CA2309424A1 - Apoptotic entities for use in treatment of neurodegenerative and other neurological disorders - Google Patents
Apoptotic entities for use in treatment of neurodegenerative and other neurological disorders Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/14—Blood; Artificial blood
- A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P21/00—Drugs for disorders of the muscular or neuromuscular system
- A61P21/04—Drugs for disorders of the muscular or neuromuscular system for myasthenia gravis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/02—Drugs for disorders of the nervous system for peripheral neuropathies
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
- A61P25/16—Anti-Parkinson drugs
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/24—Antidepressants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
Abstract
Treatment and/or prophylaxis, in mammalian patients, of neurodegenerative and other neurological medical disorders is effected by administering to the patient effective amounts of apoptotic bodies and/or apoptotic cells, preferably those derived from the patient's own white blood cells, e.g. by extracorporeal treatment of the patient's blood cells to induce apoptosis and administration of the apoptotic bodies and/or cells so formed to the patient.
Description
APOPTOTIC ENTITIES FOR USE IN TREATMENT OF
NEURODEGENERATIVE AND OTHER NEUROLOGICAL DISORDERS.
Field of the Invention This invention relates to biochemical and biological compositions and to the uses thereof in the treatment andlor prophylaxis of various neurodegenerative and other neurological disorders in mammalian patients. More particularly, it relates to treatment and prophylaxis of neurodegenerative and other neurological disorders by administration of compositions containing mammalian cellular materials and fragments thereof, and to the compositions containing the mammalian cellular materials and fragments themselves, and to processes for preparing such compositions.
Background of the Invention Two mechanisms of cell death in the body are recognized, necrosis and apoptosis. Apoptosis is the process of programmed cell death, first described by Kerr et al in 1972 [Kerr JFR, Wyllie AH, Currie AR (1992).
"Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. "British Journal of Cancer 26: 239-257", by which steady-state levels of the various organ systems and tissues in the body are maintained as continuous cell division and differentiation takes place. Cells undergoing apoptosis often exhibit distinctive morphological changes such as pronounced decrease in cell volume, modification of the cytoskeletons resulting in pronounced membrane blebbing, a condensation of the chromatin, and degradation of the DNA into oligonucleosomal fragments. Following these morphological changes, an apoptotic cell may break up into a number of small fragments known as apoptotic bodies, consisting essentially of membrane-bound bodies containing intact organelles, chromatin etc. Apoptotic bodies are normally rapidly removed from the body by phagocytosis principally by macrophages, before they can become lysed and release their potentially pro-inflammatory intracellular contents.
In simple outline, apoptosis is thought to proceed as follows.
Three phases can be identified in the apoptotic mechanism of programmed cell death:
Induction phase Effector phase Degradation phase.
The induction phase is dependent on specific interactions of death-inducing signals at the cell surface membrane. One common signal is initiated by the binding of specific ligands to receptors of the TNF receptor family present on the cell membrane. One important such receptor is Fas (APO-1, CD95), which interacts with Fas-ligand to initiate apoptosis.
The effector phase, activated by the binding of receptors and ligands of the induction phase, leads to the activation of caspases, cystinyl-aspartate-requiring proteinases (proteolytic enzymes), including caspases 1 and 8. This activation is associated with a change in the permeability of mitochondria, allowing the release of cytochrome-c which is involved in caspase activation. Activated caspases initiate a chain of lethal proteolytic events culminating in the changes in chromatin and cytoskeletal components seen in apoptosis.
Many cells undergoing apoptosis can be identified by a characteristic 'laddering' of DNA seen on agarose gel electrophoresis, resulting from cleavage of DNA into a series of fragments. These changes occur a few hours before death of the cell as defined by the ability of a cell to exclude vital dyes. The appearance of DNA laddering on agarose gel electrophoresis following extraction of DNA from cells is one recognised method of identification of apoptosis in cells [Loo, D.T. and Rillema, J.R. (1998) "Measurement of Cell Death," Mefhods in Cell Biology 57: 251-264], although it is not always sensitive enough to detect apoptosis. In situ labelling of nuclear DNA fragmentation for example using commercially available terminal dUTP nick end labelling (TUNEL) assays, are an alternative and more reproducible measure for the determination of fragmented DNA in apoptotic cells and cells undergoing apoptosis [Gavrieli Y, Sherman Y, Ben-Sasson SA (1992)", Identification of programmed cell death in situ via specific labelling of nuclear DNA
fragmentation". Journal of Cell Biology 119: 493-501 ].
During apoptosis, phosphatidylserine becomes exposed externally on the cell membrane [Fadok VA, Voelker DR, Campbell PA, Cohen JJ, Bratton DL, Henson PM (1992), "Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages". Journal of Immunology 148: 2207-2216] and this exposed phosphatidylserine binds to specific receptors to mediate the uptake and clearance of apoptotic cells in mammals [Fadok VA, Bratton DL, Rose DM, Pearson A, Ezekewitz RAB, Henson PM (2000), "A receptor for phosphatidylserine-specific clearance of apoptotic cells", Nature 405: 85-90].
The surface expression of phosphatidylserine on cells is another recognised method of identification of apoptotic cells.
Changes in mitochondria) integrity are intimately associated with apoptosis, resulting in alterations in mitochondria) membrane permeability and the release of cytochrome-c from the mitochondria into the cell cytoplasm [Susin, S.A., Lorenzo, H.K., Zamzami, N., Marzo, I, Brenner, C., Larochette, N., Prevost, M.C., Alzari, P.M. and Kroemer, G. (1999) "Mitochondria) Release of Caspase-2 and -9 during the Apoptotic Process", Journal of Experimental Medicine, 189: 381 - 394]. Measurement of changes in mitochondria) membrane potential, reflecting changes in mitochondria) membrane permeability, is another recognised method of identification of apoptotic cells.
