of the propofol binding site of the GABAR, are useful neuroactive steroid binding site. Neuroactive steroids are negative modulators of GABAR-mediated inhibition, in endogenous compounds that can be synthesized in the brain specific embodiments. from cholesterol or steroidal precursors or by metabolism of I0083. Thus, in specific embodiments one or more nega adrenal steroids or gonadal steroids, such as testosterone tive modulators of GABA receptors, whether acting allos (Mellon and Griffin, 2002). Examples of neuroactive ste terically, blocking the channel, or blocking the receptor for roids include pregnenolone and dehydroepiandrosterone, GABA itself, are utilized in therapeutic methods of the their sulfates, and their reduced metabolites. Allopreg disclosure, for example to produce a robust antidepressant nanolone and tetrahydrodeoxycorticosterone are positive action in unipolar and bipolar forms of depression, in allosteric modulators of GABAR-mediated chloride cur posttraumatic stress disorder, and in postpartum depression, rents, whereas pregnenolone Sulfate and dehydroepiandros by elevating mood and/or reducing Suicidal ideation. In terone (DHEA) sulfate are negative allosteric modulators. specific embodiments, the mechanism of action of these Neuroactive steroids bind to the delta subunit of the compounds is a strengthening of excitatory synapses that are GABAR, which is expressed heavily in the forebrain and pathologically weakened in depressed patients. In specific Ventral striatum; brain regions implicated in the genesis of embodiments, these compounds act more rapidly than con depression. There is evidence that pregnancy is associated ventional antidepressants, such as inhibitors of monoamine with decreases in the expression of delta Subunits and neurotransmitter reuptake and catabolism. In particular elevation of neurosteroid levels (Maguire et al., 2009), in embodiments, depression results from a pathological weak specific embodiments implicating dysregulation in their ening of excitatory synapses in multiple brain regions affect interactions as a contributing factor in post-partum depres ing the function of reward circuits in the ventral midbrain, S1O. and the methods of the disclosure provide a therapy that 0078 Drugs have been developed to mimic the positive addresses this physiological event. allosteric effects of neuroactive steroids and are used clini I0084. In one embodiment of this disclosure, GABAR cally as sedatives and anesthetics, such as alphaXolone, negative modulators are given alone at a therapeutically alphadolone, hydroxydione and minaxolone. Because of the effective dose. In another embodiment, GABAR negative presence of endogenous neuroactive steroids in the brain, an modulators are given at a therapeutically effective dose antagonist of this site can also be predicted to weaken together with one or more conventional antidepressant treat GABAR-mediated inhibition. Because some selective ments. In this embodiment, the conventional antidepressant serotonin reuptake inhibitors (SSRIs), such as fluoxetine, are (s) can be given (for example, orally) at current FDA reported to elevate levels of endogenous neuroactive Ste approved standard dosing regimens. roids in the brain (Pinna et al., 2006), in specific embodi I0085. In an embodiment of the disclosure, GABAR ments an antagonist at this site also amplifies the effects of negative modulators are given at a therapeutically effective such SSRIs. Drugs interacting at this site are useful in the dose, fixed or varying, over an unlimited period of time to treatment of postpartum depression, in specific embodi treat depression and related conditions. In specific embodi mentS. ments, an individual is provided an initial dosing regimen 0079 4. Competitive antagonists of the GABA.R. that has a stronger dose of the treatment than a later dosing Numerous compounds have been developed that act to block regimen that has a dose that is less than the initial dose. In the binding of GABA to the GABAR in a competitive Some embodiments, an individual is provided an initial manner. A well-known example is bicuculline. Competitive dosing regimen that has a smaller dose of the treatment than antagonists decrease the ability of synaptically released a later dosing regimen that has a dose that is greater than the GABA to activate the channel. Potent members of this class initial dose. of compound reduce inhibition so much that they are often I0086. In an embodiment of the disclosure, GABAR used to elicit epileptiform discharge in preclinical studies. A negative modulators are given at a therapeutically effective proconvulsive action would need to be greatly minimized to dose on a limited number of occasions (e.g., once per week render a member of this class of compounds clinically or once every 2-3 days) for a limited period of time (e.g., 1-4 useful, however an antagonist with low affinity is useful, in months). The modulator may be given 1, 2, 3, or 4 or more certain embodiments. times a day for a particular period of time, such as 1, 2, 3, US 2017/002O892 A1 Jan. 26, 2017
4, 5, 6, 7, 8, 9, 10, 11, 12 or more weeks, or 1, 2, 3, 4, 5, 6, these may be employed with compositions of the disclosure. 7, 8, 9, 10, 11, 12, or more months. Conventional antide Doses are mg/day per os (p.o.) unless otherwise noted. pressant treatments may be given continuously, as per cur 0093 Monoamine oxidase inhibitors (MAOJs) include rent dosing regimens approved by the FDA and other Selegiline -1.25-12 mg/day; Tranylcypromine 10-60 regulatory authorities. The depressive status of the indi mg/day: Moclobemide 100-600 mg/day (reversible MAOI. vidual may or may not be ascertained throughout the course approved outside the US); Phenelzine 15-90 mg/day; Iso of treatment. carboxazid; and a combination thereof. 0087. In specific embodiments, a GABAR negative (0094. Selective Serotonin reuptake inhibitors (SSRis) modulator acts on alpha 5 but also acts on other subunits of include Citalopram 10-20 mg/day: Escitalopram 10-20 the GABA.R. mg/day: Fluoxetine 10-40 mg/day, or 90 mg/week slow 0088. In particular embodiments, one or more analogs or release; Paroxetine 10-40 mg/day; Sertraline 25-200 derivatives of a GABAR negative modulator are utilized. mg/day: Fluvoxamine 50-300 mg/day; and a combination One of skill in the art recognizes how to modify a modulator thereof. and test its activity in methods described elsewhere herein, 0.095 Selective Serotonergic and Noradrenergic Such as in mouse models. Reuptake Inhibitors (SSRI/SNRI) include Venlafaxine XR 0089. In certain embodiments, the formulation of the one 37.5-225 mg/d; Desvenlafaxine 50-100 mg/day; Duloxetine or more modulators is tailored toward the need of the 20-120 mg/day; Milnacipran 12.5-200 mg/day: Levomll individual. For example, in cases wherein an individual has nacipran; Amoxapine; Protriptyline; Nefazodone (moderate a chronic depression problem, the formulation may need to antagonist at 5HT-2 and alpha1-adrenergic receptors); and a be such that the amount in the individual is kept at a plateau, combination thereof. yet the modulator for an individual with imminent severe 0096. Stimulants (off-label use) include Methylphenidate depression and/or Suicide may need to be formulated so that 2.5-20 mg/day p.o., or 10-30 mg transdermal; Modafinil it enters the bloodstream quickly. 100-400 mg; and a combination thereof. 0097 Examples of antagonists of NMDA-type glutamate V. Combination Therapy receptors include ketamine 6.5-13 mg/kg intramuscular 0090. In certain embodiments, methods of the present injection, or 1-4.5 mg/l
Autry et al., 2011), and restoration of normal synaptic definitive proof-of-concept test of a completely innovative strength. These results indicate that it is ultimately the mild class of potential medications, with potential for rapid increase in global activity in forebrain neural circuits that is implementation in the clinic. The outcome of these studies a mechanism via which rapid therapeutic AD actions are also tests the consideration that dysfunction of excitatory exerted. synapses contributes to the genesis of depression. One can 0128. One can test another means of producing such utilize both standard experimental approaches and invalu changes, namely mild and selective weakening of the able existing transgenic mouse models that are well Suited to strength of inhibition mediated by gamma-aminobutyric demonstrate effectiveness. One can also employ a variety of acid type-A receptors (GABARs or GABARs). GABARs chronic stress paradigms with face, construct, and predictive are heteropentameric ion channels, containing alpha, beta, validity with respect to depression and antidepressant devel and gamma Subunits in a 2:2:1 stoichiometry (Sieghart and opment, as well as a battery of behavioral, electrophysi Sperk, 2002). The interface between a gamma2 subunit and ological, and biochemical assays. The studies thus span an a subunit forms the benzodiazepine receptor, a positive levels of analysis from behavior to circuits to synapses to allosteric modulator of channel gating that increases molecules, as encouraged by the NIMH's Research Domain GABAR function. In addition to benzodiazepine agonists, Criteria initiative, in order to accelerate the translation of the there are drugs (e.g. beta- carboline) that act as inverse findings from preclinical research in rodents to human agonists at the benzodiazepine site to decrease GABAR clinical trials. function. The molecular identity of the alpha subunit in the I0132) Depression is a purely human condition, but behav benzodiazepine receptor site determines its pharmacological ioral changes can be induced in rodents in response to profile. GABARs containing alpha1 subunits mediate the environmental conditions, like chronic stress, that promote sedative, anticonvulsant and amnestic effects of benzodiaz depression in humans. Many of the behaviors affected by epines preferentially, whereas GABARS containing alpha2 chronic stress, such as reward, are analogous to human and alpha3 subunits mediate their anxiolytic effects (Ru behaviors that are symptomatic of human depression, Such dolph et al., 1999; Mohler et al., 2002). Partial inverse as anhedonia. Furthermore, Susceptibility to several chronic agonists offer the potential advantage of a wider therapeutic stress paradigms is associated with genes known to have a concentration range and a lower likelihood of producing high correlation with depression (Hasler et al., 2004). It is negative side effects, such as anxiety or epileptiform dis important to assess validity in relating the effects of chronic charge. stress in rodents to human depression. The behavioral and 0129. Negative allosteric modulators of GABARs con electrophysiological phenotypes of rodents subjected to taining alpha5 subunits, or alpha5 GABA-NAMs, have been chronic stress paradigms resemble human depression developed recently (Quirk et al., 1996; Atack et al., 2006; because they 1) include changes in affective state, cognition, 2009). Whereas non-selective benzodiazepine inverse ago and motivation; 2) result from a factor implicated in human nists are anxiogenic in humans, alpha5 GABA-NAMs are depression; and 3) respond to chronic, but not acute, admin not. alpha5-subunit mRNA is abundant in pyramidal cells in istration of SSRIs (Willner and Mitchell, 2002), as do the the hippocampus and cortex (Allen Brain Atlas) and C.5-con symptoms of depressed humans. Understanding how taining GABARS are localized to synaptic and extrasynaptic chronic stress affects the brain in rodents reveals insights sites in the dendrites (Fritschy and Mohler, 1995; Wain into the genesis and treatment of human depression. wright et al., 2000; Serwanski et al. 2006). C.5-containing 0.133 Chronic stress paradigms. One can use four differ GABARs mediate tonic inhibition and lower the excitability ent chronic stress paradigms in rats or mice to determine of pyramidal cells (Bonin et al., 2006). Interestingly, C.5 whether the effects of the compounds are paradigm-inde Subunits are up-regulated in mice after chronic stress (Mat pendent. One can employ chronic unpredictable stress sumoto et al., 2007). Because disinhibition promotes induc (CUS), which involves repeated delivery of mild stressors tion of long-term potentiation (LTP), the presumptive cel twice per day for 3 weeks (Wilner et al., 1987). One can use lular basis of memory, alpha5 GABA-NAMs are being chronic social defeat stress (SDS), a form of psychosocial developed clinically as cognitive enhancers (Ballard et al., stress in which animals chronically lose social stature to a 2009). It has been shown, for example, that alpha5 GABA more dominant animal (Malatynska and Knapp. 