WO1998023742A1 - A NOVEL CLONED GABA-RECEPTOR SUBUNIT cDNA SEQUENCE AND STABLY CO-TRANSFECTED CELL LINES - Google Patents
A NOVEL CLONED GABA-RECEPTOR SUBUNIT cDNA SEQUENCE AND STABLY CO-TRANSFECTED CELL LINES Download PDFInfo
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- WO1998023742A1 WO1998023742A1 PCT/GB1997/003159 GB9703159W WO9823742A1 WO 1998023742 A1 WO1998023742 A1 WO 1998023742A1 GB 9703159 W GB9703159 W GB 9703159W WO 9823742 A1 WO9823742 A1 WO 9823742A1
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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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- This invention concerns the cloning of a novel cDNA sequence encoding a particular subunit of the human GABAA receptor.
- the invention relates to a stable cell line capable of expressing said cDNA and to the use of the cell hne in a screening technique for the design and development of subtype-specific medicaments.
- GABA Gamma-amino butyric acid
- This receptor comprises a multime ⁇ c protein of molecular size 230-270 kDa with specific binding sites for a variety of drugs including benzodiazepmes, barbiturates and ⁇ -carbolmes, in addition to sites for the agonist ligand GABA (for reviews see MacDonald and Olsen, Ann. Rev
- the present invention relates to a new class of GABA receptor subunit, which we initially referred to as the kappa subunit (K subunit) but will now hereinafter refer to as the epsilon subunit ( ⁇ subunit).
- K subunit kappa subunit
- ⁇ subunit epsilon subunit
- the present invention accordingly provides, in a first aspect, a DNA molecule encoding the epsilon subunit of the human GABA receptor comprising all or a portion of the sequence depicted in Figure 1, or a modified human sequence.
- modified human sequence referes to a variant of the DNA sequence depicted in Figure 1. Such variants may be naturally occuring allelic variants or non-naturally occuring or
- allelic variation is well known in the art in which the nucleotide sequence may have a substitution, deletion or addition of one or more nucleotides without substantial alteration of the function of the encoded polypeptide. Particularly preferred allelic variants arise from nucleotide substitution based on the degeneracy of the genetic code.
- the sequencing of the novel cDNA molecules in accordance with the invention can conveniently be carried out by the standard procedure described in accompanying Example 1; or may be accomplished by alternative molecular cloning techniques which are well known in the art, such as those described by Maniatis et al. in Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, New York, 2nd edition, 1989.
- the present invention also relates to polynucleotides (for example, cDNA, genomic DNA or synthetic DNA) which hybridize under stringent conditions to the DNA molecule depicted in Figure 1.
- stringent conditions will be understood to require at least 95% and preferably at least 97% identity between the hybridized sequences.
- Polynucleotides which hybridize under stringent conditions to the DNA molecule depicted in Figure 1 preferably encode polypeptides which exhibit substantially the same biological activity or function as the polypeptide depicted in Figure 1.
- the present invention further relates to a GABA epsilon subunit polypeptide which has the deduced amino acid sequence of Figure 1, as well as fragments, analogs and derivatives thereof.
- fragment when referring to the polypeptide of Figure 1, means a polypeptide which retains essentially the sme biological activity or function as the polypeptide depicted in Figure 1.
- an analog may be, for example, a proprotein which can be activated by cleavage of the proprotein portion to produce an active mature polypeptide.
- the polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide or a synthetic polypeptide, preferably a recombinant polypeptide.
