Methods for ultrasonic imaging and treating diseased tissues

Abstract

A method for imaging diseased tissue is provided. The method provides delivering to the diseased tissue a liposomal imaging agent comprising liposomes containing an imaging agent. The liposomal imaging agent is bound to an antibody by an antigenic linker so that the antibody binds the liposomal imaging agent to the diseased tissue. The liposomes are broken to release the imaging agent using a catheter, and the imaging agent is viewed using an imaging technique. The tissue can also be treated by delivering a therapeutic agent to the tissue using the catheter, for example, within the liposomes of the liposomal imaging agent.

Inventors:	See; Jacko R. (Fullerton, CA)
Assignee:	SBM Biologics, Inc. (Cupertino, CA)
Appl. No.:	10/406,748
Filed:	April 3, 2003

Current U.S. Class:	600/458
Current International Class:	A61B 008/14 ()
Field of Search:	600/439,459-472,458 424/9.51,9.92,85.1

References Cited [Referenced By]

U.S. Patent Documents

Other References

Primary Examiner: Imam; Ali
Attorney, Agent or Firm: Christie, Parker & Hale, LLP

Parent Case Text

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority of U.S. Provisional Application No. 60/369,786, filed Apr. 3, 2002, the entire disclosure of which is incorporated herein by reference.

Claims

What is claimed is:

1. A method for imaging diseased tissue, comprising: delivering to the diseased tissue a liposomal imaging agent comprising liposomes containing an imaging agent, the liposomal imaging agent being bound to an antibody by an antigenic linker, wherein the antibody binds the liposomal imaging agent to the diseased tissue; breaking the liposomes to release the imaging agent using a catheter; and viewing the imaging agent using an imaging technique.

2. The method according to claim 1, wherein the liposomal imaging agent is delivered to the diseased tissue using the catheter.

3. The method according to claim 1, wherein the liposomes are broken by applying radio frequency energy or ultrasound energy with the catheter.

4. The method according to claim 1, wherein the antibody is selected from the group consisting of polyclonal antibodies, monoclonal antibodies, cytokines, growth factors, immunoglobulin, matrix metalloproteases, amino acids, peptides, extracellular matrix proteins, purified neurochemicals, chemokines, RNA fragments, DNA fragments, and nucleic acids.

5. The method according to claim 1, wherein the liposomal imaging agent comprises gas-filled liposomes.

6. The method according to claim 5, wherein the gas-filled liposomes comprise at least one gas selected from the group consisting of air, oxygen, hydrogen, nitrogen, fluorocarbons, argon, xenon, helium, and fluoropropanes.

7. The method according to claim 5, wherein the gas-filled liposomes are prepared by: preparing liposomes comprising an inner retentate surrounded by one or more outer layers; freezing and thawing the liquid liposomes to induce the inner retentate to go directly from the solid state to the gaseous state while the one or more outer layers of the liposome do not.

8. The method according to claim 1, further comprising delivering energy directly into the diseased tissue using the catheter.

9. The method according to claim 8, wherein the energy is selected from radio frequency energy and ultrasound energy.

10. The method according to claim 1, further comprising, after viewing the imaging agent, breaking the antigenic linkage using the catheter to release the liposomes from the diseased tissue.

11. A method for imaging and treating diseased tissue comprising: imaging diseased tissue according to the method of claim 1, wherein the liposomes containing the imaging agent further contain a therapeutic agent; and breaking the liposomes to release the therapeutic agent to thereby treat the diseased tissue with the therapeutic agent.

12. The method according to claim 11, wherein the therapeutic agent is selected from the group consisting of trastuzumab, infliximab and abciximab.

13. A method for imaging and treating diseased tissue comprising: imaging diseased tissue according to the method of claim 1; and introducing a therapeutic agent to the diseased tissue using the catheter to thereby treat the diseased tissue.

14. The method according to claim 13, wherein the therapeutic agent is selected from the group consisting of trastuzumab, infliximab and abciximab.

15. The method according to claim 13, wherein the therapeutic agent is contained within liposomes.

16. The method according to claim 1, wherein the diseased tissue is selected from the group consisting of tumors, areas of infection, areas of infarction, areas of scarring, areas of degeneration, and areas of tissue overgrowth of unknown etiology.

