US20100234417A1 - Thyrotropin releasing hormone receptor-orexin receptor hetero-dimers/-oligomers - Google Patents
Thyrotropin releasing hormone receptor-orexin receptor hetero-dimers/-oligomers Download PDFInfo
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- US20100234417A1 US20100234417A1 US12/514,034 US51403407A US2010234417A1 US 20100234417 A1 US20100234417 A1 US 20100234417A1 US 51403407 A US51403407 A US 51403407A US 2010234417 A1 US2010234417 A1 US 2010234417A1
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- releasing hormone
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
- G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
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- A61P25/16—Anti-Parkinson drugs
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- A—HUMAN NECESSITIES
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- A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
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- A61P3/00—Drugs for disorders of the metabolism
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Definitions
- the present invention relates to a hetero-dimeric or hetero-oligomeric receptor, comprising at least one thyrotropin releasing hormone receptor subunit associated with at least one orexin receptor subunit.
- Proteins do not act in isolation in a cell, but in stable or transitory complexes, with protein-protein interactions being key determinants of protein function (Auerbach et al., (2002), Proteomics, 2, 611-623). Furthermore, proteins and protein complexes interact with other cellular components like DNA, RNA and small molecules. Understanding both the individual proteins involved in these interactions and their interactions are important for a better understanding of biological processes.
- TRH thyrotropin-releasing hormone
- CNS central nervous system
- Narcolepsy with cataplexy is associated with low or undetectable levels of cerebrospinal fluid (CSF) orexin A levels in about 90% of patients (Baumann and Basset (2005) Sleep Medicine Reviews 9, 253-268). Mutations of the orexin receptor 2 gene lead to familial canine narcolepsy and a loss of orexin neurons and low CSF orexin A were observed with sporadic canine narcolepsy. Neurological disorders arising from acute traumatic brain injury, Guillain-Barre syndrome and advanced Parkinson's syndrome may also be linked with low or undetectable levels of CSF orexin A levels in some instances.
- Sakurai has postulated a role for the orexin system in feeding and energy homeostasis as the activity of orexin neurons is inhibited by glucose and leptin, and stimulated by ghrelin, a stomach-derived peptide which promotes feeding. This may have implications for the treatment of obesity (Sakurai (2005) Sleep Medicine Reviews 9, 231-241).
- the inventors have discovered that the orexin receptor and the thyrotropin releasing hormone receptor associate. This has important implications regarding therapies for ailments associated with either receptor.
- GPCRs may not only act as monomers but also as homo- and hetero-dimers which causes altered ligand binding, signalling and endocytosis (Rios et al. (2000) Pharmacol. Ther. 92, 71-87).
- the effect of drugs acting as agonists or antagonists of a specific receptor may therefore depend on the binding partners of this receptor. It may be desirable to limit the effect of a drug to a cellular response mediated by a specific receptor dimer.
- thyrotropin releasing hormone receptor or “TRHR” is to be understood to at least include the G protein-coupled receptor analogous to that activated by the thyrotropin releasing hormone (TRH) in the thyrotrope cells of the anterior pituitary gland, as well as a number of structures in the central nervous system (Riehl et al.
- TSH thyroid-stimulating hormone
- TRHR1 thyrotropin releasing hormone receptor 1
- thyrotropin releasing hormone receptor or “TRHR” is also to be understood to mean thyrotropin releasing hormone receptor 2 or TRHR2, a second subtype of thyrotropin releasing hormone receptor known to be expressed at least in the rat and mouse and whose function is yet to be clearly elucidated (Gershengorn (2003) Thyrotropin-releasing hormone receptor signaling, in Encyclopedia of hormones. Eds Henry H L and Norman A W. Academic Press. Vol 3; 502-510).
- TRHR thyrotropin releasing hormone receptor
- TRHR refers to TRHR1.
- orexin receptor or “OxR” is to be understood to mean either orexin receptor 1 (OxR1; OXR1; OX 1 R; hypocretin-1-receptor; hcrtr 1) or orexin receptor 2 (OxR2; OXR2; OX 2 R; hypocretin-2-receptor; hctr 2) being G protein-coupled receptors analogous to those described by Sakurai et al. to be activated by orexin A (OxA; hypocretin-1; Hcrt-1) and orexin B (OxB; hypocretin-2; Hcrt-2) (Sakurai et al. (1998) Cell 92, 573-585). “Orexin receptor” or “OxR” is to be further understood to include newly discovered orexin receptor family members.
- a hetero-dimeric or hetero-oligomeric receptor comprising at least one thyrotropin releasing hormone receptor subunit associated with at least one orexin receptor subunit.
- a method for the treatment of a patient suffering from an orexin-related ailment by administering a therapeutically effective amount of a thyrotropin-releasing hormone receptor agonist, inverse agonist or antagonist.
- the thyrotropin-releasing hormone receptor agonist, inverse agonist or antagonist may be co-administered with an orexin receptor agonist, inverse agonist or antagonist.
- a third aspect of the invention there is provided method for the treatment of a patient suffering from a thyrotropin-releasing hormone-related ailment by administering a therapeutically effective amount of an orexin receptor agonist, inverse agonist or antagonist.
- the orexin receptor agonist, inverse agonist or antagonist may be co-administered with a thyrotropin-releasing hormone receptor agonist, inverse agonist or antagonist.
- a method for the manufacture of a medicament for the treatment of a patient suffering from an orexin-related ailment comprising use of a therapeutically effective amount of a thyrotropin releasing hormone receptor agonist, inverse agonist or antagonist.
- the medicament may contain an orexin receptor agonist, inverse agonist or antagonist.
- a method for the manufacture of a medicament for the treatment of a patient suffering from a thyrotropin-releasing hormone-related ailment comprising use of a therapeutically effective amount of an orexin receptor agonist, inverse agonist or antagonist.
- the medicament may contain a thyrotropin-releasing hormone receptor agonist, inverse agonist or antagonist.
- a method for the treatment of a patient suffering from an orexin-related ailment by administering a therapeutically effective amount of a thyrotropin releasing hormone-selective binding agent, or fragment thereof.
- the thyrotropin releasing hormone-selective binding agent is an antibody, including a humanised antibody, a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a CDR-grafted antibody and/or an anti-idiotypic antibody.
- a seventh aspect of the invention there is provided a method for the treatment of a patient suffering from a thyrotropin-releasing hormone-related ailment by administering a therapeutically effective amount of an orexin-selective binding agent, or fragment thereof.
- the orexin-selective binding agent is an antibody, including a humanised antibody, a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a CDR-grafted antibody and/or an anti-idiotypic antibody.
- a test compound for thyrotropin releasing hormone receptor/orexin receptor hetero-dimer/-oligomer selective activity comprising the steps of:
- a test compound for thyrotropin releasing hormone receptor/orexin receptor hetero-dimer/-oligomer selective activity comprising the steps of:
- a test compound for thyrotropin releasing hormone receptor/orexin receptor hetero-dimer/-oligomer selective antagonism or partial agonism comprising the steps of:
- a test compound for thyrotropin releasing hormone receptor/orexin receptor hetero-dimer/-oligomer selective antagonism or partial agonism comprising the steps of:
- a method for screening a test compound for thyrotropin releasing hormone receptor/orexin receptor hetero-dimer/-oligomer selective inverse agonism comprising the steps of:
- a test compound for thyrotropin releasing hormone receptor/orexin receptor hetero-dimer/-oligomer inverse agonism comprising the steps of:
- the step of determining whether, and/or the extent to which, the test compound interacts with the thyrotropin releasing hormone receptor while the thyrotropin releasing hormone receptor is associated with the orexin receptor; and/or the step of determining whether, and/or the extent to which, the test compound interacts with the orexin receptor while the orexin receptor is associated with the thyrotropin releasing hormone receptor may be performed by way of one or more of the methods described in the applicant's co-pending international patent application “Detection System and Uses Therefor”, which derives priority from the same Australian provisional patent application 2006906292.
- a fourteenth aspect of the invention there are provided selective agonists and/or antagonists and/or inverse agonists of the thyrotropin releasing hormone receptor/orexin receptor hetero-dimer/-oligomer.
- FIGS. 1 to 3 are illustrative of the technique by which the association of thyrotropin releasing hormone receptor and the orexin receptor was detected.
- FIG. 1 shows the composition of the agents forming the basis of the system for detecting molecular associations:
- a first agent comprises a first interacting group coupled to a first reporter component;
- a second agent comprises a second interacting group coupled to a second reporter component;
- a third agent comprises a third interacting group.
- FIG. 2 shows how the administration of the modulator modulates the association of the second interacting group with the third interacting group, preferably by interacting with the third interacting group, either alone, or simultaneously with the first interacting group.
- FIG. 3 shows that if the first and third interacting groups are associated, modulation of the association of the second and third interacting groups consequently modulates the proximity of the first and second reporter components thereby modulating the signal that is able to be detected by the detector. Therefore monitoring the signal generated by proximity of the first and second reporter components by the detector constitutes monitoring the association of the first and third agents. If the first and third interacting groups are not associated, the first and second reporter components will remain spatially separated and generation of a detectable signal is unlikely.
- FIG. 4 shows the thyrotropin releasing hormone receptor (TRHR) as IG1, Rluc as RC1, beta-arrestin 2 (barr2) as IG2, Venus as RC2 and a range of different GPCRs as IG3.
- TRHR thyrotropin releasing hormone receptor
- barr2 beta-arrestin 2
- Venus RC2
- eBRET measurements at 37 C were carried out on HEK293 cells transiently co-expressing TRHR/Rluc and barr2/Venus with either pcDNA3, orexin receptor 2 (OxR2), CXC chemokine receptor 2 (CXCR2), hemagglutin epitope-tagged melanocortin receptor 3 or 4 (HA-MC3R or HA-MC4R), or dopamine D2 receptor long form (D2LR) or short form (D2SR) following the treatment of each with their respective ligands.
