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Title:  Apoptotic entities for use in treatment of neurodegenerative and other neurological disorders
United States Patent:  7,132,285
Issued: 
November 7, 2006

Inventors: 
Bolton; Anthony E. (Tideswell, GB), Mandel; Arkady (North York, CA), Sauder; Daniel (Toronto, CA)
Assignee: 
Vasogen Ireland Limited (IE)
Appl. No.:  09/871,146
Filed: 
May 25, 2001

 

Executive MBA in Pharmaceutical Management, U. Colorado


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.

SUMMARY OF THE INVENTION

This invention is directed, in part, to the novel and unexpected discovery that administration to a mammal of apoptotic cells and/or apoptotic bodies previously prepared ex vivo, can be used in the prophylaxis and/or treatment of neurodegenerative and/or other neurological disorders in the treated mammal.

Accordingly, in one of its composition aspects, this invention is directed to a pharmaceutical composition comprising a pharmaceutically acceptable excipient and an effective amount of apoptotic cells and/or apoptotic bodies.

The pharmaceutical compositions preferably employ an aqueous based pharmaceutically acceptable excipient although other excipients can be used.

As noted above, these compositions are useful in the prophylaxis and/or treatment of neurodegenerative and/or other neurological disorders in mammals. Accordingly, in one of its method aspects, this invention is directed to a 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.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

This invention is directed to the treatment and/or prophylaxis of neurodegenerative and/or other neurological disorders by the administration of apoptotic cells and/or bodies.

Neurodegenerative disorders, including Down's syndrome, Alzheimer's disease and Parkinson's disease, are associated with increased levels of reactive oxygen species (ROS), certain inflammatory cytokines, including interleukin-1.beta. (IL-1.beta.) [see Griffin W S T, Stanley L C, Ling C, White L, Macleod V. Perrot L J, White C L, Araoz C (1989). Brain interleukin 1 and S-100 immunoreactivity are elevated in Down's 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.beta. inhibits long-term potentiation in the hippocampus [Murray C A, Lynch M A (1998). Evidence that increase hippocampal expression of the cytokine interleukin-1.beta. is a common trigger for age and stress-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 T V P, Collinridge G L, (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 neuroinflammatory diseases.

Neurodegenerative and other neurological disorders treatable by the present invention include Down's syndrome, Alzheimer's disease, Parkinson's disease, senile dementia, depression and the like. In summary, it can be substantially any neurodegenerative or other neurological disorder.

"Apoptotic cells" and "apoptotic bodies," as the terms are 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; alterations in mitochondrial 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 Mitochondrial Functionality during Apoptosis," FEBS Letters 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).

The apoptotic cells and/or apoptotic bodies for use in the present invention preferably comprise not more than about 35 weight percent of necrotic cells and/or necrotic bodies based on the total weight of the apoptotic cells/bodies and necrotic cells/bodies; more preferably, not more than about 20 weight percent; and even more preferably, not more than about 10 weight percent. At these levels, the presence of such necrotic cells and/or bodies are believed not to significantly alter in vivo processes. In its most preferred embodiment, the apoptotic cells/bodies are substantially free of necrotic cells and or bodies (i.e., less than about 2 weight percent of necrotic cells/bodies).

The apoptotic cells and/or apoptotic bodies for use in the present invention are 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, more preferably from the patient's own white blood cell and even more preferably from the patient's own T lymphocytes. Even more preferably they are prepared from an established cell line. The apoptotic cells and/or apoptotic bodies are prepared extracorporeally prior to administration to the patient. Thus, in one embodiment, 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 subjection of the cells to ionizing radiation (.gamma.-rays, x-rays, etc.) and/or non-ionizing electromagnetic radiation including ultraviolet light. Apoptosis can be induced by subjecting cells to ultrasound.

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 L M, Ortego M, Alonso C, Ortiz A, Plaza J J, 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.

Yet another 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 Today, 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 oxidizing agents are preferably used, so as to reduce potential problems associated with residues and contaminations of the apoptotic cells and apoptotic bodies so formed.

The present invention is not restricted to any particular method of producing apoptotic cells and apoptotic bodies, for use herein, 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. They are suitably purified prior to use, by methods known in the art, such as differential centrifugation.

In preparing the apoptotic cells and/or 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. 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 to create apoptotic cells and/or 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 and/or bodies 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 and/or apoptotic bodies required for administration to the patient to obtain the required clinical benefit will vary depending on the source of cells, the patient's condition, the age and weight of the patient and other relevant factors which are readily determinable by the attending clinician.

Thus, an example of a preferred process according to the present invention accordingly involves extraction of an aliquot of blood from the patient to be treated, separation of the white cells therefrom, and treatment of the white cells 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 cells or bodies. Then the treated composition is administered to the patient. 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 number of viable cells selected for treatment to create apoptotic cells and/or apoptotic bodies is suitably up to about 4.times.10.sup.9, preferably from about 1,000,000 to about 1,000,000,000 and most preferably from about 50,000,000 to about 150,000,000, for each administration to a human patient. From about 10% to 90%, preferably from about 30% to 70% of the cellular composition for administration is comprised of apoptotic bodies and/or apoptotic cells, the balance being viable cells and necrotic cells. Accordingly, the preferred amounts of apoptotic cells and/or apoptotic bodies for administration are those produced by subjecting these numbers of cells to the apoptosing conditions. When whole blood is used as the source of the cells to be subjected to the apoptosis inducing conditions, these numbers of white cells are obtainable in blood aliquots of volume up to about 400 ml, preferably up to 100 ml. More specifically, 50,000,000 150,000,000 cells is equivalent to the white cells in blood aliquots of volume 10 30 ml.

The volume of the aliquot of blood withdrawn from the patient for treatment to create apoptotic cells and/or 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 and/or 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 treated apoptotic cells and/or bodies for administration to the patient 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 may enhance the benefit of the administered apoptotic cellular materials. The aliquot can be 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 and/or site by using any appropriate, known delivery system.

The compositions of this invention may optionally include a pharmaceutically acceptable excipient. Some examples of suitable excipients include sterile water, sterile saline, phosphate buffered saline, and the like.

When administered, the pharmaceutical compositions comprise an effective amount of apoptotic bodies/cells to induce a suitable prophylactic and/or therapeutic response in the patient at risk of suffering or suffering from a neurodegenerative disease. Preferably, the composition administered to the mammalian patient comprises from about 10,000 to 10,000,000 apoptotic cells or bodies per kilogram of body weight, more preferably from about 500,000 to 5,000,000 and most preferably from about 1,500,000 to about 4,000,000 apoptotic cells and/or bodies per kg body weight. The specific dose employed will, of course, be dependent upon the age, weight and severity of the disease in the treated patient all of which are within the skill of the attending clinician.

For most effective treatment and/or prophylaxis of mammalian disorders involving a neurodegenerative or neurological disorder, the patient may be given a course of treatments with apoptotic cells and/or bodies according to the invention. Each course of treatment may involve administration to the patient of from 1 to 6 aliquots of suspended cellular material, 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 and/or 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, Down's Syndrome, Alzheimer's disease, Parkinson's disease, senile dementia, depression, multiple sclerosis, Huntington's disease, peripheral neuropathies, spinal cord diseases, neuropathic joint diseases, chronic inflammatory demyelinating disease (CIPD), neuropathies including mononeuropathy, polyneuropathy, symmetrical distal sensory neruopathy, cystic fibrosis, neuromuscular junction disorders and myasthenias. In summary, it can be substantially any neurodegenerative or other neurological disorder.
 


Claim 1 of 4 Claims

1. A unit dosage composition for administration to a human patient, comprising a liquid suspension of cellular material including from about 10,000 to 10,000,000 apoptotic cells and/or apoptotic bodies per kilogram of patient body weight, wherein said apoptotic bodies and/or apoptotic cells exhibit at least two characteristics comprising DNA fragmentation, surface exposure of phosphatidylserine, or altered mitochondrial membrane permeability.

 

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If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.

 

 

     
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