Description

BACKGROUND OF THE INVENTION

The therapeutic use of antibodies has been recently approved. In fact, therapeutic imaging targets can be reached without allergic reactions. However, such procedures involve the intravenous injection of antibodies, which circulate throughout the body and hopefully attached to an antigen of a tumor, infection or site of scarring.

However, the ability to concentrate enough antibody at the target site is a problem. Antibodies are quite large molecules of complex, folded sequences of amino acids that have difficulty in penetrating the diseased tissue. The leakiness and varied vascularization and intralesional pressure of each type of tumor, infection or area of scarring due to ischemia will limit the application of antibody therapies. Antibodies that are hooked or linked to contrast imaging agents make an even larger molecule, which the further complicates the problem of penetration of the imaging or therapeutic agent into the tissue to be treated, i.e., target tissue. This problem results irrespective of whether the imaging agent is an isotope, a PET, an MRI, or a CT-type imaging agent. Further, this problem exists despite the different leakiness of different diseased tissues due to different disease entities.

With the advent of pulse inversion imaging, double resonance ultrasound imaging, and dual harmonic echo Doppler imaging, as well as 3-dimensional reconstruction, areas of imaging throughout the body for diagnosis and treatment can be satisfactorily obtained simultaneously if a contrast agent is use. Such ultrasound contrast agents typically contain an air or gas infiltrate core to increase reflectance of the diseased tissue to be studied.

DESCRIPTION OF THE INVENTION

The present invention is directed to methods for imaging and treating diseased tissue, including tumors, areas of infection, areas of infarction, areas of scarring, and areas of degeneration or tissue overgrowth of unknown etiology. In accordance with the invention, a catheter delivery system is used to deliver a liposomal ultrasound imaging agent directly into the area of interest.

Ultrasound imaging catheters are generally known in the art. In accordance with the invention, the catheter has an ultrasound device, e.g., a piezoelectric device, mounted near its distal end. A lumen passes through the length of the catheter and is open at its distal end for introduction of the imaging agent and other agents into the patient’s body, discussed further below. The lumen is preferably sufficiently large to permit the passage of a moveable guidewire through the lumen for introduction of the catheter into the patient’s body. The catheter preferably also includes an electrode capable of delivering radio frequency energy or other suitable energy, as discussed further below. The electrode is preferably a tip electrode mounted at the very distal end of the catheter, but alternatively could be a ring electrode or have any other suitable design.

The liposomal imaging agent preferably comprises gas-filled liposomes. A preferred method for making gas-filled liposomes involves using known agents to first make liquid liposomes. The liposomes are repeatedly frozen and thawed to induce the inner retentate to go directly from the solid state to the gaseous state while the outer layers of the liposome do not. Lyophilization is repeatedly used in cycles to aerosolize the inner portion while keeping the outer layers intact. Preferred gases for incorporation into the liposomes include air, oxygen, hydrogen, nitrogen, fluorocarbons, argon, xenon, helium, and fluoropropanes.

The liposomes of the present invention may be made of any suitable phospholipid, glycolipid, derived lipid, or the like. Examples of suitable phospholipids include phosphatidyl choline, phosphatidyl serine, phosphatidic acid, phosphatidyl glycerin, phosphatidyl ethanolamine, phosphatidyl inositol, sphingomyelin, dicetyl phosphate, lysophosphatidyl choline and mixtures thereof, such as soybean phospholipids, and egg yolk phospholipids. Suitable glycolipids include cerebroside, sulphur-containing lipids, ganglioside and the like. Suitable derived lipids include cholic acid, deoxycholic acid, and the like. The liposomes may be formed by any of the known methods for forming liposomes and may be loaded with an agent according to known procedures. Known methods for forming liposome-encapsulated agents are described, for example, in U.S. Pat. No. 4,235,871 to Papahadjopoulos, et al., and Oral Microbiology and Immunology, 1994, 9:146-153, the disclosures of which are incorporated herein by reference.

