WO1994012530A1 - Composition d'un peptide et d'un acyle gras polyinsature - Google Patents

Composition d'un peptide et d'un acyle gras polyinsature Download PDF

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WO1994012530A1
WO1994012530A1 PCT/HU1993/000065 HU9300065W WO9412530A1 WO 1994012530 A1 WO1994012530 A1 WO 1994012530A1 HU 9300065 W HU9300065 W HU 9300065W WO 9412530 A1 WO9412530 A1 WO 9412530A1
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Prior art keywords
peptide
fatty acyl
seq
dha
hormone
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PCT/HU1993/000065
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English (en)
Inventor
György Kéri
Robert S. Hodges
Paul J. Cachia
Ferenc SZEDERKÉNYI
Anikó HORVÁTH
Ágnes BALOGH
Zsolt Vadász
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Biosignal Kutató Fejlesztó Kft.
S.P.I. Synthetic Peptides Incorporated
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Priority to AU55740/94A priority Critical patent/AU5574094A/en
Publication of WO1994012530A1 publication Critical patent/WO1994012530A1/fr

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    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
    • C07K1/1077—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
    • 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
    • C07K14/635—Parathyroid hormone, i.e. parathormone; Parathyroid hormone-related peptides
    • 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
    • C07K14/655—Somatostatins
    • 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
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • 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
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • 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
    • C07K7/23—Luteinising hormone-releasing hormone [LHRH]; Related peptides
    • 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 present invention relates to polyunsaturated fatty acyl-peptide composition useful in inhibiting cell proliferation.
  • Uncontrolled cell proliferation is a characteristic of a number of diseased states. Such growth is observed, for example, in benign and malignant tumors, certain virally- induced diseases and psoriasis.
  • drugs used to treat cellular abnormalities characterized by uncontrolled cell growth target important biochemical steps or processes that are part of the cell growth cycle. However, ⁇ such drugs lack selectivity and inhibit the growth of both diseased and healthy cells. Therefore, development of chemotherapeutic agents having relatively high selectivity for the diseased cells would be advantageous.
  • Agents based on peptide hormones, steroid hormones, hormone-releasing factors, and their respective antagonists and agonists are relatively specific to their target cells.
  • Proliferative cells such as neoplastic cells or tumors, which arise from hormone-sensitive tissues generally are found to have hormonal requirements that are similar to those of their healthy counterparts. By altering the amount of hormone in the blood circulation it may be possible to selectively restrict the growth of these cells. However, due to the relatively high levels of hormone required for such treatment, use of hormonal chemotherapeutic agents is still limited in vivo by toxic side effects to normal cells .
  • cell proliferation may also be inhibited by targeting one or more of the cellular signal transduction systems implicated in the regulation of cell division.
  • tyrosine kinase signal transduction pathway a) the tyrosine kinase signal transduction pathway, b) the phospholipid metabolism/protein kinase C signal transduction pathway, and c) the cAMP protein kinase A signal transduction pathway.
  • tyrosine kinase signal transduction pathway a) the tyrosine kinase signal transduction pathway, b) the phospholipid metabolism/protein kinase C signal transduction pathway, and c) the cAMP protein kinase A signal transduction pathway.
  • Protein kinases have been found to be particularly important regulators of these pathways.
  • tyrosine kinases are known to play a critical role in the regulation of cell division. High levels of tyrosine kinase activity have been measured in highly proliferative cells, such as neoplastic cells. Inhibition of such phosphorylation activity can be correlated with a reduction in cell division, in some cases.
  • the current invention is directed to fatty acyl- peptide compositions having enhanced biological activity, compared to the peptide alone.
  • Peptides used to form compositions directed to the inhibition of cell proliferation include peptide hormones, peptide hormone analogues, and protein kinase peptide substrates or peptide inhibitors.
  • linking such peptides to the polyunsaturated fatty acids lowers the concentration at least several-fold of such peptides required to inhibit cell proliferation.
  • Such low chemotherapeutic drug concentrations confer the advantage of reducing toxicity to healthy cells.
  • One general object of the invention is to provide a fatty acyl-peptide composition which is useful in inhibiting cell proliferation, such as neoplastic cell proliferation.
  • the composition includes a peptide having antiproliferative activity and conjugated to the peptide, a polyunsaturated fatty acyl moiety.
  • the composition is characterized by a cell proliferative inhibitory activity which is several-fold greater than that of the antiproliferative peptide alone.
  • the fatty acyl moiety of the composition is a doco ⁇ ahexae ⁇ oyl or an eicosapentaenoyl moiety.
  • the fatty acyl moiety is a ci ⁇ -4, 7, 10, 13, 16, 19-docosahexa enoyl (DHA) or cis- 5, 8, 11, 14, 17-eicosapentae ⁇ oyl (EPA) moiety.
  • DHA 7, 10, 13, 16, 19-docosahexa enoyl
  • EPA cis- 5, 8, 11, 14, 17-eicosapentae ⁇ oyl
  • the fatty acyl moiety is preferably conjugated to the peptide through an amide linkage.
  • the peptide portion of the composition is a peptide hormone, and in a preferred embodiment, the peptide hormone is a somatostatin analog or a gonadotropin releasing hormone (GnRH) analog.
  • the peptide hormone has the sequence of SEQ ID NO: 4, and in another preferred' embodiment, the peptide hormone of the composition has the peptide sequence of SEQ ID NO: 5.
  • the peptide used in forming the fatty acyl-peptide antiproliferative composition can also be a protein kinase modulatory peptide.
  • the protein kinase modulatory peptide has a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 6.
  • Another general object of the invention is to provide a method for producing enhanced biological activity of a peptide.
  • the invention is used for producing a several-fold enhancement of cell proliferative inhibitory activity in a peptide composition. According to the invention, this enhancement is achieved by conjugating the peptide to a polyunsaturated fatty acid. Preferred peptides and fatty acyl moieties in the method are described above.
  • the invention provides fatty acyl-peptide compositions having enhanced biological activity, compared to the activity of the peptide alone.
  • Such enhanced biological activity includes such activities as enhanced hormone activity, anti-tumor activity, enhanced immunogenic activity and other peptide-specific activities.
