WO2007127813A2 - Modulation de transduction de signal par livraison d'un agent ultrasons de composés à action transmembranaire spécifique d'un site - Google Patents

Modulation de transduction de signal par livraison d'un agent ultrasons de composés à action transmembranaire spécifique d'un site Download PDF

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WO2007127813A2
WO2007127813A2 PCT/US2007/067438 US2007067438W WO2007127813A2 WO 2007127813 A2 WO2007127813 A2 WO 2007127813A2 US 2007067438 W US2007067438 W US 2007067438W WO 2007127813 A2 WO2007127813 A2 WO 2007127813A2
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cell
signal transduction
echogenic
site specific
microcapsule
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PCT/US2007/067438
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WO2007127813A3 (fr
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Margaret Wheatley
Kelleny Oum
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Drexel University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/40—Transferrins, e.g. lactoferrins, ovotransferrins

Definitions

  • This invention relates to a site specific transmembrane acting ultrasound imaging agent suitable for inducing and modulating a signal transduction cascade in a targeted cells and to methods of inducing and modulating signal transduction in targeted cells together with imaging the site of the attachment of the ultrasound contrast agent.
  • anoikis isolation
  • apoptosis programmed cell death
  • Tumor necrosis factor belongs to the class of cytokines, which are substances which are produced and secreted by a large number of cell types and which regulate, as intercellular mediators, a large number of cellular processes.
  • cytokines cytokines
  • TNF-alpha oligomers must bind to the membrane receptors for it to be possible to induce at least in physiological solutions some of the various biological activities of TNF-alpha.
  • TNF-alpha occurs as oligomer in solution, it has emerged, however, that these non-covalently linked TNF-alpha oligomers are unstable and are converted into inactive forms.
  • TNF-alpha was originally identified as an endotoxin-induced factor able to induce the necrosis of tumors in mice.
  • the antitumor effect of TNF-alpha not only results from the cytoxic effects on tumor cells but is also based on activation of antitumor effector immune cells in the blood, for example macrophages, cytotoxic lymphocytes and neutrophils.
  • the antitumor effect of TNF-alpha is additionally based on a specific damage to tumor blood vessels.
  • TNF-alpha has very low stability and exerts pleiotropic effects in vivo
  • attempts to date to employ TNF-alpha as systemic antitumor agent have been unsuccessful. It has emerged on administration of TNF-alpha that extremely severe systemic side effects, such as fever and low blood pressure, occur before therapeutically effective doses are reached.
  • systemic side effects such as fever and low blood pressure
  • biologically active proteins such as cytokines
  • biologically active proteins very often display low stability and short half-lives.
  • the proteins are rapidly removed from the blood by the liver, kidneys and other organs, with the rate of clearance depending on the size of the molecules and the extent of proteolysis. Plasma proteases in particular cause the degradation of such proteins and bring about rapid loss of the biological activity.
  • systems for immobilizing biological active proteins on or in carrier systems have been developed.
  • controlled release systems for therapeutic active ingredients or drugs are known, with drugs for example being immobilized by inclusion in a matrix. After administration of such controlled release systems there is then controlled release of the drugs through degradation processes within the living body.
  • the conformation of the active ingredient in the immobilized state is not crucial in these systems. On the contrary, the important point is that the active ingredient is in the active state after release.
  • TNF tumor necrosis factor
  • ultrasound imaging agent can be found in U.S. Pat. Nos. 6,261 ,537 and 6,680,047 to Klaveness et al., International Patent Publication WO 2007/008220 to Grayburn, U.S. Patent No. 5,585,112 to Unger et al., U.S. Patent No. 5,955,143 to Wheatley et al. and International Patent Publication WO 2002/07861 1, however, these references do not describe inducing and modulating a signal transduction cascade in a targeted cells.
  • the invention is a method of modulating a signal transduction cascade in a cell, the method comprising: (1) providing a site specific transmembrane acting ultrasound imaging agent which comprises (a) an echogenic microcapsule or nanocapsule, (b) a signal transduction inducing ligand associated with the echogenic microcapsule or nanocapsule and optionally (c) a therapeutic agent; (2) providing a cell comprising a cell-surface receptor with an affinity to the signal transduction inducing ligand; (3) initiating the signal transduction cascade in the cell by contacting the cell with the site specific transmembrane acting ultrasound imaging agent such that signal transduction inducing ligand selectively binds to the cell-surface receptor and remains associated with the echogenic microcapsule or nanocapsule and thereby modulating the signal transduction cascade in the cell; and (4) detecting selective binding of the site specific transmembrane acting ultrasound imaging agent by using an ultrasonic imaging system.
