Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
  • G01N33/5014—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing toxicity
• • 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
  • A61K47/543—Lipids, e.g. triglycerides; Polyamines, e.g. spermine or spermidine
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
  • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K49/00—Preparations for testing in vivo
  • A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
  • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
  • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
  • G01N33/5035—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects on sub-cellular localization
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
  • G01N33/5076—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving cell organelles, e.g. Golgi complex, endoplasmic reticulum

Definitions

• This invention relates to compositions and methods for delivery of phosphatidylinositol polyphosphates (phosphoinositides, PIP n s) and inositol polyphosphates (IP n s) into cells of both eukaryotes and prokaryotes. Included among these cells are cells of animals, including parasites such as protozoa and helminths, plants, fungi, and bacteria. More particularly, the invention relates to delivery of PIP n s, IP n s, and derivatives thereof into cells using polyamine shuttles.
• PIP n s phosphatidylinositol polyphosphates
• IP n s inositol polyphosphates
• Phosphatidylinositol polyphosphates serve as signaling molecules in numerous and diverse eukaryotic cellular processes. T.F.J. Martin, Phosphoinositide lipids as signaling molecules: Common themes for signal transduction, cytoskeletal regulation, and membrane trafficking. 14 Annu. Rev. Cell Dev. Biol. 231-264 (1998); A. Toker, The synthesis and cellular roles of phosphatidylinositol 4,5-bisphosphate. 10 Curr. Opin. Cell Biol. 254-261
• Activation of cellular signaling pathways can result from specific changes in membrane phosphoinositide phosphorylation in response to stimuli.
• the number and location of phosphate monoesters on the inositol head group are controlled by specific kinases and phosphatases and determines the role of each PIP n .
• Phosphoinositides as regulators in membrane traffic, 271 Science 1533-9 (1996).
• Phosphoinositide composition is dynamic in time and space, and to locate specific signaling events, it is necessary to devise a technique to study the localization of newly-synthesized PIP n s in cellular membrane domains.
• An attractive approach would be to have a cell-permeant form of any given PIP n , which would permit direct observation of PIP n changes and redistribution in specific cellular compartments following activation by extracellular signals.
• U.S. Patent No. 5,783,662 to P.A. Janmey et al. describes covalent conjugates of N- terminal-blocked polyphosphoinositide binding peptides for use in facilitating transport of a membrane-impermeable extracellular agent across the membrane of the cell.
• the extracellular agent such as a peptide, oligonucleotide, or antibiotic, is covalently coupled to the carboxyl terminus of a transport mediating peptide to form a prodrug having the formula
• X-P-A wherein X is an amine derivatizing agent, P is a polybasic transport-mediating peptide, and A is the extracellular agent, which is transported across the cell membrane.
• X is a fluorescent molecule, such as rhodamine or fluorescein
• P is a peptide domain of human gelsolin.
• U.S. Patent No. 5,693,521 to R.Y. Tsien & C. Schultz discloses acyloxyalkyl esters of phosphate-containing second messengers, such as cAMP, cGMP, inositol triphosphate, and inositol tetraphosphate, which are capable of permeating cell membranes. Once inside the cell, the ester derivatives undergo enzymatic conversion to the biologically active form of the second messenger.
• WO 98/15629 by M.P. Czech & J.K. Klarlund discloses binding proteins for phosphatidylinositides. These binding proteins, referred to as "general receptors for phosphoinositides” or “GRPs”exhibit a functionally and structurally modular form that includes a subdomain homologous to the yeast SEC7 gene product and a pleckstrin homology (PH) domain. GRP proteins preferably exhibit high affinity binding to products of the lipid kinase, phosphatidylinositol-3-OH kinase (i.e., PI 3-kinase), e.g., phosphatidylinositol-3,4,5- trisphosphate.
• PI 3-kinase phosphatidylinositol-3,4,5- trisphosphate.
• GRP proteins may also be used for screening test compounds for modulators of an interaction between a PI 3-kinase product and a GRP polypeptide receptor.
• WO 97/46688 by B. Vanhasebroeck & M.D. Waterfield discloses the lipid kinase, pi lO ⁇ , which is part of the PI 3-kinase family.
• the PI 3-kinase pi lO ⁇ interacts with p85, has a broad phosphoinositide specificity, and is sensitive to the same kinase inhibitors as PI 3- kinase 110a.
• pi lO ⁇ is selectively expressed in leukocytes.
• pi lO ⁇ shows enhanced expression in most melanomas tested and, therefore, may play a role in regulating the metastatic property exhibited by melanomas.
• compositions and methods for delivery into cells of phosphoinositides and their chemical derivatives would be a significant advancement in the art.
• compositions and methods for delivery of phosphoinositides into cells Another object of the invention is to provide compositions and methods for visualizing the location of fluorescently labeled phosphoinositides during changes in cell physiology.
• Still another object of the invention is to provide a method for screening inhibitors of changes in cell physiology.
