EP0441889A1 - Modifiziertes menschliches wachstumshormon - Google Patents

Modifiziertes menschliches wachstumshormon

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Publication number
EP0441889A1
EP0441889A1 EP89913122A EP89913122A EP0441889A1 EP 0441889 A1 EP0441889 A1 EP 0441889A1 EP 89913122 A EP89913122 A EP 89913122A EP 89913122 A EP89913122 A EP 89913122A EP 0441889 A1 EP0441889 A1 EP 0441889A1
Authority
EP
European Patent Office
Prior art keywords
growth hormone
human growth
modified
hgh
treatment
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.)
Withdrawn
Application number
EP89913122A
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English (en)
French (fr)
Inventor
Joseph Martial
Corine Lecomte
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.)
Commission Des Communautes Europeennes
Original Assignee
Universite de Liege
Commission Des Communautes Europeennes
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 Universite de Liege, Commission Des Communautes Europeennes filed Critical Universite de Liege
Publication of EP0441889A1 publication Critical patent/EP0441889A1/de
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/61—Growth hormone [GH], i.e. somatotropin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates to a protein derived from human growth hormone (hereinafter abbreviated as hGH), which no longer has the first two amino acids at the amino terminus and which retains its main biological activity. stimulation of bone and somatic growth, but which no longer shows the transient and diabetogenic insulin effects generally observed during the administration of hGH.
  • hGH human growth hormone
  • growth hormone is a protein synthesized and secreted by the somatotropic cells of the anterior lobe of the pituitary gland.
  • the important role of this hormone in human growth is well known and in therapy it is capable of filling the pituitary deficit responsible for certain forms of dwarfism.
  • Human growth hormone is a polypeptide molecule of 191 amino acids and 22,000 daltons of molecular weight, having two disulfide bridges between residues 53 and 165 on the one hand, and 182 and 189 on the other hand ( Figure 1).
  • Human growth hormone is characterized by the multiplicity of its biological activities. Its major property is to stimulate bone and somatic growth by increasing chondrogenesis, protein synthesis and cell proliferation. These somatogenic effects are mediated by growth factors ( somatomedins or IGF factors "Insulin-li e Growth Factors "), secreted under the influence of hGH mainly in the liver.
  • growth hormone exerts a wide range of metabolic effects which we can summarize as follows: the" diabetogenic "effect, anti-insulin acting on the carbohydrate metabolism, corresponds to a reduction in the assimilation of glucose, associated with a decrease in insulin sensitivity.
  • hGH in a subject with insufficient pituitary or growth hormone deficiency, the administration of human hGH exerts a transient insulin-like effect resulting in transient hypoglycemia (Merimee, 1972; Goodman, 1981). Like insulin, hGH is able in this case to increase the entry and use of glucose in the muscles (Park et al., 1952) and in fat cells (Goodman, 1967), as well as it shows a significant anti-lipolytic effect (Birnbaum and Goodman, 1976). All of these effects only last a few hours, after which the tissues become refractory to this insulin effect of growth hormone and, on the other hand, become sensitive to its anti-insulin effect described above.
  • Figure 2 schematically summarizes the modes of action of hGH as well as the overall system for regulating its secretion from the antehypophysis.
  • hGH hGH
  • authentic recombinant hormone which is identical to the natural hormone isolated from pituitary extracts and et-hGH
  • a recombinant hormone possessing an additional methionine at its amino terminal end a recombinant hormone possessing an additional methionine at its amino terminal end.
  • These two recombinant proteins have properties identical to those of the natural hormone, indicating that the lipolytic and diabetogenic activity, and the transient insulin effect, are intrinsic properties of human growth hormone.
  • the development of growth hormones devoid of diabetogenic activity would have very important therapeutic interests for the treatment of certain clinical cases for which the metabolic effects of hGH on tolerance to carbohydrates are to be avoided: cachexic patients, newborns, people elderly.
  • a modification of the primary structure of hGH in particular by genetic means, results in a modified hGH reflecting different biological activities and improved properties compared to the natural hormone or to the recombinant hormones.
  • the subject of the invention is a modified human growth hormone demonstrating a characterized somatid growth stimulation activity.
  • n being greater than or equal to 2 and not going beyond that which causes a modification of the somatid growth stimulating activity compared to natural human growth hormone
  • n is different from 13.
  • the growth hormone is characterized in that the number n of amino acids deleted does not go beyond this which causes a modification of the natural three-dimensional configuration relative to the natural human growth hormone.
  • the subject of the invention is a modified growth hormone characterized in that it comprises a deletion of the first n N-terminal amino acids of human growth hormone, n taking any of the values 2 to 24, and being different from 13.
  • - human growth hormone is characterized in that n takes any one of the values from 2 to 15, for example 15.
  • the human growth hormone is characterized in that n takes any one of the values from 2 to 14, for example 14. According to another preferred embodiment of the invention, the human growth hormone is characterized in that n takes any one of the values from 2 to 12, for example 12.
  • the human growth hormone is characterized in that n takes any one of the values from 2 to 9, for example 9.
  • the human growth hormone is characterized in that n takes any one of the values from 2 to 7, for example 7.
  • the human growth hormone is characterized in that n takes any one of the values from 2 to 5, for example 5.
  • the human growth hormone is characterized in that n is equal to 2.
  • the elimination of the first two amino acids from natural hGH was carried out by genetic engineering by planing the first two codons of the hGH gene. This results in a modified hGH protein retaining all of its growth-stimulating activity but devoid of side effects on the metabolism of carbohydrates.
  • the invention also relates to the recombinant DNAs coding for the modified growth hormone of the invention defined above.
  • the invention also relates to the process for producing a modified hGH protein according to the invention. It includes in particular: the culture of a competent cellular host, previously transformed with a nucleic acid containing a nucleotide sequence coding for the modified hGH protein itself placed under the control of regulatory elements, including in particular a promoter recognized by the polymerases of this competent cellular host, the elements of initiation and termination of translation, and the recovery of the modified protein produced from expression products of this cellular host.
  • nucleic acids described above preceded by a nucleotide sequence comprising the regulatory elements including the promoter under whose control transcription of the modified hGH gene sequence and the initiating elements are carried out and translation terminators.
  • the invention also relates to the recombinant vectors of this type, cosmids, plasmids, phages modified by a nucleic acid coding for the hGH modified in one of their sites which are not essential for their replication.
  • the invention relates to vectors modified by an insert consisting of one of the recombinant DNAs defined above, under the control of a promoter and of appropriate regulatory elements allowing the expression of this recombinant DNA in a host. competent cell transformed by said recombinant vector.
  • the above recombinant vector is an expression vector containing, at one of its sites not essential for its replication, elements necessary to promote expression, in a host cell, of a nucleic acid encoding the modified hGH of the invention, a promoter compatible with said cell host, in particular an inducible promoter.
  • the invention also relates to cellular hosts transformed by a recombinant vector as described above, a vector capable of replicating in said microorganism and comprising the regulatory elements allowing the expression of the nucleic sequence coding for the modified protein of the invention in this host.
  • a first preferred cellular host consists of E. coli transformed with a recombinant vector as will be described in the examples which follow.
  • the invention relates more particularly to cell cultures transformed under the above-mentioned conditions and capable of synthesizing the modified hGH according to the invention.
  • the invention relates to pharmaceutical compositions containing as active ingredient the modified human growth hormone of the invention.
  • compositions of the invention are suitable:
  • the invention also relates to the use of the growth hormone of the invention, in the preparation of medicaments intended for the treatment of pituitary deficiency or growth hormone deficiencies, in the treatment of growth retardation or growth retardation, in the treatment obesity, treatment of scars after accident or operation, treatment of the phenomena of aging, without showing a diabetogenic effect and / or an insulin effect, especially in cachexic patients, the elderly or newborns.
  • the invention also relates to the use of growth hormone comprising a deletion of the first 13 N-terminal amino acids of natural human growth hormone, for the preparation of medicaments intended for the treatment of pituitary deficiency or hormone deficiencies of growth, to the treatment of stunted or insufficient growth, to the treatment of obesity, to the treatment of scars after, accident or operation, to the treatment of the phenomena of aging, without presenting a diabetogenic effect and / or insulin effect , in cachexic patients, the elderly or newborns.
  • the invention also relates to a method for improving the physical appearance of a human being having scars, comprising the administration of the growth hormone of the invention, in an amount suitable for regenerating the tissues and the renewal of the dose. administered until attenuation or disappearance of scars, improving the aesthetics of the subject.
  • the invention also relates to the application as a cosmetic product of the growth hormone of the invention.
  • the invention also relates to the cosmetic compositions containing the growth hormone of the invention.
  • the cosmetic application of the products of the invention results from the tissue regeneration properties of the modified growth hormone of the invention, whether in the reduction or disappearance of scars or the fight against the effects of aging, such as than wrinkles.
  • hGH human growth hormone
  • the cDNA library was screened using probes obtained from the plasmid pchGH ⁇ OO containing the hGH gene (Martial et al., 1979). The cDNAs of the positive clones were then cloned into the plasmid pBR322, and the nucleotide sequence of the cDNAs was determined.
  • the expression system that was used to express the modified hGH gene is a prokaryotic system, in two cistrons: the first cistron consists of the cDNA coding for human Cu / Zn superoxide dismutase (hSOD), the second by the cDNA -hGH.
  • the two genes are dependent on a single promoter, in this case the tac promoter.
  • Figure 4a shows the pSOD vector used. This plasmid is derived from the vector ptac ⁇ in which was cloned the cDNA coding for human Cu / Zn superoxide dismutase or hSOD (Hallewell et al., 1985).
  • the modified hGH gene was constructed from an Xba I - Sal I fragment of the hGH cDNA isolated from the plasmid pDMIOO-hGH. This cDNA fragment is 580 bp long and its 5 'end corresponds to the second nuleotide of the codon of amino acid 7 of hGH. It is represented in part b of FIG. 5.
  • the modified hGH gene was constructed by adding to this fragment, on the 5 ′ side, a synthetic oligonucleotide represented in part a of FIG.
  • the sequence of which comprises: a 5 'end compatible with an Nco I end; the AGGA sequence from Shine-Dalgarno; a TAA translation termination codon determining the end of the first cistron; a second messenger translation initiation ATG codon; codons for amino acids 3, 4, 5 and 6 of hGH; a 3 'end compatible with an Xba I end. Consequently, the resulting modified hGH gene has a deletion of the first two codons of the natural hGH gene, and an additional methionine initiator codon at the end
  • the total proteins expressed in the transformed bacteria were analyzed by electrophoresis on polyacrylamide gel in the presence of SDS after induction of the tac promoter.
  • the appearance of two additional proteins is observed in comparison with an uninduced sample: hSOD and a second protein having the size of that expected for the modified hGH.
  • the second step in the construction of the modified hGH expression vector consisted in deleting the internal region of the hSOD gene while keeping its 5 'and 3' ends intact, in order not to affect the level of expression of hGH modified.
  • 2 unique or infrequent restriction sites making it possible to delete a part of this cDNA, without modifying the reading phase: the Stu I site at the level of codon 41 of cDNA-hSOD, and the Nco I site at codon 153 of cDNA-hSOD.
  • the plasmid was circularized by the action of DNA ligase and transformed into bacteria E. coli D1210 ( Figure 7).
  • the plasmids present in the clones of transformed bacteria and among 12 were analyzed. clones taken at random, 10 contained the deleted plasmid, called pSGHD.
  • the proteins expressed in the clones containing the plasmid pSGHD by electrophoresis of the total proteins expressed in these clones were analyzed. This electrophoresis test clearly shows that the bacteria which possess the plasmid pSGHD produce only one of the two proteins previously induced: the one whose size corresponds to the expected size for the modified human growth hormone (FIG. 8a). The rate was estimated expression of the hormone to 8% of total bacterial proteins ⁇ s. This high expression yield will allow a simplified procedure to be used to purify the expressed protein.
  • the partial purification of the modified hGH protein expressed by the plasmid pSGHD was carried out in two stages: the isolation and the solubilization of the inclusion bodies on the one hand, and their purification by HPLC chromatography on the other hand.
  • the modified hGH protein produced after induction with IPTG is not soluble, but is present in precipitated form in inclusion bodies.
  • These inclusion bodies consist essentially of growth hormone (Figure 8b), the solubilization of which was carried out by incubating them at room temperature in an 8 M urea solution dissolved in a 50 mM phosphate buffer, pH 7.0. The solution is then dialyzed against a large volume of 50 mM NH4HC03, then lyophilized.
  • the second purification step is HPLC chromatography on anion exchange resin, DEAE. This chromatography was carried out according to the conditions established for hGH of extractive origin. As the results shown in Figure 9 show, this procedure provides sufficiently purified modified hGH protein fractions. Characterization of the modified hGH:
  • radioimmunoassays were performed in the presence of a polyclonal or monoclonal antibody specific for pituitary hGH 22K, (2) experiments of binding to preparations of hepatic receptors isolated from the liver of pregnant rabbits and (3) test of somatogenic activity ÎJ vivo.
  • Radioimmunoassays three series of RIA assays were carried out, differing by the nature of the antibody used: a rabbit serum directed against the natural hormone, a monoclonal antibody directed against its NH2-terminal end, and a second monoclonal antibody directed against an internal region of the hormone.
  • the assays were carried out on the basis of the competition between the modified hGH protein and the labeled pituitary growth hormone ( 125 I-hGH).
  • modified hGH hormones necessary to shift the binding of the tracer to the antibody by 50% (ED50) were compared to the dose of pituitary hormone causing the same displacement. So we have. evaluated, for each antibody, the competition capacities of the modified hGH protein vis-à-vis the 125 I-hGH tracer, expressed as a percentage of the competition capacity of the pituitary hormone.
  • RIA assays show that the purified modified hGH is a good competitor of the pituitary tracer for the antiserum directed against the hormone.
  • pituitary and especially for the monoclonal directed against an internal region of this hormone: in the latter case, the modified hGH protein has in fact a capacity for competition vis-à-vis the tracer equivalent to 80% of that of the hGH standard.
  • the modified hGH does not compete at all with the tracer for the NH2-terminal monoclonal antibody. This result is not surprising since the modified hGH protein is deleted from amino acids 1 and 2 of the natural hormone.
  • Binding to hepatic receptors it is well established that growth hormone acts mainly in the liver, where it stimulates the synthesis of somatomedins and is involved in the regulation of metabolic systems such as gluconeogenesis and the metabolism of fatty acids.
  • the liver is therefore an organ rich in specific receptors for growth hormone, and in particular the liver of a pregnant rabbit (Posner et al., 1974). Consequently, the hepatic receptors used for the binding experiments are prepared from the fraction enriched in plasma and microsomal membranes of the liver of the pregnant rabbit.
  • the dosing by radio receivers of a given hormone consists in incubating a small fixed quantity of marked hormone (tracer) and increasing concentrations of cold hormone to be assayed.
  • hGH has a relative capacity of 125% compared to that of the international standard hGH to displace by 50% (ED50) the initial binding of the tracer 125 I-hGH to hepatic receptors.
  • the modified hGH therefore shows a capacity displacement greater than that of the standard pituitary hormone.
  • Somatogenic activity in vivo to verify that the modified hGH protein which normally attaches to somatogenic hormone receptors in the liver, retains its main biological activity of somatogenic stimulation, a test was used: (1) the tibia test (Greenspan et al., 1974).
  • modified hGH protein not only has a three-dimensional conformation very similar to that of the standard pituitary hGH hormone, but still retains its main biological activity to stimulate somatogenic growth at 100%. Insulin activity of modified hGH:
  • the transient insulin activity of human growth hormone can be measured in vitro by a lipogenesis stimulation test, carried out on rat adipocytes originating from hGH deficient tissues.
  • the presence of endogenous growth hormone in fact induces a resistance of adipose tissue to the insulin effects of hGH.
  • the insulin activity test of the recombinant modified hGH is based on the measurement of the incorporation of tritiated flucose into the lipids, as a function of the addition of increasing doses of hGH, ⁇ and is based on the test, described ( Moody et al., 1975) for the stimulation of lipogenesis by insulin. Insulin activity was measured as follows:
  • adipocytes Preparation of adipocytes: the rats are sacrificed by dislocation of the cervical vertebrae and the adipose tissue from the kidneys and epididymis removed. This tissue, cut into small pieces is immediately digested with collagenase (1 mg / ml) for 30 minutes at 37 ° C. with vigorous stirring, in the KRH pH 7.4 buffer containing 35 mg / ml dialyzed BSA, and 0.27 mM of glucose, using 3 ml of KRH buffer per rat. After filtration on gas, the cell suspension is centrifuged at 600 g for 30 seconds and the adipocytes, of lower density than the buffer, rise to the surface. The infranate is aspirated with a syringe with a long needle and the cells are resuspended in the fresh buffer. The cells are washed 5 times in KRH buffer pH 7.4 at 37 C containing 1% BSA.
  • Lipogenesis stimulation the protocol which was used to measure insulin activity is adapted from the lipogenesis stimulation test with insulin, de Moody et al., (1975).
  • the rat adipocytes are first reincubated for 4 hours at 37 ° C. in KRH buffer with a BSA concentration of 10 mg / ml, then washed and resuspended at a concentration of 0.8 x 10 5 cells / ml.
  • the test is carried out in triplicate in 10 ml polyethylene vials to which are successively added: - 400 ⁇ l of the adipocyte suspension, - 50 ⁇ l of KRH buffer containing increasing doses of hGH (0.1 to 10,000 ng / l) or insulin (0.05 to 100 ng / ml), - 50 ⁇ l of tritiated glucose (D- [3- 3 H] glucose) containing 50,000 to 100,000 dpm, in a total volume of 0.5 ml.
  • the final concentration of adipocytes is 1% (V / V) for the experiments on adipocytes from normal rats and 2% (V / V) for adipocytes from hypox rats.
  • modified hGH does not induce an increase in basic lipogenesis, even at concentrations up to 10 ⁇ g / l. In other words, the modified hGH does not reveal any transient insulin activity, although we are in an endogenous hGH deficiency system.
  • the test that has been used is to measure insulin resistance in dogs treated with the modified hGH hormone as described by ADER et al., 1987.
  • the diabetogenic activity of the modified hGH was also tested on rat adipocytes.
  • the stimulation of glucose transport by insulin in the presence or absence of the modified hGH was measured and compared with that of natural hGH.
  • Modified hGH unlike natural hGH does not inhibit action insulin in this test, which further shows that the modified hGH has no diabetogenic activity.
  • hGH Human growth hormone
  • SOD DNA coding for human superoxide dismutase.
  • cDNA-hSOD sequences belonging to the human superoxide dismutase gene.
  • hGHM DNA coding for the modified hGH protein.
  • cDNA-hSOP DNA coding for human superoxide dismutase.
  • hGHM DNA coding for the modified hGH protein.
  • cDNA-hSOD DNA coding for human superoxide dismutase.
  • the tac promoter a functional hybrid derived from the trp and lac promoters. Proc. Natl. Acad. Sci. 80, 21-25.
  • Hu ang growth hormone co plementary DNA cloning and expression in bacteria. Science 205, 602-607.

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  • Health & Medical Sciences (AREA)
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EP89913122A 1988-11-07 1989-11-07 Modifiziertes menschliches wachstumshormon Withdrawn EP0441889A1 (de)

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FR8814514 1988-11-07
FR8814514 1988-11-07

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EP0441889A1 true EP0441889A1 (de) 1991-08-21

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US5688666A (en) * 1988-10-28 1997-11-18 Genentech, Inc. Growth hormone variants with altered binding properties
US5534617A (en) * 1988-10-28 1996-07-09 Genentech, Inc. Human growth hormone variants having greater affinity for human growth hormone receptor at site 1
US6429186B1 (en) 1991-05-10 2002-08-06 Genentech, Inc. Ligand antagonists for treatment of breast cancer
JP3417558B2 (ja) 1991-05-10 2003-06-16 ジェネンテク，インコーポレイテッド 配位子作用薬および拮抗薬の選択
WO1994022469A1 (en) * 1993-04-02 1994-10-13 Ohio University Enhancement of learning and/or memory by a human somatotropin
AU693478B2 (en) * 1994-11-10 1998-07-02 Metabolic Pharmaceuticals Limited Treatment of obesity
DK0851925T3 (da) * 1995-09-21 2005-11-28 Genentech Inc Humane Væksthormonvarianter
SE0000837D0 (sv) 2000-03-13 2000-03-13 Pharmacia & Upjohn Ab New use
EP1290170A2 (de) 2000-06-16 2003-03-12 Asterion Limited Bindungsreagenzien: chimäre ligand/rezeptorproteine
AR036400A1 (es) 2001-08-30 2004-09-08 Stem Cell Therapeutics Inc Regulacion combinada de la produccion de celulas nerviosas.
ATE541921T1 (de) 2001-09-14 2012-02-15 Stem Cell Therapeutics Inc Prolaktin-induzierte zunahme an neuronalen stammzellen und dessen therapeutische anwendung
CA2492442A1 (en) 2002-07-31 2004-02-05 Stem Cell Therapeutics Inc. Method of enhancing neural stem cell proliferation, differentiation, and survival using pituitary adenylate cyclase activating polypeptide (pacap)
JP2006500927A (ja) * 2002-09-13 2006-01-12 ザ・ユニバーシティ・オブ・クイーンズランド コドン翻訳効率に基づく遺伝子発現系
AU2003283004A1 (en) 2002-10-22 2004-05-13 Waratah Pharmaceuticals, Inc. Treatment of diabetes
CA2556266A1 (en) 2004-02-13 2005-08-25 Stem Cell Therapeutics Corp. Use of luteinizing hormone (lh), and chorionic gonadotropin (hcg) for proliferation of neural stem cells and neurogenesis
WO2007036033A1 (en) 2005-09-27 2007-04-05 Stem Cell Therapeutics Corp. Oligodendrocyte precursor cell proliferation regulated by prolactin
WO2007106987A1 (en) 2006-03-17 2007-09-27 Stem Cell Therapeutics Corp. Continuous dosing regimens for neural stem cell proliferating agents and neural stem cell differentiating agents
CN113004388A (zh) * 2019-12-20 2021-06-22 三生国健药业（上海）股份有限公司 一种重组人神经生长因子的制备方法

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EP0103395A3 (de) * 1982-08-17 1985-05-22 Biogen N.V. DNS-Sequenzen, rekombinante DNS-Moleküle und Verfahren zur Herstellung von rinderwachstumshormonähnlichen Polypeptiden, mit grosser Ausbeute
CA1341012C (en) * 1982-09-27 2000-06-06 Biogen, Inc. Dna sequences, recombinant dna molecules and processes for producing swine growth hormone-like polypeptides
BG49718A3 (bg) * 1983-07-15 1992-01-15 Bio- Technology General Corp Метод за получаване на полипептид със супероксиддисмутазна активност
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PT83613B (en) * 1985-10-28 1988-11-21 Lilly Co Eli Process for the selective chemical removal of a protein amino-terminal residue

Non-Patent Citations (1)

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JPH04503154A (ja) 1992-06-11

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