  • Figure 1 shows exemplary peptide sequences, designated sequences 1-31 and identified by SEQ ID NO: 1-31, respectively, used in forming the novel peptide-fatty acyl compositions of the invention: STKS (sequence 1, SEQ. ID NO: 1), STKSI (sequence 2, SEQ ID NO: 2), SPKCS (sequence 3, SEQ. ID NO: 3) , ⁇ omatostatin analogue (sequence 4, SEQ. ID NO: 4 ), where lower case “d” signifies the presence of a D-amino acid residue and ' "NH2" at the C-terminal signifies the amidation of the C-terminal end, GnRH (sequence 5, DHA-SEQ. ID NO: 5), where "Glp” signifies the presence of pyroglutamate, EGFA (sequence 6, SEQ ID NO: 6); an N-terminal sequence of human PTH (sequence 7, SEQ ID NO:
  • polypeptide fragment derived from fibronectin (sequence 8, SEQ ID NO: 8), and a T cell epitope modulatory peptide (sequence 9, SEQ ID NO: 9) suitable for use in forming compositions of the present invention
  • sequences of protein kinase modulatory peptides such as tyrosine kinase inhibitory peptides (sequence 10, SEQ ID NO: 10), (sequence 11, SEQ ID NO: 11), (sequence 12, SEQ ID NO: 12) where "ACM" signifies that the cysteine ⁇ ulfhydryl group has been modified by an acetamidomethyl group, (sequence 13, SEQ ID NO: 13) and (sequence 14, SEQ
  • calmodulin-dependent protein kinase III inhibitory peptide sequence 15, SEQ ID NO: 15
  • dsDNA-dependent kinase inhibitory peptide sequence 16, SEQ ID NO: 16
  • protein kinase C modulatory peptide sequence 17, SEQ ID NO: 17
  • other protein kinase modulatory pep ⁇ tides such as for cAMP dependent kinase (sequence 18, SEQ ID NO:18) , (sequence 19, SEQ ID NO: 19) , and (sequence 20, SEQ ID NO: 20), phosphoryla ⁇ e kinase (sequence 21, SEQ ID NO: 21), calmodulin-dependent kinase I and II (sequence 22,
  • SEQ ID NO: 22 and sequence 23 SEQ ID NO: 23
  • dsRNA-dependent kinase sequence 24, SEQ ID NO: 24
  • proline-dependent kinase sequence 25, SEQ ID NO: 25 and sequence
  • SEQ ID NO: 27 casein kina ⁇ e I and II (sequence 28, SEQ ID NO:28 and ⁇ equence 29, SEQ ID NO: 29, AMP-activated protein kina ⁇ e ( ⁇ equence 30, SEQ ID NO:30), and S6 kinase
  • Figure 2 illustrates coupling of ci ⁇ -4, 7, 10, 13 , 16, 19- doco ⁇ ahexaenoic acid (DHA) to a peptide ( ⁇ equence 1, SEQ ID NO: 1) through an N-terminal amine group to form the fatty acyl-peptide compo ⁇ ition (compo ⁇ ition I, DHA-SEQ ID NO: 1) ; and
  • Figure 3 shows sequences of exemplary fatty acyl-pep ⁇ tide composition ⁇ of the invention: DHA-STKS (compo ⁇ ition I, DHA-SEQ. ID NO. : 1) , DHA-STKSI (composition II, DHA-SEQ ID NO: 2), DHA-SPKCS (composition III, DHA-SEQ. ID NO. : 3) ,
  • DHA-somatostatin analogue composition IV, DHA-SEQ. ID NO. : 4
  • D-Lys6 DHA
  • GnRH composition V
  • DHA-EGFA composition VI, DHA-SEQ ID NO: 6
  • polyunsaturated fatty acid refers to a com ⁇ pound having a carboxylic acid moiety and a long unbranched carbon chain, u ⁇ ually containing between about 8 and 24 carbon atoms, and containing two or more carbon-carbon double bonds.
  • a fatty acid i ⁇ conjugated to a peptide through an amide linkage a fatty acyl-peptide derivative is formed.
  • the disclosed invention includes fatty acyl- peptide derivatives, and, more generally, peptides linked to long unbranched, polyunsaturated carbon chains.
  • peptide hormone refers to a peptide that elicit ⁇ a biological response in a target cell. Peptide hormones are generally low molecular weight proteins ( ⁇
  • Such hormones can be isolated from biological sources, chemically synthesized or produced by recombinant methods. Generally, in their natural setting, peptide hor ⁇ mones are ⁇ ecreted from specific cells and produce biological effects in other cells. Analogues of such naturally occurring hormones are encompas ⁇ ed by the term "peptide hormone" and are produced recombinantly or synthetically.
  • Peptide hormones as described herein, are divided into two main categories, according to their known acti ⁇ vities in vivo .
  • J hormones act either directly or indirectly to produce the desired biological effect.
  • the desired effect is inhibition of cell proliferation.
  • Peptide hormones that act directly on a cell to inhibit division of the cell are referred to herein as direct peptide hormones; those pep ⁇ tides that act to, stimulate or inhibit synthesis or secretion of endogenous peptide growth regulatory hormones are referred to as indirect peptide hormones.
  • an indirect peptide hormone effect a change (increa ⁇ e or decrease) in the extracellular levels of a naturally occurring direct pep ⁇ tide hormone.
  • Two exemplary types of indirect peptide hor ⁇ mones are peptide hormone-releasing hormones and peptide hormone release-inhibiting hormones.
  • Naturally occurring indirect peptide hormones are generally short polypeptides, usually under 20 amino acids in length.
  • Peptide hormone analogs are synthetically or recombinantly prepared peptides which are structurally similar to naturally occurring peptide hormones. For the purpose ⁇ of the present invention, such peptide analogs are included by the term "peptide hormone.” Generally, to be useful in the present invention, such analogs have essentially a similar or a higher biological activity than that of the endogenous peptide hormone. Protein kinase modulatory peptides are peptides which act as inhibitor ⁇ of protein kinase activity. Such pep ⁇ tides may act, for example, as protein kina ⁇ e substrates, residue containing a phosphorylatable amino acid/in the ⁇ equence.
  • such kinase modulatory peptide ⁇ may bind to the kina ⁇ e catalytic site, to effect inhibition of kina ⁇ e activity.
  • Such kina ⁇ e modulatory peptides act to produce reduced pho ⁇ phorylation of endogenous protein kinase substrate ⁇ .
  • Peptide ⁇ and compo ⁇ itions that inhibit cell proliferation are ⁇ ometime ⁇ referred to herein as. "anti ⁇ proliferative" peptides or compositions.
  • the present invention is directed to peptide compositions having anti-proliferative activity. More generally, it is the discovery of the invention that these fatty acyl-peptide composition ⁇ have enhanced biological activity in comparison to underivatizea peptides.
  • Anti ⁇ proliferative fatty acyl-peptide compositions of the invention are effective to inhibit proliferation of highly proliferative cells, such as neoplastic cells or virally- infected cells.
  • peptides known to have anti-proliferative activity are linked to a fatty acyl moiety, ⁇ uch a ⁇ a doco ⁇ ahexae ⁇ oyl (DHA) or eico ⁇ apentaenoyl (EPA) group, a ⁇ described below.
  • Peptide hormones having cell proliferative inhibitory activity are known in the art, and may be direct or indirect hormones.
  • Such anti ⁇ proliferative peptide hormone ⁇ are u ⁇ ually, but not necessarily, specific to a particular type of cell, such as a cell with ⁇ pecific hormonal requirement ⁇ .
