EP1409519A1 - Conjugue lipides-peptides pour le traitement du cancer - Google Patents

Conjugue lipides-peptides pour le traitement du cancer

Info

Publication number
EP1409519A1
EP1409519A1 EP00952334A EP00952334A EP1409519A1 EP 1409519 A1 EP1409519 A1 EP 1409519A1 EP 00952334 A EP00952334 A EP 00952334A EP 00952334 A EP00952334 A EP 00952334A EP 1409519 A1 EP1409519 A1 EP 1409519A1
Authority
EP
European Patent Office
Prior art keywords
peptide
leu
tyr
pro
met
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Withdrawn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
EP00952334A
Other languages
German (de)
English (en)
Inventor
Anand C. Burman
Sudhanand Prasad
Rama Mukherjee
Manu Jaggi
Anu T. Singh
Rajan Sharma
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.)
Dabur Research Foundation
Original Assignee
Dabur Research Foundation
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 Dabur Research Foundation filed Critical Dabur Research Foundation
Publication of EP1409519A1 publication Critical patent/EP1409519A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/542—Carboxylic acids, e.g. a fatty acid or an amino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to novel antiproliferative and anti secrectory peptides that are inhibitory to vasoactive intestinal peptide receptor and are useful in the treatment of cancer.
  • the invention particularly relates to the synthesis of lipid-peptide conjugates containing fatty acids of different sizes, which inhibits the binding of VIP to its receptors.
  • the invention encompasses methods for generation of these peptides, composition containing these peptides and the pharmacological applications of these peptides especially in the treatment and prevention of cancer.
  • Vasoactive intestinal peptide is a 28-amino acid neuropeptide, which was first isolated from the porcine intestine (Said, S. I. and Mutt, V. , Science, 169, 1217-1218, 1970.) VIP acts as growth factor and plays dominant autocrine and paracrine role in the sustained proliferation of cancer cells. (Said, S.L, Peptides, 5, 143-150, 1984.) Gozes et al. have shown that VIP can serve as autocrine growth factor in lung tumors. (Gozes et al. Biomed. Res. 13 (suppl.2) 37, 1992).
  • the peptide sequence Leu-Met-Tyr-Pro-Thr-Tyr-Leu-Lys (SEQ ID NO: 1) is reported to be receptor binding inhibitor of vasoactive intestinal peptide (Said, & Mutt, Ann. N.Y. Acad. Sci., 1, 527, 1988).
  • the role this octapeptide as VIP receptor binding inhibitor has been described in the U.S Patent 5,217,953.
  • U.S. Patent Application 08/727,679 we have described the anti cancer role of this VIP binding receptor inhibitor in combination with other neuropeptide analogs.
  • U.S. Patent Application 09/248382 we have described the novel analogs of this VIP receptor binding inhibitor incorporating dialkylated amino acids.
  • BOP Benzotriazole- 1 -yl-oxy-tris-(dimethylamino)- phosphonium hexofluorophospate
  • DIPCD Diisopropyl carbodiimide
  • amino acids residues are designated by their standard abbreviations.
  • Amino acids denote L-configuration unless otherwise indicated by D or DL appearing before the symbol and separated from it by hyphen.
  • amino acids residues are designated by their standard abbreviations.
  • Amino acids denote L-configuration unless otherwise indicated by D or DL appearing before the symbol and separated from it by hyphen.
  • the present invention relates to peptides of the following general formula X-Leu-Met-Tyr-Pro-Thr-Tyr-Leu-Lys-Y wherein,
  • X is acetyl or straight, branched, or cyclic al anoyl group of from 3 - 16 carbon atoms.
  • Y is a carboxy terminal residue selected from OH or amino; or a pharmaceutical acceptable salt of the peptides.
  • FIGURE Figure 1 shows the anti-cancer activity of the peptide DT-B1 on PCT xenograft.
  • the present invention relates to peptides of the following general formula X-Leu-Met-Tyr-Pro-Thr-Tyr-Leu-Lys-Y wherein,
  • X is acetyl or straight, branched, or cyclic alkanoyl group of from 3 - 16 carbon atoms.
  • Y is a carboxy terminal residue selected from OH or amino; or a pharmaceutical acceptable salt of the peptides.
  • the preferred alkanoyl groups are acetyl, n-butanoyl, n-hexanoyl, n-octanoyl, lauroyl, myristoyl, palmitoyl, isohexanoyl, cyclohexanoyl, cyclopentylcarbonyl, n-heptanoyl, n-decanoyl, n-undecanoyl, and 3,7-dimethyloctanoyl.
  • Salts encompassed within the term "pharmaceutically acceptable salts" refer to non-toxic salts of the compounds of this invention.
