Patent Application
20130053435
1. Field of the Invention
The present invention generally relates to methods of using dihydromyricetin to modulate ethanol induced plasticity of γ-aminobutyric acid (A) receptors. The present invention also relates to methods of using dihydromyricetin to treat ethanol intoxication, alcohol use disorders and alcohol abuse.
2. Description of the Related Art
Alcohol dependence ranks third on the list of preventable causes of morbidity and mortality in the United States. There are more than 20,000 alcohol-induced deaths every year in the United States excluding accidents and homicides. In 2008, 11,773 people were killed in alcohol-impaired driving crashes, accounting for nearly one-third of all traffic-related deaths in the United States. According to the Centers for Disease Control and Prevention, the annual cost of alcohol-related crashes totals more than $51 billion.
Alcohol (ethanol, EtOH) interaction with γ-aminobutyric acid (A) receptors (GABAARs) plays a role in alcohol withdrawal syndrome (AWS). See Becker H C (1998) Alcohol Health Res World 22(1):25-33; Boehm S L, 2nd, et al. (2004) Biochem Pharmacol 68(8):1581-1602; Koob G F (2004) Biochem Pharmacol 68:1515-1525; Anacker A M and Ryabinin A E (2010) Int J Environ Res Public Health 7(2):473-493; and Dopico A M and Lovinger D M (2009) Pharmacol Rev 61(1):98-114.
GABAARs on synapses are formed of αβγ subunits which have low sensitivity to ethanol; while GABAARs containing α4βδ subunits are highly sensitive to low ethanol concentrations. See Liang J, et al. (2008) Alcohol Clin Exp Res 32(1):19-26; Santhakumar V, et al. (2007) Alcohol 41(3):211-221; and Jia F et al. (2005) J Neurophysiol 94(6):4491-4501. GABAARs are known to undergo allosteric modulation by ethanol, general anesthetics, benzodiazepines and neurosteroids. See Olsen R W and Homanics G E (2000) GABA
Benzodiazepines (e.g. diazepam) are classical medications for reducing symptoms of AWS. However, benzodiazepines are inactive at the alcohol-sensitive, and insensitive α4βδ subunit-containing GABAARs. In addition, benzodiazepines produce cross-tolerance to ethanol. Moreover, as a major side effect, frequent use of benzodiazepines can lead to dependence. In fact, the combination of benzodiazepines and alcohol cause even greater substance addiction problems which are more difficult to overcome as compared to alcohol dependence itself.
Besides benzodiazepines, only three medications, i.e. naltrexone, acamprosate, and disulfiram, are currently approved by the U.S. FDA for treating alcohol dependence. Naltrexone blocks opioid receptors and it may also impair thinking and reaction-time, and produce anxiety and other unhappy feelings. Acamprosate causes side effects including headache, diarrhea, flatulence and nausea and two large U.S. clinical trials failed to confirm its efficacy. Disulfiram is directed towards blocking the metabolism of alcohol, thereby causing a negative reaction to alcohol intake, and its side effects include flushing, accelerated heart rate, shortness of breath, nausea, vomiting, headaches, visual disturbances, mental confusion, and circulatory collapse. Disulfiram may also cause peripheral neuropathy.
Thus, a need exists for compositions and methods which treat, inhibit, reduce and/or reverse some or all GABAAR plasticity caused by exposure to ethanol.
In some embodiments, the present invention provides methods of treating, inhibiting, reducing and/or reversing GABAAR plasticity caused by exposure to ethanol, which comprises administering dihydromyricetin to a GABAA receptor that will be, is, and/or has been exposed to ethanol. In some embodiments, the present invention provides methods of potentiating the activity of GABAA receptors, which comprises administering dihydromyricetin to the GABAA receptor. In some embodiments, the present invention provides methods of antagonizing the activity of ethanol on GABAA receptors, which comprises administering dihydromyricetin to the brain tissue acting on central nervous system GABAA receptors before, during, and/or after exposure to the ethanol.
In some embodiments, the present invention provides methods of treating, inhibiting, and/or reducing ethanol intoxication, at least one symptom of alcohol withdrawal syndrome, alcohol use disorders and/or alcohol abuse in a subject, which comprises treating, inhibiting, reducing and/or reversing GABAAR plasticity of the GABAA receptors, potentiating the activity of the GABAA receptors, and/or antagonizing the activity of ethanol on the GABAA receptors as disclosed herein. In some embodiments, the subject is mammalian, preferably human. In some embodiments, the symptom of alcohol withdrawal syndrome is selected from the group consisting of tolerance to ethanol, increased basal anxiety, and hyperexcitability. In some embodiments, the treatment reduces or inhibits a decrease in alertness, in the subject, which is caused by the exposure to ethanol. In some embodiments, the alcohol abuse is high alcohol consumption that is induced by alcohol exposure.
In the embodiments disclosed herein, dihydromyricetin may be administered before, during and/or after the exposure to ethanol. In some embodiments, dihydromyricetin is administered during a period ranging from about 30 minutes to directly before exposure to ethanol. In some embodiments, dihydromyricetin is administered during a period ranging from directly after exposure to ethanol to about 30 minutes after exposure to ethanol. In some embodiments, dihydromyricetin may be administered in the form of a foodstuff, such as a beverage, which may or may not contain ethanol. In some embodiments, dihydromyricetin may be administered in the form of a pharmaceutical formulation. In some embodiments, dihydromyricetin is co-administered with ethanol. In the embodiments disclosed herein, dihydromyricetin may be administered in an effective amount. In some embodiments, dihydromyricetin is administered in a therapeutically effective amount. In some embodiments, dihydromyricetin is administered in a unit-dosage form. In some environments, the amount of dihydromyricetin in a unit-dosage form for a human is about 50-70 mg.
Both the foregoing general description and the following detailed description are exemplary and explanatory only and are intended to provide further explanation of the invention as claimed. The accompanying drawings are included to provide a further understanding of the invention and are incorporated in and constitute part of this specification, illustrate several embodiments of the invention, and together with the description serve to explain the principles of the invention.
This invention is further understood by reference to the drawings wherein:
=vehicle,
=EtOH,
=EtOH+DHM,
=DHM.
The present invention is directed to methods and compositions for treating, inhibiting and/or reducing alcohol (ethanol, EtOH) intoxication, withdrawal from alcohol exposure and alcohol abuse which comprises the administration of dihydromyricetin (DHM).
