(12) Patent Application Publication (10) Pub. No.: US 2011/00284.18 A1 Parker Et Al

Home , Cicutoxin, Oenanthotoxin

US 2011 002841 8A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2011/00284.18 A1 Parker et al. (43) Pub. Date: Feb. 3, 2011

(54) USE OF GABBA RECEPTOR ANTAGONISTS Publication Classification FOR THE TREATMENT OF EXCESSIVE SLEEPINESS AND DISORDERS ASSOCATED (51) Int. Cl. WITH EXCESSIVE SLEEPINESS A63L/7028 (2006.01) A 6LX 3/557 (2006.01) (75) Inventors: Kathy P. Parker, Rochester, NY A63L/335 (2006.01) (US); David B. Rye, Dunwoody, A63L/4355 (2006.01) GA (US); Andrew Jenkins, A63L/047 (2006.01) Decatur, GA (US) A6IP 25/00 (2006.01) Correspondence Address: (52) U.S. Cl...... 514/29: 514/220; 514/450, 514/291; FISH & RICHARDSON P.C. (AT) 5147738 P.O BOX 1022 Minneapolis, MN 55440-1022 (US) (57) ABSTRACT (73) Assignee: Emory University, Atlanta, GA GABA receptor mediated hypersomnia can be treated by (US) administering a GABA receptor antagonist (e.g., flumazenil; clarithromycin; picrotoxin; bicuculline; cicutoxin; and (21) Appl. No.: 12/922,044 oenanthotoxin). In some embodiments, the GABA receptor antagonist is flumazenil or clarithromycin. The GABA (22) PCT Filed: Mar. 12, 2009 receptor mediated hypersomnia includes shift work sleep disorder, obstructive sleep apnea/hypopnea syndrome, narco (86). PCT No.: PCT/USO9/37034 lepsy, excessive sleepiness, hypersomnia (e.g., idiopathic hypersomnia; recurrent hyperSonmia; endozepine related S371 (c)(1), recurrent stupor; and amphetamine resistant hyperSonmia), (2), (4) Date: Sep. 10, 2010 and excessive sleepiness associated with shift work sleep disorder, obstructive sleep apnea/hypopnea syndrome, and Related U.S. Application Data hypersomnia (e.g., idiopathic hypersomnia; recurrent hyper (60) Provisional application No. 61/036,047, filed on Mar. Somnia; endozepine related recurrent stupor; and amphet 12, 2008. amine resistant hypersomnia.

