WO1998020887A1 - Peptides se liant aux polyphosphoinositides, pour l'administration intracellulaire de medicaments - Google Patents
Peptides se liant aux polyphosphoinositides, pour l'administration intracellulaire de medicaments Download PDFInfo
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- WO1998020887A1 WO1998020887A1 PCT/US1996/018453 US9618453W WO9820887A1 WO 1998020887 A1 WO1998020887 A1 WO 1998020887A1 US 9618453 W US9618453 W US 9618453W WO 9820887 A1 WO9820887 A1 WO 9820887A1
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Classifications
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- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
- A61K49/0056—Peptides, proteins, polyamino acids
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- 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
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- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
- A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
- A61K49/0041—Xanthene dyes, used in vivo, e.g. administered to a mice, e.g. rhodamines, rose Bengal
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- 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
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4702—Regulators; Modulating activity
-
- 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
- C07K5/0812—Tripeptides with the first amino acid being neutral and aromatic or cycloaliphatic
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- 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
- C07K5/0819—Tripeptides with the first amino acid being acidic
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- 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
- C07K5/1016—Tetrapeptides with the first amino acid being neutral and aromatic or cycloaliphatic
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- 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
- C07K5/1019—Tetrapeptides with the first amino acid being basic
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- 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
- C07K5/1021—Tetrapeptides with the first amino acid being acidic
-
- 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
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- This invention relates to methods and compositions for facilitating the transport of a membrane-impermeable extracellular agent across a cell membrane. More particularly, the invention relates to covalent conjugates of N-terminal-blocked polyphosphoinositide (PPI) binding peptides and their use in pharmaceutical, diagnostic and genetic engineering applications.
- PPI polyphosphoinositide
- Transmembrane transport of membrane-impermeable molecules has long been recognized as an essential aspect of drug therapy and gene delivery into cells.
- the efficient transport of nucleic acids into cells is of particular importance for drug therapy involving the delivery of antisense oligonucleotides into cells to inhibit cellular or viral nucleic acid functions.
- transport of nucleic acids into cells is an inefficient process because of the high charge density of nucleic acids which impedes transport across a hydrophobic membrane barrier.
- methods for delivering nucleic acids into cells have been limited to carrier molecules which can accommodate the particular size and charge characteristics of the nucleic acid, yet still be capable of transporting the nucleic acid across the cell membrane.
- nucleic acids Physical methods for inserting nucleic acids into cells, e.g., microinjection, electroporation, osmotic shock, calcium-phosphate mediated transformation, scrape loading and rapid acceleration of DNA-coated, gold particles, are labor and time-intensive. Such methods frequently are cytotoxic and/or non-reproducible and are not easily adapted for in vivo drug therapy or even for large scale cell transformation in vitro.
- Biological methods for nucleic acid delivery into cells include liposome-mediated gene transfer (P. L. Feigner et al.. Proc.Natl.Acad.Sci. U.S.A. 84:7413 (1987)), cell fusion, retroviral vectors (M.A.Eglitis et al.
- N-l-(2,3-dioleyloxy)propyl] -N,N.N -trimethylammonium chloride also have been reported to enhance transfection efficiency. However, none of these methods is without limitations.
- Receptor-mediated delivery involves covalently coupling the exogenous molecule to a receptor-specific ligand to form a conjugate and allowing the conjugate to contact a (receptor-expressing) cell for a time sufficient to permit transmembrane transport of the conjugate. It is generally believed that binding of the ligand to the receptor in the cell membrane initiates movement of the receptor-ligand complex into the cell interior in the form of an endosome. Release of the contents of the endosome in the cell interior completes delivery of the exogenous agent to the intracellular matrix.
- the present invention overcomes the problems of the prior art by providing a carrier molecule which rapidly and efficiently transports a membrane-impermeable extracellular molecule into a cell.
- the carrier molecules of the present invention are effective at cold temperatures (e.g., about 4°C), suggesting that the transport mechanism does not require receptor-mediated endocytosis or active cellular metabolism.
- the carrier molecules of the instant invention are useful for transporting extracellular agents across the membranes of intact, but chemically fixed (metabolically dead) cells.
- the carrier molecules of the invention are internalized in a mechanism which involves interaction of the peptide component of the carrier molecules with cell membrane polyphosphoinositide (PPI) molecules.
- PPI molecules are present in most, if not all mammalian and vertebrate cell membranes. Accordingly, the invention advantageously provides a method for delivering an extracellular molecule to virtually any type of mammalian or vertebrate cell without regard for the particular proteins expressed on the cell surface.
- a carrier molecule for facilitating the transport of a membrane-impermeable extracellular agent across the membrane of a cell.
- the carrier molecule contains an amine derivatizing agent ("X") covalently coupled to the N-terminal (i.e., amino terminal) amine of a "transport-mediating peptide" ("P").
- X amine derivatizing agent
- P transport-mediating peptide
- transport-mediating peptides refers to peptides which, when covalently coupled to the extracellular agent in the form of a prodrug having the formula X-P-A, mediate transport of the extracellular agent across a cell membrane.
- the transport-mediating peptides of the invention are selected from the group consisting of Seq. I.D. No.
- the transport-mediating peptide is Seq. I.D. No. 1 (human gelsolin 135-142) or Seq. I.D. No. 2 (human gelsolin 160-169).
- Functionally equivalent peptides of Seq. I.D. Nos. 1 or 2 refers to ( 1) fragments, (2) homologs and (3) analogs of Sequence I.D. Nos. 1 and 2, that can be used in accordance with the methods of the invention to transport an extracellular agent into a cell.
- Functionally equivalent peptides contain between three and ten amino acids and are capable of binding to a PPI (i.e., functionally equivalent peptides of Seq. I.D. Nos. 1 and 2 are PPI-binding peptides).
- Functionally equivalent peptides are identified in screening assays which detect the ability of a putative functionally equivalent peptide (in the form of the carrier molecule) to transport a membrane-impermeable extracellular agent into a cell. Such screening assays rely upon biochemical or physical measurements or functional activity tests to determine whether transport of the extracellular agent across the cell membrane has occurred.