A number of other methods of identification of cells undergoing apoptosis and of apoptotic cells, many using monoclonal antibodies against specific markers for apoptotic cells, have also been described in the scientific literature.
Necrosis, in contrast, is cell death of a pathological nature, resulting from injury, bacterial toxin effects, inflammatory mediators, etc., and involving membrane rupture and release of intracellular contents to the surrounding tissue, often with harmful inflammatory consequences.
Summary of the Invention According to the present invention, the administration of apoptotic cells and/or apoptotic bodies previously prepared ex vivo are used in the prophylaxis and treatment of neurodegenerative and other neurological disorders.
According to the present invention, the administration of apoptotic cells andlor apoptotic bodies previously prepared ex vivo are used in the prophylaxis and treatment of neurodegenerative and other neurological disorders.
Neurodegenerative diseases, including Down's syndrome, Alzheimer's disease and Parkinson's disease, are associated with increased levels of certain cytokines, including interleukin-1 [i (IL-1 [3) [see Griffin WST, Stanley LC, Ling C, White L, Macleod V. Perrot LJ, White CL, Araoz C (1989(.
Brain interleukin 1 and S-100 immunoreactivity are elevated in Down syndrome and Alzheimer disease. Proceedings of the National Academy of Sciences USA 867611-7615; Mogi M, Harada M, Narabayashi H, Inagaki H, Minami M, Nagatsu T (1996). Interleukin (IL)-1 beta, IL-1, IL-4, IL-6 and transforming growth factor-alpha levels are elevated in ventricular cerebrospinal fluid in juvenile parkinsonism and Parkinson's disease. Neuroscience Letters 211:13-16]. It has also been shown that IL-1 [i inhibits long-term potentiation in the hippocampus [Murray CA, Lynch MA (1998). Evidence that increase hippocampal expression of the cytokine interleukin-1 [i is a common trigger for age and tress-induced impairments in long-term potentiation. Journal of Neuroscience 18:2974-2981]. Long-term potentiation in the hippocampus is a form of synaptic plasticity and is generally considered to be an appropriate model for memory and learning [Bliss TVP, Collinridge GL, (1993). A synaptic model of memory: long-term potentiation in the hippocampus, Nature 361:31-39]. Thus, inappropriate cytokine expression in the brain is currently believed to be involved in the development and progression of neurodegenerative diseases.
Neurodegenerative and other neurological disorders can be Down's syndrome, Alzheimer's disease, Parkinson's disease, senile dementia, depression. In summary, it can be substantially any neurodegenerative or other neurological disorders.
"Apoptotic cells and apoptotic bodies", as the term is used herein, means cells and cell bodies which exhibit one or more of the following apoptosis-characterizing features:
surface exposure of phosphatidylserine, as detected by standard, accepted methods of detection such as Annexin V staining, methods for which are commercially available (for example, Annexin V-FTIC kit, Strss-Gen Biotechnologies Corp, Vancouver, Canada);
alterations in mitochondria) membrane permeability measured by standard, accepted methods (e.g. Salvioli, S., Ardizzoni, A., Franceschi, C.
Cossarizza, A. (1997) "JC-1, but not DiOC6(3) or Rhodamine 123, is a Reliable Fluorescent Probe to assess Delta Psi Changes in Intact Cells: Implications for Studies on Mitochondria) Functionality during Apoptosis," FEBS Letfers 411:
77-82];
evidence of DNA fragmentation such as the appearance of DNA
laddering on agarose gel electrophoresis following extraction of DNA from the cells [Teiger, E., Dam, T.V., Richard, L., Wisnewsky, C., Tea, B.S., Gaboury, L., Tremblay, J., Schwartz, K. and Hamet, P. (1996) "Apoptosis in Pressure Overload-induced Heart Hypertrophy in the Rat," Journal of Clinical Investigation 97; 2891-2897], or by in situ labeling (see Gavrieli et al., 1992, referenced above).
Descr~~tion of the Preferred Embodiments The apoptotic cells and/or apoptotic bodies for use in the present invention are previously prepared ex vivo from mammalian cells that are compatible with those of the mammalian patient. They can be prepared from substantially any type of mammalian cell including cultured cell lines.
Preferably they are prepared from a cell type derived from the mammalian patient's own body or from an established cell line. More preferably they are prepared from white blood cells of blood compatible with that of the mammalian patient, even more preferably from the patient's own white blood cells and most preferably from the patient's own T lymphocytes. The apoptotic cells andlor apoptotic bodies are prepared extracorporeally prior to administration to the patient.
Thus, an aliquot of the patient's blood may be withdrawn, e.g. by venipuncture, and at least a portion of the white cells thereof subjected extracorporeally to apoptosis inducing conditions.
