• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
  • 同族专利
  • 引用文献
  • 法律状态
  • 优先权
    • 专利摘要:
    Human pancreatic GRF(hpGRF), rat hypothalamic GRF(rGRF) and porcine hypothalamic GRF(pGRF) have been earlier characterized and synthesized. The invention provides synthetic peptides which are extremely potent in stimulating the release of pituitary GH in animals, including humans, which have resistance to enzymatic degradation in the body, and which contain the sequence: R1-R2-R3-Ala-Ile-Phe-Thr-R8-Ser-R10- Arg-R12-R13- Leu-R15- Gln-R17-R18- Ala-Arg-Lys-Leu- R23-R24-R25- Ile- R27- R28- Arg-Gln-Gln-Gly-Glu-R34-Asn-Gln-Glu-R38-R39-R40-Arg-R42-R43-R44 wherein R1 is a Tyr, D-Tyr, Met, Phe, D-Phe, pCl-Phe, Leu, His or D-His, which has either a C alpha Me or N alpha Me substitution or is unsubstituted; R2 is Ala or D-Ala; R3 is Asp or D-Asp; R8 is Ser, Asn, D-Ser or D-Asn; R10 is Tyr or D-Tyr; R12 is Arg or Lys; R13 is Ile or Val; R15 is Gly or D-Ala; R17 is Leu or D-Leu; R18 is Tyr or Ser; R23 is Leu or D-Leu; R24 is His or Gln; R25 is Glu, Asp, D-Glu or D-Asp; R27 is Met, D-Met, Ala, Nle, Ile, Leu, Nva 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, Ala or Val; R43 is Asn or Arg; R44 is a natural amino acid; provided however that either (a) R1 has a C alpha Me or an N alpha Me substitution or (b) R17 and/or R23 is D-Leu or (c) R25 is either D-Glu or D-Asp and provided also that any or all of the residues between R29 and R44, inclusive, may be deleted; or a nontoxic salt thereof. The C-terminus is preferably amidated. These peptides as well as nontoxic salts thereof may be administered to animals, including humans and cold-blooded animals, to stimulate the release of GH and may be used diagnostically.
    公开号:SU1435157A3
    申请号:SU843791983
    申请日:1984-09-12
    公开日:1988-10-30
    发明作者:Эдвард Фредерик Ривье Жан；Уолкер Вейл Вили
    申请人:Дзе Салк Институт Фор Биолоджикал Стадиз (Фирма)；
    IPC主号:
    专利说明:

    And: the invention relates to a method for producing peptides (biologically active compounds) that can be used in medicine.
    The purpose of the invention is the synthesis of new peptide derivatives - low-toxic, with a higher ability to promote the release of growth hormone.
    Example 1. Synthesis of peptide tD-Glu, Nle-rhGRF (1-29) -NH2, H-His-Ala-Asp-Ala-Ile-Phe-Thr-Ser-Ser-Tyr Arg-Ile-Leu-Gly- Gln-Leu-Tyr-Ala-Arg-Lys-Leu-Leu-His-DGlu-Ile-Nla-Asn-Arg-NH was performed mono-stage on a Beckmann 990 peptide synthesizer using 2 g of MBHA resin having a substitution range of 0.1 - 0.5 mmol / g resin. The connection of BOC-Atg (TOS) to the resin is carried out using KF in a mixture of 50% in methylene chloride at 60 ° C for 24 hours with stirring, thus replacing 0.35 mmol of Arg by 1 g of the resin, -After releasing and neutralizing a peptide chain on the resin at the stage. All solvents used are thoroughly degassed by sparging I1 of the gaseous gas, for example gels or nitrogen, in order to guarantee the absence of oxygen, which could lead to undesirable oxidation of the sulfur of the meth residue.
