[go: up one dir, main page]

WO2005080971A1 - Systeme et methode de mesures electrophysiologiques de fixation de tension planaire sur plusieurs cellules et leur utilisation dans l'identification d'agents modulant des canaux ioniques - Google Patents

Systeme et methode de mesures electrophysiologiques de fixation de tension planaire sur plusieurs cellules et leur utilisation dans l'identification d'agents modulant des canaux ioniques Download PDF

Info

Publication number
WO2005080971A1
WO2005080971A1 PCT/SE2005/000239 SE2005000239W WO2005080971A1 WO 2005080971 A1 WO2005080971 A1 WO 2005080971A1 SE 2005000239 W SE2005000239 W SE 2005000239W WO 2005080971 A1 WO2005080971 A1 WO 2005080971A1
Authority
WO
WIPO (PCT)
Prior art keywords
agent
internal solution
seal
phase internal
ion concentration
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/SE2005/000239
Other languages
English (en)
Inventor
Andrew Davis
Andrew Hargreaves
Deborah Henthorn
Alexandra Orme
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
AstraZeneca AB
Original Assignee
AstraZeneca AB
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by AstraZeneca AB filed Critical AstraZeneca AB
Publication of WO2005080971A1 publication Critical patent/WO2005080971A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • 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
    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material
    • G01N33/48707—Physical analysis of biological material of liquid biological material by electrical means
    • G01N33/48728—Investigating individual cells, e.g. by patch clamp, voltage clamp