A variety of methods of inducing apoptosis in mammalian cells, so as to create apoptotic cells and apoptotic bodies, are known in the art and essentially any of these can be adopted in preparing apoptotic bodies for use in the present invention. One such method is the application of oxidative stress to cells extracorporeally (see for example Buttke and Sandstrom (1994) "Oxidative Stress as a Mediator of Apoptosis", Immunology Tod ~, Vol. 15:7-10). This can be achieved by treating the cells, in suspension, with chemical oxidizing agents such as hydrogen peroxide, other peroxides and hydroperoxides, ozone, permanganates, periodates, and the like. Biologically acceptable such oxidizing agents are preferably used, so as to reduce potential problems associated with residues associated with and contaminating the apoptotic cells and apoptotic bodies so formed. Another method is the subjection of the cells to ionizing radiation (y-rays, x-rays, etc.) andlor non ionizing electromagnetic radiation including ultraviolet light. Apoptosis can be induced by subjecting cells to ultrasound. Yet another method is the treatment of the cells with drugs such as non-specific protein kinase inhibitors as exemplified by staurosporine (see Bombeli, Karsan, Tait and Hirlan, (1997) "Apoptotic Vascular Endothelial Cells Become Procoagulant", Blood, Vol. 89:2429-2442). Also, certain chemotherapeutic agents used for the treatment of malignant tumours induce apoptosis, for example adriamycin, as can statin drugs (3-hydroxy-3methylglutaryl coenzyme A reductase inhibitors) [Guijarro C, Blanco-Colio LM, Ortego M, Alonso C, Ortiz A, Plaza JJ, Diaz C, Hernandez G, Edigo J (1998), "3-hydroxy-3methylglutaryl coenzyme A reductase and isoprenylation inhibitors induce apoptosis of vascular smooth muscle in culture". "Circulation Research 83: 490-500] and colcicine [Suzuki Y (1998)", "Cell death, phagocytosis and neurogenesis in mouse olfactory epithelium and vomeronasal organ after colcicine treatment". Annals of the New York Academy of Sciences 855: 252-254]. The use of ligands for death receptors on cells, such as Fas-ligand, will be apparent for inducing apoptosis from the discussion of apoptosis above. The present invention is not restricted to any particular method of producing apoptotic cells and apoptotic bodies, for use in the present invention, and any suitable, known process can be used.
Methods for the detection and quantitation of apoptosis can be used to determine the presence and level of apoptosis in the preparation to be administered to the patient in the present invention. At least one of the methods from those described in the Introduction above should be used to confirm the level of apoptosis achieved prior to administration.
In preparing the apoptotic bodies, care should be taken not to apply excessive levels of oxidative stress, radiation, drug treatment, etc., since otherwise there is a significant risk of causing necrosis of at least some of the cells under treatment. Necrosis causes cell membrane rupture and the release of cellular contents often with biologically harmful results, particularly inflammatory events, so that the presence of necrotic cells and their components along with the apoptotic bodies is best avoided. -The process of apoptosis should be conducted under conditions which cause apoptosis, or other inactivation or down-regulation, of the potentially phagocytosing cells _ g _ present in the cellular composition under treatment, such as macrophages, since otherwise the apoptotic bodies produced are liable to be phagocytosed before administration to the patient, and thereby rendering the preparation less effective. Appropriate levels of treatment of the cells to create apoptotic bodies for use in the present invention depend to some extent on the nature of the chosen cells and cellular composition, and the type of treatment chosen to induce apoptosis. Such appropriate levels are readily determinable by those skilled in the art, having regard to the available scientific literature on the subject including the above-reference articles.
One preferred process according to the present invention involves the culture of cells from the patient, or a compatible mammalian cell line.
The cultured cells may then be treated to induce apoptosis and create apoptotic cells andlor apoptotic bodies therein. The cells, suspended in the patient's plasma or another suitable suspension medium, such as saline or a balanced mammalian cell culture medium, can then be administered as indicated below.
The numbers of apoptotic cells can be determined by published methods available in the scientific literature on the subject including the above-reference articles. The numbers of such apoptotic cells andlor apoptotic bodies required for administration to the patient to obtain the required clinical benefit will vary depending on the source of cells, the patients condition etc. and may require some experimentation but are readily determinable by those skilled in the art.
A more preferred process according to the present invention accordingly involves extraction of an aliquot of blood from the patient to be treated, and treatment of the white cells thereof under apoptosis-causing conditions, so as to create a cellular composition in which significant numbers of the white cells therein have been apoptosed so as to create therein substantial numbers of apoptotic bodies. Then the treated composition is re-administered to the patient. The aliquot treated to cause apoptosis may be whole blood, but if preferably a separated white cell fraction thereof, separated from the blood by known means, and suspended in plasma or another suitable _ g _ suspension medium, such as saline or a balanced mammalian cell culture medium. More preferably, T lymphocytes, isolated from the blood by known means, and suspended as above, may be used as a source of apoptotic cells and apoptotic bodies.
The volume of the aliquot of blood withdrawn from the patient for treatment to create apoptotic cells andlor apoptotic bodies therein is suitable up to about 400 ml, preferably from about 0.1 to about 100 ml, and most preferably from about 5 to about 15 ml. Accordingly, the preferred amounts of apoptotic cells andlor apoptotic bodies for administration are those corresponding to the numbers derivable from the white blood cells, or isolated T lymphocytes, contained in such quantities of whole blood, following subjection to apoptosis-inducing conditions.
The suspension of apoptotic cells and/or apoptotic bodies, is prepared in a biologically acceptable liquid suspending medium, such as the patient's serum or plasma, saline or balanced mammalian cell culture medium.
The addition of other factors, such as cytokines, hormones, products of stressed cells or other appropriate biologically active material, including cells, may enhance the benefit of the administered apoptotic cells andlor apoptotic bodies. The aliquot can be re-introduced into the patient's body by any suitable method, most preferably intramuscular injection but also including subcutaneous injection, mini-grafting, intra peritoneal injection, intra-arterial injection, intravenous injection and oral administration. The apoptotic entities can be delivered to the specific body organ andlor site by using any appropriate delivery system including liposomes, microspheres, etc.