DHM may be obtained from the Japanese Raisin Tree, Hovenia dulcis. Herbal remedies containing Hovenia dulcis extracts and purified DHM have been used to ameliorate liver injuries induced by alcohol and other chemicals, ameliorate the symptoms of alcohol hangovers, and relive alcohol intoxication. See Kawai K, et al. (1977) Experientia 33(11):1454; Hase K, et al. (1997) Biol Pharm Bull 20:381-385; Yoshikawa, et al. (1997) Yakugaku Sasshi 117(2):108-118; Ji Y, et al. (2001) Zhong Yao Cai 24:126-128; Ji Y, et al. (2002) Zhong Yao Cai 25:190-191; Chen S H, et al. (2006) Zhongguo Zhong Yao Za Zhi 31:1094-1096; Liu X L, et al. (2006) Zhongguo Zhong Yao Za Zhi 31:1097-1100; Fang H L, et al. (2007) Am J Chin Med 35:693-703; Hussain R A, et al. (1990) J Ethnopharmacol 28(1):103-115; Yoshikawa K, et al. (1993) Phytochemistry 34:1431-1433; Yoshikawa M, et al. (1996) Chem Pharm Bull (Tokyo) 44:1454-1464; Wang Y, et al. (1994) China Trad Herbal Drugs 25:306-307; and Kim K, et al. (2000) Korean J Med Crop Sci 8:225-233.
However, prior to the present invention, it was unknown whether DHM and/or any Hovenia dulcis extracts are capable of modulating GABAAR plasticity caused by alcohol exposure. In fact, prior to the present invention, no study has examined the impact of DHM and/or any Hovenia dulcis extracts on GABAARs. In addition, the prior art studies do not necessarily involve situations of chronic alcohol exposure such that it can be said that the prior art studies inherently teach or suggest the administration of DHM and/or a Hovenia dulcis extract to treat, inhibit and/or reverse some or all GABAAR plasticity caused by alcohol exposure.
A variety of flavonoids, such as myricetin, quercitin, hovenitin, laricitrin, apigenin, etc., in addition to dihydromyricetin, are found in Hovenia dulcis and other plants, e.g. Kudzu, and extracts thereof that are used in herbal remedies for various conditions. Many of the beneficial effects of flavonoids with respect to alcohol exposure are the result of their antioxidant properties. Thus, it was unknown whether DHM or any compound or extract of Hovenia dulcis would have any effect on GABAAR plasticity caused by chronic alcohol exposure or if the beneficial effects of DHM and extracts of Hovenia dulcis are merely a result of antioxidant activity. In addition, although we, the inventors, believed that some amounts of DHM might pass through the blood-brain barrier, it was unknown whether such amounts would have any impact on the GABAARs as many flavenoids and antioxidants do not. Therefore, we conducted various experiments as described herein. As provided herein, the experiment show that:
1) DHM potently (1 mg/kg) counteracts EtOH intoxication. Therefore, the present invention provides methods for treating, inhibiting, reducing EtOH intoxication in a subject which comprises administering DHM to the subject in need thereof. In some embodiments, the DHM is administered before, during and/or after exposure to EtOH. In some embodiments, the DHM is administered with EtOH. For example, the DHM is added to a composition comprising the EtOH, e.g. a foodstuff such as a beverage, and then the composition is administered to the subject. In some embodiments, the EtOH intoxication is acute EtOH intoxication.
2) DHM ameliorates EtOH exposure/withdrawal-induced behavior changes, including a) tolerance to EtOH; b) increase in basal anxiety, and c) hypersensitivity to PTZ-induced seizures (hyperexcitability). Therefore, the present invention provides methods for treating a symptom caused by withdrawal from EtOH exposure which comprises administering DHM to the subject in need thereof. In some embodiments, the DHM is administered before, during and/or after exposure to EtOH has stopped. In some embodiments, the symptom is selected from the group consisting of tolerance to EtOH, increased basal anxiety, and hyperexcitability.
3) DHM prevents the escalation of EtOH consumption in subjects. Therefore, the present invention provides methods for inhibiting, reducing or preventing a subject from voluntarily consuming more EtOH which comprises administering DHM to the subject. In some embodiments, the DHM is administered before, during and/or after consumption of EtOH. In some embodiments, the DHM is administered with the EtOH to be consumed. For example, the DHM is added to a composition comprising the EtOH, e.g. a foodstuff such as a beverage, and then the composition is administered to the subject.
4) DHM does not cause intoxication, sedation or anesthesia. Therefore, the present invention provides methods for treating, reducing or preventing a decrease in alertness caused by exposure to EtOH in a subject which comprises administering DHM to the subject. In some embodiments, the DHM is administered before, during and/or after exposure to EtOH. In some embodiments, the DHM is administered with EtOH. For example, the DHM is added to a composition comprising the EtOH, e.g. a foodstuff such as a beverage, and then the composition is administered to the subject.
The experiments disclosed herein also show that: a) the counteracting effects of DHM are antagonized in vivo and in vitro by flumazenil, and DHM competitively inhibits [3H]flunitrazepam binding to the benzodiazepine site of GABAARs; b) DHM antagonizes acute EtOH-induced potentiation of GABAARs; c) DHM antagonizes EtOH-induced alterations in responsiveness of GABAARs to acute EtOH including loss of Itonic modulation and increased mIPSC sensitivity; d) DHM potentiates GABAARs in hippocampal slices and cultured neurons, and retains efficacy in potentiating GABAARs even after EtOH exposure/withdrawal which induces tolerance to EtOH; and e) DHM blocks EtOH exposure/withdrawal-induced increases in the amount of GABAAR α4 subunits in rat hippocampus. In other words, DHM potentiates the activity of GABAARs associated with EtOH exposure, antagonizes the actions of EtOH on the respective GABAARs, and binds to the benzodiazepine site of the GABAARs. As used herein, “potentiates” means causing an increase in the activity and/or effectiveness of the GABAARs.
Surprisingly, the experiments herein also show that DHM inhibits, reduces, and even reverses the plasticity of GABAARs caused by exposure to EtOH. As used herein, “plasticity” of a receptor means a change in the subunit composition of the receptor. With respect to the instant invention, as used herein, “GABAAR plasticity” refers to the change in the subunit composition of GABAARs. Exposure to EtOH causes GABAARs containing α4βδ subunits to be internalized. When the α4 subunit returns to the postsynaptic membrane, the position of the 6 subunit is changed such that the delta subunit is no longer associated with the α4 subunit, thereby resulting in GABAAR plasticity, i.e. an increase in the α4 subunit at the postsynaptic membrane as compared to that prior to EtOH exposure. As shown herein, DHM inhibits, reduces, reverses and/or prevents GABAAR plasticity caused by exposure to EtOH. These results are surprising because, until the present invention, there are no known compounds or compositions which inhibit, reduce, reverse and/or prevent GABAAR plasticity caused by EtOH exposure. The results of the experiments herein are especially surprising in view of the fact that other flavonoids, e.g. daidzin and quercetin, which are similar to DHM, do not exhibit activities that are the same or similar to DHM, i.e. potentiate GABAARs, antagonize EtOH actions, and bind the benzodiazepine sites of GABAARs.