  • indirect peptide hormones include those peptides which act to effect a decrease in the level of a direct cell proliferation ⁇ timulatory hormone or to effect an increase in the level of a direct cell proliferation inhibitory hormone.
  • GnRH gonadotropin relea ⁇ ing hormone
  • LH luteinizing hormone
  • FIG. 15 inhibit the growth of hormone dependent carcinomas, such as androgen-dependent prostate tumors (Schally) .
  • GnRH is also effective against such tumors a ⁇ colon and pancreatic tumors.
  • Figure 1 shows a sequence of a GnRH analog useful in treating such tumor ⁇ (5, SEQ ID NO: 5) .
  • Figure 3 ⁇ hows
  • GnRH analogues can be ⁇ creened for potential anti ⁇ proliferative activity, by assessing their abilities to
  • Indirect peptide hormones also include peptide hormone release-inhibiting hormone ⁇ .
  • Somatostatin i ⁇ a 14 amino acid peptide that inhibit ⁇ the relea ⁇ e of growth hormone (GH) .
  • GH enhances the proliferation of its cellular ⁇ 35 targets.
  • Native somatostatin peptide and analogs are used to form fatty acyl peptide derivative composition ⁇ of the invention, as described below.
  • the somatostatin fatty-acyl peptide is about 150 times more potent than the parent molecule.
  • fatty acyl-peptide compositions of the invention can be used in forming fatty acyl-peptide compositions of the invention.
  • peptides are selected based on their known anti-proliferative activity in a cell proliferation as ⁇ ay, described in Section III below. Selected peptides are then conjugated to fatty acyl moieties, and are tested for enhanced potency in such a ⁇ ays .
  • a fatty-acyl peptide derivative composition i ⁇ a useful anti-tumor or anti-neoplastic cell agent, when it is found to have at least a several-fold increase in potency, in comparison to the unconjugated peptide.
  • Direct Peotide Hormone ⁇ Also used in forming compositions of the invention are peptides which are known to have direct antiproliferative effects on cells.
  • An exemplary direct peptide hormone is parathyroid hormone (PTH) .
  • PTH parathyroid hormone
  • This peptide i ⁇ 84 amino acids in length, and can directly inhibit osteobla ⁇ t division.
  • Certain bone cancers are characterized by hyperproliferating o ⁇ teoblast ⁇ . Structure-function studies indicate that an N-terminal fragment of PTH is active in inhibiting osteobla ⁇ t proliferation (Kano) .
  • Thi ⁇ ⁇ equence 7 SEQ ID NO: 7 7) i ⁇ ⁇ hown in Figure 1.
  • a peptide which i ⁇ defined as a direct peptide hormone for purpo ⁇ es of thi ⁇ invention is a peptide fragment of fibronectin having the ⁇ equence SEQ ID NO: 8, ⁇ hown in Figure 1 as sequence 8. This fragment spans the recognition site within the fibronectin molecule to which cells bind for attachment to fibronectin.
  • This fibronectin recognition site peptide and analogues thereof are u ⁇ ed to inhibit fibronectin-mediated cell attachment and ⁇ preading in vitro. They may also be important for regulation of tumor cell proliferation and metasta ⁇ i ⁇ in vivo (Kumagai) .
  • fibronectin recognition site peptides are coupled to fatty acyl moieties to form compositions useful in inhibiting tumor cell proliferation and meta ⁇ tasis.
  • Direct peptide hormone ⁇ a ⁇ defined in the current invention, al ⁇ o encompa ⁇ s peptides derived from immunogenic polypeptides for modulating a response from ⁇ pecific immune cell populations.
  • a T-cell epitope peptide (TCEP) i ⁇ coupled to a fatty acyl moiety as described herein to form a composition useful in modulating the T- cell immune response.
  • SEQ ID NO:9 The sequence of TCEP (SEQ ID NO:9) is shown in Figure 1 as ⁇ equence 9, and ⁇ ynthe ⁇ i ⁇ of a DHA derivative of TCEP is detailed in Example 7.
  • Elevated protein kinase C and tyrosine kinase activities are associated with neoplastic cell prolif ⁇ eration or transformation (Wein ⁇ tein, Yarden) . Inhibition of such kinase activities can be effected by the presence of small peptide fragments which mimic protein ⁇ ub ⁇ trate pho ⁇ phorylation sites and/or protein kinase modulatory domains. Such protein kinase modulatory peptides are effective to compete with endogenous protein kina ⁇ e sub ⁇ trate ⁇ . Selection of ⁇ pecific protein kinase modulatory peptides for use in forming the fatty acyl-pep- tide derivatives of the invention i ⁇ de ⁇ cribed below.
  • Tyrosine Kinase Substrates and Inhibitors Receptors for a number of growth factors, including epidermal growth factor (EGF) , insulin growth factor (IGF) , and platelet derived growth factor (PDGF) , contain tyrosine kinase catalytic domains which phosphorylate specific intracellular protein substrates, including the receptor itself, in some cases, at tyrosine residues.
  • EGF epidermal growth factor
  • IGF insulin growth factor
  • PDGF platelet derived growth factor
  • Tyrosine kina ⁇ es may also play a role in the uncontrolled growth of keratinocytes which can result in psoriasi ⁇ or other highly proliferative skin di ⁇ order ⁇ . Keratinocytes pos ⁇ es ⁇ tyrosine kinase-like growth factor receptors.
  • Figure 1 shows ⁇ equence ⁇ of ⁇ everal exemplary tyrosine kinase modulatory peptide ⁇ which can be u ⁇ ed to form anti-proliferative compo ⁇ ition ⁇ of the invention, ⁇ uch a ⁇ STKS (SEQ ID NO: 1) and STKSI (SEQ ID NO: 2), ⁇ equence ⁇ 1 and 2, re ⁇ pectively.
  • tyrosine kinase modulatory peptides can be identified, based on peptide ⁇ equence ⁇ surrounding phosphorylation sites of endogenous protein kina ⁇ e ⁇ ub ⁇ trates in highly proliferating cells.
  • cellular sources used for identification of ⁇ uch substrate peptides are proliferating cells of the type to be targeted; however, it is appreciated that tyrosine kinase modulatory peptide ⁇ derived from one cell ⁇ ource may inhibit a tyrosine kinase derived from a different cell type.
  • proliferating cells such as a ⁇ a lymphoma cell line having a high level of tyrosine kinase activity, are incubated with [32p] -pho ⁇ phate, or a particulate fraction of the cells i ⁇ incubated with radiolabelled [gamma 32p] ATP.
  • Cellular polypeptide ⁇ are ⁇ eparated by denaturing gel electrophore ⁇ i ⁇ , and the pho ⁇ phorylated protein ⁇ are observed by autoradiography. Phosphoprotein- containing bands are excised from the gel, and the phosphoprotein i ⁇ eluted from the gel band.
  • the eluted protein i ⁇ ⁇ ubjected to partial hydroly ⁇ is, and the identity of the phosphorylated amino acid determined, according to methods known in the art (Casneillie) .
