WO1993005786A1 - Nouveau recepteur a gabaa presentant des sites de liaison de steroides - Google Patents

Nouveau recepteur a gabaa presentant des sites de liaison de steroides Download PDF

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Publication number
WO1993005786A1
WO1993005786A1 PCT/US1992/007613 US9207613W WO9305786A1 WO 1993005786 A1 WO1993005786 A1 WO 1993005786A1 US 9207613 W US9207613 W US 9207613W WO 9305786 A1 WO9305786 A1 WO 9305786A1
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gnr
binding
dhp
grc
receptor
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PCT/US1992/007613
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English (en)
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Kelvin Wellman Gee
Nancy Tsai-Yun Lan
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Cocensys, Inc.
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Priority to EP92920306A priority Critical patent/EP0603312A4/fr
Priority to JP5506095A priority patent/JPH06510999A/ja
Publication of WO1993005786A1 publication Critical patent/WO1993005786A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70571—Receptors; Cell surface antigens; Cell surface determinants for neuromediators, e.g. serotonin receptor, dopamine receptor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • C07K14/721—Steroid/thyroid hormone superfamily, e.g. GR, EcR, androgen receptor, oestrogen receptor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • GRC GABA A receptor-chloride ionophore complex
  • Barbiturates and benzodiazepines both classes of therapeutically useful drugs, are known to be able to modulate allosterically the GRC. It is believed that there are sites on certain GRCs for barbiturates and the benzodiazepines.
  • the present invention defines a new receptor site on the GRC that is specific for steroids. Further, it relates to a method for determining whether other receptor sites exist on the GRC, as well as a method for determining what molecules bind to any newly identified site.
  • the present invention relates to a GRC bearing a steroid recognition or binding site for the opening of the chloride channel in the GRC.
  • GNR GABA A associated neurosteroid receptor
  • Our invention relates to a GRC having fewer than all the subunits of the naturally occurring receptor but at least the alpha ( ⁇ ) and beta ( ⁇ ) subunits, and, in some instances, also the gamma ( ⁇ ) subunit.
  • the present invention also relates to a method for determining the existence of the GNR in the GRC, as well as any other recognition sites present on that complex. Allosteric modulatory assays are described for determining the ability of compounds to bind to the complex at new or at previously known sites. Competitive assays can also be used for this purpose.
  • the present invention also provides a means for screening for GNR-agonist drugs effective in treating stress, anxiety, insomnia, post natal depression (PND) , pre-menstrual syndrome (PMS) , nervous disorders such as depression, and seizures. It also provides a means for treating the above named conditions through the administration of agonists to the GNR. Additionally, the present invention provides a means for screening for drugs that bind to GNRs with different subtype specificities.
  • GABA A receptor consists of at least 3 subunits, ⁇ , ⁇ , and ⁇ ; Schofield et al., "Sequence And Functional Expression Of The GABA A Receptor Shows A Ligand-gated Receptor Super-family," Nature 328, 221 (1987); Pritchett et al., "Importance Of A Novel GABA A Receptor Subunit For Benzodiazepine Pharmacology," Nature 338, 582 (1989). In addition, variants of the a , ⁇ and ⁇ subunits also exist; Levitan et al.
  • the cDNAs encoding human GABA A receptor ⁇ ,,, ⁇ 2 or a 3 , plus ⁇ 1 and ⁇ 2 subunits were co-expressed in human embryonic kidney 293 cells and the ability of steroids to allosterically modulate benzodiazepine and GABA A binding and to inhibit the direct binding of [ 3 H]3 ⁇ -OH-DHP to the expressed receptor complex was determined.
  • This invention demonstrates that the GNR has the same structure activity relationships as have been previously demonstrated in brain ho ogenate. Gee et al. (1988) , supra. Allosteric modulation of GABA A binding through interactions at the GNR are unique from modulation through interactions at the other known receptor sites on the GRC. This invention therefore demonstrates the existence of the GNR as an independent site through confirmation of the physical independence of the GNR from other receptor sites on the GRC. This newly discovered site has high affinity for its ligand, in contrast to other sites on the GRC.
  • GNRs A heterogenous population of GNRs has been demonstrated in cortex and spinal cord based on variations in activity of GNR agonists at these two locations.
  • Gee et al. "The GABA A Receptor Complex in Rat Frontal Cortex and Spinal Cord Show Differential Responses to Steroid Modulation," Molecular Pharm. in press.
  • This heterogeneity is reflected in the different protein subunit subtype composition of the GNR, and may be associated with different functions in the CNS and different effects on the body.