2005). In NAMs increase LTP and enhance memory acquisition in this paradigm, individual juveniles (>4w old) are placed in hippocampus-dependent learning tasks (Collinson et al., the home cage of a dominant resident animal for 1 hr while 2002). Indeed, one alpha5 GABA-NAM compound is separated by a perforated protective screen, permitting already in clinical trials for Down Syndrome. The ability of olfactory, visual and auditory contact. The SDS protocol is this class of compounds to promote excitatory drive in repeated daily over 3 weeks. Third, one can use chronic regions of the brain like the hippocampus indicates their restraint stress (CRS), in which animals are held in tight utility as rapidly acting antidepressants, similar to ketamine fitting restraint tubes for 4h every day for 8-10 days (Lim et but lacking the deleterious side effects. al., 2012). Finally, one can directly elevate stress hormone 0130. In embodiments of the disclosure, partial inverse levels by administering corticosterone (Gourley et al., agonists of the benzodiazepine binding site of GABA 2008). All of these paradigms induce changes in animal receptors containing alpha5 subunits (L-665,708 and MRK behaviors that are reversed by chronic, but not acute, admin 016) produce a rapid and persistent restoration of a range of istration of SSRIs (Papp et al., 1996; Grippo et al., 2006: behaviors that are impaired by chronic stress in rats and mice Bondi et al., 2008), comparable to the 3-8 week delay in because they restore the strength of pathologically weakened patients in response to SSRIs. excitatory synapses. 0.134 Behavioral consequences of stress in rodents. What 0131 One can utilize studies that will challenge and seek behaviors in rodents most closely resemble the symptoms of to shift the current paradigm for understanding the genesis depressed humans and can also best predict effective thera and treatment of depression. Namely, one can provide a peutic treatments for depression? Ideal signs of a depressive US 2017/002O892 A1 Jan. 26, 2017
like state will 1) have ethological relevance, 2) be robust and neurons in the hippocampus receive a direct EC input, the accurately quantifiable, and 3) be responsive to chronic, but temporoammonic (TA) pathway, on their distal apical den not acute administration of SSRIs, like human depression. drites in stratum/acunosum molecular (SLM) (Remondes The forced Swim test and the tail Suspension test are con and Schuman, 2002; Steward & Scoville, 1976). The TA sidered to provide a measure of behavioral despair. While pathway is important for consolidation of long-term these tests are popular for their ease of use and reliability, memory, as well as spatial recognition tasks (Brunet et al., they lack some face validity because changes in these tasks 2002; Remondes & Schuman, 2004). Chronic stress pro can be detected following a single administration of SSRIs duces a number of changes in TA-CA 1 excitatory synapses in control animals (Detke et al., 1997; Petit-Demoulier et al., and deficits in memory consolidation (Cai et al., 2013; 2005). Another behavioral measure that is affected by Kallarackal et al., 2013). One can use this synapse as a chronic stress but is responsive only to chronic, but not convenient archetype for the changes that are known to acute, SSRIs is an assay of hedonic state called the sucrose occur at multiple sites in the brain in depression. preference test (SPT) (Rygula et al., 2006). In this test, 0.138. Determination of the persistence and generality of rodents are presented with a two bottle choice task with one alpha5 GABA-NAM’s antidepressant actions on behavior. bottle containing normal water and one bottle containing a 0.139. Rationale: A single injection of one of the two dilute sucrose solution. The naturally high preference of rats commercially available alpha5 GABA-NAM L-665,708 (1 and mice for the Sucrose solution is greatly diminished by mg/kg, intraperitoneally, IP) (Quirk et al., 1996) reverses the chronic stress and restored with chronic, but not acute, SSRI loss of sucrose preference (FIGS. 3A and 3B) and the treatment (Pothion et al., 2004). A lack of sucrose preference decrease in social interaction (FIGS. 3C and 3D) induced by is said to reveal anhedonia, or the inability to experience two forms of chronic stress within 24 hrs: chronic unpre reward, a core symptom of human depression. Defects in dictable stress and chronic restraint stress. For this class of Social reward are also a prominent symptom of human compounds to have use as effective ADS in humans, in depression. One assay of rodents’ social curiosity, the Social specific embodiments their restorative effects should 1) hold interaction test (SIT) (Berton et al., 2006), measures the true over a wide range of behaviors that are affected by amount of time animals spend interacting with a novel target chronic stress, 2) be independent of the paradigm by which animal and provides a measure of the rewarding value of the stress is applied, and 3) be persistent. Social interactions. 0140. In specific embodiments, a single injection of 0135 The use of these models allows one to reveal a L-665, 708 or another commercially available alpha5 rapid onset of action, which distinguishes embodiments of GABA-NAM, MRK-016 (Atack et al., 2009), at a maxi the present disclosure from the prior art. What has been done mally effective dose rapidly (within days) reverses stress previously using animal models is insufficient to have pre induced changes in a wide range of behaviors regardless of dicted a rapid onset, fast acting antidepressant action of how the stress is applied, and these effects persist for several these compounds. Use of other models and methods, such as days after the injection (FIG. 9). the forced Swim test and tail Suspension test, do not provide 0.141. Definition of control and susceptible animals. As predictive validity of a fast onset of therapeutic relief of an example, in this and at least Some other studies described depressive symptoms and Suicidal thinking in animals (i.e. herein, adult male rats are tested over 2 weeks using the humans). sucrose preference and social interaction tests (SPT, SIT) to 0136. The hippocampus in depression. Depression is establish baselines. Only animals that repeatedly meet pre likely caused by dysfunction in many brain regions and cell established criteria of control rats based on prior results types, including the hippocampus. Glucocorticoid receptors (sucrose preference >75%, social interaction ratio >130%) are expressed at high levels in pyramidal cells, rendering it are considered as acceptable controls. Those that do not are particularly sensitive to stress (Reul & de Kloet, 1985; excluded from further study so that a starting population is Magarinos et al., 1996). Indeed, chronic stress induces as homogeneous as possible. Rats are then Subjected to one hippocampal dysfunction, including dendritic atrophy and of the chronic stress paradigms, followed by SPT and SIT. spine Joss (McEwen, 1999), deficits in synaptic plasticity At this point, only those animals with Sucrose preference (Aifarez et al., 2003), decreased neurogenesis and synapto <65% and social interaction ratios <115% are considered to genesis (Duman and Li, 2012), and aberrant circuitry (Airan have been susceptible to the chronic stress and therefore et al., 2007). Notably, depressed patients have reduced useful for studying the effects of the test compounds and hippocampal Volume and deficits in hippocampus-depen other manipulations. dent behavioral tasks (MacQueen et al., 2003: Campbell and 0142 Experiment A. What is the alpha5 GABA-NAM MacQueen, 2004) such as visuospatial navigation (Hickie et dose-dependence? How long do its effects last? One can use al., 2005; Gould et al., 2007). the chronic restraint stress (CRS) paradigm for determining 0.137 Output from the hippocampus drives activity in the dose-dependence. A single injection of L-665,708 or MRK NAc, a key regulator of motivation and reward value, and is 016 is administered to susceptible rats at doses of 0.3, 1, 3, known to influence motivation, perhaps through its ability to 10, or 30 mg/kg IP, followed by SPT and SIT<24 hrs later. promote dopamine secretion (Kelley and Mittleman, 1999; Dose-response curves are prepared by plotting 1) the mean Boulenguez et al., 1996; Floresco et al., 2001; Tye et al., Sucrose preference and social interaction ratio as a function 2013). A dialogue between the hippocampus and the neo of alpha5 GABA-NAM dose and 2) the percentage of cortex is also important for maintaining normal cognitive animals that responded to the drug with an increase in function (Gray, 1998: Lisman and Otmakhova, 2001; Morris Sucrose preference to 75% and an increase in Social inter et al., 1982; Remondes and Schuman, 2004). The entorhinal action ratio to 130% as a function of dose. Animals are then cortex (EC) provides the principal inputs to the hippocam retested with the SPT and SIT at 5, 10, and 15 days pus, and the projection from area CA 1 to the EC is a post-injection to determine the persistence of the effects. principal output. The apical dendrites of CA1 pyramidal One set of control group of animals can undergo CRS, US 2017/002O892 A1 Jan. 26, 2017
receive an injection of vehicle, and be tested with SPT and mals are then subjected to 10 days of CRS, tested for sucrose SIT at 1, 5, 10, and 15 days post-injection. A second set of preference to identify susceptible individuals, then given control animals are not subjected to CRS, but receive an Sucrose preference, urine marking, and tube tests. In specific injection of the maximally effective dose of L-665,708 or embodiments, dominant animals lose their dominance as a MRK-016. In specific embodiments, the effects of the com consequence of stress. If so, one can administer an injection pounds are dose-dependent in both behaviors by both mea of L-665,708 or vehicle. In specific embodiments, domi sures, and the effects persist for 5-15 days after injection. In nance is restored by alpha5 GABA-NAM, but not vehicle, specific embodiments, vehicle injection does not restore injection in parallel with Sucrose preference. Controls are control responses in either behavior and the compounds do treated as above, but not subjected to CRS. 24hrs later they not affect either behavior significantly in unstressed animals. are retested in the Sucrose preference, urine marking, and One can use the maximally effective dose of the more tube tests. effective compound, as determined in these studies, in other 0147 B4. A third cohort of control rats receive an injec studies described elsewhere herein. tion of L-665,708 or vehicle then undergo forced swim tests 0143 Experiment B. How generalized are the restorative 1 hr later. Statistical comparisons of time spent immobile are behavioral effects of alpha5 GABA-NAMs? One can extend made between vehicle and drug injected animals. Acute the data on alpha5 GABA-NAM’s effects in the SPT and ketamine reduces immobility time in the forced swim test SIT to a broader range of stress-affected behaviors: novelty (Maeng et al., 2008), much like acute fluoxetine adminis Suppressed feeding, sexual place preference, social hierar tration, indicating that the alpha5 GABA-NAM compounds chy, and forced Swim. also decrease immobility in this test. 0144 B1. Novelty suppressed feeding (NSF) is a widely 0148 Experiment C. Does alpha5 GABA-NAM induce used fluoxetine-sensitive measure of chronic stress (Cal et adverse behavioral effects? Two complex behaviors that al., 2013). In the first cohort of animals, baseline latency in might be affected adversely by benzodiazepine inverse ago the NSF test is measured, as done previously. Rats are then nists are tested: anxiety and sleep. subjected to CRS and susceptible animals selected. CRS 0149 C1. One cohort of control rats are given a baseline continues for 5 more days, then half the animals are ran open field test. One week later, half of the animals receive domly selected to receive a vehicle injection and the other an injection of L-665,708 or vehicle, followed 24hrs later by half receives a single injection of L-655, 708. 