- TRHR thyrotropin releasing hormone receptor
- TRH thyrotropin releasing hormone
- OxA OXR2
- IL-8 interleukin-8
- a-MSH alpha-melanocyte-stimulating hormone
- BROM bromocriptine
- FIG. 5 shows the thyrotropin releasing hormone receptor (TRHR) as IG1, Rluc as RC1, either beta-arrestin 1 (barr1) or beta-arrestin 2 (barr2) as IG2, EGFP as RC2 and OxR2 as IG3.
- TRHR thyrotropin releasing hormone receptor
- Rluc Rluc
- barr1 beta-arrestin 1
- barr2 beta-arrestin 2
- EGFP RC2
- OxR2 OxR2 as IG3.
- eBRET measurements at 37 C were carried out on HEK293 cells transiently co-expressing TRHR/Rluc and EGFP/barr1 or EGFP/barr2 with either pcDNA3 or OXR2.
- Ligand treatments were either OxA or TRH only or both OxA and TRH combined.
- Phosphate-buffered saline (PBS) was used as a vehicle control.
- FIG. 6 shows the thyrotropin releasing hormone receptor (TRHR) as IG2, Rluc as RC1, beta-arrestin 2 (barr2) as IG2, Venus as RC2 and OxR1 or OxR2 as IG3.
- TRHR thyrotropin releasing hormone receptor
- barr2 beta-arrestin 2
- eBRET measurements were carried out at 37 C on HEK293 cells transiently co-expressing TRHR/Rluc and barr2/Venus with either pcDNA3, OxR1 or OxR2 following pretreatment with 10 ⁇ 6 M OxR1-selective antagonist, SB-334867-A, for approximately 40 minutes and then 10 ⁇ 6 M OxA (IG3 ligand; modulator) or 10 ⁇ 6 M TRH (IG1 ligand), or both, was added. Where antagonist was not preincubated, cells were treated with PBS instead for the same amount of time.
- FIG. 7 shows the thyrotropin releasing hormone receptor (TRHR) as IG1, Rluc as RC1, beta-arrestin 1 (barr1) or beta-arrestin 2 (barr2) as IG2, EGFP as RC2 and hemagglutin epitope-tagged OxR2 (HA-OxR2) as IG3.
- TRHR thyrotropin releasing hormone receptor
- barr1 beta-arrestin 1
- barr2 beta-arrestin 2
- EGFP RC2
- hemagglutin epitope-tagged OxR2 hemagglutin epitope-tagged OxR2
- Ligand treatments were either OxA or TRH only.
- Phosphate-buffered saline (PBS) was used as a vehicle control.
- FIG. 8 shows the thyrotropin releasing hormone receptor (TRHR) as IG1, Rluc as RC1, beta-arrestin 1 (barr1)) or beta-arrestin 1 phosphorylation-independent mutant R169E (barr1R169E) as IG2, EGFP as RC2 and OxR2 as IG3.
- TRHR thyrotropin releasing hormone receptor
- barr1R169E beta-arrestin 1 phosphorylation-independent mutant R169E
- barr1R169E beta-arrestin 1 phosphorylation-independent mutant R169E
- EGFP EGFP as RC2
- OxR2 IG3.
- eBRET measurements at 37 C were carried out on HEK293 cells transiently co-expressing TRHR/Rluc and EGFP/barr1 or EGFP/barr1R169E with either pcDNA3 or OxR2.
- Ligand treatments were either OxA or TRH only.
- FIG. 9 shows the thyrotropin releasing hormone receptor truncated at amino acid 335 (TRHR335) as IG1, Rluc as RC1, beta-arrestin 1 (barr1) as IG2, EGFP as RC2 and OxR2 or TRHR as IG3.
- TRHR335 thyrotropin releasing hormone receptor truncated at amino acid 335
- Rluc Rluc as RC1
- beta-arrestin 1 barr1
- EGFP EGFP
- OxR2 or TRHR IG3.
- eBRET measurements at 37 C were carried out on HEK293 cells transiently co-expressing TRHR335/Rluc and EGFP/barr1 with either OxR2 or TRHR.
- Ligand treatments were either OxA or TRH only.
- FIG. 10 shows a dose-response curve for the thyrotropin releasing hormone receptor (TRHR) as IG1, Rluc as RC1, beta-arrestin 2 (barr2) as IG2, Venus as RC2 and in the absence of IG3.
- TRHR thyrotropin releasing hormone receptor
- barr2 beta-arrestin 2
- Sigmoidal dose response curves were plotted using Prism (GraphPad), either assuming a Hill slope of 1 or allowing for variable slope.
- the EC 50 and Hill slope values for the variable slope curve are included in a table in the graph.
- FIG. 11 shows a dose-response curve for OxR2 as IG1, Rluc as RC1, barr2 as IG2, Venus as RC2 and in the absence of IG3.
- BRET measurements at 37 C were carried out on HEK293 cells transiently co-expressing OxR2/Rluc, barr2/Venus and pcDNA3 with increasing doses of OxA.
- Sigmoidal dose response curves were plotted using Prism (GraphPad), either assuming a Hill slope of 1 or allowing for variable slope.
- the EC 50 and Hill slope values for the variable slope curve are included in a table in the graph.
- FIG. 12 shows dose-response curves for the thyrotropin releasing hormone receptor (TRHR) as IG1, Rluc as RC1, beta-arrestin 2 (barr2) as IG2, Venus as RC2 and OxR2 as IG3.
- TRHR thyrotropin releasing hormone receptor
- barr2 beta-arrestin 2
- OxR2 OxR2
- BRET measurements at 37 C were carried out on HEK293 cells transiently co-expressing TRHR/Rluc, barr2/Venus and OxR2 with increasing doses of OxA.
- Sigmoidal dose response curves were plotted using Prism (GraphPad), either assuming a Hill slope of 1 or allowing for variable slope.
- the EC 50 and Hill slope values for the variable slope curves are included in a table in the graph. Curves generated using coelenterazine h and EnduRen as two forms of Rluc substrate (reporter component initiator) are shown.
- FIG. 13 shows dose-response curves for TRHR as IG1, Rluc as RC1, barr1 as IG2, EGFP as RC2 in the presence or absence of OxR2 as IG3.
- BRET measurements at 37 C were carried out on HEK293 cells transiently co-expressing TRHR/Rluc and EGFP/barr1 in the absence of OxR2 with increasing doses of TRH, as well as HEK293 cells transiently co-expressing TRHR/Rluc and EGFP/barr1 with OxR2 with increasing doses of OxA with and without 10 ⁇ 6 M TRH.
- TRHR/Rluc+EGFP/barr1+OxR2 TRH (10 ⁇ 6 M)+OxA: Data calculated).
- FIG. 14 shows dose-response curves for TRHR as IG1, Rluc as RC1, barr1 as IG2, EGFP as RC2 in the presence or absence of OxR2 as IG3.
- BRET measurements at 37 C were carried out on HEK293 cells transiently co-expressing TRHR/Rluc and EGFP/barr1 in the absence of OxR2 with increasing doses of TRH, as well as HEK293 cells transiently co-expressing TRHR/Rluc and EGFP/barr1 with OxR2 with increasing doses of OxA, or increasing doses of TRH with 10 ⁇ 6 M OxA.
- a curve mathematically generated by addition of the ligand-induced signal generated with 10 ⁇ 6 M OxA (from the OxA: TRHR/Rluc+EGFP/barr1+OxR2 curve) to each of the points generated for the TRH: TRHR/Rluc+EGFP/barr1 curve is also plotted (TRHR/Rluc+EGFP/barr1+OxR2: TRH+OxA (10 ⁇ 6 M): Data calculated).
- FIG. 15 shows dose response curves for TRHR335 as IG1, Rluc as RC1, barr2 as IG2, Venus as RC2 and OxR2 as IG3.
- BRET measurements at 37 C were carried out on HEK293 cells transiently co-expressing TRHR335/Rluc, barr2/Venus and OxR2 with increasing doses of TRH and OxA alone or in combination.
- FIG. 16 shows cumulative eBRET reads over time for each combination of receptors (IG1 and IG3; data captured over 83 mins).
- TRHR is IG1
- Rluc is RC1
- barr1 is IG2
- EGFP is RC2
- OxR2 is IG3.
- the same amount of EGFP/barr1 is transfected for each experiment.
- TRHR/Rluc is transfected at a constant amount (0.1 ⁇ g DNA/well) while OxR2 (IG3) is transfected at varying amounts of DNA (0, 0.01, 0.05, 0.1, 0.5, 0.7 ⁇ g DNA/well).
- eBRET measurements at 37 C were carried out on HEK293 cells following addition of 10 ⁇ 6 M OxA (modulator) to each well.
- the signal is only detected when OxR2 (IG3) is expressed (no signal was recorded at 0 ⁇ g OxR2).
- FIG. 17 shows dose response curves for TRHR as IG1, Rluc as RC1, barr2 as IG2, Venus as RC2 and OxR2 as IG3.
- BRET measurements at 37 C were carried out on HEK293 cells transiently co-expressing TRHR/Rluc, barr2/Venus and OxR2 with increasing doses of OxA in either 96-well or 384-well microplates.
- FIG. 18 shows OxR2 as IG1, Rluc8 as RC1, beta-arrestin 2 (barr2) as IG2, Venus as RC2 and hemagglutin epitope-tagged TRHR (HA-TRHR) as IG3.
- eBRET measurements at 37 C were carried out on HEK293 cells transiently co-expressing OxR2/Rluc8 and barr2/Venus with either pcDNA3 or HA-TRHR.
