JP5006789B2 - 表情皺及び加齢による皺に対する新規な局所適用剤 - Google Patents

表情皺及び加齢による皺に対する新規な局所適用剤 Download PDF

Info

Publication number
JP5006789B2
JP5006789B2 JP2007539437A JP2007539437A JP5006789B2 JP 5006789 B2 JP5006789 B2 JP 5006789B2 JP 2007539437 A JP2007539437 A JP 2007539437A JP 2007539437 A JP2007539437 A JP 2007539437A JP 5006789 B2 JP5006789 B2 JP 5006789B2
Authority
JP
Japan
Prior art keywords
acid
compound
composition
formula
compound according
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.)
Expired - Lifetime
Application number
JP2007539437A
Other languages
English (en)
Japanese (ja)
Other versions
JP2008518978A5 (enExample
JP2008518978A (ja
Inventor
イムフェルト，ドミニク
ジーグラー，ヒューゴ
ヴィクストローム，ペーター
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.)
DSM IP Assets BV
Original Assignee
DSM IP Assets BV
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 DSM IP Assets BV filed Critical DSM IP Assets BV
Publication of JP2008518978A publication Critical patent/JP2008518978A/ja
Publication of JP2008518978A5 publication Critical patent/JP2008518978A5/ja
Application granted granted Critical
Publication of JP5006789B2 publication Critical patent/JP5006789B2/ja
Anticipated expiration legal-status Critical
Expired - Lifetime legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0821—Tripeptides with the first amino acid being heterocyclic, e.g. His, Pro, Trp
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/05—Dipeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/06—Tripeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A61P21/02—Muscle relaxants, e.g. for tetanus or cramps
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
    • C07K5/0202—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing the structure -NH-X-X-C(=0)-, X being an optionally substituted carbon atom or a heteroatom, e.g. beta-amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0802—Tripeptides with the first amino acid being neutral
    • C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
    • C07K5/0806—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atoms, i.e. Gly, Ala
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0815—Tripeptides with the first amino acid being basic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
    • C07K5/1008—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atoms, i.e. Gly, Ala
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Immunology (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Epidemiology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Pain & Pain Management (AREA)
  • Neurology (AREA)
  • Orthopedic Medicine & Surgery (AREA)
  • Dermatology (AREA)
  • Physical Education & Sports Medicine (AREA)
  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Cosmetics (AREA)
  • Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Peptides Or Proteins (AREA)
  • Medicinal Preparation (AREA)
JP2007539437A 2004-11-02 2005-11-01 表情皺及び加齢による皺に対する新規な局所適用剤 Expired - Lifetime JP5006789B2 (ja)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
CH18092004 2004-11-02
CH1809/04 2004-11-02
PCT/CH2005/000638 WO2006047900A2 (de) 2004-11-02 2005-11-01 Neue topisch anwendbare wirkstoffe gegen mimische und alterabedingt falten

Publications (3)

Publication Number Publication Date
JP2008518978A JP2008518978A (ja) 2008-06-05
JP2008518978A5 JP2008518978A5 (enExample) 2008-10-16
JP5006789B2 true JP5006789B2 (ja) 2012-08-22

Family

ID=35841896

Family Applications (1)

Application Number Title Priority Date Filing Date
JP2007539437A Expired - Lifetime JP5006789B2 (ja) 2004-11-02 2005-11-01 表情皺及び加齢による皺に対する新規な局所適用剤

Country Status (12)

Country Link
US (1) US7964630B2 (enExample)
EP (1) EP1809652B1 (enExample)
JP (1) JP5006789B2 (enExample)
KR (1) KR101227301B1 (enExample)
CN (1) CN101061134B (enExample)
AT (1) ATE466867T1 (enExample)
BR (1) BRPI0517917B1 (enExample)
CA (1) CA2584643C (enExample)
DE (1) DE502005009544D1 (enExample)
ES (1) ES2345667T3 (enExample)
PL (1) PL1809652T3 (enExample)
WO (1) WO2006047900A2 (enExample)