  • FIG. 1 shows a graph of the percent of maximum enhancement of [ 3 H]FLU binding in the presence of GABA plotted against the log of the amount of 3 ⁇ -OH-DHP;
  • FIG. 2 is a bar graph of the time to onset of myoclonus vs. different concentrations of steroid compounds useful in the present invention;
  • 3 ⁇ -OH-AND stands for 3 ⁇ -hydroxy-androstane
  • PREG-S0 4 stands for pregnenolone sulfate
  • PREG stands for pregnenolone;
  • FIG. 3 is a plot of the time course of anticonvulsant activity for various prodrugs and synthetic derivatives of
  • FIG. 4 is a plot of oral anti-convulsant activity of neuroactive steroid prodrug and direct acting molecules;
  • FIG. 5 is a graph of the number of transitions occurring within ten minutes of administration of 3 ⁇ -OH-
  • FIG. 6 is a graph of the number of transitions occurring within ten minutes of administration of 3 ⁇ ,21- dihydroxy-5 ⁇ -pregnan-20-one (5 ⁇ -THD0C) at various doses;
  • FIG. 7 is a graph of the number of transitions occurring within ten minutes of administration of Diazepam at various doses;
  • FIG. 8 is a graph comparing the number of transitions within ten minutes of administration of carrier ( ⁇ - Cyclodextrin) , 3 ⁇ -hydroxy-5 ⁇ -pregnan-20-one (3 ⁇ -OH-DHP) AND 3 ⁇ -OH-DHP;
  • FIG. 9 is a set of graphs showing the enhancement of the number of transitions caused by 3 ⁇ -OH-DHP and Diazepam, and the effect of the experimental drug CGS-8216 on their enhancement;
  • FIG. 10 shows the percent increase in punished responding caused by 3 ⁇ -OH-DHP as compared to the increase caused by the positive control, Chlordiazepoxide
  • FIG. 11 is a plot showing the effect of progesterone metabolites and promogesterone (R5020) on [ 3 H]-R5020 binding to the progesterone receptor in rat uterus;
  • FIG. 12 shows differential responses of expressed human GRCs with various ⁇ subunits in combination with B 1 and ⁇ 2 subunits to 3 ⁇ -0H-DHP;
  • FIG.13 shows [ 3 H]-flunitrazepam ([ 3 H]-FLU) binding in response to 3 ⁇ -OH-DHP in P 2 homogenate from cerebellum and spinal cord;
  • FIG. 14 shows responses to 3 ⁇ -OH-DHP in receptors composed of ⁇ subunits.
  • the present invention utilizes bioengineering techniques to search for the hypothesized steroid site on the GRC separate from any known sites on the GRC such as the barbiturate and BZ sites.
  • cDNAs encoding human GRC ⁇ 1f ⁇ 2 or ⁇ 3 , plus ⁇ , and ⁇ 2 subunits were expressed in eukaryotic cell cultures such as cultures of mammalian cells. Additional studies were done on complexes containing ⁇ x , z , or ⁇ 3 with ⁇ .,.
  • binding assays were conducted with and without steroids to determine whether the steroids can allosterically modulate binding to the benzo ⁇ diazepine and GABA sites on the expressed receptor complex and to determine the ability of [ 3 H]3 ⁇ -OH-DHP to label directly the GNR in a stereospecific manner.
  • [ 3 H]-FLU binds to the BZ site on the GRC and can be used for monitoring that BZ site, while [ 3 H]-muscimol binding labels the GABA A site on the GRC.
  • receptor sites such as the GNR
  • these receptors can be used for screening pharmaceuticals which will have various physiological effects upon administration to patients.
  • the steroid sites of this invention can be used . to screen for agonist compounds .with anxiolytic, anti-convulsant, hypnotic, sleep inducing, anti- depressant, anti-PND, anti-PMS, and other effects.
  • Mammalian cells were selected for the cloning steps for their ability to take up vectors coding for the selected DNAs as well as to express the proteins in a functional manner.
  • expression of the genes coding for the receptor subunits must assemble on the cell surfaces in a manner that mimics their natural configuration in cells in which the genes are normally expressed, i.e. neurons.
  • a cell type is human embryonic kidney cells, used in the Example below.
  • genes can be inserted into vectors such as plasmids, cosmids or retroviruses and transferred into the cells by known methods.
  • Calcium phosphate precipitation, cell-cell fusion, electropora- tion, liposomes, lipofection and a variety of other methods are known in the field and can be implemented.
  • Human embryonic kidney 293 cells were seeded on 10-cm dishes the day before transfection.
  • the cells were trans- fected with a total of 20 to about 30 ⁇ g of DNA per dish using a modified high efficiency CaP0 4 precipitation method described previously (Pritchett et al., supra) .
  • DNA used for the transfection was a mixture of equal amounts of cloned cDNA encoding the human GABA A ⁇ .,, ⁇ 2 or ⁇ 3 , ⁇ ., and ⁇ 2 subunits constructed individually in an expression vector (Pritchett et al. , supra) .
  • the cells were harvested and washed twice with phosphate buffered saline (PBS) pH 7.2 and frozen or immediately used in [ 3 H]flunitrazepam ([ 3 H]FLU) binding assays.
  • PBS phosphate buffered saline
  • Flunitrazepam is a BZ which binds to the BZ site on the GRC. Therefore, monitoring of [ 3 H]-FLU binding allows monitoring of the BZ site.
  • the transfected cells of Example 1 were homogenized in 10 mM potassium phosphate buffer, pH 7.2, and washed once by centrifugation.
