Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1995-05-31
2001-05-08
Caputa, Anthony C. (Department: 1645)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S023500, C536S024100, C536S024300, C536S024310
Reexamination Certificate
active
06229000
ABSTRACT:
Calcium ions have a multitude of functions in every biological system. Cellular calcium homoestasis plays a particularly essential role in the physiology of nerve cells. The intracellular calcium concentration is approximately 0.1 &mgr;M, as oodosed to 1 mM outside the nerve cell. This strong concentration gradient (×10,000) is primarily regulated by voltage-operated calcium channels (VOCC) which can be blocked by certain calcium antagonists. During a cerebral ischaemia (cerebral apoplexy), the calcium homoeostasis in neurons of the affected infarct region is changed considerably. The voltage-dependent calcium channels are maintained in the open state by prolonged membrane depolarisation, which results in a massive influx of calcium ions. During this process, the intracellular calcium concentration rises by a factor of 1000. The high excess of calcium activates various calcium/calmodulin-dependent cellular enzyme systems such as, for example, kinases, proteases and phosoholioases, by binding with calmodulin. These enzyme systems together result in irreversible damage to nerve cells when activated over a prolonged period.
A therapeutic approach to the protection of nerves during cerebral ischaemia is the reversible blocking of the massive calcium influx into the nerve cell. The voltage-dependent neuronal calcium channels are thus a suitable pharmacological target. VOCCs exist in a variety of muscle cells (vascular muscles, heart muscles and skeletal muscles). neurons and secretory cells which have tissue-specific physiological properties.
Electrophysiological investigations (Tsien et al., 1988, Trends in Neurol. Sci. 11: 431-438) suggest at least three different types of VOCC (L-, N- and T-channels). The 1,4-dihydropyridines (DHPs) are potent blockers of the L-type calcium channels, which can be found in muscle cells and also in nerve cells. The rabbit skeletal muscle dihydropyridine receptor has been characterised biochemically and cloned (Tanabe et al., 1987, Nature 328: 313-318). The primary sequence of this &agr;1 subunit of the VOCC, derived from the cDNA data, is consistent with a 212 kD trans-membrane protein having give N-glycosylation sites and seven possible phosphorylation sites. The protein contains four trans-membrane domains which resemble each other, each of which has six presumably &agr;-helical membrane-penetrating segments (S1-S6). In each case the fourth trans-membrane segment (S4) of each domain contains a regular pattern of positive charges (Lys, Arg) which can form the voltage sensor of the calcium channel. The structure of this cloned &agr;1 subunit is consistent with an ion-conducting voltage-controlled unit of the DHP-sensitive calcium channel.
The cloned carp skeletal muscle DHP-R cDNA clone (Grabner et al., 1991, Proc. Natl. Aca. Sci. (USA), 88:727-731) was used as hybridisation probe to isolate related human calcium channel genes from neurons and to characterise them. This cloning strategy allowed the isolation and characterisation of a number of various homologous cDNA clones from human neuronal cDNA libraries and we have clear evidence that various calcium channel subtypes exist in human central nervous system. Further neuronal sub-types have novel receptor sites for which no ligands (agonists, antagonists) have been disclosed to date. The cloned calcium channel sub-types are to be expressed in transformed animal cells (for example cos cells, mouse L cells, CHO cells etc.) (Gluzman, 1981, Cell 23:175, and Chen. et al., 1987, Mol. Cell. Biol. 7:2745-2752) and employed in binding assays and/or functional test assays for screening novel, sub-type-specific ligands. This involves the cloning of complete or partial cDNA genes of various calcium channel subtypes (including heart channels, vascular channels and skeletal muscle channels) into suitable eukaryotic expression vectors (Sambrook et al., 1989, in: Molecular Cloning, A laboratory manual, ed. Chris Nolan, Cold Sring Harbor Laboratory Press, New York, N.Y.). Protein expression is controlled either by homologous regulatory elements (promoters and enhancers) or heterologous promoters (viral, for example SV40, BPV, CMV, etc., or inducible, for example metallothionein, cAMP, calcium, temperature, etc.) in combination with known enhancers and RNA processing signals (for example capping, polyA).
It is furthermore intended to develop with these recombinant cell systems functional calcium flux assays, with the aid of which specific ligands can be tested for their agonistic or antagonistic effects. The distructiveness and the main advantage of these recombinant assays compared with conventional assays (cerebral membrane preparations, cell lines) lie in the purity of the receotor/channel preparation since it is exclusively the recombinant expressed neuronal calcium channel sub-type which is present on the surface of tranfected animal cells in any number desired. This is an essential prerequisite for the selection of specific neuronal calcium channel ligands that do not affect calcium channels of non-neuronal tissue types.
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Franz Jürgen
Rae Peter
Unterbeck Axel
Weingartner Bernhard
Bayer Aktiengesellschaft
Caputa Anthony C.
Gucker Stephen
Sprung Kramer Schaefer & Briscoe
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