- the fragment, derivative or analog of the polypeptide of Figure 1 may be one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residues may or may not be one encoded by the genetic code; or one in which one or more of the amino acid residues includes a substituent group; or one in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol); or one m which the additional ammo acids are fused to the mature polypeptide, such as a leader or secretory sequence or a sequence which is employed for purification of the mature polypeptide or a proprotein sequence
- Such fragments, derivatives and analogs are
- polypeptides and DNA molecules of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity
- isolated means that the material is removed from its original environment (e.g , the natural environment if it is naturally occurring)
- a naturally-occurring DNA molecule or polypeptide present m a living animal is not isolated, but the same DNA molecule or polypeptide, separated from some or all of the coexisting materials m the natural system, is isolated
- DNA molecules could be part of a vector and/or such DNA molecules or polypeptides could be part of a composition, and still be isolated m that such vector or composition is not part of its natural environment
- the invention provides a recombinant expression vector comprising the nucleotide sequence of the human GABA receptor epsilon subunit together with additional sequences capable of directing the synthesis of the said human GABA receptor epsilon subunit m cultures of stably co-transfected eukaryotic cells
- expression vectors refers to DNA sequences that are required for the transcription of cloned copies of re
- Expression vectors may include, but are not limited to, cloning vectors, modified cloning vectors, specifically designed plasmids or viruses
- cloning vector refers to a DNA molecule, usually a small plasmid or bacte ⁇ ophage DNA capable of self- replication in a host organism, and used to introduce a fragment of foreign DNA into a host cell
- the foreign DNA combined with the vector DNA constitutes a recombinant DNA molecule which is derived from recombinant technology
- Cloning vectors may include plasmids, bacteriophages, viruses and cosmids
- the recombinant expression vector in accordance with the invention may be prepared by inserting the nucleotide sequence of the GABA epsilon subunit into a suitable precursor expression vector (hereinafter referred to as the "precursor vector") using conventional recombinant DNA methodology known from the art
- the precursor vector may be obtained commercially, or constructed by standard techniques from known expression vectors
- the precursor vector suitably contains a selection marker, typically an antibiotic resistance gene, such as the neomycm or ampicil n resistance gene
- the precursor vector preferably contains a neomycm resistance gene, adjacent the SV40 early splicing and polyadenylation region, an ampicilhn resistance gene; and an origin of replication, e.g.
- the vector also preferably contains an mducible promoter, such as MMTN-LTR (mducible with dexamethasone) or metallothionm (mducible with zinc), so that transcription can be controlled in the cell line of this invention This reduces or avoids any problem of toxicity in the cells because of the chloride channel intrinsic to the GABAA receptor.
- MMTN-LTR mimethylcholine
- metallothionm mducible with zinc
- precursor vector pMAMneo available from Clontech Laboratories Inc. (Lee et al, Nature, 1981, 294, 228; and Sardet et al, Cell, 1989, 56, 271).
- precursor vector pMSGneo can be constructed from the vectors pMSG and pSN2neo.
- the recombinant expression vector of the present invention is then produced by cloning the GABA receptor epsilon subunit cD ⁇ A into the above precursor vector.
- the receptor subunit cD ⁇ A is subcloned from the vector in which it is harboured, and ligated into a restriction enzyme site, e.g. the Hmd III site, in the polyhnker of the precursor vector, for example pMAMneo or pMSGneo, by standard cloning methodology known from the art, and m particular by techniques analogous to those described herein.
- a restriction enzyme site e.g. the Hmd III site
- 5' untranslated sequences for example by modifying the 5' end of the epsilon subunit D ⁇ A by addition of 5' untranslated region sequences from the ⁇ i subunit D ⁇ A.
- a stably co-transfected eukaryotic cell line capable of expressing a GABA receptor, which receptor comprises the epsilon receptor subunit at least one alpha receptor subunit and at least one beta receptor subunit
- the present invention provides a process for the preparation of a eukaryotic cell line capable of expressing a GABA receptor, which comprises stably co-transfectmg a eukaryotic host cell with at least three expression vectors, one such vector harbouring the cD ⁇ A sequence encoding the epsilon GABA receptor subunit, another such vector harbouring the cD ⁇ A sequence encoding an alpha GABA receptor subunit, and a third such vector harbouring the cD ⁇ A sequence encoding the beta GABA receptor subunit.