After the imaging agent is delivered with the catheter, the ultrasound imaging agent can be imaged to assure that it is in the desired location, e.g., just outside a tumor that has increased core pressure and dense capillary networks that do now allow therapeutic agent into it, utilizing the same catheter in the same position. If the imaging agent is the desired location, in the case of imaging agent encapsulated within liposomes, the catheter can be used to burst the liposomes to release the imaging agent, for example, using radio frequency or ultrasound energy. Other types of energy that can be used in connection with the invention include visible light, microwave energy, ultraviolet or infrared light having a wavelength of about 200 to about 1000 nm, laser energy and electromagnetic energy. Energy can also be delivered directly into the tumor or other target site to make the target more leaky, thus allowing more imaging agent into the target site. Preferably the energy is applied at a frequency ranging from 0.5 kHz to 40 kHz and an amplitude ranging from about 150 .mu.m to 500 .mu.m. Different amounts and types of energy can be used depending on the particular application. For example, one frequency of dual resonance imaging can be used to track the liposomal bubbles (i.e., gas-filled liposomes used as imaging agents) and a second different frequency can be used to burst the liposomal bubbles, as discussed further below.

In one embodiment, the liposomal imaging agent is bound to an antibody by an antigenic linker. Specifically, the antibody can be bound to an epitope that protrudes from the outer surface of the liposome. Examples of antibodies useful in connection with the present invention include polyclonal and monoclonal antibodies specific to infectious diseases and tumor antigens, cytokines, growth factors, immunoglobulin, matrix metalloproteases, amino acids, peptides, extracellular matrix proteins, purified neurochemicals, chemokines, fragments of RNA and DNA, and nucleic acids. Alternatively, a chelator may be used to bind other chemotherapeutic agents such as heparin or protamine.

The antibody targets the tumor or other target site, thereby binding the liposomal imaging agent to the target site. This mechanism better assures that the liposomal imaging agent will be in the desired location. Additionally, the use of antibodies permits smaller liposomes to bind to the target. All of these factors enhance the resulting ultrasound image.

Additionally, the catheter can be used to deliver liposomes containing therapeutic agents to the target site. The therapeutic agent-containing liposomes are preferably also bound to an antibody by a non-antigenic linker, as described above. If desired, one or more therapeutic agents can be incorporated into the same liposomes with the imaging agent or the different agents can be provided in different liposomes. Examples of suitable therapeutic agents for use in connection with the invention include trastuzumab (commercially available from Genentech under the name Herceptin.RTM.), infliximab (commercially available from Centocor under the name Remicade.RTM.), and abciximab (commercially available from Eli Lilly under the name ReoPro.RTM.).

When the imaging is finished, the catheter can then be used to break the linkage, for example, with radio frequency energy, to thereby release the liposomes so that they can pass through the patient’s system and be released. Liposomes carrying therapeutic agents can be similarly broken down using the catheter.

The preceding description has been presented with reference to presently preferred embodiments of the invention. Workers skilled in the art and technology to which this invention pertains will appreciate that alterations and changes in the described methods may be practiced without meaningfully departing from the principal, spirit and scope of this invention.

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Adult stem cell—See somatic stem cell.

Astrocyte—A type of supporting (glial) cell found in the nervous system.

Blastocoel—The fluid-filled cavity inside the blastocyst, an early, preimplantation stage of the developing embryo.

Blastocyst—A preimplantation embryo of about 150 cells produced by cell division following fertilization. The blastocyst is a sphere made up of an outer layer of cells (the trophoblast), a fluid-filled cavity (the blastocoel), and a cluster of cells on the interior (the inner cell mass).

Bone marrow stromal cells—A population of cells found in bone marrow that are different from blood cells.

Bone marrow stromal stem cells (skeletal stem cells)—A multipotent subset of bone marrow stromal cells able to form bone, cartilage, stromal cells that support blood formation, fat, and fibrous tissue.

Cell-based therapies—Treatment in which stem cells are induced to differentiate into the specific cell type required to repair damaged or destroyed cells or tissues.

Cell culture—Growth of cells in vitro in an artificial medium for research or medical treatment.

Cell division—Method by which a single cell divides to create two cells. There are two main types of cell division depending on what happens to the chromosomes: mitosis and meiosis.

Chromosome—A structure consisting of DNA and regulatory proteins found in the nucleus of the cell. The DNA in the nucleus is usually divided up among several chromosomes.The number of chromosomes in the nucleus varies depending on the species of the organism. Humans have 46 chromosomes.