Therefore, the present invention provides methods for treating, inhibiting, reducing, reversing and/or preventing GABAAR plasticity caused by exposure to EtOH which comprises administering DHM to the brain tissue acting on GABAARs. As used herein, “GABAAR plasticity caused by EtOH exposure” refers to GABAAR plasticity as described by Liang J, et al. (2007) J Neurosci. 27(45):12367-77; Zucca S and Valenzuela C F (2010) J Neurosci. 30(19):6776-81; and Shen et al. (2011) Mol Pharmacol. 79(3):432-42. In some of the embodiments of the present invention, the amount of DHM administered is an effective amount. As used herein, an “effective amount” of DHM is an amount that results in the desired effect as compared to a control—an amount that treats, inhibits, reduces and/or reverses GABAAR plasticity caused by exposure to ethanol, or potentiates the activity of a GABAA receptor, or antagonizes the activity of ethanol on a GABAA receptor. For example, in effective amount of DHM which reverses some or all GABAAR plasticity caused by exposure (including chronic intermittent exposure and single dose exposure) to EtOH is that which increases the amount of GABAARs having a composition and/or activity that is substantially similar to or the same as the corresponding naïve GABAARs.
A “therapeutically effective amount” of DHM is a quantity sufficient to, when administered to a subject, treat, inhibit, reduce and/or reverse GABAAR plasticity caused by exposure to EtOH, or potentiate the activity of a GABAAR, or antagonize the activity of ethanol on a GABAAR in the subject such that the condition of the subject is an observable improvement as compared to the condition of the subject prior to the treatment or as compared to a control subject. Also, as used herein, a “therapeutically effective amount” of DHM is an amount which when administered to the subject treats a given clinical condition, e.g. ethanol intoxication, at least one symptom of alcohol withdrawal syndrome, alcohol use disorders, or alcohol abuse, in the subject as compared to a control. Typically, therapeutically effective amounts of DHM can be orally or intravenously administered daily at a dosage of about 0.002 to about 200 mg/kg, preferably about 0.1 to about 100 mg/kg, e.g. about 1 mg/kg of body weight.
Ordinarily, a dose of 0.01 to 10 mg/kg in divided doses one to four times a day, or in sustained release formulation will be effective in obtaining the desired pharmacological effect. It will be understood, however, that the specific dose levels for any particular subject will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, and rate of excretion, drug combination and the severity of the particular disease and/or condition. Frequency of dosage may also vary depending on the particular disease and/or condition treated. It will also be appreciated that the effective dosage for treatment may increase or decrease over the course of a particular treatment. Changes in dosage may result and become apparent by standard diagnostic assays in clinical techniques known in the art. In some instances chronic administration may be required. Effective amounts and therapeutically effective amounts of DHM may be readily determined by one of ordinary skill by routine methods known in the art.
In some embodiments, an effective amount of DHM may be administered in the form of a foodstuff, such as a beverage. In some embodiments, the beverage contains alcohol which may be made from fermented grains (e.g., whiskey, bourbon, rye, vodka, gin and/or beer), fermented fruits (e.g., wine, brandy, sherry and cognac), sugar cane and/or sugar beets (e.g., rum), and/or fermented head of the agave (tequila). In some embodiments, an effective amount of DHM may be administered in the form of a chewing gum composition.
The pharmaceutical formulations of the invention comprise a divided dose or a single dose of DHM and may be prepared in a unit-dosage form and/or packaging appropriate for the desired mode of administration. The pharmaceutical formulations of the present invention may be administered for therapy by any suitable route including oral, rectal, nasal, topical (including buccal and sublingual), dermal, mucosal, vaginal and parenteral (including subcutaneous, intramuscular, intravenous and intradermal). It will be appreciated that the preferred route will vary with the condition and age of the recipient, the nature of the condition to be treated. For example, in some embodiments, a therapeutically effective amount of DHM may be administered to a subject in the form of a transdermal patch or an effervescent tablet (e.g. a tablet comprising an effective amount of DHM, a carbonate salt, such as sodium bicarbonate, and an acidic material, such as citric acid which results in effervescence when dissolved in a liquid such as water).
In some embodiments, the unit dose of DHM for a human subject is about 50-70 mg. Thus, in some embodiments, foodstuffs, transdermal patches, chewing gums, and/or effervescent tablets according to the present invention comprise about 50-70 mg per unit.
The Institutional Animal Care and Use Committee approved all animal experiments. Male and female Sprague-Dawley (SD) rats (250-300 g) were housed in the vivarium under a 12 h light/dark cycle and had ad libitum access to food and water.
Dihydromyricetin (DHM, (2R,3R)-3,5,7-trihydroxy-2-(3,4,5-trihydroxyphenyl)-2,3-dihydrochromen-4-one) was purchased from ZR Chemical, Shanghai, China (CAS No. 27200-12-0 98% purified by HPLC). Flumazenil, picrotoxin and bicuculine were purchased from Sigma.
Data were from at least three independent preparations of neuron cultures and/or rats as indicated. Sigmaplot (Windows version 10.1) and SigmaStat (Windows version 3.5) were used for data display and statistical analysis. Data were expressed as mean±SEM. One-way or two-way repeated measures (RM) ANOVA with post hoc comparison analyses based on Dunnett or Newman-Keuls, and student t-test were used to determine significant differences between treatment groups and vehicle group.
Metabolic studies performed in rats showed that there is no metabolic change (to food and water intake, urine volume and stool volume) induced by DHM (oral administration, 1 mg/kg) administration (data not shown).
The effect of DHM on EtOH-induced LORR in rats was examined using a standard LORR assay known in the art. See Kakihana R, et al. (1966) Science 154(756):1574-1575). Briefly, after drug injection, rats were placed in the supine position in a V-shaped support. LORR onset time was taken from the endpoint of drug injection (i.p.). LORR duration ended when the animal was able to flip over three times in 30 s. LORR assays were blindly performed. LORR durations were reported as mean (min)±SEM.