  • phosphoprotein ⁇ are ⁇ ubjected to peptide fragmentation, such a ⁇ by proteoly ⁇ i ⁇ or chemical means, and phosphotyrosine-containing peptides are sequenced, according to conventional methods (Casneil- lie) .
  • Short peptides (approximately 6-20 amino acids) corresponding to the peptide sequence ⁇ in the vicinity of the pho ⁇ phorylation ⁇ ite can be ⁇ ynthesized and te ⁇ ted for inhibition of tyro ⁇ ine kinase activity.
  • Tyrosine kinase modulatory peptides can be prepared by substituting for the tyrosine phosphorylation ⁇ ite residue an amino acid residue that cannot be phosphorylated, ⁇ uch as a phenylalanine residue, composition II in Figure 3 (DKA-STKSI) ⁇ how ⁇ an inhibitory fatty acyl-peptide compo ⁇ ition, the peptide portion of which ha ⁇ almo ⁇ t complete identity with the peptide portion of composition I
  • composition II a phenylalanine residue has been substituted for a tyrosine residue present in composition I.
  • Both peptides inhibit proliferation of neopla ⁇ tic cell ⁇ . Accordingly either peptide can be conjugated to a fatty acyl group, to form an anti-tumor fatty acyl-peptide compo ⁇ ition of the invention, a ⁇ described in Section III, below.
  • PKC Protein Kinase C
  • PKC Peotide Substrates.
  • PKC is a component of the phospholipid metabolism/protein kinase C signal tran ⁇ duction pathway which play ⁇ a critical role in normal cellular growth control. Activation of PKC is mediated by a family of G-protein-modulated receptors. When activated, the cyto ⁇ olic form of PKC binds to the cytoplasmic face of the plasma membrane.
  • One of the known protein targets of PKC i ⁇ the EGF receptor.
  • Pho ⁇ phoryla- tion of EGF receptor by protein kina ⁇ e C results in a decrease in the affinity of the receptor for EGF and a decrease in EGF receptor-associated tyrosine kinase activity (Berridge) .
  • Thi ⁇ illustrates the heterologous interaction ⁇ between signal transduction pathways, specifically between the tyrosine kinase pathway and the phospholipid metabolism/protein kina ⁇ e C pathway.
  • Figure 1 lists the ⁇ equence ⁇ of some of the known peptide substrate inhibitors for PKC, such as sequence 3 (SEQ ID NO: 3) and sequence 17 (SEQ ID NO: 17). These peptides are suitable PKC modulatory peptides for use in forming fatty acyl-pep- tide compositions of the present " invention. PKC peptide inhibitors can also be identified from endogenous protein sub ⁇ trates of protein kinase C, using the same general procedures described in part 1, above, for tyrosine kinases. 3. Other Protein Kinases.
  • Modulatory peptides targeting protein kinases other than tyrosine kinase and protein kina ⁇ e C can also be used to form fatty acyl-pep ⁇ tide anti-tumor compo ⁇ ition ⁇ of the invention.
  • High proliferation rates are related to the hyperpho ⁇ phorylation of an oncogenic product from a human papillomavirus which contains a casein kinase II phosphorylation consen ⁇ us ⁇ equence (Hashida) .
  • PKA cyclic AMP-dependent protein kinase
  • cyclic AMP turns on the gene that encodes the peptide hor ⁇ mone release-inhibiting hormone, somatostatin.
  • target protein kinases in conjunction with the present invention, are components of signalling cascades involved in cell proliferation. For example, when
  • epidermal growth factor binds to the epidermal growth factor receptor (EGFR), a tyrosine kinase, it stimulate ⁇ activation of myelin basic protein (MBP) kinase, through phosphorylation of MBP kinase. MBP kinase, in turn, activates S6 kinase by a phosphorylation event.
  • EGFR epidermal growth factor receptor
  • MBP myelin basic protein
  • EGFR-bearing cells ⁇ uch as adipocyte cells (Ahn) .
  • fatty acyl-peptide compositions of the invention using a ⁇ peptide co ponent ⁇ , pho ⁇ phorylation recognition ⁇ equence peptide ⁇ for one or , 35 more of EGFR kina ⁇ e, MBP kinase and S6 kinase will serve to inhibit the signalling cascade of which these kinases are a part. Sequences of exemplary peptide inhibitors of these kinases are shown in Figure 1 (e.g. , sequence 11, SEQ ID NO: 11, to sequence 31, SEQ ID NO:31) .
  • protein kinase activities involved in cell proliferation whose activities can be modulated include calmodulin-dependent kinase ⁇ I and II, cGMP-dependent protein kinase, ds-DNA-dependent protein kinase, proline-dependent kina ⁇ e, and AMP-activated kina ⁇ e.
  • Sequences of exemplary peptide modulatory peptide ⁇ directed to the ⁇ e kina ⁇ es are listed in Figure 1 (SEQ ID NOs: 18- 31) .
  • the peptide ⁇ were conjugated to fatty acyl moietie ⁇ , a ⁇ described below and illu ⁇ trated in Figure 2 and te ⁇ ted for thei ⁇ activities in cell proliferation assays, as described in Section III, below.
  • Peptides shorter than about 30 amino acids in length are conveniently prepared by methods commonly used in solid-phase peptide synthesis, as detailed below (Stewart) .
  • N-alpha-protected amino acid anhydrides are prepared in crystallized form and used for successive amino acid addition to the peptide N-terminus.
  • the growing peptide on a solid support is acid treated to remove the protective group, and washed several times to remove residual acid.
  • the peptide i ⁇ then reacted with another N-protected amino acid.
  • the amino acid addition reaction may be repeated two or three time ⁇ to increase the yield of growing peptide chains.
  • the protected pep ⁇ tide re ⁇ in i ⁇ treated with liquid hydrofluoric acid to deblock and release the peptide ⁇ from the ⁇ upport.
  • Preferred fatty acyl moietie ⁇ for use in the present invention are those with a high degree of unsaturation, and include such fatty acids as cis-5, 8, 11, 1 , 17- eico ⁇ apentae ⁇ oic (EPA) and ci ⁇ - ,7, 10, 13, 16, 19- doco ⁇ ahexaenoic acid (DHA) .
  • EPA eico ⁇ apentae ⁇ oic
  • DHA doco ⁇ ahexaenoic acid
  • uch polyunsaturated fatty acids can be prepared synthetically according to standard methods, isolated from the oils of marine fish, or obtained from commercial ⁇ ource ⁇ .
  • fatty acid ⁇ are linked to the peptide ⁇ via the terminal amine group or via internal amine groups, such as the amine group of ly ⁇ ine, in an amide linkage.
  • Figure 2 illustrates a scheme for coupling of ⁇ equence 1 to DHA by the terminal amino group of the peptide through an amide linkage to form the fatty acyl-peptide compo ⁇ ition I in
  • DHA is activated by reaction with N- hydroxy ⁇ uccinimide prior to reaction with an a ino-group containing polypeptide.