Clone— (v) To generate identical copies of a region of a DNA molecule or to generate genetically identical copies of a cell, or organism; (n) The identical molecule, cell, or organism that results from the cloning process.

1. In reference to DNA: To clone a gene, one finds the region where the gene resides on the DNA and copies that section of the DNA using laboratory techniques.

2. In reference to cells grown in a tissue culture dish:a clone is a line of cells that is genetically identical to the originating cell. This cloned line is produced by cell division (mitosis) of the original cell. <>

3. In reference or organisms: Many natural clones are produced by plants and (mostly invertebrate) animals. The term clone may also be used to refer to an animal produced by somatic cell nuclear transfer (SCNT) or parthenogenesis.

Cloning—See Clone.

Cord blood stem cells—See Umbilical cord blood stem cells.

Culture medium—The liquid that covers cells in a culture dish and contains nutrients to nourish and support the cells. Culture medium may also include growth factors added to produce desired changes in the cells.

Differentiation—The process whereby an unspecialized embryonic cell acquires the features of a specialized cell such as a heart, liver, or muscle cell. Differentiation is controlled by the interaction of a cell’s genes with the physical and chemical conditions outside the cell, usually through signaling pathways involving proteins embedded in the cell surface.

Directed differentiation—The manipulation of stem cell culture conditions to induce differentiation into a particular cell type.

DNA—Deoxyribonucleic acid, a chemical found primarily in the nucleus of cells. DNA carries the instructions or blueprint for making all the structures and materials the body needs to function. DNA consists of both genes and non-gene DNA in between the genes.

Ectoderm—The outermost germ layer of cells derived from the inner cell mass of the blastocyst; gives rise to the nervous system, sensory organs, skin, and related structures.

Embryo—In humans, the developing organism from the time of fertilization until the end of the eighth week of gestation, when it is called a fetus.

Embryoid bodies—Rounded collections of cells that arise when embryonic stem cells are cultured in suspension. Embryoid bodies contain cell types derived from all 3 germ layers.

Embryonic germ cells—Pluripotent stem cells that are derived from early germ cells (those that would become sperm and eggs). Embryonic germ cells (EG cells) are thought to have properties similar to embryonic stem cells.

Embryonic stem cells—Primitive (undifferentiated) cells derived from a 5-day preimplantation embryo that are capable of dividing without differentiating for a prolonged period in culture, and are known to develop into cells and tissues of the three primary germ layers.

Embryonic stem cell line—Embryonic stem cells, which have been cultured under in vitro conditions that allow proliferation without differentiation for months to years.

Endoderm—The innermost layer of the cells derived from the inner cell mass of the blastocyst; it gives rise to lungs, other respiratory structures, and digestive organs, or generally “the gut.”

Enucleated—Having had its nucleus removed.

Epigenetic—Having to do with the process by which regulatory proteins can turn genes on or off in a way that can be passed on during cell division.

Feeder layer—Cells used in co-culture to maintain pluripotent stem cells. For human embryonic stem cell culture, typical feeder layers include mouse embryonic fibroblasts (MEFs) or human embryonic fibroblasts that have been treated to prevent them from dividing.

Fertilization—The joining of the male gamete (sperm) and the female gamete (egg).

Fetus—In humans, the developing human from approximately eight weeks after conception until the time of its birth.

Gamete—An egg (in the female) or sperm (in the male) cell. See also Somatic cell.

Gastrulation—The process in which cells proliferate and migrate within the embryo to transform the inner cell mass of the blastocyst stage into an embryo containing all three primary germ layers.

Gene—A functional unit of heredity that is a segment of DNA found on chromosomes in the nucleus of a cell. Genes direct the formation of an enzyme or other protein.

Germ layers—After the blastocyst stage of embryonic development, the inner cell mass of the blastocyst goes through gastrulation, a period when the inner cell mass becomes organized into three distinct cell layers, called germ layers. The three layers are the ectoderm, the mesoderm, and the endoderm.

Hematopoietic stem cell—A stem cell that gives rise to all red and white blood cells and platelets.

Human embryonic stem cell (hESC)—A type of pluripotent stem cell derived from the inner cell mass (ICM) of the blastocyst.

Induced pluripotent stem cell (iPSC)—A type of pluripotent stem cell, similar to an embryonic stem cell, formed by the introduction of certain embryonic genes into a somatic cell.