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USE OF GABBA RECEPTOR ANTAGONSTS methods comprising administering to the Subject an effective FOR THE TREATMENT OF EXCESSIVE amount of a GABA receptor antagonist. In addition, pro SLEEPINESS AND DISORDERS ASSOCATED vided herein is a method of treating excessive sleepiness WITH EXCESSIVE SLEEPINESS associated with GABA receptor mediated hypersomnia in a Subject, comprising administering to the Subject an effective CROSS-REFERENCE TO RELATED amount of a GABA receptor antagonist. In some embodi APPLICATIONS ments, the GABA receptor mediated hypersomnia is 0001. This application claims priority under 35 U.S.C. selected from one or more of: shift work sleep disorder; S119 to U.S. Provisional Application Ser. No. 61/036,047, narcolepsy; obstructive sleep apnea/hypopnea syndrome; filed Mar. 12, 2008, incorporated by reference in its entirety REM behavior disorder; frontal nocturnal dystonia; restless herein. legs syndrome; nocturnal movement disorder, Kleine-Levin syndrome; Parkinson's disease; excessive sleepiness; hyper TECHNICAL FIELD Somnia; idiopathic hypersomnia; recurrent hypersomnia; endozepine related recurrent stupor; and amphetamine resis 0002 The present disclosure relates to the treatment of tant hypersomnia. In some embodiments, the GABA recep excessive sleepiness and promotion of wakefulness in a Sub tor mediated hypersomnia is a result of the production of ject. In particular, a method of treating hypersomnia (e.g., endogenous Somnogenic compounds in a Subject, e.g., exces GABA receptor mediated hypersomnia) using a GABA sive amounts of Somnogenic compounds. In some embodi receptor antagonist Such as flumazenil (formulated, for ments, the GABA receptor antagonist can be a negative example, for I.V., transdermal, transmucosal, Sublingual, or allosteric modulator. In some embodiments, the GABA Subdermal administration) is disclosed. receptor antagonist is selected from the group consisting of flumazenil; clarithromycin; picrotoxin; bicuculline; cicu BACKGROUND toxin; and oenanthotoxin. In some embodiments, the method 0003. There are two main categories of hypersomnia: pri includes administering an I.V., transdermal, transmucosal, mary hypersomnia (sometimes called idiopathic hyperSom sublingual, or subdermal formulation of the GABA receptor nia) and recurrent hypersomnia (sometimes called idiopathic antagonist to the Subject. recurrent hypersomnia). Both are characterized by similar 0008 Also provided herein is a method of treating exces signs and symptoms and differ only in the frequency and sive sleepiness in a Subject. The method comprises the steps regularity with which the symptoms occur. of determining whether the subject has an endogenously pro 0004 Primary hypersomnia is characterized by excessive duced somnogenic compound in a CSF sample of the Subject, daytime sleepiness over a long period of time. The symptoms e.g., an excessive amount of the Somnogenic compound; and are presentall, or nearly all, of the time. Recurring hyperSom administering to the Subject an effective amount of a GABA nia involves periods of excessive daytime sleepiness that can receptor antagonist, e.g., flumazenil. The step of determining last from one to many days, and recur over the course of a year whether the Subject has an endogenously produced somno or more. The primary difference between this and primary genic compound, including an excessive amount of the Som hypersomnia is that persons experiencing recurring hyper nogenic compound, includes the steps of: a) measuring the somnia will have prolonged periods where they do not exhibit potentiation of GABA receptors contacted with the CSF any signs of hypersomnia, whereas persons experiencing pri sample of the Subject in a whole cell patch clamp assay, mary hypersomnia are affected by it nearly all the time. Idio wherein the cells express benzodiazepine sensitive receptors; pathic hypersomnia is much like narcolepsy, except there is b) measuring the potentiation of GABA receptors contacted no cataplexy, no sleep paralysis, and no rapid eye movement with the CSF sample of the subject in a whole cell patch when the victim first falls asleep. clamp assay, wherein the cells express benzodiazepine insen 0005 Various treatments including prescription drugs sitive receptors; and c) comparing the response of step a) to have been used to treat hypersomnia without significant suc the response of step b), wherein a persistence of potentiation cess, and no substantial body of evidence Supports the effec in step b) to within +25% of the step a) response is indicative tiveness of any of these treatments. Stimulants are not gener of an endogenously produced somnogenic compound in the ally recommended to treat hypersomnia as they treat the CSF sample of the subject. In some embodiments, the som symptoms but not the base problem. There is a need for more nogenic compound is a non-classical benzodiazepine. In effective treatments of hypersomnia, especially using admin Some embodiments, the Somnogenic compound binds to a site istration routes that allow for better drug delivery and patient on the GABA receptor, e.g., an allosteric site. In some compliance. embodiments, the site on the GABA receptor is other than the benzodiazepine binding site. SUMMARY 0009. A method of treating excessive sleepiness of a sub 0006. The inventors have discovered that many patients ject endogenously producing a Somnogenic compound, e.g., that Suffer from excessive sleepiness or disorders associated an excessive amount of a Somnogenic compound, is also with excessive sleepiness have one or more endogenous Sub provided herein, the method comprising administering to the stances present, typically in excess, in their CSF that act as Subject an effective amount of a GABA receptor antagonist, positive allosteric modulators of the GABA receptor, poten e.g., flumazenil. Further described herein is a method of tiating the effect of GABA on the receptor. Treatment of such determining whether a subject will benefit from treatment patients with a GABA receptor antagonist thus can provide with a GABA receptor antagonist, e.g., flumazenil, wherein a method to treat the disorders, in particular the symptoms of the benefit is a reduction in excessive sleepiness, the method excessive sleepiness associated with the disorders. comprising determining whether the Subject has an endog 0007 Accordingly, provided herein are methods of treat enously produced somnogenic compound, e.g., an excess of ing GABA receptor mediated hypersomnia in a Subject, the the Somnogenic compound, in a CSF sample of the Subject, US 2011/002841 8 A1 Feb. 3, 2011 wherein the presence of the endogenously produced somno tering a wakefulness promoting agent (e.g., modafinil and genic compound is indicative that the subject will benefit armodafinil). In some embodiments, the wakefulness pro from treatment with GABA receptor antagonist, e.g., fluma moting agent is modafinil. In some embodiments, the method Zenil. In the method, the determining step comprises: a) mea comprises administering a time-release formulation of a Suring the potentiation of GABA receptors contacted with GABA receptor antagonist, such as a time-release transder the CSF sample of the subject in a whole cell patch clamp mal formulation. assay, wherein the cells express benzodiazepine sensitive 0017. Further provided herein is a method of treating a receptors; b) measuring the potentiation of GABA receptors GABA receptor mediated hypersomnia in a Subject, the contacted with the CSF sample of the subject in a whole cell method comprising: a) administering to the Subject a Sublin patch clamp assay, wherein the cells express benzodiazepine gual formulation of a GABA receptor antagonist, e.g., flu insensitive receptors; and c) comparing the response of step a) mazenil; and b) administering to the Subject a wakefulness to the response of step b), wherein a persistence of potentia promoting agent. In some embodiments, the method com tion to within +25% of the step a) response is indicative that prises: a) administering flumazenil in an amount of about 2 the subject will benefit from treatment with flumazenil. mg of flumazenil per Body Mass Index unit of the subject per 0010. In some embodiments of the methods described 24 hour period; and b) administering to the Subject a wake herein, the GABA receptor antagonist is a negative allosteric fulness promoting agent. Also provided is a method of treat modulator. In some embodiments, the GABA receptor ing a GABA receptor mediated hypersomnia in a Subject, the antagonist is selected from the group consisting of flumaze method comprising: a) administering to the Subject a GABA nil; clarithromycin; picrotoxin; bicuculline; cicutoxin; and receptor antagonist, e.g., flumazenil, using a subdermal oenanthotoxin. In some embodiments, the GABA receptor pump; and b) administering to the Subject a wakefulness antagonist is flumazenil. In some embodiments, the GABA promoting agent. In some embodiments, a method of treating receptor antagonist is clarithromycin. a GABA receptor mediated hypersomnia in a subject is 0011 Further provided herein are methods of treating dis provided, the method comprising: a) administering to the orders associated with excessive sleepiness (e.g., GABA subject an I.V. formulation of flumazenil in an amount of receptor mediated hypersomnia) and symptoms of excessive about 0.2 mg to about 2 mg; and b) administering to the sleepiness in a Subject. In some embodiments, the method Subject a wakefulness promoting agent. In some embodi includes administering an I.V., transdermal, transmucosal, ments, the methods described above further comprise admin sublingual, or subdermal formulation of a GABA receptor istration of a transdermal formulation of a GABA receptor antagonist, e.g., selected from flumazenil; clarithromycin; antagonist, e.g., flumazenil. picrotoxin; bicuculline, cicutoxin; and oenanthotoxin to the 0018. A method of treating a disorder associated with Subject. excessive sleepiness in a subject is provided, the method 0012. A disorder associated with excessive sleepiness can comprising administering a GABA receptor antagonist, e.g., be selected from one or more of: shift work sleep disorder; flumazenil, in an amount effective to decrease the subject's narcolepsy; obstructive sleep apnea/hypopnea syndrome; CSF-induced enhancement of whole cell patch clamp hypersomnia; REM behavior disorder; frontal nocturnal dys assayed GABAR responses in the presence of GABA such tonia; restless legs syndrome; nocturnal movement disorder; that the responses in the presence of GABA are within +25% Kleine-Levin Syndrome; and Parkinson's disease. In some of a control sample. In some embodiments, a method of embodiments, the disorder is hypersomnia, for example treating a disorder associated with excessive sleepiness in a GABA receptor mediated hypersomnia (e.g., idiopathic Subject is provided, the method comprising administering a hypersomnia; recurrent hypersomnia; endozepine related GABA receptor antagonist, e.g., flumazenil, in an amount recurrent stupor; and amphetamine resistant hypersomnia). effective to modulate the response of a CSF sample of the 0013. A method of treating a disorder associated with Subject as measured in a GABA whole cell patch clamp assay excessive sleepiness in a subject is provided, the method to within +25% of the response of a control sample. In some comprising administering to the Subject an effective amount embodiments, the modulation is a decrease in the response of of a transmucosal, transdermal, or I.V. formulation of a the CSF sample of the subject in the presence of a GABA GABA receptor antagonist, e.g., flumazenil. In some receptor antagonist, e.g., flumazenil. embodiments, treating a disorder associated with excessive 0019. A method of testing a subject for the presence of a sleepiness can include administering an effective amount of a positive allosteric modulator of GABA receptor function in GABA receptor antagonist, e.g., flumazenil, using a Subder a CSF or blood sample is also provided, the method compris mal pump. ing measuring the response of GABA Receptors contacted 0014. In some embodiments, a transmucosal formulation with the CSF or blood and with GABA in a whole cell patch of a GABA receptor antagonist, e.g., flumazenil, is admin clamp assay, and comparing the response to a control sample, istered. The transdermal formulation can be administered whereina greater than 50% increase in the response relative to Supralingually, Sublingually, or buccally. the control is indicative of the presence of a positive allosteric 0015. In some embodiments, the subject is administered modulator of GABA receptor function. about 2 mg flumazenil per Body Mass Index unit of the 0020. Also provided herein are methods of treating shift subject over a 24 hour period. Administration may be self work sleep disorder, obstructive sleep apnea/hypopnea Syn administered by the patient as needed, or in the case of an I.V. drome, and narcolepsy in a Subject, the methods comprising or subdermal route of administration, the flumazenil can be administering to the Subject an effective amount of a GABA administered automatically. In some embodiments, the effec receptor antagonist, e.g., flumazenil. A method of treating tive amount of flumazenil is about 6 mg per dose six times per excessive sleepiness associated with shift work sleep disor day. der, obstructive sleep apnea/hypopnea syndrome, hyperSom 0016 Independent of the formulation and route of admin nia (e.g., idiopathic hypersomnia; recurrent hypersomnia; istration, any of the methods may further comprise adminis endozepine related recurrent stupor; and amphetamine resis US 2011/002841 8 A1 Feb. 3, 2011 tant hypersomnia), or narcolepsy in a Subject is also provided, hypersomnia; recurrent hypersomnia; endozepine related the method comprising administering to the Subject an effec recurrent stupor; and amphetamine resistant hypersomnia. In tive amount of a GABA receptorantagonist, e.g., flumazenil. Some embodiments, a GABA receptor antagonist Such as In some embodiments, the a GABA receptor antagonist is an flumazenil is formulated for administration by a transdermal, I.V. formulation, a transdermal formulation, or a transmu transmucosal, or intravenous route for the uses described cosal formulation. herein. 0021. A method of altering a somnolent state of a subject 0026. The details of one or more embodiments of the is further provided herein, the method comprising adminis invention are set forth in the accompanying drawings and the tering to the subject an effective amount of a GABA receptor description below. Other features, objects, and advantages of antagonist, e.g., flumazenil. The Somnolent state is selected the invention will be apparent from the description and draw from one or more of narcolepsy, obstructive sleep apnea/ ings, and from the claims. hypopnea syndrome, shift work sleep disorder, and hyper Somnia (e.g., idiopathic hypersomnia; recurrent hyperSom DESCRIPTION OF DRAWINGS nia; endozepine related recurrent stupor; and amphetamine resistant hypersomnia). In some embodiments, the a GABA 0027 FIG. 1 illustrates whole cell patch clamp recordings receptor antagonist is an I.V. formulation, a transdermal for of GABAR function with and without flumazenil. mulation, or a transmucosal formulation. 0028 FIG.2 shows that human C.132y2s GABA receptor 0022. Also provided herein are methods for enhancing function is enhanced by the plasma of a Subject suffering from alertness or increasing regularity of sleep rhythms in a Sub hypersomnia. Enhancement is reduced following administra ject, promoting wakefulness in a Subject; improving cogni tion of 12 mg of a Sublingual formulation of flumazenil. tive dysfunction in a Subject; and restoring a normal sleep 0029 FIG. 3 is a graph illustrating that potentiation of pattern and improving the quality of psychosocial life and GABA function is evident in human controls absent sleep relationships in a subject, each method comprising adminis related complaints, non-human (rhesus) primates, and in tering to the subject an effective amount of a GABA receptor excess in many hypersomnic patients. antagonist, e.g., flumazenil. In some embodiments, the a 0030 FIG. 4 shows the power spectrum analyses results GABA receptor antagonist is an I.V. formulation, a transder obtained from processing 27 minutes of non-artifactual data mal formulation, or a transmucosal formulation. for subject DS122 after infusion with 2.0 mg flumazenil. 0023. A method of characterizing the phenotypic spec 0031 FIG. 5 shows the power spectrum analyses results trum of GABA receptor mediated hypersomnia is also pro obtained from processing 19 minutes of non-artifactual data vided, the method comprising measuring the potentiation of for subject DT74 after infusion with 2.0 mg flumazenil. GABA receptor function of a CSF or plasma sample of at 0032 FIG. 6 shows a histogram displaying the results of least one Subject having a disorder associated with excessive the psychomotor vigilance task (PVT) performance before sleepiness, and correlating the potentiation with at least one administration of I.V. flumazenil for case 74. measure of sleep or sleepiness of the Subject, wherein a posi 0033 FIG. 7 shows a histogram displaying the results of tive correlation is indicative that the subject's disorder is the psychomotor vigilance task (PVT) performance after 2.0 within the phenotypic spectrum of a GABA receptor medi mg dose of I.V. flumazenil for case 74. ated hypersomnia. In some embodiments, the measure of 0034 FIG. 8 shows a histogram displaying the results of sleep and sleepiness is a behavioral assessment, an electro the psychomotor vigilance task (PVT) performance before encephalographic assessment, or a subjective assessment. administration of I.V. flumazenil for case 102. The method can further comprise quantifying GABA recep 0035 FIG. 9 shows a histogram displaying the results of tor function. the psychomotor vigilance task (PVT) performance after 2.0 0024. Further provided herein are uses of a GABA recep mg dose of I.V. flumazenil for case 102. torantagonist Such as flumazenil for the manufacture of medi caments for the treatment of the following disorders and 0036 FIG. 10 shows a histogram displaying the results of conditions: obstructive sleep apnea/hypopnea syndrome; the psychomotor vigilance task (PVT) performance before shift work sleep disorder, narcolepsy; hypersomnia; and administration of I.V. flumazenil for case 122. excessive sleepiness associated with shift work sleep disor 0037 FIG. 11 shows a histogram displaying the results of der, obstructive sleep apnea/hypopnea syndrome, hyperSom the psychomotor vigilance task (PVT) performance after 2.0 nia, or narcolepsy. In some embodiments, the hypersomnia is mg dose of I.V. flumazenil for case 122. selected from one or more of idiopathic hypersomnia; recur 0038 FIG. 12 shows a histogram displaying the results of rent hypersomnia; endozepine related recurrent stupor; and the psychomotor vigilance task (PVT) performance before amphetamine resistant hypersomnia. administration of I.V. flumazenil for case 124. 0025. Also provided herein are uses of a GABA receptor 0039 FIG. 13 shows a histogram displaying the results of antagonist Such as flumazenil for the manufacture of medica the psychomotor vigilance task (PVT) performance after 1.2 ments for altering a Somnolent state of a subject; enhancing mg dose of I.V. flumazenil for case 124. alertness or increasing regularity of sleep rhythms in a Sub 0040 FIG. 14 shows a graph displaying the results of the ject, promoting wakefulness in a Subject; improving cogni psychomotor vigilance task (PVT) performance before and tive dysfunction in a Subject; and restoring a normal sleep after treatment with I.V. flumazenil for case 74. pattern and improving the quality of psychosocial life and 0041 FIG. 15 shows a graph displaying the results of the relationships in a Subject. In some embodiments, the Somno psychomotor vigilance task (PVT) performance before and lent state is selected from one or more of narcolepsy; obstruc after treatment with I.V. flumazenil for case 102. tive sleep apnea/hypopnea syndrome; shift work sleep disor 0042 FIG. 16 shows a graph displaying the results of the der; and hypersomnia. In some embodiments, the psychomotor vigilance task (PVT) performance before and hypersomnia is selected from one or more of idiopathic after treatment with I.V. flumazenil for case 122. US 2011/002841 8 A1 Feb. 3, 2011