  • DHA i ⁇ coupled by the free amine group of a lysine residue in the ⁇ equence to form the fatty acyl composition, ⁇ uch a ⁇ compo ⁇ ition V in Fig. 3.
  • Example ⁇ 2-6 detail preparation of fatty acyl-peptide compositions in which acylation of peptides i ⁇ carried out u ⁇ ing activated e ⁇ ter or acyl chloride derivative ⁇ of DHA or EPA.
  • Activated esters useful in preparing composition ⁇ of the current invention include DHA-O-benztriazole ester (DHA-OBT) , DHA-O-pentafluorophenyl ester (DHA-Opfp) , and DHA-O- ⁇ uccin midyl ester (DHA-O-N-Succ) .
  • the acylation is carried out as detailed in Example 2. Crude products, obtained after acylation, are purified by HPLC. The purity of the final product ⁇ l ⁇ characterized by analytical HPLC and TLC data, while the chemical characterization l ⁇ accompli ⁇ hed by mass ⁇ pectrometry (MS) .
  • MS mass ⁇ pectrometry
  • peptides can be selected for use in anti-proliferative fatty acyl-peptide composition ⁇ of the invention, ba ⁇ ed on their abilities to inhibit cell proliferation or to inhibit component ⁇ , such as protein kinase component ⁇ , of cell proliferative ⁇ timulu ⁇ pathway ⁇ .
  • component ⁇ such as protein kinase component ⁇
  • conjugating to ⁇ uch a peptide, a polyun ⁇ aturated long chain carbon, ⁇ uch as a polyunsaturated fatty acyl moiety described above enhances the biological activity of the peptide.
  • conjugation of a polyunsaturated fatty acyl moiety to an anti-proliferative peptide enhances its ability to inhibit neoplastic cell proliferation.
  • This aspect of the invention will be better appreciated from the discussion below.
  • tyrosine kinase modulatory peptide ⁇ selected a ⁇ de ⁇ cribed above are covalently linked to polyunsaturated fatty acids to form compositions which are effective to inhibit proliferation of neoplastic cells.
  • DHA-STKS DHA-SEQ. ID
  • Fatty acid-tyro ⁇ ine kinase inhibitory peptide compo ⁇ sitions can also be prepared by substituting for the tyro ⁇ sine residue an amino acid re ⁇ idue that cannot be pho ⁇ - phorylated.
  • DHA-STKSI DHA-SEQ ID NO: 1
  • composition II the tyrosine residue of tyrosine kinase sub ⁇ trate DHA-STKS (DHA-SEQ ID NO: 1; compo ⁇ ition I) ha ⁇ been ⁇ ub ⁇ tituted by a phenylalanine residue.
  • DHA-STKS DHA-SEQ ID NO: 1; compo ⁇ ition I
  • Both fatty acid-protein kinase peptide sub ⁇ trate and peptide inhibitor compositions inhibit the proliferation of neoplastic cells, as detailed in Example 8 and shown in
  • DHA-EGFA DHA-SEQ ID NO: 6, composition VI
  • a PKC peptide sub ⁇ trate-fatty acid compo ⁇ ition u ⁇ ed in the experiment ⁇ de ⁇ cribed in Example 8 include ⁇ a peptide fragment from myelin ba ⁇ ic protein, and has the structure shown in Figure 3 as compo ⁇ ition III (DHA-SEQ. ID NO: 3) . Additionally, endogenous substrates of PKC may be identified as described above for the tyrosine kinase substrates. The polypeptide fragments that contain PKC phosphorylation sites are then sequenced and u ⁇ ed to form the fatty acid peptide compositions of the invention.
  • fatty acyl- peptide derivatives are prepared as described in Section II, above, and inhibition of neoplastic cell proliferation by such compositions is measured, according to one or more standard cell proliferation assays.
  • inhibition of cell proliferation is mea ⁇ ured directly, by measuring the number of surviving cells after exposure of the cells to a test composition, ⁇ uch a ⁇ a fatty acyl-peptide compo ⁇ ition di ⁇ clo ⁇ ed herein.
  • an activity which correlate ⁇ to cell proliferation can be mea ⁇ ured to determine indirectly the effect of a te ⁇ t compound on cell proliferation.
  • activitie ⁇ ⁇ uch as tyrosine kina ⁇ e activity or relea ⁇ e of a growth-promoting hormone, which are known to correlate with neopla ⁇ tic cell proliferation, are u ⁇ ed a ⁇ indicators of anti-proliferative activity.
  • polyunsaturated fatty acyl-peptide anti-proliferative compositions may exhibit one or more of the following in vi tro activities: (a) inhibition of cell proliferation, (b) inhibition of tyrosine kinase activity, (c) stimulation of release of a growth-inhibiting hormone, such as lu- teinizing hormone, and (d) inhibition of release of growth- ⁇ timulating hormone ⁇ , ⁇ uch a ⁇ inhibition of growth-hormone relea ⁇ e.
  • vi tro assay ⁇ which can be correlated with uncontrolled cell growth may also serve as assay ⁇ for ⁇ electing and determining the activities of fatty acyl-peptide compo ⁇ ition ⁇ of the invention. More ⁇ pecifically, ⁇ uch assay ⁇ can ⁇ erve to determine the relative potencie ⁇ of compositions of the invention as anti-proliferative agents.
  • Example 8 An as ⁇ ay for mea ⁇ uring the inhibitory effect ⁇ of polyun ⁇ aturated fatty acyl-peptide derivative ⁇ on cell proliferation is described in detail in Example 8.
  • a number of tumor cell lines including a human prostatic adenocarcinoma cell line, a human breast adenocarcinoma cell line and a human colon adenocarcinoma cell line, were used to test compound ⁇ of the invention for effect ⁇ on cell proliferation.
  • DHA-STKS tyrosine kinase synthetic peptide ⁇ ub ⁇ trate fatty acid derivative DHA-STKS
  • DHA-SEQ ID NO: 3 protein kina ⁇ e C peptide ⁇ ubstrate fatty acid derivative DHA-STKCS
  • DHA-SEQ ID NO: 5 gonadotropin relea ⁇ e hormone fatty acid derivative DHA-D- Ly ⁇ 6-GnRH
  • Solution ⁇ of the peptide derivative ⁇ were added in culture media to test cells for an incubation period of 2.5 hours. At the end of the test period, cells were centrifuged, the pellet diluted with 1% BSA in saline and viable tumor cell counts were determined by the trypan blue exclusion method. Results of tests using peptide fatty acid derivatives DHA-STKS (DHA-SEQ. ID NO: 1) and DHA-D- lys6-GnRH (DHA-SEQ. ID NO: 5) in various transformed cell lines are ⁇ hown in Table 1.