In vitro—Latin for “in glass”; in a laboratory dish or test tube; an artificial environment.

In vitro fertilization—A technique that unites the egg and sperm in a laboratory instead of inside the female body.

Inner cell mass (ICM)—The cluster of cells inside the blastocyst. These cells give rise to the embryo and ultimately the fetus. The ICM cells are used to generate embryonic stem cells.

Long-term self-renewal—The ability of stem cells to replicate themselves by dividing into the same non-specialized cell type over long periods (many months to years) depending on the specific type of stem cell.

Mesenchymal stem cells—A term that is currently used to define non-blood adult stem cells from a variety of tissues, although it is not clear that mesenchymal stem cells from different tissues are the same.

Meiosis—The type of cell division a diploid germ cell undergoes to produce gametes (sperm or eggs) that will carry half the normal chromosome number. This is to ensure that when fertilization occurs, the fertilized egg will carry the normal number of chromosomes rather than causing aneuploidy (an abnormal number of chromosomes).

Mesoderm—Middle layer of a group of cells derived from the inner cell mass of the blastocyst; it gives rise to bone, muscle, connective tissue, kidneys, and related structures.

Microenvironment—The molecules and compounds such as nutrients and growth factors in the fluid surrounding a cell in an organism or in the laboratory, which play an important role in determining the characteristics of the cell.

Mitosis—The type of cell division that allows a population of cells to increase its numbers or to maintain its numbers. The number of chromosomes remains the same in this type of cell division.

Multipotent—Having the ability to develop into more than one cell type of the body. See also pluripotent and totipotent.

Neural stem cell—A stem cell found in adult neural tissue that can give rise to neurons and glial (supporting) cells. Examples of glial cells include astrocytes and oligodendrocytes.

Neurons—Nerve cells, the principal functional units of the nervous system. A neuron consists of a cell body and its processes—an axon and one or more dendrites. Neurons transmit information to other neurons or cells by releasing neurotransmitters at synapses.

Oligodendrocyte—A supporting cell that provides insulation to nerve cells by forming a myelin sheath (a fatty layer) around axons.

Parthenogenesis—The artificial activation of an egg in the absence of a sperm; the egg begins to divide as if it has been fertilized.

Passage—In cell culture, the process in which cells are disassociated, washed, and seeded into new culture vessels after a round of cell growth and proliferation. The number of passages a line of cultured cells has gone through is an indication of its age and expected stability.

Pluripotent—Having the ability to give rise to all of the various cell types of the body. Pluripotent cells cannot make extra-embryonic tissues such as the amnion, chorion, and other components of the placenta. Scientists demonstrate pluripotency by providing evidence of stable developmental potential, even after prolonged culture, to form derivatives of all three embryonic germ layers from the progeny of a single cell and to generate a teratoma after injection into an immunosuppressed mouse.

Polar Body—A polar body is a structure produced when an early egg cell, or oogonium, undergoes meiosis. In the first meiosis, the oogonium divides its chromosomes evenly between the two cells but divides its cytoplasm unequally. One cell retains most of the cytoplasm, while the other gets almost none, leaving it very small. This smaller cell is called the first polar body. The first polar body usually degenerates. The ovum, or larger cell, then divides again, producing a second polar body with half the amount of chromosomes but almost no cytoplasm. The second polar body splits off and remains adjacent to the large cell, or oocyte, until it (the second polar body) degenerates. Only one large functional oocyte, or egg, is produced at the end of meiosis.

Preimplantation—With regard to an embryo, preimplantation means that the embryo has not yet implanted in the wall of the uterus. Human embryonic stem cells are derived from preimplantation-stage embryos fertilized outside a woman’s body (in vitro).

Proliferation—Expansion of the number of cells by the continuous division of single cells into two identical daughter cells.

Regenerative medicine—A field of medicine devoted to treatments in which stem cells are induced to differentiate into the specific cell type required to repair damaged or destroyed cell populations or tissues. (See also cell-based therapies).