Families Citing this family (36)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100240977A1 (en) * 2007-06-20 2010-09-23 Andreas Caduff Method for measuring the response of a tissue to an electromagnetic field
AU2009215436A1 (en) 2008-02-19 2009-08-27 Myocept Inc. Postsynaptically targeted chemodenervation agents and their methods of use
WO2010003861A1 (en) * 2008-07-08 2010-01-14 Unilever Plc Antiperspirant products
EP2143418A1 (en) 2008-07-08 2010-01-13 Unilever PLC Antiperspirant compositions
EP2456457B1 (en) 2009-07-24 2014-10-22 Lipotec, S.A. Compounds which inhibit muscle contraction
JP4666537B1 (ja) * 2010-01-21 2011-04-06 株式会社資生堂 しわ改善用美容セット並びにこれを使用する美容方法
US10149812B2 (en) 2012-04-13 2018-12-11 Activen Cosmetic composition comprising a muconopeptide
ES2678793T3 (es) 2012-04-13 2018-08-17 Activen Composición cosmética que comprende un muconopéptido
RU2524428C1 (ru) * 2013-01-21 2014-07-27 Общество с ограниченной ответственностью "Синейро" Пептиды и их производные, взаимодействующие с никотиновым ацетилхолиновым рецептором и пригодные для использования в косметологии против мимических и возрастных морщин
CN103570804B (zh) * 2013-09-11 2015-04-15 深圳市维琪医药研发有限公司 一种具皮肤活性的多肽的合成方法
CA2928526A1 (en) 2013-11-01 2015-05-07 Spherium Biomed S.L. Inclusion bodies for transdermal delivery of therapeutic and cosmetic agents
RU2551276C1 (ru) * 2014-02-03 2015-05-20 Закрытое Акционерное Общество "Фарм-Синтез" Способ получения диацетата трипептида
US9649266B2 (en) * 2014-05-01 2017-05-16 Anterios, Inc. Methods to treat, prevent, and improve skin conditions
CN104327155A (zh) * 2014-10-20 2015-02-04 张嘎 一种含有15n—l—脯氨酸残基的三肽型减皱化合物及其制备方法和应用
CN104478991B (zh) * 2014-12-10 2018-06-19 深圳市维琪医药研发有限公司 一种新型多肽化合物及其制备方法和其医药应用
KR101710486B1 (ko) * 2015-01-05 2017-02-28 주식회사 앤코스메슈 올리고펩타이드 유도체 및 이를 포함하는 주름개선용 조성물
CN104592348A (zh) * 2015-01-16 2015-05-06 张嘎 一种含有3,4-脱氢-l-脯氨酸残基的三肽减皱化合物及其制备方法和应用
WO2017102588A1 (en) * 2015-12-16 2017-06-22 Dsm Ip Assets B.V. Novel use
KR102706016B1 (ko) * 2015-12-16 2024-09-13 디에스엠 아이피 어셋츠 비.브이. 신규한 용도
CN112010772A (zh) * 2016-01-26 2020-12-01 赵鸣 5-氨基酮戊酸及其衍生物的盐化合物和应用
CN106038385A (zh) * 2016-06-08 2016-10-26 广州市创美时美容化妆品有限公司 一种刺激胶原蛋白生成抵抗皱纹的化妆品组合物
CN107936108A (zh) * 2017-12-05 2018-04-20 陕西慧康生物科技有限责任公司 一种类蛇毒三肽的液相合成方法
CN107857797A (zh) * 2017-12-07 2018-03-30 陕西慧康生物科技有限责任公司 一种类蛇毒三肽的液相片段合成方法
ES2937382T3 (es) 2018-02-27 2023-03-28 Lipotrue S L Péptidos y composiciones para uso en cosmética y medicina
KR102724873B1 (ko) * 2018-05-24 2024-11-04 디에스엠 아이피 어셋츠 비.브이. 프롤린 유도체의 신규한 용도
CN118561963A (zh) 2018-10-16 2024-08-30 格罗制药公司 烟碱乙酰胆碱受体肽拮抗剂芋螺毒素组合物及相关方法
CN113045623A (zh) * 2019-12-27 2021-06-29 深圳翰宇药业股份有限公司 一种蛇毒肽syn-ake的液相合成方法
CN110950926B (zh) * 2019-12-31 2020-10-16 山东济肽生物科技有限公司 类蛇毒三肽的液相合成方法
CN114369139B (zh) * 2020-10-14 2024-04-19 成都泰莱康科技有限公司 类蛇毒三肽的制备方法
US11771637B2 (en) 2021-01-22 2023-10-03 The Procter & Gamble Company Skin care composition and method of using the same
US11571378B2 (en) 2021-01-22 2023-02-07 The Procter & Gamble Company Skin care composition and method of using the same
CN113693964B (zh) * 2021-08-16 2023-03-24 深圳市维琪医药研发有限公司 肽衍生物在制备用于皮肤修复紧致的组合物中的新用途
CN114380887A (zh) * 2021-12-09 2022-04-22 深圳翰宇药业股份有限公司 一种类蛇毒三肽的液相片段合成方法
WO2025162944A2 (en) * 2024-01-29 2025-08-07 Dsm Ip Assets B.V. Novel compounds
WO2025170081A1 (en) 2024-02-05 2025-08-14 L'oreal Stabilization of composition comprising polyhydroxy acid, beta-hydroxy acid and peptide with polyol
FR3160325A3 (fr) 2024-03-19 2025-09-26 L'oreal Stabilisation d'une composition comprenant un polyhydroxyacide, un bêta-hydroxyacide et un peptide avec du polyol