- the extracellular agent is covalently coupled to the carboxyl terminus of the transport-mediating peptide (in the form of the carrier molecule) to form a prodrug, having the formula X-P-A, which is transported across a cell membrane.
- the covalent bond between the extracellular agent and the transport-mediating peptide is a labile bond which is cleaved after the extracellular agent is delivered to the intracellular matrix, thereby releasing the extracellular agent in an underivatized form.
- cells containing the carrier molecule (X-P) and/or the prodrug (X-P-A) are provided.
- Exemplary cells to which the carrier molecules and/or prodrugs of the invention can be delivered include melanoma cells, fibroblast cells, neuronal cells, neutrophils. monocytes and blood platelets.
- the cells can be fixed according to standard procedures and the carrier molecules of the invention can be used to facilitate delivery of the extracellular agent across the membranes of fixed cells.
- a method for facilitating the transport of a membrane-impermeable extracellular agent across the membrane of a cell involves contacting the cell with a prodrug containing the extracellular agent covalently coupled to the carrier molecule of the invention for a time sufficient to permit transport of the extracellular agent across the cell membrane.
- the time sufficient for transport is on the order of five to thirty minutes. More preferably, the time for transport is on the order of 120 to 300 seconds, and most preferably, the time is on the order of 10 to 120 seconds.
- the method is useful for rapidly and efficiently facilitating the transport of an extracellular agent across a cell membrane in vitro (e.g., for delivering an oligonucleotide probe or primer to a fixed cell for in situ hybridization or for delivering an antibiotic to a bacterially-contaminated cell culture) or in vivo (e.g., for delivering an imaging agent to tumor cells in vivo).
- a cell membrane in vitro (e.g., for delivering an oligonucleotide probe or primer to a fixed cell for in situ hybridization or for delivering an antibiotic to a bacterially-contaminated cell culture) or in vivo (e.g., for delivering an imaging agent to tumor cells in vivo).
- FIG. 1A shows the fluorescence intensity of 2 ⁇ M rhodamine-SEQ. I.D. No. 2 as a function of the concentration of phosphatidylinositol 4,5-bisphosphate (PIP2);
- FIG. IB shows that the lipid effect of FIG. 1 A is specific for PIP2 since another acidic phopholipid PS (phophatidylserine) showed no effect on any of the tested rhodamine-peptide conjugates; and
- FIG. 2 shows the effect of rhodamine-SEQ. I.D. No. 2 on human blood platelet function, as determined by measuring the shear modulus of platelet rich plasma (PRP) during gel formation in the presence or absence of rhodamine-SEQ. I.D. No. 2.
- PRP platelet rich plasma
- transport-mediating peptide refers to a peptide which, when covalently coupled to the extracellular agent in the form of a prodrug, mediates transport of the extracellular agent across a cell membrane.
- a “transport-mediating peptide” is said to have a "transport- mediating activity", i.e., the ability to transport an extracellular agent across a cell membrane.
- the transport-mediating peptides of the invention include Sequence I.D. No. 1 , functionally equivalent peptides of Sequence I.D. No. 1, Sequence I.D. No. 2. and functionally equivalent peptides of Sequence I.D. No. 2.
- Transport-mediating peptides which are functionally equivalent peptides of Sequence I.D. Nos. 1 and 2 are identified in screening assays which measure the ability of a carrier molecule (containing the putative transport-mediating functionally equivalent peptide) to mediate transport of an extracellular agent across a cell or other lipid bilayer membrane.
- An exemplary screening assay to identify transport-mediating peptides that are functional equivalents of Sequence I.D. Nos. 1 or 2 is described in Example 9.
- “functionally equivalent peptides" of Sequence I.D. Nos. 1 and 2 refers to ( 1 ) fragments, (2) homologs and (3) analogs of Sequence I.D. Nos. 1 and 2, that can be used in accordance with the methods of the invention to transport an extracellular agent across a cell membrane.
- Functionally equivalent fragments, homologs and analogs are described in detail below.
- the functionally equivalent peptides contain between three and ten amino acids and are capable of binding to a polyphosphoinositide (PPI). i.e., functionally equivalent peptides of Sequence I.D. Nos. 1 and 2 are "PPI-binding peptides".
- Screening assays are of two types: ( 1 ) structural assays which detect formation of a complex containing the putative PPI-binding peptide in association with a PPI (e.g..
- the amino acid sequence of Seq. I.D. No. 1 contains the generic formula, KxxxKxKK, wherein K represents lysine (a basic amino acid) and x represents a neutral amino acid.
- the amino acid sequence of Seq. I.D. No. 2 contains the generic formula, RxxxxKxRR, wherein K represents lysine, R represents arginine (basic amino acids) and x represents a neutral amino acid. It is believed that the highly charged basic character of Sequence I.D. Nos. 1 and 2 plays an important role in the localization of the peptide to intracellular structures, e.g., nucleus. As used herein, "fragments" of Sequence I.D. No.
- Sequence I.D. Nos. 3 through 7 refers to the peptides identified as Sequence I.D. Nos. 3 through 7 and Sequence I.D. Nos. 24 through 29.
- "Fragments" of Sequence I.D. No. 2 refer to the peptides identified as sequence I.D. Nos. 8 through 13 and 30 through 36. Fragments can be synthesized without undue experimentation using standard procedures known to those of ordinary skill in the art.
- Functionally equivalent peptide fragments of Seq. I.D. Nos. 1 and 2 are identified in screening assays which measure the ability of the peptide fragment, in the form of a prodrug. to mediate transport of an extracellular agent across a cell membrane.
- Each of Sequences 3 through 13 contains the highly basic four amino acid portion of Sequence I.D. Nos. 1 or 2 (see TABLES 1 and 2) that is believed to play an important role in PPI binding.
- the invention is also useful for visualizing intracellular phosphoinositide structure, e.g., by using a biotinylated-peptide (e.g., SEQ. I.D. No. 2) to target the phosphoinositide, followed by contacting the labelled cell with gold-labelled avidin and visualizing the phosphoinositide structure by electron microscopy.