EtOH (3 g/kg, i.p.) induced 72±2 min LORR in the control group (pre-treated with saline, 20 ml/kg, i.p. 30 min prior to EtOH injection). Pre-treatment with DHM (1 mg/kg, i.p., 30 min prior to EtOH injection) the EtOH-induced LORR was reduced to 8±4 LORR (10.6±5.9% of control,
Then the effect of DHM on single-dose EtOH-intoxication and withdrawal was examined. Rats were i.p. injection with saline (20 ml/kg, vehicle), EtOH (3 g/kg), EtOH+DHM (30 min after EtOH, 1 mg/kg), or DHM (1 mg/kg) alone. After a 48 hr withdrawal period, EtOH-induced LORR assays (EtOH, 3 g/kg, i.p.) were performed. LORR duration was significantly reduced by single-dose EtOH intoxication/withdrawal, i.e. 9±3 vs. 58±5 min (vehicle). This suggests that EtOH withdrawal induces EtOH tolerance. DHM post-treatment with EtOH significantly inhibited, reduced and/or prevented a decrease in LORR duration from EtOH withdrawal (
Pentylenetetrazol (PTZ)-induced seizures were also measured in rats. After 24 hr withdrawal from vehicle (saline, 20 ml/kg, i.p.), EtOH (3 g/kg, i.p.), DHM+EtOH (1 mg/kg+3 g/kg, i.p.) or DHM (1 mg/kg, i.p.) treatment, rats were tested with PTZ-induced seizures. PTZ dose used in this study (42 mg/kg in saline) was determined as the dose that induced seizures in 75% naïve rats. Briefly, after i.p. injection of PTZ, the time to onset and the duration of tonic-clonic seizures was determined as described previously. The researchers who conducted the animal behavior experiments were blind to treatment groups. Animals were used once only for any determination.
EtOH withdrawal notably increased the PTZ-seizure duration from 1.7±0.8 (vehicle) to 8.1±1.2 min (
These findings suggest that DHM effectively inhibits, reduces, and/or prevents acute EtOH intoxication, EtOH exposure/withdrawal-induced EtOH tolerance, and EtOH withdrawal-induced hyperexcitability.
To determine whether DHM prevents EtOH intoxication-induced alterations in GABAAR sensitivity to acute EtOH, the effects of DHM on EtOH-withdrawal-induced GABAAR functional alterations with whole-cell patch-clamp recording from dentate gyms granule cells (DGCs) in rat hippocampal slices at 48 hr withdrawal was examined.
Transverse slices (400 μm) of dorsal hippocampus were obtained with a Vibratome (VT 100, Technical Products International, St. Louis, Mo.) and standard techniques known in the art. Slices were continuously perfused with artificial cerebrospinal fluid (ACSF). See Liang, J., et al. (2007) J Neurosci 27:12367-12377.
Whole-cell patch-clamp recordings were obtained at 34±0.5° C. from cells located in the DG layer at a holding potential of −70 mV, during perfusion with artificial cerebrospinal fluid (ACSF, 125 mM NaCl, 2.5 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 26 mM NaHCO3, and 10 mM D-glucose). The ACSF was continuously bubbled with 95% O2-5% CO2 to ensure adequate oxygenation of slices and a pH of 7.4. Patch electrodes were pulled from thin-wall borosilicate glass pipettes with resistances of 7.5-9 MΩ and were filled with pipette solution (i.e. 137 mM CsCl, 2 mM MgCl2, 1 mM CaCl2, 11 mM EGTA, 10 mM HEPES and 3 mM ATP, pH adjusted to 7.30 with CsOH). Signals were recorded in voltage-clamp mode with a Axopatch 700B amplifier (Molecular Devices, Sunnyvale, Calif.). Whole cell access resistances were in the range of <25 MΩ before electrical compensation by about 70%. During voltage-clamp recordings, access resistance was monitored by measuring the size of the capacitative transient in response to a 5 mV step command and the data were abandoned if changes >20% were encountered. At least 10 min was allowed for equilibration of the pipette solution with the intracellular milieu before commencing mIPSC recordings. Intracellular signal was low-pass filtered at 3 kHz and data were acquired with Digidata 1440A and software CLAMPEX 10 (Molecular Devices) at a sampling frequency of 20 kHz.
Pharmacologically-isolated GABAAR-mediated mIPSCs were recorded as previously described (Liang (2007) and Shen (2011)). For GABA concentration-response curves, evoked GABAAR-currents were recorded during acute applications of GABA, DHM, or diazepam onto neurons through a removable pipette tip using a Valvelink 8.02 fast-exchange perfusion system (AutoMate Scientific, USA). Data were analyzed using the Clampfit (Version 9.0, Molecular Devices) and the MiniAnalysis Program (versions 6.0.7, Synaptosoft, Decatur, Ga.).
The MiniAnalysis program (Synaptosoft, Decatur, Ga.) was used to analyze mIPSCs. Itonic is the averaged baseline currents of a given recording period. Itonic amplitude was calculated as the difference between the holding currents measured before and after picrotoxin (100 μM) or bicuculline (10 μM). See Wei, W., et al. (2004) J Neurosci 24, 8379-8382; Liang (2007); and Shen (2011). Briefly, the recordings were low-pass filtered off-line (Clampfit software) at 2 kHz. The mIPSCs were detected (Mini Analysis Program, version 6.0.7) with threshold criteria of 8 pA amplitude and 20 pA*ms charge transfer. The frequency of mIPSCs was determined from all automatically detected events in a given 100 s recording period. For kinetic analysis, only single event mIPSCs with a stable baseline, sharp rising phase (10 to 90% rise time), and exponential decay were chosen during visual inspection of the recording trace. Double and multiple peak mIPSCs were excluded. At least 100 individual mIPSC events were recorded under each experimental condition. The mIPSC kinetics was obtained from analysis of the averaged chosen single events aligned with half rise time in each cell. Decay time constants were obtained by fitting a double exponential to the falling phase of the averaged mIPSCs. Itonic magnitudes were obtained from the averaged baseline current of a given recording period. Itonic amplitude was calculated as the difference between the holding currents measured before and after the application of picrotoxin (50 μM) or bicuculline (10 μM). See Liang J et al (2007); Shen (2011); Hamann (2002); and Mangan P S, et al. (2005) Mol Pharmacol 67(3):775-788. The investigator performing the recordings and mIPSC analysis was blind to the treatment (vehicle, EtOH, E+D, or D) that the rats received.