0043 FIG. 17 shows a graph displaying the results of the 0050 GABA receptor mediated hypersomnia or disor psychomotor vigilance task (PVT) performance before and ders associated with excessive sleepiness are selected from after treatment with I.V. flumazenil for case 124. one or more of shift work sleep disorder, narcolepsy; 0044 FIG. 18a is an illustration of the rest-activity cycle obstructive sleep apnea/hypopnea syndrome; REM behavior of patient AS99 before treatment with flumazenil. disorder; frontal nocturnal dystonia; restless legs syndrome; 0045 FIG. 18b is an illustration of the rest-activity cycle nocturnal movement disorder; Kleine-Levin syndrome; Par of patient AS99 after treatment with flumazenil. kinson's disease; excessive sleepiness; hypersomnia; idio 0046 FIG. 19a illustrates a whole cell patch clamp record pathic hypersomnia; recurrent hypersomnia; endozepine ing from a cell expressing human C.132y2S receptors. Bars related recurrent stupor; and amphetamine resistant hyper above the traces indicate duration of GABA and CSF appli Somnia. In some embodiments, the GABA receptor medi cation. ated hypersomnia is selected from idiopathic hypersomnia; recurrent hypersomnia; endozepine related recurrent stupor; 0047 FIG. 19billustrates a whole cellpatch clamp record and amphetamine resistant hypersomnia. In some embodi ing from a cell expressing the benzodiazepine insensitive ments, the hypersomnia is idiopathic hypersomnia. In some subunit C.1 (H102R). Bars above the traces indicate duration embodiments, the hypersomnia is endozepine related recur of GABA and CSF application. rent stupor. In some embodiments, the hypersomnia is amphetamine resistant hypersomnia. DETAILED DESCRIPTION 0051. Such disorders can be characterized by many objec tive and subjective tests known in the art. For example, the 0048 Ionotropic GABA receptors (GABAR) are the Epworth Sleepiness Scale; the Stanford Sleepiness Scale; the most recognized therapeutic targets for anesthetics and seda Pittsburgh Sleep Quality Index; an Activity-Rest and Symp tive/hypnotic drugs. Mutations in the C.1, y2, and delta Sub tom Diary; Actigraphy: Psychomotor Vigilance Task; Poly units of GABA Raccount for several of the heritable epilep Somnography; Functional Magnetic Resonance Imaging; sies, endogenous positive allosteric neurosteroid modulators Profile of Mood States; Functional Outcomes of Sleep Ques contribute to fluctuations in mood due to developmental tionnaire; Medical Outcomes Study Short-Form 36; and Neu changes in expression of the C-46 GABAR, and mutation of rophysical Testing, such as the Cambridge Neurophysical the B3 subunit has been associated with chronic insomnia. The inventors have found that a naturally occurring endog Test Automated Battery (CANTAB) (e.g., physcomotor enous, positive, allosteric modulator of recombinant C.1, 32. speed, attention, working memory, and executive function). Y2 short splice variant GABAR is present in CSF plasma in 0052. In addition, GABA receptor mediated hypersom normal humans and non-human primates, and when present nia can be characterized by demonstration of enhanced in excess, produces hypersomnia and excessive daytime GABA Receptor function of a subject's CSF or plasma as sleepiness, or GABA receptor mediated hypersomnia compared to a control, e.g., see Example 1 and Example 14. (GRH) as described herein. Accordingly, treatment of such patients with a GABA receptor antagonist thus can provide II. Methods of Promoting Wakefulness and a method to treat patients having various disorders associated Enhancing Alertness in Sleepiness Associated with excessive sleepiness, and in particular treat the Symp Disorders toms of excessive sleepiness associated with the various dis 0053. Further provided herein are methods of treating orders. GABA mediated hypersomnia disorders, including shift work sleep disorder, obstructive sleep apnea/hypopnea Syn I. Methods of Treating GABA Receptor Mediated drome, narcolepsy, and excessive sleepiness associated with Hypersomnia and Disorders Associated with shift work sleep disorder, obstructive sleep apnea/hypopnea Excessive Sleepiness syndrome, hypersomnia, and narcolepsy. In some embodi ments, the GABA mediated hypersomnia is idiopathic 0049 Provided herein are methods of treating GABA hypersomnia; recurrent hypersomnia; endozepine related receptor mediated hypersomnia in a subject, the methods recurrent stupor; or amphetamine resistant hypersomnia. The comprising administering to the Subject an effective amount method comprises administering to the Subject an effective of a GABA receptor antagonist. In addition, provided herein amount of a GABA receptor antagonist, such as flumazenil. is a method of treating excessive sleepiness associated with In some embodiments, the GABA receptor antagonist is an GABA receptor mediated hypersomnia in a subject, com I.V. formulation, a transdermal formulation, or a transmu prising administering to the Subject an effective amount of a cosal formulation. GABA receptor antagonist. In some embodiments of the 0054. A method of altering a somnolent state of a subject methods described herein, the GABA receptor antagonist is is further provided herein, the method comprising adminis a negative allosteric modulator. In some embodiments, the tering to the subject an effective amount of GABA receptor GABA receptor antagonist is selected from the group con antagonist, e.g., flumazenil. The Somnolent state is selected sisting of flumazenil; clarithromycin; picrotoxin; bicu from one or more of narcolepsy, obstructive sleep apnea/ culline; cicutoxin; and oenanthotoxin. In some embodiments, hypopnea syndrome, shift work sleep disorder, and hyper the GABA receptor antagonist is flumazenil. In some Somnia (e.g., idiopathic hypersomnia; recurrent hyperSom embodiments, the GABA receptor antagonist is clarithro nia; endoZepine related recurrent stupor; and amphetamine mycin. In some embodiments, the method includes adminis resistant hypersomnia). In some embodiments, the GABA tering a I.V., transdermal, transmucosal, Sublingual, or Sub receptor antagonist is an I.V. formulation, a transdermal for dermal formulation of flumazenil to the subject. The mulation, or a transmucosal formulation. administration of flumazenil can be combined with adminis 0055 Also provided herein are methods for enhancing tration of other agents, including wakefulness promoting alertness or increasing regularity of sleep rhythms in a Sub agents and transdermal formulations of flumazenil. ject, promoting wakefulness in a Subject; improving cogni US 2011/002841 8 A1 Feb. 3, 2011

tive dysfunction in a Subject; and restoring a normal sleep 0060. In some embodiments, the formulation is a hard, pattern and improving the quality of psychosocial life and compressed, rapidly dissolving tablet adapted for direct Sub relationships in a subject, each method comprising adminis lingual dosing. The tablet includes particles made of the tering to the subject an effective amount of GABA receptor antagonist and a protective material. In some embodiments, antagonist, e.g., flumazenil. In some embodiments, the these particles are provided in an amount of between about GABA receptor antagonist is an I.V. formulation, a transder 0.01 and about 75% by weight based on the weight of the mal formulation, or a transmucosal formulation. tablet (e.g., 1%. 5%, 10%, 15%, 20%, 25%, 30%, 35%, 45%, 0056. As used herein, the term “promoting wakefulness’ 50%. 60%, 70%, and 75%). In some embodiments, the tablet refers to a decrease in sleepiness, tendency to fall asleep, or may also include a matrix made from a nondirect compres other symptoms of undesired or reduced alertness or con sion filler, a wicking agent, and a hydrophobic lubricant. In sciousness compared with sleepiness, tendency to fall asleep, Some embodiments, the tablet is adapted to dissolve sponta or other symptoms of undesired or reduced alertness or con neously in the mouth of a patient in less than about 60 seconds sciousness expected or observed without treatment. Promot (and, in Some cases, in less than about 30 seconds). ing wakefulness refers to a decrease in any stage of sleep, 0061. In some embodiments, the formulation can be a including light sleep, deeper sleep characterized by the pres compressed rapidly dissolving tablet comprising effervescent ence of high amplitude, low wave brain activity termed “slow agents. These effervescent agents allow enhanced adsorption wave sleep', and rapid eye movement (REM) sleep. of the antagonist across the mucosal membranes (e.g., 0057. A determination of whether the treatment is useful tongue, cheek, and gums) in the oral cavity. An example of in performing the methods described herein can be made, for effervescent pharmaceutical compositions suitable for use in example, by direct observation of behavioral or physiological conjunction with the methods described herein are the com properties of mammalian sleep, by self-reporting, or by Vari positions described in U.S. Pat. No. 6,200,604. ous well-known methods, including electrophysiological 0062. In some embodiments, the antagonist can be admin methods. Such methods include, for example, examining istered transmucosally using an edible film. Such films can electroencephalograph (EEG) activity amplitude and fre include a carrier comprising water-soluble polymers in com quency patterns, examining electromyogram activity, and bination with certain ingredients and provides a therapeutic examining the amount of time during a measurement time effect. In some embodiments, the film is coated and dried period, in which a mammal is awake or exhibits a behavioral utilizing existing coating technology and exhibits instant wet or physiological property characteristic of wakefulness. tability followed by rapid dissolution/disintegration upon 0058. The effectiveness of the treatments can also be char administration in the oral cavity. In some embodiments, an acterized by the objective and subjective tests described edible film can contain as the essential components a water herein, including the Epworth Sleepiness Scale; the Stanford soluble polymer or a combination of water-soluble polymers, Sleepiness Scale; the Pittsburgh Sleep Quality Index; an one or more plasticizers or Surfactants, one or more polyal Activity-Restand Symptom Diary: Actigraphy: Psychomotor cohols, and flumazenil. Non-limiting examples of edible Vigilance Task; Polysomnography; Functional Magnetic films can be found in U.S. Pat. Nos. 5,948,430; 6,177,096; Resonance Imaging; Profile of Mood States; Functional Out 6,284,264; 6,592,887; and 6,709,671. comes of Sleep Questionnaire; Medical Outcomes Study 0063. Further examples of additional pharmaceutical Short-Form 36; and Neurophysical Testing, such as the Cam compositions Suitable for transmucosal administration bridge Neurophysical Test Automated Battery (CANTAB) include those described in U.S. Pat. Nos. 5,178,878; 5,223, (e.g., physcomotor speed, attention, working memory, and 264; and 6,024,981. executive function). 0064. In some embodiments, the antagonist is combined with inactive ingredients. Such ingredients may be necessary, III. Formulation and Administration of AGABA for example, to add bulk to the pharmaceutical preparation, to Receptor Antagonist bind the preparation, to add color or flavor to the preparation, and to prevent degradation or growth of contaminants. 0059 A GABA receptor antagonist can be selected from 0065. In some embodiments, administration of the antago flumazenil; clarithromycin; picrotoxin; bicuculline; cicu nist may be performed using an implantable device, for toxin; and oenanthotoxin and can be formulated for I.V., example, an implantable, self-regulating mechanochemical transdermal, transmucosal, Sublingual, oral, and Subdermal Subdermal pump. In some embodiments, the device may administration for use with the methods described herein. A administer the antagonist on a set dosage program. In some transmucosal formulation can include Sublingual, Supralin embodiments, the device may administer the antagonist on gual, and buccal administration. For transmucosal adminis demand as determined by the Subject. In some embodiments, tration, the antagonist may be combined with one or more the device may administer the antagonist on a constant release inactive ingredients for the preparation of a tablet, packed profile. In some embodiments, the device may administer the powder, edible film Strip, Soft gel capsule, hard gel capsule, antagonist automatically. These devices are known in the art lozenge, or troches. For example, in Some embodiments, the for the treatment of other disorders, for example, diabetes. antagonists such as flumazenil may be combined with at least Non-limiting examples of various embodiments of this mode one excipient such as fillers, binders, humectants, disintegrat of administration are detailed in U.S. Pat. Nos. 5,062,841; ing agents, Solution retarders, absorption accelerators, wet 5,324,518; and 6,852,104. ting agents absorbents, or lubricating agents. According to 0066. In some embodiments, a transmucosal administra Some embodiments, the antagonist may be combined with tion of an antagonist may be combined with transdermal one or more of a polyol (e.g., lactose, Sucrose, mannitol, or administration of the same or another antagonist. Without mixtures thereof), an alcohol (e.g., ethanol), and a gum (e.g., being bound by theory, such a delivery mechanism may be acacia and guar), and then formed into a lozenge by conven useful for nocturnal application to assist the Subject with tional methods. morning wakefulness. US 2011/002841 8 A1 Feb. 3, 2011