- a biotinylated-peptide e.g., SEQ. I.D. No. 2
- Q one
- QR two
- QRL three amino acids
- Seq. I.D. No.29 FKSGLKYK TABLE 2 PEPTIDE FRAGMENTS OF SEQ.I.D. NO. 2
- conjugates can be prepared that are more efficient at transporting an extracellular agent into one particular cell type compared to another cell type, thereby permitting the enhanced delivery of the extracellular agent into a specific cell type contained in a diverse population of cell types.
- the term "homolog” refers generally to a molecule which shares a common structural feature with the molecule to which it is deemed to be an homolog.
- a "functionally equivalent peptide homolog" of Sequence I.D. Nos. 1 or 2 is a peptide which shares a common structural feature (amino acid sequence homology) and a common functional activity (transport-mediating activity) with Sequence I.D. Nos. 1 or 2.
- Functionally equivalent peptide homologs of Sequence I.D. Nos. 1 and 2 are derived from PPI-binding proteins that have sequence homology to human gelsolin.
- gelsolin is forty-five percent homologous with villin, a protein found in vertebrate brush border rnicrovilli which, like gelsolin, exhibits Ca++ dependent actin severing activity.
- Gelsolin also is thirty-three percent homologous with severin and fragmin (P.Matsudaira and P. Janmey, Cell 54: 139-140 (1988)).
- sequence homologs that have at least 50% sequence homology to Sequence I.D. Nos. 1 or 2 include. Sequence I.D. Nos. 14, 15. 16.
- the peptide homologs have at least 60% sequence homology (e.g.. Sequence I.D. Nos. 19 and 21 ) with Seq. I.D. Nos. 1 or 2, more preferably, at least 70% sequence homology (e.g.. Sequence I.D. No.
- sequence homology e.g., Sequence I.D. Nos. 17 and 20. See, e.g.. Tables 3 and 4 for the preferred functionally equivalent peptide homologs of Sequence I.D. Nos. 1 and 2. respectively.
- the functionally equivalent peptide homologs of Sequence I.D. No. 1 contain an amino acid substitution selected from the group consisting of S->I, G->L. Y->G, L->V and Y->H.
- peptide analogs refers to functionally equivalent peptide fragments and homologs of Sequence I.D. Nos. 1 and 2 that contain conservative amino acid substitutions, provided that the peptide analogs which contain the conservative substitutions bind to a PPI and have transport-mediating activity, as determined, for example, in an in vitro screening assay (see.
- b is bovine
- h is human
- c is chicken
- d is Drosophila
- rhodamine rhodamine X isothiocyanate tetramethylrhodamine-5-isothiocyanatc tetramethylrhodamine-6-isothiocyanate
- fluorescein fluorescein-5-isothiocyanate fluorescein-5 -isothiocyanate diacetate fluorescein-6-isothiocyanate
- succinimidyl ester 5 -(and 6)-carboxyfluorescein diacetate succinimidyl ester
- the derivatizing agent is rhodamine or a rhodamine derivative which includes a succinimidyl ester or an isothiocyanate reactive group.
- the derivatizing agents can be covalently coupled to the N-terminal amine group of the transport-mediating peptide using, for example, succinimide as described in the Examples.
- Exemplary amine-reactive derivatizing agents include fluorenylmethoxycarbony 1 (" F-MOC " ) .
- melanoma cells which had internalized the rhodamine-Sequence I.D. No. 2 conjugate displayed the same changes in cell morphology and function that were observed following microinjection of (uncoupled) Sequence I.D. No. 2.
- the rhodamine-Sequence I.D. No. 2 conjugate was observed to concentrate around the cell periphery at the plasma membrane, around the nuclear membrane, and especially in the nucleolus of human melanoma cells.
- a similar labeling pattern was observed in NIH-3T3 fibroblasts and neuronal cells with the growth cones of primary cultures of neurons also staining brightly.
- the carrier molecules of the invention are rapidly taken up by human blood platelets, thereby simplifying the process for delivering the PPI-binding peptides across the membranes of platelets to prevent cold-induced platelet activation.
- a preparation of platelets can be prepared which will not undergo cold-induced platelet activation but which will retain the ability to respond to a stronger activating stimulus, such as thrombin.
- the amount of carrier molecule to achieve this objective is determined, for example, using the methods described in U.S. Patent No. 5,358,844, e.g., by determining the minimal concentration of carrier molecule that can prevent cold-induced platelet activation yet permit thrombin-induced platelet activation (at physiologically relevant thrombin concentrations).
- the carrier molecules of the invention are prepared by covalently coupling the derivatizing agent to the N-terminal amine group of the transport-mediating peptide.
- the prodrugs of the invention can be prepared by covalently coupling an extracellular agent to a reactive group in the carboxyl terminus of the transport-mediating peptide.
- the extracellular agent has an intracellular function and is "membrane-impermeable".
- membrane-impermeable extracellular agent refers to a molecule that is located in or introduced to the environment external to the cell, which molecule cannot penetrate the cell membrane at a sufficient level to mediate its intracellular function.
- Intracellular functions of the extracellular include, for example, an antibiotic function, an enzymatic function, and an oligonucleotide hybridizing function (e.g., for performing PCR amplification).
- Exemplary extracellular agents include peptides, oligopeptides, proteins (e.g., apoproteins, glycoproteins, antigens and antibodies), haptens and antibodies thereto, receptors and other membrane proteins, protein analogs containing at least one non-peptide linkage in place of a peptide linkage, enzymes, enzyme modulators (e.g.. coenzymes, inhibitors), amino acids and their derivatives, hormones, Iipids, phospholipids.
- proteins e.g., apoproteins, glycoproteins, antigens and antibodies
- haptens and antibodies thereto e.g., receptors and other membrane proteins
- protein analogs containing at least one non-peptide linkage in place of a peptide linkage e.g., enzymes, enzyme modulators (e.g. coenzymes, inhibitors), amino acids and their derivatives, hormones, Iipids, phospholipids.
- nucleic acid vectors include antisense polynucleotides that can hybridize to an intracellular (cellular or viral) nucleic acid; promoters; enhancers: inhibitors; other ligands for regulating gene transcription and translation, and any other biologically active molecule that can be covalently attached to the carboxvlate group of the above-described peptides without adversely affecting the ability of the peptide to bind to a PPI and transport the extracellular agent across a cell membrane.