    The release is preferably carried out in accordance with the schedule of efficiencies A:
    Reagent
    one
    2
    60% TFA / 2% ethanedithiol 60% -. TFA / 2% ethanedithiol
    3.1RA / 1% ethanedithiol
    4.EUR (10%) in
    5. Meon
    6.Et3N (10%) in
    Mixing time, min
    ten
    15
    0.5 0.5 0.5 0.5 0.5 0.5
    LeOP (twice) 8. (twice) Attachments are preferably gp according to the schedule of operations B:
    Reagent Mixing time, min
    9. DCG1
    10, BOC-amino acid
    50-90
    0
    five
    0
    five
    0
    five
    0
    five
    0
    five
    11. Mson (twice) 0.5
    12. (twice) 0.5
    13. (3M) in
    15.0
    14.СГ-ЦСЦ0,5
    15. MeOI0.5
    16. (twice) 0.5
    In brief, 1-2 mmol). BOC-protecting the valuable amino acid in methylene chloride is used per gram of resin, one equivalent of 1.0 M DGG1 in methylene chloride is added over 2 hours for most amino acids, with the exception of BOG-Arg (TOS). BrL-sfir was used as a hydroxyl-protecting side chain group for Ser and Thr. The amide groups Asn and Gin are protected by Hap when DGC attachment is used, which is preferred. P-nitrophenyl ether (ONP) can also be used to activate the carboxyl end of Asn or Gin, for example, BOC-Asn- (OHP) can be added overnight using one equivalent of HOBt in a 50% mixture of DMF and methylene chloride In this case, DCC is not attached, 2-Chlorobenzyloxycarbonyl- (2C1-2) is used as a protective group for lateral lgS. TOS is used for guanidino. Arg and imidazole nitrogen liis, and the carboxyl group of the side chain Gin or Agr is protected with OBZ1. The phenolic hydroxyl group of TYr is protected with 2,6-dichlorobenzyl (DCB). At the end of the synthesis, a polymer resin of the following composition is obtained: BOG-N t-leHis (X) -Ala-Asp (X) -Ala-11 e-Phe-Thr (X) -Ser (X) -Ser (x 4) - Tyr (X2) - Arg (X) -Arg (Xp-Ile-Leu-Gly -Gln (X, r) Leu-Tyr, (X) -Ala-Arg (Xg) -Lys (X7) -Leu- Leu-Hi s (X) -Glu (Xj) -II e-Met-A3h (X 5-) Arg (X) -X3,
    where X-TOS, X2-DGB, X3-OBZ1 ,, Xg-Xan,, -X -, - 2Gl-Z, and X is - NH-permethylbenzhydrylamine, Hap can be partially or completely removed from the pump; TFA treatment used to unlock with -am1-1 protective group.
    To break down and remove protection, 5.4 g of the protected peptidyl resin are treated with 1.5 ml of anisole, 0.5 ml of methyl ethyl sulfide and 15 ml of hydrogen fluoride (HF) per 1 g of peptido-resin at -20 ° C for half an hour and at 0 ° C within half an hour. After
    3
    removing the fluoride in vacuum in vacuo, the remaining tarlopeptide is washed by alternating dry diethyl ether and chloroform, then the peptide is extracted. 2N degassed acetic acid gives 2.84 g (by filtration).
    The peptide cleaved and deprotected is then dissolved in 0-5% acetic acid and. subjected to purification, which may include ultragel filtration on Sephadex-50, receive 204.4 mg.
    The peptide is then purified by preparative HPLC, Cassettes mounted on a Waters Associates LC-500 preparative liquid chromatograph manufactured by Waters Associates are filled with 15-20 µm C / -Silica silica (Videk) (ZOOA). The CHARCN gradient in TEAR is created using a low pressure Eldex gradient apparatus, the chromatographic fractions are carefully controlled by the HPLC and only those with substantial purity are collected. The salting out of the purified fractions, independently tested for purity, is carried out using 15–20 µm C-18 cassettes and a phenyl 15–20 µm gradient in 0.1% TFA. The central fraction was then subjected to freeze drying, to obtain 89.2 mg of the target peptide, the purity of which exceeds 98%.
    Example 2. Synthesis of DLeu peptide, Nle hpGRF (1-29) MH2, H, Tyr-Ala-Asp-Ala-I.le-Phe-Thr-Asn-Ser-Tyr Arg-Lys-Val-Leu-Gly-Gln -Leu-Ser-Ala-Arg-Lys-Leu-DLeu-His-Gln-Tle-Hle Ser-Arg-NH is performed in a sequential step method using the Beckman 990 peptide synthesizer on the MBHA resin of Example 1.
    The trial according to TLC and .HPLC, the peptide obtained substantially pure, yield 165 mgGo -53.3 (, acetic acid).
