Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Patent
1993-07-15
1997-02-25
Sayala, Chhaya D.
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
530422, C07K 1447
Patent
active
056060203
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
This invention relates to a receptor which is specific for 6(R)-L-erythro-5,6,7,8-tetrahydrobiopterin which will hereinafter sometimes be referred to as 6R-BH4 while the one labeled with tritium at the 6-position will sometimes be referred to as [6-.sup.3 H]6R-BH4. More specifically, the invention relates to a 6R-BH4 receptor which is useful; as a tool in studying biochemical cellular reactions in response to 6R-BH4 binding to the cells; as a tool in searching for and developing agonists of the 6R-BH4 actions on the central nervous and immunological systems or antagonists capable of interrupting these actions when said search and development are conducted on the basis of the biochemical cellular reactions in response to 6R-BH4 binding to the cells and various other natures concerning the binding ability of 6R-BH4; and also as a diagnostic or therapeutic drug for diseases which are attributable to 6R-BH4.
BACKGROUND OF THE INVENTION
6R-BH4 is known to be a common coenzyme for tyrosine hydroxylase and tryptophan hydroxylase which are rate-limiting enzymes in the biosynthesis of neurotransmitters such as dopamine and serotonin. It is considered that 6R-BH4 is a regulating factor in the biosynthesis of these neurotransmitter amines since 6R-BH4 is contained in nerve endings only in an amount which is approximately the Km value of each hydroxylase. In fact, a shortage or decrease in enzymes which participate in the biosynthesis of this coenzyme from GTP will give rise to a decrease in neurotransmitter amines, thus resulting in various neuropsychiacric diseases. Actually, malignant phenylketonuria was discovered in 1974 and subsequent studies have revealed that autopsied brain specimens and cerebrospinal fluid of patients with Parkinson's disease and Alzheimer's dementia show a decrease In 6R-BH4 content. It has also been found that supplementation therapy by 6R-BH4 is effective in the treatment of malignant phenylketonurla, juvenile Parkinson's disease and, in accordance with recent studies, infantile autism of the dysbolism type. Based on these facts, over a hundred 6R-BH4 derivatives have been drug-designed in Switzerland, the United States and Japan and screened for their coenzyme action analogous to 6R-BH4. However, there has not been discovered any derivative which is superior to 6R-BH4 in terms of its action. In addition, no finding has been made hitherto relating to the biochemical cellular responses of 6R-BH4.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is an HPLC chromatogram of standard samples provided for identification of the labeled compound shown in FIG. 2;
FIG. 2 is an HPLC chromatogram carried out for confirming the radiochemical purity of the purified synthetic [6-.sup.3 H]6R-BH4, i.e., a specimen finally employed in the binding test;
FIGS. 3a and 3b are graphs showing the binding abilities of fractions prepared from various regions of rat brain and other tissues, wherein (a) shows the results of P2 fraction and (b) shows the results of cytosol fraction;
FIG. 4 is a graph showing the radioactivities which bound to the receptor when [6-.sup.3 H]6R-BH4 was added at various concentrations to a determined amount of the solubilized receptor;
FIG. 5 is a graph drawn by Scatchard-plot analysis of the data of the specific binding shown in FIG. 4;
FIG. 6 is a graph showing that the receptor of the present invention has a specific binding ability to 6R-BH4; and
FIG. 7 is a gel filtration chromatogram of the solubilized fraction containing the receptor of the present invention.
DISCLOSURE OF THE INVENTION
Our recent studies have resulted in a novel finding which demands a great change in the concepts relying on previous knowledge.
That is to say, it has been discovered that 6R-BH4 enhances the release and liberation of neurotransmitter amines such as dopamine (DA), norepinephrine and serotonin, and that it also enhances the liberation of glutamir acid, aspartic acid or .gamma.-aminobutyric acid (GABA) via DA and the release of acetylcholine via serotonin. On the other hand,
REFERENCES:
Chemical Abstracts, vol. 115, No. 19, Nov. 11, 1991; Bailey et al: "Role of C6 Chirality of tetrahydropterin cofactor in catalysis and regulation of tyrosine and phenylalanine hydroxylases", Abs. No. 201845x, 2nd pt., p. 443.
Chemical Abstracts, vol. 115, No. 19, Nov. 11, 1991; Ichikawa et al: :"Expression of mouse tyrosine hydroxylase in Escherichia coli", Abs. No. 2002245, 1st pt., p. 290.
Miwa et al., Nippon Yakurigaku Zasshi, vol 100(5), Nov. 1992, pp. 367-381. (Translated).
Miwa, Burein Saiensu Kenkyu Hokokushu, vol 5, pp. 83-93, 1992. (Translated).
Harris et al., "Protein Purification Methods", 1RL Press, 1989, pp. 59-64 and 87-97.
Koshimura et al., The 6R-L erythrotetrahydropbiopterin receptor for regulation of dopamine release studied by brain microdialysis, The Japanese Journal of Pharmacology, vol. 52, No. Sup. I, 1990, p. 76P.
Watanabe et al., Molecular mechanisms of tetrahydrobiopterin action on neurotransmitter release, Pteridines, vol. 3, No. 1-2, 1992, pp. 63-64.
Miwa et al., A novel function of tetrahydrobiopterin, Nippon Yakurigaku Zasshi, vol. 100, No. 5, Nov. 1992, pp. 367-381.
Soichi, Miwa "6R-L-Erythro-5,6,7,8-tetrahydrobiopterin receptor . . . ", Burein Saiensu Kenkyn Hokokushu, vol. 5, pp. 83-93, 1992; Ca 118(7):163975r.
Soichi, Miwa et al., "A novel function of tetrahydropioptein," Folia Pharmacological Japonica, vol 100(5), pp. 367-381 (1992), EMBASE An:93 010881.
Watanabe, Y. et al., "Tetrahydiobiopterin and dopamine release", Jpn J. Psychiatry Neurol., vol 45(2), pp. 513-514, 1991 Medline An: 92106606.
Hayashi Toshio
Morii Hiroshi
Watanabe Yasuyoshi
Watanabe Yumiko
Osaka Bioscience Institute
Sayala Chhaya D.
Suntory Limited
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