  • the signal transduction inducing ligand is a cell damage inducing ligand.
  • the cell damage inducing ligand is a member of TNF family.
  • the cell damage inducing ligand is TRIAL (SEQ. ID. NO. 1).
  • the cell damage inducing ligand is HER-2 antibody.
  • the site specific transmembrane acting ultrasound imaging agent comprises a therapeutic agent.
  • the therapeutic agent is at least one of doxorubicin, docetaxel or herceptin.
  • the therapeutic agent is released from the site specific transmembrane acting ultrasound imaging agent by a signal emitted by the ultrasonic imaging system.
  • the signal transduction inducing ligand is covalently associated with the echogenic microcapsule or nanocapsule. In certain embodiments, the signal transduction inducing ligand is non- covalently associated with the echogenic microcapsule or nanocapsule.
  • the echogenic microcapsule or nanocapsule comprise (a) an outer surface including (1) a hardened non water soluble polymer and (2) at least one hollow area formed by removal of a non-water soluble sublimable substance; and (b) an inner surface comprising at least one hollow area formed by removal of a water soluble sublimable substance, wherein said echogenic polymer microcapsule or nanocapsule are made from (i) an outer surface forming mixture comprising a non water soluble polymer and the non-water soluble sublimable substance dissolved in one or more volatile non-polar solvents and (ii) an inner surface forming mixture comprising the water soluble sublimable substance dissolved in water.
  • the echogenic microcapsule or nanocapsule comprise a gas-containing or gas- generating material.
  • the outer surface of the echogenic microcapsule or nanocapsule comprises at least one of poly(lactide), poly(glycolide), a copolymer of lactide and lactone, a poly(anhydride), a poly(styrene), a poly(alkylcyanoacrylate), a poly(amide), a poly(phosphazene), a poly(methylmethacrylate), a poly(urethane), a copolymer of methacrylic acid and acrylic acid, a copolymer of hydroxyethylmethacrylate and methylmethacrylate.
  • the site specific transmembrane acting ultrasound imaging agent is administered to a composition of cells, wherein only a fraction of cells comprise cells having the cell-surface receptor with an affinity to the signal transduction inducing ligand and therefore abnormal or tumorous cells can be distinguished from normal cells by the site specific transmembrane acting ultrasound imaging agent selectively binding to the cell-surface receptor of abnormal or tumorous cells.
  • the site specific transmembrane acting ultrasound imaging agent is administered to a mammal in an aqueous carrier.
  • the site specific transmembrane acting ultrasound imaging agent comprises a plurality of signal transduction inducing ligands.
  • a mixture of site specific transmembrane acting ultrasound imaging agents having different signal transduction inducing ligands is administered to a mammal in an aqueous carrier.
  • Fig. 1 is a scheme demonstrating a process of TRAIL triggering cell death (e.g., apoptosis)
  • Fig. 2 (prior art) is a scheme demonstrating a process of normal apoptosis induction in a cell.
  • Fig. 3 is a graph demonstrating dose and time response curves measured for anti-HER2 scFv (C6.5cys)-ligated contrast agent (Example 3).
  • Fig. 4 is a bar graph demonstrating attachment of C6.5cys scFv HER2 Antibody-PLA microspheres (Example 4). DETAILED DESCRIPTION OF THE INVENTION
  • an ultrasound contrast agent can be used for triggering a specific biological change within a cell, i.e., inducing signal transduction within a cell, when conjugated with a ligand known to induce such change, a signal transduction inducing ligand.
  • the ultrasound contrast agent of the invention can induce a cell damage or cell death (e.g., an apoptosis) of particular cells when associated (e.g., by covalent or affinity binding) with a cell damage inducing ligand.
  • a signal transduction inducing ultrasound contrast agent of the invention comprises an echogenic microcapsule or nanocapsule and a signal transduction inducing molecule associated with the echogenic microcapsule or nanocapsule for example by being covalently attached to the outer surface.