  • the cell membrane pellet (100,000 x g) was homogenized in a mixture of 10 mM potassium phosphate, pH 7.2, and 100 mM potassium chloride. Aliquots (100 ⁇ l) of cell membrane were incubated with 2 nM of [ 3 H]FLU (75 - 90 ci/mmol, . duPont, New England Nuclear) in the presence of GABA (Sigma Chem. Co., St. Louis, MO) with or without steroids (Steraloids, Wilton, NH) .
  • GABA Sigma Chem. Co., St. Louis, MO
  • steroids Steraloids, Wilton, NH
  • Figure 1 shows the potentiation of [ 3 H]FLU binding by 3 ⁇ -OH-DHP in the presence of 1 ⁇ K GABA and various concentrations of 3 ⁇ -OH-DHP. Each point on the graph represents an average of 6 separate experiments; the bars represent the SEM. [ 3 H]FLU binding in the absence of steroid was defined as control. The percentage of maximum enhancement was defined as a percent of the enhancement produced by 1 ⁇ ,M of 3 ⁇ -OH-DHP which varied between 57% and 195% above control in these experiments.
  • Muscimol is a specific GABA A agonist, which binds to the GABA A site on the GRC resulting in the opening of the Cl-channel. It has been shown in rat brain homogenate that steroids are capable of modulating (i.e., enhancing) [ 3 H]-muscimol binding with specificities similar to that observed for the enhancement of [ 3 H]FLU binding. Therefore, we studied the binding of muscimol in the receptors expressed from cDNA. The experiments involving the expression of the GABA A receptor subunit cDNAs indicate that only the a and ⁇ subunits are needed to reconstruct the muscimol binding site.
  • [ 3 H]-muscimol binding was performed under conditions similar to that for [ 3 H]FLU binding except 10 ⁇ K (+)bicu- culline was used to define nonspecific binding.
  • 10 ⁇ K (+)bicu- culline was used to define nonspecific binding.
  • 5 ⁇ -pregnan-3 ⁇ ,20 ⁇ -diol appears to have limited efficacy similar to that demonstrated in in vivo and in vitro assays using brain homogenate preparations.
  • 3 ⁇ -OH-DHP is a direct agonist at the GNR.
  • [ 3 H]3 ⁇ -OH- DHP was used in the following experiments to directly bind to and label the GNR.
  • Example 1 The transfected cells of Example 1 were used to determine the presence of specific [ 3 H]3 ⁇ -OH-DHP binding in a manner similar to that described for [ 3 H]FLU binding in Example 2.
  • [ 3 H]3 ⁇ -OH-DHP (2 nM) was used to label the steroid site.
  • Nonspecific binding was defined as binding in the presence of 3 ⁇ K of the pharmacologically active stereoisomer, 3 ⁇ -0H-DHP.
  • Binding to detergent solubilized steroid sites was determined by solubilizing the P 2 fraction from rat cere ⁇ bral cortex under conditions described in Gee et al. , Modulation Of The Chloride lonophore By Benzodiazepine Receptor Ligands: Influence Of GABA And Ligand Efficacy, Mol. Pharmacol. 30:218-255 (1986) . Ammonium sulfate (20%) was added to the soluble fraction and the resultant pre ⁇ cipitate was pelleted by centrifugation at 100,000 x g.
  • the pellet was washed three times in 50 mM Na/K phosphate buffer (pH 7.4) and 200 mM NaCl by centrifugation; the final pellet was resuspended in the buffer as a 10% (original wet wt./vol) homogenate.
  • 50 mM Na/K phosphate buffer pH 7.4
  • 200 mM NaCl by centrifugation
  • the final pellet was resuspended in the buffer as a 10% (original wet wt./vol) homogenate.
  • One hundred ⁇ l aliquots of the homogenate were incubated with 5 nM [ 3 H]3 ⁇ - OH-DHP and buffer in a total reaction volume of 1 ml for 60 min. at 25°C.
  • Control binding is defined as 2 nM [ 3 ]FLU binding in the absence of GABA. The data are the average of two independent transfection experiments. % Potentiation is defined as the % above control binding.
  • Steroids that are active on the GNR include the steroids disclosed in patent application Serial No. 379,049 filed July 13, 1989, as well as Serial No. 521,724, filed May 10, 1990, and Serial No. unknown, filed August 13, 1991.
  • Other active steroids include the steroids disclosed in U.S. Patent Nos.
  • mice were injected with various doses of the test compounds, as indicated in FIG. 2, 10 minutes prior to the injection of TBPS.
  • the time to onset of myoclonus (presence of forelimb clonic activity) induced by TBPS was determined by observing each mouse, for a period of 45 minutes.
  • Significant differences between the time to onset in control mice vs. steroid-treated mice were determined by Student's t-test.
  • the relative rank order potency and efficacy of these steroids in vivo were well correlated with those values determined in vitro.
  • mice were injected with various doses of 3 ⁇ -OH-DHP or vehicle (dimethyl-sulfoxide) 10 minutes prior to the administration of the following chemical convulsants: metrazol (85 mg/kg) ; (+)bicuculline (2.7 mg/kg) ; picrotoxin (3.15 mg/kg); strychnine (1.25 mg/kg); or vehicle (0.9% saline).