For most effective treatment and prophylaxis of mammalian disorders involving endothelial dysfunction, the patient may be given a course of treatments with apoptotic cells andlor apoptotic bodies according to the invention. Each course of treatment may involve administration to the patient of from 1 to 6 aliquots of suspended apoptotic cells andlor apoptotic bodies, as described above. No more than one such aliquot should be administered per day, and the maximum rest period between any two consecutive administrations should be not greater than about 21 days. Booster treatments as described below may advantageously be used. To maintain the desired effects, the patient may undergo booster treatments, with a further course of administration of aliquots of suspended apoptotic cells andlor apoptotic bodies as described above, at intervals of three to four months.
As noted, the present invention is applicable to the treatment and prophylaxis of a wide variety of mammalian neurodegenerative and other neurological disorders. These include, but are not limited to, Downs syndrome, Alzheimer's disease, Parkinson's disease, senile dementia, depression, multiple sclerosis, Humtingdon's disease, peripheral neuropathies, spinal cord diseases, neuropathic joint diseases, chronic inflammatory demyelinating disease (CIPD), nueropathies including mononeuropathy, polyneuropathy, symmetrical distal sensory neuropathy, cystic fibrosis, neuromuscular junction disorders and myasthenias. In summary, it can be substantially any neurodegenerative or other neurological disorder.
NEURODEGENERATIVE AND OTHER NEUROLOGICAL DISORDERS.
Field of the Invention This invention relates to biochemical and biological compositions and to the uses thereof in the treatment andlor prophylaxis of various neurodegenerative and other neurological disorders in mammalian patients. More particularly, it relates to treatment and prophylaxis of neurodegenerative and other neurological disorders by administration of compositions containing mammalian cellular materials and fragments thereof, and to the compositions containing the mammalian cellular materials and fragments themselves, and to processes for preparing such compositions.
Background of the Invention Two mechanisms of cell death in the body are recognized, necrosis and apoptosis. Apoptosis is the process of programmed cell death, first described by Kerr et al in 1972 [Kerr JFR, Wyllie AH, Currie AR (1992).
"Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. "British Journal of Cancer 26: 239-257", by which steady-state levels of the various organ systems and tissues in the body are maintained as continuous cell division and differentiation takes place. Cells undergoing apoptosis often exhibit distinctive morphological changes such as pronounced decrease in cell volume, modification of the cytoskeletons resulting in pronounced membrane blebbing, a condensation of the chromatin, and degradation of the DNA into oligonucleosomal fragments. Following these morphological changes, an apoptotic cell may break up into a number of small fragments known as apoptotic bodies, consisting essentially of membrane-bound bodies containing intact organelles, chromatin etc. Apoptotic bodies are normally rapidly removed from the body by phagocytosis principally by macrophages, before they can become lysed and release their potentially pro-inflammatory intracellular contents.
In simple outline, apoptosis is thought to proceed as follows.
Three phases can be identified in the apoptotic mechanism of programmed cell death:
Induction phase Effector phase Degradation phase.
The induction phase is dependent on specific interactions of death-inducing signals at the cell surface membrane. One common signal is initiated by the binding of specific ligands to receptors of the TNF receptor family present on the cell membrane. One important such receptor is Fas (APO-1, CD95), which interacts with Fas-ligand to initiate apoptosis.
The effector phase, activated by the binding of receptors and ligands of the induction phase, leads to the activation of caspases, cystinyl-aspartate-requiring proteinases (proteolytic enzymes), including caspases 1 and 8. This activation is associated with a change in the permeability of mitochondria, allowing the release of cytochrome-c which is involved in caspase activation. Activated caspases initiate a chain of lethal proteolytic events culminating in the changes in chromatin and cytoskeletal components seen in apoptosis.
Many cells undergoing apoptosis can be identified by a characteristic 'laddering' of DNA seen on agarose gel electrophoresis, resulting from cleavage of DNA into a series of fragments. These changes occur a few hours before death of the cell as defined by the ability of a cell to exclude vital dyes. The appearance of DNA laddering on agarose gel electrophoresis following extraction of DNA from cells is one recognised method of identification of apoptosis in cells [Loo, D.T. and Rillema, J.R. (1998) "Measurement of Cell Death," Mefhods in Cell Biology 57: 251-264], although it is not always sensitive enough to detect apoptosis. In situ labelling of nuclear DNA fragmentation for example using commercially available terminal dUTP nick end labelling (TUNEL) assays, are an alternative and more reproducible measure for the determination of fragmented DNA in apoptotic cells and cells undergoing apoptosis [Gavrieli Y, Sherman Y, Ben-Sasson SA (1992)", Identification of programmed cell death in situ via specific labelling of nuclear DNA
fragmentation". Journal of Cell Biology 119: 493-501 ].
During apoptosis, phosphatidylserine becomes exposed externally on the cell membrane [Fadok VA, Voelker DR, Campbell PA, Cohen JJ, Bratton DL, Henson PM (1992), "Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages". Journal of Immunology 148: 2207-2216] and this exposed phosphatidylserine binds to specific receptors to mediate the uptake and clearance of apoptotic cells in mammals [Fadok VA, Bratton DL, Rose DM, Pearson A, Ezekewitz RAB, Henson PM (2000), "A receptor for phosphatidylserine-specific clearance of apoptotic cells", Nature 405: 85-90].
The surface expression of phosphatidylserine on cells is another recognised method of identification of apoptotic cells.