• Yet another object of the invention is to visualize the uptake and localization of aminoglycosides.
• a still further object of the invention is to develop antibiotic selectivity assays by observing selective uptake of fluorescent aminoglycosides in pathogens versus human or animal cells.
• Another object of the invention is to provide a high-throughput screen for natural products and synthetic compounds that affect cellular uptake and targeting of both aminoglycosides and phosphoinositides to cells from vertebrate tissues versus cells of pathogenic organisms.
• Still another object of the invention is to use PIP n -fluorescent aminoglycoside uptake for the visualization of uptake and subcellular localization of aminoglycosides.
• Yet another object of the invention is to develop antibiotic selectivity assays by observing selective uptake of fluorescent aminoglycosides in pathogens, including bacteria, protozoal parasites, and the like, versus human or animal cells.
• composition of matter comprising a mixture of a phosphoinositide polyphosphate, inositol polyphosphate, or mixture thereof and a polyamine.
• a composition of matter comprising a mixture of a phosphoinositide polyphosphate, inositol polyphosphate, or mixture thereof and a polyamine.
• each of the phosphoinositide polyphosphate, inositol polyphosphate, or mixture thereof and polyamine is labeled with a detectable label.
• Preferred labels include fluorescent labels, radiolabels, chemiluminescent labels, spin labels, photophores, chromophores, biotin, nanogold particles, and the like, and mixtures thereof. Fluorescent labels are especially preferred.
• Preferred polyamines include aminoglycosidic aminocyclitols (e.g., aminoglycoside antibiotics), dendrimeric polyamines, histones, polybasic polypeptides, lipidic polyamines, polyethyleneimine, steroidal polyamines, and the like, and mixtures thereof.
• a method for facilitating uptake of a phosphoinositide polyphosphate, inositol polyphosphate, or mixture thereof into a cell comprises contacting the cell with a composition of matter comprising a mixture of the phosphoinositide polyphosphate, inositol polyphosphate, or mixture thereof and a shuttle.
• a shuttle is a polyamine such as aminoglycosidic aminocyclitols (e.g., aminoglycoside antibiotics), dendrimeric polyamines, histones, polybasic polypeptides, lipidic polyamines, polyethyleneimine, steroidal polyamines, and the like, and mixtures thereof.
• a method for facilitating uptake of an aminoglycoside antibiotic into a cell comprises contacting the cell with a composition of matter comprising a mixture of (a) a phosphoinositide polyphosphate, inositol polyphosphate, or mixture thereof, and (b) the aminoglycoside antibiotic.
• the cell is a pathogenic cell, such a prokaryotic cell or cell of a parasite, such as a protozoal cell, helminthic cell, or fungal cell.
• a method for visualizing the uptake and localization of an aminoglycoside in a cell comprises:
• the cell can be a eukaryotic cell, such as an animal, plant, protozoal, helminthic, or fungal cell.
• the cell could also be a prokaryotic cell.
• a method for screening for a compound that minimizes cytotoxicity of aminoglycoside antibiotics to mammalian cells comprises:
• a further aspect of this method comprises determining whether uptake of the aminoglycoside into a bacterial cell is substantially unaltered in the presence of the compound.
• a composition of matter comprises an aminoglycoside antibiotic covalently bonded to a fluorescent compound.
• the aminoglycoside antibiotic is covalently bonded to the fluorescent compound through a linker moiety, such as an isothiocyanate or succinimidyl ester group.
• the aminoglycoside antibiotic is neomycin and the fluorescent compound is rhodamine B or XR.
• a method for monitoring calcium flux in a cell comprises:
• the calcium indicator is calcium crimson or Fluo-3 and the change in intensity is detected as a change in fluorescence.
• FIGS, la-d show the structures of fluorescently labeled phosphoinositides, inositol polyphosphates, and aminoglycosides according to the present invention.
• FIG. lb shows the structure of Ins(l,4,5)P 3 - XRITC, a fluorescent analog of IP 3 .
• FIGS, lc-d show structures of two fluorescent neomycin derivatives, Neomycin-XRITC (Neo-XR) and Neomycin-Rhodamine B (Neo-RB), respectively; the open arrows indicate that either of the two primary aminomethyl substituents could bear the thiourea-linked fluorophore.
• FIGS. 2a-c show delivery by polyamines of fluorescently labeled phosphoinositides and inositol polyphosphates into mammalian cells.
• FIG. 2a shows NIH 3T3 mouse fibroblast cells with internalized PtdIns(4,5)P 2 -NBD/histone; in the no-carrier (histone) control shown in the inset, the PtdIns(4,5)P 2 -NBD fluorescence is extracellular.
• FIG. 2a shows NIH 3T3 mouse fibroblast cells with internalized PtdIns(4,5)P 2 -NBD/histone; in the no-carrier (histone) control shown in the inset, the PtdIns(4,5)P 2 -NBD fluorescence is extracellular.
• FIG. 2a shows NIH 3T3 mouse fibroblast cells with internalized PtdIns(4,5)P 2 -NBD/histone; in the no-carrier (histone) control shown in the inset
• FIG. 2b shows Ins(l,4,5)P 3 was delivered to NIH 3T3 cells using histone protein as a carrier; in the inset, no histone was added to the Ins(l,4,5)P 3 , and essentially all of the Ins(l,4,5)P 3 fluorescence is extracellular.
• FIG. 2c PtdIns(3,4,5)P 3 -NBD was delivered into NIH 3T3 fibroblast cells using histone as a shuttle. PtdIns(3,4,5)P 3 -NBD was mixed with histone and added to NIH 3T3 mouse fibroblast cells grown on coverslips. Low magnification (FIG. 2c) and high magnification (FIG.