  • metrazol 85 mg/kg
  • (+)bicuculline 2.7 mg/kg
  • picrotoxin 3.15 mg/kg
  • strychnine (1.25 mg/kg
  • vehicle 0.9% saline
  • RR Rotorod
  • MES maximal electroshock
  • MTZ metrazol
  • BIC bisulfide
  • the route of administration for steroids and convulsants was i.p. and s.c, respectively.
  • ED 50 values include the 95% confidence limits.
  • ⁇ Anticonvulsant data are from Swinyard & Woodhead, General principles: experimental detection, quantification and evaluation of anticonvulsants, in Antie i1eptic Drugs_ D.M. Woodbury, J.K. Penry, and C.E. Pippenger, eds., p. 111, (Raven Press, New York), 1982.
  • Example 5 Metrazol-Induced Seizures
  • Adult male CF1 mice (20-30g) were used in these studies. Anti-convulsant activities were assessed as previously described by Swinyard and Woodhead (1982) supra. Percent protection against metrazol-induced seizures was plotted against time after administration of the compound.
  • mice were injected with metrazole (85 mg/kg s.c.) at various times after administration of the compound (3 ⁇ -OH-DHP at 30 mg/kg, 3 ⁇ -acetyl-5o.-pregnan-20- one (3 ⁇ -AC-DHP) , 3 ⁇ -propyl-5 ⁇ -pregnan-20-one (3 ⁇ -PR-DHP) , and 3 ⁇ -butyryl-5 ⁇ -pregnan-20-one (3 ⁇ -BU-DHP) all at 60 mg/kg) .
  • mice Ten to twelve mice were used per dose of test drug.
  • mice were injected (i.p.) with the compound dissolved in DMSO or 2-hydroxypropyl- ⁇ -cyclodextrin, or with vehicle alone (for DMSO, 5 ⁇ -L/g body weight) , at various times prior to the administration (s.c.) of a CD 97 (dose at which 97% of the animals have seizures from Swinyard and Woodhead, 1982) dose of metrazol (85 mg/kg) or vehicle (0.9% saline, 5 ⁇ /g body weight) .
  • the mice were observed for a period of 30-45 min. The number of animals with tonic and/or clonic convulsions was recorded.
  • the ability of the steroid to abolish the tonic- clonic component was defined as the endpoint. Sedative potential was determined by a rotorod test where the number of mice staying on a rotating (6 rpm) rod for > 1 min. in each of three trials was determined. The acute LD 50 was determined by counting survivors 48 h. after the administration (i.p.) of the anti-convulsant compound. All median effective doses were determined by the method of Litchfield and Wilcoxon (1949) . Figure 3 shows the results of these experiments. Modification of the basic compound 3 ⁇ -OH-DHP at the 3 ⁇ position with an acetate, propionate or butyrate group increased the time of protection provided by the compound. Thus, compounds that bind to the GNR can be modified to provide anti-convulsant activity over a period of time, with varying degrees of protection.
  • Figure 4 shows oral anticonvulsant activity of a GNR agonist prodrug and a direct acting GNR agonist.
  • Example 6 Oral Administration
  • the GNR is useful in screening for. and selecting pharmaceuticals effective in treating anxiety.
  • the following experiments demonstrate that the progesterone metabolite and GNR agonist 3 ⁇ -OH-DHP is an effective anxiolytic in three animal models of human anxiety that measure the behavioral effects of anxiolytic compounds.
  • they show that the anxiolytic effects of 3 ⁇ -OH-DHP were mediated through a mechanism that is separate from that of the BZs.
  • the light/dark transition test (Crawley and Goodwin, 1980) is based on the observation that rodents naturally tend to explore novel environments, but open, brightly lit arenas are aversive to the rodents and inhibit exploratory behavior (Christmas and Maxwell, 1970; File, 1980) .
  • mice Male N.I.H. Swiss-Webster mice (Harlan) weighing 15-20 g were used in all experiments. The mice were housed 4/cage in polyethylene cages with sawdust bedding. The colony room was environmentally controlled (22°C) with a 12 hr. light/dark cycle (0600-1800 hrs.). The mice had free access to food and water. The experiments were run from 0700-1500 hrs. and groups were counterbalanced for time of day effects. a) Light/Dark Transitions
  • the method used was previously described by Crawley and Goodwin (1980) .
  • the apparatus included a large box (26 x 33 x 24 cm) connected to a small box (15 x 22 x 14 cm) through an opening (5 x 6 cm) .
  • the large box was brightly lit with a standard 100 W light bulb, while the small box was kept dark.
  • mice were placed in the center of the large box and the number of transitions between the large and small boxes was counted for 10 min.
  • Drug pretreatment times were as follows: diazepam (30 min.); 3 ⁇ -OH-DHP (10 min.); and 5 ⁇ -THDOC (10 min.).
  • CGS-8216 was administered 30 min. prior to the test drug.
  • the activity monitor is linked to a computer through a Digiscan Analyzer (Omnitech Electronics) and the data is analyzed using the Integrated Lab Animal Monitoring System (Omnitech Electronics) .
  • the mice were administered drugs as described for light/dark transitions.