Definitions

  • the present invention relates to improved electrophysiological methods.
  • the present invention relates to, ter alia, improved assay methods involving planar voltage-clamp electrophysiological measurements on multiple cells in parallel and their use in identifying, testing and/or screening of agents that modulate ion channels.
  • the compounds bind to the apeture- forming alpha sub-units of the channel protein carrying this current - sub-units that are encoded by the human ether-a-go-go-related gene (hERG). Since IKr plays a key role in repolarisation of the cardiac action potential, its inhibition slows repolarisation and this is manifested as a prolongation of the QT interval. Whilst QT interval prolongation is not a safety concern per se, it carries a high risk of cardiovascular adverse effects and in a small percentage of people it can lead to TdP and degeneration into ventricular fibrillation.
  • the hERG channel contains a binding site for the class III antiarrhythmic methanesulphonanilides (dofetilide, E-4031 and MK-449) and many drugs that show prolonged QT in the clinic share this site. Consequently they have either warning labels (e.g. pimozide) or have been withdrawn from the market (e.g. terfenadine).
  • warning labels e.g. pimozide
  • the lack of an appropriate therapeutic margin between activity at the hERG channel and the desired target will prevent a potential candidate drug from progressing further. It is therefore necessary to evaluate the hERG. activity of candidate drugs as early as possible to allow reduction in this activity for novel compound classes.
  • the cells can subsequently be permeablised locally at the apeture or pore by suction, electroporation or by applying a solution containing a permeablising compound - such as amphotericin. Noltage- clamp electrophysiological recordings can then be made on all or a subset of the attached cells simultaneously.
  • the present invention is based, in part, upon the surprising finding that exchanging a low ion concentration internal solution that is used in an electrophysiological measurement apparatus for another internal solution - such as a low or a high ion concentration internal solution - leads to improved assay methods.
  • the inventor's have found that exchanging a low ion concentration internal solution for another internal solution - such as a low or a high ion concentration internal solution - leads to optimised seal quality in the electrophysiological measurement apparatus and/or an optimised potency estimate for an agent being assayed.
  • the invention described herein may be used, inter alia, in the development of assays, which utilise parallel planar voltage clamp electrophysiology to determine the potency at which compounds block ion channels.
  • the present invention relates to an assay method for identifying an agent that modulates the electrical property of a membranous sample comprising the steps of: (a) providing an electrophysiological measurement apparatus; (b) contacting one or more membranous samples with a compartment of the electrophysiological measurement apparatus containing an aperture; (c) forming a seal between the membranous samples and the aperture in the presence of a low ion concentration seal phase internal solution; (d) exchanging the low ion concentration seal phase internal solution for an access phase internal solution; (e) establishing electrical access, via the aperture, to the internal compartment of the membranous sample; and (f) taking ele'ctrophysiological recordings before and after incubation of the cell with one or more agents; wherein a difference between: (i) the electrophysiological recording in the presence of the agent; and (ii) the electrophysiological recording in the absence of the agent is indicative of an agent that modulates the electrical property
  • the present invention relates to a method for maintaining the optimum seal quality of a compartment in an electrophysiological measurement apparatus comprising the steps of: (a) providing an electrophysiological measurement apparatus; (b) contacting one or more membranous samples with a compartment of the electrophysiological measurement apparatus containing an aperture; (c) forming a seal between the membranous samples and the aperture in the presence of a low ion concentration seal phase internal solution; and (d) exchanging the low ion concentration seal phase internal solution for an access phase internal solution.
  • the present invention relates to a method for maintaining optimum seal quality and determining the potency of a non-lip ophillic agent that modulates the electrical property of a membranous sample comprising the steps of: (a) providing an electrophysiological measurement apparatus; (b) contacting one or more membranous samples with a compartment of the electrophysiological measurement apparatus containing an aperture; (c) forming a seal between the membranous samples and the aperture in the presence of a low ion concentration seal phase internal solution; (d) exchanging the low ion concentration seal phase internal solution for a high ion concentration access phase internal solution; (e) establishing electrical access, via the aperture, to the internal compartment of the membranous sample; and (f) taking electrophysiological recordings before and after incubation of the cell with one or more agents; wherein a difference between: (i) the electrophysiological recording in the presence of the agent; and (ii) the electrophysiological recordings in the absence of the agent is indicative of the potency estimate for the non
  • the present invention relates to a method for determining the potency of an agent comprising the steps of: (a) providing an electrophysiological measurement apparatus; (b) contacting one or more membranous samples with a compartment of the electrophysiological measurement apparatus containing an aperture using a reduced number of cells per compartment; (c) forming a seal between the membranous samples and the aperture in the presence of a low ion concentration internal solution; (d) exchanging the low ion concentration internal solution for an access phase internal solution; (e) establishing electrical access, via the aperture, to the internal compartment of the membranous sample; and (f) taking electrophysiological recordings before and after incubation of the cell with one or more agents.
  • the present invention relates to an assay method for determining the potency of an agent comprising the steps of: (a) providing an electrophysiological measurement apparatus; (b) contacting one or more membranous samples with a compartment of the electrophysiological measurement apparatus containing an aperture using a reduced number of cells per compartment; (c) forming a seal between the membranous samples and the aperture in the presence of a low ion concentration internal solution; (d) exchanging the low ion concentration internal solution for an access phase internal solution; (e) establishing electrical access, via the aperture, to the internal compartment of the membranous sample; and (f) taking electrophysiological recordings before and after incubation of the cell with one or more agents; wherein a difference between: (i) the electrophysiological recording in the presence of the agent; and (ii) the electrophysiological recordings in the absence of the agent is indicative of the potency estimate for the agent.
  • the present invention relates to a low ion concentration seal phase internal solution.
  • the present invention relates to a process comprising the steps of: (i) performing the assay method according to the first aspect of the present invention; (ii) identifying an agent capable of modulating the electrical property of a membranous sample; and (iii) preparing a quantity of that agent.
  • the present invention relates to a process comprising the steps of: (i) performing the assay according to the first aspect of the present invention; (ii) identifying an agent capable of modulating the electrical property of a membranous sample; (iii) preparing a quantity of that agent; and (iv) preparing a pharmaceutical composition comprising that agent.
  • the present invention relates to a process comprising the steps of: (i) performing the assay according to the first aspect of the present invention; (ii) identifying an agent capable of modulating the electrical property of a membranous sample; (iii) modifying said agent; and (iv) preparing a pharmaceutical composition comprising said modified agent.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising an agent identified by the assay method according to the first aspect of the present invention or the process according to the sixth, seventh and eighth aspects of the present invention admixed with a pharmaceutically acceptable carrier, diluent, excipient or adjuvant and/or combinations thereof.
  • the present invention relates to a process of preparing a pharmaceutical composition
  • a process of preparing a pharmaceutical composition comprising admixing an agent identified by the assay method according to the first aspect of the present invention or the process according to the sixth, seventh and eighth aspects with a pharmaceutically acceptable diluent, carrier, excipient or adjuvant and/or combinations thereof.
  • the present invention relates to a method of treating a disease in a human or animal which method comprises administering to an individual an effective amount of a pharmaceutical composition comprising an agent identified by the assay method according to the first aspect of the present invention or the process according to the sixth, seventh and eighth aspects, wherein the agent is capable of modulating the disease and wherein said composition is optionally admixed with a pharmaceutically acceptable carrier, diluent excipient or adjuvant and/or combinations thereof.
  • the present invention relates to an agent identifiable, preferably identified by the assay method according to the first aspect of the present invention.
  • the present invention relates to an agent identifiable preferably identified by the assay method according to the first aspect of the present invention for use in the treatment and/or prevention of disease.
  • the present invention relates to a kit for performing the method according to the first, second, third of fourth aspects of the present invention comprising: (a) a first vessel comprising a seal phase internal solution according to the sixth aspect of the present invention; and (b) a second vessel comprising an access phase internal solution.
  • the present invention relates to the use of a seal phase internal solution according to the sixth aspect of the present invention for measuring an electrical property of a membranous sample.
  • the present invention relates to the use of a seal phase internal solution according to the sixth aspect of the present invention for identifying an agent that modulates the electrical property of a membranous sample.
  • the present invention relates to the use of a seal phase internal solution according to the sixth aspect of the present invention for maintaining the optimum seal quality of a compartment in an electrophysiological measurement apparatus.
  • the present invention relates to the use of a seal phase internal solution according to the sixth aspect of the present invention for determining the potency of a non- lipophillic agent that modulates the electrical property of a membranous sample.
  • the present invention relates to the use of a seal phase internal solution according to the sixth aspect of the present invention for determining the potency of an agent.
  • the assay method according to the first aspect of the present invention comprises the step of exchanging the low ion concentration seal phase internal solution for a low or a high ion concentration access phase internal solution.
  • the assay method according to the first aspect of the present invention comprises the step of exchanging the low ion concentration seal phase internal solution for a high ion concentration access phase internal solution.
  • the agent is a lipophillic agent.
  • the lipophillic agent is astemizole or pimozide.
  • each compartment of the electrophysiological measurement apparatus containing
  • an aperture contains from 1-900 cells in a volume of 10.5 ⁇ l buffer solution.
  • the membranous samples comprises an ion channel.
  • the ion channel is a myocardial I ⁇ r ion channel.
  • the myocardial I ⁇ r ion channel is encoded by ERG, including hERG.
  • the electrophysiological measurement apparatus is a Molecular Devices IonWorksTMHT or an Axon Instruments PatchXpress.
  • the access phase internal solution is a low or high ion concentration solution.
  • the number of membranous samples in each compartment is modulated such that the pIC 5 Q value is at a level comparable to that obtained in conventional voltage clamp electrophysiology apparatus.
  • the low ion concentration internal solution is a seal phase internal solution.
  • the low ion concentration internal solution is exchanged for a low or a high ion concentration access phase internal solution.
  • the ion in the low ion concentration seal phase internal solution is a chloride ion.
  • the ion is present at a concentration of about 40 mM.
  • the low ion concentration seal phase internal solution comprises about 40mM KCl, about lOOmM K gluconate, about 3.2 mM MgCl 2 , about 3.0mM EGTA, and about 5.0mM HEPES.
  • one or more agents are formulated into one or more compositions for use in medicine.
  • the access phase internal solution has a chloride ion concentration of about 40 mM or about 140 mM.
  • the low ion concentration seal phase internal solution comprises about 40mM KCl, about lOOmM K gluconate, about 3.2 mM MgCl 2 , about 3.0mM EGTA, and about 5.0mM HEPES and a permeabilising agent.
  • the high ion concentration access phase internal solution comprises about 140mM KCl, about l.O ⁇ -M MgCl 2 , about l.OmM EGTA, about 20mM HEPES and a permeabilising agent.