• FIG. 2a inset and FIG. 2b inset 30 minutes (FIG. 2a inset and FIG. 2b inset), 5 minutes (FIG. 2a), or 10 minutes (FIG. 2b and FIG. 2c) after dye solutions were added to the cells.
• the micrographs were obtained with a laser scanning confocal microscope.
• the bright cellular structures are due to the fluorescence of the PtdIns(4,5)P 2 -NBD (FIG. 2a); Ins(l,4,5)P 3 -XRITC (FIG. 2b); or PtdIns(3,4,5)P 3 -NBD (FIG. 2c).
• FIGS. 3a-b show phosphoinositide-mediated delivery of neomycin-rhodamine into NIH 3T3 cells.
• Neomycin-rhodamine was added to the medium of NIH 3T3 cells attached to cover slips, but remained almost exclusively extracellular for more than 10 minutes, as observed in FIG. 3a wherein the bright extracellular medium results from the neomycin- rhodamine fluorescence.
• neomycin-rhodamine was mixed with
• FIGS. 4a-d show delivery of neomycin and phosphoinositides into prokaryotic and non-mammalian eukaryotic cells.
• a fluorescent neomycin (FITC-labeled) was delivered into E. coli cells as a complex with PtdIns(4,5)P 2 .
• PtdIns(4,5)P 2 -NBD was delivered to E. coli cells as a complex with histone protein.
• FIG. 4c a neomycin- rhodamine/PtdIns(4,5)P 2 complex entered the protozoan pathogen, Cryptosporidium parvum
• FIG. 4d a PtdIns(4,5)P 2 -NBD/histone complex rapidly entered C. parvum.
• the micrographs were obtained with a laser scanning confocal microscope 5 minutes after addition of the neomycin-FITC/PtdIns(4,5)P 2 ; PtdIns(4,5)P 2 -NBD/histone; or neomycin-rhodamine/PtdIns(4,5)P 2 complexes to the cell media.
• the C. parvum cells were cultured on cover slips coated with bovine epithelial cells.
• the bright fluorescence is from the fluorophores (FITC, NBD, rhodamine) attached to neomycin (FIGS. 4a and 4c) or PtdIns(4,5)P 2 (FIGS. 4b and 4d).
• a phosphoinositide polyphosphate includes reference to two or more of such phosphoinositide polyphosphates
• reference to “an inositol polyphosphate” includes reference to two or more of such inositol polyphosphates
• reference to “an aminoglycoside” includes reference to two or more of such aminoglycosides.
• shuttle means a compound, polymer, complex, or mixture thereof that facilitates transport of phosphoinositides, inositol polyphosphates, and mixtures thereof into cells.
• Preferred shuttles comprise polyamines.
• polyamines includes aminoglycosidic aminocyclitols (e.g., aminoglycoside antibiotics), synthetic "spherical” dendrimeric polyamines, polybasic nuclear proteins (histones), polybasic polypeptides, lipidic polyamines, polyethyleneimine, steroidal polyamines, and the like, and mixtures thereof.
• a "polybasic protein” or “polybasic polypeptide” contains sufficient lysine, arginine, and/or histidine residues to complex an anionic ligand, such as an IP n or PIP n .
• the polybasic polypeptide may also contain unnatural or non-protein amino acids, N-acylglycine groups, and any of a known group of amide group replacements known as peptide bond isosteres.
• aminoglycosidic aminocyclitols or “aminoglycosides” that can be used according to the present invention include, without limitation, neomycin, gentamicin, geneticin, streptomycin, kanamycin, tobramycin, spectinomycin, formicidin, streptamine, deoxystreptamine, epistreptamine, fortamine, validamine, valienamine, hydroxyvalidamine, valiciamine, validoxylamine A, validoxylamine B, validoxylamine G, and the like, and mixtures thereof.
• the primary or secondary amino groups of aminoglycosidic aminocyclitols can be reacted with isothiocyanato groups, with succinimidyl esters, with sulfonyl halides, and with other amine-reactive derivatives of fluorescent compounds according to the guidelines set out herein.
• suitable fluorescent compounds that can be used according to the present invention include chemically activated, tetherable analogs of acrylodan, AMCA, BODIPY, Cascade-Blue, CINERF, dansyl, dialkylaminocoumarin, eosin, erythrosine, fluorescein (FITC), hydroxycoumarin, NBD, Oregon green, PyMPO, pyrene, rhodamine, Rhodol Green, TMR, Texas Red, X-Rhodamine, and the like.
• FITC fluorescein
• eukaryotic cells and similar terms mean cells of animals and plants, as well as other cells containing membrane-bound nuclei and mitochondria.
• prokaryotic cells and similar terms mean cells of bacteria, blue- green algae, and other cells lacking nuclei and mitochondria.
• bacteria includes both Gram-negative and Gram-positive species.
• Gram-negative bacteria are species of the genera Escherichia, Pseudomonas, Acinetobacter, Francisella, Bordetella, Shigella, Salmonella, Proteus, Yersinia, Klebsiella, Enterobacter, Serratia, Vibrio, Haemophilus, Pasteurella, Streptobacillus, Bacteriodes, Fusobacterium, Neisseria, and the like.
• Gram-positive bacteria are species of the genera Staphylococcus, Streptococcus, Corynebacterium, Bacillus, Clostridium, and the like.
• actinomycetes such as the genera Nocardia and Actinomyces, mycobacteria (i.e., Mycobacterium), obligately intracellular bacteria such as rickettsiae and chlamydiae (e.g., Rickettsia and Chlamydia), spiral bacteria (e.g., Spirillum, Campylobacter, Treponema, Borrelia, Leptospira, and Helicobacter), and mycoplasmas (e.g., Mycoplasma).
• actinomycetes such as the genera Nocardia and Actinomyces, mycobacteria (i.e., Mycobacterium), obligately intracellular bacteria such as rickettsiae and chlamydiae (e.g., Rickettsia and Chlamydia), spiral bacteria (e.g., Spirillum, Campylobacter, Treponema, Borrelia, Lepto
