• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
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  • 引用文献
  • 法律状态
  • 优先权
    • 专利摘要:
    Rat hypothalamic GRF has been synthesized and has the formula: H-His-Ala-Asp-Ala-Ile-Phe-Thr-Ser-Ser-Tyr-Arg-Arg-Ile-Leu-Gly-Gln-Leu- Tyr-Ala-Arg-Lys-Leu-Leu-His-Glu-Ile-Met-Asn-Arg-Gln-Gln-Gly-Glu-Arg-As n-Gln-Glu-Gln-Arg-Ser-Arg-Phe-Asn-OH. The peptide is believed to be and is hereinafter referred to as rhGRF (for rat hypothalamic GH releasing factor). The invention provides synthetic peptides which are extremely potent in stimulating the release of pituitary GH in animals and which have the formula: H-R1-Ala-Asp-Ala-Ile-Phe-Thr-R8-Ser-R10-Arg-R12-R13-Leu-R15-Gln-Leu-R1 8-Ala-Arg-Lys-Leu-Leu-R24-R25-Ile-R27-R28-Arg-Gln-Gln-Gly-Glu-R34-Asn- Gln-Glu-R38-R39-R40-Arg-R42-R43-R44-Y wherein R1 is Tyr, Met, D-Tyr, Leu, D-His or His; R8 is Ser or Asn; R10 is Tyr or D-Tyr; R12 is Arg or Lys; R13 is Ile or Val; R15 is Gly or D-Ala; R18 is Tyr or Ser; R24 is His or Gln; R25 is Glu or Asp; R27 is Met, Ala, Nle, Ile, Leu or Val; R28 is Asn or Ser; R34 is Arg or Ser; R38 is Gln or Arg; R39 is Arg or Gly; R40 is Ser or Ala; R42 is Phe or Ala; R43 is Asn or Arg; R44 is a natural amino acid or des-R44; and Y signifies the carboxyl moiety of the amino acid residue at the C-terminus and is the radical -COOR,-CRO,-CONHNHR,-CON(R)(R') or -CH2OR, with R and R' being lower alkyl, fluoro lower alkyl or hydrogen. These peptides or biologically active fragments thereof, or analogs thereof having well-known substitutions and/or additions, as well as nontoxic salts of any of the foregoing, may be administered to animals, including humans, to stimulate the release of GH and may be used diagnostically.
    公开号:SU1477248A3
    申请号:SU843692330
    申请日:1984-01-12
    公开日:1989-04-30
    发明作者:Эдуард Фредерик Ривьер Жан；Васкер Вэйл Вили (Младший)；Шписс Иохим
    申请人:Дзе Салк Институт Фор Биолоджикал Стадиз (Фирма)；
    IPC主号:
    专利说明:

     I
    Isobre-nsh refers to the method of production and wholesale of idon - new biologically active compounds that can be used in medicine and veterinary medicine.
    The purpose of the invention is to obtain chemically by new peptides that promote the growth of the pituitary gland growth hormone, more active and toxic compounds.
    Example 1 Synthesis of rh GRF (1-4 having the formula H-His-Ala-Asp-Ala- -Ile-Phe-Thr-Ser-Ser-Tyr-Arg-Arg- -Ile-Leu-Gly-Glu-Leu-Tyr-Ala -Arg-
    -Lys-Leu-Leu-His-Glu-I eD-Met-Asn-Arg-Glu-Gln-Gly-Glu-Arg-Asn-Gln-Gl-Glu-Gln-Arg-Ser-Arg-Phe-Asn- OH, was staged using a Beckmann-990 model peptidator on a chloromethylated resin having an interval of -substitution of 0.1-0.5 mmol / g of resin. The coupling (Hap) with the resin is carried out according to the general procedure using KF in DMF at 60 ° C for 24 hours with stirring. This leads to the replacement of 0.35 mmol Asn per 1 g of resin.
    After deblocking and neutralization, the peptide chain is gradually increased on the resin. The release, neutralization and addition of each amino acid is carried out in general accordance with the procedure. All solvents used are thoroughly degassed by bleeding an inert gas, such as gels or nitrogen, to ensure that there is no oxygen that could oxidize the sulfur of Met residue undesirably.

    The release is preferably carried out in accordance with scheme A: Reagent
    1.60% TFA / 2%
    tandiol 2.60% TFA / 2%
    ethanedithiol Z. TRA / 1% (anditpol 4.Et, N (10%) in Z.MeON
    6.Eg3M (10%) to 7 .MePN (twice)
    8.CM, Cli в i to specify scheme B:
    (twice)
    Mixing time, min
    ten
    15
    0.5
    0.5
    0.5
    0.5
    0.5
    0.5
    re. tsyuch i ntelno provopi g
    R e p he n t
    Mixing time, min
    16.SN ,, (twice)
    0.5
    five
    50
    0
    five
    0
    five
    0
    1-2 mmol of BOC-protected amino acid in methylene chloride are used for 1 g of resin plus one equivalent of 1.0 molar DCC1 in methylene chloride for 2 hours. When binding BOC-Arg (TOS), a mixture of 50% DMF in methylene chloride is used . Bzl ester is used as a protective group for the hydroxyl side chain in the case of Ser and Thr. The amido group of the Asn or Gin residue is protected with Hap using DSS binding in the preferred embodiment, p-Nitrophenyl ester (ONp) can also be used to activate the carboxyl end of the Asn or Gin residue, for example, BOC-Asn (ONp) can be Overnight binding using one equivalent of HOBt in a 50% mixture of DMF and methylene chloride, in which case DCC is not added. The 2-chlorobenzyloxycarbonyl () is used as a protecting group for the side chain of Lys, Tos, as well as for protecting the guanidine group of the Arg residue, and the imidazole nitrogen of the His, Glu or Asp-carboxyl group is protected as Bzl ether (OBzl). The phenyl hydroxyl group of the residue. The protecting state is protected by 2,6-dichlorobenzene DCB. At the end of the synthesis, the following composition is obtained: (X) - -A1a-A8p (X3) -A1a-11e-Rpe-Tpg- (Hf) - -Ser (X4) -Ser (X4) -Tug (X2) -Arg (X6 ) - -Arg (X,) - Ile-Lue-Gly-Gln- (Xf) -Leu- -Tyr- (Xz) -Ala-Arg- (X6) -Lys (X7) -Leu- -Leu-His ( X) -Glu- (X3) (X5) -Gln (Xp- -Glu (X,) - Gln (Xy) -Arg (X6) -Ser (X ,,) - -Arg (X6) -Phe-Asn ( X) -x ",, where
    X0 - substrate silt OG.NOVR -f-CH cm Oly,
    Hap can be partially or completely removed by treatment with TFA used to unblock from the / -amino-protecting group.
    After the final residue is coupled to the resin, His BOC is removed with 60% TFA in CH2Cl2. To remove and remove protection from the remaining protected peptide resin, it is treated with I, 5 ml of anisole, 0.5 ml of methyl ethyl sulfide and 1 5 ml of hydrogen fluoride (HF) per 1 g of peptide-resin at -20 ° C for half an hour and at 0 ° C for half an hour. After removing HF under vacuum, the remaining peptide-resin is washed in turn with dry diethyl ether and chloroform, and then the peptide is extracted with degassed 2N. aqueous acetic acid and separated from the resin by filtration.
    lle-Met-Asn (X, r) -Arg (Xe) -GluCX, -) - -Gln (X) -Gly-Glu (X,) - Arg (X6) The cleaved and deprived of protection of the peptide is dissolved in 0-5 % acetic acid and purified, which may include fine gel filtration on a Sephadex G-50.
    Then the peptide is purified by preparative or semi-preparative high-performance liquid chromatography. The sleeves for Waters Associates LC-500 are filled with C1g silica, 15-20 µm, supplied by Vydas (300A). The acetonitrile (CH3CN) gradient in TEAR is created using an Eldex low pressure gradient apparatus. Chromatographic fractions are carefully controlled using highly efficient. liquid chromatography; and only fractions exhibiting sufficient purity are combined. The desalting of the purified fractions, independently tested for purity, is carried out using gradients in 0.1% trifluoroacetic acid. The central section is then diophilized to obtain the desired peptide, the purity of which is above 98%.
    Using thin layer chromatography and high performance liquid chromatography, it is determined that
    the free acid peptide is substantially pure. For all purifications using high performance liquid chromatography, a 5 micron Vydac C, s, analytical column was used. The peptide was removed from the column using a mixture of buffer A (0.1% aqueous trifluoroacetic acid) and
    JQ buffer B (60% acetonitrile in 0.1% aqueous trifluoroacetic acid). At 51% by volume of buffer B, the peptide is eluted at 8.9 ml. Optical rotation is measured on a photovoltaic field. It is 52.4% +1 (with 1, 1% acetic acid, not corrected).
    The synthesis is repeated using an MBHA resin to obtain rh GRF (1-43) 0 NH2, using the technique commonly used in the art to bind Asn to an MBHA resin. The peptide is determined to be substantially pure by thin layer chromatography and high performance liquid chromatography. Optical rotation is measured on a photoelectric field. It is G -52.2 +1 (C 1, 1% acetic acid
    n acid, not adjusted).
    Example 2. Synthesis of Nle Jrh GRF (l-32) -NH2, having the formula H-His-Ala-Asp-Ala-Ile-Phe-Thr-Ser-Ser-Tyr-Arg-Arg-Ile-Leu-Gly- Gln-Leu-Tyr- -Ala-Arg-Lys-Leu-Leu-His-Glu-Ile-Nle-Asn-Arg-Gln-Gln-Gly-NH4, is carried out in a stepwise manner using the peptide synthesizer Beckmann-990 on MBHA resin (as in Example 1). This analogue is determined to be substantially pure by thin layer chromatography and high performance liquid chromatography, eluted from a high performance liquid chromatograph at 8.4 ml with 55% buffer B. The optical rotation is -55.8 +1 (C 1, 1% acetic acid). Example 3. Synthesis of-1 10 0 -rh GRF (l-29) -NH2, having the formula
    HD-Tyr-Ala-Asn-Ala-Ile-Phe-Thr-Ser-Ser-D-Tyr-Arg-Arg-Ile-Leu-Gly-Gln--Leu-Tyr-Ala-Arg-Lys-Leu- Leu-His- -Glu-Ile-Met-Asn-Arg-NH2
    five
    0
    five
    is carried out in a stepwise manner using a Beckmann-990 peptide synthesizer in a MBHA resin (as in Example 1). It is determined that using a thin layer of 15
    20
    chromium i or raffia and high purity uio / in-line liquid chromine raffia with elution. from a column of gravy-effluent liquid chromium gtrfo at 9.0 ml with 61% buffer B, it is rolled out that the peptide is almost pure. The optical rotation is -63, b, and (C 1, 1% acetic acid).
    Example 4. Synthesis of Tug1 -rhGRF 10 (l-29-NHz, having the formula H-Tug-A1a-Asp-Ala-Ile-Phe-The-Ser-Ser-Tyr-Arg- - Arg-Ile-Leu- Gly-Gly-Leu-Tyr-Ala-Arg-Lys-Leu-Leu-Gln-Glu-Ile-Met-Asn-Arg-NH2 is carried out in a stepwise manner using the Beckman-990 peptide synthesizer (as in Example 1). It is determined that the peptide is substantially pure by thin layer chromatography and high performance liquid chromatography by elgoing from a high performance liquid chromatograph column at 7.8 ml with 58% buffer B. Optical rotation is -49.6 +1 (C 1, 1% acetic acid).
    Example b. Synthesis of rh (l-43) -OH of the formula R-His-Ala-Asp-Ala-lie-Phe-Thr-Ser-Ser-Tyr-Arg-Arg-Ile-Leu- -Gly-Gln-Leu-Tyr- Ala-Arg-Lys-Leu-Leu-His-Glu-Ile-Met-Asn-Arg-Gln-Gln-Gly-Glu-Arg-Asn-Glu-Glu-Gln-Arg-Ser-Arg-Phe- Asn-OH was carried out in a stepwise manner using a Beckman-990 peptide synthesizer on a chloromethylated resin having a substitution interval of 0.1-0.5 mmol / g resin, as described in Example 1.
    When using TLC and HPLC, the peptide was found to be substantially pure. Optical rotation is measured on a photoelectric field. It is -50.8 + 1 (C 1,
    -Ala-Tle-Phe-lhr-Ser-Ser-Tyr-Arg-Arg-Tle-Leu-GJy-Gln-Leu-Tyr-Ala-Arg-Lys-Leu-Leu-His-Glu-Ile-Nle - -Asn-Arg-Gln-Gln-Gly-NH.j, wire by the step method using the Beckmann-99 peptide synthesizer on MBHA-resin, as in Example 1. Using TLC and HPLC analog pn is essentially pure.
    Example 7. Synthesis of fragments analog hp GRF, i.e. D-Tyr1 ° J -rh GRF (l-29) -NH2. having the formula H-His-Ala-Asp-Ala-Ile-Phe-Thr-Ser-Ser-D-Tyr-Arg-Arg-Ile-Leu-Gly-Gl-Leu-Tyr-Ala-Arg-Lys-Leu- Leu-His- -Glu-He-Met-Asn-Arg-NH2, using a stepwise method using the Beckmann-99 peptide synthesizer on MBHA-resin (as in Example 1). Using TLC and HPLC, the peptide p nan is substantially pure.
    The synthesis is repeated with the substitution of D-Tyr instead of His at the N-end with the field 25 D-Tyr1 10 -rh GRF () -29) -NH2
    Example 8. Synthesis of Nle21-r GRF (l-32) -NHa, having the formula H-His-la-Asp-Ala-Ile-Phe-Thr-Ser-Ser-Tyr-Arg-Arg-Ile-Leu-Gly -Gln- Leu-Tyr-Ala-Arg-Lys-Leu-Leu-His-G-Il-Nle-Asn-Arg-Gln-Gln-Gly-NHt, conducted step by step using the Beckman-99 peptide synthesizer on the MBGA- resin (as in example 1)
    When using TLC and HPLC, the product was found to be substantially pure. Optical rotation was measured on a photo electric field. It is G ° 0vg -52.6 ° + 1 (C 1 1% acetic acid, not corrected)
    Example 9. Synthesis of rh GRF-β-NHZ having the formula H —His-Ala-Asp-Ala-Ile-Phe-Thr-Ser30
    35
    40
    1% acetic acid, not skorrek-45 Ser-Tyr-Arg-Ile-Leu-Gly-Gln-Letiirovano)
    The synthesis is repeated using MBGA resin to obtain rh GRF) -. In this case, Asn is used as the starting amino acid. When using 1CX and HPLC, the peptide is recognized as substantially pure. Optical rotation was measured on the tricar field, it is ED -51.7 ° + 1 (C 1, 1% acetic acid, corrected).
    Example b. (ipgee hp GRF - analogue, i.e. Nle17 rh GRF (I-32) - -NH2, having the formula H-His-Ala-Achr50
    -Tyr-Ala-Arg-Lys-Leu-Leu-His-Glu-I-Met-Asri-Arg-Gln-Gln-Gly-Glu-Arg-Asn-Gln-Glu-Gln-Arg-Ser-Arg- Phe-Asn-Val-NH2 was performed stepwise using a Beckmann-990 peptide synthesizer on an MBHA resin (as in Example I). When using TLC and HPLC, the peptide is recognized to be substantially pure. The rotation of the plane 55 of the light polarization is -50.8 (CI, 1% acetic acid lot), separation using high-resolution liquid chromatography was carried out with the same
    five
    0
    -Ala-Tle-Phe-lhr-Ser-Ser-Tyr-Arg-Arg-Tle-Leu-GJy-Gln-Leu-Tyr-Ala-Arg-Lys-Leu-Leu-His-Glu-Ile-Nle - -Asn-Arg-Gln-Gln-Gly-NH.j, is carried out in a stepwise manner using a Beckmann-990 peptide synthesizer on MBHA resin, as in Example 1. When using TLC and HPLC, the analogue was found to be substantially pure.