0067 Transdermal administration of the antagonist can be (e.g., 10-30 cm). The amount of drug delivered into the skin accomplished by mixing the antagonist with Suitable phar can be controlled by a number of factors including skin patch maceutical carriers, preservatives, optional penetration size, degree of drug loading, the use of rate controlling mem enhancers, and optional gelling agents to form ointments, branes, permeation enhancers, and the like. emulsions, lotions, solutions, creams, gels, patches or the 0075. In some embodiments, the transmucosal and/or the like, wherein a fixed amount of the preparation is applied onto transdermal formulation may be a time-release or slow-re a certain area of skin. lease formulation. In some embodiments, the transdermal 0068. By the term "suitable pharmaceutical carrier' is formulation may be a time-release or slow-release formula meant a non-toxic pharmaceutically acceptable vehicle tion. The transmucosal or transdermal formulation described including, for example, polyethylene glycol, propylene gly herein may also be formulated so as to provide slow or con col, isopropanol, ethanol, oleic acid, N-methylpyrrolidone, trolled release of the antagonist using, for example, hydro sesame oil, olive oil, wood alcohol ointments, Vaseline, and propylmethyl cellulose in varying proportions to provide the paraffin or a mixture thereof. desired release profile, other polymer matrices, gels, perme 0069 Suitable penetration enhancers include, for able membranes, osmotic systems, multilayer coatings, example, Saturated and unsaturated fatty acids and their microparticles, liposomes and/or microspheres. In general, a esters, alcohols, monoglycerides, diethanolamines, N.N- controlled-release preparation is a pharmaceutical composi dimethylamines such as linolenic acid, linolenyl alcohol, tion capable of releasing the active ingredient at the required oleic acid, oleylalcohol, Stearic acid, Stearyl alcohol, palmitic rate to maintain constant pharmacological activity for a desir acid, palmityl alcohol, myristic acid, myristyl alcohol, able period of time. Such dosage forms provide a Supply of a 1-dodecanol, 2-dodecanol, lauric acid, decanol, capric acid, drug to the body during a predetermined period of time and octanol, caprylic acid, 1-dodecylazacycloheptan-2-one sold thus maintain drug levels in the therapeutic range for longer under the trademark AZONE (Nelson Research and Devel periods of time than conventional non-controlled formula opment; Irvine, Calif.), ethyl caprylate, isopropyl myristate, tions. hexamethylene lauramide, hexamethylene palmitate, capryl (0076 U.S. Pat. No. 5,591,767 describes a liquid reservoir alcohol, decyl methylsulfoxide, dimethylsulfoxide, salicylic transdermal patch for the controlled administration of ketoro acid and its derivatives, N,N-diethyl-m-toluamide, crotami lac, a non-steroidal anti-inflammatory agent with potent anal ton, 1-substituted azacycloalkan-2-ones, polyethylene glycol gesic properties. U.S. Pat. No. 5,120.548 discloses a con monolaurate and any other compounds compatible with trolled-release drug delivery device comprised of swellable medetomidine and its optically active enantiomers and the polymers. U.S. Pat. No. 5,073.543 describes controlled-re packages and having transdermal permeation enhancing lease formulations containing a trophic factor entrapped by a activity. ganglioside-liposome vehicle. U.S. Pat. No. 5,639,476 dis 0070 Suitable gelling agents include, for example, closes a stable solid controlled-release formulation having a hydroxy methyl cellulose, hydroxypropyl cellulose sold coating derived from an aqueous dispersion of a hydrophobic under the trademark KLUCEL HF (Hercules Inc.; Wilming acrylic polymer. Biodegradable microparticles are known for ton, Del.), tragacanth, Sodium alginate, gelatin, methylcellu use in controlled-release formulations. U.S. Pat. No. 5,354, lose, Sodium carboxymethylcellulose, and polyvinyl alco 566 discloses a controlled-release powder that contains the hols. Suitable preservatives include, for example, parabens, active ingredient. U.S. Pat. No. 5,733,566 describes the use of benzoic acid, and chlorocresol. polymeric microparticles that release antiparasitic composi 0071 Antioxidants can be included in the formulations tions. described herein. Suitable antioxidants include, for example, 0077. The controlled-release of the active ingredient may ascorbyl palmirate, butylated hydroxyanisole, butylated be stimulated by various inducers, for example, pH, tempera hydroxytoluene, potassium Sorbate, Sodium bisulfate, Sorbic ture, enzymes, water, or other physiological conditions or acid, propyl gallate, and sodium metabisulfite. compounds. Various mechanisms of drug release exist. For 0072. In some embodiments, the antagonist is adminis example, in one embodiment, the controlled-release compo tered by a transdermal patch. Adhesives for making transder nent may swell and form porous openings large enough to mal patches for use in the methods described herein include release the antagonist after administration to a patient. The polyisobutylene, silicone based adhesives, and acrylic poly term "controlled-release component’ means a compound or mers. The adhesive polymers can be mixed with other excipi compounds, such as polymers, polymer matrices, gels, per ents such as waxes and oils (e.g., mineral oil). A protective meable membranes, liposomes and/or microspheres that liner can be placed in contact with the adhesive layer to facilitate the controlled-release of the active ingredient in the protect against drug release from the patch prior to applica pharmaceutical composition. In another embodiment, the tion. Liners for use with the transdermal patches described controlled-release component is biodegradable, induced by herein include, for example, polyethylene terephthalate film, exposure to the aqueous environment, pH, temperature, or polyester membrane, and polycarbonate film. enzymes in the body. 0073. The backing membrane of the transdermal patch for 0078. The specific dose of an antagonist required to obtain use with the methods described herein constitutes the top face therapeutic benefit in the methods of treatment described Surface of the transdermal patch. It may be made of a single herein will, usually be determined by the particular circum layer or film of polymer, or be a laminate of one or more stances of the individual patient including the size, weight, polymer layers and metal foil. Examples of polymers suitable age, and sex of the Subject, the nature and stage of the disorder for use in making backing films include, for example, poly being treated, the aggressiveness of the disorder, and the route ester films, ethyl vinyl acetate, polypropylene, polyethylene, of administration of the compound. and polyvinyl-chloride. 007.9 For transmucosal administration (e.g., sublingual 0.074. In some embodiments, the administration rate of the administration), for example, a daily dosage of flumazenil. drug is 0.1-1000 ugh through a skin area of about 2-90 cm for example, can range from about 0.5 mg to about 10 mg per US 2011/002841 8 A1 Feb. 3, 2011

Body Mass Index (BMI) unit (e.g., about 0.5 mg to about 5 per mL, and about 4 mg to about 8 mg per mL). In some mg; about 1 mg to about 3 mg; about 1.5 mg to about 4 mg. embodiments, the transdermal formulation is formulated to a about 2 mg to about 6 mg; about 1.25 mg to about 8 mg; and concentration of about 4 mg per mL. In some embodiments, about 4 mg to about 10 mg). In some embodiments, a daily the transdermal formulation is administered once daily (e.g., dosage of flumazenil can range from about 1 mg per BMI to before bed). In some embodiments, the transdermal formula about 5 mg per BMI. In some embodiments, a daily dosage of tion is administered at least twice daily. In some embodi flumazenil can be about 1.5 mg per BMI. In some embodi ments, the transdermal formulation is administered about ments, a daily dosage of flumazenil can be about 2 mg per every eight to about twenty-four hours (e.g., about every eight BMI unit. In some embodiments, a daily dosage offlumazenil hours; about every ten hours; about every twelve hours; about can be about 3 mg per BMI unit. For example, a subject with every sixteen hours; about every twenty hours; about every a BMI of 20 could be administered a daily dosage of about 40 twenty-two hours; and about every twenty-four hours). mg of flumazenil, in other words, a daily dosage of 2 mg per I0084. A transdermal formulation may be formulated in a BMI unit. Higher or lower doses are also contemplated, as it unit dosage form, each dosage containing from about 0.5 to may be necessary to use dosages outside these ranges in some about 10 mg of flumazenil per unit dosage (e.g., about 0.5 mg CaSCS. to about 8 mg; about 1 mg to about 5 mg; about 1.5 mg to 0080. The transmucosal formulation can be administered about 4 mg, about 2 mg to about 6 mg; about 3 mg to about 7 in one single dosage or the daily dosage may be divided. Such mg; about 4 mg to about 8 mg, and about 5 mg to about 10 as being divided equally into two to six times per day daily mg). In some embodiments, each dosage can contain about 1 dosing. In some embodiments, the transmucosal formulation to about 4 mg of flumazenil per unit dosage. In some embodi is administered at least twice daily. In some embodiments, the transmucosal formulation is administered at least three times ments, each dosage contains about 2 mg of flumazenil. The daily. In some embodiments, the transmucosal formulation is term “unit dosage form” refers to physically discrete units administered about every one to six hours (e.g., about every Suitable as a unitary dosage for human Subjects and other one hour; about every two hours; about every three hours: mammals, each unit containing a predetermined quantity of about every three and a half hours; about every four hours: active material calculated to produce the desired therapeutic about every five hours; and about every six hours). In some effect, in association with a suitable pharmaceutical excipi embodiments, the transmucosal formulation is administered ent by the Subject as needed, e.g., patient controlled titration to a I0085. The components used to formulate the pharmaceu desired end effect (e.g., wakefulness or reduced sleepiness). tical compositions described above are of high purity and are 0081. A transmucosal formulation may beformulated in a Substantially free of potentially harmful contaminants (e.g., unit dosage form, each dosage containing from about 0.5 to at least National Food grade, generally at least analytical about 20 mg of the antagonist, e.g., flumazenil, per unit dos grade, and more typically at least pharmaceutical grade). age (e.g., about 0.5 mg to about 15 mg; about 1 mg to about 10 Particularly for human consumption, the composition is pref mg; about 1.5 mg to about 8 mg; about 2 mg to about 7 mg: erably manufactured or formulated under Good Manufactur about 3 mg to about 6 mg; about 4 mg to about 8 mg; about 5 ing Practice standards as defined in the applicable regulations mg to about 10 mg; about 6 mg to about 12 mg, and about 8 of the U.S. Food and Drug Administration. For example, mg to about 20 mg). In some embodiments, each dosage can suitable formulations may be sterile and/or substantially iso contain about 5 to about 10 mg of the antagonist per unit tonic and/or in full compliance with all Good Manufacturing dosage. In some embodiments, each dosage contains about 6 Practice regulations of the U.S. Food and Drug Administra mg of the antagonist. The term “unit dosage form” refers to tion. physically discrete units Suitable as a unitary dosage for I0086. The antagonist can be administered in combination human Subjects and other mammals, each unit containing a with other agents. In one embodiment, the antagonist is predetermined quantity of active material calculated to pro administered with a wakefulness promoting agent (e.g., duce the desired therapeutic effect, in association with a Suit modafinil and armodafinil). In some embodiments, the wake able pharmaceutical excipient. fulness promoting agent is modafinil. In some embodiments, 0082 For transdermal administration, for example, a daily the Subject may be resistant to one or more wakefulness dosage of flumazenil can range from about 0.5 mg to about 10 promoting agents prior to administration of the antagonist. mg (e.g., about 0.5 mg to about 5 mg; about 1 mg to about 3 The wakefulness promoting agent can be administered in an mg; about 1.5 mg to about 4 mg; about 2 mg to about 6 mg: amount less than about 600 mg per day (e.g., less than about about 1.25 mg to about 8 mg; and about 4 mg to about 10 mg). 100 mg per day; less than about 200 mg per day; less than In some embodiments, a daily dosage of transdermal fluma about 300 mg per day; less than about 400 mg per day; less Zenil can range from about 1 mg to about 5 mg. In some than about 500 mg per day; and less than about 600 mg per embodiments, a daily dosage of transdermal flumazenil can day). The specific dose of a wakefulness promoting agent be about 1.5 mg. In some embodiments, a daily dosage of required to obtain therapeutic benefit in the methods of treat transdermal flumazenil can be about 2 mg. In some embodi ment described herein will usually be determined by the ments, a daily dosage of transdermal flumazenil can be about particular circumstances of the individual Subject including 3 mg. Higher or lower doses are also contemplated as it may the size, weight, age, and sex of the Subject, the nature and be necessary to use dosages outside these ranges in some stage of the disorder being treated, the aggressiveness of the CaSCS. disorder, and the route of administration of the compound. In 0083. The transdermal formulation can be administered in Some embodiments, the wakefulness promoting agent can be one single dosage or the daily dosage may be divided, such as administered twice daily. In some embodiments, the wake being divided equally into two to six times per day daily fulness promoting agent can be administered in an amount of dosing. In some embodiments the transdermal formulation is 5 mg per BMI unit. In some embodiments, the wakefulness formulated to a concentration of about 0.5 mg to about 10 mg promoting agent can be administered in an amount of 100 mg per mL (e.g., about 0.5 mg to about 8 mg per mL, about 1 mg per dose. In some embodiments, the Subject exhibits resis to about 6 mg per mL, about 1.5 mg to about 5 mg per mL. tance to a wakefulness promoting agent prior to administra about 3 mg to about 7 mg per mL, about 4 mg to about 10 mg tion of the antagonist. In some embodiments, administration US 2011/002841 8 A1 Feb. 3, 2011