  • Representative salts and esters include following: acetate, ascorbate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, camsylate, carbonate, citrate, dihydrochloride, methanesulfonate, ethanesulfonate, p-toluenesulfonate, cyclohexylsulfamate, quinate, edetate, edisylate, estolate, esylate, fumarate, gluconate, glutamate, glycerophophates, hydrobromide, hydrochloride, hydroxy- naphthoate, lactate, lactobionate, laurate, malate, maleate, mandelate, mesylate, mucate, napsylate, nitrate, n-methylglucamine, oleate, oxalate, pahnoates, pamoate (embonate), palmitate
  • salts include Ca, Li, Mg, Na, and K salts; salts of amino acids such as lysine or arginine; guanidine, diethanolamine or choline; ammonium, substituted ammonium salts or aluminum salts.
  • the salts are prepared by conventional methods.
  • the preferred lipo-peptide analogs are:
  • novel compounds of the present invention have important pharmacological applications. They are potent anti-neoplastic agents and thereby possess therapeutic potential in a number of human cancers.
  • the lipopeptides in the present invention have been generated by using solid phase techniques or by a combination of solution phase procedures and solid phase techniques or by fragment condensation.
  • the methods for the chemical synthesis of polypeptides are well known in the art (Stewart and Young, 1969 Solid
  • the peptides were synthesized using the Fmoc strategy, on a semi automatic peptide synthesizer (CS Bio, Model 536), using optimum side chain protection.
  • the peptides were assembled from C-terminus to N-terminus.
  • Peptides amidated at the carboxy-terminus were synthesized using the Rink Amide resin.
  • the loading of the first Fmoc protected amino acid was achieved via an amide bond formation with the solid support, mediated by Diisopropylcarbodiimide (DIPCDI) and HOBt.
  • DIPCDI Diisopropylcarbodiimide
  • Substitution levels for automated synthesis were preferably between 0.2 and 0.6 mmole amino acid per gram resin.
  • the steps involved in the synthesis of the peptide analogs employed the following protocol: TABLE I
  • the N-terminal amino group was protected by 9-fluorenylmethoxy- carbonyl (Fmoc) group.
  • the hydroxyl groups of Threonine and Tyrosine were preferably protected by t-butyl group (tBu). Leu, Met and Pro were used unprotected.
  • Fmoc protected amino acid per resin nitrogen equivalent 2-8 equivalents of Fmoc protected amino acid per resin nitrogen equivalent were used.
  • the activating reagents used for coupling amino acids to the resin, in solid phase peptide synthesis are well known in the art. These include DCC, DIPCDI, DIEA, BOP, PyBOP, HBTU, TBTU, and HOBt. Preferably, DCC or DIPCDI / HOBt or HBTU/HOBt and DIEA were used as activating reagents in the coupling reactions.
  • the protected amino acids were either activated in situ or added in the form of preactivated esters known in the art such as N-hydroxy succinamide esters, pentafluorophenyl esters etc.
  • the coupling reaction was carried out in DMF, DCM or NMP or a mixture of these solvents and was monitored by Kaiser test [Kaiser et al., Anal. Biochem., 34, 595-598 (1970)]. In case of a positive Kaiser test, the appropriate amino acid was re-coupled using freshly prepared activated reagents.
  • the amino-terminal Fmoc group was removed using steps 1-6 of the above protocol and then the peptide-resin was washed with methanol and dried.
  • the analogs were then deprotected and cleaved from the resin support by treatment with trifluoroacetic acid, crystalline phenol, ethanedithiol, thioanisole and de-ionized water for 1.5 to 5 hours at room temperature.
  • the crude peptide was obtained by precipitation with cold dry ether, filtered, dissolved, and lyophilized.
  • the resulting crude peptide was purified by preperative high performance liquid chromatography (HPLC) using a LiChrOCART® C 18 (250. Times.
  • An analog of the present invention can be made by exclusively solid phase techniques, by partial solid phase/solution phase techniques and/or fragment condensation.
  • Preferred, semi-automated, stepwise solid phase methods for synthesis of peptides of the invention are provided in the examples discussed in the subsequent section of this document.
  • a typical preparation of the Fmoc-Lys-Wang Resin was carried out using 1.0 g of 4-Hydroxymethylphenoxy Resin 1% DVB cross-linked resin (0.7 mM/g) (100-200 mesh), procured from Advanced Chemtech, Louisville, KY, U.S.A. Swelling of the resin was typically carried out in dichloromethane measuring to volumes 10-40ml/g resin. The resin was allowed to swell in methylene chloride (2 X 25 ml, for 10 min.). It was washed once in dimethylformamide (DMF) for 1 min. All solvents in the protocol were added in 20 ml portions per cycle.