- Ligand treatments were either OxA or TRH only.
- Phosphate-buffered saline (PBS) was used as a vehicle control. Data presented as ligand-induced BRET ratios.
- FIG. 19 shows the thyrotropin releasing hormone receptor (TRHR) as IG1, Rluc8 as RC1, beta-arrestin 2 (barr2) as IG2, Venus as RC2 and hemagglutin epitope-tagged OxR2 (HA-OxR2) as IG3.
- TRHR thyrotropin releasing hormone receptor
- barr2 beta-arrestin 2
- HA-OxR2 hemagglutin epitope-tagged OxR2
- FIG. 20 shows the thyrotropin releasing hormone receptor (TRHR) as IG1, Rluc8 as RC1, beta-arrestin 2 (barr2) as IG2, Venus as RC2 and hemagglutin epitope-tagged OxR2 (HA-OxR2) as IG3.
- TRHR thyrotropin releasing hormone receptor
- barr2 beta-arrestin 2
- HA-OxR2 hemagglutin epitope-tagged OxR2
- FIG. 21 shows z-factor data for the thyrotropin releasing hormone receptor (TRHR) as IG1, Rluc8 as RC1, beta-arrestin 2 (barr2) as IG2, Venus as RC2 and hemagglutin epitope-tagged OxR2 (HA-OxR2) as IG3.
- TRHR thyrotropin releasing hormone receptor
- barr2 beta-arrestin 2
- Venus Venus
- eBRET measurements at 37 C were carried out on HEK293 cells transiently co-expressing TRHR/Rluc8 and barr2/Venus with HA-OxR2 aliquoted into all wells of a 96-well plate.
- Phosphate-buffered saline PBS was added to the first two rows and the last two rows of the 96-well plate (48 wells in total) as a vehicle control.
- OxA was added to the middle four rows of the 96-well plate (48 wells in total). Data presented as fluorescence/luminescence.
- eBRET extended BRET BRET monitored over extended time periods.
- ECFP Enhanced Cyan Fluorescent Protein which is a variant of the Aequorea victoria green fluorescent protein gene (GFP).
- EGFP Enhanced Green Fluorescent Protein is a red-shifted variant of wild-type GFP.
- GPCRs G-protein coupled receptors.
- His(6) Histidine tag consisting of 6 consecutive histidine residues.
- CCR2 selective ligand Monocyte chemoattractant protein 1 (CCR2 selective ligand).
- MIP1b Macrophage inflammatory protein 1b (CCR5 selective ligand).
- Rluc8 An improved Renilla luciferase.
- the invention described herein may include one or more ranges of values (e.g. size, concentration etc).
- a range of values will be understood to include all values within the range, including the values defining the range, and values adjacent to the range that lead to the same or substantially the same outcome as the values immediately adjacent to that value which defines the boundary to the range.
- the invention relates, inter alia, to hetero-dimeric or hetero-oligomeric receptor, comprising at least one thyrotropin releasing hormone receptor subunit associated with at least one orexin receptor subunit.
- hetero-dimer and “hetero-oligomer”, and variations such as “hetero-dimeric” and “hetero-oligomeric”, as used herein, refer to an entity within which at least one thyrotropin releasing hormone receptor is associated with at least one orexin receptor.
- the phrase “associated with”, as used herein, refers to combination via any known direct or indirect stabilising atomic or molecular level interaction or any combination thereof, where the interactions include, without limitation, bonding interactions such as covalent bonding, ionic bonding, hydrogen bonding, co-ordinate bonding, or any other molecular bonding interaction, electrostatic interactions, polar or hydrophobic interactions, or any other classical or quantum mechanical stabilising atomic or molecular interaction.
- hetero-dimeric or hetero-oligomeric receptor comprising at least one thyrotropin releasing hormone receptor subunit associated with at least one orexin receptor subunit represents a novel drug target.
- association of the thyrotropin releasing hormone receptor with orexin receptor enables the use of ligands of one receptor (be they agonists, inverse agonists or antagonists) in the treatment of ailments related to the other receptor.
- the present invention encompasses a method for the treatment of a patient suffering from an orexin-related ailment by administering a therapeutically effective amount of a thyrotropin-releasing hormone receptor agonist, inverse agonist or antagonist.
- the thyrotropin-releasing hormone receptor agonist, inverse agonist or antagonist may be co-administered with an orexin receptor agonist, inverse agonist or antagonist.
- the present invention further encompasses a method for the treatment of a patient suffering from a thyrotropin-releasing hormone-related ailment by administering a therapeutically effective amount of an orexin receptor agonist, inverse agonist or antagonist.
- the present invention further encompasses a method for the manufacture of a medicament for the treatment of a patient suffering from an orexin-related ailment by administering a therapeutically effective amount of a thyrotropin releasing hormone receptor agonist, inverse agonist or antagonist.
- the medicament may further contain an orexin receptor agonist, inverse agonist or antagonist.
- the present invention further encompasses a method for the manufacture of a medicament for the treatment of a patient suffering from a thyrotropin-releasing hormone-related ailment by administering a therapeutically effective amount of an orexin receptor agonist, inverse agonist or antagonist.
- the medicament may further contain a thyrotropin-releasing hormone receptor agonist, inverse agonist or antagonist.
- the present invention encompasses a method for the treatment of a patient suffering from an orexin-related ailment by administering a therapeutically effective amount of a thyrotropin-releasing hormone-selective binding agent, or fragment thereof.
- the thyrotropin-releasing hormone-selective binding agent may be an antibody, including a humanised antibody, a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a CDR-grafted antibody and/or an anti-idiotypic antibody.
- the present invention further encompasses a method for the treatment of a patient suffering from a thyrotropin-releasing hormone-related ailment by administering a therapeutically effective amount of an orexin-selective binding agent, or fragment thereof.
- the orexin-selective binding agent may be an antibody, including a humanised antibody, a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a CDR-grafted antibody and/or an anti-idiotypic antibody.
- the present invention further encompasses a method for the treatment of a patient suffering from a thyrotropin-releasing hormone-related ailment or an orexin-related ailment by administering a therapeutically effective amount of a thyrotropin releasing hormone receptor/orexin receptor hetero-dimer/-oligomer selective agonist, inverse agonist or antagonist.
- the present invention further encompasses the use of a therapeutically effective amount of a thyrotropin releasing hormone receptor/orexin receptor hetero-dimer/-oligomer selective agonist, inverse agonist or antagonist for the manufacture of a medicament for the treatment of a patient suffering from a thyrotropin-releasing hormone-related ailment or an orexin-related ailment.
- the present invention further encompasses a method for the treatment of a patient suffering from a thyrotropin-releasing hormone-related ailment by administering a therapeutically effective amount of a selective orexin receptor/thyrotropin-releasing hormone receptor hetero-dimer/-oligomer agonist, inverse agonist or antagonist.
- the selective orexin receptor/thyrotropin-releasing hormone receptor hetero-dimer/-oligomer agonist, inverse agonist or antagonist may be co-administered with a thyrotropin-releasing hormone receptor agonist, inverse agonist or antagonist.
- the selective orexin receptor/thyrotropin-releasing hormone receptor hetero-dimer/-oligomer agonist, inverse agonist or antagonist may be co-administered with an orexin receptor agonist, inverse agonist or antagonist.
- the present invention further encompasses a method for the treatment of a patient suffering from a orexin-related ailment by administering a therapeutically effective amount of a selective orexin receptor/thyrotropin-releasing hormone receptor hetero-dimer/-oligomer agonist, inverse agonist or antagonist.
- the selective orexin receptor/thyrotropin-releasing hormone receptor hetero-dimer/-oligomer agonist, inverse agonist or antagonist may be co-administered with a thyrotropin-releasing hormone receptor agonist, inverse agonist or antagonist.
- the selective orexin receptor/thyrotropin-releasing hormone receptor hetero-dimer/-oligomer agonist, inverse agonist or antagonist may be co-administered with an orexin receptor agonist, inverse agonist or antagonist.
- the present invention further encompasses a method for the manufacture of a medicament for the treatment of a patient suffering from an thyrotropin-releasing hormone-related ailment comprising use of a therapeutically effective amount of a selective orexin receptor/thyrotropin-releasing hormone receptor hetero-dimer/-oligomer agonist, inverse agonist or antagonist.
- the medicament may contain an orexin receptor agonist, inverse agonist or antagonist.
- the medicament may contain an thyrotropin-releasing hormone receptor agonist, inverse agonist or antagonist.
- the present invention further encompasses a method for the manufacture of a medicament for the treatment of a patient suffering from an orexin-related ailment comprising use of a therapeutically effective amount of a selective orexin receptor/thyrotropin-releasing hormone receptor hetero-dimer/-oligomer agonist, inverse agonist or antagonist.
- the medicament may contain an orexin receptor agonist, inverse agonist or antagonist.
- the medicament may contain an thyrotropin-releasing hormone receptor agonist, inverse agonist or antagonist.
- Thyrotropin releasing hormone-related ailments include aliments that are related to increased or decreased production of thyrotropin releasing hormone, and/or increased or decreased responsiveness of cells to thyrotropin releasing hormone.
- the following list (Gary, Keith A., et al., The Thyrotropin-Releasing Hormone (TRH) Hypothesis of Homeostatic Regulation: Implications for TRH-Based Therapeutics, JPET 305:410-416, 2003) provides some examples of TRH-related ailments:
- thyrotropin releasing hormone-related ailment is not limited thereto.
- Orexin-related ailments include aliments that are related to increased or decreased production of orexin, and/or increased or decreased responsiveness of cells to orexin.