24 hrs later a second open field test in a new arena. In a specific they again undergo the NSF test. embodiment, a mixed design analysis of variance reveals no (0145 B2. Conditioned place preference is a well-estab difference of either alpha5 GABA-NAM or vehicle injection lished assay of reward valuation and affective state. There on the amount of time spent in the center Zone, an indicator fore, a second cohort of animals undergoes the same pro of anxiety, or in total line crossings, an indicator of loco cedure but is tested with a sexual place preference test. motion, compared to either their own baseline or across Control male rats are conditioned to associate sexually groups. receptive females with specific olfactory and visual cues, 0150 C2. A second cohort is tested for altered daily then given a probe test of environmental preference (Cama activity rhythms, as an initial indicator of altered sleep. Rats cho et al., 2004). They are then subjected to CRS and are housed singly in a cage with multiple crossed infrared susceptible animals selected. Half of the rats receive vehicle beams (SmartCage, Afasci, Inc). Frequency of beam breaks injection and the other half are given an injection of L-665, is recorded continuously over a 48hr-period prior to an 708. 24 hrs later the place preference probe test is repeated. injection of L-665,708 or vehicle. Activity is then recorded For both cohorts, two way comparisons are then made over the following 48 hrs. Total number of beam breaks and between behavioral scores (before vs. after injectionxdrug the duration of periods of inactivity are compared in indi vs. Vehicle). Mount and intromission latencies, which are vidual animals before and after injection, and between known to be sensitive to chronic stress (Gronli et al., 2005), treatment and vehicle groups in another mixed ANOVA. are also measured as indicators of sexual motivation (Hull, 0151 Experiment D. Can alpha5 GABA-NAMs reverse 2002). In specific embodiments, only rats receiving L-665, the effects of multiple stress paradigms? One can complete 708 display NSF latencies and place preferences that are initial studies with CRS and CUS and extend them to rats different from their own scores before injection and different subjected to social defeat stress (SDS) (Papciak et al., 2013) from vehicle-injected rats. Control rats are given NSF or and direct corticosterone administration (Gourley et al., sexual place preference tests. One week later they receive an 2008). injection of L-665,708 and are retested 24 hrs later. In 0152 D1. One cohort of controls experience social defeat specific embodiments, the injections have no effect on either daily for 1hr each day for 3 weeks. behavior in unstressed control rats, as was observed with 0153 D2. Another cohort of controls receives corticos SPT and SIT. terone hemisuccinate (50 pg/ml) via the drinking water in 0146 B3. Rodents display a prominent innate social their home cage for 4 weeks, to produce elevated blood hierarchy that is known to be sensitive to chronic stress and concentrations correlating with the animals diurnal cycle. is restored by chronic fluoxetine (Lehmann et al., 2013). Previous studies have shown that peak levels are comparable Urine scent marking, a form of social communication in to those elicited in rats during chronic stress procedures rodents, correlates with dominance status, with socially (Gourley et al., 2008). Susceptible rats in each group then dominant rats making more and larger marks than those that undergo another week of SDS or corticosterone administra are socially subordinate. Another robust test of social domi tion. Half of the rats in each group receive an injection of nance, the tube test (Wang et al., 2011), may be used. L-665,708 and half vehicle. 24 hrs later they again undergo Control rats are housed in pairs for one week, then given a SPT and SITs. Within- and between-subjects statistical com series of tube tests and a urine marking test to identify the parisons of Sucrose preference and Social interaction ratios dominant and Subordinate rat in each pair. Dominant ani are made for individuals before and after vehicle or drug US 2017/002O892 A1 Jan. 26, 2017 injection in a mixed design ANOVA. In specific embodi entorhinal cortex (Source of the temporoammonic pathway), ments, injection of the test compound, but not the vehicle, or the shell of the NAc. After recovery, they are trained to produces a significant change in the animals' Sucrose pref remain still in a plastic tube without anesthesia with their erence and social interaction ratios in both SDS and corti heads secured. costerone-treated rats. 016.0 A1. A 60 min period of continuous recording of 0154. In cases wherein one needs to detect small differ spontaneous field potential activity is made under dim ences in behaviors in response to stress or alpha5 GABA illumination. Rats are then injected with either L-665,708 or NAM administration, studies may be designed with stan vehicle, and placed back in the tube for an additional 60-90 dardized starting behaviors and evidence of susceptibility so min of recording. Discrete fast Fourier transforms are made as to reduce variability due to unknown events during the on 60s-long recording segments for preparation of spectro breeding and delivery of animals, resilient individuals, etc. grams. Oscillation power (area under the curve) is integrated The studies may also be designed to allow use of repeated for different frequency bandwidths for power spectral den measures and paired comparisons wherever possible, thus sity plots (0–3-7 Hz; C–8-12 Hz; B=13-29 Hz; Y=30-80 Hz) increasing the ability to detect small effects. Initial experi (Buhl et al., 1998; Raver et al., 2013). Just as with ketamine ments on the effect of CRS and alpha5 GABA-NAM treat (e.g. Kittleberger et al., 2012: Lazarewicz et al., 2010; ment gave robust effect sizes for the SPT and SIT (f=0.73 Hinman et al., 2013), in specific embodiments increases in and f-0.43, respectively). Using the smaller SIT effect size, power are observed by pairwise comparison of spectra and a.=0.05, 8 animals/group are required to have a 95% before and after drug injection. Initial studies are consistent chance of observing significant differences in future SPTs in with this consideration (FIG. 2). a mixed-design ANOVA format (calculated with G*Power 0.161 A2. One can next test whether alpha5 GABA and IBM SPSS software). These group sizes are consistent NAMs exert their influence on activity through binding to with prior experience and published results for similar the benzodiazepine site on the GABAR. The experiments experiments (i.e. Lim et al., 2012). Stress effects on social above are repeated in a second cohort of animals with the dominance in rats have not been demonstrated, but it is co-injection of the pharmacological antagonist of the ben adversely affected in mice and restored by fluoxetine (Leh Zodiazepine receptor, flumazenil (20 mg/kg IP) (Tietz et al., mann et al., 2013). If dominance is not achieved with CRS 1999), 30 min prior to L-665,708 injection. In specific in rats, then the studies are performed in C57BL6/J mice, as embodiments, L-665,708 produces no significant change in in earlier published work. oscillatory activity under these conditions because it cannot 0155. Upon completion of the aforementioned studies, bind to the GABAR. one establishes whether alpha5 GABA-NAM antidepres (0162 Experiment B. Do alpha5 GABA-NAMs actions Sant-like restorative actions on stress-sensitive behaviors are depend on serotonin signaling? One way in which alpha5 highly generalizable across a range of the motivational GABA-NAM-induced oscillatory activity might restore behavioral phenotype (feeding, social, sexual) and whether stress-altered behavior is by promoting serotonin release they are independent from the means in which the stress was from raphe neurons. applied (direct vs. physical vs. Social). In specific embodi 0163 Bi. One can use Pet1cre-Lmx1bflox mice (Zhao et ments, a useful antidepressant compound is broadly effec al., 2006), in which adults lack any serotonergic neurons in tive, regardless of the proximate cause of the altered behav the CNS, to characterize this. Surprisingly, these mice have ior, and has few side effects. not yet been characterized in many depression-related 0156 Determination of the mechanism of action of behaviors, but it is possible that these behaviors are abnor alpha5 GABA-NAM at the level of synaptic circuits. mal. One can first characterize these mice in the SPT and 0157 Rationale: Ketamine induces a rapid increase in SIT. If their behavioral responses are within the same range neural oscillations in circuits in the hippocampus, entorhinal as wild type littermate mice, then one can subject them to 10 cortex, NAc, and PFC in rodents (e.g. Kittleberger et al., days of CRS. One week later, they receive a single injection 2012: Middleton et al., 2008; Hunt et al., 2011) and in of L-665,708 or vehicle, followed 24 hrs later by a second humans (Driesen et al., 2013). It is widely accepted that this round of SPT and SIT. In specific embodiments, alpha5 increase in circuit activity underlies the induction of ket GABA-NAMs restorative action on these behaviors is inde amine's antidepressant-like actions because oscillatory pendent of serotonin release and serotonin receptor activa activity at these frequencies promotes not only LTP (e.g. tion. In specific embodiments, L-655,708 triggers an Huerta and Lisman, 1993) but also other forms of activity increase in both measures so that they are different compared dependent strengthening of excitatory synapses that are to levels before drug injection in repeated measures com critical to the reversal of the stress phenotype. One can parisons, whereas in certain embodiments vehicle-treated examine whether alpha5 GABA-NAM’s ability to reverse animals do not have different measures in a mixed ANOVA the stress phenotype results from triggering similar oscilla design. If these mice do not have sucrose preferences >75% tory activity. and social interaction ratios >130%, then one does not 0158. In specific embodiments, alpha5 GABA-NAMs pursue the experiments further. bind to benzodiazepine receptors to induce rapid activation 0164 B2. One can select control and CRS susceptible of neural circuits in the reward circuitry and restore stress rats. Half of the susceptible rats are injected twice with the altered behaviors because of their ability to activate activity toxin PCPA (300 mg/kg., IP, 24 hrs apart) (Dewar et al., dependent synaptic plasticity. 1992) to deplete serotonin acutely. The other half is injected 0159 Experiment A. Does alpha5 GABA-NAM admin with saline. 24 hrs after the second PCPA injection, both sets istration produce activation of neuronal circuits in vivo as a of rats are given a single injection of L-665,708. 24 hrs later result of binding to benzodiazepine receptors? Rats have they are given SPT and SITs. Animals are sacrificed and electrodes placed stereotaxically into str. pyramidale of area sections stained with an antibody against serotonin to verify CA1 (the output of the hippocampus), layer II of the PCPA-induced serotonin depletion, as has been done previ US 2017/002O892 A1 Jan. 26, 2017