  • DMF dimethylformamide
  • the first amino acid was weighed in three to six fold excess, along with a similar fold excess of HOBt, in the amino acid vessel of the peptide synthesizer. These were dissolved in dimethylformamide (A.C.S. grade) (J.T.Baker,, New Jersey, U.S.A.) and activated with DIPCDI and 4-dimethyl amino pyridine (DMAP)just prior to the addition to the resin in the reaction vessel of the peptide synthesizer. The coupling reaction was carried out for a period ranging from 6 hours. The loading of the amino acid on the resin was confirmed by the weight gain of the resin. The loading efficiency was ascertained by the increase of weight of the resin after the addition of the amino acid.
  • dimethylformamide A.C.S. grade
  • DMAP 4-dimethyl amino pyridine
  • EXAMPLE 6 The cytotoxic effect of Lipo peptide analogs, DT-A1 (SEQ ID NO: 2), DT-Bl (SEQ ID NO: 3), DT-Ol (SEQ ID NO: 4), DT-Ml (SEQ ID NO: 5) and DT-P1 (SEQ ID NO: 6) was studied by MTT assay which is based on the principle of uptake of MTT [3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide], a tetrazolium salt by the metabolically active cells where it is metabolized by active mitochondria into a blue colored formazan product which can be read spectrophoto- metrically.
  • tumor cells PTC primary colon
  • KB Oral squamous
  • U87MG Gaoblastoma
  • HBL100 Breast
  • HeP2 Longephrine
  • ECV304 Endothelial
  • PA-1 Longephrine
  • LI 32 LI 32
  • the formazan crystals formed inside the cells were dissolved with a detergent comprising 10% Sodium dodecyl sulfate and 0.01 N HCl and optical density read on a multiscan ELISA reader. The optical density was directly proportional to the number of proliferating and metabolically active cells. Percent cytotoxicity of peptide analogs is shown in the following Tables.
  • the antitumor activity of DT-Bl was studied in human colon adenocarcinoma (PTC) xenografts in nude mice.
  • PTC tumor xenografts were grown in Balb/c a thymic mice by subcutaneous inoculation of a single cell suspension of PTC cells (15 X 10 6 cells/100 ⁇ L).
  • the tumor bearing animals were divided into 2 groups of three animals each including one group comprising untreated control animals.
  • Treatment with DT-Bl was initiated when the average tumor volumes, as measured using a vernier caliper, were between 1.3 cm 3 .
  • Solutions of DT-Bl was prepared at a concentration of 126 ⁇ g/ml and intravenously administered to the assigned group of tumor bearing animals at a dose of 12.6 ⁇ g/100 ⁇ L twice a day so that the total dose of 25.2 ⁇ g/day was administered to each animal. The treatment was continued for a period of 14 days.
  • the percentage inhibition of tumor growth was calculated using the formula (1- tumor volume-treated/tumor volume-control) * 100.
  • Figure 1 shows the tumor kinetics till day 20 in the treated and untreated animals.
  • DT-Bl showed a significant antitumor activity on PTC xenografts.
  • the percentage inhibition of tumor growth caused by DT-Bl as compared to controls on day 20 was 95.85%. All publications referenced are incorporated by reference herein, including the amino acid sequences listed in each publication. All the compounds disclosed and referred to in the publications mentioned above are incorporated by reference herein, including those compounds disclosed and referred to in articles cited by the 10 publications mentioned.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Organic Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Public Health (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Molecular Biology (AREA)
  • Epidemiology (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention porte sur de nouveaux peptides antiprolifération et antisécrétoires inhibiteurs du récepteur des peptides intestinaux vasoactifs (VIP) et utilisables pour le traitement du cancer, et en particulier sur la synthèse de conjugués lipides-peptides contenant des acides gras de différentes tailles, et qui inhibent la fixation des VIPs à leurs récepteurs. L'invention porte en outre sur les procédés d'élaboration de ces peptides, sur des préparations les contenant et sur des applications pharmacologiques de ces peptides spécialement destinées au traitement et à la prévention du cancer.
EP00952334A 2000-07-31 2000-07-31 Conjugue lipides-peptides pour le traitement du cancer Withdrawn EP1409519A1 (fr)