  • PC 3 human prostatic adenocarcinoma cell line
  • MCF 7 human brea ⁇ t adenocarcinoma cell line
  • HT 29 human colon adenocarcinoma cell line.
  • Additional method ⁇ are available for monitoring the viability of cells, including as ⁇ ays based on the differential dye uptake by viable cells in comparison to that taken up by non-viable cells.
  • viable cells take up diacetyl fluore ⁇ cein and hydrolyze it to fluore ⁇ cein, to which the cell membrane of live cells is impermeable. Live cells fluoresce green.
  • Nonviable cells may be counter-stained with ethidium bromide and will fluoresce red.
  • LH Luteinizing Hormone
  • fatty acyl derivatives of ⁇ uch compound ⁇ are effective to inhibit neoplastic cell proliferation at concentrations which are several-fold lower than the concentration of peptide alone required to inhibit such cell proliferation.
  • an analog of gonado- tropin releasing hormone analog conjugated to the poly ⁇ unsaturated fatty acid, DHA, (D-Lys 6 (DHA) -GnRH; DHA-SEQ ID NO: 5) was tested in an a ⁇ ay of LH release by anterior pituitary cell suspensions as detailed in Example 9.
  • luteinizing hormone level ⁇ were quantitated by a double antibody radioimmunoa ⁇ ay procedure.
  • Peptide hormone-release inhibiting hormones decrease the extracellular levels of peptide hormones which interact with ⁇ pecific cellular receptor ⁇ . Generally, binding of peptide hormone to the receptor trigger ⁇ a ca ⁇ cade of biochemical events mediated through second me ⁇ enger ⁇ . In some cases, an end-result of such hormonal activity is cellular proliferation. Peptide hormone-release inhibiting hormone ⁇ which act to inhibit such hormone ⁇ are useful in forming fatty acid-peptide compo ⁇ ition ⁇ described by the pre ⁇ ent invention. Analogue ⁇ of ⁇ uch peptide hormone- release inhibiting hormones are also u ⁇ eful in forming ⁇ uch compo ⁇ ition ⁇ .
  • Somato ⁇ tatin i ⁇ an exemplary peptide hormone-release inhibiting hormone analogue which inhibits hormonally activated cell proliferation.
  • Somatostatin i ⁇ a growth hormone relea ⁇ e inhibitory hormone which inhibit ⁇ relea ⁇ e of growth hormone.
  • Growth hormone (GH) binds to specific cell-surface receptors distributed widely throughout the body. Binding of GH agonists to GH receptor ⁇ re ⁇ ult ⁇ in increa ⁇ ed cellular division, and hence, cell proliferation.
  • a ⁇ omato ⁇ tatin-fatty acyl analogue (SEQ ID NO: 4) was ⁇ ynthesized as detailed in Example 5. Inhibition of growth hormone relea ⁇ e by the DHA-acylated somatostatin analogue was measured, as de ⁇ cribed in Example 10. GH levels were determined by a double-antibody radio- immunoassay for the hormone. In thi ⁇ a ⁇ ay, the DHA- somato ⁇ tatin analogue wa ⁇ 1000 time ⁇ more effective in stimulating release of growth hormone than was either unacetylated somatostatin analogue or somato ⁇ tatin.
  • tyrosine kinase activity can be measured in cell ⁇ from the human brea ⁇ t adenocarcinoma line MDA-MB-231. Briefly, the ⁇ e cell ⁇ are incubated with a te ⁇ t compound, ⁇ uch as the polyunsaturated fatty acid-tyro ⁇ ine kinase peptide inhibitor DHA-STKSI (DHA-SEQ ID NO: 2), then harvested and homogenized.
  • DHA-STKSI DHA-SEQ ID NO: 2
  • Tyrosine kinase activity is measured by incorporation of radiolabeled pho ⁇ phate into an endogenous protein substrate in the cell ⁇ .
  • the endogenou ⁇ protein substrate is the EGF receptor, and the pho ⁇ phorylation event i ⁇ an autopho ⁇ phorylation event.
  • STKSI the underivatized peptide inhibitor
  • DHA-STKSI DHA-SEQ ID NO:2
  • DHA pre ⁇ ent by it ⁇ elf at 20 microgram ⁇ /ml decrea ⁇ ed the extent of pho ⁇ phorylation by only 5%. It can be appreciated, in accordance with the pre ⁇ ent invention, that similar cellular as well as in vi tro tyrosine kina ⁇ e assays can be used to monitor activity and to test compounds for their abilities to inhibit ⁇ uch activity.
  • Fatty acyl-peptide compositions of the invention are useful in inhibiting uncontrolled proliferation of cell ⁇ , such a ⁇ benign and malignant tumor cell ⁇ , virally-infected cell ⁇ , p ⁇ oriatic cells, and the like.
  • cell ⁇ such as a ⁇ benign and malignant tumor cell ⁇ , virally-infected cell ⁇ , p ⁇ oriatic cells, and the like.
  • the use of the composition of the invention for inhibition of such cell proliferation is illustrated by experiments summarized in Table 1.
  • peptides are selected for use in forming compo ⁇ ition ⁇ of the invention, ba ⁇ ed on their known or experimentally determined activities in inhibiting cell proliferation. Selected peptides are then used to form polyunsaturated fatty acyl-peptide composition ⁇ , according to the general method ⁇ described in Section II, above and detailed in Example ⁇ 1-7.
  • polyunsaturated fatty acyl-peptide compositions of the pre ⁇ ent invention are effective to produce a ⁇ everal-fold enhancement of anti-proliferative activity, in compari ⁇ on to peptides alone.
  • fatty acyl-GnRH and -GnRH analogues will find use in treating androgen-dependent prostate adenocarcinoma ⁇ .
  • Te ⁇ t GnRH compound ⁇ can be screened in an LH relea ⁇ e as ⁇ ay, a ⁇ described in Example 9, then tested in an experimental animal model, such as a rat bearing the Dunning R-3327-H prostate adenocarcinoma.
  • GnRH derivative ⁇ of the invention are expected to find usefulne ⁇ in treatment of e ⁇ trogen-dependent mammary tumors, and their efficacy can.be mea ⁇ ured in rats ( ⁇ uch a ⁇ Wi ⁇ tar Furth rat ⁇ ) carrying a mammary tumor ⁇ uch a ⁇ the MT/W9A mammary tumor, according to methods known in the art.
  • LHRH analog composition ⁇ will inhibit growth of certain pituitary tumor ⁇ , chondro ⁇ arcoma ⁇ , and o ⁇ teo ⁇ arcoma ⁇ (Schally) .
  • An anti-neopla ⁇ tic or anti-tumor treatment method, a ⁇ described herein, include ⁇ expo ⁇ ing target neopla ⁇ tic cell ⁇ to a concentration of fatty acyl-peptide compound effective to inhibit neopla ⁇ tic cell proliferation at lea ⁇ t about 50%, and preferably about 90%.