- the prodrug is formed by allowing the peptide to react with the derivatizing agent to form the carrier molecule X-P (see, e.g.. Example 2), and then allowing the carrier molecule to react with the extracellular agent A to form the prodrug X-P-A (see, e.g.. Example 7).
- An exemplary procedure for forming an antibiotic prodrug is shown in Example 10.
- the extracellular agent contains a functional group that is reactive with a functional group in the carboxyl terminus of the transport-mediating peptide (e.g., the terminal carboxyl group).
- the prodrugs are formed by allowing the functional groups of the extracellular agent and the peptide to form a covalent linkage using coupling chemistries known to those of ordinary skill in the art. Numerous art-recognized methods for forming a covalent linkage can be used. See, e.g., March, J., Advanced Organic Chemistry, supra.
- the covalent bond between the extracellular agent and the transport-mediating peptide is selected to be sufficiently labile (e.g., to enzymatic cleavage by an enzyme present in the cell) so that it is cleaved following transport of the extracellular agent into the cell, thereby releasing the free extracellular agent to the cell interior.
- Art-recognized biologically labile covalent linkages e.g., imino bonds, and "active" esters can be used to form prodrugs where the covalently coupled extracellular agent is found to exhibit reduced intracellular activity in comparison to the intracellular activity of the extracellular agent alone.
- Exemplary labile linkages are described in U.S. Patent No. 5,108,921, issued to Low et al. If the polypeptide does not have a free amino-or carboxyl-terminal functional group that can participate in a coupling reaction, such a group can be introduced, e.g., by introducing a cysteine (containing a reactive thiol group) into the peptide by site directed mutagenesis.
- reactive functional groups that are present in the amino acid side chains of the transport-mediating peptide preferably are protected, e.g., with protecting groups such as those shown in TABLE 7, to minimize unwanted side reactions prior to coupling the peptide to the derivatizing agent and/or to the extracellular agent.
- protecting group refers to a molecule which is bound to a functional group and which may be selectively removed therefrom to expose the functional group in a reactive form.
- the protecting groups are reversibly attached to the functional groups and can be removed therefrom using, for example, chemical or other cleavage methods.
- the peptides of the invention can be synthesized using commercially available side-chain-blocked amino acids (e.g., FMOC-derivatized amino acids from Advanced Chemtech.Inc, Louisville, KY).
- side-chain-blocked amino acids e.g., FMOC-derivatized amino acids from Advanced Chemtech.Inc, Louisville, KY.
- the peptide side chains can be reacted with protecting groups after peptide synthesis, but prior to the covalent coupling reaction.
- the carrier molecules and prodrugs of the invention can be prepared in which the amino acid side chain do not participate to any significant extent in the coupling reaction of the peptide to the derivatizing agent or to the extracellular agent.
- conjugates e.g., pyrene-Cys-P2 domain, rhodamine-B-Cys-P2 domain, and fluorescein-Cys-P2 domain
- pyrene-Cys-P2 domain did not enter intact human blood platelets, NIH-3T3 fibroblasts or melanoma cells (as determined by functional activity assays).
- the rhodamine-Sequence I.D. No. 2 conjugate was rapidly and efficiently taken up by the above-identified cells (as determined using fluorescence measurements and functional assays). For example, at concentrations significantly less than 5 uM, the rhodamine-Sequence I.D. No. 2 conjugate resulted in punctate staining of melanoma cells without altering the surface protrusion activity of these cells, indicating that the conjugate was not cytotoxic at these concentrations. That contacting the cells with the rhodamine-Sequence I.D. No.
- the invention also provides a method for facilitating the transport of a membrane-impermeable extracellular agent across the membrane of a cell.
- the method involves contacting the cell with the extracellular agent covalently coupled to the above-described carrier molecule of the invention, for a time sufficient to permit transport of the extracellular agent across the cell membrane.
- the time sufficient for transport is determined using routine optimization. In general, the time sufficient for transport is on the order of 10 to 300 seconds.
- the derivatizing agent is a fluorescent molecule (e.g., rhodamine or fluorescein) and the transport-mediating peptide is selected from the group consisting of Sequence I.D. Nos. 1-36.
- the carrier molecule contains rhodamine covalently coupled to Sequence I.D. Nos. 1 or 2.
- the carrier molecules of the invention are particularly useful for delivering an extracellular agent to cells in vitro.
- Exemplary procedures for delivering an extracellular agent that is an antibiotic or an oligonucleotide to cells in vitro are provided in Examples 10 and 1 1 , respectively.
- the methods of the invention for delivering the extracellular agent to cells in vitro utilize art-recognized protocols with the only substantial procedural modification being the substitution of the prodrug for the drug used in the art-recognized protocol.
- the extracellular agent is an antibiotic and the invention is used to deliver the antibiotic to the cytoplasm of cells in vivo or in vitro. Bacterial contamination of cells in culture is frequently associated with large scale production of recombinant molecules.
- the invention overcomes this problem by providing a carrier molecule for delivering the antibiotic to the cell cytoplasm, thereby eliminating intracellular bacterial contamination.
- the methods of the invention for reducing bacterial contamination of cells in culture are identical to methods that use conventional antibiotic treatment with the exception that the antibiotic prodrug is substituted for the conventional antibiotic drug in the standard procedure.
- the extracellular agent is an oligonucleotide and the carrier molecule of the invention is used to deliver the oligonucleotide to the cell to effect in vitro or in vivo cellular transformation.
- the extracellular agent is an antisense RNA that is capable of hybridizing to a target intracellular nucleic acid (e.g., a cell or viral nucleic acid) and the carrier molecules of the invention are used to deliver the antisense RNA into the cell to modulate transcription of the target intracellular nucleic acid in vitro or in vivo.
- the extracellular agent is an oligonucleotide probe or primer and the carrier molecules of the invention are used to deliver the oligonucleotide probe or primer to the cell for in situ hybridization to a target nucleic acid and subsequent amplification using, for example, the polymerase chain reaction (PCR).
- PCR polymerase chain reaction
- U.S. Patent No. 4,683,202 issued to Mullis and Example 1 1.