  • the present invention has a number of advantages. These advantages will be apparent in the following description.
  • the present invention is advantageous since it provides commercially useful methods and assay methods.
  • the present invention is advantageous since it provides a commercially useful method for assaying lipophillic and non-lipophillic agents.
  • the present invention is advantageous since it provides a commercially useful method for obtaining optimum seal quality in an electrophysiological measurement apparatus.
  • the present invention is advantageous since it provides a commercially useful method for obtaining an optimum potency estimate of an agent that is assayed in an electrophysiological measurement apparatus.
  • the present invention is advantageous since it provides a commercially useful method for obtaining both an optimum seal quality and also an optimum potency estimate for an agent that is assayed in an electrophysiological measurement apparatus.
  • Potency was assessed using an 8-point dose-response curve.
  • Devation from conventional pICso was assessed by comparing the pIC 50 value obtained using conventional, pipette-based electrophysiology recordings against the pIC 50 value obtained using the IonWorksTM ⁇ T system (conventional pIC 50 minus IonWorksTMHT pIC 5 o).
  • the x- axis represents cells/ml in the stock cell suspension (xlOOO). 3.5ul of this suspension is added to each well on the IonWorksTMHT system. For all compounds, there is higher deviation from conventional pICso at higher cell concentrations than at lower cell concentrations (see Table
  • the y- axis represents the percentage of cells on a PatchPlate which gave a seal resistance of greater than 50Mohms as measured during Scan #2 on the IonWorksTMHT system.
  • the x-axis represents cells/ml in the stock cell suspension ( lOOO). 3.5ul of this suspension is added to each well on the IonWorksTMHT system.
  • the graph shows that the number of usable cells falls off significantly if the stock cell suspension contains less than 0.25x10 6 cells/ml. ..
  • Low ion concentration seal-phase internal solution 40mM KCl, lOOmM K gluconate, 3.2 mM MgCl 2 , 3.0mM EGTA, and 5.0mM HEPES
  • high ion concentration access-phase internal solution 140mM KCl, l.OmM MgCl 2 , l.OmM EGTA, 20mM HEPES.
  • the graph shows that using the preferred assay conditions on the IonWorksTMHT system (fig c) gives the best correlation between potency estimated using the IonWorksTMHT system and that estimated using conventional whole-cell voltage clamp techniques.
  • IonWorksTMHT system compared to the potency estimated using rubidium (Rb + ) efflux and atomic absorption spectroscopy (x-axis).
  • Potency (PIC 5 Q) was assessed using an 8- point dose-response curve on the IonWorksTMHT system and the following assay conditions; low ion concentration seal phase internal solution (40mM KCl, lOOmM K gluconate, 3.2 mM MgCl 2 , 3. OmM EGTA, and 5.
  • OmM HEPES low ion concentration seal phase internal solution
  • high ion concentration access-phase internal solution 140mM KCl, 1.OmM MgCl 2 , 1.OmM EGTA, 20mM HEPES.
  • Cell number was 0.25x10° cells/ml in the stock cell suspension.
  • the graph shows that using the preferred assay conditions on the IonWorksTMHT system gives approximately 10-fold higher estimate for potency than Rb + efflux / atomic absorption
  • the term "ion” refers to any atom or other molecular entity that has acquired an electric charge by loss or gain of one or more electrons.
  • the ion may be a cation or an anion.
  • the ion is an anion
  • the ion is a chloride ion.
  • low ion concentration preferably low chloride ion concentration, means that the ion concentration is from 0 to about 60 mM.
  • the low ion concentration is from 0 to about 50 mM - such as about 5-50mM, about 10-50mM, about 15-50mM, about 20-50mM, about 25-50mM, about 30-50mM,about 35-50mM, about 40-50mM, about 45-50mM, about 45-49mM, about 45- 48mM, or about 45-47mM or any suitable combination of start or end points, for example, from about 37-48mM.
  • the low ion concentration is about 46 mM, preferably, 46.4 mM.
  • the low ion concentration will be such that the osmolarity of the final solution will be within normal physiological parameters (about 250 to 310 mOsm/liter).
  • the low ion concentration may be from 0 to about 40 mM - such as about 5-40mM, about 10- 40mM, about 15-40mM, about 20-40mM, about 25-40mM, about 30-40mM or about 35- 40mM or any suitable combination of start or end points, for example, from about 37-39mM.
  • ion substitutes - such as non-chloride ion substitutes - may be used in the low ion concentration solutions, which allow the ion (preferably, chloride ion) concentration to be lowered without changing the overall ionic strength of the solution.
  • the ion substitute may be in the form of potassium chloride or magnesium chloride and the like.
  • the following may also be used as ion substitutes to maintain osmolarity: gluconate, aspartate, methanesulfonate, isethionate, cyclamate, glutamate, sulphate, methanesulphonate, formate, acetate, iodide, bromide, SCN “ , C(CN) 3 " , N(CN) 2 " , ClO 4 " , NO 3 ' , ClO 3 " , BrO 3 " or HCO 3 " or combinations thereof.
  • the substitute is a non-chloride ion potassium salt - such as potassium gluconate, potassium aspartate, potassium methanesulfonate, potassium isethionate, potassium cyclamate, potassium glutamate, potassium sulphate, potassium methanesulphonate, potassium formate, potassium acetate, potassium iodide, potassium bromide, K SCN “ , K + C(CN) 3 -, K + N(CN) 2 " , K + ClO 4 " , ⁇ NOs " , K + ClO 3 " , K + BrO 3 " or K + HCO 3 " or combinations thereof
  • the concentration of these salts - such as potassium gluconate - will be in the range of from 70-180mM, preferably from 80-170mM, more preferably from 80-160mM, more preferably from 80-150mM, more preferably from 80-140mM, more preferably from 80- 130mM, more preferably from 80-120mM, more preferably from 80-1 lOmM, or more preferably from 90-1 lOmM potassium gluconate or any suitable start or end point - such as from 90-160mM.
  • high ion concentration preferably high chloride ion concentration
  • the term "high ion concentration" means that the ion concentration is from about 120 to about 180 mM, preferably, from about 130 to about 170 mM.
  • the high ion concentration is from about 130 to about 160 mM - such as from about 130-160mM , about 135-150 mM, about 135-145 mM, about 138-142mM or any suitable combination of start or end points, for example, from about 136-149mM. In a highly preferred embodiment, the high ion concentration is about 140 mM, preferably, about 142mM.
  • the high ion concentration will be such that the osmolarity of the final solution will be within normal physiological parameters (about 250 to 310 mOsm/liter).
  • the high ion concentration may be from about 140 to about 160 mM - such as from about 145-160mM , about 150-160 mM, or about 155-159 mM or any suitable combination of start or end points, for example, from about 156-160mM. ,
  • access-phase relates to the period during which whole-cell access is being obtained and maintained and where the portion of cell membrane sealed to the apeture is exposed to an internal solution containing a permeablisation compound. This includes the time when the electrophysiological recordings are made and compounds are applied to the cells.
  • an "access phase internal solution” in the context of the present invention refers to a solution containing a permeablisation compound that is used during the period when whole-cell access is being obtained and maintained, including the time when the electrophysiological recordings are made and compounds are applied to the cells.
  • the access phase internal solution may have either a high or a low ion concentration.
  • the access phase internal solution may be a low chloride ion access phase internal solution containing from 0 to about 40mM chloride ions, preferably, 40mM chloride ions.
  • the access phase internal solution may be a low chloride ion access phase internal solution containing from 0 to about 60mM chloride ions.
  • the access phase internal solution may be a high chloride ion seal phase internal solution containing from about 120 to about 180 mM chloride ions, preferably, 140mM chloride ions.
  • the access phase internal solution will comprise additional reagents - such as potassium gluconate and/or magnesium chloride or the like, and/or EGTA and/or a buffer.
  • additional reagents - such as potassium gluconate and/or magnesium chloride or the like, and/or EGTA and/or a buffer.
  • the access phase internal solution may comprise 70-180mM potassium gluconate, preferably from 80-170mM, more preferably from 80-160mM, more preferably from 80-150mM, more preferably from 80-140mM, more preferably from 80-130mM, more preferably from 80- 120mM, more preferably from 80-1 lOmM, or more preferably from 90-1 lOmM potassium gluconate or any suitable start or end point - such as from 90-160mM.
  • the access phase internal solution may comprise 0-20mM MgCl 2 , preferably from 0-15mM, more preferably from 0-1 OmM, more preferably from 0-5mM, more preferably from 0-4mM, more preferably from l-4mM, more preferably from 2-4mM or more preferably 3mM MgCl 2 or any suitable start or end point - such as from 2-13mM.
  • the access phase internal solution may comprise 0-40mM EGTA, preferably from 0-3 OmM, more preferably from 0-2 OmM, more preferably from 0-1 OmM, more preferably from 0- 5mM, more preferably from l-5mM or more preferably from 2-4mM or any suitable start or end point - such as from l-9mM.
  • the access phase internal solution may comprise 0-40mM HEPES, preferably from 0-30mM, more preferably from 0-20mM, more preferably from 0-1 OmM, more preferably from 2- lOmM, more preferably from 4-8mM or more preferably from 4-6mM or any suitable start or end point - such as from 2-18mM.
  • the access phase internal solution may comprise range 0.01 to lmg/ml permeabilising agent, preferably from 0.05- 1 mg/ml, more preferably from 0.1-1 mg/ml, more preferably from 0.5- 1 mg/ml or more preferably from 0.8-1 mg/ml or any suitable start or end point - such as from 0.06-0.8 mg/ml.
  • the low ion concentration access phase internal solution comprises or consists of about 40mM KCl , about lOOmM K gluconate, about 3.2mM MgCl 2 , about 3. OmM EGTA about 5.
  • OmM HEPES, and a permeabilising agent - such as Amphotericin B at about 0. lmg/ml.
  • the high ion concentration access phase internal solution comprises or consists of about 140mM KCl, about l.OmM MgCl 2 , about l.OmM EGTA, about 20mM HEPES and a permeabilising agent- such as Amphotericin B at about 0. lmg/ml.
  • the composition of the access phase internal solution, aside from the presence of the permeablisation compound, may be the same or substantially the same as the seal phase internal solution.
  • An important part of the electrophysiological measurement apparatus used in accordance with the present invention is the ability to form an electrical seal between the surface of a substrate and a biological membrane.
  • the membranous sample is placed in suspension in a compartment, and drawn to an aperture - such as pore or a hole - through the use of differential pressure applied between two compartments, thus creating an electrical seal.
  • Any chemical that is capable of permeabilising a membranous sample such that it is permeable to monovalent ions - such as chloride ions - may be used.
  • permeabilisation is achieved by using certain antibiotics - such as Nystatin and Amphotericin B. These antibiotics work by forming chemical pores in the cell membrane that are permeable to monovalent ions - such as chloride.
  • permeabilising agents - such as gramicidin or ⁇ -escin - may also be used.
  • the term "seal-phase” relates to the period within which one or more cells are sealed to the apeture of the electrophysiological measurement apparatus, between addition of the cell suspension to the wells and up to the point where the internal solution is exchanged for one containing a permeablisation compound.
  • a "seal phase internal solution” in the context of the present invention refers to a solution that does not contain a permeablisation compound and is used during the period in which one or more cells are sealed to the apeture.
  • the seal phase internal solution has a low ion concentration.
  • the seal phase internal solution may be a low chloride ion seal phase internal solution containing, for example, from 0 to about 50mM chloride ions, preferably, 46mM chloride ions
  • the seal phase internal solution will comprise additional reagents - such as sodium gluconate and/or magnesium chloride or the like, and or EGTA and/or a buffer.
  • additional reagents - such as sodium gluconate and/or magnesium chloride or the like, and or EGTA and/or a buffer.
  • the access phase internal solution may comprise 0-60mM potassium chloride, preferably from 10-60mM, more preferably from 20-60mM, more preferably from 30-60mM, more preferably from 30-50mM or any suitable start or end point - such as from 1 l-59mM.
  • the access phase internal solution may comprise 70-180mM potassium gluconate, preferably from 80-170mM, more preferably from 80-160mM, more preferably from 80-150mM, more preferably from 80-140mM, more preferably from 80-130mM, more preferably from 80- 120mM, more preferably from 80-1 lOmM, or more preferably from 90-1 lOmM potassium gluconate or any suitable start or end point - such as from 90-160mM.
  • the access phase internal solution may comprise 0-20mM MgCl 2; preferably from 0-15mM, more preferably from 0-1 OmM, more preferably from 0-5mM, more preferably from 0-4mM, more preferably from l-4mM, more preferably from 2-4mM or more preferably from 3mM MgCl 2 or any suitable start or end point - such as from 2-13mM.