- a major example of an orexin-related ailment is narcolepsy with cataplexy. This is associated with low or undetectable levels of cerebrospinal fluid (CSF) orexin A levels in about 90% of patients (Baumann and Bassetti (2005) Sleep Medicine Reviews 9, 253-268). Mutations of the orexin receptor 2 gene lead to familial canine narcolepsy and a loss of orexin neurons and low CSF orexin A were observed with sporadic canine narcolepsy.
- CSF cerebrospinal fluid
- orexin receptor modulators include orexin A (OxA; hypocretin-1; Hcrt-1), orexin B (OxB; hypocretin-2; Hcrt-2) and fragments thereof (Lang et al. (2004) J Med Chem 47, 1153-1160).
- OxR1 and OxR2 include 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline analogues (Hirose M et al. (2003) Bioorg. Med. Chem. Lett. 13, 4497-4499), Almorexant ((2R)-2- ⁇ (1S)-6,7-dimethoxy-1-[2-(4-trifluoromethylphenyl)-ethyl ⁇ -3,4-dihydro-1H-isoquinolin-2-yl]-N-methyl-2-phenyl-acetamide; ACT-078573; Actelion Pharmaceuticals Ltd., Allschwil, Switzerland; Brisbare-Roch et al. (2007) Nature Medicine 13, 150-155).
- OxR1 antagonists include SB-334867-A (1-(2-methylbenzoxazol-6-yl)-3-[1,5]naphthyridin-4-yl urea hydrochloride), SB-674042 (1-(5-(2-fluoro-phenyl)-2-methyl-thiazol-4-yl)-1-((S)-2-(5-phenyl-(1,3,4)oxadiazol-2ylmethyl)-pyrrolidin-1-yl)-methanone), SB-408124 (1-(6,8-difluoro-2-methyl-quinolin-4-yl)-3-(4-dimethylamino-phenyl)-urea) and SB-410220 (1-(5,8-difluoro-quinolin-4-yl)-3-(4-dimethylamino-phenyl)-urea) (Haynes et al. (2000) Regulatory Peptides 96, 45-51; Langmead et al.
- OxR2 antagonists include N-Arylmethyl tert-leucyl 6,7-dimethoxy-1,2,3,4-tetrahydroiso-quinoline analogues and N-acyl 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline analogues (Hirose M et al. (2003) Bioorg. Med. Chem. Lett.
- thyrotropin releasing hormone receptor thyrotropin releasing hormone
- TRH thyrotropin releasing hormone
- TRF thyrotropin releasing hormone
- pGlu-His-Pro-NH 2 thyrotropin releasing hormone
- [Glu2]TRH [Glu2]TRH with the amino-terminal pyroglutamyl residue replaced with a pyridinium moiety
- RX-77368 pGlu-His-(3,3′-dimethyl)-Pro-NH 2 ; Ferring Pharmaceuticals, Feltham, Middlesex, UK), CG-3509 (Grunenthal GmBH, Stolberg, Germany), MK-771 (1-pyro-2-aminoadipyl-L-histidyl-L-thiazolidine-4-carboxamide; Merck, Rahway, N.J.), posatirelin (RGH 2202; L-6-ketopiperidine-2-carbonyl-L-leucyl-L-proline amide; Gedeon Richter Pharmaceuticals, Budapest, Hungary), Ro 24-9975 (1S,3R,5(2S),5S)-5-[(5-oxo-1-phenylmethyl)-2-pyrrolidinyl]-methyl]-5-[(1H-imidazol-5-yl)methyl]-cyclohexaneacetamide; Hoffman-La Roche,
- TRH analogs may be useful for the treatment of excessive daytime sleepiness in narcolepsy (Nishino et al. (1997) The Journal of Neuroscience 17, 6401-6408).
- the TRH analogs CG-3703 and TA-0910 significantly reduced slow wave sleep (SWS) and rapid eye movement (REM) sleep in a dose- and time-dependent manner.
- SWS slow wave sleep
- REM rapid eye movement
- Serum T 3 and T 4 did not change significantly “suggesting that the anticataplectic and alerting effects of TRH and analogs of TRH are mediated by neuromodulatory CNS properties and not by indirect effects on the thyroid axis.” (Nishino et al. (1997) The journal of neuroscience 17, 6401-6408). These observations were supported by a further study in 2000 (Riehl et al. (2000) Neuropsychopharmacology 23, 34-45). The mode of action of TRH and orexins (and analogs thereof) in the pathophysiology of narcolepsy remains to be elucidated, however, the hetero-dimer/-oligomer interaction identified in this invention contributes to the integration of these receptor systems. Riehl et al.
- hypocretin [orexin]-containing neurons are exclusively localized in the lateral hypothalamus (Sakurai et al. 1998 [ Cell, 92, 573-585]; Peyron et al. 1998 [ J. Neurosc. 18, 9996-10015]), an area that is rich in TRH neurons (Kreider et al. 1985 [ Peptides 6, 997-1000]).
- hypocretin [orexin] and TRH receptors are G-protein coupled receptors for neuropeptides, and that the TRH receptor exhibits the second highest (25%) homology (with the Y2 neuropeptide Y receptor having the highest homology) to the hypocretin [orexin] receptors (Sakurai et al. 1998 [Cell, 92 , 573 -585]), suggesting that TRH may play an important role in the pathophysiology of narcolepsy through an unknown specific interaction with the hypocretin [orexin] system.” (Riehl et al. (2000) Neuropsychopharmacology 23, 34-45). The authors have identified the likelihood of TRH and orexin system integration without identifying that such integration could occur as a result of the receptor hetero-dimerization/-oligomerization identified in this invention.
- the TRH and orexin receptor systems may integrate with regard to the control of feeding and metabolic homeostasis. Thyroid hormone secretion is suppressed during starvation, whereas preprohypocretin (the precursor of orexin peptides) mRNA is upregulated in the lateral hypothalamus.
- preprohypocretin the precursor of orexin peptides
- mRNA is upregulated in the lateral hypothalamus.
- the present invention provides a method for the treatment of a patient suffering from an orexin-related ailment other than narcolepsy by administering a therapeutically effective amount of a thyrotropin-releasing hormone receptor agonist, inverse agonist or antagonist.
- the present invention provides a method for the treatment of a patient suffering from an orexin-related ailment by administering a therapeutically effective amount of a thyrotropin-releasing hormone receptor agonist, inverse agonist or antagonist other than TA0910 (Ceredist).
- the present invention provides a method for the treatment of a patient suffering from an orexin-related ailment by administering a therapeutically effective amount of a thyrotropin-releasing hormone receptor agonist, inverse agonist or antagonist other than TA0910 (Ceredist), CG3703 and CG3509.
- the present invention provides a method for the treatment of a patient suffering from an orexin-related ailment by administering a therapeutically effective amount of a thyrotropin-releasing hormone receptor agonist, inverse agonist or antagonist selected from the group: thyrotropin releasing hormone (TRH; thyroliberin; TRF; pGlu-His-Pro-NH 2 ), [Glu2]TRH, [Glu2]TRH with the amino-terminal pyroglutamyl residue replaced with a pyridinium moiety (Prokai-Tatrai et al. (2005) Med. Chem.
- JTP-2942 N alpha -[(1S,2R)-2-methyl-4-oxocyclopentylcarbonyl]-L-histidyl-L-prolinamide monohydrate; Japan Tobacco, Inc., Tokyo, Japan), azetirelin (YM-14673; Yamanouchi Pharmaceutical Co., Ltd, Tokyo, Japan), DN-1417 (Gamma-butyrolactone-gamma-carbonyl-histidyl-prolinamide citrate; Miyamoto M et al. (1981) Life Sci.
- RX-77368 (pGlu-His-(3,3′-dimethyl)-Pro-NH 2 ; Ferring Pharmaceuticals, Feltham, Middlesex, UK), CG-3509 (Grunenthal GmBH, Stolberg, Germany), MK-771 (1-pyro-2-aminoadipyl-L-histidyl-L-thiazolidine-4-carboxamide; Merck, Rahway, N.J.), posatirelin (RGH 2202; L-6-ketopiperidine-2-carbonyl-L-leucyl-L-proline amide; Gedeon Richter Pharmaceuticals, Budapest, Hungary), Ro 24-9975 (1S,3R,5(2S),5S)-5-[(5-oxo-1-phenylmethyl)-2-pyrrolidinyl]-methyl]-5-[(1H-imidazol-5-yl)methyl]-cyclohexaneacetamide; Hoffman-La Roche, Basel, Switzerland), pro
- the present invention provides a method for the treatment of a patient suffering from an orexin-related ailment by administering a therapeutically effective amount of a thyrotropin-releasing hormone receptor agonist, inverse agonist or antagonist selected from the group: thyrotropin releasing hormone (TRH; thyroliberin; TRF; pGlu-His-Pro-NH 2 ), [Glu2]TRH, [Glu2]TRH with the amino-terminal pyroglutamyl residue replaced with a pyridinium moiety (Prokai-Tatrai et al. (2005) Med. Chem.
- JTP-2942 N alpha -[(1S,2R)-2-methyl-4-oxocyclopentylcarbonyl]-L-histidyl-L-prolinamide monohydrate; Japan Tobacco, Inc., Tokyo, Japan), azetirelin (YM-14673; Yamanouchi Pharmaceutical Co., Ltd, Tokyo, Japan), DN-1417 (Gamma-butyrolactone-gamma-carbonyl-histidyl-prolinamide citrate; Miyamoto M et al. (1981) Life Sci.