- In situ hybridization coupled with PCR amplification is useful for detecting a variety of cellular and non-cellular (e.g., viral) nucleic acids.
- the method for in situ PCR involves transporting the oligonucleotide primer in the form of a prodrug into the cell, allowing the oligonucleotide primer to hybridize to an intracellular target nucleic acid, amplifying the target nucleic acid in situ and detecting the amplified target nucleic acid.
- the extracellular agent (such as a DNA probe) can be delivered to fixed or unfixed intact cells for performing PCR amplification and detecting intracellular nucleic acids.
- the carrier molecules are used for delivering an extracellular agent to cells in vivo.
- a method for manufacturing a pharmaceutical composition for delivering a carrier molecule or extracellular agent having an intracellular activity to a cell in vivo involves placing the above-described carrier molecule or prodrug in a pharmaceutically acceptable carrier to form a pharmaceutical composition and administering the pharmaceutical composition containing a therapeutically effective amount of the prodrug to the recipient.
- a therapeutically effective amount of carrier molecule or prodrug is between about 1 ug and about 100 mg/ ' kg.
- the carrier molecule or prodrug may be used alone or in appropriate association, as well as in combination with other pharmaceutically active compounds.
- the carrier molecule or prodrug may be formulated into preparations in solid, semisolid, liquid or gaseous form such as tablets, capsules, powders, granules, ointments, solutions, suppositories, and injections, in usual ways for oral, parenteral, or surgical administration.
- the carrier molecule or prodrug of the invention can be formulated as ointments or creams.
- Exemplary pharmaceutically acceptable carriers for peptide drugs, described in U.S. 5,21 1 ,657, are useful for containing the carrier molecules and prodrugs of the invention.
- the carrier molecules of the invention are useful as agents for modulating PPI-mediated signal transduction.
- the PPI-binding peptides of the instant invention are presented in a membrane permeable form (e.g., covalently coupled to rhodamine or a rhodamine derivative), thereby eliminating the necessity for. e.g., microinjection or liposome encapsulation, to deliver the PPI-binding peptide to the cell interior in order to mediate signal transduction.
- the invention advantageously provides PPI-binding peptides in the form of carrier molecules that can be administered in accordance with art-recognized methods for drug delivery in vivo.
- the carrier molecules can be formulated into a topical pharmaceutic preparation to deliver to local cells an amount of PPI-binding peptide sufficient to inhibit PPI-related signal transduction pathways such as those involved in cell trafficking and proliferation.
- the topical application to the skin of a PPI-binding peptide in the form of a carrier molecule is useful for inhibiting cell proliferation associated with conditions such as psoriasis.
- Topical application of the carrier molecules of the invention by catheter to an arterial wall is useful for preventing post-angioplasty thrombosis or restenosis.
- the instant invention provides methods and compositions for facilitating the transport of an exogenous agent across a cell membrane.
- the following examples illustrate representative utilities of the instant invention.
- a standard active ester coupling procedure is used to synthesize X-P.
- the rhodamine B N-hydroxysuccinimide is coupled with the N-terminal amino group of the peptide.
- the reaction is carried out in dimethylformamide at room temperature for several hours. See, e.g., Molecular Probes Handbook 1992-1994, Part IIB.5 for a list of exemplary references describing reactions of rhodamine "succinimidyl esters.”
- Example 3 Selecting an agent "X”: A Procedure to Determine whether the (uncoupled) amine derivatizing agent "X" is significantly less soluble in a lipid bilaver compared to a derivatizing agent that is covalently coupled to a peptide of the invention
- Partitioning of a putative derivatizing agent into a lipid bilayer is determined using any of the following lipid vehicles: ( 1 ) unilammellar or multilammeller vesicles (liposomes) of known composition containing, for example, phosphatidyl choline (PC), phosphatidyl glyceride (PG) and phosphatidyl inositide (PI); micelles of lysophospholipids, PIP2 or PIP; and (3) mixed micelles of sodium dodecyl sulfate (SDS), triton and other phospholipids.
- PC phosphatidyl choline
- PG phosphatidyl glyceride
- PI phosphatidyl inositide
- micelles of lysophospholipids, PIP2 or PIP micelles of lysophospholipids, PIP2 or PIP
- SDS sodium dodecyl sulfate
- PPIs
- PC Dioleoyl-L-alpha-phosphatidylcholine
- PIP phosphatidylinositol 4-monophosphate
- PIP2 phosphatidylinositol 4,5-bisphosphate
- Partitioning of the putative derivatizing agent into the lipid bilayer is determined without undue experimentation using any of the following methods. Selection of a particular type of detection method to determine the extent of partitioning is dependent upon the nature of the putative derivatizing agent.
- solubility of the derivatizing agent in the lipid bilayer is determined by observing the predicted changes in fluorescence spectra or intensity (quantum yield) that one of ordinary skill in the art would expect when the fluorophore partitions from an aqueous phase into a lipid phase. If the fluorophore is not environmentally-sensitive, partitioning into the lipid bilayer is detected by fluorescence quenching using acrylamide or similar molecules which reduce the fluorescence of the water soluble fluorophore. Such quenching reagents do not reduce the fluorescence of fluorophores bound in a lipid phase or partitioned within liposomes or micelles.
- the partitioning into a lipid bilayer of non-fluorescent derivatizing agents that contain ester bonds or sulfhydryl groups is determined by observing changes in the susceptibility of such agents to esterases or reducing agents (e.g., Ellman's reagent or
- the partitioning of radiolabeled derivizating agents into a lipid bilayer is determined by separating lipid particles from soluble components using, for example, ultracentrifugation, and measuring the relative proportions of radiolabel present in the lipid particle fraction compared with the soluble fraction.
- Non-functional procedures refers to tests which do not rely upon the functional activities of the derivatizing agent (X) or transport-mediating peptide (P) for detection.