  • conjugation of the signal transduction inducing molecule to a much larger particle such as echogenic microcapsule or nanocapsule did not affect binding capacity or functionality of the signal transduction inducing molecule.
  • molecules of a high molecular weight e.g. TRAIL (SEQ. ID. NO. 1) has molecular weight 34kDa) retained signal transduction activity when covalently attached to microcapsules of contrast agent, even when using methods that used conjugation via a lysine amino group, a group that is located near the active site.
  • the inflicted cell death was slower and more global when the cell damage inducing ligand was conjugated to the echogenic microcapsule or nanocapsule as compared to the unconjugated ligand, and that the rate of killing can be controlled by the ligand density attached to the contrast agent.
  • Advantages of the invention include ability to visualize target cells, focusing energy exactly at the site and at the point of interaction between the signal transduction inducing ligand and the cell, utilization of the interaction between the echogenic contrast agent and the ultrasound energy, and utilizing the force produced by the ultrasound for release of a desired compound (e.g., a therapeutic agent).
  • a desired compound e.g., a therapeutic agent.
  • bioactive agents e.g., proteins and peptides
  • extracellular signaling molecules include hormones (e.g., melatonin), growth factors (e.g., epidermal growth factor), extra-cellular matrix components (e.g., fibronectin), cytokines (e.g., interferon-gamma), chemokines (e.g., RANTES), neurotransmitters (e.g., acetylcholine), and neurotrophins (e.g., nerve growth factor).
  • the signal transduction cascade involves generation of second messages (e.g., cAMP, or protein kinase activity.)
  • Examples of cellular responses to extracellular stimulation that require signal transduction include gene activation, metabolism alterations, continued proliferation and death of a cell, and stimulation or suppression of locomotion.
  • cell receptors are preferably cell-surface receptors, however, ligand gated ion channel receptors can be involved also.
  • cell-surface receptors include G- protein coupled receptors (e.g., chemokine receptors), receptor tyrosine kinases (RTKs) (e.g., growth factor receptors), integrins, and toll-like receptors (TLRs).
  • RTKs receptor tyrosine kinases
  • TLRs toll-like receptors
  • ligand gated ion channel receptors include Cys-loop receptors, ionotropic glutamate receptors, and ATP-gated channels.
  • a signal transduction inducing molecule or "a signal transduction inducing ligand” are used interchangeable herein and encompass molecular species which, when interacting with cell surface receptors, trigger an intracellular cascade of events, for example, TNF-related apoptosis-inducing ligand (APO2L), epinephrine and norepinephrine, glucagon, luteinizing hormone, follicle stimulating hormone, thyroid-stimulating hormone, calcitonin, parathyroid hormone, antidiuretic hormone, insulin, growth hormone, prolactin, oxytocin, erythropoietin, angiotensin II, antidiuretic hormone, gonadotropin-releasing hormone, thyroid- releasing hormone, atrial naturetic hormone, nitric oxide, active fragments thereof such as the 19KDa fragment of TRAIL known as sTRAIL, and synthetic species such as monoclonal antibody engineered through biotechnology for example Herceptin ® or
  • a cell damage inducing ligand denotes a ligand capable of triggering cell damage or cell death, both apoptic and non-apoptic.
  • Non-limiting examples of such cell damage inducing ligands include ligands to receptors on cancer cell surfaces such as, for example, (a) an antibody to a receptor on a particular cell surface, the receptor capable of stimulating the cell to divide and grow (e.g., Her-2 specific antibody to Her-2 protein found on the surface of certain cancer cells such as MDA-MB 231) and (b) tumor necrosis (TNF)-related apoptosis-inducing ligand (TRAIL), which is a member of the TNF family of cytokines that promotes apoptosis; TRAIL induces apoptosis via death receptors (DR4 and DR5) in cancer cells but not in normal cells.
  • TNF tumor necrosis
  • TRAIL tumor necrosis-related apoptosis-inducing ligand
  • ultrasound imaging agent is used interchangeably with “contrast agent (CA)” and includes the terms “echogenic nanocapsule” and “echogenic microcapsule”.
  • echogenic microcapsules is used interchangeably with the term “microbubbles.”
  • Any ultrasound contrast agent can be used for covalent modification with a signal transduction inducing ligand and specifically, for modification with an apoptosis inducing ligand, if it has accessible functional groups of its outer surface with can react with corresponding functional groups of such ligands.