Recordings from neurons of EtOH-treated rats revealed a loss of Itonic potentiation by acute EtOH (60 mM) application (
Parallel Western blots from rat hippocampus were examined to determine whether EtOH induced changes in total protein of GABAAR α4 subunits. Hippocampal tissues from rats were lysed in RIPA-buffer containing 1% Triton X-100, 0.1% sodium dodecyl sulfate (SDS), 50 mM Na3PO4, 150 mM NaCl, 2 mM EDTA, 50 mM NaF, 10 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride (PMSF) and Complete protease inhibitor cocktail (Roche). The lysate was centrifuged for 15 min (14,000×g, 4° C.) and the supernatant collected for Western blot analysis. Western blots were performed using rabbit anti-GABAAR α4 (aa 379-421) and mouse anti-β-actin (Sigma) followed by HRP-conjugated secondary antibodies. Bands were detected using ECL detection kit (Amersham) and analyzed by densitometric measurements using ImageQuant 5.2 (Molecular Dynamics). Bands were stripped with buffer containing 62.5 mM Tris-HCl, 100 mM β-mercaptoethanol and 2% SDS (pH 6.7) and reprobed several times. Protein concentrations were determined with BCA Protein Assay Kit (Pierce) according to the manufacturer instructions.
Western blots of hippocampal tissue GABAAR α4 subunit after 48 hr withdrawal from rats gavaged with vehicle, EtOH, E+D or DHM are shown in
DHM Enhances GABAAR-Mediated Currents, and Antagonizes their Potentiation by Acute EtOH in DGCs from Naïve Rats
To determine whether the anti-alcoholic effects of DHM result from its interaction with GABAARs which represent a major target of alcohol actions, the effects of acute DHM on GABAAR function in DGCs in hippocampal slices from naïve rats were examined as described above. Acute DHM (0.3 μM) enhanced GABAAR-mediated Itonic from 17.5±4.9 to 29.0±6.7 pA, prolongs mIPSC decay time and enhances mIPSC total charge transfer (area) in DGCs (area increased from 571±61 to 615±22 fC), in a concentration-dependent manner (
Both EtOH and DHM potentiate GABAAR-mediated currents when applied separately. However, EtOH-induced Itonic potentiation was concentration-dependently decreased by DHM in the presence of EtOH (decreased from 43.8±1.8 to 32.0±2.0 pA by 1 μM DHM,
These data indicate that DHM antagonizes EtOH intoxication-induced GABAAR plasticity by interfering with EtOH-induced potentiation of GABAARs. Therefore, the present invention provides methods of antagonizing EtOH-induced GABAAR plasticity by the co-administration of DHM and EtOH. The present invention also provides methods of antagonizing EtOH-induced GABAAR plasticity by administering DHM prior to exposure to EtOH. In some embodiments, the present invention provides methods for potentiating GABAAR-mediated currents which comprises administering DHM.
To determine whether DHM inhibit and/or prevent EtOH-induced GABAAR plasticity in cultured neurons in vitro, the following experiment was conducted.
Hippocampal neurons from embryonic day 18 rats were prepared by papain dissociation (Worthington Biochemical, Lakewood, N.J.) and cultured in neurobasal medium and B27 supplement (Invitrogen). Cultures were kept at 37° C. in a 5% CO2 humidified incubator as described previously. See Shen, Y., et al. (2011) Mol Pharmacol 79:432-442.
Hippocampal neurons from embryonic day 18 SD rats were prepared by papain dissociation (Worthington Biochemical, Lakewood, N.J.) and cultured in neurobasal medium (Invitrogen) and B27 supplement as previously reported. See Stowell J N and Craig A M (1999) Neuron 22(3):525-536. Briefly, embryos were removed from maternal rats anesthetized with isoflurane and euthanized by decapitation. Hippocampus were dissected and placed in Ca2+- and Mg2+-free HEPES-buffered Hank's buffered salt solution (pH 7.45). Tissues were dissociated by papain digestion followed by trituration through a Pasteur pipette and papain inhibitor treatment. Cells were pelleted and resuspended in neurobasal medium containing 2% B27 serum-free supplement, 100 U/ml penicillin, 100 μg/ml streptomycin, 0.5 mM glutamine (all from Invitrogen), and 10 μM glutamate (Sigma).
Dissociated neurons were then plated at a density of 0.3×105 cells/cm2 onto 12 mm round coverslips in 24-well plates (for patch-clamp recording) and/or at a density of 0.5×105 cells/cm2 in 6-well plates (for Western blot and biotinylation assays) coated with poly-D-lysine (Sigma, 50 μg/ml). Cultures were kept at 37° C. in a 5% CO2 humidified incubator. Thereafter, one third to half of the medium was replaced twice a week with neurobasal culture medium containing 2% B27 supplement, and 0.5 mM glutamine.
After DIV13-14 neurons (cultured in vitro for 13-14 days), half of the medium of cultured neurons was replaced with neurobasal culture medium containing 120 mM EtOH (final EtOH concentration was 60 mM), 0.2 μM DHM plus 120 mM EtOH, or 0.2 μM DHM only (DHM control, i.e. without EtOH) for 30 min and then replaced all medium with half fresh neurobasal culture medium plus half original medium, respectively. Control neurons were treated with corresponding vehicle as same procedure as EtOH-treated neurons. The concentration of 60 mM EtOH was selected in view of prior experiments. See Liang J et al (2007). Specifically, the concentration of 60 mM EtOH used to treat cultured neurons was chosen to match blood levels measured in adult rats after intoxication with gavage of 5 g/kg, which produced about 60 mM blood peak plasma [EtOH] lasting for about 2 to 3 hr and induced significant plasticity in GABAARs and tolerance.
DIV14 neurons (cultured in vitro for 14 days) were treated with either vehicle, EtOH, EtOH+DHM or DHM alone, followed by 24 h withdrawal. Then, immediately before electrophysiological recording, cells grown on coverslips were transferred to a perfusion chamber (Warner Instruments) and visualized with an inverted microscope (TE200, Nikon). Whole-cell patch-clamp recordings were obtained from cultured neurons under voltage-clamp mode at room temperature (22-25° C.), at a holding potential of −70 mV. Cells were perfused with an extracellular solution (137 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 20 mM glucose and 10 mM HEPES (310-320 Osm, pH adjusted to 7.40 with NaOH)). Glass pipettes were filled with the same internal solution as that in slice recordings, with an input resistance of 4-7 MΩ. GABAAR-mediated mIPSCs were recorded using the same pharmacological method as mentioned above. For GABA concentration-response curve, evoked GABAAR-mediated currents were recorded by acute applications of GABA and/or DHM onto the cultured neurons through a removable tip that were positioned close to the soma of the neuron with a Valvelink 8.02 fast-exchange perfusion system (AutoMate Scientific, USA). Electrical signals were amplified using a Multiclamp 200 B amplifier (Molecular Devices, USA). After establishing the whole-cell configuration, at least 10 min were allowed to elapse before the application of drug to allow the membrane patch to stabilize and exchange of ions between the recording electrode and the cytosol to occur. Data were acquired with pClamp software (Version 10.0, Molecular Devices, USA), digitized at 20 kHz (Digidata 1440A, Molecular Devices), and analyzed using the Clampfit software (Version 10.0, Molecular Devices) and the Mini Analysis Program (versions 6.0.7, Synaptosoft, Decatur, Ga.) using methods known in the art. See Hamann M, et al. (2002) Neuron 33(4):625-633; Stell B M and Mody I (2002) J Neurosci 22(10): RC223; and Liang (2007).