  • mice were first habituated to the open-field apparatus for 15 min. The following day, mice were pretreated with a test drug and placed in the center of the activity chambers. The total distance traveled was measured for 10 min. c) Vogel Paradigm
  • An Anxio-Monitor (Omnitech Electronics, Columbus, OH) was used for measuring lick suppression.
  • the testing chamber consisted of a clear plexiglass box (29 cm x 29 cm x 23 cm) with a metal drinking tube located 2.5 cm from the floor and extending 2 cm into the box. The shock was applied through the drinking spout and was controlled by the Anxio-Monitor. The number of licks was counted and displayed by the Anxio-Monitor.
  • the reinforcer was 0.1 M sucrose.
  • mice were allowed to explore the test apparatus and drink without punishment for 10 min. or 100 licks. The following day (48 hr. after the start of water deprivation) , mice were pretreated with 3 ⁇ -OH-DHP
  • the steroids 3 ⁇ -0H-DHP, 3 ⁇ -OH-DHP, and 5 ⁇ -THDOC were synthesized as described in U.S. Patent Application Serial No. unknown filed August 13, 1991, which is incorporated herein by reference.
  • 2-Hydroxypropyl ⁇ -cyclodextrin ( ⁇ -cyclodextrin) is available from Aldrich (Milwaukee, WI) .
  • Diazepam and chlordiazepoxide are available from Sigma, Co. (St. Louis, MO) .
  • CGS-8216 was obtained from Ciba-Geigy (Summit, NJ) . All drugs were dissolved in 20% ⁇ -cyclodextrin in water and sonicated overnight. All drugs were administered intraperitoneally in a volume of 100 ⁇ L/20 g.
  • CGS-8216 was administered subcutaneously in a volume of 100 /L/20 g.
  • Dose-response curves for 3 ⁇ -OH-DHP, 5 ⁇ -THDOC and diazepam generated in the light/dark transition test were run over several days.
  • the vehicle control data were analyzed across test days using a 1-way analysis of variance (ANOVA) . Because the vehicle data were not significantly different across days, the control data were collapsed for each test drug.
  • the dose-response curves were then analyzed using a 1-way ANOVA, followed by Dunnett's t-test for individual comparisons between doses and control.
  • the open-field and habituated locomotor data were analyzed using ANOVA followed by Dunnett's t-test. 3 ⁇ -OH-DHP and CDP were tested on separate days in the lick-suppression test.
  • the control groups were significantly different, therefore the data was analyzed using a Student's t-test (2-tailed) and for comparison is graphically displayed as percent of corresponding control. All data is expressed as the mean ⁇ S.E.M.
  • the GNR agonist 3 ⁇ -OH-DHP produced anxiolytic effects as seen by an increase in the number of transitions in the light/dark paradigm (Fig. 5) .
  • the number of transitions was increased by 3 ⁇ -OH-DHP significantly (p ⁇ .01) at 10, 20, and 40 mg/kg.
  • 3C.-QH-DHP reached maximal effect at a dose of 20 mg/kg with an average of .70.2 ⁇ 4.3 transitions in a 10 min. period.
  • the highest dose tested, 40 mg/kg started a trend towards a reduction in the number of transitions.
  • Several compounds have been shown to produce an inverted U-shaped dose-response curve in the light/dark transition paradigm (Crawley et al., 1986).
  • 5 ⁇ -THDOC, at a dose of 20 mg/kg was significantly different from vehicle (p ⁇ .01).
  • 5 ⁇ -THD0C did produce a greater number of transitions at 10 mg/kg compared with vehicle (49.4 ⁇ 2.0 vs. 35.2 ⁇ 2.0), the difference did not reach significance (p ⁇ .06) .
  • 5 ⁇ -THD0C 40 mg/kg
  • 5 ⁇ -THD0C produced a significant decrease (p ⁇ .05) in the number of transitions as compared with vehicle control.
  • Diazepam's effects on light/dark transitions is shown in Fig. 7.
  • Diazepam's maximal response was at 10 mg/kg with 86.4 ⁇ 5.4 transitions. Though significant at 20 mg/kg, diazepam's effects were diminished as compared with the effects at 10 mg/kg. These results are similar to the inverted U-shaped curves seen with the two GNR agonists, 3 ⁇ -OH-DHP and 5 ⁇ -THDOC.
  • the specific BZ antagonist CGS-8216 (10 mg/kg) was unable to block the anxiolytic effect of 3 ⁇ -OH-DHP (Fig. 9A) .
  • 3 ⁇ -0H-DHP (10 mg/kg) produced significant (p ⁇ .01) increases alone and in the presence of CGS-8216 (Fig. 9A) .
  • CGS-8216 was able to block the anxiolytic effect of diazepam (Fig. 9B) .
  • Diazepam (1.0 mg/kg) alone produced a significant (p ⁇ .01) increase in transitions as compared to control (Fig. 9B) .
  • CGS-8216 did not demonstrate any intrinsic activity and was not significantly different from vehicle control (p>.4) .