- RX-77368 (pGlu-His-(3,3′-dimethyl)-Pro-NH 2 ; Ferring Pharmaceuticals, Feltham, Middlesex, UK), MK-771 (1-pyro-2-aminoadipyl-L-histidyl-L-thiazolidine-4-carboxamide; Merck, Rahway, N.J.), posatirelin (RGH 2202; L-6-ketopiperidine-2-carbonyl-L-leucyl-L-proline amide; Gedeon Richter Pharmaceuticals, Budapest, Hungary), Ro 24-9975 (1S,3R,5(2S),5S)-5-[(5-oxo-1-phenylmethyl)-2-pyrrolidinyl]-methyl]-5-[(1H-imidazol-5-yl)methyl]-cyclohexaneacetamide; Hoffman-La Roche, Basel, Switzerland), protirelin (5-oxo-L-prolyl-L-histi
- the present invention comprises a method for screening a test compound for thyrotropin releasing hormone receptor/orexin receptor hetero-dimer/-oligomer selective activity, the method comprising the steps of:
- the present invention comprises a method for screening a test compound for thyrotropin releasing hormone receptor/orexin receptor hetero-dimer/-oligomer selective activity, the method comprising the steps of:
- the step of determining whether, and/or the extent to which, the test compound interacts with the thyrotropin releasing hormone receptor while the thyrotropin releasing hormone receptor is associated with the orexin receptor; and/or the step of determining whether, and/or the extent to which, the test compound interacts with the orexin receptor while the orexin receptor is associated with the thyrotropin releasing hormone receptor are performed by way of the methods described in the applicant's co-pending international patent application “Detection System and Uses Therefor”, which derives priority from the same Australian provisional patent application 2006906292.
- the present invention includes selective agonists and/or antagonists and/or inverse agonists of the thyrotropin releasing hormone receptor/orexin receptor hetero-dimer/-oligomer.
- the term “patient” refers to any animal that may be suffering from one or more of orexin- or thyrotropin releasing hormone-related ailments. Most preferably the animal is a mammal. The term will be understood to include for example human, farm animals (i.e., cattle, horses, goats, sheep and pigs), household pets (i.e., cats and dogs) and the like.
- terapéuticaally effective amount refers to an amount sufficient to modulate a biological activity associated with the interaction of orexin receptor agonist, inverse agonist or antagonist with the orexin receptor or thyrotropin releasing hormone receptor agonist, inverse agonst or antagonist with the thyrotropin-releasing hormone receptor or of orexin receptor/thyrotropin-releasing hormone receptor hetero-dimer/oligomer-specific agonist, inverse agonist or antagonist with an orexin receptor/thyrotropin-releasing hormone receptor hetero-dimer/oligomer.
- a therapeutically effective amount of a thyrotropin-releasing hormone receptor agonist, inverse agonist or antagonist or a therapeutically effective amount of an orexin receptor agonist, inverse agonist or antagonist in combination may be lower than therapeutically effective amounts of thyrotropin-releasing hormone receptor agonist, inverse agonist or antagonist or orexin receptor agonist, inverse agonist or antagonist when administered alone.
- a thyrotropin-releasing hormone receptor agonist, inverse agonist or antagonist and a orexin receptor agonist, inverse agonist or antagonist in combination may generate a therapeutic effect at what would otherwise be sub-therapeutic doses of either.
- Medicaments of the invention may be administered by injection, or prepared for oral, pulmonary, nasal or for any other form of administration.
- the medicaments are administered, for example, intravenously, subcutaneously, intramuscularly, intraorbitally, ophthalmically, intraventricularly, intracranially, intracapsularly, intraspinally, intracisternally, intraperitoneally, buccal, rectally, vaginally, intranasally or by aerosol administration.
- the mode of administration must, however, be at least suitable for the form in which the medicament has been prepared.
- the mode of administration for the most effective response may need to be determined empirically and the means of administration described below are given as examples, and do not limit the method of delivery of the composition of the present invention in any way. All the above formulations are commonly used in the pharmaceutical industry and are commonly known to suitably qualified practitioners.
- the medicaments of the invention may include pharmaceutically acceptable nontoxic excipients and carriers and administered by any parenteral techniques such as subcutaneous, intravenous and intraperitoneal injections.
- the formulations may optionally contain one or more adjuvants.
- the pharmaceutical forms suitable for injectable use include sterile aqueous solutions (where water-soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
- the compounds of the invention may be encapsulated in liposomes and delivered in injectable solutions to assist their transport across cell membrane.
- Such preparations may contain constituents of self-assembling pore structures to facilitate transport across the cellular membrane.
- the carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
- Proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
- Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
- Sterile injectable solutions may be prepared by incorporating the active compounds in the required amount in an appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilisation.
- dispersions are prepared by incorporating the various sterilised active ingredient into a sterile vehicle that contains the basic dispersion medium and the required other ingredients from those enumerated above.
- the preferred methods of preparation are vacuum drying and freeze-drying techniques that yield a powder of the active ingredient plus any additional desired ingredient from previously sterile-filtered solution thereof.
- Solid dosage forms include tablets, capsules, pills, troches or lozenges, cachets or pellets.
- liposomal or proteinoid encapsulation may be used to formulate the present compositions (as, for example, proteinoid microspheres reported in U.S. Pat. No. 4,925,673).
- Liposomal encapsulation may be used and the liposomes may be derivatised with various polymers (E.g., U.S. Pat. No. 5,013,556).
- the formulation will include the compounds described as part of the invention (or a chemically modified form thereof), and inert ingredients which allow for protection against the stomach environment, and release of the biologically active material in the intestine.
- the location of release may be the stomach, the small intestine (the duodenum, the jejunum, or the ileum), or the large intestine.
- the small intestine the duodenum, the jejunum, or the ileum
- the large intestine One skilled in the art has available formulations that will not dissolve in the stomach, yet will release the material in the duodenum or elsewhere in the intestine.
- the release will avoid the deleterious effects of the stomach environment, either by protection of the composition or by release of the compounds beyond the stomach environment, such as in the intestine.
- a coating impermeable to at least pH 5.0 is essential.
- examples of the more common inert ingredients that are used as enteric coatings are cellulose acetate trimellitate (CAT), hydroxypropylmethylcellulose phthalate (HPMCP), HPMCP 50, HPMCP 55, polyvinyl acetate phthalate (PVAP), Eudragit L30D, Aquateric, cellulose acetate phthalate (CAP), Eudragit L, Eudragit S, and Shellac. These coatings may be used as mixed films.
- a coating or mixture of coatings can also be used on tablets, which are not intended for protection against the stomach. This can include sugar coatings, or coatings that make the tablet easier to swallow.
- Capsules may consist of a hard shell (such as gelatin) for delivery of dry therapeutic i.e. powder; for liquid forms, a soft gelatin shell may be used.
- the shell material of cachets could be thick starch or other edible paper. For pills, lozenges, moulded tablets or tablet triturates, moist massing techniques can be used.
- the therapeutic can be included in the formulation as fine multiparticulates in the form of granules or pellets of particle size about 1 mm.
- the formulation of the material for capsule administration could also be as a powder, lightly compressed plugs or even as tablets.
- the therapeutic could be prepared by compression.
- Colourants and flavouring agents may all be included.
- compounds may be formulated (such as by liposome or microsphere encapsulation) and then further contained within an edible product, such as a refrigerated beverage containing colorants and flavouring agents.
- diluents could include carbohydrates, especially mannitol, alpha-lactose, anhydrous lactose, cellulose, sucrose, modified dextrans and starch.
- Certain inorganic salts may be also be used as fillers including calcium triphosphate, magnesium carbonate and sodium chloride.
- Some commercially available diluents are Fast-Flo, Emdex, STA-Rx 1500, Emcompress and Avicell.
- Disintegrants may be included in the formulation of the therapeutic into a solid dosage form.
- Materials used as disintegrants include but are not limited to starch including the commercial disintegrant based on starch, Explotab. Sodium starch glycolate, Amberlite, sodium carboxymethylcellulose, ultramylopectin, sodium alginate, gelatin, orange peel, acid carboxymethyl cellulose, natural sponge and bentonite may all be used.
- Another form of the disintegrants are the insoluble cationic exchange resins.
- Powdered gums may be used as disintegrants and as binders and these can include powdered gums such as agar, Karaya or tragacanth. Alginic acid and its sodium salt are also useful as disintegrants.
- Binders may be used to hold the therapeutic compounds together to form a hard tablet and include materials from natural products such as acacia, tragacanth, starch and gelatin. Others include methylcellulose (MC), ethyl cellulose (EC) and carboxymethyl cellulose (CMC). Polyvinyl pyrrolidone (PVP) and hydroxypropylmethyl cellulose (HPMC) could both be used in alcoholic solutions to granulate the therapeutic.
- MC methylcellulose
- EC ethyl cellulose
- CMC carboxymethyl cellulose
- PVP polyvinyl pyrrolidone
- HPMC hydroxypropylmethyl cellulose
- Lubricants may be used as a layer between the therapeutic and the die wall, and these can include but are not limited to: stearic acid including its magnesium and calcium salts, polytetrafluoroethylene (PTFE), liquid paraffin, vegetable oils and waxes. Soluble lubricants may also be used such as sodium lauryl sulfate, magnesium lauryl sulfate, polyethylene glycol of various molecular weights, and Carbowax 4000 and 6000.
- the glidants may include starch, talc, pyrogenic silica and hydrated silicoaluminate.
- a surfactant might be added as a wetting agent.
- Surfactants may include anionic detergents such as sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate.
- anionic detergents such as sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate.
- Cationic detergents might be used and could include benzalkonium chloride or benzethomium chloride.
- nonionic detergents that could be included in the formulation as surfactants are lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrose fatty acid ester, methyl cellulose and carboxymethyl cellulose. These surfactants could be present in the formulation of the compounds either alone or as a mixture in different ratios.
- Additives which potentially enhance uptake of the compounds are for instance the fatty acids oleic acid, linoleic acid and linolenic acid.
- Controlled release formulation may be desirable.
