Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Having -c- – wherein x is chalcogen – bonded directly to...
Reexamination Certificate
2001-11-06
2004-04-27
Aulakh, Charanjit S. (Department: 1625)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Having -c-, wherein x is chalcogen, bonded directly to...
C546S084000, C514S290000
Reexamination Certificate
active
06727260
ABSTRACT:
TECHNICAL FIELD
This invention relates to the use of compounds capable of inhibiting the activity of a proton-gated cation channel in the treatment or alleviation of diseases or disorders associated with, or mediated by a drop in extracellular pH.
BACKGROUND ART
For almost 20 years, proton-gated cation channels have been known to exist in membranes of sensory neurons, where they are assumed to participate in nociception. Recently, several Acid Sensing Ion Channels (ASIC) have been cloned, and while some of these like ASIC3 (DRASIC) and ASIC-&bgr; are selectively expressed in sensory ganglia and/or the spinal cord, others, like ASIC1 and ASIC2, are expressed also in the brain [see e.g. Waldmann R, Champigny G, Bassilana F, Heurteaux C, Lazdunski M: A proton-gated cation channel involved in acid-sensing;
Nature
1997 386 (6621)173-177; Lingueglia E, De Weille J R, Bassilana F, Heurteaux C, Sakai H, Waldmann R, Lazdunski M: A modulatory subunit of acid sensing ion channels in brain and dorsal root ganglion cells;
J. Biol. Chem
. 1997 272 (47) 29778-29783; Bassilana F, Champigny G, Waldmann R, De Weille J R, Heurteaux C, Lazdunski M: The acid-sensitive ionic channel subunit ASIC and the mammalian degenerin MDEG form a heteromultimeric H+-gated Na+ channel with novel properties;
J. Biol. Chem
. 1997 272 (46) 28819-28822; Olson T H, Riedl M S, Vulchanova L, Ortiz-Gonzalez X R, Elde R: An acid sensing ion channel (ASIC) localizes to small primary afferent neurons in rats; NeuroReport 1998 9 (6) 1109-1113; Chen C -C, England S, Akopian A N, Wood J N: A sensory neuron-specific, proton-gated ion channel;
Proc. Natl. Acad. Sci. USA
1998 95 (17) 10240-10245; and Coscoy S, de Weille J R, Lingueglia E, Lazdunski M: The pre-transmembrane 1 domain of acid—sensing ion channels participates in the ion pore;
J. Biol. Chem
. 1997 274 (15) 10129-101321].
The proton-gated channels cloned all belong to the amiloride-sensitive Na-channel/degenerin family of ion channels, and like for ionotropic purinergic receptors, the topology presumably is two transmembrane domains flanking a long extracellular loop.
Compounds inhibiting the proton-gated cation channels have been suggested useful for the treatment of pain.
WO 98/35034 discloses mammal neuronal acid sensing cationic channels, which are considered useful for screening for analgesic drugs and for the treatment of degeneration of periferic neurons
Since the demonstration that the level of extracellular glutamate in the brain is greatly elevated under ischaemia, much pharmaceutical research has been focused on design of glutamate receptor antagonists. There has, however, been discrepancy between in vitro and in vivo studies: especially with respect to the effect of antagonists of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptors. In vitro, neuro degeneration due to oxygen/glucose deprivation is blocked by the NMDA receptor antagonist MK-801, but not by &agr;-amino-3-hydoxy-5-methyl4-isoxazolepropionic acid (AMPA) receptor antagonists. Strikingly, the case is opposite in in vivo models of global cerebral ischaemia: AMPA receptor antagonists reduces infarct volume, while NMDA receptor antagonists are ineffective.
Isatin derivatives like those described herein, for use as AMPA antagonists have been disclosed in e.g. WO 94/26747, WO 96/08494 and WO 96/08495. However, the use of these isatin derivatives as ASIC antagonising compounds have never been disclosed.
SUMMARY OF THE INVENTION
The present work shows the presence of ASIC in cultured mouse cortical neurons, a preparation widely used for studying native ion channels and in vitro models of neuro degeneration after ischaemia.