  • mice When placed in a novel, brightly lit open-field, mice demonstrate a low level of activity (i.e., exploration, locomotion, etc.) , whereas anxiolytics increase the amount of activity in a novel environment (Lister, 1990) .
  • anxiolytics increase the amount of activity in a novel environment (Lister, 1990) .
  • the GNR agonists 3 ⁇ -OH-DHP and 5 ⁇ -THDOC produced significant (p ⁇ .01) increases in activity as compared with control.
  • diazepam produced a significant (p ⁇ .01) increase in activity as compared with ⁇ -cyclodextrin vehicle control. Consistent with the light-dark transition paradigm, 3 ⁇ -OH-DHP did not show any effect in the open-field test.
  • mice were pretreated 10 min or 30 min (diazepam) prior to being placed in the center of the open-field apparatus. Total distance travelled was measured for 10 min (see
  • mice were treated with 9-10 mice.
  • 3 ⁇ -OH-DHP also produced a significant increase in locomotor activity (p ⁇ .01) in mice that were acclimated to the test chambers.
  • 3 ⁇ -OH-DHP 20 mg/kg
  • CDP 10 mg/kg
  • the agonists of the GNR on the GRC will also be effective in treating other conditions affected by the opening and closing of the chloride channel on this receptor complex.
  • Agonist BZs and barbiturates that bind at the GRC induce the hypnotic state and sleep.
  • a correlation is shown by this invention between the modulation of GABA A binding by BZs and barbiturates and the modulation o'f GABA A binding by agonists at the GNR.
  • some compounds now known to be agonists of the GNR have been shown to induce the hypnotic state and sleep. Therefore it is shown by this invention that GNR agonists will likewise induce the hypnotic state and/or sleep.
  • BZs and barbiturates are routinely used in the treatment of mood disorders and acute anxiety attacks such as are caused by panic disorders. Their mode of action is through modulation of the GABA A receptor.
  • the data presented here demonstrate that agonists of the GNR are useful in the treatment of mood disorders such as depression and acute anxiety attacks such as are caused by panic disorders.
  • FIG. 11 The data plotted in FIG. 11 were obtained by performing assays in accordance with the receptor binding assay described in Gee et al. (1988) , supra. incorporated herein by reference, to determine the effect of GNR agonists and the progestin R5020 on the binding of [ 3 H]R5020 to the progesterone receptor in rat uterus. All points on the plot of FIG. 11 represent the mean of triplicate determinations. The following compounds are those listed in FIG.
  • the hormonal activity of agonists of the novel steroid receptor was further studied through testing their potential estrogenic, progestinic, mineralocorticoid and glucocorticoid activities. These activities were analyzed by monitoring the ability.of the drugs to inhibit binding of the steroid hormones to their respective hormone receptors. The results are shown in Tables 6-9. They are expressed as percent inhibition of 3 H-ligand binding to the various steroid hormone receptors for the drugs at 10 "6 and 10 '5 M. Control values are represented by the binding in the absence of drugs.
  • mice were adrenalectomized 3 days prior to sacrifice.
  • brain cytosol fractions were prepared as follows. Brains from male Sprague-Dawley rats were removed immediately following sacrifice and the cerebral cortices dissected over ice. A P 2 homogenate was prepared as previously described (Gee et al., "Modulation of the chloride ionophore by benzodiazepine receptor ligands: influence of gamma-aminobutyric acid and ligand efficacy," Molecular Pharmacology. 30. 218 (1986)).
  • the cortices were gently homogenized in 0.32 M sucrose followed by centri- fugation at 1000 x g for 10 minutes. The supernatant was collected and centrifuged at 9000 x g for 20 minutes. The resultant P 2 pellet was suspended as a 10% (original wet weight/volume) suspension in 50 mM Na/K phosphate buffer (pH 7.4) 200 mM NaCl to form the homogenate.
  • the drugs were incubated with 3 nM of 3 H-aldosterone (the specific ligand for the mineralocorticoid receptor) in the presence of the selective type II agonist RU28362 (0.5 ⁇ K) which blocks 3 H-aldosterone binding to the type II (glucocorticoid) receptors.
  • the specific binding was 1756 cpm/fraction.
  • brain cytosol fractions were prepared as for Table 6, and the drugs were incubated with 3 nM of 3 H- dexamethasone (the specific ligand for the glucocorticoid receptor) .
  • the specific binding was 1174 cpm/fraction.
  • Estrogen Receptor Table 8 shows the inhibition of 3 H-estradiol (the specific ligand for the estrogen receptor) binding to bovine uteri cytosol, prepared as previously described. Two concentrations of 3 H-estradiol; (A) 0.15 nM and (B) 0.25 nM, were incubated with the cytosol in the presence of the drugs. The specific binding for 3 H-estradiol at 0.15 nM and 0.25 nM was 1241 cpm/fraction and 1951 cpm/fraction, respectively.