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/US2000/020876 WO2002010193A1 (fr) 2000-07-31 2000-07-31 Conjugue lipides-peptides pour le traitement du cancer

Publications (1)

Publication Number Publication Date
EP1409519A1 true EP1409519A1 (fr) 2004-04-21

Family

ID=21741642

Family Applications (1)

Application Number Title Priority Date Filing Date
EP00952334A Withdrawn EP1409519A1 (fr) 2000-07-31 2000-07-31 Conjugue lipides-peptides pour le traitement du cancer

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EP (1) EP1409519A1 (fr)
AU (1) AU2000265054A1 (fr)
CA (1) CA2405728A1 (fr)
WO (1) WO2002010193A1 (fr)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1633380A4 (fr) * 2003-06-19 2010-08-11 Yeda Res & Dev Lipopeptides antimicrobiens et anticancereux
US10487115B2 (en) * 2016-02-01 2019-11-26 University Of Canberra Proteinaceous compounds and uses therefor

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
IL87055A (en) * 1988-07-08 1994-06-24 Illana Gozes Conjugates of vasoactive intestinal peptide (vip) and of fragments thereof and pharmaceutical compositions comprising them
US5217953A (en) * 1990-11-30 1993-06-08 The United States Of America As Represented By The Secretary Of The Department Of Health & Human Services Vasoactive intestinal peptide antagonist
US5565424A (en) * 1994-02-07 1996-10-15 Ramot - University Authority For Applied Research And Industrial Development Ltd. Superactive VIP antagonists
JP2003530297A (ja) * 1998-10-16 2003-10-14 ザ ガバメント オブ ザ ユナイテッド ステイツ オブ アメリカ， リプリゼンテッド バイ ザ セクレタリー オブ ザ デパートメント オブ ヘルス アンド ヒューマン サービシーズ Ｖｉｐアンタゴニストを用いる併用療法

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
See also references of WO0210193A1 *
SINGH H. ET AL: "A SYNTHETIC PEPTIDE L-8-K AND ITS ANTIBODY BOTH INHIBIT THE SPECIFIC BINDING OF VASOACTIVE INTESTINAL PEPTIDE TO HAMSTER PANCREATIC CANCER CELLS", ANNALS OF THE NEW YORK ACADEMY OF SCIENCES, vol. 527, 1988, pages 679 - 681 *

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CA2405728A1 (fr) 2002-02-07
AU2000265054A1 (en) 2002-02-13
WO2002010193A1 (fr) 2002-02-07

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