of the antagonist can reverse or decrease a subjects resistance 0.095 GABARs are enhanced by many chemicals and to a wakefulness promoting agent. drugs. General anesthetics, as already noted, enhance inhibi 0087. In some embodiments, treatment of a disorder asso tion by making the channels stay open for longer periods of ciated with excessive sleepiness can include the following: time, increasing the duration of inhibition. This is also true for 0088 a) transmucosal, e.g., Sublingual, administration of many neurosteroids (e.g., progesterone metabolites) and for an antagonist, e.g., flumazenil; and ethanol. GABARs are also a critical binding site for benzo 0089 b) administration of awakefulness promoting agent. diazepines, such as Valium. These important anxiolytic and In some embodiments, the treatment can further include: sedative drugs cause the receptors to bind GABA more 0090 c) transdermal administration of an antagonist, e.g., tightly, also enhancing inhibition by the receptor. flumazenil. 0096. It is important to note that all of these compounds do 0091 For example, in some embodiments, a sublingual not activate the channel. They are all “allosteric modulators'. formulation of flumazenil is administered about every 2 to 4 They bind to sites separate from the GABA binding sites and hours during the waking hours of the day (e.g., every about 3 simply enhance or amplify the effect of GABA. In the to 3.5 hours). In some embodiments, a wakefulness promot absence of GABA, physiologic and/or therapeutic concentra ing agent is administered from one to three times during the tions of these different compounds have no effect on the waking hours of the day (e.g., about every 4 hours). In some channel. GABA must be present for them to have an effect. embodiments, the wakefulness promoting agent is modafinil. Similarly, the benzodiazepine antagonist flumazenil is not a In some embodiments, a transdermal or time-release formu GABAR blocker. It occupies the benzodiazepine binding lation of flumazenil is administered once daily (e.g., before site, thus blocking drugs like Valium from acting on the chan bed). nel. Although it is bound to the receptor, flumazenil does not have an effect on the channel. Its functional effect can only be IV. Assay for GABA Receptor Mediated observed when both GABA and a benzodiazepine are present. Hypersomnia (0097. Developed in the late 1970s, the single cell electro physiology method known as patch clamp is a standard for 0092. The GABA receptors are one of several classes of measuring the function of ion channels in research laborato chemically gated ion channels that incorporate the features of ries. The techniques takes advantage of the high electrical both “receptors' and “ion channels' into one membrane pro resistance between a cell Surface and specially constructed tein. These chemically gated channels (ligand gated ion chan microelectrodes, and capacitative feedback electronics which nel: LGIC) can detect extracellular chemical signals such as combine to give ultra low noise (<100 fa) recordings of ions neurotransmitters released from neighboring cells and in flowing through single ion channels. response will open an ion channel to allow specific ions to 0.098 Provided herein is a method of diagnosing and treat enter or leave the cell. When this results in a net movement of ing a patient Suffering from hypersomnia associated with the positive charge into the cell, the cell becomes more electri endogenous production of GABA receptor modulators, e.g., cally positive and thus more excitable. Conversely, when this excessive production of Such modulators. There are many results in a net flow of negative ions into the cell, the neuron reports of hypersomnia disorders in Subjects who do not becomes more electrically negative and thus more inhibited. respond well to conventional stimulant (e.g., amphetamine) In this way, LGICs act as chemical-to-voltage converters and therapies. These subjects may be suffering from a form of are fundamental to cell-to-cell communication and neuronal hypersomnia referred to as amphetamine resistant hyperSom activity. Drugs and chemicals that enhance or block these nia, from an increased production of endozepines (e.g., hemin functions have profound effect on brain circuits and ulti and protoporphyrin IX), or from an increased production of mately human behavior. For example, most general anesthet another substance that binds to the GABA receptor. Without ics render patients unconscious by enhancing the function of being bound by theory, the Subject may be producing endog inhibitory LGICs, the most common of which is the GABA enous benzodiazepines (i.e. "endozepines') or other Somno receptor. genic compound(s) that interact directly or indirectly with the 0093. The most common inhibitory neurotransmitter in benzodiazepine binding site on the GABAR, enhancing the human nervous system is Y-aminobutyric acid or GABA. receptor function as classic benzodiazepines Such as Valium. It is released by neurons at Synapses, the specialized junctions 0099. A method of diagnosing a patient suffering from between 2 neurons that permits rapid cell-to-cell communi GABA mediated hypersomnia associated with increased cation. After leaving the presynaptic neuron and crossing the production of endozepines or other Somnogenic Substance(s) synaptic gap, the molecules of GABA arrive at the postsyn can be performed by measuring the effect of a subjects aptic membrane where they can interact with a LGIC, the cerebral spinal fluid (CSF) or blood or plasma on recombi Type-A GABA receptor (GABAR). After GABA binds to nant GABAR function under whole cell patch clamp condi the receptor, the LGIC changes shape and allows the flow of tions (see, e.g., FIG. 1 and Example 1 or FIGS. 19A and 19B negatively charged chloride ions into the neuron, which and Example 14). In some embodiments, the effect of the CSF results in the neuron becoming inhibited and unable to pass a or blood or plasma can be compared to the effect observed message onto another neuron, until GABA unbinds and the when the CSF or blood or plasma is co-applied with a GABA inhibition passes. receptor antagonist Such as flumazenil. In some embodi 0094. If GABAR function is blocked, then the brain cir ments, application of the antagonist Such as flumazenil can cuits in which they are imbedded experience less inhibition. modulate the response of a CSF or blood sample of a subject This can cause the circuits to become hyper-excitable, exhib as measured in a GABA whole cell patch clamp efficacy assay iting much more excitation than normal. This will result in to within 25% of a control sample response. In some embodi convulsions and seizures if the block is not removed. This can ments, the modulation is a decrease in the response of the CSF occur in the presence of a GABAR channel blocker toxin or sample of the Subject in the presence of the antagonist Such as a GABA antagonist. This can also occur in Some patients who flumazenil. In some embodiments, the effect of the CSF or have inherited forms of epilepsy. In these patients, a blood or plasma in an assay expressing benzodiazepine sen GABAR gene has mutated to make a dysfunctional sitive receptors can be compared to the effect observed of the GABAR that does not function as well as it should. CSF or blood or plasma in an assay expressing benzodiaz US 2011/002841 8 A1 Feb. 3, 2011

epine insensitive receptors. In some embodiments, the Sub cellular medium, a motor-driven solution exchange device; stance in the CSF or blood or plasma sample of a subject and instructions for use of the kit. potentiates the response of GABA as measured in a GABA whole cell patch clamp efficacy assay. In some embodiments, VI. Definitions the potentiation of the GABA response in the benzodiazepine sensitive receptors and the potentiation of the GABA (0103 Unless defined otherwise, all technical and scien response in the benzodiazepine insensitive receptors are tific terms used herein have the same meaning as is commonly within +25% of each other. In some embodiments, the per understood by one of ordinary skill in the art to which this sistence of potentiation within +25% of the GABA responses disclosure belongs. All patents, applications, published appli in benzodiazepine sensitive and insensitive receptor assays is cations, and other publications are incorporated by reference indicative that the subject would benefit from treatment with in their entirety. In the event that there is a plurality of defi a GABA receptor antagonist. In some embodiments, the nitions for a term herein, those in this section prevail unless GABA receptor antagonist is flumazenil. stated otherwise. 0100 Further, a method of diagnosing a patient suffering 0104. As used herein, the singular forms “a,” “an,” and from GABA mediated hypersomnia associated with “the include plural referents unless the context clearly dic increased production of endoZepines or other somnogenic tates otherwise. Substances can be performed by measuring the effect of a 0105. A “subject' can include both mammals and non subjects cerebral spinal fluid (CSF) or blood or plasma on mammals. Mammals include, for example, humans; nonhu recombinant GABAR function under whole cell patch man primates, e.g. apes and monkeys; cattle, horses; sheep; clamp conditions. rats; mice; pigs; and goats. Non mammals include, for example, fish and birds. 0106 The expression “effective amount', when used to V. Kits describe an amount of compound in a method, refers to the amount of a compound that achieves the desired pharmaco 0101 Also provided herein are kits for treating disorders logical effect or other effect, for example an amount that associated with excessive sleepiness. Akit can include an I.V. transdermal, oral, or transmucosal (e.g., Sublingual, Supralin results in reduced sleepiness. gual, and buccal) formulation of a GABA receptor antago 0107 The terms “treating and “treatment’ mean causing nist. In some embodiments, the GABA receptorantagonist is atherapeutically beneficial effect, Such as ameliorating exist flumazenil. In some embodiments, the kit can further includes ing symptoms, preventing additional symptoms, ameliorat one or more of a Wakefulness promoting agent (e.g., modafi ing or preventing the underlying metabolic causes of symp nil) and a transdermal formulation of a GABA receptor toms, postponing or preventing the further development of a antagonist. In some embodiments, a kit can include one or disorder and/or reducing the severity of symptoms that will or more delivery systems and directions for use of the kit (e.g., are expected to develop. instructions for treating a subject). In some embodiments, a kit can include a Sublingual formulation of flumazenil and a EXAMPLES transdermal formulation of flumazenil. In another embodi ment, a kit can include a Sublingual formulation offlumazenil Example 1 and a wakefulness promoting agent. In some embodiments, the kit can include a Sublingual formulation of flumazenil and Endozepine Modulation of GABAR Function a label that indicates that the contents are to be administered 0.108 HEK293 cells transiently expressing human C.1, B2, to a Subject resistant to amphetamines. In another embodi and Y2s subunits were Superfused at 1 mL/min with an extra ment, the kit can include a Sublingual formulation of a cellular solution (ACSF) containing 145 mm NaCl, 3 mm GABA receptor antagonist Such as flumazenil and a label KCl, 1.5 mm CaCl2, 1 mm MgCl2, 6 mm d-glucose, and 10 that indicates that the contents are to be administered to a mm HEPES-NaOH adjusted to pH 7.4. Whole cell patch Subject positive for increased production of endozepines or clamp recordings from cells Voltage clamped at-60 mV were other Somnogenic compounds, as described herein. In a fur made using the Multiclamp 700B amplifier (Molecular ther embodiment, a kit can include a Sublingual formulation Devices, Sunnyvale, Calif.). The resistance of the patch of flumazenil and a label that indicates that the contents are to pipette was 4-6M when filled with intracellular solution (145 be administered with a wakefulness promoting agent and/or a mm N-methyl-d-glucamine hydrochloride, 5 mm dipotas transdermal formulation of flumazenil. sium ATP 1.1 mm. EGTA, 2 mm MgCl, 5 mm HEPES-KOH, 0102 Also provided herein are kits for performing a diag and 0.1 mm CaCl adjusted to pH 7.2). In addition to the nostic assay. In some embodiments, the diagnostic assay can continuous bath perfusion with extracellular medium, Solu be used to diagnose Subjects Suffering from a GABA recep tions including AS99-CSF (as described below). GABA and/ tor mediated hypersomnia and/or to determine Subjects that or flumazenil were applied rapidly to the cell by local perfu would benefit from treatment with a GABA receptor antago sion using a motor-driven Solution exchange device (Rapid nist. In some embodiments, a kit for use as a diagnostic assay Solution Changer RSC-160; Molecular Kinetics, Indianapo is provided with the components for carrying out a patch lis, Ind.). Solutions were exchanged within approximately 50 clamp assay as described herein. In some embodiments, the ms. Laminar flow out of the rapid solution changer head was kit can include a GABA receptor antagonist and cells which achieved by driving all solutions at identical flow rates (1.0 transiently or stably express human C.132y2S GABA recep mL/min) via a multichannel infusion pump (KD Scientific, tors. In some embodiments, the kit can include cells which Holliston, Mass.). The solution changer was driven by proto transiently and stably express human C.1?2y2S GABA cols in the acquisition program of pCLAMP version 9.2 (Mo receptors and cells which transiently and stably express a lecular Devices, Sunnyvale, Calif.). AS-CSF was isolated benzodiazepine insensitive subunit (e.g., C.1 (H102R). In from AS99, a patient experiencing hypersomnia. The patient Some embodiments, the kit further comprises one or more of also exhibited apparent resistance to amphetamine treatment. an extracellular Solution that can function as a control sample, All other compounds were obtained from the Sigma-Aldrich e.g., a control CSF sample; an intracellular solution; an extra Co. US 2011/002841 8 A1 Feb. 3, 2011