  • the access phase internal solution may comprise 0-40mM EGTA, preferably from 0-3 OmM, more preferably from 0-20mM, more preferably from 0-1 OmM, more preferably from 0- 5mM, more preferably from l-5mM or more preferably from 2-4mM or any suitable start or end point - such as from l-9mM.
  • the access phase internal solution may comprise 0-40mM HEPES, preferably from 0-30mM, more preferably from 0-20mM, more preferably from 0-1 OmM, more preferably from 2- lOmM, more preferably from 4-8mM or more preferably from 4-6mM or any suitable start or end point - such as from 2-18mM.
  • the low ion concentration access phase internal solution comprises or consists of about 40mM KCl, about 1 OOmM K gluconate, about 3.2mM MgCl 2 , about 3.OmM EGTA and about 5.0mM HEPES.
  • any electrophysiological measurement apparatus may be used that utilises a single, small (eg., several micron diameter) hole, aperture or pore in an at least substantially planar substrate to provide the sealing function resulting in a high- resistance electrical seal.
  • the substrate may include, for example, thin plastic films in which small holes have been machined or silicon substrates, where standard photolithographic/wet etching techniques are used to make the holes.
  • the electrophysiological measurement apparatus is a Molecular Devices IonWorksTMHT or an Axon Instruments PatchXpress.
  • Nanion Port-a-Patch System Another planar patch systems that is commercially available is the Nanion Port-a-Patch System.
  • planar patch systems in development include: Sophion Q Patch and Cytocentrics Cytopatch, for example.
  • MEMBRANOUS SAMPLE The term "membranous sample” as used herein, has its natural meaning.
  • the membranous sample may be or may be derived from a mammal.
  • the membranous sample is or is derived from an animal or a human.
  • the membranous sample is or is derived from a human.
  • the membranous sample may be or may be derived from biological material.
  • the membranous sample may include living cells, isolated cell fragments - such as organelles, and/or artificial membranes - such as vesicles
  • the membranous sample comprises one or more ion channels.
  • the present invention may be used to study membrane components that are associated with or capable of bringing about measurable voltage changes and/or current flows across biological membranes.
  • Suitable membrane components may include ion channels and ion transporters, ligand-gated channels and transporters.
  • Ion channels are membrane proteins that allow ions to flow across biological membranes, including the plasma membrane and organelle membranes. Ion channels are believed to create water-filled pores through which ions and some small hydrophilic molecules can pass by diffusion.
  • Ligand-gated channels open or close in response to a binding, reaction, and/or other association of signalling molecules, known as "ligands". These channels may be gated by the binding of extracellular or intracellular ligands. In either case, the ligand is different than the substance that is transported when the channel opens.
  • External ligands gate a variety of ion channels, including ATP gated- channels, glutamate- activated cationic channels, and cys-loop superfamily channels, for example.
  • External ligands are commonly neurotransmitters — such as acetylcholine, amino acids, catecholamines, miscellaneous monoamines, and peptides. .
  • Internal ligands also gate a variety of ion channels - such as G-protein coupled receptors, chloride channels, and calcium-gated potassium channels. These channels generally are controlled by second messengers, which are small signalling molecules - such as cyclic AMP (cAMP), cyclic GMP (cGMP), and calcium ions.
  • second messengers which are small signalling molecules - such as cyclic AMP (cAMP), cyclic GMP (cGMP), and calcium ions.
  • Ion transporters are membrane proteins that use energy - such as that derived from ATP to force ions or small molecules though the membrane via an electrochemical gradient.
  • the transporters may be direct active transporters, binding ATP directly and using the energy of its hydrolysis to drive active transport, or indirect active transporters, using ATP indirectly by using the downhill flow of a different type of ion to drive active transport, where the gradient of the different type of ion is created by a direct active transporter, allowing another transporter to create a gradient of a different type of ion, and then using.
  • Indirect transporters may be further subdivided into symporters and antiporters depending on whether the driving ion and the pumped ion (or other molecule) pass through the membrane in the same or opposite directions, respectively.
  • Exemplary direct active transporters include the Na+/K+ ATPase and the H+ ATPase.
  • Exemplary indirect active transporters include (1) symporters such as the Na+/glucose transporter, the various amino acid/Na+ transporters, and the NaAiodide transporter, and (2) antiporters such as the Na+/K+ ATPase.
  • the ion channel is a cardiac ion channel - such as SCN5A, KvLQTl, Kvl.5, Kv4.2, Kv4.3, Kir2.1, Kir2.2, Kir2.3, Kir2.4, CaV1.2, CaV3.1, CaV3.2, HCN1, HCN4 or , CFTR.
  • cardiac ion channel - such as SCN5A, KvLQTl, Kvl.5, Kv4.2, Kv4.3, Kir2.1, Kir2.2, Kir2.3, Kir2.4, CaV1.2, CaV3.1, CaV3.2, HCN1, HCN4 or , CFTR.
  • the ion channel is a myocardial I ⁇ r channel - such as a myocardial I r ion channel encoded by the ERG channel, including hERG.
  • a novel human cDNA from a hippocampal cDNA library was isolated by homology to the Drosophila 'ether-a-go-go' (eag) gene, which encodes a Ca(2+)-modulated potassium channel.
  • the authors called the cDNA HERG (human ether-a-go-go-related gene).
  • the HERG gene was localised to human chromosome 7.
  • the HERG gene contains 15 exons spanning approximately 19 kb on chromosome 7q35.
  • Oligonucleotide primers have been synthesised to cover the entire coding region and
  • the HERG gene contains 16 exons (which includes an alternative exon lb),
  • the HERG gene has been expressed Xenopus laevis oocytes and the potassium channel's biophysical properties and its sensitivity to various pharmacological agents
  • subunit may be required for certain drug sensitivities. Since blockage of I(Kr) is a known
  • Torsade de pointes can degenerate into ventricular fibrillation, which can lead to sudden
  • hERG has also been demonstrated to encode an inwardly rectifying potassium channel.
  • Inward rectifiers are a large class of potassium channels that preferentially conduct inward
  • these channels also have small outward conductances that regulate the resting
  • HERG channels show gating properties consistent with many of the outwardly rectifying potassium channels, but they also have an inactivation mechanism that attenuates efflux during depolarization.
  • the HERG potassium channel is unusual in that it seems to have the architectural plan of the depolarization-activated K+ channel family (6 putative transmembrane segments), yet it exhibits rectification like that of the inward-rectifying K+ channels, a family with different molecular structure (2 transmembrane segments).
  • HERG channels expressed in mammalian cells have been studied and it has been found that this inward rectification . ⁇ .. arises from a rapid and voltage-dependent inactivation process that reduces conductance at positive voltages.
  • the inactivation gating mechanism resembles that of C-type inactivation, often considered to be the 'slow inactivation' mechanism of other K+ channels.
  • the characteristics of this gating suggested a specific role for this channel in the normal suppression of arrhythmias.
  • the role for HERG in suppressing extra beats might help explain the increased incidence of cardiac sudden death in patients that lack
  • HERG currents either because they carry a genetic defect (familial long QT syndrome type 2) or because they are being treated with class III antiarrhythmics that block HERG channels.
  • NAB domain A subunit interaction domain, termed the NAB domain, in the hydrophilic cytoplasmic N terminus of HERG has also been identified. This domain is responsible for the
  • Truncated HERG proteins Truncated HERG proteins
  • deletion mutant at position 1261 contain the NAB domain but lack the rest
  • LQT may be the result of decreased expression of a functional HERG potassium channel in the heart.
  • LQT-related arrhythmias suggested that mutations in cardiac- specific ion channel genes (or genes that modulate cardiac ion channels) cause delayed myocellular repolarization. Delayed myocellular repolarization could promote reactivation of L-type Ca(2+) channels, resulting in secondary depolarizations. These secondary depolarizations are the likely cellular mechanism of torsade de pointes arrhythmias.
  • ASSAY METHOD The assay methods described herein may be used with single samples, for example, using pipette-based or planar-substrate-based measurement devices.
  • the methods are used with planar-substrate-based measurement devices.
  • the methods are used with multiple samples, sequentially and/or simultaneously, thereby enabling screening, including high-throughput screening.
  • the assay may be performed as follows.
  • Cells - such as CHOkl cells - heterologously expressing an ion channel - such as the hERG K + channel - may be prepared in accordance with known methods.
  • the PatchPlateTM may be a 384 well plate in an 8 x 48 well configuration.
  • the well format may be that of a 1536 well (32 x 48 well) plate and the PatchPlateTM resembles a 1536 well plate cut into four
  • each well is approximately 15 ⁇ l.
  • the plate consists of a proprietary membrane into which a single hole per well (approx. 2
  • a PatchPlateTM is manually placed into the system and sealed into place with a manifold.
  • Negative pressure is applied to the bottom surface of the plate and about 3.5 ⁇ l 'external
  • solution' is added automatically via the fluidics head to each well of the plate.
  • Suitable external solutions are described in Kiss et al. (2003) Assay and Drug Development Technologies 1, 1-2, 127-135 and also by Schroeder et al. (2003) Journal of Biochemical Screening 8(1), 50-64, and may comprise, for example, 0.9 mM CaCl 2j 2.67 mM KCl, 1.47 mM K 3 PO 4 , 0.5 mM MgCl 2 , 138 mM NaCI, 8.10 mM Na 3 PO 4 , pH 7.4
  • the bottom surface of the plate is primed by filling the plenum beneath the PatchPlateTM with a low ion concentration internal solution - such as low chloride ion seal phase internal
  • each well represents an individual electrophysiologically-enabled chamber.
  • the quality of the PatchPlateTM apetures may be tested using, for example, a lOmV test pulse applied sequentially to 48 wells at a time using the electronic measurement head.
  • apeture is required to reach a threshold of 2.8 M ⁇ ⁇ 0.5 M ⁇ . Wells that fall outside this are
  • the sub-plate negative pressure is maintained at a level more negative than atmospheric pressure to suck the cells onto the apetures.
  • Seal formation is monitored sequentially across the plate in batches of 48 wells while holding at about -70mV by applying a lOmV voltage pulse. This equates to the cell-attached mode in conventional voltage-clamp electrophysiology.
  • Agents may be added via, for example, the automated pipette system from a 96 or 384 well compound plate.
  • the methods described herein may be used to identify agents that do not, or substantially do not interact with certain ion channels - such as hERG ion channels.
  • ion channels - such as hERG ion channels.
  • This has been extremely difficult to achieve in a modern drug discovery environment since to do so by design requires testing large numbers of compounds to check for activity at these ion channels and, where appropriate, to screen out the activity.
  • the inability to do this reliably relates to conventional methodologies for ion channel screening: the assays either have a throughput that is far too low to make testing large numbers of compounds for activity tenable, or they lack the ability to reliably test for effects on channel function.
  • the term "modulating" may refer to preventing, suppressing, blocking, alleviating, restorating, elevating, increasing or otherwise affecting an electrical property of a membranous sample.
  • the term refers to preventing, suppressmg or blocking an electrical property of a membranous sample.
  • the term refers to preventing, suppressing or blocking an electrical property of an ion channel — such as hERG ion channel - such that an agent does not, or substantially does not interact with an ion channel.
  • the methods described herein may also be used to identify agents that block ion channels - such as hERG ion channels - in, for example, toxicological screening.
  • the methods of the present invention may also be used to obtain optimal potency estimates, for example, for determining the potency at which the agents block ion channels.
  • a method for determining the potency of an agent comprising the steps of: (a) providing an electrophysiological measurement apparatus; (b) contacting one or more membranous samples with a compartment of the electrophysiological measurement apparatus containing an aperture using a reduced number of cells per compartment; (c) forming a seal between the membranous samples and the aperture in the presence of a low ion concentration internal solution; (d) exchanging the low ion concentration internal solution for an access phase internal solution; (e) establishing electrical access, via the aperture, to the internal compartment of the membranous sample; and taking electrophysiological recordings before and after incubation of the cell with one or more agents.
  • optimal potency estimates are obtained for lipophilic and/or non-lipophilic agents.