- the compounds could be incorporated into an inert matrix that permits release by either diffusion or leaching mechanisms i.e., gums.
- Slowly degenerating matrices may also be incorporated into the formulation.
- Another form of a controlled release of this therapeutic is by a method based on the Oros therapeutic system (Alza Corp.), i.e. the drug is enclosed in a semipermeable membrane which allows water to enter and push drug out through a single small opening due to osmotic effects. Some enteric coatings also have a delayed release effect.
- Film coating may be carried out in a pan coater or in a fluidized bed or by compression coating.
- the compounds may be delivered to the lungs of a mammal while inhaling and traverses across the lung epithelial lining to the blood stream.
- Contemplated for use in the practice of this invention are a wide range of mechanical devices designed for pulmonary delivery of therapeutic products, including but not limited to nebulizers, metered-dose inhalers, and powder inhalers, all of which are familiar to those skilled in the art.
- Ultravent nebulizer manufactured by Mallinckrodt, Inc., St. Louis, Mo.
- Acorn II nebulizer manufactured by Marquest Medical Products, Englewood, Colo.
- the Ventolin metered dose inhaler manufactured by Glaxo Inc., Research Triangle Park, N.C.
- the Spinhaler powder inhaler manufactured by Fisons Corp., Bedford, Mass.
- each formulation is specific to the type of device employed and may involve the use of an appropriate propellant material, in addition to the usual diluents, adjuvants and/or carriers useful in therapy. Also, the use of liposomes, microcapsules or microspheres, inclusion complexes, or other types of carriers is contemplated.
- Formulations suitable for use with a nebulizer will typically comprise the compounds suspended in water.
- the formulation may also include a buffer and a simple sugar (e.g., for protein stabilization and regulation of osmotic pressure).
- the nebulizer formulation may also contain a surfactant, to reduce or prevent surface induced aggregation of the compounds caused by atomization of the solution in forming the aerosol.
- Formulations for use with a metered-dose inhaler device will generally comprise a finely divided powder containing the compounds suspended in a propellant with the aid of a surfactant.
- the propellant may be any conventional material employed for this purpose, such as a chlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or a hydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane, or combinations thereof.
- Suitable surfactants include sorbitan trioleate and soya lecithin. Oleic acid may also be useful as a surfactant.
- Formulations for dispensing from a powder inhaler device will comprise a finely divided dry powder containing the compound and may also include a bulking agent, such as lactose, sorbitol, sucrose, or mannitol in amounts which facilitate dispersal of the powder from the device, e.g., 50 to 90% by weight of the formulation.
- the compounds (or derivative) should most advantageously be prepared in particulate form with an average particle size of less than 10 microns, most preferably 0.5 to 5 microns, for most effective delivery to the distal lung.
- Nasal delivery of the compounds is also contemplated. Nasal delivery allows the passage of the protein to the blood stream directly after administering the therapeutic product to the nose, without the necessity for deposition of the product in the lung.
- Formulations for nasal delivery include those with dextran or cyclodextran.
- the medicaments of the invention may be given as a single dose schedule, or preferably, in a multiple dose schedule.
- a multiple dose schedule is one in which a primary course of delivery may be with 1 to 10 separate doses, followed by other doses given at subsequent time intervals required to maintain or reinforce the treatment.
- the dosage regimen will also, at least in part, be determined by the need of the individual and the judgement of the practitioner.
- the IGs are provided in the form of the two receptors (TRHR and OxR).
- One of the two is attached to an RC (IG1-RC1, IG3).
- a second IG (IG2-RC2) is derived from a molecule that interacts with the receptors upon ligand binding (e.g. beta-arrestin, or a mutant thereof).
- the detection system not only detects the formation of the TRHR-OxR heterodimer but can distinguish whether a ligand or drug acts as an agonist, partial agonist, antagonist, inverse agonist or partial inverse agonist at the receptor hetero-dimer.
- HEK293FT embryonic fibroblasts
- COS-7 approximately 150,000 cells/well
- DMEM Complete Media
- FCS fetal calf serum
- Transient transfections were carried out 24 h after seeding using GeneJuice (Novagen) or Metafectene (Biontex) according to manufacturer instructions.
- the BRET signal observed between interacting proteins can be shown in conjunction with (as oppose to being subtracted by) the background BRET ratio to evaluate error associated with the BRET signal observed between interacting proteins and the error associated with the background BRET ratio independently.
- data are shown as ‘fluorescence/luminescence’ being the ratio of light emission through the ‘acceptor wavelength window’ over the 400-475 nm emission for a particular cell sample. Unless otherwise stated, BRET signals were measured in 96-well microplates.
- eBRET signals were measured from cells transiently co-expressing TRHR/Rluc and barr2/Venus with either pcDNA3, OxR2, CXCR2, HA-MC3R, HA-MC4R, D2LR or D2SR following the treatment of each with their respective ligands.
- a signal is not detected when IG3 is CXCR2, HA-MC3R, HA-MC4R, D2LR or D2SR and agonists specific for these IG3s modulate the association of IG2 and IG3, demonstrating the specificity of the signal for the combination with OxR2 as IG3.
- eBRET signals were measured from cells transiently co-expressing TRHR/Rluc and EGFP/barr1 or EGFP/barr2 with either pcDNA3 or OXR2. Ligand treatments were either OxA or TRH only or both OxA and TRH combined.
- This example demonstrates that a signal resulting from the proximity of RC1 and RC2 is detected for the combination where the thyrotropin releasing hormone receptor (TRHR) is IG1, Rluc is RC1, either beta-arrestin 1 (barr1) or beta-arrestin 2 (barr2) is IG2, EGFP is RC2 and OxR2 is IG3 when the modulator, OxA, modulates the association of IG2 and IG3 as a result of interacting specifically with IG3.
- TRHR thyrotropin releasing hormone receptor
- Rluc is RC1
- beta-arrestin 1 barr1
- beta-arrestin 2 barr2
- OxR2 is IG3 when the modulator, OxA, modulates the association of IG2 and IG3 as a result of interacting specifically with IG3.
- this example demonstrates signal detection using an alternative combination from that shown in example 1, including use of a different IG2 and RC2.
- this example demonstrates the delayed kinetic profile observed for the signal resulting from RC1 and RC2 proximity due to modulation of the association of IG2 and IG3, in this case by OxA, as distinct from the more rapid kinetic profile observed for an eBRET signal resulting from RC1 and RC2 proximity due to association of IG1 and IG2 when this IG1-IG2 association is modulated by ligand, in this case TRH, interacting specifically with IG1.
- ligand in this case TRH
- this example demonstrates the additive effect of combined treatment with IG1 ligand (TRH) and IG3 ligand (OxA; modulator).
- this example provides further and distinct evidence for the molecular association of the thyrotropin releasing hormone receptor with the orexin receptor, as this additive effect is indicative of RC1 and RC2 proximity as a result of IG1-IG2 association in addition to IG2-IG3-IG1 association.
- This provides evidence against signals originating from non-specific IG1-IG2 association in the absence of an IG1-specific ligand.
- this additive effect may also be partly due to IG1 ligand acting as a modulator to modulate the association of IG2 and IG3 via allosteric effects on IG3.
- this additive effect may also be partly due to an active IG conformation (one that is bound to agonist) being more favourable for signal generation, perhaps enabling increased proximity of RC1 and RC2, or more favourable relative orientation of RC1 and RC2.
- eBRET signals were measured from cells transiently co-expressing TRHR/Rluc and barr2/Venus with either pcDNA3, OxR1 or OxR2 following pretreatment with 10 ⁇ 6 M OxR1-selective antagonist, SB-334867-A, for approximately 40 minutes prior to addition of 10 ⁇ 6 M OxA (IG3 ligand; modulator) or 10 ⁇ 6 M TRH (IG1 ligand), or both.
- Cells not pretreated with antagonist were pretreated with PBS instead for the same amount of time.
- This example shows a signal resulting from the proximity of RC1 and RC2 detected for the combination where the thyrotropin releasing hormone receptor (TRHR) is IG1, Rluc is RC1, beta-arrestin 2 (barr2) is IG2, Venus is RC2 and OxR1 or OxR2 is IG3 when the modulator, OxA, modulates the association of IG2 and IG3 as a result of interacting specifically with IG3.
- TRHR thyrotropin releasing hormone receptor
- Rluc is RC1
- beta-arrestin 2 barr2
- Venus is RC2
- OxR1 or OxR2 is IG3 when the modulator, OxA, modulates the association of IG2 and IG3 as a result of interacting specifically with IG3.
- eBRET signals were measured from cells transiently co-expressing TRHR/Rluc and EGFP/barr1 or EGFP/barr2 with either pcDNA3 or HA-OxR2. Ligand treatments were either OxA or TRH only.
- TRH stimulated a rapid increase in eBRET signal reaching a peak in the first few minutes, the signal then drifted down slightly over the remainder of the recording period (grey squares). No increase in eBRET signal above baseline was observed following OxA addition to cells lacking HA-OxR2 (grey triangles).
- This example shows a signal resulting from the proximity of RC1 and RC2 detected for the combination where the thyrotropin releasing hormone receptor (TRHR) is IG1, Rluc is RC1, beta-arrestin 1 (barr1) or beta-arrestin 2 (barr2) is IG2, EGFP is RC2 and hemagglutin (HA) epitope-tagged OxR2 (HA-OxR2) is IG3 when the modulator, OxA, modulates the association of IG2 and IG3 as a result of interacting specifically with IG3.
