Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1995-10-11
2002-06-04
Pak, Michael (Department: 1646)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S320100, C435S325000, C530S350000, C536S023500
Reexamination Certificate
active
06399325
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to novel galanin receptor proteins and partial peptides thereof; novel DNAs containing a galanin receptor protein or partial peptide-encoding DNA; processes for producing said galanin receptor protein (or partial peptide); use of said receptor protein (or partial peptide) and said protein (or partial peptide)-encoding DNA; a method of measuring the physiological actions of galanin using a galanin receptor protein-expressing cell or the galanin receptor protein; a method of screening galanin receptor agonists/antagonists using the galanin receptor protein-expressing cell or galanin receptor protein; a kit for said screening; an agonist or antagonist obtained by said screening method; and a pharmaceutical composition containing said agonist or antagonist.
The present invention also relates to a novel mouse pancreatic &bgr; cell line MIN6-derived galanin receptor protein and a partial peptide thereof; a novel DNA coding for said mouse galanin receptor protein or its partial peptide; processes for producing said mouse galanin receptor protein or its partial peptide; use of said mouse galanin receptor protein and said protein or peptide-encoding DNA; a method of measuring the physiological actions of galanin using a mouse-derived cell line MIN6 or the mouse galanin receptor protein; and a method of screening a galanin receptor agonist/antagonist using said mouse-derived cell line MIN6 or the receptor protein.
The present invention also relates to a novel human galanin receptor protein; a partial peptide of the human galanin receptor protein; a novel DNA which codes for the galanin receptor protein or partial peptide; a vector carrying said DNA; a transformant harboring said vector; a process for producing the human galanin receptor protein (or its partial peptide); a method of screening a galanin receptor agonist/antagonist using the human galanin receptor protein or a human galanin receptor protein-expressing cell (including the transformant); a kit for said screening; an agonist or an antagonist, obtained by said screening method; and a pharmaceutical composition containing said agonist or antagonist.
BACKGROUND OF THE INVENTION
A variety of hormones, neurotransmitters and the like control, regulate or adjust the functions of living bodies via specific receptors located in cell membranes. Many of these receptors mediate the transmission of intracellular signals via activation of a guanine nucleotide-binding protein (hereinafter, sometimes referred to as “G protein”) with which the receptor is coupled and possess the common (homologous) structure, i.e. seven transmembranes (membrane-spanning regions (domains)). Therefore, such a receptor is generically referred to as “G protein coupled receptor” or “seven transmembrane (membrane-spanning) receptor”.
G protein coupled receptor proteins which are widely distributed in the functional cellular surface of cells and organs in the living bodies have a very important role as targets for molecules such as hormones, neurotransmitters and physiologically active substances, which molecules control, regulate or adjust the functions of living bodies.
The pancreas plays an important role of carrying out the carbohydrate metabolism by secreting not only a digestive fluid but also glucagon and insulin. Insulin is secreted from the &bgr; cells and its secretion is promoted chiefly by glucose. It has been known that a variety of receptors exist in the &bgr; cells, and the secretion of insulin is controlled by various factors such as peptide hormones (galanin, somatostatin, gastric inhibitory polypeptide, glucagon, amylin, etc.), sugars (mannose, etc.), amino acids, and neurotransmitters in addition to glucose. As for the galanin and amylin, however, there has not yet been reported any discovery concerning the structure of their receptor protein cDNA. It is not known whether there exist any unknown receptor proteins or receptor protein subtypes.
It is a very important means in investigating development of new pharmaceuticals to clarify the relation between substances controlling the complicated functions of pancreas and specific receptors thereto. In order to develop new pharmaceuticals by conducting an effective screening of agonists and antagonists to the receptor proteins for controlling the functions of pancreas, it was necessary to investigate the function of receptor protein genes and also to express them in a suitable expression system.
By utilizing the fact that a G protein coupled receptor protein exhibits homology in part of the structure thereof at the amino acid sequence level, an experiment of looking at DNAs coding for novel receptor proteins relying upon a polymerase chain reaction (hereinafter simply referred to as “PCR”) has recently been made.
Galanin is a peptide existing in central and peripheral areas and, in central area, it shows an action of inhibition of liberation of neurotransmitter (acetylcholine) (European Journal of Pharmacology, vol.164, 355-360, 1989) and an action of antagonizing foreign acetylcholine (Proceedings of National Academy of Sciences, U.S.A., vol.85, 9841-9845, 1988) while, in pancreas, it shows a pharmacological action such as inhibition of insulin secretion (Diabates, vol. 34, 192-196, 1985). It has been also confirmed that galanin has an effect of inhibiting the behavior of learning (Neuroscience Letters, vol.88, 331-335, 1988) and of inhibiting the feeling of fullness after a meal. Such findings suggest a possibility that, if pharmaceuticals which inhibit the action of galanin are developed, they may be used as intelligence tropic agents and as remedies for obesity and for diabetes.
All of the pharmacological actions of galanin take place via a specific galanin receptor existing in target tissues. Accordingly, the simplest means for inhibiting the action of galanin is to develop pharmaceuticals which specifically inhibit the reaction of galanin with the receptor, i.e. galanin receptor antagonists. In the development of galanin receptor antagonists, it is usually necessary to conduct a receptor binding experiment. In the case of galanin, experiments on galanin receptor binding using membrane fractions of brain hippocampal formation (European Journal of Biochemistry, vol. 181, 269-276, 1989) and of stomach and duodenum (Peptides, vol. 11, 333-338, 1990) have been reported already.
It has been also reported that there is a specific galanin receptor in Rin-m-5F cells obtained from rat pancreas (Endocrinology, vol. 124, 2635-2641, 1989). According to the above-mentioned reports, it is already possible to conduct a galanin receptor binding experiment. However, the amount of the galanin receptor in those membrane fractions is as low as around 50 fmol/mg and, therefore, it was necessary to use a large amount of cell fractions for one measurement.
Galanin exhibits the above-mentioned pharmacological actions in living body and, if the actions can be easily measured in vitro, that will be meaningful for the process of developing the receptor antagonists. It has been reported already that the action of inhibiting the insulin secretion by galanin can be substituted with an in vitro measurement using Langerhans islet isolated from pancreas (European Journal of Pharmacology, Vol. 203, 111-114, 1991). However, Langerhans island is required to be isolated upon each experiment and, therefore, this method is not easily accomplished.
As easier means, several methods using pancreatic &bgr;-cell strains (Rin-m-5F cells) have reported. They are, for example, a method in which an effect of galanin receptor to a second messenger system (i.e. an activity of inhibiting the adenylate cyclase) is measured (European Journal of Biochemistry, Vol. 177, 147-152, 1988) and a method in which an activity of opening the potassium channel is measured (Proceedings of National Academy of Sciences, U.S.A., Vol. 85, 1312-1316, 1988). A method in which the activity of inhibiting the insulin secretion of galanin using said cell strain has been reported too. However, those methods are applicab
Fujii Ryo
Fukusumi Shoji
Hinuma Shuji
Hosoya Masaki
Ohgi Kazuhiro
Conlin David G.
Edwards & Angell LLP
Pak Michael
Takeda Chemical Industries Ltd.
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