Changes in mitochondria) integrity are intimately associated with apoptosis, resulting in alterations in mitochondria) membrane permeability and the release of cytochrome-c from the mitochondria into the cell cytoplasm [Susin, S.A., Lorenzo, H.K., Zamzami, N., Marzo, I, Brenner, C., Larochette, N., Prevost, M.C., Alzari, P.M. and Kroemer, G. (1999) "Mitochondria) Release of Caspase-2 and -9 during the Apoptotic Process", Journal of Experimental Medicine, 189: 381 - 394]. Measurement of changes in mitochondria) membrane potential, reflecting changes in mitochondria) membrane permeability, is another recognised method of identification of apoptotic cells.
A number of other methods of identification of cells undergoing apoptosis and of apoptotic cells, many using monoclonal antibodies against specific markers for apoptotic cells, have also been described in the scientific literature.
Necrosis, in contrast, is cell death of a pathological nature, resulting from injury, bacterial toxin effects, inflammatory mediators, etc., and involving membrane rupture and release of intracellular contents to the surrounding tissue, often with harmful inflammatory consequences.
Summary of the Invention According to the present invention, the administration of apoptotic cells and/or apoptotic bodies previously prepared ex vivo are used in the prophylaxis and treatment of neurodegenerative and other neurological disorders.
According to the present invention, the administration of apoptotic cells andlor apoptotic bodies previously prepared ex vivo are used in the prophylaxis and treatment of neurodegenerative and other neurological disorders.
Neurodegenerative diseases, including Down's syndrome, Alzheimer's disease and Parkinson's disease, are associated with increased levels of certain cytokines, including interleukin-1 [i (IL-1 [3) [see Griffin WST, Stanley LC, Ling C, White L, Macleod V. Perrot LJ, White CL, Araoz C (1989(.
Brain interleukin 1 and S-100 immunoreactivity are elevated in Down syndrome and Alzheimer disease. Proceedings of the National Academy of Sciences USA 867611-7615; Mogi M, Harada M, Narabayashi H, Inagaki H, Minami M, Nagatsu T (1996). Interleukin (IL)-1 beta, IL-1, IL-4, IL-6 and transforming growth factor-alpha levels are elevated in ventricular cerebrospinal fluid in juvenile parkinsonism and Parkinson's disease. Neuroscience Letters 211:13-16]. It has also been shown that IL-1 [i inhibits long-term potentiation in the hippocampus [Murray CA, Lynch MA (1998). Evidence that increase hippocampal expression of the cytokine interleukin-1 [i is a common trigger for age and tress-induced impairments in long-term potentiation. Journal of Neuroscience 18:2974-2981]. Long-term potentiation in the hippocampus is a form of synaptic plasticity and is generally considered to be an appropriate model for memory and learning [Bliss TVP, Collinridge GL, (1993). A synaptic model of memory: long-term potentiation in the hippocampus, Nature 361:31-39]. Thus, inappropriate cytokine expression in the brain is currently believed to be involved in the development and progression of neurodegenerative diseases.
Neurodegenerative and other neurological disorders can be Down's syndrome, Alzheimer's disease, Parkinson's disease, senile dementia, depression. In summary, it can be substantially any neurodegenerative or other neurological disorders.
"Apoptotic cells and apoptotic bodies", as the term is used herein, means cells and cell bodies which exhibit one or more of the following apoptosis-characterizing features:
surface exposure of phosphatidylserine, as detected by standard, accepted methods of detection such as Annexin V staining, methods for which are commercially available (for example, Annexin V-FTIC kit, Strss-Gen Biotechnologies Corp, Vancouver, Canada);
alterations in mitochondria) membrane permeability measured by standard, accepted methods (e.g. Salvioli, S., Ardizzoni, A., Franceschi, C.
Cossarizza, A. (1997) "JC-1, but not DiOC6(3) or Rhodamine 123, is a Reliable Fluorescent Probe to assess Delta Psi Changes in Intact Cells: Implications for Studies on Mitochondria) Functionality during Apoptosis," FEBS Letfers 411:
77-82];
evidence of DNA fragmentation such as the appearance of DNA
laddering on agarose gel electrophoresis following extraction of DNA from the cells [Teiger, E., Dam, T.V., Richard, L., Wisnewsky, C., Tea, B.S., Gaboury, L., Tremblay, J., Schwartz, K. and Hamet, P. (1996) "Apoptosis in Pressure Overload-induced Heart Hypertrophy in the Rat," Journal of Clinical Investigation 97; 2891-2897], or by in situ labeling (see Gavrieli et al., 1992, referenced above).
Descr~~tion of the Preferred Embodiments The apoptotic cells and/or apoptotic bodies for use in the present invention are previously prepared ex vivo from mammalian cells that are compatible with those of the mammalian patient. They can be prepared from substantially any type of mammalian cell including cultured cell lines.
Preferably they are prepared from a cell type derived from the mammalian patient's own body or from an established cell line. More preferably they are prepared from white blood cells of blood compatible with that of the mammalian patient, even more preferably from the patient's own white blood cells and most preferably from the patient's own T lymphocytes. The apoptotic cells andlor apoptotic bodies are prepared extracorporeally prior to administration to the patient.
Thus, an aliquot of the patient's blood may be withdrawn, e.g. by venipuncture, and at least a portion of the white cells thereof subjected extracorporeally to apoptosis inducing conditions.