- TRHR thyrotropin releasing hormone receptor
- Rluc is RC1
- beta-arrestin 1 barr1
- beta-arrestin 2 barr2
- EGFP RC2
- hemagglutin (HA) epitope-tagged OxR2 HA-OxR2
- IG3 can be tagged, such as by the addition of a hemagglutin (HA) epitope-tag, however, this tag does not constitute a reporter component and does not interfere with and/or contribute to the signal generated by the proximity of RC1 and RC2. Such tagging enables additional information to be ascertained, such as the relative expression level of IG3.
- HA hemagglutin
- eBRET signals were measured from cells transiently co-expressing TRHR/Rluc and EGFP/barr1 or EGFP/barr1 phosphorylation-independent mutant R169E (EGFP/barr1R169E) with either pcDNA3 or OxR2. Ligand treatments were either OxA or TRH only.
- TRH stimulated a rapid increase in eBRET signal reaching a peak in the first few minutes, the signal then drifted down slightly over the remainder of the recording period.
- the signal for the EGFP/barr1R169E (white triangles) was lower than that for EGFP/barr1 (white circles), which may reflect lower expression levels of this protein.
- This example shows a signal resulting from the proximity of RC1 and RC2 detected for the combination where the thyrotropin releasing hormone receptor (TRHR) is IG1, Rluc is RC1, barr1 or barr1R169E is IG2, EGFP is RC2 and OxR2 is IG3.
- TRHR thyrotropin releasing hormone receptor
- a detectable signal can be generated when using a mutant beta-arrestin, such as the beta-arrestin 1 phosphorylation-independent mutant R169E, as one of the interacting groups.
- eBRET signals were measured from cells transiently co-expressing TRHR335/Rluc and EGFP/barr1 with either OxR2 or TRHR. Ligand treatments were either OxA or TRH only.
- This example shows a signal resulting from the proximity of RC1 and RC2 detected for the combination where the thyrotropin releasing hormone receptor truncated at amino acid 335 (TRHR335) is IG1, Rluc is RC1, beta-arrestin 1 (barr1) is IG2, EGFP is RC2 and OxR2 or TRHR is IG3.
- this example provides further and distinct evidence for the molecular association of the thyrotropin releasing hormone receptor with the orexin receptor, as the inability of IG1 to interact with IG2 is indicative of RC1 and RC2 proximity as a result of IG2-IG3-IG1 association and not IG1-IG2 association. This provides further evidence against signals originating from non-specific IG1-IG2 association in the absence of an IG1-specific ligand.
- this example demonstrates that the signal results from IG2-IG3-IG1 association as opposed to IG3 activation causing transactivation of IG1, which then associates with IG2, thereby bringing RC1 and RC2 into close proximity without IG2 and IG3 associating.
- BRET signals were measured from cells transiently co-expressing: TRHR/Rluc and barr2/Venus with pcDNA3 (treated with increasing doses of TRH; FIG. 10 ); OxR2/Rluc and barr2/Venus with pcDNA3 (treated with increasing doses of OxA; FIG. 11 ); and TRHR/Rluc and barr2/Venus with OxR2 (treated with increasing doses of OxA; FIG. 12 ).
- This example shows: a TRH dose-response curve for TRHR as IG1, Rluc as RC1, barr2 as IG2, Venus as RC2 and in the absence of IG3 ( FIG. 10 ); an OxA dose-response curve for OxR2 as IG1, Rluc as RC1, barr2 as IG2, Venus as RC2 and in the absence of IG3 ( FIG. 11 ); and OxA dose-response curves for the TRHR as IG1, Rluc as RC1, barr2 as IG2, Venus as RC2 and OxR2 as IG3 ( FIG. 12 ).
- the EC 50 values for signals resulting from the modulator (OxA) acting on IG3 (OxR2) and consequent proximity of IG1-RC1 (TRHR/Rluc) and IG2-RC2 (barr2/Venus; FIG. 12 ) are comparable to those from OxA activation of IG1 (OxR2) resulting in proximity of IG1-RC1 (OxR2/Rluc) and IG2-RC2 (barr2/Venus; FIG. 11 ), and distinct from those from TRH activation of IG1 (TRHR) resulting in proximity of IG1-RC1 (TRHR/Rluc) and IG2-RC2 (barr2/Venus; FIG. 10 ).
- this example further demonstrates that the signal results from IG2-IG3-IG1 association as opposed to IG1-IG2 association.
- the dose-response Hill slopes for modulator (OxA) acting on IG3 (OxR2) resulting in proximity of IG1-RC1 (TRHR/Rluc) and IG2-RC2 (barr2/Venus; FIG. 12 ) are substantially greater than 1.
- this example demonstrates the potential for identifying and monitoring specific molecular associations using the Hill slope as an indicator.
- Rluc substrate reporter component initiator
- coelenterazine h and EnduRen can be used to generate data with similar EC 50 values ( FIG. 12 ).
- BRET signals were measured from cells transiently co-expressing TRHR/Rluc and EGFP/barr1 in the absence of OxR2 with increasing doses of TRH, as well as cells transiently co-expressing TRHR/Rluc and EGFP/barr1 with OxR2 with increasing doses of OxA with and without 10 ⁇ 6 M TRH, or increasing doses of TRH with 10 ⁇ 6 M OxA.
- This example shows a curve mathematically generated by addition of the ligand-induced signal generated with 10 ⁇ 6 M TRH (from the TRH: TRHR/Rluc+EGFP/barr1 curve) to each of the points generated for the OxA: TRHR/Rluc+EGFP/barr1+OxR2 curve (TRHR/Rluc+EGFP/barr1+OxR2: TRH (10 ⁇ 6 M)+OxA: Data calculated) overlain on a curve generated from data observed for the TRHR/Rluc+EGFP/barr1+OxR2: TRH (10 ⁇ 6 M)+OxA combination ( FIG. 13 ).
- this example shows a curve mathematically generated by addition of the ligand-induced signal generated with 10 ⁇ 6 M OxA (from the OxA: TRHR/Rluc+EGFP/barr1+OxR2 curve) to each of the points generated for the TRH: TRHR/Rluc+EGFP/barr1 curve (TRHR/Rluc+EGFP/barr1+OxR2: TRH+OxA (10 ⁇ 6 M): Data calculated) overlain on a curve generated from data observed for the TRHR/Rluc+EGFP/barr1+OxR2: TRH+OxA (10 ⁇ 6 M) combination ( FIG. 14 ).
- this example clearly demonstrates the additive effect of combined treatment with IG1 ligand (TRH) and IG3 ligand (OxA; modulator) in a dose dependent manner.
- this example provides further evidence for the molecular association of the thyrotropin releasing hormone receptor with the orexin receptor, as this additive effect is indicative of RC1 and RC2 proximity as a result of IG1-IG2 association in addition to IG2-IG3-IG1 association.
- This provides further evidence against signals originating from non-specific IG1-IG2 association in the absence of an IG1-specific ligand.
- this additive effect may also be partly due to IG1 ligand acting as a modulator to modulate the association of IG2 and IG3 via allosteric effects on IG3.
- this additive effect may also be partly due to an active IG conformation (one that is bound to agonist) being more favourable for signal generation, perhaps enabling increased proximity of RC1 and RC2, or more favourable relative orientation of RC1 and RC2.
- BRET signals were measured from cells transiently co-expressing TRHR335/Rluc, barr2/Venus and OxR2 with increasing doses of TRH and OxA alone or in combination.
- this example further demonstrates that co-treatment of IG1 and IG3 can result in additional signal generation and/or information compared to treatment of IG3 alone and that such co-treatment is encompassed by the present invention.
- eBRET signals were measured from cells transiently co-expressing TRHR/Rluc, EGFP/barr1 and OxR2 following addition of 10 ⁇ 6 M OxA.
- This example shows cumulative eBRET reads over time for each combination of receptors (IG1 and IG3; data captured over 83 mins).
- the same amount of EGFP/barr1 (IG2-RC2) is transfected for each experiment.
- TRHR/Rluc IG1-RC1
- OxR2 IG3
- the signal is only detected when OxR2 (IG3) is expressed (no signal was recorded at 0 ⁇ g OxR2).
- this example demonstrates that increasing the amounts of OxR2 DNA in each transfection results in increases in the detectable signal.
- the largest detectable signal is observed at a 1:1 ratio of DNA concentration (0.1:0.1 ⁇ g DNA/well). Further increases in the OxR2 DNA concentration (0.5 or 0.7 ⁇ g DNA/well) with levels higher than the amount of TRHR/Rluc DNA results in a lower signal being detected.
- this example provides further and distinct evidence for the molecular association of the thyrotropin releasing hormone receptor with the orexin receptor, as such decreases in signal with increases in IG3 concentration beyond that of IG1 concentration would not be expected to occur if the signal was not dependent upon specific molecular association of IG1 and IG3.
- BRET signals were measured from cells transiently co-expressing TRHR/Rluc, barr2/Venus and OxR2 with increasing doses of OxA in 96-well and 384-well microplates.
- BRET measurements were carried out using the same concentration of cells expressing the same concentration of agents, the same concentration of Rluc substrate (reporter component initiator) and the same concentration of ligand (modulator).
- the total volume added to each well of the 384-well plate was approximately half that added to each well of the 96-well plate.
- This example demonstrates measurement of a detectable signal indicative of the molecular association of TRHR with OxR2 in a dose-dependent manner in 384-well plates in addition to 96-well plates.
- this example demonstrates that the method described in the invention is able to be scaled down, thereby making it amenable to high-throughput screening applications.
- eBRET signals were measured from cells transiently co-expressing OxR2/Rluc8 and barr2/Venus either with HA-TRHR or pcDNA3. Ligand treatments were either OxA or TRH.
- This example demonstrates that a signal resulting from the proximity of RC1 and RC2 is detected specifically for the combination where OxR2 is IG1, Rluc8 is RC1, beta-arrestin 2 (barr2) is IG2, Venus is RC2 and HA-TRHR is IG3, and when the modulator, in this case TRH, modulates the association of IG2 and IG3 as a result of interacting specifically with IG3.