- the stable cell-line which is established expresses an ⁇ GABA receptor.
- GABA receptor Each receptor thereby expressed, comprising a unique combination of ⁇ , ⁇ and ⁇ subunits, will be referred to hereinafter as a GABA receptor "subunit combination".
- Pharmacological and electrophysiological data confirm that the recombinant ⁇ receptor expressed by the cells of the present invention has the properties expected of a native GABA receptor.
- Expression of the GABA receptor may be accomplished by a variety of different promoter-expression systems in a variety of different host cells.
- the eukaryotic host cells suitably include yeast, insect and mammalian cells.
- the eukaryotic cells which can provide the host for the expression of the receptor are mammalian cells.
- Suitable host cells include rodent fibroblast lines, for example mouse Ltk", Chinese hamster ovary (CHO) and baby hamster kidney (BHK); HeLa; and HEK293 cells. It is necessary to incorporate at least one ⁇ subunit, at least one ⁇ and the ⁇ subunit into the cell line in order to produce the required receptor. Within this limitation, the choice of receptor subunit combination is made according to the type of activity or selectivity which is being screened for. In order to employ this invention most effectively for screening purposes, it is preferable to build up a library of cell lines, each with a different combination of subunits. Typically a library of 5 or 6 cell line types is convenient for this purpose. Preferred subunit combinations include: cti ⁇ i ⁇ .
- Another preferred subunit combination is ⁇ 2 ⁇ .
- Cells are then co-transfected with the desired combination of three expression vectors.
- Calcium phosphate precipitation of DNA is most commonly used (Bachetti et al, Proc. Natl Acad. Sci. USA, 1977, 74, 1590-1594: Maitland et al, Cell, 1977, 14, 133-141). and represents a favoured technique in the context of the present invention.
- a small percentage of the host cells takes up the recombinant DNA. In a small percentage of those, the DNA will integrate into the host cell chromosome.
- neomycin resistance gene will have been incorporated into these host cells, they can be selected by isolating the individual clones which will grow in the presence of neomycin. Each such clone is then tested to identify those which will produce the receptor. This is achieved by inducing the production, for example with dexamethasone, and then detecting the presence of receptor by means of radioligand binding. Alternatively, expression of the GABA receptor may be effected in
- Xenopus oocytes see, for instance, Hadingham et al Mol. Pharmacol. , 1993, 44, 1211-1218. Briefly, isolated oocyte nuclei are injected directly with injection buffer or sterile water containing at least one alpha subunit, at least one beta subunit, and the epsilon subunit engineered into a suitable expression vector. The oocytes are then incubated.
- the expression of subunit combinations in the transfected oocytes may be demonstrated using conventional patch clamp assay.
- This assay measures the charge flow into and out of an electrode sealed on the surface of the cell.
- the flow of chloride ions entering the cell via the GABA gate ion channel is measured as a function of the current that leaves the cell to maintain electrical equilibrium within the cell as the gate opens.
- the present invention provides protein preparations of GABA receptor subunit combinations, especially human GABA receptor subunit combinations, derived from cultures of stably transfected eukaryotic cells of the present invention.
- the protein preparations of GABA receptor subunit combinations can be recovered and purified from recombinant cell cultures by methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Protein refolding steps can be used, as necessary, in completing configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps.
- HPLC high performance liquid chromatography
- polypeptides of the present invention may be a naturally purified product, or a product of chemical synthetic procedures, or produced by recombinant techniques from a prokaryotic or eukaryotic host (for example, by bacterial, yeast, higher plant, insect and mammahan cells in culture). Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. Polypeptides of the invention may also include an initial methionine amino acid residue.
- the GABA epsilon subunit polypeptide of the present invention is also useful for identifying other subunits of the GABA receptor.
- An example of a procedure for identifying these subunits comprises raising high titre polyclonal antisera against unique, bacterially expressed GABA epsilon polypeptides. These polyclonal antisera are then used to immunoprecipitate detergent-solubilized GABA receptors from a mammalian brain, for example, a rat brain.