A variety of methods of inducing apoptosis in mammalian cells, so as to create apoptotic cells and apoptotic bodies, are known in the art and essentially any of these can be adopted in preparing apoptotic bodies for use in the present invention. One such method is the application of oxidative stress to cells extracorporeally (see for example Buttke and Sandstrom (1994) "Oxidative Stress as a Mediator of Apoptosis", Immunology Tod ~, Vol. 15:7-10). This can be achieved by treating the cells, in suspension, with chemical oxidizing agents such as hydrogen peroxide, other peroxides and hydroperoxides, ozone, permanganates, periodates, and the like. Biologically acceptable such oxidizing agents are preferably used, so as to reduce potential problems associated with residues associated with and contaminating the apoptotic cells and apoptotic bodies so formed. Another method is the subjection of the cells to ionizing radiation (y-rays, x-rays, etc.) andlor non ionizing electromagnetic radiation including ultraviolet light. Apoptosis can be induced by subjecting cells to ultrasound. Yet another method is the treatment of the cells with drugs such as non-specific protein kinase inhibitors as exemplified by staurosporine (see Bombeli, Karsan, Tait and Hirlan, (1997) "Apoptotic Vascular Endothelial Cells Become Procoagulant", Blood, Vol. 89:2429-2442). Also, certain chemotherapeutic agents used for the treatment of malignant tumours induce apoptosis, for example adriamycin, as can statin drugs (3-hydroxy-3methylglutaryl coenzyme A reductase inhibitors) [Guijarro C, Blanco-Colio LM, Ortego M, Alonso C, Ortiz A, Plaza JJ, Diaz C, Hernandez G, Edigo J (1998), "3-hydroxy-3methylglutaryl coenzyme A reductase and isoprenylation inhibitors induce apoptosis of vascular smooth muscle in culture". "Circulation Research 83: 490-500] and colcicine [Suzuki Y (1998)", "Cell death, phagocytosis and neurogenesis in mouse olfactory epithelium and vomeronasal organ after colcicine treatment". Annals of the New York Academy of Sciences 855: 252-254]. The use of ligands for death receptors on cells, such as Fas-ligand, will be apparent for inducing apoptosis from the discussion of apoptosis above. The present invention is not restricted to any particular method of producing apoptotic cells and apoptotic bodies, for use in the present invention, and any suitable, known process can be used.
Methods for the detection and quantitation of apoptosis can be used to determine the presence and level of apoptosis in the preparation to be administered to the patient in the present invention. At least one of the methods from those described in the Introduction above should be used to confirm the level of apoptosis achieved prior to administration.
In preparing the apoptotic bodies, care should be taken not to apply excessive levels of oxidative stress, radiation, drug treatment, etc., since otherwise there is a significant risk of causing necrosis of at least some of the cells under treatment. Necrosis causes cell membrane rupture and the release of cellular contents often with biologically harmful results, particularly inflammatory events, so that the presence of necrotic cells and their components along with the apoptotic bodies is best avoided. -The process of apoptosis should be conducted under conditions which cause apoptosis, or other inactivation or down-regulation, of the potentially phagocytosing cells _ g _ present in the cellular composition under treatment, such as macrophages, since otherwise the apoptotic bodies produced are liable to be phagocytosed before administration to the patient, and thereby rendering the preparation less effective. Appropriate levels of treatment of the cells to create apoptotic bodies for use in the present invention depend to some extent on the nature of the chosen cells and cellular composition, and the type of treatment chosen to induce apoptosis. Such appropriate levels are readily determinable by those skilled in the art, having regard to the available scientific literature on the subject including the above-reference articles.
One preferred process according to the present invention involves the culture of cells from the patient, or a compatible mammalian cell line.
The cultured cells may then be treated to induce apoptosis and create apoptotic cells andlor apoptotic bodies therein. The cells, suspended in the patient's plasma or another suitable suspension medium, such as saline or a balanced mammalian cell culture medium, can then be administered as indicated below.
The numbers of apoptotic cells can be determined by published methods available in the scientific literature on the subject including the above-reference articles. The numbers of such apoptotic cells andlor apoptotic bodies required for administration to the patient to obtain the required clinical benefit will vary depending on the source of cells, the patients condition etc. and may require some experimentation but are readily determinable by those skilled in the art.
A more preferred process according to the present invention accordingly involves extraction of an aliquot of blood from the patient to be treated, and treatment of the white cells thereof under apoptosis-causing conditions, so as to create a cellular composition in which significant numbers of the white cells therein have been apoptosed so as to create therein substantial numbers of apoptotic bodies. Then the treated composition is re-administered to the patient. The aliquot treated to cause apoptosis may be whole blood, but if preferably a separated white cell fraction thereof, separated from the blood by known means, and suspended in plasma or another suitable _ g _ suspension medium, such as saline or a balanced mammalian cell culture medium. More preferably, T lymphocytes, isolated from the blood by known means, and suspended as above, may be used as a source of apoptotic cells and apoptotic bodies.
The volume of the aliquot of blood withdrawn from the patient for treatment to create apoptotic cells andlor apoptotic bodies therein is suitable up to about 400 ml, preferably from about 0.1 to about 100 ml, and most preferably from about 5 to about 15 ml. Accordingly, the preferred amounts of apoptotic cells andlor apoptotic bodies for administration are those corresponding to the numbers derivable from the white blood cells, or isolated T lymphocytes, contained in such quantities of whole blood, following subjection to apoptosis-inducing conditions.
The suspension of apoptotic cells and/or apoptotic bodies, is prepared in a biologically acceptable liquid suspending medium, such as the patient's serum or plasma, saline or balanced mammalian cell culture medium.