  • an assay method for determining the potency of an agent comprising the steps of: (a) providing an electrophysiological measurement apparatus; (b) contacting one or more membranous samples with a compartment of the electrophysiological measurement apparatus containing an aperture using a reduced number of cells per compartment; (c) forming a seal between the membranous samples and the aperture in the presence of a low ion concentration internal solution; (d) exchanging the low ion concentration internal solution for an access phase internal solution; (e) establishing electrical access, via the aperture, to the internal compartment of the membranous sample; and (f) taking electrophysiological recordings before and after incubation of the cell with one or more agents; wherein a difference between: (i) the electrophysiological recording in the presence of the agent; and (ii) the electrophysiological recordings in the absence of the agent is indicative of the potency estimate for the agent.
  • the low ion concentration internal solution - such as the low ion concentration seal phase internal solution - is exchanged for a high ion concentration internal solution - such as a high ion concentration access phase internal solution.
  • the low ion concentration internal solution is a low ion concentration seal phase internal solution comprising about 40 mM chloride ions.
  • the low ion concentration seal phase internal solution may comprise about 40mM KCl, about lOOmM K gluconate, about 3.2 mM MgCl 2 , about 3.0mM EGTA, and about 5.0mM HEPES.
  • the high ion concentration internal solution is a high ion concentration access phase internal solution comprising about 140 mM chloride ions.
  • the access phase internal solution may comprise about 140mM KCl, about l.OmM MgCl 2 , about l.OmM EGTA, about 20mM HEPES and a permeabilising agent.
  • an optimum seal quality is also obtained using these assay conditions.
  • a low or a high ion concentration internal solution — such as a low or a high ion concentration seal phase internal solution — is exchanged for a high ion concentration internal solution - such as a high ion concentration access phase internal solution.
  • the methods of the present invention may be used for determining the potency at which lipophillic agents block ion channels.
  • the pIC 5 o estimates for compounds - such as lipophillic compounds are up to 2 log units less than the ICso estimates obtained using conventional voltage-clamp electrophysiological experiments.
  • the drop-off in potency is positively correlated to the measures of compound lipophillicity, ACDlogP, ClogP and % non-polar surface area.
  • each compartment will contain approximately 3500 cells in a volume of 10.5 ⁇ l buffer
  • the present inventors have found that by reducing or adjusting the cell number in each compartment, the pIC 5 o estimates for the compounds - such as lipophillic compounds - can be increased to a level comparable to those obtained in conventional voltage-clamp electrophysiology experiments.
  • concentration of lipid (cell membrane) in the well will also increase linearly giving a greater volume of lipid for compound to partition into and reducing the effective concentration in the well. This causes the potency of these compounds to be underestimated. Reducing the cell number also reduces the lipid:buffer ratio and hence the amount of compound that partitions into the membrane. The potency estimate is thus closer to that obtained with conventional voltage-clamp electrophysiology experiments.
  • well - such as about 1-875 cells per well, about 50-875 cells per well, about 100-875 cells per well, about 150-875 cells per well, or about 175-875 cells per well, or any combination of start or end points - such as about 173-854 cells per well.
  • the optimal cell number :buffer ratio is in the range about 1-90 cells per 1 ⁇ l buffer - such as
  • start or end points- such as 20-82 cells per 1 ⁇ l buffer.
  • reducing or adjusting the number of membranous samples in each compartment advantageously provides for an optimal potency estimate for the agent.
  • the access phase internal solution may typically comprise about 40mM KCl, about lOOmM K gluconate, about 3.2 mM MgCl 2 , about 3. OmM EGTA, about 5.0 mM HEPES and a permeabilising agent or about 140mM KCl, about l.OmM MgCl 2 , about l.OmM EGTA, about 20mM HEPES and a permeabilising agent.
  • the number of membranous samples - such as cells - in each compartment is
  • the number of membranous samples in each compartment is the number of membranous samples in each compartment.
  • compartment is between about 17 and 83 in a volume of 1 ⁇ l buffer solution.
  • improved pIC 50 estimates for agents can also be obtained using an access phase internal solution that does not contain a permeabilising agent. Therefore, adjusting the number of cells in each compartment can be used to improve pIC 50 estimates when used in conjunction with permeabilisation methods other than those that include a permeablisation agent.
  • 'whole cell access' or 'electrical access' can be induced in many ways.
  • access may be achieved using membrane permeabilisation with voltage pulses of sufficient strength and duration that the membrane inside the pipette physically breaks down. This approach is well known in the field and is commonly referred to as "zapping".
  • the membrane may also be 'permeabilised' by adding negative pressure (suction) to the back of the pipette, or to the side of the planar substrate containing the internal solution.
  • the maintenance of an electrical seal between the surface of a substrate and a biological membrane is particularly important since this results in a high-resistance electrical seal. Maintaining the optimum seal quality throughout the assay period is therefore important to obtain an optimum assay result.
  • the inventors have surprisingly found that the optimum seal quality can be obtained by using a low ion concentration internal solution - such as a low ion concentration seal phase internal solution - and can be maintained by exchanging the low ion concentration internal solution for a high or a low ion concentration internal solution - such as a high or a low ion concentration access phase internal solution.
  • the seal phase internal solution may typically comprise about 40mM KCl, about l.OmM MgCl 2 , about l.OmM EGTA, and about 20mM HEPES
  • the access phase internal solution may typically comprise about 40mM KCl, about l.OmM MgCl 2 , about OmM EGTA, about 20mM HEPES and a permeabilising agent, or about 140mM KCl, about 1.OmM MgCl 2 , about 1.OmM EGTA, about 20mM HEPES and a permeabilising agent.
  • the seal quality of the aperture, hole or pore may be tested using a lOmV test pulse applied sequentially to the chambers or wells using the electronic measurement head of an electrophysiological measurement apparatus.
  • each apeture is required to reach a threshold of 2.8 M ⁇ --- 0.5 M ⁇ .
  • the sub-plate negative pressure is maintained at a level more negative than atmospheric pressure to suck the cells onto the apetures.
  • Seal formation is monitored sequentially across the plate in batches - such as in batches of 48 wells - while holding at, for example, -70mV by applying a lOmV voltage pulse. This equates to the cell-attached mode in conventional voltage-clamp electrophysiology.
  • the seal- phase internal solution in the bottom reservoir solution is exchanged for an access-phase internal solution in order to produce a permeabilised patch configuration.
  • Cells are then tested by the application of a voltage protocol. This process equates to the whole-cell mode in conventional patching.
  • Dysfunctional hERG ion channels may lead to the development of LQT.
  • Dysfunctional hERG ion channels causing LQT may occur due to mutations in specific genes and/or treatment with any of a variety of drugs. These drugs include, but are not limited to those being taken to treat cardiac arrhythmias and also other drugs including antihistamines and some antibiotics - such as erythromycin.
  • the LQT is a result of mutations or drag induced, it is due to an effect on an ion channel.
  • the drugs interact with the major subunit of the hERG ion channel, thereby affecting the flow of potassium ions in cells - such as cardiac cells.
  • Mutations in hERG also can affect potassium ion flow through this channel. This can result in LQT syndrome and may lead to torsade de pointes.
  • the agents of the present invention may be useful in the treatment of the disorders listed in WO-A-99/52890.
  • the agents of the present invention may be useful in the treatment of the disorders listed in WO-A-98/05635.
  • diabetes including Type II diabetes, obesity, cancer, inflammation or inflammatory disease, dermatological disorders, fever, cardiovascular effects, haemorrhage, coagulation and acute phase response, cachexia, anorexia, acute infection, HIV infection, shock states, graft- versus-host reactions, autoimmune disease, reperfusion injury, meningitis, migraine and aspirin-dependent anti-thrombosis; tumour growth, invasion and spread, angiogenesis, metastases, malignant, ascites and malignant pleural effusion; cerebral ischaemia, ischaemic heart disease, osteoarthritis, rheumatoid arthritis, osteoporosis, asthma, multiple sclerosis, neurodegeneration, Alzheimer's disease, atherosclerosis, stroke, vasculitis, Crohn's disease and ulcerative colitis; periodontitis, gingivitis; psoriasis, atopic dermatitis, chronic ulcers, epidermolysis bullosa; corneal ulceration,
  • the agents of the present invention may be useful in the treatment of disorders listed in WO-A-98/07859.
  • cytokine and cell proliferation/differentiation activity e.g. for treating immune deficiency, including infection with human immune deficiency virus; regulation of lymphocyte growth; treating cancer and many autoimmune diseases, and to prevent transplant rejection or induce tumour immunity
  • regulation of haematopoiesis e.g. treatment of myeloid or lymphoid diseases
  • promoting growth of bone, cartilage, tendon, ligament and nerve tissue e.g.
  • follicle-stimulating hormone for healing wounds, treatment of bums, ulcers and periodontal disease and neurodegeneration; inhibition or activation of follicle-stimulating hormone (modulation of fertility); chemotactic/chemokinetic activity (e.g. for mobilising specific cell types to sites of injury or infection); haemostatic and thrombolytic activity (e.g. for treating haemophilia and stroke); antiinflammatory activity (for treating e.g. septic shock or Crohn's disease); as antimicrobials; modulators of e.g. metabolism or behaviour; as analgesics; treating specific deficiency disorders; in treatment of e.g. psoriasis, in human or veterinary medicine.
  • chemotactic/chemokinetic activity e.g. for mobilising specific cell types to sites of injury or infection
  • haemostatic and thrombolytic activity e.g. for treating haemophilia and stroke
  • antiinflammatory activity for treating e.g. s
  • agents of the present invention may be useful in the treatment of disorders listed in WO-A-98/09985.
  • macrophage inhibitory and/or T cell inhibitory activity and thus, anti-inflammatory activity i.e.
  • inhibitory effects against a cellular and/or humoral immune response including a response not associated with inflammation; inhibit the ability of macrophages and T cells to adhere to extracellular matrix components and fibronectin, as well as up-regulated fas receptor expression in T cells; inhibit unwanted immune reaction and inflammation including arthritis, including rheumatoid arthritis, inflammation associated with hypersensitivity, allergic reactions, asthma, systemic lupus erythematosus, collagen diseases and other autoimmune diseases, inflammation associated with atherosclerosis, arteriosclerosis, atherosclerotic heart disease, reperfusion injury, cardiac arrest, myocardial infarction, vascular inflammatory disorders, respiratory distress syndrome or other cardiopulmonary diseases, inflammation associated with peptic ulcer, ulcerative colitis and other diseases of the gastrointestinal tract, hepatic fibrosis, liver cirrhosis or other hepatic diseases, thyroiditis or other glandular diseases, glomerulonephritis or other renal and urologic diseases, otitis or other oto-rhino-
  • retinitis or cystoid macular oedema retinitis or cystoid macular oedema, sympathetic ophthalmia, scleritis, retinitis pigmentosa, immune and inflammatory components of degenerative fondus disease, inflammatory components of ocular trauma, ocular inflammation caused by infection, proliferative vitreo-retinopathies, acute ischaemic optic neuropathy, excessive scarring, e.g.
  • Alzheimer's disease and other degenerative diseases, conditions or disorders of the CNS inflammatory components of stokes, post-polio syndrome, immune and inflammatory components of psychiatric disorders, myelitis,, encephalitis, subacute sclerosing pan- encephalitis, encephalomyelitis, acute neuropathy, subacute neuropathy, chronic neuropathy, Guillaim-Barre syndrome, Sydenham chora, myasthenia gravis, pseudo-tumour cerebri, Down's Syndrome, Huntington's disease, amyotrophic lateral sclerosis, inflammatory components of CNS compression or CNS trauma or infections of the CNS, inflammatory components of muscular atrophies and dystrophies, and immune and inflammatory related diseases, conditions or disorders of the central and peripheral nervous systems, post-traumatic inflammation, septic shock, infectious diseases, inflammatory complications or side effects of surgery, bone marrow transplantation or other transplantation complications and/or side effects, inflammatory and/or immune complications and side effects of gene therapy, e.g
  • monocyte or leukocyte proliferative diseases e.g. leukaemia
  • monocytes or lymphocytes for the prevention and/or treatment of graft rejection in cases of transplantation of natural or artificial cells, tissue and organs such as cornea, bone marrow, organs, lenses, pacemakers, natural or artificial skin tissue.
  • agent may be a single entity or it may be a combination of entities.
  • the agent may be an organic compound or other chemical.
  • the agent may be a compound, which is obtainable from or produced by any suitable source, whether natural or artificial.