- the invention also provides preparations of membranes containing subunit combinations of the GABA receptor, especially human GABA receptor subunit combinations, derived from cultures of stably transfected eukaryotic cells of the present invention.
- the cell line, and the membrane preparations therefrom, according to the present invention have utility in screening and design of drugs which act upon the GABA receptor, for example benzodiazepmes, barbiturates, ⁇ -carbolines and neurosteroids.
- the present invention accordingly provides the use of stably cotransfected cell lines described above, and membrane preparations derived therefrom, in screening for and designing medicaments which act upon GABA receptors comprising the ⁇ subunit Of particular interest m this context are molecules capable of interacting selectively with GABA receptors made up of varying subunit combinations
- the cell line in accordance with the present invention, and the membrane preparations derived therefrom provide ideal systems for the study of structure, pharmacology and function of the various GABA receptor subtypes
- preferred screens are functional assays utilizing the pharmacological properties of the GABA receptor subunit combinations of the present invention
- a method for determining whether a ligand, not known to be capable of binding to a human GABAA receptor comprising the epsilon subunit, can bind to a human GABAA receptor comprising the epsilon subunit which comprises contacting a mammalian cell comprising DNA molecules encoding at least one alpha receptor subunit, at least one beta receptor subunit and the epsilon receptor subunit with the ligand under conditions permitting binding of gands known to bind to the GABAA receptor, detecting the presence of any of the ligand bound to the GABAA receptor comprising the epsilon subunit, and thereby determining whether the ligand binds to the GABAA receptor comprising the epsilon subunit
- the epsilon subunit-encodmg DNA in the cell may have a coding sequence substantially the same as the coding sequence shown m Figure 1
- the mamm substantially the same as the coding sequence shown m Figure 1
- This response system may be based on ion flux changes measured, for example, by scintillation counting (where the ion is radiolabelled) or by interaction of the ion with a fluorescent marker
- Particularly suitable ions are chloride
- the mammalian cell is non-neuronal in origin
- a non-neuronal mammalian cell is a fibroblast cell such as an Ltk " cell Drug candidates are identified by choosing chemical compounds which bind with high affinity to the expressed GABAA receptor protein m transfected cells, using radiohgand binding methods well known in the art Drug candidates are also screened for selectivity by identifying compounds which bind with high affinity to one particular GABAA receptor combination but do not bind with high affinity to any other GABAA receptor combination or to any other known receptor site Because selective, high affinity compounds interact primarily with the target
- Ligands or drug candidates identified above may be agonists or antagonists at human GABAA receptors comprising the epsilon subunit, or may be agents which allosterically modulate a human GABAA receptor comprismg the epsilon subunit
- These ligands or drug candidates identified above may be employed as therapeutic agents, for example, for the modulation of appetite behaviours, hormonal interactions and cognition
- the ligands or drug candidates of the present invention thus identified as therapeutic agents may be employed in combination with a suitable pharmaceutical carrier
- Such compositions comprise a therapeutically effective amount of the agonist or antagonist, and a pharmaceutically acceptable carrier or excipient
- the compositions containing the ligand or drug candidate identified according to the methods of the present invention are m unit dosage forms such as tablets, pills, capsules, wafers and the like
- the therapeutic agent may be presented as granules or powders for extemporaneous formulation as volume defined solutions or suspensions Alternatively, the therapeutic agent may be presented m ready-prepared volume defined solutions or
- the principal active ingredient is mixed with a pharmaceutical carrier, e g conventional tabletmg ingredients such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, and other pharmaceutical diluents, e.g water, to form a solid preformulation composition containing a homogeneous mixture of a compound of the present invention, or a non-toxic pharmaceutically acceptable salt thereof
- a pharmaceutical carrier e g conventional tabletmg ingredients such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, and other pharmaceutical diluents, e.g water
- a pharmaceutical carrier e g conventional tabletmg ingredients such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate
- the two components can be separated by an enteric layer which serves to resist disintegration in the stomach and permits the inner component to pass mtact into the duodenum or to be delayed in release
- an enteric layer which serves to resist disintegration in the stomach and permits the inner component to pass mtact into the duodenum or to be delayed in release
- enteric layers or coatings such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol and cellulose acetate.