    Example 3. Under the same conditions, peptides were obtained: DLeu, Nle rGRf (1-29) H-His-Ala-Asp-Ala-Ile-Phe-Thr-Ser-Ser-Tyr-Arg-Arg-Ile-Leu- Gly-Gln-DLeu-Tyr-Ala-Arg-Lys-Leu-DLeu-His-Gln-Jle-Nle-Asn-ArgNH, yield 226.7 MG, -51.2 (acetic acid). TLC and HPLC confirmed the purity of the peptide.
    ju 15 20 25 so
    4B

    50
    55
     1) G 1 n, NI g b pCR F (I-29) Kfj,: Ntug-All-Lcr-L1 -Tle-Phc-Thr-Asn-Ser-Tyr-Ari-l.-ys-Vfll-Leu- Gly-Gln-Leu-Ser-Ab4-Arg-Lys-V.eu-Leu-Gl ii-DOln-I lo-N1 o-Ser-Ari JJH Output 204.4 mg, ofjp -50.0 ° (C 1, acetic acid).
    N "MeTyg DGln JrhGrF (1-29) NHg: hHmeTug-A a-Asp-Ala-Ile-Phe-Thr-Ser-Ser-Tyr-Arg-Arg-Ile-Leu-Gly-Gln-Leu-Tyr-Ala -Arg-Lys-Leu-Leu-His-DGln-Ile-Met-Asn-ArgNH, yield 52 mg, -51, Y (acidic acid). TLC and HPLC data confirmed the substantial purity of peptzilla.
    In vitro tests were performed using synthetic hpGRF (1-40) -OH as a standard to determine the effectiveness of the resulting synthetic peptides in promoting growth hormone secretion. Peptides were tested at equimolar concentrations. The cultures used included rat pituitary cells removed 3-5 days before the experiment. For comparative tests, cultures that are considered optimal for secreting growth hormone were used.
    Incubation with the test substance was carried out for 3–4 hours, and the A / 1C-turns of the culture medium were taken and transferred to measure their contents in an immunoactive GH (ir GH) and tested well-characterized by a radioimmune method.
    The results of this comparison with hpGRF (1-40) OH for equimolar concentrations are presented in the table.
    The in vitro tests of these synthetic peptides show that each of them exhibits the full internal biological activity of hpGRF (1-40) -OH.
    The maximum effective concentration for tDLeu 3, hpGRF (1-29) NH2 is 1 nmol.
    In addition to the iti vitro tests of growth hormone secretion, in vivo experiments were carried out by introducing synthetic peptides through an implanted catheter into a normally free moving American rat after pretreatment with FLA-63, a dopamine hydroxylase inhibitor that suppresses spontaneous GH secretion without affecting reaction to exogenous GRF. Through the same catheter immediately, 5 and 20 minutes after the injection,
    514
    take blood samples Blood GH levels, measured radioimmunologically, show that synthetic peptides that are polygenic under the conditions of the method are powerful stimulants of secretion of the pituitary GH and, besides, show longer duration of action. Other known in vivo GRF tests that are effective in detecting GH secretion have been used to confirm these results. Doses ranging from 1 kg to 50 µg of these peptides per kg of live weight were considered, which are effective in affecting GH secretion.
    Conducted tests have established that the compound of the invention is classified as low toxic.
    Thus, the compounds obtained under the CONDITIONS of this method have a higher activity in promoting growth hormone secretion than the known hpGRF (1-40) OH promoter, and they also have a longer effect.
    权利要求:
    Claims (5)
    [1]
    1.hpGRF (1-40)
    [2]
    2.t N MeTvr Nle JrGP.Fd29),
    [3]
    3. D-Leu-f, Nle hpGRF (129) HH2
    [4]
    4.tD-Leu, Nle hpGRF (129) NHj
    [5]
    5. D-Glu2, Nle 1hpGRF (129)
    2.2
    9.3
    7.2.