  • Example 11 Progesterone Receptor For the data presented in Table 9, bovine uteri cytosol was isolated as they were for Table 7 and used for following binding to progesterone receptors by following the inhibition of 3 H-progesterone , the natural ligand. Two 3 H-progesterone concentrations, (A) 0.15 nM and (B) 0.25 nM were incubated with the cytosol in the presence of the drugs. The specific binding for 3 H-progesterone at 0.15 nM and 0.25 nM was 2893 cpm/fraction and 4222 cpm/fraction, respectively. This data reinforces our findings shown in Fig. 11 that indicates that GNR agonists have no activity at the progesterone receptor. TABLE 9 Inhibition of 3 H-Progesterone Binding to the Bovine Uteral Progesterone Receptors
  • Subtypes of the subunits ( ⁇ ,, ⁇ , ⁇ .,_ 3 , ⁇ .,_ 2 , etc.) of the steroid receptor described herein occur in a variety of combinations in vivo. Studies have shown that the spinal cord may be devoid of the ⁇ , subunit, whereas the cerebellum is rich in ⁇ , subunits. (Vicini S., (1991) Pharmacologic significance of the structural heterogeneity of the GABA. receptor-chloride ion channel complex. Neuropsychopharmacology 4, 9-15) . Regionally selective expression of receptor subunit subtypes may account for differential responsiveness of tissues to GABAergic inhibition and neurosteroid modulatory effects.
  • subunits of the GNR described herein can be expressed in a variety of combinations. Experiments were performed to analyze binding of GNR agonists to various combinations of the expressed subunits. The results demonstrate that there is a differential response to agonists as a function of the different subtypes.
  • Example 12 Binding to Different Subtypes a) Expression of GABA A receptor complexes Previous transfection procedures were followed (Pritchett et al, (1989) Type I and type II GABA A -receptors produced in transfected cells. Science 245, 1389-1392) except that cells were seeded for 36 hours instead of 24 hours prior to transfection.
  • the human embryonic kidney 293 cells were transfected with a total of 20 ⁇ g of DNA per dish using a modified high efficiency calcium phosphate precipitation method described previously (Chen and Okayama, (1987) High efficiency transformation of mammalian cells by plasmid DNA. Mol. Cell. Biol 7, 2745- 2752) .
  • DNA used for the transfection was a mixture of equal amounts of cloned cDNA encoding the human GABA A ⁇ 1# ⁇ 2 or ⁇ 3 , ⁇ 1 and ⁇ 2 -subunits constructed individually in an expression vector (Chen and Okayama, supra) .
  • the cells were harvested and washed twice with phosphate buffered saline (PBS) pH 7.2 and frozen or immediately used in the binding assays, b) [ 3 H]FLU and [ 3 H]Muscimol Binding Assays
  • PBS phosphate buffered saline
  • the transfected cells or rat (male Sprague-Dawley, 150-2OOg) brain tissue from selected regions were homogenized in 50 mM potassium phosphate buffer pH 7.2 and washed once by centrifugation.
  • the cell membrane pellet(10,000 x g) was homogenized in a mixture of 10 mM potassium phosphate, pH 7.2 and 100 mM NaCl.
  • Nonspecific binding was defined as binding in the presence of 1 ⁇ K clonazepam (Sigma Chemical Co.) and 10 ⁇ K GABA in [ 3 H]FLU and [ 3 H]muscimol binding assays, respectively. Assays were terminated after a 90 min (t 3 H]FLU binding) or 60 min ([ 3 H]muscimol binding) incubation at 25°C by rapid filtration through Schleicher and Schuell (Keene, NH) No. 32 glass fiber filters. The filters were washed twice with 5 ml of ice-cold PBS. Filter-bound radioactivity was quantified by liquid scintillation spectrophotometry.
  • Y [Ymax* X/ (X + EC 50 ) ] + control
  • Y % of control binding
  • X log [3 ⁇ -0H-DHP] .
  • Control 100%
  • EC 50 concentration of 3 ⁇ -0H-DHP required to produce half-maximal enhancement.
  • Differential responses to 3 ⁇ -OH-DHP of expressed human GRCs with various a subunits in combination with ⁇ 1 and ⁇ 2 subunits are shown in Figure 12. Maximal enhancement and EC 50 values are for [ 3 H]FLU (2nM) binding to a ⁇ 2 - C
  • the steroid clearly enhanced [ 3 H]FLU binding to each of the expressed receptor subtypes.
  • assembled receptors with the ⁇ 3 subunit consistently gave a much greater maximal response to steroid stimulation than those composed of other subunits.
  • 3 ⁇ -OH-DHP produced a 200% enhancement of [ 3 H]FLU binding
  • t ⁇ B z and a z B ⁇ 2 complexes showed less than 100% enhancement above the control.
  • a similar differential response of the GRC to GABA with various ⁇ subunits has also been demonstrated previously (Pritchett et al, supra) .
  • the GABA concentration (l M) used in the present study did not produce a significant differential enhancement when compared to binding in the absence of GABA (Pritchett et al, supra) and was used in the control conditions in the present study.
  • the percentage enhancement by the steroid remained constant regardless of the level of control binding which varied among different transfection experiments.
  • GRC subtypes expressed in Xenopus oocytes were also differentially modulated by 3 ⁇ -OH-DHP dependent upon subunit composition (Shingai et al, (1991)
  • Fig. 14 shows potentiation of [ 3 H]muscimol binding by 3a:- OH-DHP in transfected cell membranes.