- This example also demonstrates the use of a second type of luciferase, Rluc8, which in this case is used as RC1 with Venus as RC2.
- this example demonstrates that IG3 can be tagged, such as by the addition of a hemagglutin (HA) epitope-tag, however, this tag does not constitute a reporter component and does not interfere with and/or contribute to the signal generated by the proximity of RC1 and RC2.
- HA hemagglutin
- eBRET signals were measured from cells transiently co-expressing TRHR/Rluc8 and barr2/Venus with HA-OxR2 aliquoted into all wells of a 96-well plate.
- PBS Phosphate-buffered saline
- OxA was added to the middle four rows of the 96-well plate (48 wells in total). Data are presented as fluorescence/luminescence.
- OxA treatment of cells co-expressing TRHR/Rluc8 and barr2/Venus with HA-OxR2 resulted in an increase in the fluorescence/luminescence ratio ( FIG. 20 ) that was not observed following treatment with phosphate-buffered saline (PBS) vehicle control ( FIG. 19 ).
- PBS phosphate-buffered saline
- This example further demonstrates a third method of representing BRET data that can be used in representing a detectable signal indicative of the molecular association of the thyrotropin-releasing hormone receptor and the orexin receptor.
- this example demonstrates that IG3 can be tagged, such as by the addition of a hemagglutin (HA) epitope-tag, however, this tag does not constitute a reporter component and does not interfere with and/or contribute to the signal generated by the proximity of RC1 and RC2.
- HA hemagglutin
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Families Citing this family (15)
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---|---|---|---|---|
BRPI0717416A2 (pt) | 2006-09-21 | 2013-11-12 | Prometheus Lab Inc | Método para realizar um imunoensaio complexo de alta produtividade, e, arranjo |
BRPI0813583A2 (pt) * | 2007-07-13 | 2014-12-30 | Prometheus Lab Inc | Métodos para selecionar um medicamento anticâncer, para identificar a resposta de um tumor pulmonar, e para prognosticar a resposta de um paciente, e, arranjo |
EP2210104A4 (de) * | 2007-11-01 | 2011-02-23 | Univ Arizona State | Zellfreie verfahren zum nachweis von protein-ligand-wechselwirkungen |
WO2009108637A1 (en) | 2008-02-25 | 2009-09-03 | Prometheus Laboratories, Inc. | Drug selection for breast cancer therapy using antibody-based arrays |
JP2012521967A (ja) * | 2009-03-27 | 2012-09-20 | ディメリックス バイオサイエンス ピーティーワイ リミテッド | 新規な受容体のヘテロ二量体/ヘテロオリゴマー |
ES2627909T3 (es) | 2009-07-15 | 2017-08-01 | Diatech Holdings, Inc. | Selección de fármacos para el tratamiento del cáncer gástrico usando matrices basadas en anticuercuerpos |
US8956859B1 (en) | 2010-08-13 | 2015-02-17 | Aviex Technologies Llc | Compositions and methods for determining successful immunization by one or more vaccines |
CA2821985C (en) | 2011-01-11 | 2019-07-09 | Dimerix Bioscience Pty Ltd | Combination therapy |
US9719995B2 (en) | 2011-02-03 | 2017-08-01 | Pierian Holdings, Inc. | Drug selection for colorectal cancer therapy using receptor tyrosine kinase profiling |
KR101851425B1 (ko) | 2011-09-02 | 2018-04-23 | 네스텍 소시에테아노님 | 치료 효능 결정을 위한 신호 경로 단백질의 프로파일링 |
IE20120542A1 (en) * | 2012-12-19 | 2014-07-02 | Provost Fellows Foundation Scholars And Other Members Of Board Of The College Of The Holy And Undivi | Novel TRH binding site in human CNS |
KR102192554B1 (ko) | 2013-03-13 | 2020-12-18 | 에스케이바이오팜 주식회사 | 탈력발작의 치료 |
JP6863897B2 (ja) | 2015-01-09 | 2021-04-21 | アダルタ・リミテッド | Cxcr4結合分子 |
KR102085554B1 (ko) * | 2018-02-21 | 2020-03-06 | 영남대학교 산학협력단 | 사카로마이세스 세레비시애의 g-단백질 연결 수용체에 대한 알파 팩터의 친화도 측정방법 |
CN111089971B (zh) * | 2019-12-11 | 2021-01-15 | 浙江大学 | 一种膜电位调控下的蛋白相互作用定量检测装置 |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4925673A (en) * | 1986-08-18 | 1990-05-15 | Clinical Technologies Associates, Inc. | Delivery systems for pharmacological agents encapsulated with proteinoids |
US5013556A (en) * | 1989-10-20 | 1991-05-07 | Liposome Technology, Inc. | Liposomes with enhanced circulation time |
Family Cites Families (19)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4391904A (en) * | 1979-12-26 | 1983-07-05 | Syva Company | Test strip kits in immunoassays and compositions therein |
US4671958A (en) * | 1982-03-09 | 1987-06-09 | Cytogen Corporation | Antibody conjugates for the delivery of compounds to target sites |
US4751180A (en) * | 1985-03-28 | 1988-06-14 | Chiron Corporation | Expression using fused genes providing for protein product |
US4935233A (en) * | 1985-12-02 | 1990-06-19 | G. D. Searle And Company | Covalently linked polypeptide cell modulators |
US5891646A (en) | 1997-06-05 | 1999-04-06 | Duke University | Methods of assaying receptor activity and constructs useful in such methods |
EP1137940A4 (de) * | 1998-10-23 | 2004-06-02 | Glaxo Group Ltd | Verfahren zur identifizierung von liganden von nukleären rezeptoren |
DE60026603T2 (de) | 1999-12-22 | 2007-03-01 | PerkinElmer BioSignal Inc., Montréal | Biolumineszenz-Resonanz-Energie-Transfer(BRET)-System mit großer spektraler Auflösung zwischen der Donator- und der Akzeptoremissionswellenlänge sowie dessen Verwendun |
US6893827B1 (en) * | 2000-02-07 | 2005-05-17 | Applera Corporation | Receptor function assay for G-protein coupled receptors and orphan receptors by reporter enzyme mutant complementation |
US20030096299A1 (en) * | 2001-07-09 | 2003-05-22 | Valerie Wittamer | Natural ligand of G protein coupled receptor ChemR23 and uses thereof |
NZ554913A (en) * | 2001-12-14 | 2009-01-31 | Targacept Inc | Methods and compositions for the treatment of central nervous system disorders |
FR2836475B1 (fr) * | 2002-02-26 | 2007-02-23 | Aventis Pharma Sa | Procede de detection de ligands du recepteur de la leptine |
DE10218785A1 (de) | 2002-04-26 | 2003-11-13 | Infineon Technologies Ag | Halbleiterspeichereinrichtung und Betriebsverfahren für eine Halbleiterspeichereinrichtung |
DE10230141B4 (de) * | 2002-07-04 | 2004-07-15 | Priontype Gmbh | Verfahren und Kit zur Anreicherung und zum Nachweis von veränderten Prion-Proteinen (PrPSc) |
JP2004093527A (ja) | 2002-09-04 | 2004-03-25 | Center For Advanced Science & Technology Incubation Ltd | 蛍光共鳴エネルギー転移を用いた蛋白質の分析方法 |
ATE500270T1 (de) * | 2003-05-20 | 2011-03-15 | Univ Glasgow | Materialien und methoden die sich auf g-protein gekoppelte rezeptor oligomere beziehen |
US7049076B2 (en) * | 2003-07-09 | 2006-05-23 | Sentigen Biosciences, Inc. | Method for assaying protein—protein interaction |
US7488583B2 (en) | 2003-09-25 | 2009-02-10 | Odyssey Thera, Inc. | Fragment complementation assays for G-protein-coupled receptors and their signaling pathways |
WO2005050182A1 (en) * | 2003-11-19 | 2005-06-02 | Dimerix Biosciences Pty Ltd | Resonance energy transfer assay system for multi-component detection |
CN1627072A (zh) * | 2003-12-11 | 2005-06-15 | 中国人民解放军军事医学科学院卫生学环境医学研究所 | 一种蛋白质芯片,其制备方法及用途 |
-
2007
- 2007-11-09 AU AU2007317204A patent/AU2007317204A1/en not_active Abandoned
- 2007-11-09 WO PCT/AU2007/001722 patent/WO2008055313A1/en active Application Filing
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- 2007-11-09 CA CA002669185A patent/CA2669185A1/en not_active Abandoned
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- 2007-11-09 AU AU2007317203A patent/AU2007317203B2/en not_active Ceased
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- 2007-11-09 US US12/442,899 patent/US8283127B2/en active Active
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- 2007-11-09 WO PCT/AU2007/001723 patent/WO2008055314A1/en active Application Filing
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- 2009-05-07 IL IL198639A patent/IL198639A/en active IP Right Grant
- 2009-05-21 ZA ZA200903529A patent/ZA200903529B/xx unknown
-
2012
- 2012-09-10 US US13/608,855 patent/US8568997B2/en active Active
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4925673A (en) * | 1986-08-18 | 1990-05-15 | Clinical Technologies Associates, Inc. | Delivery systems for pharmacological agents encapsulated with proteinoids |
US5013556A (en) * | 1989-10-20 | 1991-05-07 | Liposome Technology, Inc. | Liposomes with enhanced circulation time |
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IL198639A0 (en) | 2010-02-17 |
KR20090083925A (ko) | 2009-08-04 |
EP2605015A1 (de) | 2013-06-19 |
JP5313149B2 (ja) | 2013-10-09 |
AU2007317203B2 (en) | 2010-12-16 |
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