  • maleimide groups on a surface of a contrast agent can react with a thiol group of a cystine and therefore a thioester linkage will be formed between the contrast agent and the ligand.
  • a person skilled in the art would readily ascertain a combination of functional groups needed for formation of a covalent bond between the two entities.
  • Any ultrasound contrast agent can be used for surface adsorption modification with a signal transduction inducing ligand and specifically, for modification with an apoptosis inducing ligand, if it has accessible functional groups of its outer surface with can react electrostatically or by hydrophobic-interaction with corresponding functional groups of such ligands.
  • Any ultrasound contrast agent can be used for modification with a signal transduction inducing ligand and specifically, for modification with an apoptosis inducing ligand by incorporation during fabrication, if it he fabrication process does not destroy the activity of such ligands.
  • ultrasound imaging agent examples include U.S. Pat. Nos. 6,261 ,537 and 6,680,047 to Klaveness et al., International Patent Publication WO 2007/008220 to Grayburn, U.S. Patent No. 5,585,1 12 to Unger et al., U.S. Patent No. 5,955,143 to Wheatley et al. and International Patent Publication WO 2002/07861 1 and references described therein.
  • the ultrasound imaging agent is made based on methods described in US 2004/0161384 Al to Wheatley et al.
  • Such echogenic microcapsule or nanocapsule comprise (a) an outer surface including (1) a hardened non water soluble polymer and (2) at least one hollow area formed by removal of a non-water soluble sublimable substance; and (b) an inner surface comprising at least one hollow area formed by removal of a water soluble sublimable substance, wherein said echogenic polymer microcapsule or nanocapsule are made from (i) an outer surface forming mixture comprising a non water soluble polymer and the non-water soluble sublimable substance dissolved in one or more volatile non-polar solvents and (ii) an inner surface forming mixture comprising the water soluble sublimable substance dissolved in water.
  • the echogenic microcapsules or nanocapsules comprise a gas-containing or gas-generating material.
  • the outer surface of the echogenic microcapsule or nanocapsule is made of liposomes, lipid coatings, and polymers.
  • Lipids which may be used to create lipid microspheres include but are not limited to: lipids such as fatty acids, lysolipids, phosphatidylcholine with both saturated and unsaturated lipids including dioleoylphosphatidylcholine; dimyristoylphosphatidylcholine; dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dioleoylphosphatidylcholine, dipalmitoylphosphatidylcholine; distearoyl phosphatidylcholine; phosphatidylethanolamines such as dioleoylphosphatidylethanolamine; phosphatidylserine; phosphatidyl glycerol; phosphatidylinositol, sphingolipids such
  • Lipids bearing hydrophilic polymers such as polyethyleneglycol (PEG), including and not limited to PEG 2,000 MW, 5,000 MW, and PEG 8,000 MW, are particularly useful for improving the stability and size distribution of the gaseous precursor-containing liposomes.
  • PEG polyethyleneglycol
  • Polymers useful for this invention are preferably biodegradable.
  • outer surface of the echogenic microcapsule or nanocapsule comprises at least one of polylactide, polyglycolide, a copolymer of lactide and lactone, a polyanhydride, a polystyrene, a polyalkylcyanoacrylate, a polyamide, a polyphosphazene, a poly(methylmethacrylate), a polyurethane, a copolymer of methacrylic acid and acrylic acid, a copolymer of hydroxyethylmethacrylate and methylmethacrylate.
  • Signal Transduction Inducing Ligand Signal Transduction Inducing Ligand
  • signal transduction inducing ligands include TNF-related apoptosis- Inducing ligand (A.K.A. APO2L), epinephrine and norepinephrine, glucagon, luteinizing hormone, follicle stimulating hormone, thyroid-stimulating hormone, calcitonin, parathyroid hormone, antidiuretic hormone, Insulin, growth hormone, prolactin, oxytocin, erythropoietin, Epinephrine and norepinephrine, angiotensin ⁇ , antidiuretic hormone, gonadotropin-releasing hormone, thyroid-releasin hormone, Atrial naturetic hormone, nitric oxide, active fragments thereof such as the 19KDa fragment of TRAIL known as sTRAIL, and synthtic species such as monoclonal antibody engineered through biotechnology for example Herceptin ® or synthetic drugs such as Gleevec ® which has been implicated in dendritic cell
  • Second messengers include Cyclic AMP, Protein kinase activity, calcium and/or phosphoinositides and Cyclic GMP.