• parasite includes protozoans, such as Crypto sporidium spp., Toxoplasma spp., Trypanosoma spp., Giardia spp., Trichomonas spp., Entamoeba spp.,
• Plasmodium spp. Balantidium coli, Pneumocystis spp., Leishmania spp., Acanthamoeba spp., Naegleria spp., and the like; and helminths such as Enterobius spp., Trichinella spp., Schistosoma spp., Ascaris spp., Trichuris spp., Ancyclostoma spp., Necator spp., Strongyloides spp., Trichostrongylus spp., Diphyllobothrium spp., Hymenolepsis spp., Wuchereria spp., Onchocerca spp., Loa spp., Taenia spp., Draculuncus spp., Paragonimus spp., Clonorchis spp., Fasciola spp., Fasciolopsis spp.
• fungi includes Microsporum spp., Trichophyton spp., Epidermophyton spp., Candida spp., Microsporum spp., Cryptococcus spp., Aspergillus spp., Malassezia spp., Coccidioides spp., Histoplasma spp. Mucor spp., Blastomyces spp., Sporotrichum spp., Torulopsis spp., Rhodotorula spp., and the like.
• substantially unaltered means that uptake of an aminoglycoside antibiotic into a prokaryotic cell in the presence of a compound being tested for ability to reduce cytotoxicity to a mammalian cell is 90% or greater of the uptake of the aminoglycoside in the absence of the compound.
• NBD and PtdIns(3,4,5)P 3 -NBD the illustrative fluorescent inositol polyphosphate Ins(l,4,5)P 3 -XRITC, and two illustrative fluorescent polyamine shuttles, neomycin-XRITC (Neo-XR) and neomycin-Rhodamine B (Neo-RB), are shown in FIGS. la-d.
• the fluorescent PIP-S are fully water-soluble at the concentrations employed.
• the attachment of the fluorophore to the acyl chain allows full recognition by target proteins of both the inositol polyphosphate head group and the glycerol backbone proximal to the headgroup.
• G.D the attachment of the fluorophore to the acyl chain allows full recognition by target proteins of both the inositol polyphosphate head group and the glycerol backbone proximal to the headgroup.
• Prestwich et al. in Phosphoinositides: Chemistry, Biochemistry and Biomedical Applications 24-37 (K.S. Bruzik ed. 1999). All fluorescent and non-fluorescent shuttle and PIP n derivatives have been synthetically prepared, G.D. Prestwich, Touching all the bases: Inositol polyphosphate and phosphoinositide affinity probes from glucose, 29 Ace. Chem. Res. 503- 513 (1996), and are readily available (Echelon Research Laboratories, Salt Lake City, Utah), thus facilitating implementation of the present invention.
• polyamine-PIP n complexes are preferably formed prior to addition to the extracellular medium of the cells.
• the molar ratio of PIP n to polyamine is preferably in the range of about 1000:1 to 1 :1000, more preferably 50:1 to 1 :50, and most preferably 5:1 to 1 :5.
• the localization of both the polyamine shuttle and the cargo PIP n can be followed by monitoring one- or two-color fluorescent tags using laser scanning confocal microscopy.
• Dendrimeric amines were nearly as efficient as neomycin, but caused cell detachment over several hours, perhaps due to disruption of intracellular PIP n - cytoskeletal protein interactions.
• the histones were the least efficient shuttles, but did not cause any gross change in cell physiology; both cell division and redistribution of labeled PIP n s was directly observable.
• Other polyamines examined were less efficient, but could still cause measurable but slow intracellular uptake in attached or detached cells.
• FIG. 2a illustrates the histone-facilitated entry of PtdIns(4,5)P 2 -NBD into NIH 3T3 fibroblast cells after 5 minutes.
• FIG. 2b shows histone-facilitated entry of Ins(l,4,5)P 3 , and FIG. 2b (inset) is the control without histone.
• FIG. 2a illustrates the histone-facilitated entry of PtdIns(4,5)P 2 -NBD into NIH 3T3 fibroblast cells after 5 minutes.
• FIG. 2b shows histone-facilitated entry of Ins(l,4,5)P 3
• FIG. 2b (inset) is the control without histone.
• Endocytosis was altered in cells by incubation at lowered pH (5.5-6.0), at low temperature (4°C) or in the presence of GTP ⁇ S. Under these conditions, the lipophilic styryl dye FM 4-64 remained in the plasma membrane of MDCK cells with no detectable intracellular accumulation. Upon release from the endocytic block by returning cells to standard growth medium, FM 4-64 was detectably endocytosed within 5-10 min. In CHO cells, a kinetic delay in endocytic vesicle movement was realized with the different treatments. Thus, for each of the three treatments, FM 4-64 movement into CHO cells was significantly delayed but not prevented, and, upon release from the block, FM 4-64 was rapidly internalized in the CHO cells.
• Ptdlns(4,5)P 2 -NBD and the fluorophore-conjugated aminoglycoside was confirmed by co- staining with organelle-specific fluorophores.
• the fluorescent aminoglycoside derivative, Neo-RB requires PtdIns(4,5)P 2 to achieve intracellular uptake.
• the fluor was concentrated in the endoplasmic reticulum (ER) and the nucleoli, consistent with its complexation to transfer RNA and ribosomal RNA subunits or their precursors.
• the PIP n analog, PtdIns(4,5)P 2 -NBD could be seen in the plasma membrane and in intracellular patterns consistent with its presence in the ER, Golgi, and nuclear membrane, and substructures within the nucleus (including the nucleoli).
• the co-localization of PtdIns(4,5)P 2 -NBD and Neo-RB in nucleoli and in the ER is noteworthy. Indeed, these images suggest a method (described in more detail below) for screening for compounds that would minimize mammalian cytotoxicity of aminoglycoside antibiotics and maximize bacterial toxicity, since for the first time it is possible to visually monitor both uptake and binding to high-affinity RNA targets in the ER and the nucleolus. This method would be complementary to the identification of RNA aptamers selected for binding to aminoglycosides.