- compositions of the present invention may be incorporated for administration orally include aqueous solutions, suitably flavoured syrups, aqueous or oil suspensions, and flavoured emulsions with edible oils such as cottonseed oil, sesame oil, coconut oil, peanut oil or soybean oil, as well as elixirs and similar pharmaceutical vehicles
- Suitable dispersing or suspending agents for aqueous suspensions include synthetic and natural gums such as tragacanth, acacia, algmate, dextran, sodium carboxymethylcellulose, methylcellulose, polyvmyl-pyrrohdone or gelatin
- Compositions of the present invention may also be administered via the buccal cavity using conventional technology, for example, absorption wafers
- compositions m the form of tablets, pills, capsules or wafers for oral administration are particularly preferred.
- a minimum dosage level for the ligand or drug candidate identified according to the methods of the present invention is about O.O ⁇ mg per day, preferably about 0.5mg per day and especially about 2.5mg per day.
- a maximum dosage level for the ligand or drug candidate is about 3000mg per day, preferably about 1500mg per day and especially about 500mg per day
- the compounds are administered on a regimen of 1 to 4 times daily preferably once or twice daily, and especially once a day It will be appreciated that the amount of the therapeutic agent required for use therapy will vary not only with the particular compounds or compositions selected but also with the route of administration, the nature of the condition being treated, and the age and condition of the patient, and will ultimately be at the discretion of the patient's physician or pharmacist
- Figure 3 Effects of pentobarbital on oocytes expressing oti ⁇ i, ⁇ i72s and ⁇ i ⁇ i ⁇ GABAA receptors a) Potentiation of the response to a GABA EC20 by increasing concentrations of pentobarbital on oocytes expressing ⁇ i ⁇ i, ⁇ i 2S and cti ⁇ j ⁇ GABAA receptors b) Direct activation of the GABAA receptor by increasing concentrations of pentobarbital on oocvtes expressing ⁇ i ⁇ i, ⁇ i72S and ⁇ i ⁇ i ⁇ GABAA receptors
- Figure 4 Effects of propofol and etomidate on e-contammg receptors a) Potentiation of the response to a GABA EC20 by lO ⁇ M propofol on oocytes expressing ⁇ i ⁇ i, ⁇ i7 2 s and ⁇ i ⁇ i ⁇ GABAA receptors b) Potentiation of the response to a GABA EC20 by etomidate on oocytes expressing ⁇ i ⁇ i, ⁇ r ⁇ 2S, ⁇ i ⁇ i ⁇ , ⁇ 2 and ⁇ 2 ⁇
- R07883 contained sequences homologous to the TM1-TM3 spanning domain of other GABAA receptor subunits.
- R49718 contained sequences homologous to the TM4 domain of GABAA receptors, a putative stop codon and putative 3' untranslated region.
- PCR Polymerase chain reaction
- cDNA sequences 5' of the R07883 sequence were obtained by 5' anchored PCR using human bram Marathon cDNA cloning kit (Clontech) according to manufacturer's protocols
- the nested antisense primers used were derived from the R07883 sequence (ASl, 5' catcgtggtcacggaagaagggac 3', AS2, 5' gccaaaccgcctgctcacattgaa 3') PCR products were subcloned into pMos vector (Amersham) using standard techniques, and sequenced using an Apphed Biosystems 373 DNA sequencer and dye terminator chemistry.