    Example 7. Synthesis of an analog fragment of hp GRF, i.e. D-Tyr1 ° J -rh GRF (l-29) -NH2. having the formula H-His-Ala-Asp-Ala-Ile-Phe-Thr-Ser-Se-D-Tyr-Arg-Arg-Ile-Leu-Gly-Gln-Le-Tyr-Ala-Arg-Lys- Leu-Leu-His- -Glu-He-Met-Asn-Arg-NH2 was performed in a stepwise manner using a Beckman-990 peptide synthesizer on an MBHA resin (as in Example 1). When using TLC and HPLC, the peptide was found to be substantially pure .
    The synthesis is repeated with the substitution of D-Tyr instead of His at the N-terminus to obtain 5 D-Tyr1 10 -rh GRF () -29) -NH2.
    Example 8. Synthesis of Nle21 -rh GRF (l-32) -NHa, having the formula H-His-la-Asp-Ala-Ile-Phe-Thr-Ser-Ser-Tyr-Arg-Arg-Ile-Leu-Gly -Gln- Leu-Tyr-Ala-Arg-Lys-Leu-Leu-His-Glu-Ile-Nle-Asn-Arg-Gln-Gln-Gly-NHt, carried out stepwise using the Beckman-990 peptide synthesizer at MBGA - resin (as in example 1)
    When using TLC and HPLC, the peptide was found to be substantially pure. Optical rotation was measured on a photoelectric field. It is G ° 0vg -52.6 ° + 1 (C 1, 1% acetic acid, not corrected)
    Example 9. Synthesis of rh GRF-β-NHZ having the formula H-β-His-Ala-Asp-Ala-Ile-Phe-Thr-Ser0
    five
    0
    Ser-Tyr-Arg-Ile-Leu-Gly-Gln-Leu
    -Tyr-Ala-Arg-Lys-Leu-Leu-His-Glu-Ile-Met-Asri-Arg-Gln-Gln-Gly-Glu-Arg-Asn-Gln-Glu-Gln-Arg-Ser-Arg -Phe-Asn-Val-NH2 was carried out in a stepwise manner using a Beckmann-990 peptide synthesizer on an MBHA resin (as in Example I). When using TLC and HPLC, the peptide is found to be substantially pure. The rotation of the plane of polarization of light is -50.8 (CI, 1% acetic acid), separation using high-resolution liquid chromatography was carried out under similar conditions outlined earlier, and using 567th buffer b, retention volume 7.2 ml.
    Biological testing
    Obtained in various examples are compared with purified synthetic hp GRF (1-40) -OH or purified synthetic hp GRF (1-40) Phe-Gln-NH during in vitro tests to determine their effectiveness in stimulating the release of growth hormone (GR).
    In direct comparison, equimolar concentrations of various synthesized analogs are used. Cultures are used which include pituitary cells of rats removed previously for 4-5 days. Cultures that are considered optimal for isolating growth hormones are used for comparative testing. Incubation with the substance to be tested is carried out for 3-4 hours, and aliquots of the culture medium are removed and processed to measure their composition by immunoreactive GR (IR-GR) with good resolution immunoassay.
    The results of comparative tests using equimolar concentrations are given in Table. one.
    An in vitro test of these synthetic peptides shows that the EC50 varies in the range of 20-100 pmol, and the lowest effective concentration is 3-8 pmol. The maximum effective concentration for phGRF (1-40) MNg is 1 nmol.
    The results of the comparative test for equimolar concentrations of different hp GRF analogs based on rh GRF are given in Table 2.