• G. Werstuck & M. Green Controlling gene expression in living cells through small molecule-RNA interactions, 282 Science 296-
• Neo-RB clearly stained the cell wall and to a lesser degree the plasma membrane, while the Ptdlns(4,5)P 2 -NBD moved on into cell plasma membranes. Importantly, omission of the shuttle resulted in very slow and inefficient uptake of the PtdIns(4,5)P 2 -NBD. Finally, delivery of the Ptdlns(4,5)P 2 -NBD compound to the budding yeast, Saccharomyces cerevisiae, using a neomycin shuttle, has also been obtained, but uptake was slower than in animal and plant cells.
• NeoRB fluorescently labeled aminoglycosides
• FIG. 4a shows that fluorescent neomycin (FITC-labeled) was delivered into E. coli cells as a complex with PtdIns(4,5)P 2 .
• FIG. 4b shows PtdIns(4,5)P 2 -NBD was delivered into E. coli cells as a complex with histone protein.
• FIG. 4c a neomycin-rhodamine/PtdIns(4,5)P 2 complex entered the protozoan pathogen, Cryptosporidium parvum (bright spots among the field of epithelial cells).
• FIG. 4a neomycin-rhodamine/PtdIns(4,5)P 2 complex entered the protozoan pathogen, Cryptosporidium parvum (bright spots among the field of epithelial cells).
• the bright fluorescence is from the fluorophores (FITC, NBD, and Rhodamine) attached to neomycin (FIGS. 4 a and c) or PtdIns(4,5)P 2 (FIGS. 4 b and d).
• bradykinin works differentially on cell types containing distinct seven membrane pass (G-protein coupled) receptors (Bl, B2, B3), the results were variable, with some cells types responding to 1 ⁇ M bradykinin with a modest relocalization of intracellular
• Ptdlns(4,5)P 2 -NBD and significant loss of fluorescent signal within minutes.
• Other cell types showed little or no response to bradykinin.
• the results obtained with mastoparan were similar to those obtained with bradykinin.
• Studies with human neutrophils also establish changes in intracellular PIP n s introduced by the shuttle system occur in predicted fashion upon stimulation with growth factors and peptides such as formyl-Met-Leu-Phe.
• Ptdlns(4,5)P 2 -NBD redistribution experiment was conducted in which insulin was employed to activate cells.
• 3T3-L1 adipocytes serum starved
• Ptdlns(4,5)P 2 -NBD/C6-histone complex was pre-loaded with an Ptdlns(4,5)P 2 -NBD/C6-histone complex.
• PtdIns(4,5)P 2 -NBD was chosen because activation of receptor tyrosine kinases and PI 3-kinase could lead to conversion of Ptdlns(4,5)P 2 to Ptdlns(3,4,5)P 3 by PI 3-kinase, or PtdIns(4,5)P 2 could act as a substrate for phospholipase C isozymes ( ⁇ or ⁇ ). Both enzymatic activities are known to be modulated by PIP n binding in these cells.
• the histone protein was chosen as the polyamine shuttle because high concentrations of aminoglycoside antibiotics are known to perturb cellular phospholipase activity.
• NBD moves from the plasma membrane to intracellular sites.
• the second hypothesis is favored, in which the diffuse perinuclear-localized fluorophore would be diacylglyceryl-NBD, which is derived from phospholipase C action on Ptdlns(4,5)P 2 -NBD.
• diacylglyceryl-NBD which is derived from phospholipase C action on Ptdlns(4,5)P 2 -NBD.
• the presently claimed method can also be used to introduce Ins(l,4,5)P 3 into cells for observing calcium flux.
• Cover slip-attached 3T3-L1 preadipocytes were loaded with the calcium indicator, calcium crimson, using a 15 -minute incubation period. This dye exhibits a red fluorescence that increases in intensity upon calcium binding. Medium was exchanged and cells were observed until no fluorescence changes were evident. Then, a complex of Ins(l,4,5)P 3 and histone was added to the medium. A time-course collection of images showed an increase in red fluorescence within 10 seconds in the cytosol and nuclei of the treated cells.
• the use of the shuttle-cargo system to ferry affinity-tagged or native phosphoinositides into living cells provides a simple method to examine the action, localization, and metabolism of phosphoinositides involved in signal transduction pathways in the context of changes in cell physiology.
• the availability of cell-permeant PIP n s allows direct observation in time and space of specific phosphoinositide signaling events occurring in eukaryotic cells during growth and differentiation, following oncogenic transformation, and during pathogen infection.
• PIP n s can be used to shuttle aminoglycosides and other biologically active polyamines specifically into eukaryotic cells. Given the current critical need for new antibiotic strategies, this new technique should stimulate the pursuit of molecules that could reclaim the use of many aminoglycosides for which toxicity or resistance has reduced their clinical utility.
• HTS high throughput screening
• Fluorescent derivatives of polyamines were synthesized from the amine and either the isothiocyanate or succinimidyl ester derivatives of the fluorescent reagent in 1 N TEAB (aqueous triethylammonibicarbonate, pH 7.5). All compounds were purified by HPLC and their structures were confirmed by NMR and Electron Spray Mass Spectrometry (ES-MS).
• Neomycin trisulfate 110 mg, 0.121 mmol
• rhodamine B isothiocyanate 8.2 mg, 0.015 mmol; Aldrich Chemical Co., St. Louis, Missouri
• TEAB triethylammonium bicarbonate, pH 8.6
• DMF dimethylformamide
• the solution was stirred for 72 hr at 25 °C.
• the solution was then concentrated in vacuo, the residue was dissolved in water (10 mL), and byproducts were removed with two 10-mL methylene chloride extractions.