- One of the PCR products was found to extend far enough to contain sequence encoding a putative initiating methionme and 5' untranslated region
- a full length cDNA was generated by PCR using primers derived from sequences in the 5' UT of the anchored PCR product, and the 3' UT sequences m R49718 (5' caggtggtgcggccgctctccgcggaaatgttgt 3 and 5'tccacagggcggccgctggtacctacaagttaag 3', both incorporating a Notl site for subclonmg)
- the PCR product (1550bp) was subcloned into pCDNAI Amp
- the oocyte nuclei were directly injected with 10-20 nl of injection buffer (88 mM NaCl, 1 mM KCl, 15 mM HEPES, at pH 7, filtered through mtro-cellulose) or sterile water containing different combinations of human GABA subunit cDNAs (20 ng ml l ) engineered into the expression vector pCDM8 or pcDNAI/Amp.
- injection buffer 88 mM NaCl, 1 mM KCl, 15 mM HEPES, at pH 7, filtered through mtro-cellulose
- sterile water containing different combinations of human GABA subunit cDNAs (20 ng ml l ) engineered into the expression vector pCDM8 or pcDNAI/Amp.
- oocytes were placed in a 50 ⁇ l bath and perfused at 4-6 ml mm * with modified Barth's medium (MBS) consisting of 88 mM NaCl, 1 mM KCl, 10 mM HEPES, 0.82 mM MgSO 4 , 0.33 mM Ca(NO 3 ) 2 , 0 91 mM CaCl 2 , 2 4 mM NaHC0 3 , at pH 7 5 Cells were impaled with two 1-3 M ⁇ electrodes containing 2 M KCl and voltage-clamped between -40 and -70 mV
- Antisense ohgonucleotide probes to the human ⁇ subunit sequence were generated on an Applied Biosystems Automated DNA synthesiser Probe 1 5 5' TGT-GCC-CGC-CAA-CAT-CAG-GAA-GCT-TTT-GTG-TGC-CAG-ATT-
- Each ohgonucleotide was 3 ' -end labelled with [ r 'S] deoxyadenosine ⁇ '-(th ⁇ ot ⁇ phosphate) in a 30.1 molar ratio of 35 S- ⁇ sotope:ohgonucleot ⁇ de using terminal deoxynucleotidyl transferase for 15 mm at 37°C in the reaction buffer supplied.
- Radiolabelled ohgonucleotide was separated from unincorporated nucleotides using Sephadex G50 spin columns. The 5 specific activities of the labelled probes in several labelling reactions varied from 1.2-2.3 x 10 9 cpm/mg. Monkey brains were removed and fresh frozen m 1 cm blocks. 12 ⁇ m sections were taken and fixed for m situ hybridisation. Hybridisation of the sections was carried out according to the method of Sirinathsmg i and Dunnett (Imaging gene expression m 0 neural graft; Molecular Imaging in Neuroscience: A Practical Approach,
- the localisation appears to be very restricted; residing mamly in the hypothalamus and the arcuate nucleus Weak expression could also be seen in the paraventricular nucleus and in the hilus of the hippocampus There was no detectable message the caudate, putamen, l ⁇ globus palhdus, dorsal thalamus, amygdala, brain stem or cerebellum
- the discrete distribution implies a possible function m "appetite behaviours" (hunger, thirst, sex) and possible hormonal interactions (vasopressm, oxytocm, adrenocorticotrophm hormone and gonadotrophms)
- ⁇ i ⁇ i ⁇ all benzodiazepmes tested at l ⁇ M except CL-218872 which was tested at lO ⁇ M.
- ⁇ 372s all benzodiazepmes tested at approximately 100 x their Ki. Data shown are the mean ⁇ sem of four determinations. The results demonstrate that ⁇ i ⁇ i ⁇ receptors are not modulated by benzodiazepmes.
- Etomidate potentiation has been shown to depend on the ⁇ subunit isoform (Hill-Venning et al. Br. J. Pharmacol. 1997, 120, 749-7 ⁇ 6). When the ⁇ 2 isoform is coexpressed with ⁇ i ⁇ a 10 fold increase in etomidate potency is observed compared to ⁇ i ⁇ i ⁇ .
- 2 ⁇ subunit does not alter the ⁇ subunit selectivity with etomidate.
- Small direct effects were observed with both propofol and etomidate on ⁇ i ⁇ i ⁇ receptors which were absent on ⁇ i ⁇ i and ⁇ 2S receptors. This correlates with the higher potency for direct activation observed with pentobarbital.
Abstract
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Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
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JP52438998A JP2001505423A (en) | 1996-11-25 | 1997-11-18 | Novel cloned GABA receptor subunit cDNA sequences and stably co-transfected cell lines |
EP97912375A EP0948617A1 (en) | 1996-11-25 | 1997-11-18 | A NOVEL CLONED GABA-RECEPTOR SUBUNIT cDNA SEQUENCE AND STABLY CO-TRANSFECTED CELL LINES |
CA002271953A CA2271953A1 (en) | 1996-11-25 | 1997-11-18 | A novel cloned gaba-receptor subunit cdna sequence and stably co-transfected cell lines |
AU49606/97A AU4960697A (en) | 1996-11-25 | 1997-11-18 | A novel cloned gaba-receptor subunit cdna sequence and stably co-transfected cell lines |
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GB9624442.1 | 1996-11-25 | ||
GBGB9624442.1A GB9624442D0 (en) | 1996-11-25 | 1996-11-25 | Nucleic acids |
GB9720995.1 | 1997-10-03 | ||
GBGB9720995.1A GB9720995D0 (en) | 1997-10-03 | 1997-10-03 | Nucleic acids |
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Cited By (2)
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WO1999060118A1 (en) * | 1998-05-15 | 1999-11-25 | Centre National De La Recherche Scientifique (Cnrs) | GABAA RECEPTOR ε2 SUBUNIT |
US7029870B1 (en) | 1997-07-03 | 2006-04-18 | Human Genome Sciences, Inc. | Gabaa receptor epsilon subunits |
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WO1994013799A1 (en) * | 1992-12-10 | 1994-06-23 | Merck Sharp & Dohme Limited | Stably transfected cell lines expressing gaba-a receptors |
WO1996006862A1 (en) * | 1994-08-26 | 1996-03-07 | Human Genome Sciences, Inc. | Gabaa receptor epsilon subunit |
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1997
- 1997-11-18 EP EP97912375A patent/EP0948617A1/en not_active Withdrawn
- 1997-11-18 AU AU49606/97A patent/AU4960697A/en not_active Abandoned
- 1997-11-18 WO PCT/GB1997/003159 patent/WO1998023742A1/en not_active Application Discontinuation
- 1997-11-18 CA CA002271953A patent/CA2271953A1/en not_active Abandoned
- 1997-11-18 JP JP52438998A patent/JP2001505423A/en active Pending
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WO1994013799A1 (en) * | 1992-12-10 | 1994-06-23 | Merck Sharp & Dohme Limited | Stably transfected cell lines expressing gaba-a receptors |
WO1996006862A1 (en) * | 1994-08-26 | 1996-03-07 | Human Genome Sciences, Inc. | Gabaa receptor epsilon subunit |
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Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7029870B1 (en) | 1997-07-03 | 2006-04-18 | Human Genome Sciences, Inc. | Gabaa receptor epsilon subunits |
US7365163B2 (en) | 1997-07-03 | 2008-04-29 | Human Genome Sciences, Inc. | GABAA receptor epsilon subunits |
WO1999060118A1 (en) * | 1998-05-15 | 1999-11-25 | Centre National De La Recherche Scientifique (Cnrs) | GABAA RECEPTOR ε2 SUBUNIT |
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JP2001505423A (en) | 2001-04-24 |
EP0948617A1 (en) | 1999-10-13 |
AU4960697A (en) | 1998-06-22 |
CA2271953A1 (en) | 1998-06-04 |
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