01.09 Results indicated that AS-CSF had no intrinsic was formed, a flavor was added. The flavor added was GABA efficacy, but it enhanced the amplitude of response to selected from the following (quantities given are per 50 tab EC concentrations of GABA (see FIG. 1a). In a second lets): experiment, 4 uM flumazenil was co-applied with AS-CSF. 0119 a)2 drops lemon, 1 drop marshmallow, 4 milli The flumazenil immediately reversed the enhancing effect of grams yellow color the AS-CSF (see FIG. 1b). 0110 Positive modulation of GABA receptor function by 0120 b) 2 drops creme de mint, 4 mg green color 100% or more is normal for concentrations of general anes 0121 c) 2 drops tangerine, 1 drop marshmallow, 4 mg thetic drugs that would anesthetize a human. The results Orange indicate that AS-CSF contains a positive allosteric modulator 0.122 d) 5 drops cherry, 2 drops vanilla, 4 mg red color. of GABA Receptor that would have potent sedative effects The formulation provided 100 tablets. in a human. The reversal of this effect by flumazenil Suggests that the positive modulator likely acts directly or indirectly at Example 4 the benzodiazepine binding site on the GABA receptor. 0111. Accordingly, patients experiencing disorders asso Formulation of Tablet Triturate Base 20%/80% Pow ciated with excessive sleepiness (e.g., idiopathic or amphet der amine resistant hypersomnia) who test positive for a positive allosteric modulator of GABA receptor function may likely (0123 benefit from administration of flumazenil. Example 2 Ingredient: Amount added: Formulation of Flumazenil as Tablet for Sublingual Dosing Sucrose powdered (confectioners) 20 grams Lactose monohydrate (hydrous) 80 grams 0112 0.124 Procedure: The sucrose and lactose monohydrate were sieved through 120 or smaller mesh. After adding the Ingredient: Amount added: active ingredient (e.g., flumazenil), the mixture was wetted with an excipient of 40% distilled water and 60% alcohol. The Flumazenil 0.3 grams Tablet triturate base (20%80% powder) 4.7 grams formulation provided 100 grams of table triturate base 20%/ Tablet triturate exipient (flavorless) 2 milliliters 80% powder. Flavor, PCCA Bittershop 4 drops Stevia concentrate (250 mg/mL) 2 drops Example 5 0113 Procedure: The ingredients were combined and Formulation of Stevia Concentrate Solution (250 mixed to formathick paste. After the thick paste was formed, mg/ml) a flavor was added. The flavor added was selected from the following: 0.125 0114 a)2 drops lemon, 1 drop marshmallow, 4 milli grams yellow color 0115 b) 2 drops creme de mint, 4 mg green color Ingredient: Amount added: 0116 c) 2 drops tangerine, 1 drop marshmallow, 4 mg Stevia powder extract 25 grams Orange. Sodium benzoate 0.6 grams The formulation provided 50 tablets. Water preserved liquid 100 milliliters Example 3 0.126 Procedure: The stevia powder and sodium benzoate Formulation of Flumazenil as Tablet Triturate for were dissolved in the water preserved. See Example 6 for Sublingual Dosing waterpreserved liquid formulation. The mixture was warmed to aid in dissolution. The formulation prepared 100 mL of 0117 Stevia concentrate solution. Example 6 Ingredient: Amount added: Formulation of Water Preserved (Paraben) Liquid Flumazenil 0.6 grams Tablet triturate base (20%80% powder) 9.4 grams O127 Tablet triturate exipent (flavorless) 4 milliliters Flavor, PCCA Bittershop 8 drops Stevia concentrate solution (250 mg/mL) 4 drops Ingredient: Amount added: 0118 Procedure: The ingredients were combined and Water preserved concentrate liquid 10 milliliters mixed to form a thick paste. See Example 4 for tablet triturate Water distilled liquid 3780 mL. base (20%/80% powder) formulation and Example 5 for ste via concentrate solution formulation. After the thick paste US 2011/002841 8 A1 Feb. 3, 2011 11

0128 Procedure: The liquids were mixed to prepare the at the GABA receptor with behavior in 70 individuals suf water preserved (paraben) liquid. See Example 7 for water fering from sleepiness or hypersomnia. Ten, age and sex preserved concentrate liquid formulation. matched controls deemed affected or unaffected by sleepi ness will also be studied. Initial identification, recruitment, Example 7 and biological sample procurement will take place in the Formulation of Water Preserved Concentrate Liquid outpatient clinic and diagnostic sleep laboratory which share dedicated space. After satisfying inclusion/exclusion criteria 0129 and upon providing consent, additional behavioral, wake? sleep, and rest-activity cycle assessments will be conducted along with quantification endogenous GABA receptor bio Ingredient: Amount added: activity. Subjects will then be admitted to a clinical setting for 24 hours and their clinical response to single-blind intrave Methylparaben 19 grams Propylparaben NF 9.6 grams nous delivery of saline, 0.5, and 2.0 mg flumazenil will be Propylene glycol USP 100 mL. determined. Other known causes of hypersomnia, Such as hypocretin deficient narcolepsy, exogenous BZD use, iatro 0130 Procedure: The ingredients were mixed together genic effects of common medications known to positively or and stirred until the methylparaben and propylparaben NF negatively modulate GABAR (e.g., Steroids, methylxan thines and many antibiotics), and metabolic disorders (e.g., were completely dissolved. urea cycle disorders) will be excluded. Finally, to offer some Example 8 further sense of the commonality and phenotypic spectrum associated with plasma potentiation of GABAR function, Formulation of Flumazenil as Cream for Transder this activity will be quantified in a population-based sample mal Dosing of subjects.

0131) Inclusion/Exclusion Criteria 0.136 Patients complaining of daytime sleepiness/hyper somnia with an Epworth Sleepiness Scale or >15 and who Ingredient: Amount added: exhibit either objective sleepiness (MSL.<8 minutes), REM Flumazenil 0.04 grams sleep propensity during their diagnostic evaluation, or treat Prophlene glycol USP 0.1 milligrams Food color, pink (powder) 0.03 milligrams ment resistant sleepiness will be recruited. Patients with Versabase cream 10 grams DSM-IV Axis I disorders such as depression, bipolar disease, serious medical co-morbidities such as stroke, congestive heart failure, active cancer, severe obstructive pulmonary dis 0132) Procedure: The ingredients were combined and ease, asthma, or uncontrolled type I or II diabetes will be mixed. The formulation provided 10 milliliters of cream. excluded. Any patient with a history of CNS trauma, infec tion, or neurodegenerative condition will be excluded. Example 9 Patients with, treated or untreated sleep disordered breathing Formulation of Flumazenil as Cream for Transder (AHD-10) will also be excluded. Subjects with chronic health mal Dosing conditions otherwise well-controlled with medication (e.g., hypertension, hypothyroidism, arthritis) will be allowed to 0133) participate. Potential controls and subjects will be excluded if they are ingesting psychoactive medications including seda tive-hypnotics, anxiolytics, mood-stabilizers presumed to act via GABAergic mechanisms, neuroleptics, and anti-depres Ingredient: Amount added: sants. In addition, given the known ability of steroids, meth Flumazenil 0.25 grams ylxanthines, and many antibiotics to allosterically modulate Prophlene glycol USP 0.25 milliliters Food color, red (powder) 0.0075 milligrams GABAR, potential Subjects taking gluco- or mineralo-cor Versabase cream 25 grams ticoids, theophylline, or certain antibiotics will be excluded (at least while they are ingesting these agents). Three mls each of plasma and urine will be sent to MedTox. Laboratories 0134) Procedure: The ingredients were combined and (Burlington, N.C.) to be analyzed for classic BZDs and their mixed. The formulation provided 25 milliliters of cream. metabolites by gas chromatography (GC) and high perfor mance liquid chromatography (HPLC). The specific agents Example 10 and respective reporting limits (i.e., thresholds for detection) will include: Desalkylflurazepan (flurazepam metabolite) 10 Characterization of the Spectrum of GABA Recep ng/ml, Nordiazepam 50 ng/ml, oxazepam 50 ng/ml. tor Mediated Hypersomnia lorazepam 10 ng/ml, diazepam 50 ng/ml, hydroxyflurazepam 0135 An organized, multidimensional approach to char 10 ng/ml, temazepam 50 ng/ml, chordiazepoxide, 50 ng/ml. acterizing the phenotypic spectrum of GRH will be employed midazolam 10 ng/ml, flurazepam 10 ng/ml, alpha-hydroxy to determine who is affected, and how it manifests with spe alprazolam 50 ng/ml, alproZolam, 13 ng/ml, hydroxytriaz cific attention to overlap with ICSD-2 defined sleep and cir olam 10 ng/ml, triazolam 10 ng/ml and estazolam 10 ng/ml. cadian rhythm disorders. This will involve recruiting and Additional, individual samples will be sent to NMS Labs extensively characterizing and correlating biological activity (Willow Grove, Pa.) for GC quantification of Zolpidem (4-5 US 2011/002841 8 A1 Feb. 3, 2011