The addition of other factors, such as cytokines, hormones, products of stressed cells or other appropriate biologically active material, including cells, may enhance the benefit of the administered apoptotic cells andlor apoptotic bodies. The aliquot can be re-introduced into the patient's body by any suitable method, most preferably intramuscular injection but also including subcutaneous injection, mini-grafting, intra peritoneal injection, intra-arterial injection, intravenous injection and oral administration. The apoptotic entities can be delivered to the specific body organ andlor site by using any appropriate delivery system including liposomes, microspheres, etc.
For most effective treatment and prophylaxis of mammalian disorders involving endothelial dysfunction, the patient may be given a course of treatments with apoptotic cells andlor apoptotic bodies according to the invention. Each course of treatment may involve administration to the patient of from 1 to 6 aliquots of suspended apoptotic cells andlor apoptotic bodies, as described above. No more than one such aliquot should be administered per day, and the maximum rest period between any two consecutive administrations should be not greater than about 21 days. Booster treatments as described below may advantageously be used. To maintain the desired effects, the patient may undergo booster treatments, with a further course of administration of aliquots of suspended apoptotic cells andlor apoptotic bodies as described above, at intervals of three to four months.
As noted, the present invention is applicable to the treatment and prophylaxis of a wide variety of mammalian neurodegenerative and other neurological disorders. These include, but are not limited to, Downs syndrome, Alzheimer's disease, Parkinson's disease, senile dementia, depression, multiple sclerosis, Humtingdon's disease, peripheral neuropathies, spinal cord diseases, neuropathic joint diseases, chronic inflammatory demyelinating disease (CIPD), nueropathies including mononeuropathy, polyneuropathy, symmetrical distal sensory neuropathy, cystic fibrosis, neuromuscular junction disorders and myasthenias. In summary, it can be substantially any neurodegenerative or other neurological disorder.
Claims (7)
1. The use of apoptotic bodies and/or apoptotic cells in treatment and/or prophylaxis in mammalian patients of neurodegenerative and other neurological medical disorders.
2. The use of apoptotic bodies and/or apoptotic cells in the preparation of a medicament for the treatment and/or prophylaxis of neurodegenerative and other neurological medical disorders in mammalian patients.
3. Method for the treatment of or prophylaxis against neurodegenerative and other neurological medical disorders in a mammalian patient, which comprises administering to the patient an effective amount of apoptotic bodies and/or apoptotic cells.
4. Uses and methods according to any preceding claim wherein the apoptotic bodies and/or cells derive from extracorporeal treatment of blood cells compatible with those of the mammalian patient.
5. Uses and methods according to claim 4 wherein the blood cells are white blood cells of blood compatible with that of the mammalian patient.
6. Uses and methods according to claim 5 wherein the blood cells are the patient's own white blood cells.
7. Uses and methods according to claim 6 wherein the blood cells are the patient's own T lymphocytes.
Priority Applications (16)
Application Number | Priority Date | Filing Date | Title |
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CA 2309424 CA2309424A1 (en) | 2000-05-25 | 2000-05-25 | Apoptotic entities for use in treatment of neurodegenerative and other neurological disorders |
JP2001585781A JP2003534282A (en) | 2000-05-25 | 2001-05-25 | Apoptotic bodies for use in treating neurodegenerative and other neurological disorders |
US09/871,146 US7132285B2 (en) | 2000-05-25 | 2001-05-25 | Apoptotic entities for use in treatment of neurodegenerative and other neurological disorders |
DK01935897T DK1289535T3 (en) | 2000-05-25 | 2001-05-25 | Apoptotic devices for use in the treatment of neurodegenerative and other neurological diseases |
ES01935897T ES2290134T3 (en) | 2000-05-25 | 2001-05-25 | APOPTOTIC ENTITIES THAT ARE USED IN THE TREATMENT OF NEURO-DEGENERATIVE DISORDERS AND OTHER NEUROLOGICAL DISORDERS. |
DE2001629322 DE60129322T2 (en) | 2000-05-25 | 2001-05-25 | APOPTOTIC BODIES FOR USE IN THE TREATMENT OF NEURODE GENERATIVE AND OTHER NERVE DISEASES |
EP20010935897 EP1289535B1 (en) | 2000-05-25 | 2001-05-25 | Apoptotic entities for use in treatment of neurodegenerative and other neurological disorders |
PT01935897T PT1289535E (en) | 2000-05-25 | 2001-05-25 | Apoptotic entities for use in treatment of neurodegenerative and other neurological disorders |
PCT/CA2001/000759 WO2001089537A2 (en) | 2000-05-25 | 2001-05-25 | Apoptotic entities for use in treatment of neurodegenerative and other neurological disorders |
AT01935897T ATE366581T1 (en) | 2000-05-25 | 2001-05-25 | APOPTOTIC BODY FOR USE IN THE TREATMENT OF NEURODEGENERATIVE AND OTHER NERVOUS DISEASES |
AU2001261987A AU2001261987A1 (en) | 2000-05-25 | 2001-05-25 | Apoptotic entities for use in treatment of neurodegenerative and other neurological disorders |