  • the signal transduction inducing ligand is a cell damage inducing ligand, a ligand capable of inducing a cell death of a particular cell when bound to a receptor on the cell surface.
  • cell damage inducing ligands include ligands to receptors on cancer cell surfaces such as, for example, (a) an antibody to a receptor on a particular cell surface, the receptor capable of stimulating the cell to divide and grow (e.g., Her-
  • Her-2 specific antibody to Her-2 protein found on the surface of certain cancer cells such as MDA-
  • TRAIL tumor necrosis-related apoptosis-inducing ligand
  • Tumor necrosis factor belongs to the class of cytokines, which are substances which are produced and secreted by a large number of cell types and which regulate, as intercellular mediators, a large number of cellular processes. Cytokines include, for example, lymphokines, interleukins, monokines and growth factors. Tumor necrosis factor (TNF, also called TNF-alpha or cachectin) is the eponymous member of a large gene family of cytokines which are structurally homologous and have diverse biological properties, some of which overlap.
  • TNF Tumor necrosis factor
  • the members known at present include TNF itself and, inter alia, lymphotoxin alpha (LT-alpha) which, like TNF, binds to the same receptors (TNFRl and TNFR2) and displays very similar signaling properties, and LT-beta, FasL, CD27L, CD30L, CD40L, TWEAK, OX40L, EDA, AITRL, VEGI, LIGHT, 4- 1 BBL, APRIL, BLYS , RANKL and TRAIL, for each of which one or more specific membrane receptors exist (Locksley et aL, Cell, 104:487-501, 2001).
  • LT-alpha lymphotoxin alpha
  • TNF tumor necrosis factor
  • the molecules of the tumor necrosis factor (TNF) ligand family are involved via their complementary receptors, which are collected into the TNF receptor family in a large number of in particular immunoregulatory processes.
  • the members of the TNF ligand family are membrane proteins of type II (Locksley et al., see above).
  • insoluble and membrane-associated forms of members of the TNF ligand family may differ considerably in their bioactivity (Grell et ah, Cell, 1995).
  • TNF lymphotoxin
  • LT-alpha lymphotoxin
  • TNF is, like most other members of the TNF family, produced by the producing cell always initially as membrane-associated TNF from which the classical soluble cytokine TNF is formed only on proteolytic cleavage.
  • This membrane TNF is a genuine transmembrane protein which exists in trimeric form and is biologically active. It has been possible to show in this connection that membrane TNF has a special range of cellular effects which are not attained by soluble TNF and by LT-alpha (Grell et ah, Cell, 83 ( 1995), 793-802).
  • TNF TNF-like protein
  • FasL TRAIL
  • CD40L FasL, TRAIL and CD40L
  • TNF is formed primarily by macrophages/monocytes, lymphocytes and mast cells and influences inflammations, sepsis, lipid and protein metabolism, blood formation, angiogenesis, wound healing and immune defenses and exerts cytotoxic or cytostatic effects on certain tumor cells. Lymphotoxin a likewise has cytotoxic effects on certain tumor cell lines. Relatively little is known about the molecular basis of the diverse effects of TNF-alpha in vivo and in vitro. Analysis of the crystal structure of the recombinant soluble TNF-alpha molecule has revealed that this substance is an oligomeric protein consisting of three identical subunits each of 17.5 kDA.
  • TNF-alpha oligomers must bind to the membrane receptors for it to be possible to induce at least in physiological solutions some of the various biological activities of TNF-alpha. Although it has been shown that TNF-alpha occurs as oligomer in solution, it has emerged, however, that these non-covalently linked TNF-alpha oligomers are unstable and are converted into inactive forms. It is, however, assumed that the structural changes of TNF-alpha also take place in vivo because monomers and oligomers are found in different proportions in the cerebrospinal fluid of patients with meningitis.
  • the site specific transmembrane acting ultrasound imaging agent comprises a therapeutic agent.
  • therapeutic agents can be found in U.S. Pat. Nos. 6,261 ,537, columns 39-60 (incorporated herein).
  • the therapeutic agent is at least one of doxorubicin, docetaxel or herceptin.
  • the therapeutic agent is released from the site specific transmembrane acting ultrasound imaging agent by a signal emitted by the ultrasonic imaging system.