    6.2
    5.8
    类似技术:
    公开号 | 公开日 | 专利标题
    SU1435157A3|1988-10-30|Method of producing peptides
    US4737487A|1988-04-12|VIP type peptides
    SU1575944A3|1990-06-30|Method of obtaining peptides
    CA1111841A|1981-11-03|Cyclopeptides
    HU191263B|1987-01-28|Process for producing factor for promoting letting out growth hormone of human pancreatic origine
    DK162649B|1991-11-25|ANALOGY PROCEDURE FOR PREPARATION OF PGRF OR PGRF-ANALOGUE PEPTIDES, PHARMACEUTICAL ACCEPTABLE ADDITIONAL SALTS THEREOF AND INTERMEDIATE USE OF THE PROCEDURE
    KR900002681B1|1990-04-23|Combinations having synergistic pituitary growth hormone releasing activity
    EP0212432A2|1987-03-04|Calcitonin gene related peptide derivatives
    CA2085362A1|1991-12-30|Histidine substituted growth hormone releasing factor analogs
    FI87080B|1992-08-14|FOERFARANDE FOER FRAMSTAELLNING AV GRF -ANALOGER.
    US5416073A|1995-05-16|Growth hormone-releasing peptides and method of treating animals, therewith
    US5049654A|1991-09-17|Calcitonin gene related peptide derivatives
    EP0365779A2|1990-05-02|Cyclic GRF-analogs II
    HU199508B|1990-02-28|Process for producing grf analogues and pharmaceutical compositions comprising same as active ingredient
    SU1598881A3|1990-10-07|Method of producing peptides
    Fujii et al.1981|Total synthesis of bovine pancreatic ribonuclease A. Part 1. Synthesis of the protected pentadecapeptide ester |
    EP0225020A2|1987-06-10|Peptides comprising, in sequence, units selected from the amino-acid residues 17 to 24 of ViP
    EP0188214A2|1986-07-23|Novel growth hormone-releasing peptides and method of treating mammals therewith
    EP0283956B1|1992-11-11|Fluorine containing atrial natriuretic peptides
    IE842199L|1985-02-28|Grf and analogs
    WO1989010935A1|1989-11-16|Atrial peptide derivatives
    CA1243301A|1988-10-18|Crf and analogs
    EP0271041A2|1988-06-15|Atrial peptide derivatives
    EP0268297A2|1988-05-25|Peptides with sulfate ester groups
    EP0101929B1|1986-01-22|Polypeptide-diesters, their production and use
    同族专利:
    公开号 | 公开日
    PT79189B|1986-08-14|
    HU200192B|1990-04-28|
    ES535846A0|1985-10-01|
    PH22310A|1988-07-22|
    CA1271599A|1990-07-10|
    DK165749B|1993-01-11|
    JPS60100596A|1985-06-04|
    PT79189A|1984-10-01|
    IE842324L|1985-03-13|
    MX161018A|1990-07-05|
    DK433684A|1985-03-14|
    YU157984A|1988-04-30|
    NZ209515A|1988-08-30|
    EP0146210B1|1987-03-11|
    AU3259484A|1985-03-21|
    NO843592L|1985-03-14|
    ES8600217A1|1985-10-01|
    HUT35696A|1985-07-29|
    US4518586A|1985-05-21|
    DK433684D0|1984-09-11|
    NO166944B|1991-06-10|
    IN160247B|1987-07-04|
    JPH0689035B2|1994-11-09|
    AU577057B2|1988-09-15|
    FI88402B|1993-01-29|
    YU45968B|1992-12-21|
    FI88402C|1993-05-10|
    FI843579L|1985-03-14|
    NO166944C|1991-09-18|
    IE57774B1|1993-04-07|
    IL72799D0|1984-11-30|
    EP0146210A1|1985-06-26|
    GR80335B|1985-01-11|
    IL72799A|1987-10-30|
    DK165749C|1993-06-14|
    DE3462603D1|1987-04-16|
    FI843579A0|1984-09-12|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    NZ204456A|1982-06-16|1987-05-29|Salk Inst For Biological Studi|Synthetic pancreatic growth-hormone releasing factor|
    IL70530A|1983-01-13|1986-09-30|Salk Inst For Biological Studi|Synthetic peptides having growth hormone releasing factor activity and compositions containing them|
    EP0121764B1|1983-03-07|1990-05-30|F. Hoffmann-La Roche Ag|Polypeptides|
    AU575843B2|1983-08-10|1988-08-11|The Administrators Of The Tulane Eductional Fund|Growth hormone releasing peptides|