CA002409992A CA2409992A1 (en) | 2000-05-25 | 2001-05-25 | Apoptotic entities for use in treatment of neurodegenerative and other neurological disorders |
TW90113346A TWI239843B (en) | 2000-05-25 | 2001-06-01 | Apoptotic entities for use in treatment of neurodegenerative and other neurological disorders |
HK03106461A HK1055080A1 (en) | 2000-05-25 | 2003-09-10 | Apoptotic entities for use in treatment of neurodegenerative and other neurological disorders |
US11/583,986 US20070087010A1 (en) | 2000-05-25 | 2006-10-17 | Apoptotic entities for use in treatment of neurodegenerative and other neurological disorders |
US11/927,094 US20080131416A1 (en) | 2000-05-25 | 2007-10-29 | Apoptotic entities for use in treatment of neurodegenerative and other neurological disorders |
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CA 2309424 CA2309424A1 (en) | 2000-05-25 | 2000-05-25 | Apoptotic entities for use in treatment of neurodegenerative and other neurological disorders |
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CA2309518A1 (en) * | 2000-05-25 | 2001-11-25 | Vasogen Ireland Limited | Apoptotic entities for use in treatment of t-cell-mediated and inflammatory disorders |
TWI281407B (en) * | 2000-09-18 | 2007-05-21 | Vasogen Ireland Ltd | Apoptosis-mimicking synthetic entities and use thereof in medical treatment |
CA2333494A1 (en) * | 2001-02-01 | 2002-08-01 | Vasogen Ireland Limited | Blood brain barrier modulation |
WO2003024423A1 (en) * | 2001-09-18 | 2003-03-27 | Vasogen Ireland Limited | Apoptosis-mimicking synthetic entities and use thereof in medical treatment |
US20060008517A1 (en) * | 2004-07-09 | 2006-01-12 | Lynch Marina A | Treatment of age-related memory impairment |
US11318163B2 (en) | 2015-02-18 | 2022-05-03 | Enlivex Therapeutics Ltd | Combination immune therapy and cytokine control therapy for cancer treatment |
US11497767B2 (en) | 2015-02-18 | 2022-11-15 | Enlivex Therapeutics R&D Ltd | Combination immune therapy and cytokine control therapy for cancer treatment |
US11596652B2 (en) | 2015-02-18 | 2023-03-07 | Enlivex Therapeutics R&D Ltd | Early apoptotic cells for use in treating sepsis |
US11304976B2 (en) | 2015-02-18 | 2022-04-19 | Enlivex Therapeutics Ltd | Combination immune therapy and cytokine control therapy for cancer treatment |
US11000548B2 (en) | 2015-02-18 | 2021-05-11 | Enlivex Therapeutics Ltd | Combination immune therapy and cytokine control therapy for cancer treatment |
EP3258943B1 (en) | 2015-02-18 | 2021-05-12 | Enlivex Therapeutics Ltd. | Combination immune therapy and cytokine control therapy for cancer treatment |
EP3285877B1 (en) | 2015-04-21 | 2022-10-19 | Enlivex Therapeutics Rdo Ltd | Therapeutic pooled blood apoptotic cell preparations and uses thereof |
US11730761B2 (en) | 2016-02-18 | 2023-08-22 | Enlivex Therapeutics Rdo Ltd | Combination immune therapy and cytokine control therapy for cancer treatment |
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NZ249176A (en) * | 1992-02-07 | 1996-11-26 | Vasogen Inc | Method of increasing concentration of nitric oxide in blood using ozone and uv radiation |
US5980954A (en) | 1992-02-07 | 1999-11-09 | Vasogen Ireland Limited | Treatment of autoimmune diseases |
DE60002979T2 (en) | 1999-01-12 | 2004-05-19 | Vasogen Ireland Ltd., Shannon | TREATMENT AGAINST CELL DEAD |
CA2271190A1 (en) * | 1999-05-06 | 2000-11-06 | Vasogen Ireland Limited | Improved method for treating mammals with modified mammalian blood |
AU2000269903B2 (en) | 2000-03-07 | 2004-04-22 | E.F.P. Floor Products Fussboden Gmbh | Mechanical connection of panels |
CA2309417A1 (en) * | 2000-05-25 | 2001-11-25 | Anthony E. Bolton | Apoptotic entities for use in treatment of endothelium dysfunction disorders |
CA2309518A1 (en) * | 2000-05-25 | 2001-11-25 | Vasogen Ireland Limited | Apoptotic entities for use in treatment of t-cell-mediated and inflammatory disorders |
US7122208B2 (en) * | 2001-04-06 | 2006-10-17 | Vasogen Ireland Limited | Compositions containing apoptotic entities |
EP1469733A4 (en) * | 2001-11-29 | 2008-07-23 | Therakos Inc | Methods for pretreating a subject with extracorporeal photopheresis and/or apoptotic cells |
US7255880B2 (en) * | 2003-04-03 | 2007-08-14 | Vasogen Ireland Limited | Treatment of endothelin-related disorders |
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- 2001-05-25 JP JP2001585781A patent/JP2003534282A/en not_active Withdrawn
- 2001-05-25 PT PT01935897T patent/PT1289535E/en unknown
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- 2001-05-25 AU AU2001261987A patent/AU2001261987A1/en not_active Abandoned
- 2001-05-25 AT AT01935897T patent/ATE366581T1/en not_active IP Right Cessation
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WO2001089537A2 (en) | 2001-11-29 |
HK1055080A1 (en) | 2003-12-24 |
US20020044924A1 (en) | 2002-04-18 |
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WO2001089537A3 (en) | 2002-08-01 |
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JP2003534282A (en) | 2003-11-18 |
DE60129322D1 (en) | 2007-08-23 |
US7132285B2 (en) | 2006-11-07 |
US20080131416A1 (en) | 2008-06-05 |
DK1289535T3 (en) | 2007-11-05 |
DE60129322T2 (en) | 2008-03-13 |
AU2001261987A1 (en) | 2001-12-03 |
PT1289535E (en) | 2007-10-19 |
US20070087010A1 (en) | 2007-04-19 |
ES2290134T3 (en) | 2008-02-16 |
ATE366581T1 (en) | 2007-08-15 |
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