    In addition to the in vitro growth hormone secretion tests, in vivo experiments are also performed: the synth peptide is introduced through a constant catheter to free-moving normal male rats. Animals are pretreated with FLA-63, a dopamine hydroxylase inhibitor that suppresses spontaneous secretion of growth hormone, without affecting the response to exogenous GRF. Blood samples are taken through the same catheter immediately before injections and after 5 and 20 minutes after them, hormone levels are measured by radioimmunoassay.
    five
    0
    five
    0
    five
    0
    five
    0
    a hundred in the blood. The result shows that synthetic hp GRF analogs are potent stimulators of secretion of the growth hormone of the pituitary gland. A dosage of 20-25 mcg per kg body weight is considered effective. The compounds are classified as low toxic.
    权利要求:
    Claims (3)
    [1]
    1. Nle27 -rh GRF (l-32) -NH23.21 (2.02-5.43)
    [2]
    2. GRF (l-32) -NH22.15 (1.12-4.41)
    [3]
    3., Nle21 -rhGRF (l-32) -NHa 3.22 (2.12-5.33)
    table 2
    Comparative activity of compounds obtained under the conditions of the proposed method with the activity of hp GRF (1-40) -OH taken as I 00%
    Peptide
    Relative activity
    E
    Comparison,%
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    同族专利:
    公开号 | 公开日
    NZ207658A|1988-07-28|
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    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    US4732972A|1983-03-07|1988-03-22|Hoffmann-La Roche Inc.|Polypeptides having growth hormone releasing activity|
    US4689318A|1985-08-29|1987-08-25|The Salk Institute For Biological Studies|GRF analogs|
    US4880778A|1986-05-12|1989-11-14|Eastman Kodak Company|Combinations having synergistic growth hormone releasing activity and methods for use thereof|
    US5002931A|1987-05-22|1991-03-26|The Salk Institute For Biological Studies|GRF analogs VII|
    US4839344A|1987-06-12|1989-06-13|Eastman Kodak Company|Polypeptide compounds having growth hormone releasing activity|
    US4801456A|1987-07-09|1989-01-31|International Minerals & Chemical Corp.|Growth hormone-releasing factor analogs|
    USRE33699E|1987-07-09|1991-09-24|International Minerals & Chemical Corp.|Growth hormone-releasing factor analogs|
    US4880777A|1987-09-01|1989-11-14|Eastman Kodak Company|Synthetic peptides having growth hormone releasing activity|
    EP0400051B1|1988-01-28|1995-05-10|Polygen Holding Corporation|Polypeptide compounds having growth hormone releasing activity|
    US5043322A|1988-07-22|1991-08-27|The Salk Institute For Biological Studies|Cyclic GRF analogs|
    US5756458A|1989-06-16|1998-05-26|Pharmacia & Upjohn Company|Stabilized potent GRF analogs|
    AU656144B2|1990-06-29|1995-01-27|F. Hoffmann-La Roche Ag|Histidine substituted growth hormone releasing factor analogs|
    DE69108192T2|1990-12-10|1995-07-20|Hoffmann La Roche|Process for the enzymatic production of GRFNH2.|
    JPH05507939A|1991-04-09|1993-11-11|
    US5246920A|1992-06-15|1993-09-21|University Of South Florida|Treatment of hyperprolactinemia|
    US5811074A|1992-06-29|1998-09-22|University Of South Florida|Method of diagnosing pituitary dependent growth hormone deficiency|
    US7268113B2|2001-02-02|2007-09-11|Conjuchem Biotechnologies Inc.|Long lasting growth hormone releasing factor derivatives|
    US20060128615A1|2002-09-18|2006-06-15|Pierrette Gaudreau|Ghrh analogues|
    US20090088380A1|2007-07-12|2009-04-02|Pierrette Gaudreau|Ghrh analogs and therapeutic uses thereof|
    法律状态:
    优先权:
    申请号 | 申请日 | 专利标题
    US06/488,748|US4595676A|1983-04-26|1983-04-26|Rat hypothalamic GRF|
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