    PT79094B|1983-08-29|1986-08-14|Salk Inst For Biological Studi|Grf analogs|US4728726A|1982-10-04|1988-03-01|The Salk Institute For Biological Studies|GRF analogs IIIb|
    US4528190A|1983-10-25|1985-07-09|The Salk Institute For Biological Studies|GRF Analogs IV|
    AU575843B2|1983-08-10|1988-08-11|The Administrators Of The Tulane Eductional Fund|Growth hormone releasing peptides|
    US4626523A|1983-09-13|1986-12-02|The Salk Institute For Biological Studies|GRF analogs II|
    US4617149A|1983-09-21|1986-10-14|Eli Lilly And Company|Growth hormone release factor analogs|
    US4734399A|1985-08-06|1988-03-29|Hoffmann-La Roche Inc.|Growth hormone releasing factor analogs|
    US4649131A|1984-09-24|1987-03-10|Hoffmann-La Roche Inc.|Growth hormone releasing factor analogs|
    CA1271600A|1985-01-07|1990-07-10|David Howard Coy|Growth hormone-releasing peptides and method oftreating mammals therewith|
    EP0193910A3|1985-03-06|1988-11-23|Sumitomo Pharmaceuticals Company, Limited|Synthesis of a derivative of grf and intermediate peptides|
    JPS62187490A|1985-08-12|1987-08-15|Syntex Inc|Bovine growth hormone release factor derivative|
    US4689318A|1985-08-29|1987-08-25|The Salk Institute For Biological Studies|GRF analogs|
    US4870054A|1985-09-27|1989-09-26|Recker Robert R|Treatment for osteoporosis using GRF or a biologically active analog thereof|
    US4880778A|1986-05-12|1989-11-14|Eastman Kodak Company|Combinations having synergistic growth hormone releasing activity and methods for use thereof|
    IL84758A|1987-01-13|1992-03-29|Salk Inst For Biological Studi|Peptides stimulating the release of pituitary growth hormone in fish and amphibians,and pharmaceutical compositions containing them|
    IL86102A|1987-05-11|1994-04-12|Univ Tulane|Alkylated growth hormone-releasing peptides and 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|
    US5023322A|1988-08-31|1991-06-11|Mta Kutatas-Es Szervezetelemzo Intezete|Analogs of growth hormone releasing factorand a method for the preparation thereof|
    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|
    US5550212A|1993-12-17|1996-08-27|The Administrators Of The Tulane Educational Fund|Analogues of hGH-RHNH2 having antagonistic activity|
    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|
    WO2008095063A1|2007-01-31|2008-08-07|Dana-Farber Cancer Institute, Inc.|Stabilized p53 peptides and uses thereof|
    KR101525754B1|2007-03-28|2015-06-09|프레지던트 앤드 펠로우즈 오브 하바드 칼리지|Stitched polypeptides|
    US20090088380A1|2007-07-12|2009-04-02|Pierrette Gaudreau|Ghrh analogs and therapeutic uses thereof|
    EP2603600B1|2010-08-13|2018-11-21|Aileron Therapeutics, Inc.|Peptidomimetic macrocycles|
    WO2012142706A1|2011-04-21|2012-10-26|Theratechnologies Inc.|Growth hormone releasing factoranalogs and uses thereof|
    JP6342808B2|2011-10-18|2018-06-13|エイルロン セラピューティクス，インコーポレイテッド|Peptidomimetic macrocycle|
    WO2013123267A1|2012-02-15|2013-08-22|Aileron Therapeutics, Inc.|Triazole-crosslinked and thioether-crosslinked peptidomimetic macrocycles|
    CN112500466A|2012-02-15|2021-03-16|艾瑞朗医疗公司|Peptidomimetic macrocycles|
    AU2013337388B2|2012-11-01|2018-08-02|Aileron Therapeutics, Inc.|Disubstituted amino acids and methods of preparation and use thereof|
    WO2016049359A1|2014-09-24|2016-03-31|Aileron Therapeutics, Inc.|Peptidomimetic macrocycles and uses thereof|
    EP3294318A4|2015-03-20|2019-04-03|Aileron Therapeutics, Inc.|Peptidomimetic macrocycles and uses thereof|
    法律状态:
    优先权:
    申请号 | 申请日 | 专利标题
    US53217083A| true| 1983-09-13|1983-09-13|
    US06/545,094|US4518586A|1983-01-13|1983-10-25|GRF Analogs III|
    [返回顶部]