ously (Cai et al., 2013). If alpha5 GABA-NAMs act inde require NMDAR-dependent activity-dependent strengthen pendently of serotonin, in specific embodiments both PCPA ing of excitatory synapses for their expression. and saline-injected rats show normalization of stress-sensi 0169. Determination of the molecular and synaptic tive behaviors in response to alpha5 GABA-NAM treat mechanisms of alpha5 GABA-NAM action. ment. (0170 Rationale: The inventors and others have observed (0165 Experiment C. Do alpha5 GABA-NAM’s actions a weakening of specific excitatory synapses after chronic depend on NMDAR-dependent plasticity or GluA1 phos stress and in specific embodiments this contributes to the phorylation? Ketamine's actions probably depend on block genesis of depression. Chronic fluoxetine administration ade of interneuron NMDARs, with the subsequent increase reverses these deficits and in specific embodiments these in activity driving strong activation of pyramidal cell effects underlie the restoration of normal behavior by effec NMDARs. One can now test whether alpha5 GABA-NAM tive ADS. Qualitative and quantitative changes in the converges on this latter effector mechanism (FIG. 1). response of TACA 1 synapses to activation of 5-HT18Rs 0166 C1. One can use CxNR1KO mice (Iwasato et al., following chronic stress, which are reversed by AD treat 2000), in which NMDARs are genetically deleted in hip ment (Cai et al., 2013), are described. Here one can test the pocampal and cortical pyramidal cells. These mice are also effects of alpha5 GABA-NAMs on these stress-induced uncharacterized in many depression-related behaviors and it phenotypes. The TA-CA1 synapse can be used as an arche is possible that these behaviors are abnormal (see however: type for changes that may occur in multiple brain regions. Rompala et al., 2013). One can first characterize these mice One can also begin to identify the signaling pathways in SPT and SIT. If their behavior responses are within the through which these changes are mediated. same range as wild type littermate controls, then one can 0171 The rapid reversal of stress-induced changes in subject them to 10 days of CRS and repeat SPT and SITs. behavior by alpha5 GABA-NAMs, as described above, is After an additional 5 days of CRS, they receive a single accompanied by a rapid 1) strengthening of AMPAR-medi injection of L-665,708 or vehicle, followed 24 hrs later by ated excitatory synaptic transmission, 2) normalization of a third round of SPT and SIT. In specific embodiments, 5-HT1BR-mediated synaptic potentiation, and 3) increase in alpha5 GABA-NAM’s restorative action on these behaviors GluA1 expression levels. is dependent on pyramidal cell NMDAR receptor activation (0172 Experiment A. Do alpha5 GABA-NAMs restore and therefore, in the absence of these NMDARs, alpha5 AMPAR-mediated synaptic responses? Control and CRS GABA-NAMs are unable to restore stress-induced behav susceptible rats are given a single injection of L-665, 708 or ioral changes. Paired comparisons in either alpha5 GABA vehicle, followed 24 hrs later by a second round of SPT and NAM or vehicle-injected rats reveal no significant differ SIT. Hippocampal brain slices are then prepared for elec ences in Sucrose preference or Social interaction compared to trophysiological recording. Because TA-CA1 synapses are before injection, in specific embodiments. If these mice do electrotonically remote from CA1 cell somata, one can use not display responses in the normal range of responses in extracellular recording of local field excitatory postsynaptic SPT and SIT under control conditions, then one can admin potentials (fEPSPs) in SLM. AMPAR-mediated responses ister a single injection of L-665, 708 or vehicle at the are compared using two independent methods (fiber Volley maximal dose, followed by a second round of SPT and SIT amplitude and NMDAR-mediated responses) to control for 24 hrs later. In specific embodiments, their behavior is not the intensity of stimulation and the health of the slices, as has significantly different in either test after injection, If alpha5 been done previously (Kallarackal et al., 2013). One can first GABA-NAM is effective in the CxNR1 KO mice; in which normalize AMPAR responses to the amplitude of fiber NMDARs in the NAc are not deleted, then in specific Volley over a range of stimulation intensities. One can also embodiments activation of inputs from the cortex and hip normalize responses by comparing the slopes of the pocampus by alpha5 GABA-NAM are crucial in its AD-like AMPAR- and NMDAR-mediated components at a modest actions. stimulation intensity producing fiber volleys of -0.2 mV 0167 C2. One can next use S831A mice in which serine before and after application of DNQX. Schaffer collateral 831 of the GluA1 subunit of the AMPAR has been mutated responses are measured as a control because they are not to alanine, rendering them incapable of being phosphory affected by chronic stress by this measure (Kallarackal et al., lated by CaM kinase. Previous studies have shown that 2013). In specific embodiments, AMPAR-mediated excita NMDAR-dependent LTP is abnormal in these mice (Lee et tion is decreased in slices from vehicle-injected CRS rats al., 2010). These mice have a behavioral phenotype that compared to unstressed, vehicle-injected controls. Injection resembles that produced by chronic stress, although social of alpha5 GABA-NAMs restores control levels of excitation interaction was not yet tested. One can therefore give in slices from CRS rats, but has no effect in control rats. This baseline SPT and SITs to naive mice. Because in particular is what has been observed in initial studies (FIGS. 10A, embodiments their responses are not in the normal range in 10C). these tests, 5 days later one can give a single injection of (0173 Experiment B. Do alpha5 GABA-NAMs restore L-665,708 or vehicle, followed by a second round of SPT 5-HT1BR-mediated potentiation? One can consider whether and SIT 24 hrs later. In specific embodiments, their behavior alpha5 GABA-NAMs reverse another phenotypic alteration is not significantly different in either test. Hippocampal brain of stressed TA-CA 1 synapses: amplification and persistence slices may be harvested upon completion for electrophysi of responses to the 5-HT1BR agonist anpirtoline (FIG. 3A) ological and biochemical analyses, as described below, to (Cai et al., 2013). Slices are prepared from the four groups test correlations between behavior and synaptic function. described in Experiment A. Anpirtoline (501.1M) is bath 0.168. In specific embodiments, alpha5 GABA-NAM's applied for 60 min, followed by a 90 min washout period. In antidepressant-like restorative actions on stress-sensitive specific embodiments, anpirtoline potentiation of TA-CA1 behaviors 1) are induced by a benzodiazepine receptor fEPSPs is of a larger magnitude and is persistent in slices dependent increase in oscillatory circuit activity and 2) from vehicle-injected CRS animals compared to unstressed, US 2017/002O892 A1 Jan. 26, 2017
vehicle-injected controls and that injection of alpha5 BDNF increases in tissue from alpha5 GABA-NAM-in GABA-NAM restores control levels of reversible potentia jected rats, compared to tissue from vehicle-injected rats. In tion in slices from CRS rats, but has no effect in control rats the hippocampal samples, the changes in phospho-eEF2 and (see FIG. 3B). BDNF are greater in dendritic tissue than in somatic tissue. (0174 Experiment C. Do alpha5 GABA-NAM restore 0.178 D3. Nestler and colleagues have shown that induc GluA1 phosphorylation and/or expression? Tissue punches tion of the transcription factor AFos3 in D1 R-expressing are obtained from the SLM region (where the stress-sensi medium spiny neurons in the NAc in response to chronic tive TA-CA1 synapses are) in the slices prepared for Experi stress is an important mediator of resilience and may play a ment A. Control punches are taken from Str. radiatum (where role in the antidepressant actions of fluoxetine by changing the stress-insensitive Schaffer collateral synapses are). West gene expression and synaptic strength (Perrotti et al., 2004; ern blotting is used to compare expression of GluA1 and Lobo et al., 2013: Grueter et al., 2013). It is considered S831 phosphorylation, GluA2, PSD-95, and NR1, as has whether alpha5 GABA-NAM injection also affects delta been done previously (Cai et al., 2013; Kallarackal et al., Fosb expression. Control and CRS-susceptible rats receive 2013). In specific embodiments, expression of GluA1 and an injection of L-665, 708. One hour later, rats are sacrificed PSD-95, but not GluA2 or NR1, is decreased in the SLM in and tissue harvested. Tissue punches are taken from the vehicle-injected CRS animals compared to unstressed, NAc, area CA1, EC, and PFC and subjected to Western vehicle-injected controls and injection of alpha5 GABA blotting for quantification of deltaFosB levels. Sections are NAM increases GluA1 S831 phosphorylation and restores prepared from these same regions and stained for AFosB in control levels of expression in CRS rats, but has no effect in order to count immunoreactive nuclei with Stereological control rats (see FIG. 3). techniques (Vialou et al., 2010). Sections through the ventral 0175 Experiment D. What signaling pathway does striatum are co-stained with an antibody against the D1R, alpha5 GABA-NAM activate to mediate its beneficial since the expression of AFosB in these cells may be par actions? ticularly important for SSRI actions (Lobo et al., 2013). 0176 D1. Duman and colleagues (Liet al., 2011) showed Similar to fluoxetine, in specific embodiments in stressed that ketamine increases expression of several genes associ and unstressed animals, alpha5 GABA-NAM affects AFosB ated with synaptic function through rapid activation of the expression in the NAc and other brain regions, consistent mTOR signaling pathway. One can use the same approach to with initial studies (FIG. 5). test the role of this pathway in alpha5 GABA-NAM's 0179. With regard to the sensitivity of the assays, power actions. Cannulae are positioned in the cerebral ventricles. analysis based on previously published data on the effects of One week later, CRS-susceptible rats are identified. Rats are GUS on AMPAR:NMDAR ratios (Kallarackal et al., 2013) then divided into three groups. Group one receives rapamy suggests that detection of an effect size with p=0.6 would cin (0.2 nmol, ICV) 30 min prior to an injection of L-665, require a total n=21 to have 0.95 power. These studies are 708, group two receives saline ICV 30 min prior to an designed to establish a correlation between restoration of injection of L-665,708, and group three receives rapamycin stress-induced behavioral and synaptic phenotypes. Such an ICV prior to an IP injection of vehicle. 24 hrs later they are initial step may lead to more synapse- and behavior-specific retested in SPT and SITs. If mTOR signaling is required for studies, and one can then characterize other synapses elec the effects of alpha5 GABA-NAMs, then in specific embodi trophysiologically and biochemically, particularly in the ments pairwise comparisons show that alpha5 GABA-NAM NAc. The results of the studies on signaling pathways are +saline rats have increased Sucrose preference and social valuable in designing effective experimental interventions, interactions as compared to before injection, whereas the Such as targeted knockdown and overexpression of delta alpha5GABA-NAM+rapamycin and vehicle+rapamycin Fosb (Grueter et al., 2013), to establish causality. rats do not. Rats are then sacrificed for electrophysiological 0180 Thus, it is determined whether alpha5 GABA and biochemical analyses, as above. In specific embodi NAM's rapid reversal of stress-induced behavioral pheno ments, the restorative alpha5 GABA-NAMs-induced synap type is accompanied by rapid changes in the stress-induced tic and biochemical changes observed in Experiments A-C synaptic phenotype. One can also identify the signaling are also blocked by rapamycin. One can also use Western blotting to look for increases in phospho-mTOR and BDNF pathways through which these changes are mediated. after alpha5 GABA-NAM injection in the NAc, area CA1, 0181. It is determined that partial inverse agonists of the EC, and PFC. benzodiazepine site of GABARs containing C5 subunits (for 0177 D2. Kavalali and Monteggia (2012) have advanced example) have antidepressant efficacy. In one embodiment, an mTOR-independent model of ketamine action in which stress-induced synaptic changes cause stress-induced behav inhibition of NMDARs de-phosphorylates eukaryotic elon ioral changes. In other embodiments, there is mapping of gation factor eEF2, allowing rapid de-suppression and trans specific synapses affected by stress and the corresponding lation of dendritic mRNAs, including BDNF. One can use behavioral consequence of that dysfunction, such as the the same approach to test this mechanism of alpha5 GABA correlation between TA-CA1 synaptic strength and memory NAM action. Control and CRS-susceptible rats are given an consolidation (Kallarackal et al., 2013). injection of L-665, 708. One hour later, rats are sacrificed 0182. In specific embodiments, one tests whether and tissue harvested. Tissue punches are taken from the 5-HT1BR-induced potentiation and activity-dependent syn NAc, area CA 1 (separating dendritic and somatic layers), aptic plasticity share common signaling mechanisms at the EC, and the PFC and subjected to Western blotting for TA-CA1 synapses. 