  • Therapeutic agents can be added during the stage of making the echogenic particle depending on the solubility of the therapeutic agent and desired formulation, a particular therapeutic agent can be present in the outer surface of the echogenic particle or in the inner surface. Methods of formulating each particular imaging agent with the therapeutic agent would be apparent to a person skilled in the art.
  • the site specific transmembrane acting ultrasound imaging agent of the invention comprises (a) an echogenic microcapsule or nanocapsule, (b) a signal transduction inducing ligand associated with the echogenic microcapsule or nanocapsule and optionally (c) a therapeutic agent.
  • Association of the echogenic microcapsule or nanocapsule with the signal transduction inducing ligand can be covalent or non covalent.
  • Both echogenic microcapsules/nanocapsules and ligands have complementary functional groups capable of binding or reacting with each other.
  • Description of covalent and non-covalent attachment is provided in U.S. 2004/0265392A1 to Tovar et al. (incorporated therein) for immobilizing TNF as at each monomer or as a trimer based on interaction of functional groups on the surface of TNF and the nanoparticle surface matrix. It is required that the activity (i.e., ability to affect signal transduction cascade in a cell) of the attached ligand remains substantially comparable to the activity of the free ligand.
  • an affinity pair can be used with each of the component of the affinity pair being present on each of the molecules being joined, e.g., biotin-avidin affinity pair.
  • the site specific transmembrane acting ultrasound imaging agent of the invention can be made by first making echogenic microcapsule or nanocapsule and then attaching signal transduction inducing ligand either covalently or non-colalently. Covalent attachment is preferred.
  • Another way of making the site specific transmembrane acting ultrasound imaging agent involves adding the signal transduction inducing ligand to the outer surface making phase such that the ligand.
  • the method is described in detail in Examples below, e.g., surface ligation of TRAIL to microbubbles (see Examples 4 and 7).
  • the biodegradable polymeric, hard-shelled microbubbles are produced using a double emulsion method that results in a hollow gas-filled sphere which reflects impinging ultrasound beams to enhance ultrasound images.
  • TRAIL-bound microbubbles When exposed to the TRAIL-bound microbubbles, cell death was observed in both MCF-7 and MDA-MB-231 human breast cancer cell models over a 48 hour period. Cell death was markedly lower in cells incubated with unmodified microbubbles. The killing abilities of TRAIL were not hindered by its conjugation to the microbubbles.
  • Potential applications of this approach may include the simultaneous ultrasound imaging and treatment of malignant tumors, especially if further adapted to a nano-scale contrast agent platform or the co-localize (on the contrast agent), simultaneous imaging and delivery of drug and Trail, a combination which has been shown to be synergistic.
  • Normal co administration suffers form all the disadvantages of drug toxicity, but co localization within the contrast agent would circumvent this problem.
  • Another embodiment of the method is directed to a Her-2 specific antibody conjugated with microbubble contrast agents (Examples 1-3).
  • Her-2 is a protein found on the surface of certain cancer cells. When human epidermal growth factor attaches itself to Her-2 receptors on the breast cancer cells, it stimulates the cells to divide and grow.
  • BT-474 tumor cell line that overexpresses Her-2, is used to test the antibody conjugated microcapsule contrast agent in vitro.
  • the epidermal growth factor receptor type 2 (ErbB-2 / Her2) tyrosine kinase is a carcinoma-associated receptor whose activation is responsible for tumor cell survival, proliferation, and metastasis in many human cancers. ErbB receptor-binding peptides block the signaling pathway.
  • PLA poly (lactic acid)
  • BMPH ⁇ -Maleimidopropionic acid hydrazide
  • EDC l-Ethyl-3-[3-dimethylaminopropyl]carbodiimide Hydrochloride
  • This solution was immediately added to the microbubbles: 1 mL BMPH solution (25 ⁇ mol BMPH) and 1 mLEDC solution (50 ⁇ mol EDAC). The mixture was incubated for 30 minutes at room temperature with gentle agitation using a rotatory shaker. The reacted microbubbles were purified by centrifugation at 1000 X g for 10 min. The pellet was resuspended in MES buffer. Washing was repeated 2 additional times. In the final wash step, the pellet was resuspended in 4 mL PBS buffer, and labeled "activated microbubble solution".