  • control binding values for ⁇ .- ⁇ .,, ⁇ 2 ⁇ 1 and ⁇ J ⁇ 1 ranged from 28 - 101, 39 - 112, and 21 - 130 fmol/mg protein, respectively. Consistent with that observed with the ⁇ receptor complex, ⁇ 3 ⁇ 1 showed a greater enhancement of [ 3 H]muscimol binding by 3 ⁇ -OH-DHP than those composed of ⁇ , ⁇ 1 or ⁇ 2 ⁇ 1 subunits, although the difference from ⁇ 2 ⁇ ., is less pronounced, thus suggesting that site of interaction with the steroid is probably similar in both ⁇ or ⁇ receptor complexes. It is not known whether or not differential responses to GNR agonists will occur when the receptor is composed of a single subunit (homooligomer) .
  • the distribution of the ⁇ subunit variants is heterogenous in the CNS and variants appear to respond differentially to these GNR agonists, it is possible then that the magnitude of the responses to GNR agonists may vary regionally. Upon further characterization, this property may allow for the design of more subtype selective compounds directed at this novel steroid recognition site.
  • the GNR agonists of this invention are prepared for delivery to an individual in conventional dosage unit forms by incorporating a GNR agonist or a mixture of GNR agonists with a nontoxic pharmaceutical carrier according to accepted procedures in a nontoxic amount sufficient to produce the desired pharmacodynamic activity in a subject, animal or human.
  • the composition contains the active ingredient in an active, but nontoxic amount, selected from about 5 mg to about 500 mg of active ingredient per dosage unit. This quantity depends on the affinity of the compound for the GNR, the specific biological activity desired and the condition of the patient.
  • Desirable objects of this invention are the treatment of stress, anxiety, PMS, PND, and seizures such as those caused by epilepsy to ameliorate or prevent the attacks of anxiety, muscle tension, and depression common with patients suffering from these central nervous system abnormalities. Additional desirable objects of this invention are to prevent insomnia and produce hypnotic activity.
  • the pharmaceutical carrier employed may be, for example, either a solid, liquid, or time release (see e.g.
  • Representative solid carriers are lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid, microcrystalline cellulose, polymer hydrogels and the like.
  • Typical liquid carriers are propylene glycol, aqueous solutions of ⁇ -cyclodex- trins, syrup, peanut oil, and olive oil and the like emulsions.
  • the carrier or diluent may include any time-delay material well known to the art, such as glycerol monostearate or glycerol distearate alone or with wax, microcapsules, microspheres, liposomes, and/or hydrogels.
  • the preparation when using a solid carrier, can be plain milled micronized, in oil, tableted, placed in a hard gelatin or enteric-coated capsule in micronized powder or pellet form, or in the form of a troche, lozenge, or suppository.
  • a liquid carrier When using a liquid carrier, the preparation can be in the form of a liquid, such as an ampule, or as an aqueous or nonaqueous liquid suspension.
  • Liquid dosage forms also need pharmaceutically acceptable preservatives and the like.
  • nasal spray, sublingual administration and timed release skin patches are also suitable pharmaceutical forms for topical administration.
  • the method of producing anxiolytic, anticonvulsant, mood altering (such as anti-depressant) or hypnotic activity comprises administering to a subject in need of such activity an agonist at the GNR, usually prepared in a composition as described above with a pharmaceutical carrier, in a nontoxic amount sufficient to produce said activity.
  • GNR active progesterone metabolite i.e., GNR agonists
  • Plasma levels of active and major metabolites are monitored during pre-menses and post-menses of the patient.
  • the amount of the GNR agonists administered, either singly or as mixtures thereof, are thus calculated to supplement the levels of GNR active progesterone metabolites during the premenses state.
  • the route of administration may be any route that effectively transports the active compound to the GRCs that are to be stimulated. Administration may be carried out parenterally, enterally, rectally, intravaginally, intradermally, sublingually, or nasally; the oral and dermal routes are preferred. For example, one dose in a skin patch may supply the active ingredient to the patient for a period of up to one week.

Abstract

Complexe (GRC) de récepteur à GABAA-ionophore de chlorure dans lequel GABA est de l'acide gamma-aminobutyrique, ayant un nouveau site de liaison, le GNR, indépendant d'autres sites connus sur le GRC. Un procédé destiné à la recherche d'agonistes du GNR par formation d'un GRC exprimé par co-expression d'ADN complémentaire codant les sous-unités GRC dans des cellules et à déterminer la capacité d'un stéroïde de moduler de manière allostérique la liaison de la benzodiazépine au complexe récepteur exprimé. La figure 1 montre l'amélioration de la liaison d'un analogue de benzodiazépine au GRC en tant que fonction de la concentration d'un modulateur stéroïde qui se lie au GNR.
PCT/US1992/007613 1991-09-13 1992-09-09 Nouveau recepteur a gabaa presentant des sites de liaison de steroides WO1993005786A1 (fr)

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EP0603312A4 (fr) 1995-06-07
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JPH06510999A (ja) 1994-12-08
AU2657292A (en) 1993-04-27

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