  • effective concentration ⁇ can be determined in an in vi tro a ⁇ ay, a ⁇ de ⁇ cribed in Section IV, above.
  • the method of the invention ha ⁇ general utility in enhancing biological activity of a biologically active peptide, by attaching to the peptide a polyun ⁇ aturated fatty acyl moiety.
  • Example 1 Synthesis of Peotide ⁇ Solid-phase synthesis was carried out according to standard method ⁇ .
  • the ⁇ ynthesi ⁇ i ⁇ performed u ⁇ ing a Beckman model 900 peptide ⁇ ynthe ⁇ izer.
  • Side chain protecting group ⁇ include for cysteine, 4- Met-Benzyl; for lysine, 2-chlorobenzyloxycarbonyl; for serine, benzyl; for arginine, tosyl; for threonine, benzyl; for a ⁇ partate, benzyl; and for tyrosine, 2-bromobenzyl- oxycarbonyl.
  • a mixture of protected peptide re ⁇ in (1.32 g) , 2- mercaptopyridine (0.5 g) , p-cre ⁇ ol (2.6 g) , and liquid hydrogen fluoride (HF) (25 ml) is stirred at 0 degrees with a rapid stream of nitrogen ga ⁇ , first below 0 degree ⁇ , then at 24 degree ⁇ .
  • the mixture i ⁇ ⁇ tirred in ethyl acetate (25 ml) until a finely divided ⁇ olid i ⁇ obtained.
  • the ⁇ olid i ⁇ filtered, washed with ethyl acetate, and air dried.
  • the solid is stirred in 50% aqueous acetic acid (10 ml) to dis ⁇ olve the peptide, filtered and wa ⁇ hed with 20 ml water.
  • the mixture was ⁇ tirred overnight under He atmo ⁇ phere for the completion of the transe ⁇ terification reaction.
  • This reaction mixture can be used directly for the N-terminal acylation of the required peptide or worked up for the preparation of pure DHA-O-N-Succ .
  • the reaction mixture was diluted to 30 ml with water and extracted with 3 x 10 ml of peroxide-free ether (treated and stored over alumina) .
  • the combined ether phase was back-extracted with 3 x 5 ml of water.
  • the ether solution wa ⁇ dried over anhydrou ⁇ ⁇ odium ⁇ ulphate, evaporated to dryne ⁇ s and taken up in 4 ml of acetone.
  • Example 3 Preparation of DHA-STKSI (DHA-SEQ ID NO. 2) A procedure similar to that described in Example 2 was used to generate the activated ester group of DHA (DHA-O-N- ⁇ uccinimide) . Then, 0.03 mM of STKSI (36.2 mg) was acylated with 0.05 mM of DHA-O-N-Succ ester in the presence of 0.15 mM of DIEA according to Example 2. The product wa ⁇ eluted from the same HPLC gradient a ⁇ described in Example 2, the product eluted at 64.5% B. The final yield was 82%. The chemical characterization of the compound wa ⁇ carried out by mass ⁇ pectrometry. MH+theoretical: 1517.4, MH+mea ⁇ ured:1515.5.
  • Example 4 Preparation of DHA-SPKCS (DHA-SEQ ID NO: 3) SPKC-(Ly ⁇ /ep ⁇ ilon-TFA/)2,9 (54.8 mg, 0.04 mM, final concentration) wa ⁇ ⁇ u ⁇ pended in 3 ml of water-acetone (1:1) containing 0.08 mM DIEA. 'Crude DHA-O-N- Succ activated ester in DMF solution (0.04mM), a ⁇ de ⁇ cribed in Example 2, wa ⁇ added, and the suspension was stirred overnight under a He atmo ⁇ phere. The reaction mixture was evaporated to dryness and the product was TFA-deprotected in the following fashion.
  • the product was eluted at 11.5 minutes, the yield was 65%, the purity of the product was 97%.
  • the product was analyzed by mas ⁇ spectrometry, with the following result ⁇ : MH+theoretical:1700.8; MH+measured: 1700.8
  • DHA-SEQ ID NO: 4 360 ul each of 0.5 mM ⁇ olution ⁇ of DHA, N,N'-dii ⁇ o- propyl carbodiimide (DIC) , and pentafluorophenol in dimethylformamide (DMF) were mixed and kept at 25. oC.
  • Step 1 I ⁇ ocratic elution with 50 ml of 2-propa- nol-acetic acid-water (20:40:40) .
  • Step 2 Gradient elution with 400 ml of elution mixture applied in ⁇ tep 1 and 400 ml of 2-propanolacetic acid-water (35:32.5:32.5) .
  • the purity of the fraction ⁇ was checked by HPLC, the purest fraction ⁇ were pooled, evaporated, lyophilized and yielded 57% product. The purity of the product wa ⁇ 92%. The composition of the product was analyzed by mas ⁇ ⁇ pectrometry.
  • Example 7 Svnthe ⁇ is of DHA-T Cell Epitope Peptide (DHA-SEQ ID NO: 12) T-cell epitope peptide (TCEP) (132 mg, 0.075 mmole) , with TFA and formyl protecting groups, was di ⁇ olved in DMSO (2 ml) containing DIEA (40 ⁇ l, 0.30 mmole) and DHA ⁇ uccinyl ester (0.075 mmole). The reaction mixture was stirred for 3 hours at room temperature at which time acetonitrile/water (1:1, v/v), 0.5 ml) and TFA (0.5 ml) were added.
  • DHA-SEQ ID NO: 12 T-cell epitope peptide
  • reaction mixture was then filtered through a 0.2 ⁇ m filter and this solution was applied directly to a preparative rever ⁇ ed-pha ⁇ e HPLC column for purification.
  • DHA-TCEP was collected and identified by mas ⁇ ⁇ pectro ⁇ copy.
  • the purified peptide was treated in 1 M aqueous piperidine for 8 hour ⁇ at room temperature at which time the product wa ⁇ i ⁇ olated by lyophilization.
  • Example 8 A ⁇ av of the Inhibition of Cell Proliferation The growth inhibitory effect of peptide hormone analogue ⁇ , ⁇ uch a ⁇ DHA-STKS, DHA-STKCS and D-Ly ⁇ (DHA)-
  • GnRH was evaluated on the basis of changes in the total cell number.
  • Cells were cultivated in RPMI 1640 medium (GIBCO BRL, Eggenstein, Germany, Cat. No. : 074-01800) ⁇ upplemented with 10% fetal calf ⁇ erum.
  • Tumor cell ⁇ were wa ⁇ hed with pho ⁇ phate-buffered ⁇ aline, then 0.25% tryp ⁇ in wa ⁇ added to detach them.
  • the cell ⁇ were ⁇ uspended in RPMI 1640 medium and after resu ⁇ pending the cell ⁇ were ⁇ tained with trypan blue and counted in a Burker chamber.
  • 0.1 ml cell ⁇ u ⁇ pension and 0.1 ml 0.1% trypan blue were mixed.