  • BT-474 cells were cultured in T-75 tissue flasks in Dulbeco's Modified Eagle Medium with Earle's salts (DMEM) with Penicillin Streptomycin, Fetal Bovine Serum, and L-DMEM
  • Glutamine The cells were incubated at 37°C in 95% air and 5% CO2 and passaged regularly at about full confluence. During static attachment studies, the cells were seeded in 12 well culture plates at passage 6. Statistical analyses were performed using the Prism software (San Diego,
  • CA Graphpad Software
  • the cells were first washed with media and then incubated with the 1 ml of contrast agent containing media for the specified time point (0, 5, 10, 15 min). All studies were done in triplicate, one sample set was pre-blocked for an hour prior to experimentation with the same ligand on the surface of the ligand-modified contrast agent at a concentration of 150 ⁇ g/ml in 1 ml of medium, one sample set of non-conjugated PLA, and a control with PLA microspheres that went through the conjugation process but without the linker. At the specified time point, the medium was removed and the cells were washed three times with 1 ml of growth media solution. The cells were then viewed under a phase contrast microscope and pictures were taken.
  • the static attachment study was performed at time 0, 5, 10, and 15 min at 0.5 mg/ml concentration of conjugated microspheres with 1 ml volume of microcapsule and media mixture in each well of a 12 well plate.
  • Dried microcapsules (100 mg) were combined with 5 mg EDC (l-Ethyl-3-[3- dimethylaminopropyl]carbodiimide Hydrochloride) (1 :1 molar ratio of -COOH groups in microcapsules to EDC), 2.7 mg ofNHS (N-hydroxysuccinimide), (1:2 molar ratio to EDC), and 10ml of buffer (0.1 M MES (2-[N-morpholino]ethanesulfonic acid sodium salt), 0.3 M NaCl, pH 6.5,) and shaken on a rotary shaker for 15 min.
  • EDC l-Ethyl-3-[3- dimethylaminopropyl]carbodiimide Hydrochloride
  • TRAIL 35 ⁇ l, 1 :2 molar ratio of COOH groups on the polymer to TRAIL
  • TRAIL 35 ⁇ l, 1 :2 molar ratio of COOH groups on the polymer to TRAIL
  • the dye was aspirated and the cells were resuspended in 50 ⁇ l in fresh HBSS.
  • a 4% glutaraldehyde solution in HBSS was freshly prepared and added to the cells and incubated at room temperature for 15minutes. Cells were observed under a Nikon Eclipse TE2000-U fluorescent microscope.
  • EXAMPLE 6 Demonstration of Potency of TRAIL-Ligated Contrast Agent Using the LIVE/DEAD assay from Molecular Probes the amount of dead cells versus live cells was evaluated qualitatively.
  • the basis for the viability test is differential permeability of live and dead cells to a pair of fluorescent stains.
  • SYTO® 10 a green fluorescent nucleic acid stain, is a highly membrane-permeable dye and labels all cells, including those with intact plasma membranes.
  • DEAD Red® is a cell- impermanent red fluorescent nucleic acid stain that labels only cells with compromised membranes.
  • With breast cancer cell line MCF-7 TRAIL was placed on cells at concentrations ranging from 10-50ng/ml and analyzed using the LIVE/DEAD assay.
  • the concentrations of the ligand can be varied to control the amount of cell death. This can be seen as the concentration of TRAIL on the capsules increases, more of the cells appear red under microscopic examination. When no TRAIL was present cell death did not occur.
  • Microbubbles were prepared by a double emulsion (W/O)/W solvent evaporation process.
  • Poly (D,L-lactic acid) (PLA) or Poly (D,L lactic-co-glycolic acid) (PLGA) (0.5Og) was dissolved in methylene chloride (10ml), and ammonium carbamate (ImI) (IM) solution was added.
  • the polymer solution was probe sonicated at 1 1 OW for 30s.
  • the resulting (W YO) emulsion was then poured into cold (4°C), polyvinyl alcohol solution (5%) and homogenized for 5 min at 9000 rpm.
  • the double emulsion was then poured into a 2% isopropanol solution and stirred at room temperature for 1 hour.
  • the capsules were then collected by centrifugation and washed three times with hexane.
  • the capsules were flash frozen, lyophilized for 48 hours, and stored in a desiccator at -20 0 C until used.