5-HT1BRs potentiate TA-CA1 synapses quantification of BDNF and phosphorylated and total eEF2 by a strengthening of postsynaptic AMPAR responses medi expression, as in Autry et al. (2011). If dephosphorylation of ated by phosphorylation of AMPARs by CaMK (Cal et al., eEF2 contributes to alpha5 GABA-NAM’s actions, then in 2013). Another parallel signaling pathway, involving ras and specific embodiments phosphorylated eEF2 decreases and activation of ERK, is also activated. Inhibition of ERK US 2017/002O892 A1 Jan. 26, 2017 20 blocks 5-HT1BR-mediated potentiation by preventing 0189 Western blotting. Performed as previously (Kal CaMK from being able to phosphorylate GluA1Rs. larackal et al., 2013) with commercial antibodies. 0183) One can determine how potentiation of TA-CA 1 0.190 Chronic unpredictable stress and social defeat synapses by 5-HT1BRs is enhanced after GUS. The mag stress is as described in Cai et al. (2013). nitude of 5-HT1BR-induced potentiation at TA-CA1 syn 0191 Chronic restraint stress. Animals are placed in tight apses is greater after CUS and in specific embodiments this fitting restraint tubes and set in cages with normal bedding was because of a low initial strength of excitatory synapses in room light for 4 hrs daily over 10 days (Lim et al., 2012), (Cai et al., 2013). This was confirmed with electrophysiol then returned nightly to their home cages. ogy and biochemistry (Kallarackal et al., 2013). 5HT1BR (0192 alpha5 GABA-NAM L-655,708 and MRK-0106 mediated potentiation is also persistent in slices from CUS (Tocris) are dissolved in DMSO and then diluted in saline animals, unlike controls, and this is mediated by a persistent for injection. phosphorylation of S831 of GluA1Rs. In specific embodi 0193 Sucrose preference and novelty suppressed feeding ments, phosphorylation of S845 in GluR1 subunits iss tests were as described in Cai et al. (2013). critical and. One can compare changes in S845 phosphory 0194 Social interaction test. Test rats are first placed lation in control and CUS tissue. Administration of corti alone for 3 min in an arena with a small holding pen at one costerone is necessary and Sufficient to mimic both of these end empty, then removed for 60 min and placed back in the consequences of CUS. arena with a novel juvenile rat in the holding pen. 0184 One can determine why potentiation of TA-CA1 0.195 Video recordings are made of the position of the rat synapses by 5-HT1BRs is absent after chronic SSRI treat in the arena and then analyzed post-hoc to compute an ment. Long-term SSRI treatment abolishes 5-HT1BR-in interaction ratio, defined as the ratio of the time the rat duced potentiation (Cai et al., 2013). There is no evidence spends in an interaction Zone near the holding pen with the that chronic fluoxetine treatment in non-CUS animals juvenile present and absent (Berton et al., 2006). Multiple caused upregulation of AMPARs or persistent increases in SITs are performed >5 days apart. synaptic strength, indicating that the lack of serotonin 0196. Sexual conditioned place preference. Rats are first responses in animals chronically treated with SSRis is not given a pretest, 3 weeks of conditioning, and then a prefer due to prior maximization of synaptic strength. One can ence test (Camacho et al., 2004). In the test sessions, each rat study changes in the activation of various second messenger is placed in the middle compartment, and the time spent in pathways for evidence of disruptions that can explain the 2 side compartments, each with unique visual cues and results. LTP is absent in naive rats given fluoxetine, Sug bedding, is measured over 30 min to determine side pref gesting that CaMK activation may be impaired. erence. Three times per week for 3 weeks, males are then 0185. One can determine whether activation of placed in the preferred compartment for 30 min directly 5-HT1BRS and potentiation of glutamatergic synapses are from their home cage with a receptive female (ovariecto necessary for the behavioral therapeutic effects of ADs. mized and given 10mg estradiol benzoate and 2mg proges SSRIs cannot reverse stress-induced anhedonia if terone, 48 and 4h before tests) until ejaculation. Latency to 5-HT1BRs are blocked pharmacologically or knocked out. the first mount and to intromission are scored. 48 hrs after Furthermore, SSRIs cannot reverse anhedonia in GluA1 the last copulation, preference is tested as above. S831A mice, implicating serotonin-mediated potentiation. 0.197 Tube test. Rats are housed as pairs and receive 2 S831A transgenic mice, but not 5-HT1BR knock-out mice, training sessions in which they are placed at one end of alm were anhedonic even in the absence of CUS, Supporting a long, 5cm diameter tube, with a food reward at the far end. model of depression in which cognitive and emotional Ten times per day for 5 days, they are placed at opposite disturbances result from a dysfunction of excitatory Syn ends of the tube. The rat that comes out at the opposite end apses, rather than a dysfunction of serotonergic signaling of the tube is declared the winner. A score is assigned to each (Cai et al., 2013; Kallarackal et al., 2013). rat in the pair based on the number of aggregate wins. Only 0186 GENERAL METHODS. All studies are per pairs in which one rat wins-75% of trials are considered to formed, and all data are analyzed, with the experimenter have a sufficiently strong dominance to test the effects of blinded to the drug treatment, genotype, or behavioral CRS. protocol used. Unless otherwise noted, male Sprague-Daw 0198 Urine scent marking. Rat pairs are placed in oppo ley rats of 4-6 weeks old are used. site sides of a cage divided by a screen with sheets of filter 0187 Electrophysiology in vitro. Performed as in Kal paper underneath. Dried sheets are stained with ninhydrin, larackal et al. (2013). For AMPA:NMDA ratios, Mg2+-free which stains amines in urine, and photographed. The num saline is used to produce a robust NMDAR-mediated ber and area of the marks are quantified using Image.J response. AMPA responses are quantified as the initial rising (Lehmann et al., 2013). phase of the response (FIG. 10A). NMDA responses are 0199 Forced swim test. Rats are placed in a 50cm determined after application of DNQX (50 uM). diameter tank filled with room temperature water. The 0188 Electrophysiology in vivo. Tungsten microelec duration of immobility during the last 4 min of the 6-min test trodes (tip impedances=0.5M.S2) are implanted stereotaxi is scored (Maeng et al., 2008). cally in str. pyramidale of area CA1, layer II of the EC, layer 0200. Sucrose preference test. Rats are administered a V of the medial PFC, or the shell of the NAc and secured to sucrose preference test before chronic unpredictable stress the skull with cement. After implantation, rats will be or no stress. The left bar of the pairs (black) reflects CUS rats habituated to stay still under light restraint (a metal pole and the right bar of the pairs (white) reflects unstressed rats. connecting the headstage to a rigid arm) in a narrow tube. After 4 weeks, mean Sucrose preference following 4 weeks On-going electrical activity will be recorded in the absence of CUS+vehicle injection was significantly lower than the of anesthesia. Field potentials will be analyzed using Spike2 unstressed group, indicating anhedonia, (FIG. 8. 3x2 software. repeated-measures ANOVA group-time interaction F(2.18) US 2017/002O892 A1 Jan. 26, 2017
=12.309, p=0.006: *p-0.05 vs all other groups, Bonferroni containing GABA receptors are useful as rapidly acting and test) N=5 CUS rats and N=7 unstressed rats. After another clinically viable antidepressant compounds. week of stress, rats treated with L-655,708 24 hours earlier had a restored Sucrose preference, indicating antidepressant Introduction action. Unstressed rats did not have significantly different 0204 The present standard of care for treating major sucrose preference after 4 or 5 weeks of no stress. depressive disorder is to increase the concentration of mono 0201 MRK-016 rapidly reverses loss of sucrose prefer amine neurotransmitters by inhibiting their reuptake with ence and social interaction behaviors after chronic restraint medications such as selective serotonin reuptake inhibitors stress (FIG. 9). After two weeks of chronic restraint stress, (SSRIs). SSRIs are fully effective in only half of depressed rats had a reduction in Sucrose preference, which was not patients (Gaynes and Warden, 2009), however. In addition, reversed after vehicle treatment (F(3.33)—20.63, p<0.0001, the 3-8 week latency to achieve a therapeutic effect com n=12 rats, p<0.05 compared to pre-CUS baseline, Tukey's plicates optimization of medication and delays symptomatic post-hoc test). 24 hours after treatment with MRK-016, relief. More effective and faster acting therapies are clearly Sucrose preference was not significantly different than the needed to reduce the socioeconomic burden of this debili baseline before stress exposure, and this effect persisted for tating condition. 7 days. Similarly, Social interaction scores decreased sig (0205 The discovery that inhibitors of NMDA-type glu nificantly after chronic stress and vehicle treatment (F=(2. tamate receptors (NMDARs), such as ketamine, exert a 14)=11.84, p=0.0009, n=8 rats). *, p<0.05 compared to rapid antidepressant action (Trullas and Skolnick, 1990; pre-CUS baseline, Tukey's post-hoc test). However, 24 Berman et al., 2000; Zarate et al., 2006) has triggered a hours after MRK-016 (3 mg/kg) treatment social interaction reevaluation of the causes of depression and potential targets scores were not significantly different than the pre-stress for antidepressants. Unfortunately, there are concerns that condition, indicating rapid antidepressant action. This per ketamine's addictive and psychotomimetic properties will sisted for 7 days despite further stress exposure. and in the hinder it from reaching its potential to treat human depres same animals after an additional week of CRS and 7 days sion (Machado-Vieira et al., 2009). Ketamine also produces after the injection of MRK-016. a rapid antidepressant-like behavioral response in rodents subjected to chronic stress (Maeng et al., 2008: Autry et al., Example 3 2011: Li et al., 2010; 2011). This has provided critical mechanistic insights into how rapid antidepressant actions 0202 Alpha5-selective negative allosteric modulators of may be produced and raised hopes that new drugs can be GABAA receptors exert a rapid antidepressant action and developed that target the same effector mechanisms, without restore stress-induced impairment of excitatory synaptic ketamine's disadvantages. strength 0206. A common element linking the therapeutic actions of antidepressants, including SSRIs (Cai et al., 2013), ket Overview amine (Pittenger and Duman, 2008; Kavalali and Monteg gia, 2012; Abdallah et al., 2014), and scopolamine (Voleti et 0203 Selective serotonin reuptake inhibitors (SSRIs) are al., 2013), is their shared effects on excitatory synapses in the first line of pharmacological treatment for depression, cortico-mesolimbic reward circuits. Chronic stress produces but SSRIs are effective in only half of patients and typically depression-like changes in behavior as well as deleterious take several weeks to relieve symptoms. The NMDA recep effects on excitatory synaptic structure and function in tor antagonist ketamine exerts a rapid antidepressant action, multiple brain regions that are associated with cognition, but has troubling side-effects. In the present disclosure it was reward, and mood, including the hippocampus (McEwen, considered that partial inverse benzodiazepine agonists 2000; Kallarackal et al., 2013), prefrontal cortex (PFC) would exert similar effects on brain activity as ketamine, but (Yuen et al., 2012; Pittenger and Duman, 