ng/ml), HPLC quantification for Zaleplon (3 ng/ml), and 0.88) and REM onset scores (kappa coefficients of 0.78-0. HPLC tandem mass spectrometry (LC-MS/MS) quantifica 88). The stability of the MSL on repeat testing in known tion of eSZopiclone. narcoleptics is high (r=0.81, p<0.01) with test-retest reliabil 0.137 In order to more carefully delineate a provisional ity improving visa Vis diagnostic certainty with the additional diagnosis of GRH and to provide additional potentially ICSD-2 requirement of two or more sleep onset REM-sleep important biochemical data relevant to the spectrum of hyper periods (Kappa=0.95; variance=0.08; Z=2.33; p<0.05). Somnolence disorders such as narcolepsy with cataplexy, CSF for hypocretin (HCRT-1) will be assayed using a com Blood Collection mercially available RIA (Orexin ARIA kit, Phoenix Pharma 0142. Thirty mL of venous blood will be drawn for: 1) ceuticals, Belmont, Calif.). This assay has an intra-assay vari lymphoblastoid cell line generation to establish a permanent ability of <5%. Other recognized metabolic causes of source of DNA and cells for future investigations; 2) clear hypersomnolence will also be screened. For example, disor plasma aliquoted and frozen for future analytic studies; and 3) ders of the urea cycle and the catabolic enzymes for GABA buffy coat and purified DNA banked for future genetic stud (e.g., GABA-transaminase and Succinic semialdehyde dehy ies. The DNA will be purified from 200 ul of buffy coat using drogenase) have been associated with lassitude and hyper a Qiagen kit protocol (Qiagen, Valencia, Calif.). The cell somnia, albeit, incompletely characterized by MSLT or lines, buffy coat, plasma and purified DNA will all be labeled ICSD-2. These must be ruled out as a potential contributors to with barcode compatible labels and banked at -80°C. within hypersomnia by assessing arterial ammonia and urine and CRIN dedicated resources. De-identified DNA from all par plasma organic and amino acids. The latter analyses will be ticipants will be assigned a 6-digit reference number. Aliquots performed in a CLIA certified laboratory employing ion with this reference number will be forwarded to a laboratory exchange chromatography. for testing. Flumazenil Infusion Collection of Lumbar CSF 0.143 All patients and family members (afflicted, unaf 0.138. The subject will be instructed in the proper use and flicted) will provide Informed Consent for collection of cere care of the Activatch and completion of the sleep/wake diary brospinal fluid (CSF). Lumbar punctures (LP) will be per within one month following the lumbarpuncture. The subject formed under sterile conditions using standard procedures, will complete all study inventories (see below), and within Subcutaneous administration of 4% lidocaine, and collection two weeks, will undergo 48-hours of ambulatory polysom of 15-20 ml CSF with a 22 gauge spinal needle inserted at nography (see below). Five home/clinic visits will be made L3/L4 or L4/L5. One ml fractions will be labeled with the during that period to hook-up the Subject, check the integrity participants 6-digit reference number and frozen immediately of the electrodes, and to disconnect the subject from the upon dry ice and then stored at -80 degrees Centigrade for equipment. Within two weeks, subjects will be scheduled for future analyses. LPs will be performed between 0830 and a 24-hour admission to the ACTSI and after a full-night of 0930 after completion of the first MSLT nap. This will obviate recorded sleep receive saline (control), 0.5 mg, and 2.0 mg the need to control for subsequent daytime activity levels and flumazenil at roughly 2.5 hour intervals while undergoing extent of food intake which hypothetically could affect continuous EEG monitoring and hourly monitoring of vital endogenous activity at the GABA.R. signs. All subjects will complete a baseline Stanford Sleepi ness Scale (SSS) and Psychomotor Vigilance Task (PVT) (see Questionnaire Assessments below) which will be repeated at 10,30, 60,90, 120, and 150 0144. Administered questionnaires serve as both screen minutes following each injection. ing instruments and as predictors in the regression models described below. Subjective sleepiness as a trait variable will Polysomnographic Recording be assessed using the Epworth Sleepiness Scale (ESS) and 0139 Diagnostic nocturnal polysomnography (NPSG) overall quality of sleep will be assessed using the Pittsburgh and subsequent daytime testing will be recorded with the Sleep Quality Index (PSQI). State and trait anxiety will be Embla digital PSG system (Medcare Corporation, Buffalo, assessed with the State-Trait Anxiety Inventory (STAI) and N.Y.) with a sampling rate of 512 HZ, as this allows for Fast mood will be assessed with the Beck Depression Inventory Fourier Transform of EEG signals. The system employs a (BDI). These are all standardized scales with population Windows XP platform and uses proprietary software (Som based norms. Data on functional impairments related to nologica Science). Spectral analyses, or the Welch method of sleepiness using the Functional Outcomes of Sleep Question FFT smoothing, that provide an average of several FFT's will naire (FOSQ) will also be collected. The FOSQ is a self be useful to more fully characterize the signature of finger report measure designed to assess the impact of excessive print of endogenous GABAAR like activity given the known sleepiness on multiple activities of daily living. effects of GABA on corticothalamic excitability as manifest Actigraphy in the EEG. (0145 The Activatch wrist-worn monitor, manufactured Multiple Sleep Latency Testing by Respironics (Murrysville, Pa.), will be used to assess char acteristic sleep durations in patients for two weeks prior the 0140 Daytime sleepiness will be objectively assessed infusion protocol. Patients will also be provided a sleep log to with the MSLT, which is a clinical and research tool that uses keep during the two weeks to generate data on timing of sleep standard guidelines for testing and scoring. Sleep latencies and napping. and number of REM onsets will be determined according to standard criteria. Ambulatory Polysomnography 0141. The MSLT displays excellent interrater and 0146 Ambulatory PSG over a 48 hour period will be con intrarater reliabilities for sleep latency (coefficients of 0.81 ducted using the same equipment cited above which can be US 2011/002841 8 A1 Feb. 3, 2011 adapted for this use to document the degree of hypersomnia 60 cases would yield 99% power to reject the null of hypoth suggested by actigraphy. Only EEG, EOG, Submental EMG esis of the contribution for any single domain to GABA lead, ECG, and pulse oxymetry will be conducted. No limb potentiation. It is fully recognized that, in multivariate models leads will be used for patient safety reasons. Sleep stages, encompassing each of the five domains listed above, actual episodes of desaturation, and ECG will be analyzed. power might be somewhat reduced because of the contribu tion of multiple variables to the prediction. Nonetheless, Psychomotor Vigilance Task (PVT) given the substantial effects observed in FIG. 3, sufficient power to understand how different variables may predict 0147 The Psychomotor Vigilance Task (PVT) provides a potentiation when considered simultaneously should be sensitive marker of minute-to-minute fluctuations in alertness retained. Regression models will also be used to determine during the flumazenil infusion protocol. The PVT is a what factors may predict change in GABA potentiation under 10-minute, simple, portable reaction time test (finger button flumazenil infusion. Each patient's Baseline potentiation press response to light) designed to evaluate the ability to level (measured under saline infusion) will be forced and it is Sustain attention and respond in a timely manner to salient determined whether either low or high dosage of flumazenil signals. Data to be generated include: 1) frequency of lapses, predicts change Subsequent to infusion. Domain variables which refer to the number of times the subjects fail to respond selected for entry into these models are limited only to those to the signal or fail to respond in a timely manner; 2) the shown to relevant to the prediction of Baseline potentiation, median reaction time (RT) over the 10-minute interval. Addi thus saving degrees of freedom whenever possible. This mod tionally, as a measure of state sleepiness, the Stanford Sleepi eling allows the determining of the extent that other variables ness Scale (SSS) will be administered immediately prior to (demographic, recent sleep history, etc) may have to moder each trial. The PVT/SSS will be administered at 10, 30, 60, ate or mediate the GABA-mediated response to flumazenil. 90, 120, and 150 minutes following each infusion of saline or The behavioral response to flumazenil (performed separately flumazenil. by dose) will also be examined, defined as the mean of the median RTs for the 4 PVT measurements closest to point of Statistical Analysis/Power Calculations infusion. Each patient's Baseline median RT (mean of 4 Base line/saline measurements) (see FIGS. 6, 8, 10, and 12) and 0148. The relationship between the extent of GABA Baseline GABA potentiation levels will be forced initially in potentiation and behavioral outcomes will be examined using these regressions, followed by entry of significant predictors regression models. Separate models will be run for each type of Baseline potentiation found in the analyses described of specimen source (e.g., CSF and plasma derived markers of above. potentiation). A simple bivariate relationship between the two 0149 Further, the plasma-measured GABA potentiation measures of GABA potentiation using correlational models will be examined as the dependent variable among 227 indi will be examined, relying on non-parametric alternatives viduals, all of whom will have received two nights of PSG and (Spearman) should the measure present with a non-normal an intervening day of MSLT. The hypersomnolence demon distribution. The extent of GABA potentiation in the Baseline condition among patients will be predicted using predictors strated by the index cases will represent a more extreme form Such as standard demographics (e.g., gender, age), psycho of a continuous trait present in segment of the population metrics (e.g., STAI. BDI), recent sleep history (e.g., cumula generally. To that end, the initial review of the data indicated tive sleep over the preceding 2 weeks as measured with actig that 58 had mean MSLT-defined sleep latencies of less than 5 raphy, daytime naps on sleep log), and laboratory-based minutes that could not be accounted for by known sleep measurements of nocturnal sleep (e.g., FFT derived relative disorders. If the MSLTs across all 227 cases show a bimodal delta power or beta power) or daytime alertness (e.g., MSLT distribution, the analyses would be limited to only those cases sleep latency. PVT-derived median reaction time). Because at the extremes (e.g., mean latencies.<5 minutes versus mean multiple measurements in each domain and the sheer number latencies 15 minutes) and employing an ANCOVA of domains increase the likelihood of Type I error, such error approach. However, the distribution of mean sleep latencies is will be minimized by first carefully examining the intercor more continuous and, as is often the case of studies using relations among measures within each domain. Substantial MSLT, sharply skewed to the right. In this case, log trans collinearity is expected to be among many of these. For forms are performed on these mean values before proceeding. example, trait anxiety (STAI) and depressed mood (BDI) are The overall approach will be similar to those described above, likely to be highly intercorrelated, as are Baseline PVT though they are somewhat more limited by the range of vari median reaction times and MSLT-defined sleep latency. The ables collected. specific approach to deriving variables to employ in the regression might include a selection of a single variable from Example 11 each domain chosen on the basis of a more normally distrib Electroencephalography (EEG) Power Spectrum uted range of scores across Subjects. Alternatively, the data Analysis reduction techniques can be relied on Such as principal com ponents analyses (PCA) to determine a single measure in 0150. Quantitative analysis of delta (0.4-3.99 Hz), theta each domain that best captures variance within that domain. (4.00-7.99 Hz), alpha (8.00-12.99 Hz) and beta power (13. Thus, a single score (or composite score, if PCA was used) 00-16.00 Hz) was obtained from EEG spectral analyses of the from each domain will be entered in the regression predicting C4-M1 electrode. Manual and automated artifact removal potentiation. Based on the data presented in FIG. 3, large methods were utilized prior to EEG spectral analyses to pre effects will be displayed. GABA potentiation differences vent erroneous results. Spectral analyses were conducted uti between controls and patients will be substantial (d=3.095). lizing the computational software program MATLAB v 7.1. Assuming effects of this size are maintained in the work The Welch method of FFT smoothing was employed to obtain proposed here, and assuming a 2-tailed alpha of 0.01, an N of power spectrum values from an average of several FFT's. The US 2011/002841 8 A1 Feb. 3, 2011

FFT contained a minimum of 512 data samples with a 50% Example 12 overlap moving window of the Subsequent 512 data samples. EEG data collected at 200 Hz provided an FFT window Psychomotor Vigilance Task (PVT) comprised of approximately 2.56 seconds of data. Parameters 0153. The dose and temporal reversibility of the sleepiness for Welch spectral analyses were user adjustable within the of patients to intravenous flumazenil were determined MATLAB program such that user defined frequency bands employing the PVT/SSS paradigm as described in Example for specific frequency resolutions were obtained. Power val 10. Five hypersomnic patients demonstrated dose-dependent ues for the defined frequency bands were represented by improvements in vigilance and Subjective alertness with mV/Hz (microvolts squared divided by hertz). intravenous delivery of flumazenil as shown in Table 3.