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3966701A (en) * 1973-11-07 1976-06-29 The Dow Chemical Company Fibrinogen peptide derivatives
US4563305A (en) * 1981-01-07 1986-01-07 University Of Miami Radiolabelled substrates for assaying mammalian enzymes
US7408027B1 (en) * 1991-12-06 2008-08-05 Max-Planck-Gesellschaft Zur Forderung Der Wissenchaften Tools for the diagnosis and treatment of Alzheimer's disease
AU2334897A (en) 1996-03-18 1997-10-10 Regents Of The University Of California, The Peptide inhibitors of neurotransmitter secretion by neuronal cells
ES2160485B1 (es) * 1999-04-23 2002-05-16 Lipotec Sa Peptidos inhibidores de la exocitosis neuronal, composiciones cosmeticas y farmaceuticas que los contienen.

Also Published As

Publication number Publication date
KR101227301B1 (ko) 2013-01-29
KR20070085372A (ko) 2007-08-27
PL1809652T3 (pl) 2010-10-29
US20090111731A1 (en) 2009-04-30
ATE466867T1 (de) 2010-05-15
WO2006047900A3 (de) 2006-07-27
BRPI0517917B1 (pt) 2020-11-10
US7964630B2 (en) 2011-06-21
CN101061134A (zh) 2007-10-24
CA2584643A1 (en) 2006-05-11
CN101061134B (zh) 2011-05-11
JP2008518978A (ja) 2008-06-05
DE502005009544D1 (de) 2010-06-17
WO2006047900A2 (de) 2006-05-11
ES2345667T3 (es) 2010-09-29
EP1809652B1 (de) 2010-05-05
CA2584643C (en) 2013-01-29
BRPI0517917A (pt) 2008-10-21
EP1809652A2 (de) 2007-07-25

Similar Documents

Publication Publication Date Title
JP5006789B2 (ja) 表情皺及び加齢による皺に対する新規な局所適用剤
US8304393B2 (en) Epithelial cell growth promoter
US8722852B2 (en) Cosmetic composition for stimulating the synthesis of proteins of the basement membrane
KR20040062602A (ko) 피부약학적 및 화장품학적 활성 올리고펩타이드
KR20120105450A (ko) 피부, 점막 및/또는 모발의 치료 및/또는 관리에 사용되는 펩티드 및 화장품학적 또는 약제학적 조성물에 있어서의 이의 용도
JPH09512806A (ja) 新規なアミノ酸誘導体、その製造方法及びこれらの化合物を含む医薬組成物（ｉｉ）
US20250019400A1 (en) Polypeptide and cosmetic composition or pharmaceutical composition and use thereof
WO2007087738A1 (de) Biologisch aktive tripeptide, deren kupferkomplexe und salze
CN104478991B (zh) 一种新型多肽化合物及其制备方法和其医药应用
CN107207566A (zh) 稳定的肽缀合抗坏血酸衍生物、用于制备其的方法和包含其的化妆品组合物
CA2587284C (en) Tripeptide and tetrapeptide thioethers
KR101224809B1 (ko) 레티노산 유도체, 이의 제조 방법 및 이를 포함하는 화장품 조성물
WO2015111857A1 (ko) 펩타이드 유도체 및 이를 함유하는 기능성 화장품 조성물
KR20180108988A (ko) 인간 섬유아세포에서 콜라겐 합성 촉진 및 콜라겐 분해 효소 활성화 억제를 유도하는 펩타이드를 함유한 화장품 조성물 및 제조방법
CN104592348A (zh) 一种含有3,4-脱氢-l-脯氨酸残基的三肽减皱化合物及其制备方法和应用
HK1237798B (zh) 稳定的肽缀合抗坏血酸衍生物、用於制备其的方法和包含其的化妆品组合物
HK1028409A (en) Phenethylamine derivatives
HK1237798A1 (en) Stable peptide-conjugated ascorbic acid derivative, method for preparing same, and cosmetic composition containing same