• the aqueous solution was again concentrated in vacuo, and reversed-phase (C 18 ) HPLC fractions containing purified Neo-RB were collected using an acetonitrile gradient in 0.06% trifluoroacetic acid, and were lyophilized.
• ES-MS revealed an M +2 peak for the tetraprotonated species: mz 511.4 (calculated for M+4H, 1023.3).
• Neo-XR (FIG. 1 c) was prepared according to the procedure of
• Example 1 except that X-RITC (Molecular Probes, Inc., Eugene, Oregon) was used as the activated fluorescent reagent instead of Rhodamine B isothiocyanate.
• X-RITC Molecular Probes, Inc., Eugene, Oregon
• Example 3 In this example, a dendrimer amine with 12 primary amines was fluorescently labeled according to the procedure of Example 1 except that the dendrimer amine was substituted for neomycin.
• the dendrimer amine was prepared by the reduction of 12-cascade:amino(3):(l- azapropylidene):(l-azabutylidene):2-propanenitrile, R. Moors & F.Vogtle, Dendrimere polyamine, 126 Chem. Ber. 2133-2135 (1993) (hereby incorporated by reference), with di- isobutylaluminum hydride in methylene chloride.
• Example 4 In this example, the procedure of Example 1 was followed except that the dendrimer amine, DAB-Am-32 (Aldrich Chemical Co., St. Louis, Missouri), was substituted for neomycin.
• Example 5 In this example, the procedure of Example 1 was followed except that histone Type III-S from calf thymus (Sigma Chemical Co., St. Louis, Missouri) was substituted for neomycin.
• Example 6 In this example, the procedure of Example 1 was followed except that fluorescein isothiocyanate (FITC) was substituted for Rhodamine B isothiocyanate and the solution was stirred for 1 hr and kept standing for 36 hr at room temperature.
• FITC fluorescein isothiocyanate
• ES-MS m/z 1003.63 Calcd 1004.2.
• Example 7 In this example, the procedure of Example 6 was followed except that SNAFL-NHS was substituted for FITC and the solution was stirred for 12 hr and kept standing for 7 days. ES-MS, m/z 1002.19 Calcd 1022.
• Example 8 In this example, the procedure of Example 6 was followed except that geneticin disulfate was substituted for neomycin, and the solution was stirred for 1 day. ES-MS, m/z 887.21 Calcd 885.9.
• Example 9 In this example, the procedure of Example 6 was followed except that gentamicin (mixture of gentamicin C diligent C 2 , C la ) monosulfate was substituted for neomycin.
• Example 10 In this example, the procedure of Example 6 was followed except that kanamycin was substituted for neomycin. The results were substantially similar to those obtained with neomycin.
• Example 1 1 In this example, the procedure of Example 6 was followed except that paromomycin was substituted for neomycin. The results were substantially similar to those obtained with neomycin.
• Example 12 In this example, the procedure of Example 6 was followed except that streptomycin was substituted for neomycin. The results were substantially similar to those obtained with neomycin.
• Example 13 In this example, the procedure of Example 6 was followed except that tobramycin was substituted for neomycin. The results were substantially similar to those obtained with neomycin.
• Example 14 Synthesis of fluorescent PtdlnsP- derivatives. s ⁇ -2-O-Stearoyl Ptdlns(4,5)P 2 -NBD (C 18 )was synthesized as previously described, J. Chen et al., Synthesis of photoactivatable 1 ,2-O-diacyl-stt-glycerol derivatives of 1 -L-phosphatidyl-D-myo-inositol 4,5-bisphosphate and 3,4,5-trisphosphate, 61 J. Org. Chem. 6305-6312 (1996) (hereby incorporated by reference). NBD-X-SE was purchased from Molecular Probes, Inc. (Eugene, Oregon).
• Example 15 In this example, the procedure of Example 14 was followed except a shorter chain sn- l-O-(6-aminohexanoyl) ⁇ «-2-O-hexanoyl derivative was prepared. Reaction with NBD-X-SE was performed as described to produce the PtdIns(4,5)P 2 -NBD (C 6 ), which was characterized by proton NMR.
• Example 16 In this example, the procedure of Example 14 was followed except that an aminoacyl derivative of PtdIns(3,4,5)P 3 was substituted for the aminoacyl derivative of PtdIns(4,5)P 2 .
• Example 17 In this example, the procedure of Example 14 was followed except that an aminoacyl derivative of PtdIns(3,5)P 2 was substituted for an aminoacyl derivative of PtdIns(4,5)P 2 , and BODIPY-FL or BODIPY-5G1 was substituted for NBD.
• Example 18 In this example, the procedure of Example 14 was followed except that Texas Red was substituted for NBD.
• COS-7 Madin-Darby canine kidney (MDCK), NIH 3T3 mouse fibroblasts, and 3T3-L1 adipocyte cells were cultured (DMEM 90%, calf serum 10%) on 10- mm diameter cover slips for 12-24 hr prior to phosphoinositide delivery experiments.
• Chinese hamster ovary (CHO) cells were cultured on cover slips in Ham's F12 medium with 5% calf serum.
• dyes were added to the medium (approximately 20 ⁇ l) on cover slips that were mounted directly to slides or to glass plates with a 200- ⁇ l capacity well. Immediately following addition of the dyes, the glass plate or slide was mounted on the microscope stage for observation.
• the 488 nm laser line was used for excitation of NBD-conjugated PIP n s.
• the 568 nm laser line was used for Neo-XR and Neo-RB. Images of animal cells were collected using a 60X oil immersion objective. No post-acquisition enhancement of images was performed, except for gray-scale conversion to color using Laser Sharp software or Confocal Assistant software. When no polyamine shuttle was added to the Ptdlns(4,5)P 2 -NBD, individual cells appeared as dark ghosts or multicellular clusters (FIG. 2a (inset)), indicating that the PIP n analog failed to enter the cells to detectable levels within 30 minutes.
• Example 20 In this example, the procedure of Example 19 was followed except that Ins(l,4,5)P 3 was substituted for PtdIns(4,5)P 2 -NBD.
• Example 19 the procedure of Example 19 was followed except that XRITC- Ins(l,4,5)P 3 was substituted for PtdIns(4,5)P 2 -NBD.
• Example 22 In this example, the procedure of Example 19 was followed except that
• PtdIns(3,4,5)P 3 was substituted for PtdIns(4,5)P 2 -NBD.
• Example 23 To determine whether movement of PIP n -polyamine shuttle into cells was due to endocytosis or crossing the membrane passively, endocytosis was slowed or blocked.
• CHO and MDCK cells were incubated in low pH medium (5.5-6.0), at low temperature (4°C), or in the presence of GTP ⁇ S to alter endocytosis.
• Localization of the lipophilic styryl dye, FM 4- 64, in the plasma membrane and its movement into cells was monitored via time course optical section collection by confocal microscopy. When cells were released from one of the endocytic blocks by returning them to standard growth medium, FM 4-64 was detectably endocytosed within 5-10 min.
• FM 4-64 movement was monitored concomitantly with phosphoinositide analog/shuttle solutions.
• MDCK cells endocytosis was blocked and the phosphoinositide analog/shuttle complexes still rapidly entered cells.
• CHO cells endocytosis was slowed and phosphoinositide analog/shuttle complexes still rapidly entered cells.
• the lipophilic styryl dye FM 4-64 remained in the plasma membrane of MDCK cells with no detectable intracellular accumulation.
• FM 4-64 was detectably endocytosed within 5-10 min.
• a kinetic delay in endocytic vesicle movement was realized with the different treatments.
• FM 4-64 movement into CHO cells was significantly delayed but not prevented; upon release from the block, FM 4-64 rapidly internalized in the CHO cells.
• Example 24 Organelle-specific stains and endocytosis. Intracellular localization of the phosphoinositide-conjugated fluorophores and aminoglycoside-conjugated fluorophores was determined in five animal cell lines and plant root tip cells by co-localization with commercially available organelle-specific fluorescent dyes. Staining of organelles was performed simultaneously or in side-by-side experiments using organelle-specific fluorophores. All organelle stains were obtained from Molecular Probes. In this study, DiOC 6 (ER), BODIPY TR ceramide (Golgi), Acridine orange (nucleic acids), DAPI (DNA), and Sytol 1 (nucleic acids) were used to stain intracellular organelles. FM 4-64 was used to stain the plasma membrane, endosomes, and lysosomes.
• Neo-RB was documented for CHO cells and of the 1 : 1 complex of PtdIns(4,5)P 2 -NBD with Neo-XR into 3T3-L1 adipocytes. The location of the fluorescent dyes appears to reach equilibrium within 5 min incubation for most cells and complexes tested. The red Neo-RB appears concentrated in the endoplasmic reticulum (ER) and the nucleoli, consistent with its complexation to transfer RNA and ribosomal RNA subunits. The green Ptdlns(4,5)P 2 -NBD can be seen in the plasma membrane and in intracellular patterns consistent with its presence in the ER, Golgi, the nuclear membrane, and substructures within the nucleus (including the nucleoli).
• ER endoplasmic reticulum
• the green Ptdlns(4,5)P 2 -NBD can be seen in the plasma membrane and in intracellular patterns consistent with its presence in the ER, Golgi, the nuclear membrane, and substructures within the nu
• Example 25 Whole Arabidopsis thaliana plants were grown in liquid medium (0.5X Murashige and Skoog, IX B5 vitamins, 2% glucose). For imaging experiments, intact one- to two-week- old whole plants were placed in the glass plate wells (total volume 200 ⁇ l) and fluorescent shuttle-cargo complexes were added. Plants were kept submerged and intact for all experiments, by covering the top of the well with a cover slip after fluorophore addition. Images of plant cells were collected 10-15 min following addition of the fluorescent PIP n - fluorescent neomycin complex. Microscopy was carried out according to the procedure described above; a 40X objective was used for some of the plant cell images. The results of this experiment showed that PtdIns(4,5)P 2 -NBD was delivered to the plasma membrane of the plant cells in less than 10 minutes and did not move into other intracellular membranes during a 60-minute observation period.
• liquid medium 0.5X Murashige and Skoog, IX B5 vitamins, 2% glucose.
• Example 26 In this example, the procedure of Example 24 was carried out, except the cells used were the yeast, Saccharomyces cerevisiae. Uptake was achieved, but the rate of uptake was slower than with animal or plant cells.
• Example 27 In this example, the procedure of Example 24 was carried out, except the cells used were E. coli cells and the shuttle was histone. The results obtained from this experiment showed that the carrier polyamine facilitated entry of Ptdlns(4,5)P 2 -NBD into bacterial cells (FIGS. 4a and 4b), and vice versa, that PtdIns(4,5)P 2 facilitated uptake of polyamines, such as histone or fluorescently labeled aminoglycosides, into bacterial cells.
• Example 24 the procedure of Example 24 was carried out, except the cells used were Cryptosporidium parvum.
• the results obtained from this experiment showed that the shuttle facilitated entry of the Ptdlns(4,5)P 2 -NBD into protozoal cells in preference to human epithelial cells (FIGS. 4c and 4d).
• the labeled aminoglycoside was selectively delivered to pathogen cells in comparison to human host cells.