TABLE 3 Baseline, Saline FLU 0.325-0.5 mg FLU 1.2-2 Ing % Reaction Stanford RTS RTS GABA time (RT) Sleepiness in in Case potentiation in ms lapses Scale (SSS) ms lapses SSS ms lapses SSS 74 200+f- 21.7 432.3 +f- 84.8 31.0 6 236.8 +f- 47.7 0.7 4 207+f- 4.97 0.7 3 99 160+f- 9.2 285.5 +f- 13 NA 6 255.8 +f- 6.3 NA 3 225.5 +f- 2.3 NA 1 102 189+f- 24.3 1962 +f- 1478 16.4 6 1642 +/- 1036 3.1 3 363.2 +f- 38.2 3.8 4 122 149+f- 20.4 369.5 +f- 78.9 17.2 5 297.8 +f- 16.2 O.6 1 269.3 +f- 6.8 O6 1 124 58.5 +f-3.5 327.8+f- 22.96 5.8 6 259.8 +f- 3.8 1.3 2 250 +/- 1.2 1.O 2

0151. The EEG power spectrum analyses for two subjects 0154 Administration of flumazenil (FLU) was associated (DS122 and DT74) were obtained (see FIGS. 4 and 5, respec with dramatic and Substantial improvement in reaction time tively). Table 1 provides the corresponding sampling fre performance on the PVT and subjective alertness on the SSS. quency and FFT window size for each patient data set. There Relative to baseline, median RTs decreases at low (t=2.56, p=0.063) dose, and number of lapses decreased both at low was a spectral change approximately five minutes after intra (t=3.03, p=0.056) and high (t=3.51, p=0.039) dose. When venous infusion of 2.0 mg flumazenil that manifested as dimi compared to the worst Baseline measure for each case, SSS nution of delta frequencies and emergence of higher EEG showed significant improvement for both low (t=8.55, p=0. frequencies emblematic of improved vigilance/arousal. 001) and high (t=7.06, p=0.002) dose. Raw histograms for cases 74, 102, 122, and 124 displaying baseline PVT perfor TABLE 1. mance and PVT performance after 2.0 mg are shown in FIGS. 6-17. Patient Sampling Frequency FFT size

DS SOOHz 1024 Example 13 DT 200 Hz 512 Clinical Study of GABA Receptor Mediated Hyper somnia (GRH) 0152 Table 2 displays mean relative band power results (O155 Patient AS99 with a diagnosis of “narcolepsy” and obtained from EEG power spectrum (i.e., delta, theta, alpha, restless legs syndrome (RLS) complained of "craving sleep, beta) analyses of subject ED102 and DS122 for each clinical and of long, unrefreshing sleep periods. Polysomnography treatment (i.e., Saline, 0.5 mg flumazenil, and 2.0 mg fluma revealed periodic leg movements (31 per hour), but was oth Zenil). Ten minute data segments were selected 30 minutes erwise normal (TST=444 min). A mean sleep nap latency of following each clinical treatment and were analyzed via a 2.6 minutes absent intrusion of REM sleep confirmed patho three second processing window to obtain the relative power logical sleepiness and a diagnosis of idiopathic hypersomnia. spectrum results provided in Table 2. Patient AS99’s examination was normal with a BMI of 22.3, and urine drug screens (repeatedX3), serum ammonia (n=2). TABLE 2 thyroid functions (n=2), complete blood counts (n=5), vita min B12, and comprehensive metabolic screens (n=2) were Delta Theta Alpha Beta Gamma normal. Ferritin (23 ng/ml) and % transferrin saturation Subject Treatment Power Power Power Power Power (13%) were low with otherwise normal serum iron. The RLS ED102 Saline O.S314 0.1824. O.1090 O.12O7 O.OS6S was successfully treated with iron Supplementation and ED102 0.5 mg O.S199 0.17SS O.1017 O.1387 O.O642 pramipexole; however, hypersomnia persisted despite maxi flumazenil ED102 2.0 mg O482O 0.1479 0.0866 0.1803 O.1032 mum doses of dextroamphetamine (60 mg) in combination flumazenil with modafinil (800 mg). Actigraphy confirmed resolution of DS122 Saline O4122 O.2777 O.1966 0.1011 O.O124 RLS/PLMs, yet revealed erratic rest-activity cycles with DS122 0.5 mg O.3515 0.2995 O.21.83 O.1165 O.O142 sleep periods varying from 5 to 10 hours per night. Patient flumazenil DS122 2.0 mg O.3128 0.3776 O.1798 0.1141 O.O157 AS99's condition progressed and weight decreased flumazenil (BMI=20), and patient AS99 developed anxiety and hyper tension requiring treatment with metoprolol attributed to Supratherapeutic doses of psychoStimulants. Affective and US 2011/002841 8 A1 Feb. 3, 2011

factitious disorders were ruled out by two independent psy tectable quantities of 3C.5 B-THP. 3C,5C-THDOC, 3C.5 B chiatric assessments. Weaned off all medications, CSF was THDOC, and 3C,5C.-androstandiol. obtained and hypocretin determined to be high-normal (401 0159 Further, it was shown that the substance accounting pg/ml) thus ruling out a diagnosis of narcolepsy. Electro for potentiation at GABA receptors has a molecular weight physiological analysis for bioactivity in CSF and plasma less than 3,000. Pooled CSFs from confirmed GRH subjects revealed the presence of a positive allosteric modulator of the Versus controls were fractionated with filters having approxi GABA receptor reversible with the competitive BZD mately 3,000 molecular weight cut-off. Bioactivity at antagonist flumazenil. The dose and temporal reversibility of GABA receptors in both samples was completely retained sleepiness to intravenous flumazenil were then determined within the smaller molecular weight fractions. employing the PVT/SSS paradigm. The rest-activity cycles 0160. It was also found that the substance accounting for of patient AS99 improved with chronic sublingual flumazenil potentiation at GABA receptors may act at a non-traditional administration (see FIGS. 18a and 18b). The sleep, mood, benzodiazepine site (see FIGS. 19a and 19b). In a whole cell sleepiness, and quality of life improved dramatically and are patch clamp current recording from a cell expressing human sustainable with sublingual flumazenil in patient AS99 (see C.132y2s receptors, the response to 10 uMGABA is potenti Table 4) as shown through the Pittsburgh Sleep Quality Index, ated by the co-application of a 50% CSF, indicating the pres Beck Depression Inventory, Epworth Sleepiness Scale, Func ence of a positive allosteric modulator (see FIG. 19a). A tional Outcomes of Sleep, and SF-36 Health Survey. recording from a different cell expressing the benzodiazepine insensitive subunit C.1 (H102R) shows that the enhancement TABLE 4 persists (see FIG. 19b). Not to be bound by theory, this data February 22 March 31 April 28 indicates that the Somnogenic compound is not a classical (pre- (1 month (2 months benzodiazepine, or does notact conventionally at the classical Variable treatment) post) post) high-affinity benzodiazepine binding site on the GABA Pittsburgh Sleep Quality Index 4 2 1 receptor. Beck Depression Inventory 7 2 Epworth Sleepiness Scale 18 3 3 Example 15 Functional Outcomes of Sleep General Productivity 6 23 24 Patch Clamp Analysis of CSF Bioactivity Social Activity 12 24 24 Activity 3.6 19.6 21.3 0.161 Patch clamp analyses of CSFs from non-human pri Vigilance 8 22 24 mates, drawn from animals under different conditions, was Total 29.5 886 93.3 also used to identify the Somnogenic GABAergic Substance. Total Mean 7.4 22.1 23.3 In this experiment, CSF was drawn from 4 monkeys (Canjala, SF-36 Health Survey Santiaga, Penelope, and Cricket) at 3 different time points. 1) early morning, 2) late afternoon and 3) very late evening, Physical 39.5 544 66.2 having been kept awake throughout (when they would nor Mental 49.7 64.O 56.2 mally be asleep). A whole cell patch clamp current was recorded (as described in Example 1), and the response to 10 0156 Patient AS99 continued use of sublingual flumaze uM GABA co-administered with primate CSF was deter nil for 9 months with positive results. When prescribed mined. The bioactivity of the CSF is expressed as a percent clarithromycin, patient AS99 suddenly developed 4 nights of increase, or potentiation, of the control current by the CSF insomnia that reversed promptly upon discontinuation. (see Table 5). The first two columns show the normal diurnal Clarithromycin is an antibiotic with a high incidence of hypo variation of this somnogenic compound. It appears that this mania/insomnia associated with its use, and it functions as a Substance waxes and wanes in animals as it does in humans negative allosteric modulator at GABA receptors. during the normal day night cycle. More interestingly, in 2 of the 4 animals, the bioactivity increased still further after the Example 14 animals were “wake-extended'. These promising results indicate that under sleep deprived conditions, humans may Identification of Substance Causing Potentiation at also benefit from flumzenil or other GABAergic therapy to GABA Receptors relieve the symptoms of fatigue they experience from the accumulation of this somnogenic compound. 0157. Several studies were performed in order to identify the Substance accounting for potentiation at GABA recep TABLE 5 tors. It was determined that adenosine is not the Substance, as several concentrations (1 mM, 100 uM, and 10 uM) of Morning Evening Wake Enhanced adenosine in artificial CSF exhibited no activity at GABA Canjala 79.92.7 76.6 4.6 73.4 4.7 receptors. Santiaga 49.O 2.0 S8.7 O.8 649 - 1.7 0158. In addition, it was shown that the substance account Penelope 54.63.5 77.1 - O.S 98.4 23.1 ing for potentiation at GABA receptors is not a neurosteroid. Cricket 51.3 O.7 58.1 4.2 63.9 6.9 Cerebrospinal fluid from four hypersomnic cases were tested in duplicate by quantitative HPLC for endogenous neuroac (0162. A number of embodiments of the invention have tive GABAergic steroids (i.e., neurosteroids). The controls been described. Nevertheless, it will be understood that vari revealed no differences in the levels of pregnenolone, DHEA, ous modifications may be made without departing from the 3C,5C.-THP. 3C.5 B-androstandiol. 3C,5C.-androsterone, and spirit and scope of the invention. Accordingly, other embodi 3C,53-androsterone. Controls and subjects exhibited unde ments are within the scope of the following claims. US 2011/002841 8 A1 Feb. 3, 2011

1. A method of treating GABA receptor mediated hyper 69. The method of claim 1, wherein the GABA receptor Somnia in a Subject, comprising administering to the Subject mediated hypersomnia is hypersomnia. an effective amount of a GABA receptor antagonist. 70. The method of claim 1, wherein the GABA receptor 2.-61. (canceled) mediated hypersomnia is an endozepine stupor. 71. The method of claim 1, wherein the GABA receptor 62. The method of claim 1, wherein the GABA receptor mediated hypersomnia is amphetamine resistant hyperSom mediated hypersomnia is excessive sleepiness. 18. 63. The method of claim 1, wherein the GABA receptor 72. The method of claim 1, wherein the GABA receptor mediated hypersomnia is sleep apnea. mediated hypersomnia is narcolepsy. 64. The method of claim 1, wherein the GABA receptor 73. The method of claims 1, wherein the GABA receptor mediated hypersomnia is nocturnal dystonia. antagonist is selected from the group consisting of flumaze 65. The method of claim 1, wherein the GABA receptor nil; clarithromycin; picrotoxin; bicuculline; cicutoxin; and mediated hypersomnia is restless legs syndrome. oenanthotoxin. 74. The method of claim 1, wherein the GABA receptor 66. The method of claim 1, wherein the GABA receptor antagonist is flumazenil. mediated hypersomnia is nocturnal movement disorder. 75. The use of a GABA receptor antagonist in the produc 67. The method of claim 1, wherein the GABA receptor tion of a medicament for the treatment of GABA receptor mediated hypersomnia is Kleine-Levin Syndrome. mediated hypersomnia. 68. The method of claim 1, wherein the GABA receptor mediated hypersomnia is Parkinson's disease. c c c c c