2008), and nucleus would not exert as many side effects if targeted only to accumbens (NAc) (Lim et al., 2012). Conversely, ketamine, GABA receptors containing alpha5 subunits, which are serotonin, and SSRIs promote excitatory synaptic transmis enriched in the hippocampus and prefrontal cortex. In the sion and reverse the effects of chronic stress in these same present disclosure it is shown that the alpha5-selective areas (Pittenger and Duman, 2008: Li et al., 2010; Autry et inverse agonist L-655,708 reversed the alterations in hedo al., 2011; Cai et al., 2013). nic behavior in the Sucrose preference and Social interaction 0207 Although the mechanisms underlying ketamine's tests produced by two different chronic stress paradigms in rapid antidepressant action remain under active investiga rats within 24 hrs of systemic administration. L-655,708 had tion, one hypothesis is that it suppresses NMDAR-mediated no effect on hedonic behavior in unstressed animals. L-655, excitation of inhibitory interneurons (Moghaddam et al., 708 injection also restored within 24 hrs the strength of 1997: Farber et al., 1998: Homayoun and Moghaddam, pathologically weakened excitatory synaptic transmission at 2007: Abdallah et al., 2014; cf. however: Autry et al., 2011). the stress-sensitive temporoammonic-CA1 synapse, mea This results in a mild disinhibition of the neuronal popula sured electrophysiologically, and increased levels of GluA1 tion. Presumably because of this increase in network activ subunit of the AMPA receptor, measured with Western ity, a brief period of ketamine administration triggers several blotting. In specific embodiments, the ability of L-655,708 activity-dependent processes. Such as induction of long-term to restore excitatory synaptic strength rapidly underlies its potentiation, increased expression of the immediate early ability to restore stress-induced behavioral alterations rap gene deltaFosB, and altered activity of the mTOR and/or idly, Supporting evidence that dysfunction of multiple excit eEF2 signaling pathways. This ultimately leads to the rapid atory synapses in cortico-mesolimbic reward pathways con induction of synapse-related genes, increased synthesis of tributes, in part, to the genesis of depression. Partial inverse synaptic proteins (Li et al., 2010; Autry et al., 2011), agonists of the benzodiazepine receptor site of C5 subunit restoration of normal synaptic strength, and persistent ame US 2017/002O892 A1 Jan. 26, 2017 22 lioration of depressive signs and symptoms. These results 0213 Social interaction test (SIT). As described previ indicate that it is ultimately the mild increase in activity in ously (Berton et al., 2006), rats were placed in a plastic forebrain neural circuits that is the key mechanism via which enclosure (82x82 cm) with a 5x5 grid of squares (16.4x 16.4 ketamine exerts its rapid therapeutic antidepressant actions. cm) visible underneath the clear plastic flooring. A Small 0208 Based on this consideration, the inventors sought to translucent perforated plastic box (20x16 cm) was placed identify other compounds that could trigger mild increases against the center of one wall of the arena. During the testing in neuronal activity, like ketamine. Partial inverse agonists at conditions white overhead fluorescent lighting was used to the benzodiazepine site of y-aminobutyric acid type-A illuminate the arena. A video camera positioned 170 cm receptors (GABARs) are negative allosteric modulators above the floor of the arena was used to track the movements that have been shown to promote coherent network activity of the rats during the experiment. (Hajos et al., 2004). GABARs containing the C5 subunit 0214. Each test consisted of a “target-absent trial fol are exclusively expressed by prefrontal cortical neurons and lowed by a “target-present trial. During testing, an indi hippocampal pyramidal cells, offering the means for selec vidual test rat was placed in the center of the arena and, after tively targeting cortical inputs to mesolimbic circuits, 30s of adaptation, the rat’s movements were recorded with thereby minimizing psychotomimetic and sedative side the video camera for 2.5 minutes. After the “target-absent effects. L-655,708 is one such partial inverse agonist of trial, the test rat was removed from the arena and placed GABAR benzodiazepine sites with a 10-100 fold selectiv back in its home cage. For the “target-present trial, a novel ity for GABARs containing the alpha5 subunit (Atack et juvenile (3-6 weeks old; always younger than the rat being al., 2005; 2006). alpha5-selective inverse agonists of the tested) rat was placed in the internal box at one end of the benzodiazepine site are not epileptogenic, hallucinogenic, or arena and, after three minutes, the test rat was returned to the anxiogenic in humans (Atack et al., 2009). center of the arena and filmed for 2.5 minutes. Both rats 0209. It was considered that L-655,708 would exert a were removed and the testing arena and internal box were rapid antidepressant action via convergence on ketamine's cleaned with 70% ethanol before the next rat was tested. No downstream effector mechanisms, and therefore predicted that a single treatment of L-655,708 would rapidly (<24 hrs) test rat ever encountered the same target rat more than once. reverse chronic stress-induced changes in both hedonic 0215 Results were quantified as the percentage of time behavior and excitatory synaptic transmission at the arche spent in the “interaction Zone' (the five grid squares imme typical stress-sensitive synapse between temporoammonic diately surrounding the plastic cage) as measured for both (TA) afferents and the distal dendrites of CA1 pyramidal the “target-absent” and “target-present trials. These per cells. centages were then used to calculate an interaction ratio, which is the percent time spent in the “interaction Zone' Materials and Methods during the “target-present trial divided by that of the “target-absent”. 0210 Chronic restraint stress (CRS). Male Sprague Daw ley rats (45 weeks-old; Harlan Laboratories) were placed in 0216 Acute slice electrophysiology. Standard methods appropriately sized restraint tubes, highly restricting their were used to prepare 400-um-thick transverse hippocampal movement for periods of four hours in a brightly lit lab, slices. Dissection and recording saline contained: 124 mM every day for ten days during the light phase of their diurnal NaCl, 3 mM KC1, 1.25 mM NaH2PO, 1.5 mM MgSO, 2.5 cycle (Lim et al., 2012). mM CaCl2, 26 mM NaHCO, and 10 mM glucose, bubbled 0211 Chronic unpredictable stress (CUS). Male with 95% O2/5% CO. Slices were then transferred to a Sprague-Dawley rats (45 weeks-old; Harlan Laboratories) Submersion-type recording chamber and perfused at 0.5 2 were randomly divided into control and CUS groups. Rats in ml/min at 20-22° C. Picrotoxin (100 uM) and CGP52432 (2 the CUS group were individually housed. CUS animals were uM) were included to block GABA and GABA receptors, treated with two mild stressors every day for 5-6 weeks and the CA3 region was removed with a scalpel. Because during their light phase (Willner et al., 1987). The stressors TA-CA1 synapses are electrotonically remote from CA1 cell were randomly cycled to enhance unpredictability. The Somata, extracellular recording of local field excitatory stressors were: forced Swim, in which rats were placed in a postsynaptic potentials (fEPSPs) was used. Recording basin containing cold water for 5 minutes; Strobe lighting, in pipettes (3-5 MSG2) contained extracellular saline and were otherwise total darkness for 30 minutes; restraint, during placed in Stratum lacunosum-moleculare (SLM) to record which rats were put into appropriately sized restraint tubes TA-CA1 responses. fBPSPs were amplified 1000x, filtered for 30 minutes; exposure to white noise for 30 minutes; and at 3 kHz, and digitized at 10 kHz. Concentric bipolar food or water deprivation for 14 hours spanning the dark tungsten electrodes were placed >500 um from the stimu phase. lating electrodes in SLM for TA afferents. Stimuli (100 us) 0212 Sucrose preference test (SPT). Rats were given a were delivered at 0.05 HZ. choice between two bottles containing tap water or a 1% 0217. As in previous work (Kallarackal et al., 2013), Sucrose solution (Rygula et al., 2006). Testing was con Mg2+-free saline was used to produce a robust NMDAR ducted overnight (16 hours), including the full duration of mediated component of the fBPSP for quantification of their dark phase. Animals were first trained with both bottles AMPAR-mediated synaptic excitation. 3-5 consecutive while group-housed. For Subsequent tests, including during responses were averaged and fFPSP slope was calculated the baseline period, animals were individually housed. over a 1-3 ms window. For AMPAR-mediated responses, the Sucrose preference was calculated as amount of the Sucrose window was fixed in the initial rising phase of the response, Solution consumed as a percentage of the total amount of 2-5 ms after its initiation. For NMDA responses, DNQX (50 liquid consumed. For determining the effects of L-655,708. uM) was bath applied for 15 min and the slope was calcu only unstressed rats that demonstrated a Sucrose preference lated over a 3-5 ms window in the rising phase of the >75% at baseline were used. response, 5-10 ms after its initiation. US 2017/002O892 A1 Jan. 26, 2017
0218 Responses were elicited over a range of stimulation sures ANOVA: F(1,11)=7.514, p=0.019) (FIG. 6B). These intensities before and after application of DNQX. Responses behavioral changes are consistent with previous descriptions were first compared across slices by normalizing them to the of chronic SSRIs (Cai et al., 2013) and acute ketamine (Li amplitude of the fiber volley. The linear portion of the et al., 2011). relationship between response slope and FV amplitude was 0223) In order to determine if the rapid reversal of fit with a straight line and the slopes of the fitted lines were stress-induced changes in behavior by L-655,708 was spe compared across conditions, as has been done previously cific to the CRS procedures, these experiments were (Kallarackal et al., 2013). The inventors also normalized the repeated using another chronic psychological stress para slope of AMPAR-mediated responses to the slope of the digm, chronic unpredictable stress (CUS) (Willner et al., NMDAR-mediated response, choosing traces that had a 1987). Social interaction tests were conducted at three fiber volley closest to 0.2 mV in amplitude. sequential time points (FIG. 7): before CUS (baseline), after 0219 Western blotting. SLM tissue punches (1 mm 4 weeks of CUS and an injection of the vehicle solution 24 diameter) were dissected from area CA1 in hippocampal hrs earlier, and after an additional week of CUS and an slices and pooled (3-4 punches/sample). Membranes were injection of L-655,708 24 hrs earlier (0.7 mg/kg, i.p.). The probed with antibodies directed against GluA1 (Millipore mean Social interaction ratio was significantly lower in rats Bioscience Research Reagents) and B-actin (Cell Signaling subjected to CUS that were given a vehicle injection com Technology). Levels of proteins are expressed as the ratio of pared to both baseline and unstressed rats (3x2 repeated intensity normalized to B-actin intensity, as done previously measures ANOVA group-time interaction: F(2.50)=6.538, (Kallarackal et al., 2013). p=0.005; n=18 CUS, 9 unstressed rats) (FIG. 10A), consis 0220 Drugs/stats. L-655,708 was purchased from Tocris tent with previous descriptions of stress-induced behavioral Bioscience (R&D Systems, Minneapolis, Minn.) and pre changes (Berton et al., 2006). 24 hrs after a single injection pared at a concentration of 5 mM in 75% saline/25% of L-655,708, social interaction in 14 of 18 CUS rats were DMSO. The dose used in this study (0.7 mg/kg i.p.) was reversed to levels that were not significantly different than taken from the study of Martin et al (2009), in which pre-stress baseline or responses in unstressed animals (FIG. L-655,708 was shown to reverse the amnestic properties of 7B, black symbols). The four CUS rats injected with L-655, the anesthetic etomidate; a positive sign of its efficacy in 708 that failed to display an increase in social interaction brain. This dose is sufficient to occupy about 70% of appeared largely resilient to the CUS procedures (FIG. 7B, forebrain binding sites (Atack et al., 2005) gray symbols). 