Reproductive cloning—The process of using somatic cell nuclear transfer (SCNT) to produce a normal, full grown organism (e.g., animal) genetically identical to the organism (animal) that donated the somatic cell nucleus. In mammals, this would require implanting the resulting embryo in a uterus where it would undergo normal development to become a live independent being. The first animal to be created by reproductive cloning was Dolly the sheep, born at the Roslin Institute in Scotland in 1996. See also Somatic cell nuclear transfer (SCNT).

Signals—Internal and external factors that control changes in cell structure and function. They can be chemical or physical in nature.

Somatic cell—Any body cell other than gametes (egg or sperm); sometimes referred to as “adult” cells. See also Gamete.

Somatic cell nuclear transfer (SCNT)—A technique that combines an enucleated egg and the nucleus of a somatic cell to make an embryo. SCNT can be used for therapeutic or reproductive purposes, but the initial stage that combines an enucleated egg and a somatic cell nucleus is the same. See also therapeutic cloning and reproductive cloning.

Somatic (adult) stem cells—A relatively rare undifferentiated cell found in many organs and differentiated tissues with a limited capacity for both self renewal (in the laboratory) and differentiation. Such cells vary in their differentiation capacity, but it is usually limited to cell types in the organ of origin. This is an active area of investigation.

Stem cells—Cells with the ability to divide for indefinite periods in culture and to give rise to specialized cells.

Stromal cells—Connective tissue cells found in virtually every organ. In bone marrow, stromal cells support blood formation.

Subculturing—Transferring cultured cells, with or without dilution, from one culture vessel to another.

Surface markers—Proteins on the outside surface of a cell that are unique to certain cell types and that can be visualized using antibodies or other detection methods.

Teratoma—A multi-layered benign tumor that grows from pluripotent cells injected into mice with a dysfunctional immune system. Scientists test whether they have established a human embryonic stem cell (hESC) line by injecting putative stem cells into such mice and verifying that the resulting teratomas contain cells derived from all three embryonic germ layers.

Therapeutic cloning—The process of using somatic cell nuclear transfer (SCNT) to produce cells that exactly match a patient. By combining a patient’s somatic cell nucleus and an enucleated egg, a scientist may harvest embryonic stem cells from the resulting embryo that can be used to generate tissues that match a patient’s body. This means the tissues created are unlikely to be rejected by the patient’s immune system. See also Somatic cell nuclear transfer (SCNT).

Totipotent—Having the ability to give rise to all the cell types of the body plus all of the cell types that make up the extraembryonic tissues such as the placenta. (See also Pluripotent and Multipotent).

Transdifferentiation—The process by which stem cells from one tissue differentiate into cells of another tissue.

Trophectoderm—The outer layer of the preimplantation embryo in mice. It contains trophoblast cells.

Trophoblast—The outer cell layer of the blastocyst. It is responsible for implantation and develops into the extraembryonic tissues, including the placenta, and controls the exchange of oxygen and metabolites between mother and embryo.

Umbilical cord blood stem cells—Stem cells collected from the umbilical cord at birth that can produce all of the blood cells in the body (hematopoietic). Cord blood is currently used to treat patients who have undergone chemotherapy to destroy their bone marrow due to cancer or other blood-related disorders.

Undifferentiated—A cell that has not yet developed into a specialized cell type.

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Adult stem cells are the stem cells found in all tissues and fluids that are not embryonic.

Bone marrow from another individual results in rejection usually via graph vs. host disease. To prevent this rejection, the patient is treated with X-Rays and chemotherapy. This is done prior to the transfusion. This also results in the patient taking anti rejection drugs the rest of their life. Using one’s own adult stem cells does not produce a reaction/graph vs. host disease. This is autologous (self) stem cells from your body and prevents this rejection.

Incorporated in Texas in 2008, StemTroniX, Inc. began as a concept of seasoned entrepreneurial individuals with over a total of 100 years of experience in the biotechnology, biopharmaceutical, proteomic and financing fields.

In addition to appreciating the great need for individuals in the USA and the world to have the opportunity for a premier company focusing on the total process of adult stem cell therapy, they decided to remain in the background of the company to secure that the focus of the company is to remain providing adult stem therapy at a reasonable price to patients in a certified center with technology developed by StemTroniX, Inc. and others.

Mission:

To develop and commercialize adult stem cell therapies, as well as remaining committed to helping people live fuller, healthier lives and to continue to be the premier provider of adult stem cell therapy to treat diseases in the USA.

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