- An exemplary method for determining whether the carrier molecule (X-P) has been transported across a cell membrane and into the intracellular matrix involves: (1) contacting a cell (e.g., HL60 or U937 cells grown in suspension) with a carrier molecule containing a labeled derivatizing agent (e.g., a fluorescent or radiolabeled derivatizing agent) and/or labeled transport-mediating peptides (e.g., radiolabeled peptides) for a time sufficient for the carrier molecule to enter the cell; (2) separating the cell from the soluble carrier molecule (e.g., by centrifugation, gel filtration chromatography); and (3) determining the relative proportions of the labeled carrier molecule in the cell fraction and the soluble fraction, e.g., by FACS (fluorescence activated
- Transport of a carrier molecule into a cell also can be determined in an analogous manner to any of the methods disclosed in Example 3 for determining partitioning of the derivatizing agent into a lipid vehicle.
- the partitioning of a carrier molecule which contains an ester bond or a sulfhydryl group into a cell can be determined by: (1) contacting the cell with the carrier molecule for a time sufficient for the carrier molecule to enter the cell; (2) separating the cell from the soluble carrier molecule which has not penetrated the cell (e.g., by centrifugation); and (3) determining the proportion of the carrier molecule present in the soluble fraction by exposing an aliquot of the soluble fraction to an esterase or reducing agent and determining the susceptibility of the soluble fraction to the esterase or reducing agent as a measure of the amount of carrier molecule present in the soluble fraction.
- This assay is similar to that described for studying PIP2 binding of profilin (Goldschmidt-Clermont P.J., et al.. Science 251 : 1231 -1233 (1991 ); Machesky. L.M.. et al.. Cell Reg. l :937-950 (1990)) and CapZ (Heiss and Cooper. Biochemistry 30:8753-8758 (1991 )). It is based on the fact that PIP2 micelles are large (90 kDa) compared to the transport-mediating peptides of the invention (or carrier molecules containing these peptides), and polypeptide/PIP2 complexes will elute from a size exclusion gel filtration column earlier than the unbound polypeptide.
- the putative transport-mediating peptides are incubated with PIP2 micelles for 5 min. at room temperature, and 100 ul of the mixture is chromatographed at room temperature on a Superose 12 HR 10/30 column (FPLC system, Pharmacia, Piscataway, NJ) equilibrated with a buffer containing 5 mM Tris-HCI, pH 7.5, 75 mM KCI and 0.1 mM NaN 3 . P1P2 is not included in the elution buffer. The elution is performed at 0.5 ml/min., and 0.5 ml fractions are collected. The elution profile is monitored by absorbance at 280 nm. 300 ul of selected fractions are dried down in a rotoevaporator
- the amount of peptide bound to PIP is determined by measuring the decrease in the peptide absorbance peak.
- the sensitivity of this assay can be improved by using a labeled (e.g., radiolabeled) peptide and measuring the amount of label in the eluted fractions.
- X-P carrier of the invention for the rhodamine-SEQ. I.D. No. 2 conjugate in the assay using a broad concentration range, e.g., 0.01 ⁇ M to 1000 ⁇ M, of the putative carrier).
- fluorophores change emission spectra or intensities when bound to hydrophobic (lipid) ligands. Accordingly, the following assay is useful for determining whether an exemplary X-P or X-P-A has associated with a membrane or hydrophobic layer.
- the targets of an exemplary X-P are phosphoinositides (PPIs like PIP or PIP2) by adding increasing amounts of PPIs or control Iipids to the exemplary X-P and measuring fluorescence changes to evaluate whether the X-P had bound to the hydrophobic ligand.
- Figure 1 shows the fluorescence intensity of 2 ⁇ M rhodamine-GS 160-169 (i.e., rhodamine-SEQ. I.D. No.
- the carrier molecule can be introduced into the cell using conventional procedures (e.g., liposome delivery, microinjection). Each carrier molecule is tested over a wide concentration range to select the most effective carrier molecules for coupling to an exogenous agent to form a prodrug. Determination of transport of the rhodamine-Sequence I.D. No. 2 conjugate into human melanoma cells
- the first assay employs a two compartment Boyden chamber. Migration through a membrane in response to a chemoattractant is assayed using a 48-well chamber (Nucleopore, Pleasanton, CA) with a 5 ⁇ m polycarbonate filter. Cells (either fibroblasts, melanoma cells or neutrophils) are trypsinized, counted, and 5 x 10 4 cells per well are loaded in the top wells.
- wound healing assays can be used to measure cell motility.
- a dish of confluent cells is scored with the tip of a rubber policeman.
- the width of the wound is measured by observation through an Olympus CK inverted tissue culture microscope with a grid reticule in the eyepiece. This allows measurement of the width of the wound when first made and at intervals thereafter, as the migrating cell edges close the wound. Care is taken to measure the wound in the same locations at each interval.
- the rate of closure of the wound edge reflects the locomotory rate of the cells along the edge.
- This assay can be performed in the presence or absence of the peptide to determine the effects on motility, and therefore of peptide transport into the cells.
- Example 6 Selecting an agent "A”: A Procedure to Determine whether the (uncoupled) agent is membrane-impermeable, e.g.. by determining solubility of "A" (coupled vs. uncoupled forms) in a lipid bilayer
- Membrane-impermeability is determined by comparing the solubilities of an unconjugated and a conjugated putative membrane-impermeable exogenous agent in a lipid bilayer or other hydrophobic layer.
- membrane-impermeable in reference to an exogenous agent means an exogenous agent which cannot penetrate the cell membrane at a sufficient level to mediate its intracellular function.
- polyphosphoinositides are included in the lipid bilayer that is used to evaluate partitioning of the exogenous agent into a lipid bilayer.
- PPIs polyphosphoinositides
- Partitioning of the exogenous agent into the lipid bilayer is determined without undue experimentation using any of the following methods. Selection of a particular type of detection method to determine the extent of partitioning is dependent upon the nature of the exogenous agent. For example, for an exogenous agent that is labeled with a fluorescent molecule, solubility in the lipid bilayer is determined by observing the predicted changes in fluorescence spectra or intensity (quantum yield) that one of ordinary skill in the art would expect when the fluorophore partitions from an aqueous phase into a lipid phase.
- the N-terminal amino acid of the extracellular agent can be coupled to the C-terminal of the carrier molecule and the remaining amino acids of the extracellular peptide can be added sequentially until the complete prodrug is formed.