The presence of ASIC in central neurons is of particular interest because tissue acidosis is a well established feature of cerebral ischaemia. While the acidic pH in general has been regarded as neuro-protective due to proton inhibition of NMDA receptors, it may also have adverse effects by reason of activation of acid sensing ion channels contributing to membrane depolarisation, subsequent Ca
2+
accumulation and neuro-degeneration.
Therefore, in a first aspect, the invention relates to the use of a compound capable of inhibiting a mammalian proton-gated cation channel for the manufacture of a medicament for the treatment, prevention or alleviation of a disease, disorder or condition associated with, or mediated by a drop in extracellular pH.
In another aspect the invention provides a method for the treatment, prevention or alleviation of a disease, disorder or condition in a subject, including a human, which disease, disorder or condition is associated with, or mediated by a drop in extracellular pH, said method comprising administering to the subject a pharmaceutically effective amount of a compound capable of inhibiting a mammalian proton-gated cation channel.
Other objects of the invention will be apparent to the person skilled in the art from the following detailed description and the examples.
DETAILED DISCLOSURE OF THE INVENTION
ASIC Antagonising Compounds
In its first aspect the invention relates to the use of compounds capable of inhibiting a mammalian proton-gated cation channels for the manufacture of medicaments for the treatment, prevention or alleviation of a disease, disorder or condition associated with, or mediated by a drop in extracellular pH.
In the context of this invention compounds capable of inhibiting a mammalian proton-gated cation channel is also designated ASIC antagonising compounds.
Method of Screening
The ASIC antagonising compounds of the invention may be identified using the following screening method, which method comprises the subsequent steps of
(i) subjecting a proton-gated cation channel containing cell to the action of protons by adjustment of the pH to an acidic level;
(ii) subjecting a proton-gated cation channel containing cell to the action of the chemical compound; and
(iii) monitoring the change in membrane potential or the current induced by protons on the proton-gated cation channel containing cell.
In a preferred embodiment, the proton-gated cation channel is ASIC1 (also known as human brain sodium channel channel 2 (hBNaC2)), ASIC1A, ASIC1B, ASIC2 (also known as human brain sodium channel channel 1 (hBNaC1), or MDEG1), ASIC2A, ASIC2B, ASIC3 (also known as DRASIC), or ASIC-&bgr;.
The proton-gated cation channel may or may not be endogenous to the cell in question, i.e. be a channel naturally occurring in the cell.
Cells for use in the method of the invention, in which proton-gated cation channel are naturally present includes cortical neuronal cells, in particular mouse or rat cortical neuronal cells, and human embryonic kidney (HEK) cells, in particular HEK 293 cells.
Alternatively the proton-gated cation channel may be exogenous to the cell in question, and may in particular be introduced by recombinant DNA technology, such as transfection or infection. Such cells include Chinese hamster ovary (CHO) cells,
Xenopus laevis
oocytes, or any other cell lines capable to express proton-gated cation channels.
The proton-gated cation channel containing cells may be subjected to the action of protons by adjustment of the pH to an acidic level using any convenient acid or buffer, including organic acids such as formic acid, acetic acid, citric acid, ascorbic acid and lactic acid, and inorganic acids such as hydrochloric acid, hydrobromic acid and nitric acid, perchloric acid and phosphoric acid.
In the method of the invention, the current flux induced by protons over the membrane of the proton-gated cation channel containing cell may be monitored by patch clamp techniques.
Alternatively, the change in membrane potential induced by protons of the proton-gated cation channel containing cells may be monitored using fluorescence methods. When using fluorescence methods, the proton-gated cation channel containing cells are incubated with a membrane potential indicating agent, that allow for a determination of changes in the membrane potential of the cells, caused by the added protons. Such membrane pot
Varming Thomas
Wätjen Frank
Aulakh Charanjit S.
Birch & Stewart Kolasch & Birch, LLP
Neurosearch A/S
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