Landscapes

• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Engineering & Computer Science (AREA)
• Biomedical Technology (AREA)
• Immunology (AREA)
• Chemical & Material Sciences (AREA)
• Molecular Biology (AREA)
• Urology & Nephrology (AREA)
• Hematology (AREA)
• Bioinformatics & Cheminformatics (AREA)
• General Health & Medical Sciences (AREA)
• Medicinal Chemistry (AREA)
• Toxicology (AREA)
• Pathology (AREA)
• Tropical Medicine & Parasitology (AREA)
• Food Science & Technology (AREA)
• General Physics & Mathematics (AREA)
• Biochemistry (AREA)
• Analytical Chemistry (AREA)
• Biotechnology (AREA)
• Cell Biology (AREA)
• Physics & Mathematics (AREA)
• Microbiology (AREA)
• Epidemiology (AREA)
• Public Health (AREA)
• Veterinary Medicine (AREA)
• Animal Behavior & Ethology (AREA)
• Pharmacology & Pharmacy (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Rheumatology (AREA)
• Diabetes (AREA)
• Endocrinology (AREA)
• Biophysics (AREA)
• Gastroenterology & Hepatology (AREA)
• Investigating Or Analysing Biological Materials (AREA)
• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
• Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)

Applications Claiming Priority (5)

Publications (2)

Family

ID=26799415

Family Applications (1)

Country Status (4)

Families Citing this family (8)

Citations (1)

• 1999
  • 1999-09-29 EP EP99952984A patent/EP1119315A4/de not_active Withdrawn
  • 1999-09-29 CA CA002345532A patent/CA2345532A1/en not_active Abandoned
  • 1999-09-29 WO PCT/US1999/022594 patent/WO2000018949A2/en not_active Application Discontinuation
  • 1999-09-29 AU AU65026/99A patent/AU6502699A/en not_active Abandoned

Patent Citations (1)

Non-Patent Citations (2)

Also Published As

Similar Documents

Legal Events