- the prodrug is formed by allowing the transport-mediating peptide to first react with the derivatizing agent to form the carrier molecule X-P, and then allowing the carrier molecule to react with the extracellular agent A to form the prodrug X-P-A.
- An angiotensin prodrug is formed using a solid phase synthesis method in which the peptide chain is step-wise elongated as described above.
- Appropriate spacer molecules e.g., episilon-amino-capronic acid "Fmoc-Aca"
- Fmoc-Aca episilon-amino-capronic acid
- Various other linkers can be used to insert molecules of known composition and size between any of the derivatizing agent, the transport-mediating peptide and the extracellular agent.
- Such agents are commerically available (see, e.g., Sigma Chemical Co. catalog, St. Louis, MO (1992)).
- Antibiotic prodrugs which have a free hydroxyl group are prepared using Fmoc-Aca to derivatize the antibiotic and thereafter couple the derivatized antibiotic to a carrier molecule of the invention.
- Antibiotics which already include a reactive functional group, for example, ampicillin (Sigma, St. Louis, MO, product A-9393) are directly attached to the carrier molecule or can be coupled via a spacer molecule if so desired using the above-described chemistries.
- Example 8 Non-Functional Procedures to Determine whether X-P-A has been transported across the cell membrane
- Strains of bacteria that are known to invade mammalian cells are cultured in Luria broth (LB).
- LB Luria broth
- 1 x 10 7 bacteria are added to 1 x 10 6 adherent cells cultured in 24-well microtiter plates without antibiotics.
- the monolayers are washed 3x with phosphate-buffered saline (PBS) and extracellular bacteria are killed by the addition of fresh tissue culture media to which 1 OO ⁇ g/ml gentamycin is added. Efficient bacterial killing requires a further 2 hour incubation at 37°C, 5% C0 2 .
- PBS phosphate-buffered saline
- the monolayers are then washed 3x with PBS, lysed with 0.1 ml 1% Triton X-100, and the number of viable internal bacteria are determined by titration of the cell lysate on LB agar with 100 ⁇ g/ml ampicillin.
- a concentration of the compound sufficient to achieve intracellular levels of antibiotic equal to the measured MIC (minimal inhibitory concentration) of the antibiotic is added (with the extracellular gentamycin) to the monolayer.
- the number of viable intracellular bacteria is measured in the presence or absence of X-P-A.
- X-P (without A) serves as an additional negative control. Other concentrations of X-P-A are tested to confirm that the MIC is significantly less if the antibiotic is delivered intracellularly.
- Example 5 The results of the functional assay in Example 5 are used to determine an effective intracellular MIC and that concentration of X-P-A is incubated with the cells for 2 to 24 hours to determine the effect of incubation time with X-P-A on Mycoplasma contamination.
- Example 1 Protocol for In situ hybridization using an X-P-oligonucleotide
- An oligonucleotide probe having a nucleotide sequence complementary to, for example, a nucleic acid sequence encoding a viral-specific protein is prepared according to standard procedures.
- the target nucleic acid can contain a sequence encoding a unique protein product of the HIV virus.
- the oligonucleotide is covalently coupled to rhodamine-SEQ. I.D. No.2, according to the protocol presented above in Example 7 to form an "oligonucleotide prodrug".
- the cells which are intended for in situ hybridization analysis are prepared according to standard procedures.
- the cells may be fixed on unfixed prior to contacting the cells with the oligonucleotide prodrug.
- the oligonucleotide prodrug is incubated in the presence of cells which are being tested for the presence of a target nucleic acid for a time sufficient for the oligonucleotide prodrug to be transported across the cell membrane. Routine optimization of transport conditions such as time, temperature, concentration, are performed according to standard practice.
- the oligonucleotide prodrug facilitates transport of the oligonucleotide probe or primer that is complementary to a target nucleic acid across the cell membrane, thereby facilitating in situ hybridization of the probe or primer to intracellular target nucleic acid. In situ hybridization is performed and the results are analyzed according to standard procedures (see, e.g., Molecular Cloning, 2nd edition, ed. Sambrook et al., Cold Spring Harbor Laboratory Press (1989)).
- Exemplary imaging agents that are attached to a carrier molecule include those identified in U.S. Patent No. 4,861 ,581 , issued to Epstein et al. These include (1) radiolabels; (2) radiopaque materials; and (3) magnetic resonance enhancing materials. Exemplary radiolabels include: Tc-99m, 1-123, 1-125, In-1 1 1. In-1 13m, Ga-67, or other suitable gamma-emitters. Exemplary radiopaque materials include iodine compounds, barium compounds, gallium compounds, thallium compounds, and the like.
- radiopaque materials include: barium, diatrizoate, ethiodized oil, gallium citrate, iocarmic acid, iocetamic acid, iodamide, iodipamide, iodoxamic acid, iogulamide. iohexol, iopamidol, iopanoic acid, ioprocemic acid, iosefamic acid, ioseric acid, iosulamide meglumine, iosumetic acid, iotasul.
- Magnetic resonance enhancing materials refer to materials that can be detected by or that enhance the effects of magnetic resonance imaging equipment.
- Exemplary magnetic resonance enhancing materials include gadolinium, copper, iron, and chromium. It is preferred that these metal atoms be prepared in the form of a conventional organometallic chelates. which are then bound to the carrier.
- imaging agents are coupled to the carrier molecule (X-P) using conjugation procedures known to those of ordinary skill in the art. Exemplary procedures are described in U.S. Patent No. 4,861 ,581.
- Example 13 Protocol for Using X-P-therapeutic agent for Treatment of a Medical
- Exemplary therapeutic agents are disclosed above. Such agents are conjugated to a carrier using conjugation reactions that are known to those of ordinary skill in the art (see, e.g., U.S. Patent No. 4,861,581 and Example 3. above). As would be apparent to one of ordinary skill in the art, the exact dose of prodrug to be given to an individual is determined by consideration of the nature of the condition being treated, whether the condition is acute or chronic, the biological characteristics of the individual being treated (e.g., body weight, size and general health characteristics). In general, dosages are determined in accordance with standard practice for optimizing the correct dosage for treating the medical disorder, i.e., improving the condition and/or delaying progression of a disease or disorder. The therapeutic agent prodrug is administered according to standard practice.