  • the viability of tumor cells was estimated on the basis of trypan blue (0.4%) exclusion using a hematocytometer.
  • Example 9 Luteinizing Hormone (LH) Release A ⁇ ay Anterior pituitarie ⁇ were obtained from adult (male or female) Wi ⁇ tar ⁇ train rats according to the method previously de ⁇ cribed (Shaw) .
  • the pituitarie ⁇ were cut into ⁇ mall piece ⁇ , incubated with collagena ⁇ e then di ⁇ per ⁇ ed mechanically to ⁇ ingle cell ⁇ .
  • the cell- ⁇ u ⁇ pen ⁇ ion wa ⁇ mixed with Sephadex G-10 bead ⁇ a ⁇ ⁇ upport material and tran ⁇ ferred into a ⁇ uperfusion chamber.
  • the cell ⁇ were continuou ⁇ ly perfu ⁇ ed with oxygenated medium or with a medium containing the peptide to be te ⁇ ted, ⁇ uch a ⁇ D- Lys6 (DHA) -GnRH.
  • the biological potency of a given analogue was determined based on the pituitary hormone responses (peaks) to the peptide stimulation over the baseline secretion.
  • Example 10 As ⁇ ay for Growth Hormone (GH)
  • GH Growth Hormone
  • Myers ⁇ uperfu ⁇ ion method
  • the hypophyses are cut into small piece ⁇ , incubated with collagena ⁇ e, and di ⁇ per ⁇ ed to single cell ⁇ .
  • the cells in a ⁇ uperfu ⁇ ion chamber are continuou ⁇ ly perfu ⁇ ed with oxygenated medium or with medium containing the peptide to be te ⁇ ted.
  • the level ⁇ of GH are determined by a double-antibody radioimmunoa ⁇ ay for the hormone.
  • the following methodology can be u ⁇ ed.
  • Antibody is produced by dissolving an appropriate amount of growth hormone (2-3 mg) in 200 microliters 0.01 N sodium hydroxide. The solution is diluted to 1 mg/ml with protein free PBS and mixed 1:1 with Freund' ⁇ adjuvant. 0.5-1 mg growth hormone i ⁇ in ⁇ jected into three ⁇ ubcutaneou ⁇ ⁇ ites of a young guinea pig, and repeated at two- to three-week interval ⁇ .
  • the hGH radioimmunoa ⁇ ay i ⁇ performed in the following manner.
  • the ⁇ tandard' ⁇ concentrations in hGH assay ⁇ are the following: 5, 2.5, 1.25, 0.625, 0.313, 0.156, and 0.078 ng/ml. 50 ml of the unknown growth hormone concentration i ⁇ added to tube ⁇ not containing the standard.
  • the next step is to add the guinea pig anti-hGH serum (fir ⁇ t anti ⁇ body) .
  • the radiolabeled hGH i ⁇ added to every tube.
  • the a ⁇ ay tube ⁇ are ⁇ haken gently, left at room temperature for two hour ⁇ , and then placed in a refrigerator at 4'C for at lea ⁇ t three day ⁇ .
  • the tubes are removed from the refrigerator and the appropriate dilution of the goat anti-guinea pig serum added (second antibody) .
  • the second antibody is diluted in 1% BSA PBS buffer to obtain approximately 50% precipitation of antibody-bound labeled hGH. Aliquot ⁇ of 100 microliter ⁇ of the ⁇ econd antibody are added to every tube. The tube ⁇ are gently agitated, left at room temperature for about two hour ⁇ , and then placed at 4'C. for at least eight hours. Following incubation with the second antibody, the tubes are centrifuged at 2000 g for 30 minutes at 4 degrees in a refrigerated IEC-PR6 centrifuge.
  • the ⁇ upernatant ⁇ are a ⁇ pirated by vacuum suction, with care taken not to disturb the precipitates packed in the bottom of tube ⁇ .
  • the pellet i ⁇ resuspended in 2 ml cold PBS and recentrifuged as described above. Washing the precipitates with buffer increase ⁇ the reproducibility.
  • Example 11 Measurement of Autophosphorylation of the EGF Receptor Autophosphorylation of the EGF receptor wa ⁇ mea ⁇ ured according to Bellot et al . (Bellot) .
  • Cells were washed once with binding buffer. The plates were then placed in a water bath at 37 degrees and different concentrations of the analogues were added to the cells for ⁇ pecific period ⁇ of time.
  • the buffer wa ⁇ removed and cells were scraped off the plates with 0.5 ml of Laemmli's ⁇ ample buffer, boiled for 5 minutes, and ⁇ onicated for 10 seconds. Aliquot ⁇ of each ⁇ ample were run on two different SDS-polyacrylamide gel ⁇ (7%) and each gel was transferred to nitrocellulose paper.
  • the cell ⁇ were incubated with the ⁇ ub ⁇ trate analogue ⁇ , ⁇ uch as DHA-SPRCS or DHA-STRS for 24 hour ⁇ , then harve ⁇ ted and homogenized.
  • the reaction volume 100 microliter ⁇ , and homogenized in a Dounce homogenizer 30 time ⁇ in 5 vol buffer (50 mM Tri ⁇ -HCl, pH 7.8, 50 mM MgCl2, 10 icromolar ⁇ odiumvanadate, 1 mM EDTA, and 50 microgram/ml aprotinin.
  • the reaction volume of 100 ml contained 50 mM Tris-HCl, pH 7.8, 50 mM MgCl2, 10 micromolar sodium vanadate, 0.1% nonidet P-40, 5 micromole gamma 32p-ATP, ImM substrate and 60 microliter homogenate.
  • the as ⁇ ay wa ⁇ initiated by addition of the ATP. After incubation the reaction wa ⁇ stopped by addition of trichloroacetic acid, and the supernatant was spotted on a 2 x 2 cm pho ⁇ phocellulo ⁇ e paper (Whatman P-81) .
  • the paper ⁇ quare ⁇ were wa ⁇ hed with pho ⁇ phoric acid and acetone, and the dried paper ⁇ were counted for radioactivity in ⁇ cintillation fluid.
  • Example 12 Preparation of DHA-EGFA (SEP. ID. NO: 6) 0.035 mM of EGFA was ⁇ u ⁇ pended in 2.5 ml of water- acetone (1:1), then 0.035 mM DIEA (6.5 ul) and 0.035 mM of

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Abstract

Composition d'un peptide et d'un acyle gras, utilisable en tant qu'inhibiteur de la prolifération cellulaire. La composition comprend une fraction d'acyle gras polyinsaturé liée de manière covalente à un peptide. En outre, on a prévu un procédé d'amélioration de l'activité antiproliférative d'un peptide au moyen d'une liaison covalente entre celui-ci et un acide gras polyinsaturé.
PCT/HU1993/000065 1992-11-30 1993-11-29 Composition d'un peptide et d'un acyle gras polyinsature WO1994012530A1 (fr)

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