0221) All quantification and analyses of behavioral, elec 0224 L-655,708 had no significant effect on the time rats trophysiological, and Western blotting results were per spent in the interaction Zone in the target absent condition formed with the experimenter blinded to the condition of the after either CUS (F(2,23)=1.484, p=0.248) or CRS (F(1,10) animal, tissue, or protein sample. Data were first compared =0.971, p=0.348), as well as in unstressed animals, indicat with an analysis of variance, followed by pairwise post-hoc ing that it did not induce lasting, nonspecific effects on comparison tests using SPSS software. locomotor behavior that could account for its effects on the social interaction ratio (FIG. 12). Exemplary Results 0225 Unstressed rats displayed no change in either Sucrose preference or social interaction 24 hrs after injection Rapid Reversal of Stress-Induced Changes in Hedonic with either L-655,708 or saline (FIGS. 7C and 7D). Behavior by L-655,708 0226 Taken together, these data show that L-655,708 0222 Several hedonic behaviors in rodents are sensitive reversed chronic stress-induced depressive-like behavioral to chronic stress and are restored by chronic, but not acute, changes in less than 24 hrs in a model-independent manner. administration of SSRIs, providing one means to Screen for L-655,708 administration produced no change in the behav potential antidepressant compounds. First, it was determined ior of unstressed animals in these tests. whether L-655,708 would rapidly (<24 hrs) restore normal 0227 Rapid reversal of stress-induced weakening of hedonic behavior following chronic restraint stress (Wa excitatory synaptic transmission by L-655,708 tanabe et al., 1992; Lim et al., 2012), consistent with the 0228 Chronic stress induces a reduction in excitatory rapid antidepressant-like actions of ketamine (Li et al., synaptic transmission in many cortico-mesolimbic nuclei. 2011). Baseline measurements were first made of social including TA-CA1 synapses in the hippocampus (Kal interaction and Sucrose preference, and then 4-5 week old larackal et al., 2013). Chronic, but not acute, administration rats were subjected to ten days of CRS. At the culmination of fluoxetine restores their strength with a time course that of CRS treatment, rats were injected with either vehicle is comparable with behavioral restoration (Cai et al., 2013). (75% saline, 25% DMSO; i.p.) or L-655,708 (0.7 mg/kg, Therefore, TA-CA1 synaptic transmission was examined to i.p.). Social interaction and Sucrose preference tests were ask whether L-655,708 would reverse a stress-induced syn then repeated 24 hrs post-injection. Chronic restraint stress aptic phenotype as rapidly as it reversed stress-induced produced a decrease in Social interaction ratio in vehicle behavioral changes. injected CRS rats compared to all other groups, whereas the 0229. The inventors used extracellular electrophysiologi social interaction ratio in the L-655,708 injected rats was not cal recording offEPSPs at TA-CA1 synapses in SLM of area significantly different than baseline (F(1,12)=2.85, p=0.115; CA1 in brain slices prepared from unstressed rats, rats n=7 vehicle-injected, 7 L-655,708 injected) (FIG. 6A). subjected to ten days of CRS and given a vehicle injection Similarly, there was a significant decrease in Sucrose pref 24 hrs earlier, and rats subjected to ten days of CRS and erence in rats that received a vehicle injection compared to given an L-655,708 injection 24 hrs earlier (0.7 mg/kg, i.p.). their pre-stress baseline, whereas stressed rats administered These were the same rats used for the behavioral analyses in L-655,708 showed levels of sucrose preference that were not FIG. 6. fEPSPs were recorded in ACSF lacking added Mg2+ different from their pre-stress baseline (2x2 repeated-mea so as to unblock NMDA receptors. The slope was first US 2017/002O892 A1 Jan. 26, 2017 24 quantified of the AMPAR component of the fEPSP elicited tive inverse benzodiazepine agonist L-655,708 thus displays over a range of Stimulation intensities as a function of the antidepressant efficacy at behavioral, electrophysiological, amplitude of the fiber volley (FV), a measure of the number and molecular endpoints. of synapses activated. DNQX was then applied to block AMPAR-mediated transmission and elicited a series of Significance of Certain Embodiments NMDAR-mediated responses over the same range of stimu 0236. It is described herein that L-655,708, a partial lation intensities, as in a previous study (Kallarackal et al., inverse agonist at the benzodiazepine site of the GABAR 2013). that is highly selective for receptors containing alpha5 0230. There was a decrease in the slope of the AMPAR Subunits, reverses the behavioral and synaptic phenotypes mediated component of the fBPSP across all stimulation produced by two different chronic stress paradigms within intensities in slices from rats subjected to CRS and given a 24 hrs of a single systemic administration. vehicle injection, compared to slices from unstressed rats 0237. The motivated, appetitive behaviors assayed in the and slices from rats subjected to CRS and given an L-655, Social interaction and Sucrose preference tests assay the 708 injection (One-way ANOVA: F(2,17), 3.675, p=0.047: hedonic properties of two distinct stimuli, food and sociality. n=5 unstressed, 8 CRS+vehicle, 7 CRS+L-655,708) (FIGS. In both the Sucrose preference and Social interaction tests, 10A and 10B). There was no corresponding difference in the rats must respond in an active manner to display the high slope of the NMDA component of the fBPSP under these levels of Sucrose preference and social interaction that are conditions (F(2,17), 0.549, p=0.588) (FIGS. 10A and 10B). normally observed. Chronic stress is known to decrease the An AMPA:NMDA ratio was computed for each slice using rewarding properties of a variety of natural and unnatural the slope of the response elicited when the FV was ca. 0.2 stimuli in rodents, including their high preference for pal mV in amplitude before and after application of DNQX. The atable sucrose-containing solution (Pothion et al., 2004) and AMPA:NMDA ratio was significantly higher in slices from the naturally rewarding value of social interactions (Berton CRS rats injected with L-655,708 24 hrs earlier, compared et al., 2006). The loss of the rewarding properties of these to vehicle-treated animals subjected to CRS (p<0.05 LSD stimuli is considered to be analogous to anhedonia, or the post-hoc), and was not different than responses in tissue inability to experience reward, a core symptom of human from unstressed rats (One-way ANOVA: F(2,17), 4.345, depression. Importantly, normal behavior in both the Sucrose p=0.03) (FIG. 10C). preference test (Rygula et al., 2006) and social interaction 0231. These results replicate the earlier observations that test (Berton et al., 2006) are restored with chronic, but not AMPAR-mediated signaling, but not NMDAR-mediated acute, administration of SSRIs, thus providing a strong signaling, is impaired by chronic stress at TA-CA1 synapses predictive indication of the potential human antidepressant and it is now demonstrated that this impairment can be efficacy of L-655,078 or other alpha5-selective inverse ago reversed rapidly by treatment with L-655,708. The behav nists of the benzodiazepine site. ioral antidepressant efficacy of L-655,708 is associated with 0238 alpha5-containing GABA receptors as an antide a restoration of excitatory neurotransmission at this stress pressant drug target. GABA receptors are heteropentameric sensitive synapse. ion channels, most commonly containing alpha, beta, and 0232 Rapid reversal of stress-induced downregulation of gamma Subunits in a 2:2:1 stoichiometry (Sieghart and GluA1 expression by L-655,708 Sperk, 2002). The interface between a gamma2 subunit and 0233 Restoration of stress-impaired AMPAR-mediated an C. Subunit forms the benzodiazepine receptor, a site of transmission produced by treatment with either chronic allosteric modulation that alters channel gating and SSRIs (Kallarackal et al., 2013) or ketamine (Liet al., 2011) GABAR function. In addition to benzodiazepine agonists is associated with an increase in the expression of the GluA1 (positive modulators), there are inverse agonists that act at subunit of the AMPAR. It was considered whether L-655, the benzodiazepine site to decrease GABA receptor func 708 would also restore GluA1 expression as rapidly as it tion (negative modulators). The molecular identity of the a reverses the stress-induced behavioral and synaptic pheno subunit in the benzodiazepine receptor site determines its types. pharmacological profile. GABARS containing al Subunits 0234 SLM tissue was harvested from slices taken from mediate the sedative and amnestic effects of benzodiaz unstressed rats, rats Subjected to ten days of CRS and given epines preferentially, whereas GABARS containing C2 and a vehicle injection 24 hrs earlier, and rats Subjected to ten O3 subunits mediate their anxiolytic effects (Rudolph et al., days of CRS and given an injection of L-655,708 24 hrs 1999); Mohler et al., 2002). Partial inverse agonists with low earlier (0.7 mg/kg, i.p.). These were the same rats used for affinity for these receptor subtypes offer the potential advan FIG. 6. As reported previously for CUS, rats subjected to tage of a wider therapeutic concentration range and a lower CRS and injected with vehicle displayed a significantly likelihood of producing negative side effects, such as anxiety lower level of GluA1 protein in SLM as compared to or epileptiform discharge. unstressed control rats (Kruskal-Wallis H test: 202)=10.62, 0239 O.5-subunit mRNA is abundant in pyramidal cells p=0.0049; n=6 unstressed rats, 6 CRS+ vehicle, 8 CRS+L- in the hippocampus and deep layers of the neocortex, and 655,708) (FIG. 11). In contrast, stressed rats injected with C.5-containing GABARS are localized at Synaptic and L-655,708 24 hrs earlier displayed levels of GluA1 in that extrasynaptic sites in the dendrites (Fritschy and Mohler, were not significantly different than levels in unstressed 1995; Wainwright et al., 2000; Serwanski et al., 2006). controls. C.5-containing GABARs mediate tonic inhibition (Carais 0235. These results demonstrate that the stress-induced cos et al., 2004) and lower the excitability of pyramidal cells decrease in GluA1 protein expression, which is correlated (Bonin et al., 2006), so negative allosteric modulators of with both weakened excitatory synaptic transmission at C.5-containing GABARS should promote coherent activity TA-CA1 synapses and altered hedonic behaviors, is reversed (Hajos et al., 2004; Towers et al., 2004). Drugs targeting rapidly by treatment with L-655,708. The C5 subunit-selec these receptors are thus an attractive means to selectively US 2017/002O892 A1 Jan. 26, 2017