Legal Events

Date Code Title Description
A521 Request for written amendment filed

Free format text: JAPANESE INTERMEDIATE CODE: A523

Effective date: 20080805

A621 Written request for application examination

Free format text: JAPANESE INTERMEDIATE CODE: A621

Effective date: 20080918

A711 Notification of change in applicant

Free format text: JAPANESE INTERMEDIATE CODE: A711

Effective date: 20081009

A131 Notification of reasons for refusal

Free format text: JAPANESE INTERMEDIATE CODE: A131

Effective date: 20110531

A601 Written request for extension of time

Free format text: JAPANESE INTERMEDIATE CODE: A601

Effective date: 20110818

A602 Written permission of extension of time

Free format text: JAPANESE INTERMEDIATE CODE: A602

Effective date: 20110825

A601 Written request for extension of time

Free format text: JAPANESE INTERMEDIATE CODE: A601

Effective date: 20110922

A602 Written permission of extension of time

Free format text: JAPANESE INTERMEDIATE CODE: A602

Effective date: 20110930

A601 Written request for extension of time

Free format text: JAPANESE INTERMEDIATE CODE: A601

Effective date: 20111018

A602 Written permission of extension of time

Free format text: JAPANESE INTERMEDIATE CODE: A602

Effective date: 20111025

A521 Request for written amendment filed

Free format text: JAPANESE INTERMEDIATE CODE: A523

Effective date: 20111129

A131 Notification of reasons for refusal

Free format text: JAPANESE INTERMEDIATE CODE: A131

Effective date: 20120117

A521 Request for written amendment filed

Free format text: JAPANESE INTERMEDIATE CODE: A523

Effective date: 20120406

TRDD Decision of grant or rejection written
A01 Written decision to grant a patent or to grant a registration (utility model)

Free format text: JAPANESE INTERMEDIATE CODE: A01

Effective date: 20120508

A01 Written decision to grant a patent or to grant a registration (utility model)

Free format text: JAPANESE INTERMEDIATE CODE: A01

A61 First payment of annual fees (during grant procedure)

Free format text: JAPANESE INTERMEDIATE CODE: A61

Effective date: 20120525

FPAY Renewal fee payment (event date is renewal date of database)

Free format text: PAYMENT UNTIL: 20150601

Year of fee payment: 3

R150 Certificate of patent or registration of utility model

Ref document number: 5006789

Country of ref document: JP

Free format text: JAPANESE INTERMEDIATE CODE: R150

Free format text: JAPANESE INTERMEDIATE CODE: R150

R250 Receipt of annual fees

Free format text: JAPANESE INTERMEDIATE CODE: R250

R250 Receipt of annual fees

Free format text: JAPANESE INTERMEDIATE CODE: R250

R250 Receipt of annual fees

Free format text: JAPANESE INTERMEDIATE CODE: R250

R250 Receipt of annual fees

Free format text: JAPANESE INTERMEDIATE CODE: R250

R250 Receipt of annual fees

Free format text: JAPANESE INTERMEDIATE CODE: R250

R250 Receipt of annual fees

Free format text: JAPANESE INTERMEDIATE CODE: R250

R250 Receipt of annual fees

Free format text: JAPANESE INTERMEDIATE CODE: R250

R250 Receipt of annual fees

Free format text: JAPANESE INTERMEDIATE CODE: R250

R250 Receipt of annual fees

Free format text: JAPANESE INTERMEDIATE CODE: R250

R250 Receipt of annual fees

Free format text: JAPANESE INTERMEDIATE CODE: R250

R250 Receipt of annual fees

Free format text: JAPANESE INTERMEDIATE CODE: R250