  • Ammonium carbamate sublimes in the lyophilizer, leaving a gas-filled void in its place thereby producing echogenic polymeric microcapsules of less than 3 microns in size as confirmed by particle size analysis (Malvern Instruments).
  • microbubbles were prepared by a double emulsion (W/0)/W solvent evaporation process using camphor (0.05g) and PLA/PLGA (0.50g) dissolved in methylene chloride (10ml), and ammonium carbonate (ImI) (4% (w/v)) solution was added.
  • the polymer solution was probe sonicated at 1 1OW for 30s.
  • the resulting (W/O) emulsion was then poured into cold (4°C), 5% polyvinyl alcohol solution and homogenized for 5 min at 9000 rpm.
  • the double emulsion was then poured into a 2% isopropanol solution and stirred at room temperature for 1 hour.
  • the capsules were then collected by centrifugation and washed three times with hexane.
  • the capsules were flash frozen, lyophilized for 48 hours, and stored in a desiccator at -20 0 C until used.
  • Camphor and ammonium carbonate sublime in the lypophilizer, leaving a void in their place and producing echogenic polymeric microcapsules of less than 3 microns in size. b) Echogenicity of microbubbles
  • the pulse-echo setup was used to determine acoustic performance of the drug loaded agents.
  • a 12.7 mm diameter, 50.8 mm spherically focused transducer was used with a center frequency of 5 MHz.
  • the transducer had a -6 dB bandwidth of 91 % and a pulse length of 1.2 mm.
  • the transducer was placed in a 25°C water bath with 18.6 M ⁇ -cm deionized water.
  • the transducer was then focused through an acoustically transparent window in the sample holder at a depth of 14 cm from the top of the surface.
  • a pulser/receiver 5072 PR Panamterics Inc.
  • Received signals were then amplified 40 dB and read in an oscilloscope (Lecroy 9350 A). Data was then stored and analyzed using Lab View 7 Express (National Instruments).
  • TRAIL conjugated microspheres were collected via centrifugation (1000xg)(10 min), flash frozen, and lyophilized for 48 hrs. d) Inducing apoptosis in breast cancer cells via ultrasound contrast agents MCF-7 and MDA-MB-231 cells were cultured in 48 well- plates.
  • MDA-MB-231 and MCF-7 cells both showed little apoptosis after 3, 24, and 48 hr incubation with Mb and TRAIL-Mb-noXlink.
  • TRAIL-Mb had significant apoptosis effect on both cell lines starting at 3 hours and continuing for 48 hours. Compared to TRAIL alone on the cells, in which full apoptosis of cell populations was seen starting at 24 hrs, TRAIL-Mb continued to induce significant apoptosis the entire experimental period.
  • TNF-Selectokine a novel prodrug generated for tumor targeting and site-specific activation of tumor necrosis factor. Oncogene 21, 4257-4265 (2002)

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Abstract

La présente invention concerne un agent d'imagerie par ultrasons à action transmembranaire spécifique d'un site qui convient pour induire et moduler une cascade de transduction de signal dans des cellules visées, et des méthodes pour induire et moduler une transduction de signal dans des cellules visées tout en imageant le site d'attachement de l'agent de contraste par ultrasons.
PCT/US2007/067438 2006-04-25 2007-04-25 Modulation de transduction de signal par livraison d'un agent ultrasons de composés à action transmembranaire spécifique d'un site WO2007127813A2 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3897590A4 (fr) * 2018-12-23 2022-09-14 B.G. Negev Technologies and Applications Ltd., at Ben-Gurion University Microsphères stables, leurs procédés de fabrication et leur utilisation

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7083572B2 (en) * 1993-11-30 2006-08-01 Bristol-Myers Squibb Medical Imaging, Inc. Therapeutic delivery systems
US7238786B2 (en) * 2002-06-14 2007-07-03 Immunomedics, Inc. Monoclonal antibody cPAM4

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7083572B2 (en) * 1993-11-30 2006-08-01 Bristol-Myers Squibb Medical Imaging, Inc. Therapeutic delivery systems
US7238786B2 (en) * 2002-06-14 2007-07-03 Immunomedics, Inc. Monoclonal antibody cPAM4

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3897590A4 (fr) * 2018-12-23 2022-09-14 B.G. Negev Technologies and Applications Ltd., at Ben-Gurion University Microsphères stables, leurs procédés de fabrication et leur utilisation

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