- topical formulations of the prodrug are formulated in an ointment or cream for surface administration.
- exemplary prodrugs for topical application include antisense prodrugs which are directed to inhibiting transcription of viruses such as those associated with, for example, genital herpes.
- Such topical formulations further can include chemical compounds such as dimethylsulfoxide (DMSO) to faciliate surface penetration of the active ingredient, e.g., the antisense prodrug.
- DMSO dimethylsulfoxide
- compositions of the invention are also useful for delivering a therapeutic agent such as an antibiotic that is normally unable to cross the cell membrane but that is highly active against an intracellular pathogen (Tulkens 1990; Tulkens 1991 ; van, den et al. 1991; Van, der et al. 1991).
- a therapeutic agent such as an antibiotic that is normally unable to cross the cell membrane but that is highly active against an intracellular pathogen (Tulkens 1990; Tulkens 1991 ; van, den et al. 1991; Van, der et al. 1991).
- antibiotic prodrugs are administered intravenously.
- CORRESPONDENCE ADDRESS (A) ADDRESSEE: WOLF, GREENFIELD & SACKS, P.C.
- MOLECULE TYPE protein
- FRAGMENT TYPE internal
- Lys lie Leu Val Lys Asn Lys Lys 1 5
- MOLECULE TYPE peptide (v) FRAGMENT TYPE: internal
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Abstract
Procédés et compositions utilisés pour faciliter le transport d'un agent extracellulaire ne pénétrant pas dans la membrane, ayant une activité intracellulaire dans la membrane d'une cellule. Les procédés de l'invention consistent à coupler de manière covalente un groupe de blocage à extrémité N-terminale à un peptide induisant le transport, pour la formation d'une molécule porteuse. Ladite molécule porteuse est couplée de manière covalente à l'agent extracellulaire de sorte qu'un promédicament pouvant pénétrer dans la membrane soit formé. Les peptides induisant le transport sont fortement basiques et se lient aux polyphosphoinositides.
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU77371/96A AU7737196A (en) | 1996-11-14 | 1996-11-14 | Polyphosphoinositide binding peptides for intracellular drug delivery |
PCT/US1996/018453 WO1998020887A1 (fr) | 1996-11-14 | 1996-11-14 | Peptides se liant aux polyphosphoinositides, pour l'administration intracellulaire de medicaments |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
PCT/US1996/018453 WO1998020887A1 (fr) | 1996-11-14 | 1996-11-14 | Peptides se liant aux polyphosphoinositides, pour l'administration intracellulaire de medicaments |
Publications (1)
Publication Number | Publication Date |
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WO1998020887A1 true WO1998020887A1 (fr) | 1998-05-22 |
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Application Number | Title | Priority Date | Filing Date |
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PCT/US1996/018453 WO1998020887A1 (fr) | 1996-11-14 | 1996-11-14 | Peptides se liant aux polyphosphoinositides, pour l'administration intracellulaire de medicaments |
Country Status (2)
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AU (1) | AU7737196A (fr) |
WO (1) | WO1998020887A1 (fr) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE19935302A1 (de) * | 1999-07-28 | 2001-02-08 | Aventis Pharma Gmbh | Konjugate und Verfahren zu deren Herstellung sowie deren Verwendung zum Transport von Molekülen über biologische Membranen |
US7101967B2 (en) * | 1998-11-13 | 2006-09-05 | Cyclacel Limited | Transport vectors |
WO2022156531A1 (fr) * | 2021-01-19 | 2022-07-28 | 中国人民解放军军事科学院军事医学研究院 | Peptide de liaison à la dynéine capable de traverser une barrière biologique, et son utilisation |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4454065A (en) * | 1982-05-18 | 1984-06-12 | Smithkline Beckman Corporation | Oligopeptide prodrugs |
WO1991017170A1 (fr) * | 1990-05-04 | 1991-11-14 | Biogen, Inc. | Constructions de fusion de gelsoline multimeres |
US5108921A (en) * | 1989-04-03 | 1992-04-28 | Purdue Research Foundation | Method for enhanced transmembrane transport of exogenous molecules |
WO1993025564A1 (fr) * | 1992-06-15 | 1993-12-23 | Brigham And Women's Hospital | Peptides de fixation de phosphoinositides derives des sequences de gelsoline et villine |
WO1995018221A1 (fr) * | 1993-12-28 | 1995-07-06 | Chugai Seiyaku Kabushiki Kaisha | Gene codant pour les adseverines |
-
1996
- 1996-11-14 WO PCT/US1996/018453 patent/WO1998020887A1/fr active Application Filing
- 1996-11-14 AU AU77371/96A patent/AU7737196A/en not_active Abandoned
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4454065A (en) * | 1982-05-18 | 1984-06-12 | Smithkline Beckman Corporation | Oligopeptide prodrugs |
US5108921A (en) * | 1989-04-03 | 1992-04-28 | Purdue Research Foundation | Method for enhanced transmembrane transport of exogenous molecules |
WO1991017170A1 (fr) * | 1990-05-04 | 1991-11-14 | Biogen, Inc. | Constructions de fusion de gelsoline multimeres |
WO1993025564A1 (fr) * | 1992-06-15 | 1993-12-23 | Brigham And Women's Hospital | Peptides de fixation de phosphoinositides derives des sequences de gelsoline et villine |
WO1995018221A1 (fr) * | 1993-12-28 | 1995-07-06 | Chugai Seiyaku Kabushiki Kaisha | Gene codant pour les adseverines |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7101967B2 (en) * | 1998-11-13 | 2006-09-05 | Cyclacel Limited | Transport vectors |
DE19935302A1 (de) * | 1999-07-28 | 2001-02-08 | Aventis Pharma Gmbh | Konjugate und Verfahren zu deren Herstellung sowie deren Verwendung zum Transport von Molekülen über biologische Membranen |
WO2022156531A1 (fr) * | 2021-01-19 | 2022-07-28 | 中国人民解放军军事科学院军事医学研究院 | Peptide de liaison à la dynéine capable de traverser une barrière biologique, et son utilisation |
Also Published As
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AU7737196A (en) | 1998-06-03 |
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