  • the agent may be an amino acid molecule, a polypeptide, or a chemical derivative thereof, or a combination thereof.
  • the agent may even be a polynucleotide molecule - which may be a sense or an anti-sense molecule, or an antibody, for example, a polyclonal antibody, a monoclonal antibody or a monoclonal humanised antibody.
  • the agent may be attached to an entity (e.g. an organic molecule) by a linker.
  • the entity may be designed or obtained from a library of compounds, which may comprise peptides, as well as other compounds, such as small organic molecules.
  • the entity may be a natural substance, a biological macromolecule, or an extract made from biological materials such as bacteria, fungi, or animal (particularly mammalian) cells or tissues, an organic or an inorganic molecule, a synthetic agent, a semi- synthetic agent, a structural or functional mimetic, a peptide, a peptidomimetics, a peptide cleaved from a whole protein, or a peptides synthesised synthetically (such as, by way of example, either using a peptide synthesizer or by recombinant techniques or combinations thereof), a recombinant agent, an antibody, a natural or a non-natural agent, a fusion protein ' or equivalent thereof and mutants, derivatives or combinations thereof.
  • the entity will be an organic compound.
  • the organic compounds will comprise two or more hydrocarbyl groups.
  • hydrocarbyl group means a group comprising at least C and H and may optionally comprise one or more other suitable substituents. Examples of such substituents may include halo-, alkoxy-, nitro-, an alkyl group, a cyclic group etc.
  • substituents may include halo-, alkoxy-, nitro-, an alkyl group, a cyclic group etc.
  • a combination of substituents may form a cyclic group. If the hydrocarbyl group comprises more than one C then those carbons need not necessarily be linked to each other. For example, at least two of the carbons may be linked via a suitable element or group.
  • the hydrocarbyl group may contain hetero atoms. Suitable hetero atoms will be apparent to those skilled in the art and include, for instance, sulphur, nitrogen and oxygen.
  • the entity comprises at least one cyclic group.
  • the cyclic group may be a polycyclic group, such as a non-fused polycyclic group.
  • the entity comprises at least the one of said cyclic groups linked to another hydrocarbyl group.
  • the entity may contain halo groups - such as fluoro, chloro, bromo or iodo groups.
  • the entity may contain one or more of alkyl, alkoxy, alkenyl, alkylene and alkenylene groups - which may be unbranched- or branched-chain.
  • the agent of the present invention may have known therapeutic properties and a reduced, diminished or decreased activity towards an electrical property of a membranous sample - such as an ion channel eg. a hERG ion channel.
  • the ideal position is for compounds not to interact with ion channels - such as hERG.
  • the agent of the present invention may have known therapeutic properties and have no or substantially no activity towards an electrical property of a membranous sample - such as an ion channel eg. hERG.
  • TdP Torsade de Pointes
  • TdP Measurable incidence of TdP in humans has been reported with include aprindine, bepridil, chlorpromazine, flecainide, halofantrine, haloperidol, lidoflazine, maprotiline, pentamidine, pimozide and thioridazine.
  • TdP in humans has been reported in isolated cases with amantadine, amitriptyline, chloral hydrate, chloroquine, ciprofloxacin, clarithromycin, cocaine, desipramine, diphenhydramine, domperidone, doxepin, erythromycin i.v., fexofenadine, fluoxetine, furosemide, imipramine, ketanserin, mexiletine, mibefradil, nifedipine, papaverine, perhexiline, probucol, propafenone, sparfloxacin, spiramycin, sultopride, tacrolimus and zimeldine.
  • the agent may be a modified form of astemizole, cisapride, droperidol, grepafloxacin, levomethadyl, prenylamine, terfenadine, terodiline, aprindine, bepridil, chlorpromazine, flecainide, halofantrine, haloperidol, lidoflazine, maprotiline, pentamidine, pimozide and thioridazine, amantadine, amitriptyline, chloral hydrate, chloroquine, ciprofloxacin, clarithromycin, cocaine, desipramine, diphenhydramine, domperidone, doxepin, erythromycin i.v., fexofenadine, fluoxetine, furosemide, imipramine, ketanserin, mexiletine, mibefradil, nifedipine, papaverine, perhexi
  • the agent may be administered in the form of a pharmaceutically acceptable salt.
  • Suitable acid addition salts are formed from acids which form non-toxic salts and include the hydrochloride, hydrobromide, hydroiodide, nitrate, sulphate, bisulphate, phosphate, hydrogenphosphate, acetate, trifluoroacetate, gluconate, lactate, salicylate, citrate, tartrate, ascorbate, succinate, maleate, fumarate, gluconate, formate, benzoate, methanesulphonate, ethanesulphonate, benzenesulphonate and p-toluenesulphonate salts.
  • suitable pharmaceutically acceptable base addition salts can be formed from bases, which form non-toxic salts and include the aluminium, calcium, lithium, magnesium, potassium, sodium, zinc, and ammonium salts such as diethanolammonium, salts.
  • a pharmaceutically acceptable salt of an agent may be readily prepared by mixing together solutions of an agent and the desired acid or base, as appropriate.
  • the salt may precipitate from solution and be collected by filtration or may be recovered by evaporation of the solvent.
  • An agent may contain one or more asymmetric carbon atoms and therefore exist in two or more stereoisomeric forms. Where an agent contains an alkenyl or alkenylene group, cis (E) and trans (Z) isomerism may also occur.
  • the present invention includes the individual stereoisomers of an agent and, where appropriate, the individual tautomeric forms thereof, together with mixtures thereof.
  • Separation of diastereoisomers or cis- and tr ⁇ r ⁇ -isomers may be achieved by conventional techniques, e.g., by fractional crystallisation, chromatography or H.P.L.C. of a stereoisomeric mixture of an agent or a suitable salt or derivative thereof.
  • An individual enantiomer of an agent may also be prepared from a corresponding optically pure intermediate or by resolution, such as by H.P.L.C. of the corresponding racemate using a suitable chiral support or by fractional crystallisation of the diastereoisomeric salts formed by reaction of the corresponding racemate with a suitable optically active acid or base, as appropriate.
  • the present invention also encompasses all suitable isotopic variations of an agent or a pharmaceutically acceptable salt thereof.
  • An isotopic variation of an agent or a pharmaceutically acceptable salt thereof is defined as one in which at least one atom is replaced by an atom having the same atomic number but an atomic mass different from the atomic mass usually found in nature.
  • isotopes that may be incorporated into an agent and pharmaceutically acceptable salts thereof include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulphur, fluorine and chlorine such as 2 H, 3 H, 13 C, 14 C, 15 N, 17 0, 18 0, 31 P, 32 P, 35 S, 18 F and 36 C1, respectively.
  • isotopic variations of an agent and pharmaceutically acceptable salts thereof are useful in drag and/or substrate tissue distribution studies. Tritiated, i.e., 3 H, and carbon- 14, i.e., 14 C, isotopes are particularly preferred for their ease of preparation and detectability. Further, substitution with isotopes such as deuterium, i.e., 2 H, may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements and hence may be preferred in some circumstances. Isotopic variations of an agent of the present invention and pharmaceutically acceptable salts thereof of this invention can generally be prepared by conventional procedures using appropriate isotopic variations of suitable reagents.
  • an agent may be derived from a prodrug.
  • prodrugs include entities that have certain protected group(s) and which may not possess pharmacological activity as such, but may, in certain instances, be administered (such as orally or parenterally) and thereafter metabolised in the body to form an agent of the present invention which are pharmacologically active.
  • pro-moieties for example as described in "Design of Prodrags" by H. Bundgaard, Elsevier, 1985 (the disclosured of which is hereby incorporated by reference), may be placed on appropriate functionalities of agents. Such prodrags are also included within the scope of the invention.
  • the present invention also includes the use of zwitterionic forms of an agent of the present invention.
  • the terms used in the claims encompass one or more of the forms just mentioned.
  • the present invention also includes the use of solvate forms of an agent.
  • An agent may be administered as a pharmaceutically acceptable salt.
  • a pharmaceutically acceptable salt may be readily prepared by using a desired acid or base, as appropriate.
  • the salt may precipitate from solution and be collected by filtration or may be recovered by evaporation of the solvent.
  • An agent may be prepared by chemical synthesis techniques.
  • any stereocentres present could, under certain conditions, be racemised, for example if a base is used in a reaction with a substrate having an optical centre comprising a base-sensitive group. This is possible during e.g., a guanylation step. It should be possible to circumvent potential problems such as this by choice of reaction sequence, conditions, reagents, protection deprotection regimes, etc. as is well-known in the art.
  • the compounds and salts of the invention may be separated and purified by conventional methods.
  • Separation of diastereomers may be achieved by conventional techniques, e.g. by fractional crystallisation, chromatography or H.P.L.C. of a stereoisomeric mixture of a compound of formula (I) or a suitable salt or derivative thereof.
  • An individual enantiomer of a compound of formula (I) may also be prepared from a corresponding optically pure intermediate or by resolution, such as by H.P.L.C. of the corresponding racemate using a suitable chiral support or by fractional crystallisation of the diastereomeric salts formed by reaction of the corresponding racemate with a suitably optically active acid or base.
  • An agent or variants, homologues, derivatives, fragments or mimetics thereof may be produced using chemical methods to synthesize an agent in whole or in part. For example, if they are peptides, then peptides may be synthesized by solid phase techniques, cleaved from the resin, and purified by preparative high performance liquid chromatography (e.g., Creighton (1983) Proteins Structures And Molecular Principles, WH Freeman and Co, New York NY). The composition of the synthetic peptides may be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure; Creighton, supra).
  • Synthesis of peptide agents may be performed using various solid-phase techniques (Roberge JY et al. (1995) Science 269: 202-204) and automated synthesis may be achieved, for example, using the ABI 43 1 A Peptide Synthesizer (Perkin Elmer) in accordance with the instructions provided by the manufacturer. Additionally, the amino acid sequences comprising an agent or any part thereof may be altered during direct synthesis and/or combined using chemical methods with a sequence from other subunits, or any part thereof, to produce a variant agent.
  • the coding sequence of a peptide agent may be synthesized, in whole or in part, using chemical methods well known in the art (see Carathers MH et al.
  • the term "mimetic” relates to any chemical, which includes, but is not limited to, a peptide, polypeptide, antibody or other organic chemical, which has the same qualitative activity or effect as a reference agent.
  • derivative or "derivatised” as used herein includes chemical modification of an agent. Illustrative of such chemical modifications would be replacement of hydrogen by a halo group, an alkyl group, an acyl group or an amino group.
  • the chemical modification of an agent may either enhance or reduce hydrogen bonding interaction, charge interaction, hydrophobic interaction, Van Der Waals interaction or dipole interaction between the agent and the target,
  • the identified agent may act as a model (for example, a template) for the development of other compounds.
  • An agent may be prepared by recombinant DNA techniques.
  • a composition may comprise other therapeutic substances in addition to the agent.
  • Agents identified by the method of the present invention may be used as therapeutic agents - i.e., in therapy applications.
  • therapy includes curative effects, alleviation effects, and prophylactic effects.
  • the therapy may be on mammals such as humans or animals.
  • the therapy may be for treating disorders associated with dysfunctional ion channels - such as the development of LQT and/or torsade de pointes.
  • compositions useful in the present invention may comprise a therapeutically effective amount of agent(s) and pharmaceutically acceptable carrier, diluent or excipient (including combinations thereof).
  • compositions may be for human or animal usage in human and veterinary medicine and will typically comprise any one or more of a pharmaceutically acceptable diluent, carrier, or excipient.
  • Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985).
  • the choice of pharmaceutical carrier, excipient or diluent may be selected with regard to the intended route of administration and standard pharmaceutical practice.
  • Pharmaceutical compositions may comprise as - or in addition to - the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s) or solubilising agent(s).
  • compositions useful in the present invention may be formulated to be administered using a mini-pump or by a mucosal route, for example, as a nasal spray or aerosol for inhalation or ingestable solution, or parenterally in which the composition is formulated by an injectable form, for delivery, by, for example, an intravenous, intramuscular or subcutaneous route.