EtOH exposure/withdrawal-neurons showed dramatic decrease in Itonic magnitude (from 13.8±1.4 pA in vehicle-neurons to 5.6±1.0 pA in EtOH-neurons) and in its responsiveness to acute EtOH (EtOH potentiation decreased from 109.6±15.7% in vehicle-neurons to 14.3±18.9% in EtOH-neurons,
Western blots of the cultured neurons were performed and examined as described herein. Biotinylation assays for GABAARs of the cultured neurons were performed as described previously. See Chung W O, et al. (2000) Infect Immun 68(12):6758-6762. Briefly, the neurons in culture dishes were placed on ice and washed twice with ice cold PBS. Then the neurons were incubated for 30 min on ice with PBS containing 1 mg/ml sulfo-NHS-LC-biotin (ProteoChem). After quenching the biotin-reaction with Tris-buffered saline (TBS), neurons were lysed in 150 μl of modified RIPA-buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na2 EDTA, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM b-glycerophosphate, 1 mM Na3VO4, and 1 μg/ml leupeptin). The homogenates were centrifuged for 15 min (14,000×g, 4° C.). An aliquot (10%) of the supernatant was removed to measure β-actin. The remaining supernatant was incubated with 60 μl of 50% neutravidin agarose (Pierce Chemical Company) for 4 hr at 4° C. and washed four times with lysis buffer. Agarose-bound proteins were taken up in 20 μl of SDS sample buffer and boiled. Western blots were performed as mentioned above.
The data from the Western blots and biotinylation data showed that DHM eliminates or reverses EtOH exposure/withdrawal-induced alterations in the cell-surface GABAAR α4 subunit in cultured neurons (
The effect of DHM on cultured hippocampal neurons was also examined. The concentration-response curves of DHM on GABAAR-mediated Itonic (
To clarify the direct effects of DHM on GABAARs, GABA (1-300 μM) was puffed onto cultured hippocampal neurons and the concentration-response curve was established. Co-application of DHM (0.3 and 1 μM) with GABA increased the amplitude of GABA-activated currents at the same concentration of GABA, producing a left shift of the GABA concentration-response curve (
Further LORR assays were conducted as follows. Rats were divided into 4 groups and intraperitoneally (i.p.) injected with saline, EtOH (3 g/kg, E), EtOH combined with DHM (1 mg/kg, E+D1), or DHM (D1). EtOH induced 69±18 LORR. E+D1 reduced LORR to 2.7±1.4 (
Additional pre-treatment and post-treatment experiments were also conducted. 30 min prior to EtOH injection (D1+E), DHM reduced LORR to 8.2±4.1 (
To examine the target of DHM's anti-EtOH effects, flumazenil, the selective benzodiazepine antagonist of modulation of GABAARs, was tested. See Hunkeler, W., et al. (1981) Nature 290:514-516. EtOH induced 69±11.3 LORR; co-injection of DHM (3 mg/kg) and EtOH reduced LORR to 2.7±1.7 (
High doses of DHM (doses hundreds-fold higher than that for its antagonistic effects on EtOH intoxication) were examined. DHM (100 and 300 mg/kg) induced only 0.9±0.8 and 4.0±2.8 LORR, respectively (
During the LORR assay, venous blood samples were taken at the various points from 5-180 min to measure plasma EtOH concentrations (plasma [EtOH]) from EtOH− and EtOH+DHM groups. Blood samples from the tail vein of rats at different time points (0, 5, 30, 60, 90, 180 min) after EtOH or E+D i.p. injections, or from the rats after the voluntary alcohol two-bottle choice procedure (EtOH− and EtOH+DHM group) were collected for plasma [EtOH] assays. See Liang (2007). The rat was put into a restraint tube and its tail was warm in about 40° C. The tail vein at the tip of the rail was punched with a sharp blade. Approximately 0.2 ml venous blood was dropped to a capillary blood collection tube containing lithium heparin (Ram Scientific Inc. Yonkers, N.Y.). Blood samples were centrifuged at 2500 rpm for 15 min. The supernatant was collected and stored at −80° C. until assay. The EtOH content of each blood sample was measured in duplicate along with EtOH standards using the alcohol oxidase reaction procedures (GM7 Micro-Stat; Analox Instruments, Lunenberg, Mass.).
EtOH induced onset of LORR within 5 min. Plasma [EtOH] rapidly increased for 5 min followed by a slower increase to around 60 min, then [EtOH] declined gradually. In E+D1 and E+D10 (DHM 1, 10 mg/kg) groups, the rise time of plasma [EtOH] was slowed at early time (
As performed previously, whole-cell patch-clamp recordings in dentate gyms granule cells (DGCs) from hippocampal slices in vitro were conducted. Bath application of EtOH (60 mM) increased Itonic from 22.0±0.7 to 46.9±1.4 pA and enhanced mIPSCs from 0.53±0.02 to 0.64±0.02 nC (
The effect of flumazenil on the anti-EtOH actions of DHM were then tested as provided herein. DHM (3 μM) decreased EtOH-potentiated Itonic from 44.8±2.3 to 21.0±0.9 pA and mIPSCs from 0.78±0.01 to 0.70±0.02 nC, while flumazenil (10 μM) reversed the DHM actions (reversed Itonic to 37.3±1.6 pA, and mIPSCs to 0.78±0.01 nC,
The effects of DHM (0.1 to 30 μM) on GABAAR-mediated Itonic and mIPSCs of DGCs in hippocampal slices. DHM (1 μM) enhanced Itonic (22.5±2.5 to 44.0±4.1 pA) and increased mIPSC area (0.59±0.01 to 0.72±0.03 nC,
To further examine the site of DHM actions on GABAARs, the flumazenil effects on DHM enhancing GABAAR function in cultured hippocampal neurons at DIV13-14 (DIV: days in vitro) was assayed. DHM (1 μM) potentiated Itonic (194.9±13.6% of control) and mIPSC area (181.8±9.2% of control,
The actions of DHM (0.03-100 μM) on the benzodiazepine sites using [3H]flunitrazepam binding in cortical membrane homogenates from naïve adult rats was examined. Standard procedures for preparation of rat cortical membranes for radioligand binding assays were conducted as previously described with modifications in speed and number of centrifugation and washes, and buffer compositions. See Li G D., et al. (2010) J Biol Chem 285:8615-8620. Naïve rat cortex was dissected from brain and homogenized in 0.32 M sucrose, 10 mM HEPES buffer (pH 7.4), and centrifuged at 650×g, 4° C. The subsequent supernatant was centrifuged at 150,000×g to collect the desired membrane-containing pellet. The pellet was washed and centrifuged two more times, first using ice-cold water and second using membrane buffer containing 50 mM KH2PO4, 1 mM EDTA, 2 mM benzamidine HCl, 0.5 mM DTT, 0.1 mM benzethonium HCl, 0.01% bacitracin, 0.2 PMSF (pH 7.4), and the resulting pellet was frozen. On the day of binding assay, the pellet was homogenized in assay buffer containing 50 mM KH2PO4, 1 mM EDTA, 200 mM KCl (pH 7.4) and centrifuged, and resuspended in fresh assay buffer to a final protein concentration of 1 mg/ml. [3H]flunitrazepam (85.2 Ci/mmol, PerkinElmer, Boston, Mass.), brain homogenate, and DHM were combined for a final assay volume of 0.5 ml, incubated on ice, and filtered by Brandel cell harvester M-24R (Brandel Co, Gaithersburg Md.). Samples were counted in a Beckman LS-3801 liquid scintillation counter. Specific binding was defined as the total amount bound (zero unlabeled ligand) minus the binding in the presence of 10 μM final concentration flurazepam (Sigma). Data was analyzed with GraphPad Prism 4.0 Software (San Diego, Calif.) to determine IC50 (One-site competition equation) and Hill slope (Sigmoidal Dose-Response equation). Experiments were conducted in triplicate.
Significant inhibition of [3H]flunitrazepam binding by DHM was observed, starting at 0.3 μM in a concentration-dependent manner, with an IC50 of 4.36 μM and Hill slope of −0.73 (
The effects of DHM on GABAAR-mediated currents in cultured hippocampal neurons were also examined. DHM concentration-dependently potentiated Itonic (from 9.5±1.5 to 21.0±2.3 pA by 0.3 μM DHM, EC50 was about 0.20 μM) and increased mIPSCs (to 128.2±8.3% of control by 1 μM DHM, EC50 was about 0.20 μM; the responses to higher than 1 μM DHM decreased slightly,
The effects of DHM on GABAAR-currents induced by focal puffs of 10 and 300 μM GABA in cultured neurons at DIV14 were examined. Co-application of DHM (0.3 and 1 M) and GABA increased the amplitude of GABA-currents and produced a left shift of the GABA concentration-response curve (
The effect of DHM on EtOH withdrawal symptoms in rats was examined. Rats were divided into 4 groups and gavaged with vehicle, EtOH (5 g/kg, E), EtOH combined with DHM (1 mg/kg, E+D) or DHM respectively. 48 hr after injection, rats were sub-divided into 3 groups to measure signs of EtOH withdrawal.
Anxiety and locomotion/ataxia associated with EtOH withdrawal was measured on an elevated plus-maze in EtOH-withdrawn rats (EPM,
Subjects belonging to the vehicle group spent 2.71±0.71 in open arms and 1.80±0.67 in closed arms. Subjects belonging to the EtOH group spent significantly shorter time in the open arms (0.88±0.32) and longer time in closed arms (3.64±0.27) than vehicle group; while subjects belonging to be EtOH+DHM (E+D) group spent similar times (open: 2.68±0.77 and closed: 1.88±0.79). DHM did not affect the time rats spent in either arm (open: 2.92±0.70 and closed: 1.52±0.56). These data suggest that (1) EtOH exposure/withdrawal produces anxiety, (2) DHM combined with EtOH inhibits, reduces and/or prevents EtOH-induced anxiety, and (3) DHM does not affect anxiety levels.
Tolerance to EtOH was measured with acute EtOH-induced LORR (in minutes,
As described herein, hyperexcitability was assayed with PTZ-induced seizures duration (
The total protein content of GABAAR α4 subunit in hippocampus 48 hr after the above 4 treatments was assayed. Western blots showed that EtOH exposure increased the total α4-protein level to 184.0±26.0% as compared to that of the vehicle group. There was no increase in the total α4-protein level in EtOH+DHM group (93.0±21.0% of control). DHM exposure had no effect on α4 subunit level (88.3±10.3% of control,
Whether DHM prevents EtOH-induced GABAAR plasticity in CNS neurons was assayed. Four groups of rats were gavaged with vehicle (vehicle group), EtOH (EtOH group), EtOH combined with DHM (1 mg/kg, E+D, EtOH+DHM group), or DHM (DHM group). After 48 hr withdrawal, whole-cell GABAAR-mediated currents were recorded on DGCs in hippocampal slices. In the vehicle group, bath application of EtOH (60 mM) enhanced Itonic from 28.8±3.1 to 62.1±3.3 pA (
The effect of zolpidem, an agonist of benzodiazepines, on DGCs in rats following the above 4 treatments. Zolpidem induced a potentiation of GABAAR-currents in the DHM group as in the vehicle group, but did not affect GABAAR-currents in EtOH group, thereby suggesting that EtOH produces cross-tolerance to zolpidem (
The effects of DHM on cultured neurons pre-exposed to EtOH were examined. Bath application of DHM enhanced Itonic and mIPSCs concentration-dependently (0.03-30 μM,
The surface expression of α4 subunit in cultured neurons was measured using cell-surface biotinylation followed by Western blot analysis. EtOH treated neurons showed increased α4 subunit surface expression (249.7±28.1% of control); while this increase was blocked (123.0±8.4% of control,
The effects of DHM on alcohol consumption were examined. All fluids were presented in 100 ml graduated glass cylinders with stainless-steel drinking spouts inserted 15 min after the lights went off in the reversed light/dark cycle room. Bottles were weighed 30 min and 24 hrs after the fluids were presented. Each rat was weighed daily to monitor health and calculate the grams of ethanol intake per kilogram of body weight. Rats were divided into 4 groups and offered intermittent access to two bottle choice of water/water, 20% EtOH/water, E+D/water, or DHM/water respectively.
Rats were trained to have free two-bottle choice access to water/water, 20% (w/v) EtOH/water, EtOH+DHM (0.05 mg/ml, E+D)/water or DHM/water for two weeks. Sweetener (2 pk/L) was added to every bottle for the first week. Sweetener (1 pk/L) was added to every bottle for the second week. After training, rats were given two-bottle choice access to water/water, EtOH/water, E+D/water, or DHM/water (without sweetener for all) for three 24-hr-sessions per week (Mondays, Wednesdays and Fridays). Rats had unlimited access to two bottles of water between the EtOH-access periods. The placement of the EtOH bottle was alternated each EtOH drinking session to control for side preferences. Rats were maintained on 20% EtOH intermittent access two-bottle choice paradigm for 7 weeks (21 EtOH-access sessions). Half of EtOH group had DHM added to the EtOH bottle beginning on the fifth week (13th session). The rest of the EtOH group continued EtOH-access sessions. EtOH consumption was expressed as grams of EtOH consumed per kilogram of body weight. Rats access to two bottles of water were taken as the control-group. There was no significant difference in body weight between the control and the EtOH-drinking rats at the end of the experiments.
Starting from the second week, EtOH consumption increased from 3.1±1.3 to 7.5±0.5 g/kg/day in EtOH/water-group. Co-administration of EtOH with DHM (E+D/water group) counteracted this increase in EtOH-intake (2.6±0.4 g/kg/day,
At the end of the fourth week, plasma [EtOH] from the group of rats exposed to E+D/water was significantly lower than that from the group exposed to E/water. Plasma [EtOH] correlated well with the measured amount of EtOH consumed. Plasma [EtOH] (mg/dl) for each animal was measured following 30, 45, 60 and 100 min of voluntary 20% EtOH started at the alcohol day of the end of the 4th week. Plasma [EtOH] in the two groups are significantly different (p<0.05,
Additional experiments were conducted that show that there was a reduction in EtOH (3 g/kg, i.p.)-induced LORR duration by combined-treatment with DHM (1 mg/kg, i.p., n=6 rats/group) in female rats that is similar to that of male rats. Similarly, in female rats, co-administration of DHM+EtOH similarly inhibits, reduces and/or prevents EtOH intoxication/withdrawal-induced increases in PTZ-induced seizure duration and seizure incidence.
The experiments herein show that in hippocampal neurons in cultured or slice, DHM concentration-dependently potentiated GABAAR-mediated mIPSCs and tonic current. With cultured neurons, DHM caused a left shift of the GABA concentration-response relationship. These results suggest DHM potentiates both synaptic and extrasynaptic GABAARs. However, DHM exposure/withdrawal did not induce long lasting GABAAR plasticity at the cellular level. DHM does not induce intoxicated symptoms such as LORR nor causes AWS such as increase in seizures susceptibility nor induces cross-tolerance to EtOH at a dose range that is adequate to ameliorate EtOH intoxication. Therefore, DHM may be used to treat acute and chronic alcohol consumption.
In rats withdrawal from EtOH exposure, behavioral experiments in vivo show decreased seizure thresholds, anxiety, and tolerance to sedative/anesthetic drugs in a manner similar to the symptoms observed in human AWS. As disclosed herein, studies with hippocampal slices, show that there is significant Itonic sensitivity to acute EtOH at 48 hr withdrawal from DHM co-administration with EtOH. Also, DHM co-administration with EtOH blocks the reduction of baseline Itonic magnitude by EtOH treatment. Similar effects of DHM were found in cultured hippocampal neurons. These results from rat hippocampus show that EtOH-induced alterations in α4-containing GABAARs are blocked by DHM. These results suggest that DHM not only antagonizes the effect of EtOH on GABAAR function but also blocks α4-containing GABAARs re-localization from extrasynapses to synapses. Therefore, DHM may be used to treat alcohol use disorders associated with GABAAR plasticity resulting from exposure to EtOH.
The experiments herein show that, with naïve GABAARs and GABAARs that have been exposed to EtOH, DHM continues to effectively potentiate synaptic and extrasynaptic GABAARs. Moreover, DHM blocks acute EtOH potentiation of synaptic GABAARs in native hippocampal neurons in brain slices. These results indicate that DHM is a modulator of GABAARs, thereby indicating that DHM may be an effective treatment for alcohol intoxication and AWS in subjects who are tolerance to other medications, such as benzodiazepines.
Comparative experiments with daidzin, quercetin, genistein, myricetin, and puerarin show that the effects of DHM on alcohol intoxication, alcohol use disorders and alcohol abuse associated with GABAARs due to EtOH exposure may be unique. In particular, patch clamp recordings of neurons from rat hippocampal slices show that A) DHM potentiates extrasynaptic GABAAR-mediated tonic current (Itonic) and post-synaptic currents (mIPSCs), whereas daidzin had no effect on Itonic and significantly potentiated mIPSCs. Quercetin had no effect on either Itonic or mIPSCs. Thus, DHM may be used to selectively modulate extrasynaptic GABAARs. Patch clamp recordings of neurons in rat hippocampal slices show that, in the presence of 60 mM EtOH, DHM dose-dependently blocks EtOH potentiation of GABAAR-mediated Itonic and mIPSCs, whereas daidzin and quercetin do not. [3H]flunitrazepam binding assay shows that DHM, daidzein and dainzin bind GABAARs, but are significantly replaced by [3H]flunitrazepam; while genistein, myricetin, puerarin and quercetin do not bind to GABAARs. These results indicate that DHM uniquely antagonizes alcohol potentiation of GABAARs in CNS neurons.
To the extent necessary to understand or complete the disclosure of the present invention, all publications, patents, and patent applications mentioned herein are expressly incorporated by reference therein to the same extent as though each were individually so incorporated.
The examples and experiments disclosed herein are intended to illustrate, but not limit the invention. Having thus described exemplary embodiments of the present invention, it should be noted by those skilled in the art that the within disclosures are exemplary only and that various other alternatives, adaptations, and modifications may be made within the scope of the present invention. Accordingly, the present invention is not limited to the specific embodiments as illustrated herein, but is only limited by the following claims.
The present invention claims the benefit U.S. Patent Application Ser. No. 61/376,528, filed 24 Aug. 2010, which is herein incorporated by reference in its entirety.
This invention was made with Government support under Grant Nos. AA007680, AA016100 and AA0017991, awarded by the National Institutes of Health. The Government has certain rights in this invention.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US11/48749 | 8/23/2011 | WO | 00 | 7/5/2012 |