alter activity within cortico-mesolimbic circuits without naling, activation of mTOR signaling, and protein synthesis altering activity in other circuits. Indeed, deletion of the C.5 (Autry et al., 2011: Li et al., 2010; 2011). An increase in gene alters learning in hippocampal-dependent tasks but not coherent circuit activity is also likely to promote the induc hippocampal-independent ones (Collinson et al., 2002). tion of long-term potentiation (e.g. Huerta and Lisman, Interestingly, alpha5 Subunits are up-regulated in mice after 1993). All of these activity-dependent processes have been chronic stress (Matsumoto et al., 2007), Suggesting a role for shown to strengthen excitatory synapses. Indeed, L-655,708 the alpha5 subunit in the cognitive deficits associated with administration triggers a rapid increase in GluA1 protein chronic stress and depression, in particular embodiments. levels in the distal dendrites of CA1 pyramidal cells, coin 0240 Mechanisms of L-655,708 action. The observation cident with an increase in AMPAR-mediated synaptic exci that L-655,708 rapidly restores pathologically weakened tation. Interestingly, chronic, but not acute, administration of AMPAR-mediated excitatory transmission at TA-CA1 syn SSRIs also promotes GluA1 protein levels in this same apses of the hippocampus indicates a likely mechanism by region (Kallarackal et al., 2013), which is densely innervated which L-655,708 exerts its antidepressant efficacy in behav with serotonergic terminals. ioral testing. SSRIs (Cai et al., 2013), ketamine (Autry et al., 2011; Li et al., 2010), and L-655,708 all enhance excitatory 0243 Therapeutic potential. Because disinhibition pro synaptic strength at multiple sites within the cortico-me motes induction of long-term potentiation, the presumptive Solimbic reward circuitry, thereby providing strong Support cellular basis of memory, partial inverse agonists of for an association between the restoration of excitatory GABARs containing C5 subunits have been developed as synaptic strength and the reversal of depressive behavioral cognitive enhancers (Ballard et al., 2009; Quirk et al., 1996; phenotypes (Duman, 2014). TA-CA1 synapses are not Atack et al., 2006: 2009). Unlike non-selective benzodiaz uniquely sensitive to stress or responsive to antidepressants, epine inverse agonists, these compounds are not anxiogenic, but they do serve as a convenient archetype of stress hallucinogenic, or epileptogenic in humans (Atack et al., induced changes that are likely occurring at many synapses 2009). in the cortico-mesolimbic reward circuitry (Lim et al., 2012: Yuen et al., 2012). The net effect of this weakening is likely 0244 Although SSRIs are reasonably safe and well tol to be a dysfunction in processing of rewarding stimuli which erated, and are not addictive, they are effective in only a may underlie common symptoms of depression, Such as Subset of patients and only after a delay of weeks-to-months. anhedonia (Nestler and Carlezon, 2006). Restoration of the Ketamine, on the other hand, exerts a rapid antidepressant strength of these synapses by effective antidepressants action in the majority of patients, but its therapeutic viability should restore normal responses to rewarding stimuli (Belu is extremely limited to its dissociative and anesthetic prop jon and Grace, 2014). erties, as well as its potential for abuse and overdose. 0241 Acute administration of ketamine improves mood L-655,708 reversed stress-induced behavioral changes and and reduces depressive symptoms within 1-2 hours in restored AMPAR-mediated excitatory synaptic strength in humans and these effects persist for up to two weeks. two well-validated rodent models of antidepressant efficacy, Similarly, ketamine restores Sucrose preference and novelty like SSRIs and ketamine. Because these effects were Suppressed feeding behaviors rapidly (24 hrs) in chronically induced within 24 hrs of a single systemic injection, in stressed animals (Li et al., 2011). Ketamine's therapeutic particular embodiments partial inverse agonists of benzodi effects are induced during the brief period (ca. 1-2 hours) azepine receptors acting on alpha5-subunit containing when it is present in the brain at Sufficient concentrations to inhibit NMDARs, triggering relief of symptoms that persist GABARS represent a novel, rapidly acting, effective, and for days after it is cleared from the body. During this clinically viable treatment for human depression. induction phase, ketamine may preferentially reduce exci 0245 Although the present invention and its advantages tation of GABAergic inhibitory interneurons (Dwyer and have been described in detail, it should be understood that Duman, 2013; Farber et al., 1998; Homayoun and Moghad various changes, Substitutions and alterations can be made dam, 2007), thereby producing a mild disinhibition of the herein without departing from the spirit and scope of the neuronal population and increased activity in the hippocam invention as defined by the appended claims. Moreover, the pus, entorhinal cortex, NAc, and PFC, as observed in Scope of the present application is not intended to be limited rodents (e.g. Kittelberger et al., 2012; Middleton et al., 2008: to the particular embodiments of the process, machine, Hunt et al., 2011) and in humans (Cornwell et al., 2012: manufacture, composition of matter, means, methods and Driesen et al., 2013). This activity is accompanied by a steps described in the specification. As one of ordinary skill neurochemically detectable Surge of glutamate release in the in the art will readily appreciate from the disclosure of the PFC and NAc (Lorrain et al., 2003: Moghaddam et al., 1997: present invention, processes, machines, manufacture, com Razoux et al., 2007). C.5-selective benzodiazepine inverse positions of matter, means, methods, or steps, presently agonists also produce mild disinhibition through their nega existing or later to be developed that perform substantially tive allosteric actions, and should therefore also trigger an the same function or achieve Substantially the same result as increased coherence in oscillatory activity, as has been the corresponding embodiments described herein may be observed following injection of nonselective benzodiaz utilized according to the present invention. Accordingly, the epine inverse agonists (Hajos et al., 2004). appended claims are intended to include within their scope 0242. In specific embodiments, the ability of both L-655, Such processes, machines, manufacture, compositions of 708 and ketamine to promote activity accounts for their matter, means, methods, or steps. Moreover, the scope of the shared antidepressant efficacy because Such activity present application is not intended to be limited to the strengthens excitatory synapses via convergence onto com particular embodiments of the process, machine, manufac mon activity-dependent signaling pathways. Potential activ ture, composition of matter, means, methods and steps ity-dependent processes include an increase in BDNF sig described in the specification. US 2017/002O892 A1 Jan. 26, 2017 26
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0307 Trullas R, Skolnick P (1990). Functional antago 5. The method of claim 1, wherein the depression-related nists at the NMDA receptor complex exhibit antidepressant disorder is major depressive disorder (MDD); dysthymia; actions. Eur J Pharmacol 18: 1-10. cyclothymic disorder; seasonal affective disorder/seasonal 0308 Yuen E. Y. Wei J, Liu W. Zhong P. Li X, Zhen Y depression; depression after traumatic brain injury; postpar (2012). Repeated stress causes cognitive impairment by tum depression; premenstrual dysphoric disorder; depres Suppressing glutamate receptor expression and function in sive symptoms associated with menopause; depression fol prefrontal cortex. Neuron 73: 962-977. lowing substance abuse? withdrawal; bipolar disorder; 0309 Voleti B, Navarria A, Liu RJ, Banasr M, Li N, bipolar disorder in remission; or depressive episodes of Terwilliger R, et al (2013). Scopolamine rapidly increases bipolar disorder. mammalian target of rapamycin complex 1 signaling, Syn 6. The method of claim 1, wherein the anxiety-related aptogenesis, and antidepressant behavioral responses. Biol disorder is general anxiety disorder, obsessive compulsive Psychiatry 74: 742-749. disorder: Impulse control disorder; anxiousness associated 0310 Wafford K A (2005) GABAA receptor subtypes: with depression; repeated episodes of anxiety, extreme any clues to the mechanism of benzodiazepine dependence? apprehension or fear of Social interaction (Social phobia); Curr Opin Pharmacol. 5: 47-52. panic disorders; posttraumatic stress syndrome or posttrau 0311 Wainwright A, Sirinathsinghji DJ, Oliver K R matic stress disorder; or separation anxiety disorder. (2000) Expression of GABA(A) receptor alpha5 subunit 7. The method of claim 1, wherein the attention-related like immunoreactivity in human hippocampus. Brain Res disorder is attention deficit hyperactive disorder; or adult Mol Brain Res. 80: 228-232. attention deficit hyperactive disorder. 8. The method of claim 1, wherein the psychosis-related 0312 Watanabe Y. Gould E. McEwen BS (1992) Stress disorder is schizophrenia, Schizophrenia-spectrum disorder induces atrophy of apical dendrites of hippocampal CA3 or psychotic depressive illness. pyramidal neurons. Brain Res 588: 341-345. 9. The method of claim 1, wherein the eating disorder is 0313 Willner P. Towell A, Sampson D, Sophokleous S, anorexia nervosa, bulimia; or obesity. Muscat R (1987). Reduction of sucrose preference by 10. The method of claim 1, wherein the individual has chronic unpredictable mild stress, and its restoration by a avoidant personality disorder, antisocial personality disor tricyclic antidepressant. Psychopharmacol (Berl) 93: 358 der; borderline personality disorder; conduct disorder; 364. dependent personality disorder; depressive personality dis 0314. Zarate CA, Singh J B, Carlson PJ, Brutsche NE, order; histrionic personality disorder, narcissistic personal Ameli R. Luckenbaugh DA et al (2006). A randomized trial ity disorder; negativistic personality disorder, obsessive of an N-methyl-D-aspartate antagonist in treatment-resistant compulsive personality disorder, paranoid personality major depression. Arch Gen Psychiatry 63: 856-864. disorder, Schizoid personality disorder; or Schizotypal per 0315 Zarate CA, et al. (2012) Replication of ketamine's Sonality disorder. antidepressant efficacy In bipolar depression: a controlled 11. The method of claim 1, wherein the individual has add-on trial. Bioi Psychiatry. 71:939-946. been diagnosed with the medical condition. 1. A method of treating or preventing or ameliorating at 12. The method of claim 1, wherein the individual has not least one symptom of a medical condition in an individual, been diagnosed with the medical condition. comprising the step of providing to the individual a thera 13. (canceled) peutically effective amount of one or more negative modu 14. (canceled) lators of GABA receptors, wherein the medical condition is 15. (canceled) selected from the group consisting of a depression-related 16. (canceled) disorder; an anxiety-related disorder; an attention-related 17. (canceled) disorder; a psychosis-related disorder, an eating disorder, a 18. The method of claim 1, wherein the negative modu personality disorder, cognitive impairment following trau lator of GABA receptor is a partial inverse agonist of a matic brain injury; neuropathic pain; chronic muscle or bone GABA receptor comprising an O.5 subunit. pain; diabetic complications resulting in nerve injury; gen 19. The method of claim 18, wherein the partial inverse eralized attack of muscular weakness; recurring sleep epi agonist of a GABA receptor comprising an C5 subunit is sodes during the day; migraine; addiction; Suicidality; and a L-655,708, RO4938581, CP-457,920, MRK-016, or a com combination thereof. bination thereof. 2. The method of claim 1, wherein the onset of amelio 20. The method of claim 19, wherein the partial inverse ration of one or more depression-related symptoms occurs agonist is L-655,708. within hours, days, or weeks. 21. (canceled) 3. The method of claim 1, wherein the GABA receptor 22. (canceled) comprises an alpha5 subunit. 23. (canceled) 4. The method of claim 1, wherein the negative modulator 24. The method of claim 1, further comprising the step of is selected from the group consisting of a negative allosteric providing to the individual a therapeutically effective modulators acting at the benzodiazepine binding site; nega amount of another therapy. tive allosteric modulators acting at the barbiturate steroid 25. The method of claim 24, wherein the other therapy is binding site; negative allosteric modulators acting at the selected from the group consisting of monoamine oxidase neuroactive steroid binding site; competitive antagonists of inhibitors (MAOis), selective serotonin reuptake inhibitors the GABAR: negative modulators acting in the pore of the (SSRis), serotonin-norepinephrine reuptake inhibitors (SN GABAR channel; nicotinamide and related compounds; Ris), norepinephrine reuptake inhibitors (NRIs), triple inverse agonists and antagonists of the propofol binding site reuptake inhibitors, modulators of CNS acetylcholine func of the GABAR; and a combination thereof. tion, stimulants, anti-glucocorticoids, antagonists of US 2017/002O892 A1 Jan. 26, 2017 29
NMDA-type glutamate receptors, tricylic antidepressants (TCAs), and a combination thereof. 26. The method of claim 24, wherein the other therapy is an anti-depressant.