  • the formulation may be designed to be administered by a number of routes.
  • nucleotide sequences encoding said protein may be delivered by use of non- viral techniques (e.g., by use of liposomes) and/or viral techniques (e.g., by use of retroviral vectors) such that the said protein is expressed from said nucleotide sequence.
  • the components useful in the present invention may be administered alone but will generally be administered as a pharmaceutical composition - e.g., when the components are in admixture with a suitable pharmaceutical excipient, diluent or carrier selected with regard to the intended route of administration and standard pharmaceutical practice.
  • a physician will determine the actual dosage, which will be most suitable for an individual subject.
  • the specific dose level and frequency of dosage for any particular patient may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the individual undergoing therapy.
  • the component(s) may be formulated into a pharmaceutical composition, such as by mixing with one or more of a suitable carrier, diluent or excipient, by using techniques that are known in the art.
  • the term "host cell” refers to any cell that is of use in the present invention - such as cell comprising a recombinant ion channel.
  • Host cells may be transformed or transfected with a nucleotide sequence contained in a vector e.g. a cloning vector.
  • a nucleotide sequence contained in a vector e.g. a cloning vector.
  • said nucleotide sequence is carried in a vector for the replication and/or expression of the nucleotide sequence.
  • the cells will be chosen to be compatible with the said vector and may, for example, be prokaryotic (for example bacterial), fungal, yeast or plant cells.
  • E. coli The gram-negative bacterium E. coli is widely used as a host for cloning nucleotide sequences. This organism is also widely used for heterologous nucleotide sequence expression. However, large amounts of heterologous protein tend to accumulate inside the cell. Subsequent purification of the desired protein from the bulk of E. coli intracellular proteins can sometimes be difficult. In contrast to E. coli, bacteria from the genus Bacillus are very suitable as heterologous hosts because of their capability to secrete proteins into the culture medium. Other bacteria suitable as hosts are those from the genera Streptomyces and Pseudomonas.
  • eukaryotic hosts including yeasts or other fungi may be preferred.
  • yeast cells are preferred over fungal cells because yeast cells are easier to manipulate.
  • some proteins are either poorly secreted from the yeast cell, or in some cases are not processed properly (e.g. hyperglycosylation in yeast). In these instances, a different fungal host organism should be selected.
  • the host cells are mammalian cells - such as CHO cells.
  • Introduction of a vector into a host cell can be effected by various methods. For example, calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction or infection may be used. Such methods are described in many standard laboratory manuals - such as Sambrook et al, Molecular Cloning: A Laboratory Manual, 2d ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Host cells containing the expression vector can be selected by using, for example, G418 for cells transfected with an expression vector carrying a neomycin resistance selectable marker.
  • nucleotide sequence may need to be suitably modified before transformation - such as by removal of introns.
  • a host cell may be transformed with a nucleotide sequence.
  • Host cells transformed with the nucleotide sequence may be cultured under conditions suitable for the replication or expression of the nucleotide sequence.
  • Nucleotide sequences - such as nucleotide sequences encoding ion channels - may be present in a construct.
  • construct which is synonymous with terms such as “conjugate”, “cassette” and “hybrid” - includes a nucleotide sequence directly or indirectly attached to a promoter.
  • An example of an indirect attachment is the provision of a suitable spacer group such as an intron sequence including the Shl-intron or the ADH intron, intermediate to the promoter and the nucleotide sequence.
  • fused which includes direct or indirect attachment.
  • the terms do not cover the natural combination of the nucleotide sequence coding for the protein ordinarily associated with the wild type nucleotide sequence promoter and when they are both in their natural environment.
  • the construct may even contain or express a marker, which allows for the selection of the nucleotide sequence construct in, for example, a bacterium, preferably of the genus Bacillus, such as Bacillus subtilis, or plants into which it has been transferred.
  • a marker which allows for the selection of the nucleotide sequence construct in, for example, a bacterium, preferably of the genus Bacillus, such as Bacillus subtilis, or plants into which it has been transferred.
  • markers which may be used, for example those encoding mannose-6-phosphate isomerase (especially for plants) or those markers that provide for antibiotic resistance - e.g. resistance to G418, hygromycin, bleomycin, kanamycin and gentamycin.
  • Nucleotide sequence - such as nucleotide sequences encoding ion channels - may be present in a vector.
  • vector includes expression vectors and transformation vectors and shuttle vectors.
  • transformation vector means a constract capable of being transferred from one entity to another entity - which may be of the species or may be of a different species. If the constract is capable of being transferred from one species to another e.g. from anE. coli plasmid to a bacterium, such as of the genus Bacillus, then the transformation vector is sometimes called a "shuttle vector". It may even be a constract capable of being transferred from an E. coli plasmid to an Agrobacterium to a plant.
  • the vectors may be transformed into a suitable host cell as described below to provide for expression of a polypeptide.
  • the vectors may be for example, plasmid, virus or phage vectors provided with an origin of replication, optionally a promoter for the expression of the said polynucleotide and optionally a regulator of the promoter.
  • the present invention relates to a kit for performing the methods of the present invention comprising: (a) a first vessel comprising a seal phase internal solution according to the present invention; and (b) a second vessel comprising an access phase internal solution.
  • the seal phase internal solution has a chloride ion concentration of about 40 mM. More preferably, wherein the seal phase internal solution comprises about 40mM KCl, about l.OmM MgCl 2 , about l.OmM EGTA, and about 20mM HEPES.
  • the access phase internal solution has a chloride ion concentration of about 40 mM or more. More preferably, wherein the ' access phase internal solution comprises about 40mM KCl, about l.OmM MgCl 2 , about OmM EGTA, about 20mM HEPES and a permeabilising agent or about 140mM KCl, about OmM MgCl 2 , about l.OmM EGTA, about 20mM HEPES and a permeabilising agent.
  • the present invention employs, unless otherwise indicated, conventional techniques of molecular biology, microbiology, and recombinant DNA, which are within the capabilities of a person of ordinary skill in the art. Such techniques are explained in the literature. See, for example, J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Books 1-3, Cold Spring Harbor Laboratory Press; Ausubel, F. M. et . al. (1995 and periodic supplements; Current Protocols in Molecular Biology, ch. 9, 13, and 16, John Wiley & Sons, New York, N.Y.); B. Roe, J. Crabtree, and A.
  • CHOKl-hERG cells are maintained at 37°C in a humidified environment (5% CO 2 ) in the
  • HAM'S F-12 + L-glutamine SIGMA N-6658
  • 10% FBS SIGMA CR0848
  • 0.6mg/ml Hygromycin SIGMA H-5527
  • CHO-Kvl.5 cells are maintained at 37°C in a humidified environment (5% CO 2 ) in the
  • HAM'S F-12 + L-glutamine SIGMA N-6658
  • 10% FBS SIGMA CR0848
  • 0.5mg/ml Hygromycin SIGMA H-5527
  • Cells should be split every 2-3 days at a dilution of 1. TO to 1:20.
  • cells should be split at a range of dilutions between 1:6 and 1:10. These values are meant as guidelines. If cells are split early or late in the day the range of dilutions should be altered accordingly in order to get a plate density of approx. 70% after 2 days incubation.
  • a dialog box will enquire if buffer has been placed in the buffer boat, Select yes.
  • the perforation agent used is Amphotericin B (Sigma A4888). 1) Weigh out 25-30mg Amphotericin B into a 1.5ml Eppendorf tube. 2) Add 830 ⁇ l DMSO to the tube and vortex immediately.
  • the vacuum pressure should be checked. On the digital dial the pressure should increase rapidly and settle at around 9.2 in. water within a minute.
  • the F-head will aspirate PBS from the buffer station and dispense it into the patch plate. It will then move to the wash station for cleaning.
  • the E-head will take Hole Test recordings and move to the wash station for cleaning.
  • the F-head will aspirate cells from the cell boat and dispense them into the patch plate. The cells are then given a few minutes to reach and then seal at holes in the bottom of the patch plate.
  • the E-head will then take Seal Test recordings (after approx. 10 mins) and then move to the wash station for cleaning. 7) The Amphotericin is then introduced to the lines and switched for the internal solution under the plenum (you should see this in the de-bubbler). It will be cycled through the plenum in order to gain access to the cells. The fluid height in the debubbler should rest just above the inlet pipe. 8) After approx. 22 mins the E-head will start to take pre-compound recordings from half of the plate (groups 1-4).
  • the F-head will apply compound to the first half of the plate, from rows A-D of the compound plate, with washes in between each addition. 10) After 25 mins. the E-head will take post-compound recordings from the first half of the plate with washes in between each recording. It will then take pre-compound records from the remainder of the plate (groups 5-8).
  • the F-head will dispense compound into the remaining half of the plate, from rows E-H of the compound plate, with washes in between each addition. 12) After approx. 40 mins, the E-head will take post-compound recordings from the remaining half of the plate, with washes in between each recording.
  • a clean up sequence is carried out to wash both the E and F-head and drain all pipes of fluid.
  • Table 1 show the effect of four combinations of different seal- and access-phase solutions ie. 40mM and 140mM KCl, 0 and lOOmM K Gluconate.
  • a seal-phase internal solution that is low in chloride (CI " ) ions (low chloride ion seal-phase internal solution) is used.
  • the seal phase internal solution has the following composition:
  • Such a solution may have the following composition:
  • the optimal seal quality obtained using low chloride ion seal-phase internal solution can be maintained through the access-phase by using either low chloride ion seal-phase internal solution containing amphotericin B (low chloride ion access-phase internal solution) or high chloride ion seal-phase internal solution containing amphotericin B (high chloride ion access- phase internal solution) during the access phase.
  • the poor seal quality obtained using high chloride ion seal-phase internal solution is maintained through the access-phase by using high chloride ion access-phase internal solution during the access phase and cannot be improved by using low chloride ion access-phase internal solution during the access-phase.
  • a low chloride ion seal-phase internal solution must be used during the seal-phase followed by either low chloride ion access-phase internal solution or high chloride ion access-phase internal solution during the access phase.
  • the potency (log of the concentration of compound in moles/litre (M) which inhibit the current magnitude by 50%; pICso) of compounds - such as the standard hERG channel blocking agents cisapride - is around 0.5 log units less than the pIC 50 obtained using conventional voltage-clamp electrophysiological experiments.
  • the pIC 50 estimates for some lipophillic compounds are up to 2 log units less than the pIC 5 o estimates obtained using conventional voltage-clamp electrophysiological experiments.
  • the drop-off in potency is positively correlated to the measures of compound lipophillicity, ACDlogP, ClogP and % non-polar surface area.
  • Molecular Devices recommends the use of a cell suspension at lxlO 6 cells per ml for addition to the individual wells of the PatchPlate (lonWorks HT Users Guide, molecular Devices Corp. 2002, Chapter 5, page 5-4. 3.5ul of this cell suspension is added to each well and the total
  • each well will contain approximately 3500
  • the pIC 5 o estimates for the lipophillic compounds can be surprisingly increased to a level comparable to those obtained in conventional voltage- clamp electrophysiology experiments.
  • the concentration of lipid (cell membrane) in the well will also increase linearly giving a greater volume of lipid for compound to partition into and reducing the effective concentration in the well This causes the potency of these compounds to be underestimated. Reducing the cell number reduces the lipid:buffer ratio and hence the amount of compound that partitions into the membrane. The potency estimate is thus closer to that obtained with conventional voltage- clamp electrophysiology experiments.
  • the optimal range is 175-875 cells per well.
  • the optimal cell number will vary depending on the volume of buffer added, since this will alter the cell number:buffer ratio.
  • the optimal cell number:buffer ratio is in the range 17-83 cells per lul buffer.
  • the numbers on the Y axis are deviation from the pIC50 measured by conventional electrophysiology versus IonWorksHT (i.e. conventional pIC50 - lonworksHT pIC50).
  • a lipophillic agent comprising the steps of:

Landscapes

  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Food Science & Technology (AREA)
  • Urology & Nephrology (AREA)
  • Molecular Biology (AREA)
  • Hematology (AREA)
  • Medicinal Chemistry (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Biophysics (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

La présente invention concerne une méthode de dosage destinée à l'identification d'un agent qui permet de moduler la propriété électrique d'un échantillon membranaire. Ladite méthode de dosage consiste à (a) fournir un appareil de mesure électrophysiologique, (b) mettre en contact au moins un échantillon membranaire avec un compartiment dudit appareil de mesure électrophysiologique doté d'une ouverture, (c) former un joint entre les échantillons membranaires et l'ouverture en présence d'une solution interne de phase de scellement à faible concentration ionique, (d) échanger cette solution interne par une solution interne à phase d'accès, (e) établir un accès électrique, via l'ouverture, au compartiment interne de l'échantillon membranaire, et (f) effectuer des enregistrements électrophysiologiques avant et après l'incubation de la cellule avec au moins un agent. Une différence entre (i) l'enregistrement électrophysiologique en présence de l'agent et (ii) l'enregistrement électrophysiologique en l'absence de l'agent indique un agent qui module la propriété électrique d'un échantillon membranaire.
PCT/SE2005/000239 2004-02-23 2005-02-21 Systeme et methode de mesures electrophysiologiques de fixation de tension planaire sur plusieurs cellules et leur utilisation dans l'identification d'agents modulant des canaux ioniques Ceased WO2005080971A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
SE0400421-4 2004-02-23
SE0400421A SE0400421D0 (sv) 2004-02-23 2004-02-23 Assay

Publications (1)

Publication Number Publication Date
WO2005080971A1 true WO2005080971A1 (fr) 2005-09-01

Family

ID=31989589

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/SE2005/000239 Ceased WO2005080971A1 (fr) 2004-02-23 2005-02-21 Systeme et methode de mesures electrophysiologiques de fixation de tension planaire sur plusieurs cellules et leur utilisation dans l'identification d'agents modulant des canaux ioniques

Country Status (2)

Country Link
SE (1) SE0400421D0 (fr)
WO (1) WO2005080971A1 (fr)

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6063260A (en) * 1994-10-28 2000-05-16 Neurosearch A/S Patch clamp apparatus and technique having high throughput and low fluid volume requirements
WO2001059447A1 (fr) * 2000-02-11 2001-08-16 Yale University Electrodes patch-clamp planes
US20020053915A1 (en) * 2000-07-07 2002-05-09 Weaver Charles David Electrophysiology configuration suitable for high throughput screening of compounds for drug discovery
US20020164777A1 (en) * 2001-02-09 2002-11-07 Kelly Andrew J.G. Devices and methods for high throughput patch clamp assays
WO2002103354A1 (fr) * 2001-06-20 2002-12-27 Sophion Bioscience A/S Appareil et procede pour determiner et/ou surveiller les proprietes electrophysiologiques des canaux ioniques
WO2003021230A2 (fr) * 2001-09-05 2003-03-13 Essen Instruments, Inc. Systeme de mesure electrophysiologique de grande capacite
US20030146091A1 (en) * 1997-12-17 2003-08-07 Horst Vogel Multiaperture sample positioning and analysis system
WO2003104455A1 (fr) * 2002-06-07 2003-12-18 Sophion Bioscience A/S Procedes de criblage

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6063260A (en) * 1994-10-28 2000-05-16 Neurosearch A/S Patch clamp apparatus and technique having high throughput and low fluid volume requirements
US20030146091A1 (en) * 1997-12-17 2003-08-07 Horst Vogel Multiaperture sample positioning and analysis system
WO2001059447A1 (fr) * 2000-02-11 2001-08-16 Yale University Electrodes patch-clamp planes
US20020053915A1 (en) * 2000-07-07 2002-05-09 Weaver Charles David Electrophysiology configuration suitable for high throughput screening of compounds for drug discovery
US20020164777A1 (en) * 2001-02-09 2002-11-07 Kelly Andrew J.G. Devices and methods for high throughput patch clamp assays
WO2002103354A1 (fr) * 2001-06-20 2002-12-27 Sophion Bioscience A/S Appareil et procede pour determiner et/ou surveiller les proprietes electrophysiologiques des canaux ioniques
WO2003021230A2 (fr) * 2001-09-05 2003-03-13 Essen Instruments, Inc. Systeme de mesure electrophysiologique de grande capacite
WO2003104455A1 (fr) * 2002-06-07 2003-12-18 Sophion Bioscience A/S Procedes de criblage

Also Published As

Publication number Publication date
SE0400421D0 (sv) 2004-02-23

Similar Documents

Publication Publication Date Title
CN108272782B (zh) 小分子在制备治疗Dravet综合征或全面性癫痫伴热性惊厥附加症的药物中的用途
Feske Calcium signalling in lymphocyte activation and disease
Uehara et al. Store-operated Ca2+ entry uncoupled with ryanodine receptor and junctional membrane complex in heart muscle cells
EP0584446B2 (fr) Protéine microsomale de transfert des triglycérides
US8101380B2 (en) Schizophrenia-related isoform of KCNH2 and development of antipsychotic drugs
US20110065661A1 (en) Pharmacoperones for correcting disease states involving protein misfolding
JP4594924B2 (ja) Ｔｒｐｍ５モジュレーターのスクリーニング方法
Abbott et al. Phosphorylation and protonation of neighboring MiRP2 sites: function and pathophysiology of MiRP2‐Kv3. 4 potassium channels in periodic paralysis
US20080311578A1 (en) System for screening eukaryotic membrane proteins
US20120190558A1 (en) Schizophrenia-related isoform of kcnh2 and development of antipsychotic drugs
ES2272309T3 (es) 15571, una nueva molecula de tipo gpcr de la familia de tipo secretina y sus usos.
Sakaguchi et al. Hydroxyzine, a first generation H1-receptor antagonist, inhibits human ether-a-go-go–related gene (HERG) current and causes syncope in a patient with the HERG mutation
CN102272152B (zh) 通过中电导钙激活钾通道调控细胞-细胞融合的组合物及方法
EP1538217A1 (fr) Procede de criblage d'un amplificateur de la teneur en insuline
EP4608514A1 (fr) Molécules imitant tasl et leurs applications
WO2005080971A1 (fr) Systeme et methode de mesures electrophysiologiques de fixation de tension planaire sur plusieurs cellules et leur utilisation dans l'identification d'agents modulant des canaux ioniques
WO1999025832A1 (fr) Proteine toso
JP2003501029A (ja) Ｓｎｏｒｆ３３受容体をコ−ドするｄｎａ
Thompson et al. Characterization of 5-HT3 receptor mutations identified in schizophrenic patients
WO2001023565A1 (fr) P-glycoproteines provenant de macaca fascicularis et utilisations correspondantes
EP1062337B1 (fr) Isoforme de canal calcique humain de type n et utilisations
Baldwin Membrane transport: a practical approach
CN115998736B (zh) Y-27632在特异性抑制幻觉作用中的应用
JP2004522441A (ja) 破骨細胞関連骨疾患を治療するための活性を目的として化合物を探索する方法
HK1039636A1 (zh) 髓磷脂碱性蛋白肽及其应用

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NA NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): BW GH GM KE LS MW MZ NA SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LT LU MC NL PL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase