WO2009155538A1

WO2009155538A1 - Compositions and methods of depleting amyloid-beta peptides from cerebrospinal fluid to treat alzheimer's disease

Info

Publication number: WO2009155538A1
Application number: PCT/US2009/047996
Authority: WO
Inventors: Thomas L. Fare; Ajay Verma; Adam J. Simon
Original assignee: Merck & Co., Inc.; Rosetta Inpharmatics Llc
Priority date: 2008-06-20
Filing date: 2009-06-19
Publication date: 2009-12-23

Abstract

The invention provides compositions, devices, systems, and methods for removing harmful substances from the bodily fluids of a subject, such as removing amyloid beta peptides from the cerebrospinal fluid of a subject.

Description

COMPOSITIONS AND METHODS OF DEPLETING AMYLOID-BETA PEPTIDES FROM CEREBROSPINAL FLUID TO TREAT ALZHEIMER'S DISEASE

CROSS-REFERENCE TO RELATED APPLICATION This application claims the benefit of U.S. Application No. 61/074551, filed on

June 20, 2008, which is hereby incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

The present invention relates to compositions, devices, and methods for removing toxic or pathogenic substances from the bodily fluid of human subjects. BACKGROUND

Alzheimer's disease (AD) is a progressive dementia characterized by memory failure in the early stages of the disease (Selkoe, DJ. , Science 298:789-791, 2002). The pathological symptoms of Alzheimer's disease (AD) include synaptic loss, selective neuronal death, a decrease in certain neurotransmitters, and the presence of abnormal proteinaceous deposits in neurons (neurofibrillary tangles), the cerebral vasculature, and the extracellular space (diffuse and neuritic plaques) (Bateman, RJ. , et al., Nat. Med. 72:856-861, 2006). The main components of diffuse and neuritic plaques are amyloid-beta peptides (A- beta). A-beta peptides are generated from the amyloid precursor protein (APP) by proteolytic cleavage that results in multiple A-beta species, ranging in length from 38-43 amino acids, with varying amino and carboxyl termini (Golde, T.E., et al., "Biochemical Detection of Aβ Isoforms: Implications for Pathogenesis, Diagnosis, and Treatment of Alzheimer's Disease," Biochim. Biophys. Acta. 7502:172-187, 2000; Wolfe, M.S., and S.Y. Guenette, "APP at a Glance," /. Cell ScL 720:3157-3161, 2007). In humans, A-beta peptides are present in the cerebrospinal fluid and the peripheral blood. Neuritic plaques are composed of extracellular deposits of amyloid fibrils, of which A-beta42 and A-beta40 are the principal components, whereas diffuse plaques are composed primarily of A-beta42 (Selkoe, DJ., "Alzheimer's Disease: Genes, Proteins, and Therapy," Physiol. Rev. 87:741-766, 2001). The accumulation of A-beta peptides in the brain has been hypothesized to cause the pathologic and behavioral manifestations of AD, including synaptic dysfunction and loss, neurofibrillary tangle formation, neuronal degeneration, and impaired memory (Selkoe, DJ., "Alzheimer's Disease: Genes, Proteins, and Therapy," Physiol. Rev. 87:741-766, 2001). A-beta also forms soluble oligomers that are different in structure from amyloid fibrils. The A-beta oligomers, also known as A-beta derived diffusible ligands (ADDLs), are toxic to neurons and rapidly inhibit long-term potentiation (LTP), a classic experimental paradigm for memory and synaptic plasticity (Lambert, M. P., et al., Proc. Natl. Acad. ScL 95:6448-6453, 1998; Walsh, D.M., et al., Nature 416:535-539, 2002; Wang, H.W., et al., Brain Res. 924:133-140, 2002). Further, A-beta oligomers have been implicated in the physical degeneration of synapses (Mucke, L., et al., J. Neurosci. 20:4050-4058, 2000) and in age-onset memory failure in hAPP transgenic mice (Morgan, D., et al., Nature 408:982-985, 2000; Dodart, J.C., et al., Nat. Neurosci. 5:452-457, 2002; Kotilinek, L.A., et al., /. Neurosci. 22:6331-6335, 2002). The soluble oligomers also accumulate in the frontal cortex of brains from AD patients (Gong, Y., et al, Proc. Natl. Acad. ScL USA 76»6»: 10417- 10422, 2003) and bind to the dendrites and synaptic terminals of human neurons from AD brains and cultured rat hippocampal neurons (Lacor, P.N., et al., /. Neurosci. 24:10191-10200, 2004). Further support for the role of A-beta oligomers in the pathology of AD is provided by studies of hAPP transgenic mice strains passively immunized with antibodies that bind A-beta. In these studies, peripheral administration of anti- A-beta monoclonal antibodies resulted in dramatic reversal of memory failure without a corresponding reduction in amyloid plaque pathology in the brain (Dodart, J. C, et al., Nat. Neurosci. 5:452-457, 2002; Kotilinek, L. A., et al., /. Neurosci. 22:6331-6335, 2002). Further, passive immunization of hAPP transgenic mice with monoclonal antibodies that selectively bind A-beta oligomers also improved learning and memory without reducing the amount of A-beta in the brain (Lee, E.B., et al., /. Biol. Chem. 281:4292-4299, 2006). Based on the above observations, among others, the current hypothesis for the memory loss associated with AD is that synaptic failure before neuronal death is responsible for early memory loss, and that neurotoxic, soluble A-beta oligomers, rather than amyloid fibrils in plaques, are responsible for synaptic failure (Klein, W.L., et al, Trends Neurosci 24:2X9-224, 2001; Hardy, J., and DJ. Selkoe, Science 297:353-356, 2002).

In addition to the reversal of memory impairment noted above, some studies have reported that chronic peripheral administration of monoclonal antibodies specific for A-beta reduces amyloid burden in the brains of hAPP transgenic mice (Bard et al., 2000; DeMattos et al., 2001). Peripherally administered anti- A- beta antibodies were detectable in the cerebrospinal fluid, indicating that the antibodies crossed the blood-brain barrier (Dodart et al., 2002; Bard et al., 2000). However, other studies have failed to show reduced amyloid pathology following long-term peripheral administration of A-beta monoclonal antibodies in hAPP transgenic mice (Lee, E.B., et al, 2006). Further, the amount of anti-A-beta antibodies that actually enter the brain from the peripheral circulation is reported to be low, representing less than 0.1% of the total antibody injected, which is inconsistent with a mass action mechanism for removal of A-beta from the brain (see Levites, Y., et al., FASEB J. 26>:E2002-E2014, 2006). Thus, the mechanism by which passive immunization with A-beta monoclonal antibodies alters amyloid deposition and reverses learning and memory failure remains unclear (Levites et al., 2006).

Another approach for reducing the level of amyloid beta in the brain has been intracranial or intracerebral injection of anti-A-beta antibodies. Intracranial injection of anti-A-beta antibodies has been reported to promote clearance of existing amyloid plaques in APP transgenic mice (Bacskai, B.J., et al., Nat. Med. 7:369-372, 2001). In contrast, intracerebral administration of antibodies that selectively recognize A-beta oligomers in a different strain of APP transgenic mice resulted in no reduction of A-beta amyloid burden (Lee et al., 2006). Further, another study suggests that intracerebral injection of anti-A-beta antibodies prevents amyloid deposition rather than clearance of pre-existing plaques (Tucker, S. M., et al., /. Neuropathol. Exp. Neurol. «57:30-40, 2008). The above methods utilize antibodies either systemically in the peripheral blood or locally within the cerebrospinal fluid in the brain. However, in these methods the antibodies are free to circulate in the blood and/or cerebrospinal fluid. Therefore, the antibodies may not inhibit the pathogenic properties of A-beta depending on the affinity and accessibility of the antibody to A-beta in different locations within the body and central nervous system. Thus, there is a need for compositions and methods that deplete A-beta in its various forms from bodily fluids independently of the source of A-beta or the accessibility of A-beta in its pathogenic state.

SUMMARY This summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This summary is not intended to identify key features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter. In one aspect, the invention provides a composition for depleting amyloid-beta from the endogenous bodily fluid of a subject, the composition comprising a biocompatible material that binds to amyloid-beta peptides present in endogenous bodily fluid, wherein the composition is oriented such that endogenous bodily fluid contacts a surface of the biocompatible material under unidirectional flow conditions, and wherein the composition depletes at least a portion of the amyloid-beta peptides present in endogenous bodily fluid.

In another aspect, the invention provides a medical device for depleting a target substance from endogenous bodily fluid of a living subject, the medical device comprising (a) a composition comprising a biocompatible material that binds to a target substance present in the endogenous bodily fluid of a living subject, and (b) a housing enclosing the composition, the housing comprising a first end with an inlet for receiving bodily fluid comprising the target substance from a living subject and a second end with an outlet for returning the bodily fluid depleted of at least a portion of the target substance to the living subject, wherein the composition is arranged within the housing such that the endogenous bodily fluid directly contacts the composition.

In another aspect, the invention provides a medical device for depleting at least a portion of amyloid-beta from endogenous cerebrospinal fluid in a living subject, the medical device comprising (a) a composition comprising a biocompatible material that binds to amyloid-beta present in endogenous CSF of a living subject; and (b) a housing enclosing the composition, the housing comprising a first end with an inlet for receiving endogenous CSF comprising amyloid-beta from a living subject and a second end with an outlet for returning the CSF depleted of at least a portion of amyloid-beta to the living subject, wherein the composition is configured within the housing such that the endogenous CSF directly contacts the composition.

In another aspect, the invention provides a method of depleting amyloid-beta peptides from endogenous cerebrospinal fluid of a living subject, the method comprising (a) contacting endogenous cerebrospinal fluid of a living subject with a composition comprising a biocompatible material that binds to amyloid-beta peptides present in cerebrospinal fluid; and (b) returning the cerebrospinal fluid depleted of at least a portion of amyloid-beta peptides to the living subject.

In another aspect, the invention provides a system for depleting a target substance from bodily fluid of a living subject, the system comprising (a) a medical device that includes (i) an inlet port for receiving bodily fluid from a living subject, (ii) a composition comprising a biocompatible material that binds to a target substance present in the bodily fluid of a living subject, (iii) an outlet port for returning the bodily fluid depleted of the target substance to the living subject; (b) a pump that is fluidically connected to the medical device of (a); and (c) a computer comprising a memory, an analog and digital interface, and a user interface for controlling fluid movement through the system.

DESCRIPTION OF THE DRAWINGS

The foregoing aspects and many of the attendant advantages of this invention will become more readily appreciated as the same become better understood by reference to the following detailed description, when taken in conjunction with the accompanying drawings, wherein:

FIGURE IA shows a surface view of a representative embodiment of the invention, illustrating a composition that binds target substances in bodily fluids; FIGURE IB shows the device of FIGURE IA implanted in the brain of a subject in sagittal section in accordance with an embodiment of the invention;

FIGURE 2A shows a longitudinal section of a representative device comprising a composition that binds target substances in bodily fluids in accordance with an embodiment of the invention; FIGURE 2B shows the device of FIGURE 2A implanted in the brain of a subject in sagittal section in accordance with an embodiment of the invention;

FIGURE 3A shows a longitudinal section of a representative device comprising a composition that binds target substances in bodily fluids, the device comprising a plurality of inlets located along the side of the housing in accordance with another embodiment of the invention;

FIGURE 3B shows the device of FIGURE 3A implanted in the brain of a subject in sagittal section in accordance with an embodiment of the invention;

FIGURE 4A shows a longitudinal section of a representative device comprising a composition that binds target substances in bodily fluids, the device comprising a reservoir with a deformable surface in accordance with another embodiment of the invention;

FIGURE 4B shows the device of FIGURE 4A implanted in the brain of a subject in sagittal section in accordance with another embodiment of the invention; FIGURE 5 is a schematic diagram of a representative extracorporeal system for removing a target substance from the cerebrospinal fluid or other bodily fluids;

FIGURE 6 is a schematic diagram illustrating cerebrospinal fluid flow through a representative implanted device in accordance with an embodiment of the invention; FIGURE 7 is a graph showing removal of A-beta 40 and A-beta 42 peptides from artificial CSF (ACSF) by a column containing immobilized monoclonal antibody 6E10, as described in Example 2;

FIGURE 8 is a graph showing binding of A-beta 40 to a GVS 0.2 micrometer positively charged filter, as described in Example 3; FIGURE 9A is a schematic diagram of a representative cerebrospinal fluid pheresis apparatus in accordance with another embodiment of the invention, as described in Example 4;

FIGURE 9B is a schematic diagram showing use of the apparatus shown in FIGURE 9 A, as described in Example 4; and FIGURE 9C is a schematic diagram showing use of another embodiment of the apparatus shown in FIGURE 9A, as described in Example 4.

DETAILED DESCRIPTION

This section presents a detailed description of the many different aspects and embodiments that are representative of the inventions disclosed herein. This description is by way of several exemplary illustrations, of varying detail and specificity. Other features and advantages of these embodiments are apparent from the additional descriptions provided herein. The description illustrates different components and methodology useful in practicing various embodiments of the invention. The examples are not intended to limit the claimed invention. Based on the present disclosure the ordinary skilled artisan can identify and employ other components and methodology useful for practicing the present invention.

I. DEFINITIONS

Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs. Unless otherwise defined, all singular terms include the plural.

As used herein, the term "central nervous system" means the brain and spinal cord of a subject. The term central nervous system also includes the spaces that contain cerebrospinal fluid, such as the intrathecal and subarachnoid spaces of the brain and spinal cord, the ventricles and cisternea of the brain, and the spinal canal.

As used herein, the term "amyloid-beta" is interchangeable with the terms "amyloid-beta peptide," "A-beta," and "Aβ," and comprises a peptide fragment of the amyloid precursor protein (APP; Genbank Accession No. P05067), as described in Selkoe, D. J., "Alzheimer's Disease: Genes, Proteins, and Therapy," Physiol. Rev. 87:741-766, 2001, which is hereby incorporated by reference herein. The term A-beta includes naturally occurring sequences of 38-43 amino acids (A-beta [sub]x, where x is 38 to 43) derived from the carboxy-terminal region of APP, such as A-beta 1-40 (D AEFRHDSGY EVHHQKLVFF AED VGSNKG A IIGLMVGGVV, SEQ ID NO: 1) and A-beta 1-42 (D AEFRHDSGY EVHHQKLVFF AED VGSNKGA IIGLMVGGVV IA, SEQ ID NO:2), and amino -terminal truncations of A-beta 1-40 (A-beta x-40) and A-beta 1-42 (A-beta x-42). The term A-beta also includes naturally occurring mutations and/or variants associated with Alzheimer's disease, for example, missense mutations at positions 692, 693, and 694 of the APP 770 amino acid isoform, as described in Selkoe (2001) and Marks, N., and MJ. Berg, "Neurosecretases Provide Strategies to Treat Sporadic and Familial Alzheimer Disorders," N euro chemistry Int'l 52:184-215 (2008), which is hereby incorporated by reference herein. The term A-beta further includes peptides that are at least 90% identical (such as at least 95%, 98%, 99%) to A-beta 1-40 (SEQ ID NO:1) or 90% identical (such as at least 95%, 98%, 99%) to A-beta 1-42 (SEQ ID NO:2). A-beta also includes monomeric and oligomeric forms of the peptide, as described in International PCT Publication WO 2006/055178, which is hereby incorporated by reference herein.

As used herein, the term "endogenous bodily fluid" includes any fluid that is naturally produced by and may be isolated from a living subject, including without limitation, natural fluids such as blood, plasma, lymph, urine, peritoneal fluid, and cerebrospinal fluid (CSF). The term "bodily fluid" includes endogenous bodily fluids and fluids produced as the result of medical procedures, such as fluids produced by dialysis, apheresis, plasmapheresis, and CSF-pheresis. As used herein, the term "endogenous cerebrospinal fluid" means CSF that is naturally produced in the central nervous system of a subject, including CSF removed from a living subject prior to contact with a composition of the invention. As used herein, the term "biocompatible material" refers to a material that is intended to come into contact with bodily fluids and/or for implantation in the body of a living subject, such as a human or an animal. The term "sterilizable biocompatible material" refers to a biocompatible material that can be sterilized using sterilization methods known in the art. Non-limiting examples of biocompatible materials include those listed as USP Class VI biocompatible materials.

As used herein, the terms "immunoabsorption" and "immunoadsorption" are used interchangeably and refer to the process of using an immunosorbent to selectively bind, purify, or isolate a substance. As used herein, the term immunosorbent refers to a molecule that binds with high affinity, specificity, and/or selectivity to a particular antigen, and includes, but is not limited to, an antibody or fragment thereof that binds to a specific antigen present in solution or a fluid.

As use herein, the term "intrinsic surface chemistry" means those natural molecular interactions which result from the manufacture, cleaning or sterilization of a material. As used herein, the term "reactive group" means a chemical compound or molecule that has the property of binding a target substance such as amyloid-beta peptides. II. ASPECTS AND EMBODIMENTS OF THE INVENTION

Generally described, the present invention provides compositions, devices, systems, and methods for depleting target substances from the bodily fluids of a living subject and returning the bodily fluid depleted of the target substance to the living subject. The compositions, devices, systems, and methods utilize a sterilizable biocompatible material capable of depleting target substances from the bodily fluids of a living subject. As used herein, the term "depleting" includes capturing, binding, sequestering, reducing, decreasing, degrading, and/or retaining a target substance or substances, such that at least a portion of the target substance is removed from the fluid. As used herein, the term "selectively depleting" refers to depleting a target substance, wherein the biological properties and amounts of other components of the bodily fluid are not substantially depleted or otherwise substantially altered. For example, the biocompatible material may deplete less than 5%, 10%, 15%, 20%, 25%, 30%, 40%, or 50% of non-target substances from the bodily fluid. The biocompatible material may have a plurality of binding agents attached thereto, wherein the binding agents are capable of selectively binding to a target substance present in the bodily fluid of a subject. Binding agents that selectively bind to a target substance may be used to selectively deplete a target substance from the bodily fluid of a subject. The methods of the invention utilize the composition, devices, and systems of the invention to deplete target substances from the bodily fluids of a living subject. In some embodiments, the invention also provides a sensor to measure physiological parameters of the bodily fluid and/or the binding capacity of the fluid contacting surface. In some embodiments, the compositions and devices of the invention are surgically implanted in the body of a living subject. In some embodiments, the compositions, devices, and systems of the invention are located outside the body of a living subject. In some embodiments, the composition of the invention binds and depletes amyloid-beta in its various forms from bodily fluids, such as cerebrospinal fluid or peripheral blood, thereby removing A-beta regardless of its source.

THE USE OF A COMPOSITION TO REMOVE TARGET SUBSTANCES FROM BODILY FLUIDS In one aspect, the invention provides a composition that may be used to deplete target substances from bodily fluids of a living subject. In one embodiment, the composition comprises a biocompatible material that binds to amyloid-beta peptides present in endogenous bodily fluid, wherein the composition is oriented such that endogenous bodily fluid contacts a surface of the biocompatible material under unidirectional flow conditions and wherein the composition depletes at least a portion of the amyloid-beta peptides present in endogenous bodily fluid.

In some embodiments, the biocompatible material comprises reactive groups that bind to amyloid-beta peptides. In another embodiment, the biocompatible material further comprises a plurality of binding agents that each selectively binds to amyloid-beta peptides. In one embodiment, the bodily fluid is cerebrospinal fluid.

Each of the components of the invention will now be described in more detail below, wherein like components have like reference numbers.

FIGURE IA illustrates the components of an exemplary composition 10 according to one embodiment of the present invention. The composition comprises a sterilizable biocompatible material having a fluid contacting surface 20. The fluid contacting surface may further comprise a plurality of binding agents 30. It is understood that while only a limited number of binding agents are illustrated, the fluid contacting surface may include any number of binding agents. For example, the number of binding agents present on the fluid contacting surface may comprise tens, hundreds, thousands, millions, or billions of binding agents attached to the fluid contacting surface. Further, the binding agents 30 may substantially cover the entire fluid contacting surface, or only portions thereof. The composition may optionally have a sensor 40 attached thereto that measures relevant physiological parameters of the fluid, such as temperature, pH, and osmolarity. The sensor 40 may also measure the binding capacity of the fluid contacting surface 20 or the binding agents 30. In some embodiments, the sensor 40 is part of a device or system that comprises a composition of the invention and the sensor 40 may be located upstream or downstream of the composition relative to the fluid flow. In operation, the bodily fluid 50 of a subject that contains a target substance 52 contacts the fluid contacting surface of the biocompatible material 20. The target substance 52 selectively binds to the fluid contacting surface 20 and/or to the binding agents 30 attached thereto, resulting in bodily fluid 60 that is partially or completely depleted of the target substance 52. In some embodiments, the bodily fluid also contacts the sensor 40.

In operation, the composition 10 of the invention may be located external to the body of a subject or implanted in a suitable location inside the body of a subject.

FIGURE IB illustrates a representative example of one embodiment of the composition 10 of FIGURE IA implanted in the brain of a human subject. The composition 10 is surgically implanted in the subarachnoid space 510 located between the sagittal sinus 530 and the cerebral cortex 540. The natural flow of the CSF 500 brings the CSF into contact with the composition 10, resulting in depletion of amyloid beta from the CSF. In another embodiment, the composition 10 is surgically implanted in any suitable compartment of the ventricular-cisternal system of the central nervous system (CNS) that allows the CSF to contact the composition. In one embodiment, the composition is surgically implanted such that CSF from the intrathecal space of the spinal cord contacts the composition.

In some embodiments, the fluid contacting surface 20 of the composition 10 is functionalized to deplete at least a portion of a target substance present in bodily fluid of a living subject. For example, in one embodiment, the fluid contacting surface 20 comprises a polymer that binds a target substance present in bodily fluid of a living subject. In some embodiments, the polymer binds amyloid-beta peptides. Representative examples of polymer surfaces that bind amyloid-beta peptides include hydrophobic surfaces comprised of polystyrene and polysiloxane-dodecanoic acid complexes, and positively charged hydrophilic surfaces comprised of poly(allyamine hydrochloride) (PAH) as described in Rocha, S., et al., "Adsorption of Amyloid β-Peptide at Polymer Surfaces: A Neutron Reflective Study," Chem. Phys. Chem. 6:2521 -253 A, 2005, which is hereby incorporated by reference herein.

In some embodiments, the fluid contacting surface 20 comprises the interior surface of tubing. In one embodiment, the tubing is comprised of polyvinyl diflouride, nylon, polytetrafluoroethylene (PTFE), polypropylene, cellulose acetate, nitrocellulose, silica, polystyrene, polysulphone, polyethersulfone, polyethylene, cuprophane, various silicones, poly (2-hydroxyethyl methacrylate) (pHEMA) or any of a number of commercially- available hydrogels, blends of the foregoing, or the foregoing derivatized with antibodies specific to targets of interest.

In some embodiments, the fluid-contacting surface 20 is the surface of the biocompatible material. In one embodiment, the biocompatible material is a filter or membrane. Exemplary membranes include, but are not limited to, polyvinyl diflouride, nylon, polytetrafluoroethylene (PTFE), polypropylene, cellulose acetate, nitrocellulose, silica (fibers or filters), polystyrene, polyethylene, various silicones, polysulphone, polyethersulfone (PES), cuprophane, poly (2-hydroxyethyl methacrylate) (pHEMA), or any of a number of commercially-available hydrogels, blends of the foregoing or the foregoing derivatized with antibodies specific to targets of interest. The filter or membrane can have various pore sizes, including without limitation the range of 0.2 to 1.0 micrometers. The depletion of amyloid-beta by exemplary filter materials is described in Example 1.

In general, the filter or membrane may be formed to provide a tortuous, low-restriction, low-resistance flow path that presents a high- surface area means to remove targeted molecules. The membrane material can be selected to be compatible with the body fluid of interest, and, in some embodiments, comprises a surface that is chemically modified to present — for example, hydroxyl groups, succinimides, or amines to facilitate antibody binding to the membrane. The filter or membrane can have any suitable physical dimensions for incorporation into a medical device of the invention. In one embodiment, the filter or membrane is porous. In one embodiment, the membrane is in sheet form having two opposing sides, a first side and a second side that is substantially coplanar with the first side, with a central portion therebetween. In another embodiment, the sterilizable biocompatible material is formed into a tube. In some embodiments the tube may be supplemented with filters, membranes, or beads, as described below.

In another embodiment, the biocompatible material is in the form of beads, for example, agarose, sepharose, cellulose, or protein A beads. In one embodiment, the beads are cyanogen-bromide (CNBr) activated Sepharose beads (Amersham Biosciences). In another embodiment, the beads are paramagnetic beads, such as Dynabeads® (Invitrogen). The beads may be functionalized to facilitate binding of antibodies. For example, the beads may be coupled to streptavidin in order to bind biotinylated antibodies. The beads may be porous to increase the surface area available for binding. In other embodiments, the biocompatible material may be a porous ceramic or silica material. In some embodiments, the biocompatible material comprises microspheres. Microspheres may be made of any number of materials, including but not limited to glass, polystyrene, ceramic, silica, magnetic, paramagnetic (iron oxide), or super-paramagnetic materials. The beads and microspheres may be coated or functionalized with materials that facilitate attachment of the binding agent to the solid support, such as streptavidin, biotin, dextran, lectins, amine, and carboxyl groups. In another embodiment, the biocompatible material comprises nanoparticles functionalized with a binding agent. A representative example of this embodiment includes gold nanoparticles or magnetic microparticles functionalized with antibodies that bind A-beta, as described in Georganopoulou, D. G., et al., "Nanoparticle-Based Detection in Cerebral Spinal Fluid of a Soluble Pathogenic Bio marker for Alzheimer's Disease," Proc. Natl. Acad. ScL U.S.A. 702:2273-2276, 2005.

In some embodiments, the biocompatible material includes those listed and approved under U.S. Pharmacopeia USP Class VI, including nylon, silicone, and acrylic. In some embodiments, silane functionalized glass, quartz or silica is selectively activated to bind target molecules of interest. In some embodiments, the biocompatible material comprises silanes or silicones. For example, silanes can be used to selectively bind analytes of interest by altering hydrophobic or hydrophilic surface properties. Referring again to FIGURE IA, in some embodiments of the composition 10, the fluid contacting surface 20 comprises a plurality of binding agents 30 that selectively bind a target substance. In some embodiments, the binding agents 30 are antibodies, antibody fragments, diabodies, aptamers, enzymes, and peptides. In some embodiments, the plurality of binding agents 30 is substantially identical to each other-for example, a single species of monoclonal antibody that binds to a target substance. In some embodiments, the plurality of binding agents 30 includes at least two distinct binding agents, wherein the at least two distinct binding agents are different from each other-for example, two different species of monoclonal antibodies that each bind to the same target substance or that bind to different target substances, or a monoclonal antibody and a polyclonal antibody. In some embodiments, the plurality of at least two distinct binding agents is located at different regions of the fluid contacting surface 20, whereas in some embodiments, the plurality of at least two distinct binding agents is partially or completely interspersed or commingled on the surface.

In one embodiment, the binding agents 30 are attached to the fluid contacting surface 20. As used herein, the term attached includes the terms "bound" and "immobilized." Binding agents 30 may be attached to the fluid contacting surface 20 by any means known in the art. For example, the fluid contacting surface 20 may be functionalized with streptavidin, biotin, dextran, lectins, amine, carboxyl and hydro xyl groups, and succinimides to facilitate antibody binding to the surface.

In some embodiments, the binding agent 30 is an antibody that binds to a target substance such as A-beta. As used herein, the term antibody includes, but is not limited to, polyclonal or monoclonal antibodies, and chimeric, human (e.g. isolated from B cells), humanized, neutralizing, bispecific, catalytic, antigen binding fragment (Fab), single chain variable fragment (scFv), or single chain antibodies thereof. In one embodiment, an antibody of the instant invention is monoclonal. For production of antibodies, various hosts including goats, rabbits, chickens, rats, mice, humans, and others can be immunized by injection with the peptide of interest. Methods for producing antibodies are well-known in the art. See, e.g., Kohler and Milstein (Nature 25(5:495-497, 1975) and Harlow and Lane (Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory, New York, 1988).

In some embodiments, the binding agent 30 selectively binds amyloid-beta peptides and variants thereof such as A-beta 1-40 (SEQ ID NO: 1) or A-beta 1-42 (SEQ ID NO:2). In one embodiment, the binding agent 30 is an antibody that binds to A-beta peptides, including A-beta 1-40 and/or A-beta 1-42. In some embodiments, the antibody binds monomers of A-beta peptides. In another embodiment, the antibody binds oligomers of A-beta peptides. For example, exemplary humanized monoclonal antibodies that recognize A-beta-derived diffusible ligands (ADDLs), as described in PCT Publication WO 2006/055178, which is hereby incorporated by reference herein, may be used as binding agents, in accordance with various embodiments of the invention. Other examples of antibodies that specifically bind A-beta peptides include the humanized anti-A-beta monoclonal antibody Bapineuzumab (AAB-OOl) (Elan Corporation), the anti-A-beta 40 specific and anti-A-beta 42 specific monoclonal antibodies described in Levites, Y., et al. ("Anti-Abeta42 and Anti-Abeta40-Specific niAbs Attenuate Amyloid Deposition in an Alzheimer Disease Mouse Model," J. Clin. Invest. 116: 193-201, 2006) and the monoclonal antibody NAB61 that preferentially recognizes a conformational epitope present in oligomeric forms of A-beta as described in Lee, E. B., et al. ("Targeting Amyloid-Beta Peptide (A-beta) Oligomers by Passive Immunization With a Conformation-Selective Monoclonal Antibody Improves Learning and Memory in A-Beta Precursor Protein (APP) Transgenic Mice," J. Biol. Chem. 281:4292-4299, 2006). In some embodiments, the binding agent 30 is a diabody that binds to A-beta peptides. A diabody is an engineered antibody construct comprising the heavy and light chain binding domains joined by a linker that operably links the heavy and light chains on the same polypeptide chain, thereby preserving the binding function (see, Holliger, P., et al, Proc. Natl. Acad. ScL USA 90:6444, 1993; Poljak, R.J., Structure 2:1121-1123, 1994). Diabodies (dimeric antibody fragments) are bivalent and bispecific. Any art-recognized method to generate diabodies can be used. Suitable methods are described by Holliger, P., et al. (1993); Poljak, RJ. (1994); Zhu, Z., et al., Biotechnology 74:192-196, 1996; and U.S. Patent No. 6,492,123, which are incorporated herein by reference. In some embodiments, the binding agent 30 comprises fragments of an isolated antibody, wherein the fragment specifically binds to A-beta. Fragments are intended to include Fab fragments, F(ab')2 fragments, F(ab') fragments, bispecific scFv fragments, Fd fragments, and fragments produced by a Fab expression library, as well as peptide aptamers. F(ab')2 fragments are produced by pepsin digestion of the antibody, whereas Fab fragments are generated by reducing the disulfide bridges of the F(ab')2 fragments. Alternatively, Fab expression libraries can be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity (see Huse, W.D., et al., Science 254:1275-1281, 1989). In some embodiments, the antibody fragments comprise neutralizing antibodies that retain the variable region binding site thereof. Exemplary are F(ab^r)₂ fragments, F(ab') fragments, and Fab fragments. See, generally, Immunology: Basic Processes, J. Bellanti (ed.) 2^nd ed., pp. 95-97, 1985.

In another embodiment, the binding agent 30 is a nucleic acid or peptide aptamer that specifically binds to A-beta. Aptamer refers to a synthetic recognition molecule whose design is based on the structure of the target molecule. Nucleic acid aptamers consist of variable length chains of nucleic acids selected for binding affinity and specificity to a target. Peptide aptamers consist of a variable peptide loop attached at both ends to a protein scaffold selected for binding affinity and specificity to a target. This double structural constraint greatly increases the binding affinity of the peptide aptamer to levels comparable to that of an antibody (for example, nanomolar binding affinities).

In some embodiments, the binding agent 30 of the composition is an enzyme that binds to a target substance. In one embodiment, a molecule with enzymatic activity is attached to the fluid-contacting surface 20 of a biocompatible material. In operation, the enzyme depletes a target substance from the bodily fluid of a subject by binding to the substance followed by enzymatic cleavage and degradation of the substance. As used is this embodiment, it is understood that the term "binding" refers to contact between the enzyme and a target substance sufficient to allow for cleavage or degradation of the substance. In one embodiment, the enzyme is the peptidase insulysin (insulin degrading enzyme: EC 3.4.24.56), a zinc metalloprotease that degrades amyloid-beta peptides 1-40 and 1-42 (Mukherjee, A., et al., "Insulysin Hydro lyzes Amyloid-Beta Peptide to Products That Are Neither Neurotoxic Nor Deposit on Amyloid Plaques," /. Neuroscience 20:8745-49, 2000). In another embodiment, the enzyme is the zinc metallopeptidase neprilysin (neutral endopeptidase: EC 3.4.24.11). Neprilysin degrades A-beta and prevents amyloid plaque formation in vivo (Leissring, M. A., et al., "Enhanced Proteolysis of Beta-Amyloid in APP Transgenic Mice Prevents Plaque Formation, Secondary Pathology, and Premature Death," Neuron 40:1087-93, 2003); mutations in the neprilysin gene are associated with familial forms of Alzheimer's disease (Helisalmi, S., et al., "Polymorphisms in Neprilysin Gene Affect the Risk of Alzheimer's Disease in Finnish Patients," J. Neurology Neurosurgery and Psychiatry 75:1746-48, 2004); and neprilysin-deficient mice exhibit elevated brain A-beta concentrations, amyloid-like deposits, and neuronal degeneration (Madani, R., et al., "Lack of Neprilysin Suffices to Generate Murine Amyloid-Like Deposits in the Brain and Behavioral Deficit In Vivo " J. Neurosci. Res. 84:1871-78, 2006).

In some embodiments, the enzyme may be any enzyme recognized in the art that degrades a harmful, toxic, or pathogenic substance present in bodily fluids. In some embodiments, the binding agent 30 is a nucleic acid, such as DNA, RNA, or PNA, which selectively binds the target substance 52 of interest. Oligonucleotides that selectively bind specific proteins are well known in the art. In other embodiments, the binding agent is any organic molecule that binds the target substance 52 of interest.

In some embodiments, the fluid contacting surface 20 may comprise one or more polymers having antibodies attached thereto. For example, the fluid contacting surface may comprise polymers of PTFE and polypropylene, wherein the PTFE and/or the polypropylene have antibodies that selectively bind amyloid-beta attached thereto.

In some embodiments, the composition 10 comprises a sensor 40 that measures various physiological parameters of the bodily fluid of a subject including, but not limited to, temperature, pH, and osmolarity. In some embodiments, the sensor 40 measures the binding capacity of the fluid-contacting surface 20 and/or the binding capacity of the binding agents 30 attached to the surface. In one embodiment, a representative example of which is illustrated in FIGURE IA, the sensor 40 is in contact with the fluid contacting surface 20 of the composition. In some embodiments, the sensor 40 may be partially or completely covered with immobilized binding agents 30. In one embodiment, the sensor 40 is an optical fiber coated with antibodies that detects specific binding of the substance through a change in optical properties near the fiber (e.g., the dialectric constant). In another embodiment, the sensor 40 is a piezoelectric device coated with antibodies that detects specific binding of the substance through the change in mass on the piezoelectric crystal (e.g., a change in the resonant frequency). In the above embodiments, the sensor 40 may be coupled to a battery-driven, radio-frequency transmitter for communication with a receiver located outside the body of the subject. In some embodiments, the sensor 40 is activated by an external source— for example, inductively-coupled current that would activate the sensor to transmit data to a user. In some embodiments, the data collected by the sensor is analyzed by radiated stimulation, for example, by PET scan, MRI, light, and ultrasound.

In some embodiments, the sensor 40 may comprise a microelectromechanical system (MEMS) or nanoelectromechanical system (NEMS) that operates in the static or dynamic mode. Static mode sensors comprise structures functionalized on one side for binding of a specific analyte, whereby analyte binding results in unbalanced surface stresses that cause the structure to deflect, thereby indicating detection of binding. Dynamic mode sensors comprise resonant sensors that are excited at natural resonant frequencies, whereby analyte binding causes a shift in resonant frequency that indicates detection. An exemplary example of a nanomechanical resonant sensor that detects prion proteins is described in Varshney, M., et al., "Prion Protein Detection Using Nanomechanical Resonator Arrays and Secondary Mass Labeling," Anal. Chem. 80:2141-2148, 2008. In some embodiments, the target substance 52 to be depleted from bodily fluid is a toxic or pathogenic substance that has a deleterious effect on the brain or central nervous system of a subject. In one embodiment, the target substance 52 is monomeric, oligomeric, or aggregated amyloid- beta (A-beta). Amyloid-beta refers to peptides that are produced from the amyloid precursor protein (APP), such as A-beta 1-40 (SEQ ID NO: 1) or A-beta 1-42 (SEQ ID NO:2) peptides and naturally-occurring mutations and/or variants thereof. In another embodiment, the target substance 52 is a normal or infectious prion protein that induces Creutzfeldt-Jakob disease (CJD) in humans, scrapie in sheep, and bovine spongiform encephalopathy (BSE) in cattle. In another embodiment, the target substance 52 is tau, a component of neurofibrillary tangles found in brain tissue that is elevated in the CSF from Alzheimer's disease patients. (Arai et al., "Tau in Cerebrospinal Fluid: A Potential Diagnostic Marker," Ann. Neurology 38:649-652, 1995). In another embodiment, the target substance 52 is beta-2 microglobulin, a protein that reaches high levels in the CSF of Alzheimer's disease patients. (Martinez et al., "Relationship of Inter leukin-1 Beta and Beta2-Microglobulin With Neuropeptides in Cerebrospinal Fluid of Patients With Dementia of the Alzheimer Type," /. Neuroimmunology 48:235-240, 1993).

In some embodiments, the composition depletes at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the amyloid-beta peptides present in the endogenous CSF that comes into contact with the composition. In some embodiments, the composition depletes at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the soluble amyloid-beta peptides produced in endogenous CSF of a subject per day. In another embodiment, the composition depletes at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the total amyloid-beta peptides present in the endogenous CSF of a subject.

In some embodiments, the composition 10 depletes the target substance without substantially removing or adversely affecting the biological properties of other components present in the bodily fluid of a subject. For example, in those embodiments wherein the composition 10 depletes amyloid beta from the CSF, the concentration of other non-target proteins and small molecules present in CSF, such as glucose, are substantially unaltered.

In some embodiments, the bodily fluid that is contacted with the composition 10 of the invention is at least one of blood, plasma, lymph, or cerebrospinal fluid. In one embodiment, the cerebrospinal fluid that contacts the composition is naturally occurring, endogenous cerebrospinal fluid. The term endogenous is understood to include CSF that has been removed from a subject but not substantially altered prior to contacting the CSF with a composition of the invention. In another embodiment, the CSF has not been substantially modified, diluted, dialyzed, filtered, or otherwise altered in its biological or biochemical properties prior to contacting the composition of the invention.

In some embodiments, the target substance to be depleted from bodily fluid is any molecule that has an adverse, harmful, or pathogenic affect on the physiology or metabolism of a subject. For example, metabolic pathway intermediates that are harmful or cause disease when they accumulate to high levels may be depleted by the composition of the invention.

In some embodiments, the target substance to be depleted from endogenous bodily fluids consists of foreign substances such as bacteria or viruses. In another embodiment, the substance to be removed from endogenous bodily fluids consists of cells produced by the subject. For example, in some embodiments, the binding agent binds cells of the immune system such as T and B lymphocytes, macrophages, monocytes, and microglia that are present in the CSF during inflammation of the central nervous system. In some embodiments, the composition binds inflammatory mediators such as complement, cytokines, chemokines, and other molecules associated with an inflammation response that is present in the CSF.

In some embodiments, the target substance to be depleted is one or more proteins from blood that accumulate as the result of a medical procedure, such as beta2- micro globulin that accumulates in the plasma of patients with kidney disease undergoing dialysis (dialysis-related amyloidosis).

THE USE OF A MEDICAL DEVICE TO DEPLETE TARGET SUBSTANCES FROM THE BODILY FLUIDS OF A SUBJECT In another aspect, the invention provides a medical device that may be used to deplete target substances from the endogenous bodily fluids of a living subject. In one embodiment, the device comprises (a) a composition comprising a biocompatible material that binds to a target substance present in the endogenous bodily fluid of a living subject, and (b) a housing enclosing the composition, the housing consisting of a first end with an inlet for receiving bodily fluid comprising the target substance from a living subject and a second end with an outlet for returning the bodily fluid depleted of at least a portion of the target substance to the living subject, wherein the composition is arranged within the housing such that the bodily fluid directly contacts the composition.

In another embodiment, the device comprises (a) a composition comprising a biocompatible material that binds to amyloid- beta present in endogenous CSF of a living subject; and (b) a housing enclosing the composition, the housing consisting of a first end with an inlet for receiving endogenous CSF comprising amyloid-beta from a living subject and a second end with an outlet for returning the CSF depleted of at least a portion of amyloid-beta to the living subject, wherein the composition is arranged within the housing such that the endogenous CSF directly contacts the composition.

In one embodiment, the composition of the device comprises a biocompatible material having a fluid contacting surface that binds to amyloid-beta peptides present in endogenous cerebrospinal fluid of a living subject. In another embodiment, the composition of the device comprises a biocompatible material comprising a plurality of binding agents attached thereto that each selectively binds to amyloid-beta present in endogenous CSF of a living subject.

FIGURE 2A shows a longitudinal section of a representative example of one embodiment of a device of the invention. The device 100 comprises a composition comprising a biocompatible material having a fluid contacting surface 120. The fluid contacting surface may have a plurality of binding agents 130 attached thereto. The composition is encased in a housing 160 having an inlet 162 and an outlet 164. A check valve 110 may be provided to ensure unidirectional flow of bodily fluid through the housing 160. As used herein, the term unidirectional flow refers to the net or bulk flow of bodily fluid in substantially one direction through the device, such that the bodily fluid enters one end of the device and exits the other end of the device after at least a portion of the bodily fluid comes into contact with the fluid contacting surface 120. The device 100 is optionally provided with a sensor 140 for measuring physiological parameters of the bodily fluid and/or the binding capacity of the fluid contacting surface 120 or the binding agents 130.

In some embodiments, the housing 160 comprises a medical grade sterilizable biocompatible material, for example, implant grade silicone (USP Class VI), a non-reactive polymer, or a biocompatible metal or alloy. Examples of suitable metals include titanium and alloys thereof, tantalum, and stainless steel. Suitable polymers include, but are not limited to, acrylonitrile polymers such as acrylonitrile-butadiene-styrene polymer and the like; halogenated polymers such as polytetrafluoroethylene, polyurethane, polychlorotrifluoroethylene, copolymer tetrafiuoroethylene and hexafluoropropylene; polyethylene vinylacetate (EVA), polyimide; polysulfone; polycarbonate; polyethylene; polypropylene; polyvinylchloride-acrylic copolymer; polycarbonate-acrylonitrile-butadiene-styrene; polystyrene; cellulosic polymers; and the like. Further exemplary polymers are described in The Handbook of Common Polymers, Scott and Roff, CRC Press, Cleveland Rubber Co., Cleveland, Ohio. In some embodiments, the housing 160 is coated with suitable polymers, including polyurethanes as described in U.S. Patent Publication No. 2007/0276504 Al (Sparer et al).

In operation, the bodily fluid 50 of a subject containing the target substance 52 to be depleted enters the device through the inlet 162 and contacts the fluid contacting surface 120. The fluid 60 depleted of the target substance 52 exits the device through the outlet 164, thereby returning to the subject. In the operation of some embodiments, the bodily fluid further contacts the plurality of binding agents 130 and/or the sensor 140 before exiting the device.

In some embodiments, the device 100 of the invention is implantable in the central nervous system of a subject. In one embodiment, the device of the invention may be implanted in the central nervous such that the natural physiological flow of the endogenous CSF results in the CSF coming into contact with the fluid contacting surface or the binding agents of the device. The average amount of CSF in an adult human is about 150 ml, and the human body produces about 500 ml of CSF every 24 hours. The CSF originates from the choroid plexus located in the paired lateral ventricles of the brain, flows into the single midline third ventricle, and then down into the fourth ventricle. The third and fourth ventricles also produce CSF from choroidal tissues. From the fourth ventricle, the CSF flows into the subarachnoid space and fills the basal cisterns beneath the brain. The majority of the CSF is transiently sequestered within the basal cisternae or within spaces surrounding the cerebral cortex and spinal cord. From the basal cisterns, the CSF flows down the spinal cord and up and over the superior surfaces of the cerebral hemispheres. The CSF exits the brain through the arachnoid granulations (arachnoid villi) into the sagittal sinus located beneath the skull, and thus returns to the venous blood flow. Some CSF is also taken up into veins around the spinal nerve roots and into the lymphatics of the nose. (See, Vanneste, J. A., et al., "Shunting Normal- Pressure Hydrocephalus: Do the Benefits Outweigh the Risks? A Multicenter Study and Literature Review," Neurology 42:54-59, 1992; Gleason, P.L., et al., "The Neurobiology of Normal Pressure Hydrocephalus," Neurosurg. CHn. North Am. 4:667-675, 1993).

In addition to the net flow of CSF described above, the local CSF flow is influenced by the heart beat such that there is a pulsatile (back- and- forth) movement of the CSF within the brain compartments as the heart contracts and relaxes. Therefore, in some embodiments, the device may be positioned to take advantage of the net CSF flow, whereas in other embodiments, the device may be positioned to take advantage of the local flow of the CSF.

FIGURE 2B illustrates one representative example of an embodiment of the device 100 shown in FIGURE 2A implanted in the brain of a human subject. The device 100 is implanted in the brain such that the inlet is in contact with the subarachnoid space 510 and the outlet is in contact with the lateral ventricle 520. The net flow of CSF is indicated by the arrows 500. In operation, the CSF enters the device inlet 162 from the subarachnoid space 510, contacts the composition of the invention, and exits the device through the outlet 164 into the lateral ventricle 520.

FIGURE 3A shows a longitudinal view of a representative example of another embodiment of a device of the invention. In this embodiment, the device 200 comprises a composition comprising a biocompatible material having a fluid contacting surface 220. The fluid contacting surface 220 may have a plurality of binding agents 230 attached thereto. The composition is encased in a housing 260 having an inlet 262 and an outlet 264. A check valve 210 may be provided to ensure unidirectional flow of bodily fluid through the housing. The housing 260 further comprises a plurality of inlets 206 located along the lateral sides of the housing. The device 200 is optionally provided with a sensor 240 for measuring physiological parameters of the bodily fluid and/or the binding capacity of the fluid contacting surface 220 and/or the binding agents 230. In operation, the bodily fluid 50 of a subject containing the target substance 52 to be depleted enters the device through the plurality of inlets 262, 206 and contacts the fluid contacting surface 220. The fluid depleted of the target substance 60 exits the device through the outlet 264, thereby returning to the subject. In the operation of some embodiments, the bodily fluid further contacts the plurality of binding agents 230 and/or the sensor 240 before exiting the device.

FIGURE 3B shows one representative example of an embodiment of the device 200 shown in FIGURE 3A implanted in the brain of a human subject. The device 200 is implanted in the brain such that the inlet 262 is in contact with the subarachnoid space 510 and the outlet 264 is in contact with the lateral ventricle 520. The net flow of CSF is indicated by the arrows 500. In operation, the CSF enters the device 200 through one or more inlets 206, 262, contacts the composition of the invention, and exits through the outlet 264 into the lateral ventricle 520.

In some embodiments, the device housing 260 does not contain a check valve at either end. Rather, the housing 260 is open at each end to allow the endogenous flow of the CSF to enter the device 200 from either end and make contact with the fluid contacting surface 220 and/or the plurality of binding agents 230 attached to the surface.

Referring now to FIGURE 4A, in another embodiment, the device 300 comprises a composition further comprising a biocompatible material having a fluid contacting surface 320. The fluid contacting surface 320 may have a plurality of binding agents 330 attached thereto. The composition is encased in a housing 360 having an inlet 362 and an outlet 364. The device 300 further comprises a fluid reservoir 400 attached to the housing, the reservoir having a deformable top surface 402. The deformable top surface 402 comprises a flexible, resealable biocompatible material. In this embodiment, the fluid reservoir 400 is similar in design to an Ommaya reservoir, a medical device used to deliver medication directly to the CSF of a patient. The Ommaya reservoir comprises a dome- shaped reservoir made of a resealable material, such as plastic or silicone rubber, and a catheter attached to the underside of the reservoir. The fluid reservoir 400 is surgically placed under the scalp and the catheter is inserted into a ventricle of the brain. Medication is injected into the reservoir 400 and the reservoir 400 is pumped by manually pressing and releasing the deformable top surface 402, thereby forcing the medication into the ventricle. Referring again to FIGURE 4A, check valves 310, 312 are provided to ensure unidirectional flow of bodily fluid through the device 300. The device 300 is optionally provided with a sensor 340 for measuring physiological parameters of the bodily fluid and/or the binding capacity of the fluid contacting surface 320 or the binding agents 330.

In operation, the bodily fluid 50 of a subject containing the target substance 52 to be depleted enters the device through the inlet 362 and contacts the fluid contacting surface 320. In some embodiments, the bodily fluid further contacts the plurality of binding agents 330 and/or the sensor 340. In operation, the deformable top surface 402 is manually deformed and released to draw fluid into the device 300 through the inlet 362 and into the fluid reservoir 400 through the check valve 310. The fluid in the reservoir 400 exits the device 300 through the check valve 312 and the outlet 364, thereby returning fluid 60 depleted of the target substance 52 to the subject.

FIGURE 4B illustrates a representative example of one embodiment of the device shown in FIGURE 4A implanted in the brain of a subject. The device 300 is implanted such that the inlet 362 contacts the lateral ventricle 520 and the deformable top surface 402 of the reservoir 400 is located beneath the scalp of a subject. In operation, the deformable top surface 402 is depressed by manual pressure exerted on the scalp above the implanted device 300 and then released. The outlet 364 is located in the subarachnoid space 510 of the brain.

In the above embodiments, the outlet 364 of the implanted device may be placed in contact with any space in the central nervous system that is suitable for returning CSF to the subject. In some embodiments, the outlet 364 of the device 300 may be connected to the inlet of an implanted catheter, wherein the outlet of the catheter delivers the CSF to other locations in the body of a subject. In one embodiment, the catheter delivers the depleted CSF to another location within the central nervous system— for example, to the basal cisterns or the spinal cord. In another embodiment, the catheter delivers the depleted CSF to locations outside the central nervous system,— for example, to the peritoneal cavity. THE USE OF A SYSTEM TO DEPLETE -TARGET SUBSTANCES FROM THE CEREBROSPINAL FLUID OF A SUBJECT

In another aspect, the invention provides a system for depleting target substances from the bodily fluid of a living subject. In one embodiment of this aspect of the invention, a system is provided for depleting a target substance from endogenous bodily fluid of a living subject, the system comprising (a) a medical device that includes (i) an inlet port for receiving endogenous bodily fluid from a living subject, (ii) a composition comprising a biocompatible material that binds to a target substance present in the bodily fluid of a living subject, (iii) an outlet port for returning the bodily fluid depleted of at least a portion of the target substance to the living subject; (b) a pump that is fluidically connected to the medical device of (a); (c) at least one sensor for detecting the amount of the target substance in the bodily fluid; and (d) a computer comprising a memory, an analog and digital interface, and a user interface for controlling fluid movement through the system. In one embodiment, the bodily fluid is cerebrospinal fluid. In one embodiment, the target substance is amyloid-beta peptides. In other embodiments, the target substance is any pathological substance or compound that is associated with symptoms of dementia including, but not limited to, tau and prion proteins.

FIGURE 5 shows an exemplary embodiment of a system 1000 of the invention. The cerebrospinal fluid (CSF) of a subject is provided to the CSF apheresis system 1000 via a first spinal tap needle 1020 that is in fluid connection via tubing to a dual-channel fluid swivel 1100 and to an inlet pressure transducer 1200. The tubing may be any sterilizable biocompatible material known in the art, including Teflon® and silicone. Arrows indicate the flow of fluid through the system 1000. The amount of target substance present in the CSF may be monitored by a flow-through optical cell 1120 that is connected to a UV/visible light source 1140 and a fiber optic spectrometer 1160 that quantifies the absorption of light by the target substance at a given frequency. The inlet pressure transducer 1200 is connected by tubing to a peristaltic pump 1300. Peristaltic pumps are well known in the art and are commercially available. In one embodiment, the peristaltic pump may be a variable flow-rate pump, such as those available from Instech (Plymouth Meeting, Pennsylvania). The peristaltic pump 1300 is fluidically connected to a device 1400 that contains a composition 10 of the invention. The device 1400 is fluidically connected to the outlet pressure transducer 1500. Sample valves 1420, 1440 and bypass valves 1460, 1480 may be connected in line between the peristaltic pump 1300 and the device 1400 or between the device 1400 and the outlet pressure transducer 1500. The outlet pressure transducer 1500 is connected by tubing to the dual-channel fluid swivel 1100, which is connected by tubing to a second spinal tap needle 1040 for returning the CSF to the subject. In some embodiments, the CSF passes through a bubble trap 1720 located between the outlet pressure transducer 1500 and the fluid swivel 1100. The amount of target substance present in the CSF after flowing through the device 1400 and contacting the composition 10 may be quantified by a flow-through optical cell 1180 that is connected to a UV/visible light source 1140 and a fiber optic spectrometer 1160. The inlet 1200 and outlet 1500 pressure transducers transmit an inlet pressure signal 1220 or an outlet pressure signal 1520 to an Analog & Digital Interface 1600, which in turn is connected to a digital computer 1800. The Analog & Digital Interface 1600 transmits a pump speed control signal 1620 to the peristaltic pump 1300 to control the pump speed based on the input signals from the inlet and outlet pressure transducers. The sample valves 1420, 1440 collect samples of the CSF before 1420 and after 1440 the CSF has passed through the device 1400. The samples may be provided to a fraction, collector 1700 for analysis, for example, to determine the amount of target substance present in the CSF by ELISA or other immunoassays. The sample valves are controlled by pre-column 1640 and post-column 1660 control signals generated by the Analog and Digital Interface 1600, which automatically cause samples to be removed from the fluid for analysis or other downstream uses. The bypass valves 1460, 1480 are located immediately upstream 1460 and downstream 1480 of the device 1400 and allow the fluid to bypass the device 1400 when necessary. In one embodiment, the upstream bypass valve 1460 is a two position valve that allows fluid to bypass the device or flow through the device. In another embodiment, the downstream bypass valve 1480 is a four position valve that also functions as a sample valve, and allows (1) treated CSF to be returned to the subject, (2) bypassed fluid to be returned to the subject, (3) bypassed fluid to be sampled, or (4) treated fluid to be sampled.

In one embodiment, the system 1000 comprises a composition 10 having a fluid binding surface 20 that selectively binds to amyloid-beta peptides. In another embodiment, the system comprises a composition 10 having binding agents 30 that selectively bind to amyloid-beta peptides. In some embodiments, the system depletes at least about 10%, 20%, 30%, 40% 50%, 60%, 70%, 80%, 90%, 95% or 99% of the amyloid-beta peptides present in the CSF that comes into contact with the composition 10.

In operation, the flow rate of CSF through the device 1400 of the system is controlled by the peristaltic pump 1300. The flow rate may be adjusted as necessary to pass a desired amount of CSF through the device 1400 over a desired time period. Examples of flow rates are described in the method section below.

A METHOD FOR REMOVING SUBSTANCES FROM BODILY FLUIDS This aspect of the invention provides methods for depleting target substances from bodily fluids of a living subject by contacting the bodily fluid with a composition, device, or system of the invention.

In some embodiments, the invention provides methods for depleting amyloid-beta peptides from endogenous bodily fluids of a living subject. In one embodiment, the invention provides a method of depleting amyloid-beta peptides from endogenous cerebrospinal fluid of a living subject, comprising (a) contacting endogenous cerebrospinal fluid of a living subject with a composition comprising a biocompatible material that binds to amyloid-beta peptides present in cerebrospinal fluid; and (b) returning the cerebrospinal fluid depleted of at least a portion of amyloid-beta peptides to the living subject.

FIGURE 6 illustrates a representative example of one embodiment of the method of the invention. The endogenous CSF contacts an implanted device 2000 comprising a composition 2100 that comprises a plurality of binding agents that deplete amyloid-beta. The flow of CSF may be the endogenous flow that results from natural circulation of CSF in the central nervous system, or may be provided by an implanted pump 2200 located upstream or downstream of the device 2000. The CSF contacts the composition 2100 of the invention which results in depletion and removal of A-beta from the CSF. The composition 2100 is optionally removable or replaceable from the device 2000 when the composition becomes saturated with A-beta peptides. The CSF may contact an optional sensor 2300 that measures physiological and/or biochemical properties of the CSF, and may also measure the binding capacity of the composition 2100 to determine when the composition becomes saturated with A-beta. The CSF that is depleted of A-beta may be returned to the subarachnoid space and/or the ventricular-cisternal system of the central nervous system. In one embodiment, the method is a closed loop, in that CSF is continually coming into contact with a device of the invention, wherein CSF depleted of A- beta is constantly returned to the CSF pool.

In some embodiments, the method comprises contacting the CSF with a composition 10 of the invention exterior to the living subject. In one embodiment, the method comprises removing the CSF from a subject, contacting the CSF with a composition 10 of the invention, and returning the CSF depleted of the target substance to the subject. The CSF may be removed from the subject by any means recognized in the art, for example by spinal tap needles connected to catheters. In a representative example of one embodiment, the CSF is depleted of target substances and returned to the subject by the system illustrated in FIGURE 5.

In some embodiments of the method, the flow rate of CSF through an external composition, device, or system of the invention is adjusted as necessary to contact a desired amount of CSF with the composition during a desired time period. In some embodiments, 5 ml to 160 ml of CSF is contacted with the composition per treatment period. For example, in some embodiments, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150 or 160 ml of CSF is contacted with the composition per treatment period. Higher or lower amounts of CSF may be treated per treatment period if necessary or desired. The treatment period may be determined based on the medical requirements of each individual subject and may vary from minutes to hours for each treatment session. Treatment sessions may be daily, weekly, or monthly. In one embodiment, the flow rate of CSF that contacts the composition 10 ranges from about 5 mL/hour to about 160 mL/hour.

In some embodiments, the method comprises contacting the CSF with a composition 10 and/or device of the invention that is implanted within the living subject. In some embodiments, the method comprises implanting a composition 10 or device comprising the composition 10 in the CNS of the living subject in such a manner that the endogenous, physiological flow of the CSF results in the contacting of the fluid with the composition 10 and the returning of the CSF depleted of the target substance to the subject. Representative examples of these embodiments are illustrated in FIGURES 1-3. In another embodiment, the method comprises contacting the CSF with an implanted composition 10 of the invention by applying a force to a deformable portion of a device comprising the composition 10. A representative example of this embodiment is illustrated in FIGURES 4A and 4B. In another embodiment, the method comprises contacting the CSF of a subject with a device comprising the composition 10 that is implanted at locations outside of the CNS, for example, subcutaneously or in the peritoneal cavity of the subject. In one embodiment, the CSF is removed from the CNS, for example, by means of a spinal tap, and contacted with the device by means of a catheter. In one embodiment, the device comprising the composition 10 is implanted subcutaneously, for example, in the abdomen of a subject, to allow access to the device for replacing the composition when the binding capacity of the fluid contacting surface and/or binding agents becomes saturated with the target substance. In one embodiment, the flow of CSF through the implanted device is driven by endogenous physiological pressure. In another embodiment, flow through the implanted device is driven by a pump that is fluidically connected to and controls the rate of flow through the device. In some embodiments, the pump is implanted within the body of a subject. Examples of implantable pumps include diaphragm pumps, piston pumps, rotor pumps, peristaltic pumps, screw pumps, or any other suitable pump. Representative examples of implantable pumps include electrically driven piston pumps such as the MiniMed Technologies MIP series pump and peristaltic pumps such as the Medtronic SynchroMed® II infusion pump. An implantable pump of the invention may be fluidically connected to the device either upstream or downstream of the device, such that the pump is located between the CSF source and the device, or between the device and the location where CSF is returned to the body of the subject.

In some embodiments, the pump is located external to the body of a subject. Examples of external pumps include the MiniMed Paradigm® Brand of Insulin pumps (Medtronic, Inc.). In these embodiments, the externally located pump is connected to an implanted device comprising the composition 10 via a catheter that passes through the skin of the subject. The pump can be located upstream or downstream of the implanted device. After passing through the device and contacting the composition 10, the CSF is returned to the subject by dispensing the fluid into any bodily cavity suitable for reabsorption of CSF into the circulation— for example, the peritoneal cavity.

In another embodiment, the method comprises removing CSF from the central nervous system of a subject, contacting the CSF with an implanted device comprising the composition 10, passing the CSF through a catheter to a pump that is fluidically coupled to the device, the pump being located external to the body of the subject and returning the CSF to the subject. In this embodiment, the inlet of the implanted device is connected to a source of CSF and the outlet is connected to the inlet of an external pump via a first catheter that passes through an insertion site in the skin. The outlet of the pump is connected to an insertion site in the skin via a second catheter, the second catheter having a proximal end connected to the pump and a distal end disposed in the body of the subject. In operation, the external pump pulls the CSF through the device via the first catheter and discharges the depleted CSF via the second catheter into a body cavity suitable for reabsorption of CSF into the circulation— for example, the peritoneal cavity.

In some embodiments, the method comprises depleting amyloid-beta peptides from the CSF of a subject by contacting the CSF with a fluid contacting surface that selectively or no n- selectively binds A-beta. Examples of suitable fluid contacting surfaces in accordance with this embodiment include various polymers, as described above.

In some embodiments, the method comprises depleting amyloid-beta peptides from the CSF of a subject by immunoabsorption. In one embodiment, the immunoabsorbent is an antibody or fragment thereof. In one embodiment, the antibodies or fragments thereof bind to A-beta monomers. In another embodiment, the antibodies or fragments thereof bind to A-beta oligomers. For example, the antibody may bind with high affinity to multi-dimensional conformations of soluble A-beta oligomers, referred to as A-beta derived diffusible ligands or ADDLs, as described in PCT Publication WO 2006/055178, which is hereby incorporated by reference herein. Other examples include the humanized anti- A-beta monoclonal antibody Bapineuzumab (AAB-OOl) (Elan Corporation) and the anti- A-beta 40 specific and anti- A-beta 42 specific monoclonal antibodies described in Levites, Y., et al., "Anti-Abeta42 and Anti-Abeta40-Specific rriAbs Attenuate Amyloid Deposition in an Alzheimer Disease Mouse Model," J. Clin. Invest. 116: 193-201, 2006. In another embodiment, the immunoabsorbent is a diabody that specifically binds A-beta peptides. In another embodiment, the immunoabsorbent is an aptamer that specifically binds A-beta peptides.

In some embodiments, the method depletes at least about 10%, 20%, 30%, 40% 50%, 60%, 70%, 80%, 90%, 95% or 99% of the amyloid-beta peptides present in the endogenous CSF that comes into contact with the composition 10 of the invention. In some embodiments, the method depletes at least about 10%, 20%, 30%, 40% 50%, 60%, 70%, 80%, 90%, 95% or 99% of the amyloid-beta peptides produced by the CNS in a human subject per day. In another embodiment, the method depletes at least about 10%, 20%, 30%, 40% 50%, 60%, 70%, 80%, 90%, 95% or 99% of the total amyloid-beta peptides present in the CSF of a human subject.

To determine the amount of A-beta peptides removed from the CSF of a subject, the amount of A-beta protein present in the CNS can be quantified using the methods described in Bateman et al., "Human Amyloid-β Synthesis and Clearance Rates as Measured in Cerebrospinal Fluid In vivo " Nat. Med. 72:856-861, 2006; and Oe, T., et al., "Quantitative Analysis of Amyloid Beta Peptides in Cerebrospinal Fluid of Alzheimer's Disease Patients by Immuno affinity Purification and Stable Isotope Dilution Liquid Chromatography/Negative electrospray ionization tandem mass spectrometry," Rapid Commun. Mass Spectrom. 20:3723-35, 2006, which are hereby incorporated by reference herein. Alternatively, the removal of A-beta from CSF can be monitored using the nanoparticle-based detection method described in Georganopoulou, D. G., et al., "Nanoparticle-Based Detection in Cerebral Spinal Fluid of a Soluble Pathogenic Biomarker for Alzheimer's Disease," Proc. Natl. Acad. ScL 702:2273-2276, 2005. In another embodiment, the method comprises depleting prion proteins from the

CSF of a subject. Prion proteins are implicated in nerve-damaging diseases such as bovine spongiform encephalopathy (Mad Cow Disease) in cattle, scrapie in sheep, and Creutzfeldt- Jakob Disease in humans. In these diseases, the endogenous cellular prion protein is converted into a conformationally altered infectious form. Thus, in one embodiment, the method depletes endogenous and/or infectious prion proteins from the CSF of a subject.

Prion proteins may be depleted using antibodies that specifically bind to bovine, sheep and human prion proteins. Antibodies that specifically bind prion proteins are well known in the art. Examples of representative antibodies that bind bovine prion protein are described in Varshney, M., et al., "Prion Protein Detection Using Nano mechanical Resonator Arrays and Secondary Mass Labeling," Anal. Chem. 80:2141-2148, 2008. Examples of representative antibodies that bind human prion proteins are described in U.S. Patent No. 7,202,021 (Vey, M., et al.).

In some embodiments, the bodily fluid depleted of a target substance by the method is endogenous CSF of a subject. In one embodiment, the CSF has not been substantially modified, diluted, dialyzed, filtered, or otherwise altered in its biological properties prior to being depleted of the target substance by the method. In another embodiment, the method does not substantially remove, deplete, or alter the biological properties of other desirable components of the CSF.

In some embodiments, the method can be used to deplete inflammatory mediators from the CSF. For example, the method may be useful in treating a variety of inflammatory polyneuropathies such as Guillain-Barre Syndrome/Acute Inflammatory

Demyelinating Polyneuropathy, Chronic Inflammatory Demyelinating Polyneuropathy, cerebral lupus, multiple sclerosis, amyotrophic lateral sclerosis, and bacterial meningitis.

The method may be used to lower the concentrations of inflammatory mediators including inflammatory proteins/peptides such as tumor necrosis factor-alpha, interleukins, interferons, immunoglobulins, endotoxins, complement and activators of the complement cascade such as C3a and C5a and inflammatory cells in the CSF of patients with inflammatory diseases that affect the nervous system.

In another embodiment, the method is useful for removing disease agents from the CSF such as bacteria, viruses, and fungi. In this embodiment, the binding agent comprises an agent that binds to the disease causing agent, such as an antibody that binds to pathogenic bacteria, viruses, or fungi. In one embodiment, the method is useful for removing agents that cause meningitis, including bacterial, viral, and fungal meningitis, from the CSF.

In some embodiments, the method comprises contacting endogenous bodily fluids, such as blood, plasma, or peritoneal fluid, with a composition of the invention, whereby toxic, harmful, or pathogenic substances are depleted from the bodily fluid. EXAMPLE 1

This example shows the results of tests comparing the ability of different filter materials to deplete amyloid-beta from human CSF. Tables 1 and 2 show experimental results demonstrating the depletion of an exemplary A- beta peptide (A-beta 1-40, SEQ ID NO: 1) from human CSF by different materials, including polymers, nylon, and glass. Table 1 shows the percent decrease of A-beta and total protein after contacting the CSF sample with the filter material relative to untreated CSF. Table 2 shows the amount of A-beta and total protein remaining in the CSF sample after contacting the sample with the filter material. The CSF sample was contacted with each filter material up to 10 times. Binding of A-beta 1-40 to the biotin- labeled monoclonal antibody 6E10 (Catalog No. SIG-39340, Covance) conjugated to streptavidin-coupled beads (Dynabeads®, Invitrogen) was used as a positive control. Table 1. Depletion of A-beta 1-40 and

Total Protein in Human CSF by Various Filter Materials.

Data is ex ressed as ercent decrease relative to untreated CSF.

*The manufacturer and order number for each material is as follows: PVDF, polyvinyl diflouride (Millipore # SLGV-013SL, pore size 0.22 micrometers (μm)); Polystyrene (Costar, 96-well plate # 3590); Nylon (Whatman # 6786-0402, pore size 0.2 μm); PTFE, polytetraflourethylene (Fisherbrand # 09-719G, pore size 0.2 μm); polypropylene ((Whatman # UN203NPEPP, pore size 0.2 μm); PES, polyethersulfone (Whatman # 6780-1302, pore size 0.2 μm); GMF, glass micro fiber (Whatman #6902-2504, pore size 0.45 μm); CA, cellulose acetate (Nalgene # 190-2580, pore size 0.8 μm); 6E10, biotinylated monoclonal antibody (Covance # SIG-39340).

**Human CSF was passed through the filter material using a syringe filter, a syringe-less filter device, or was contacted with the material (polystyrene) up to 10 times, then diluted 1:4 with 3% PBS/BSA (bovine serum albumin). The diluted CSF filtrate was analyzed for A-beta 1-40 using an ELISA. Total protein was measured using the BCA (bicinchoninic acid) protein assay.

Table 2. The Amount of A-Beta 1-40 and Total Protein Remaining in Human CSF After Contact by Various Filter Materials.

Data is expressed as amount of A-beta (pM) and total protein (ug/ml) remaining after each pass over the filter.

Values represent mean +/- standard error of measurement (SEM).

This data shows that PTFE, polypropylene, and GMF were the most effective at depleting A-beta 1-40 after the first contact of CSF with the material. The data further shows that PVDF, polystyrene, nylon, PES, CA, and the control antibody 6E10 demonstrated little or no reduction in total protein after single or multiple passes. PTFE, polystyrene and GMF showed reductions in total protein of 8%, 25%, and 26% after the first pass, respectively. The data suggests that PTFE, polystyrene and GMF may be useful for depleting amyloid-beta peptides from CSF.

EXAMPLE 2 This example shows that immunoadsorption effectively removes amyloid- beta peptides from artificial CSF.

TM

A 1.0 ml Hi- Trap Streptavidin column (GE Healthcare) was bound with biotinylated-6E10, a monoclonal anti-Aβ antibody directed against the N-terminal region (amino acids 1-16) of amyloid-beta peptides. Artificial CSF (ACSF) was formulated to mimic the human CSF composition of ions, total protein, sAPP, and the Aβl-40 and Aβl-42 concentration of human CSF. A total volume of 120 ml of ACSF was passed through the column at a rate of 1.0 ml/min. Samples of the ACSF were collected prior to application on the column (time=0) and following filtration of every 10 ml. These samples were analyzed for Aβl-40 and Aβl-42 using an ELISA assay. As shown in FIGURE 7, the 6E10 antibody effectively removed nearly all of the Aβl-40 and Aβl-42 peptides from the ACSF. Further, the column showed high binding capacity for both Aβl-40 and Aβl-42.

This example demonstrates that immunoadsorption columns using antibodies that bind amyloid-beta peptides effectively remove amyloid-beta peptides from artificial CSF and that the columns have high binding capacity. EXAMPLE 3

This example shows that filters approved for clinical use to infuse material into the human body effectively remove amyloid-beta peptides from human CSF.

Table 3 lists commercially available filters and columns that were tested for their amyloid-beta binding capability and their overall protein binding capacity. In these experiments, Aβl-40 was measured by ELISA and protein was measured using the bicinchoninic acid assay. Aβl-42 was also measured in some assays, and binding of

Aβl-42 was similar to that of Aβ 1-40. Table 3. Filters and Columns Tested for Ability to Bind Amyloid-Beta and Total Protein in CSF.

As shown in FIGURE 8, the GVS positively charged filter (Speedflow™ Adult 0.2 μm positive, GVS Filter Technology Inc., Indianapolis, Indiana) showed reasonable binding of Aβl-40 and relatively low total protein binding. The GVS filter effectively cleared Aβl-40 from the first 4-5 ml of human CSF (Bioreclamation, Inc., New York) passed through the filter (0.5 ml per minute). The GVS filter became increasingly saturated after about 4 ml of CSF were contacted with the filter, such that after about 10 ml of CSF filtration the GVS filter no longer retained any amyloid- beta.

To confirm that the GVS filter was binding Aβ species, the GVS filter was eluted

TM with detergent (Tween-20 ) and subjected to SDS-PAGE separation and analyzed by Western blotting using the E610 N-terminal anti-Aβ antibody. The results indicated that Aβl-40 and soluble APP were eluted from the GVS filter (data not shown), demonstrating that Aβ species were binding to the filter.

This example demonstrates that a filter clinically approved for infusion of fluids into human subjects is effective at removing amyloid- beta from human CSF. EXAMPLE 4

This example describes a proposed pilot clinical study to assess the safety and utility of removing amyloid- beta from the CSF of human subjects.

A CSF pheresis apparatus comprising an amyloid-beta adsorbing filter will be used in a pilot study to demonstrate the utility of removing amyloid-beta from human

CSF. As shown in FIGURE 9A, the apparatus 900 will be attached to the end of an intrathecal (epidural) CSF catheter 902 whose lumen is in contact with the intrathecal space of the spine. CSF from the catheter will be routed by tubing and stopcocks 904

(e.g., a 3-way valve or a 4-way valve) to either a peristaltic pump 906 for sample collection 908 or to a syringe 910 controlled by a syringe pump (not shown). The syringe 910 can return CSF to the intrathecal compartment either directly or via passage through an amyloid- beta adsorbing filter 912, such as the GVS positively charged filter described in Example 3.

Human subjects will include up to two healthy volunteers (aged 18-45 years) and about six elderly patients (aged 50 to 75 years) with mild cognitive impairment (MCI).

Subjects will undergo either single (cohort 1) or multiple (cohort 2) CSF pheresis sessions. Subjects in the first cohort include one healthy volunteer and three patients with

MCI. Subjects in the second cohort include one healthy volunteer and three patients with MCI. Approximately one hour prior to insertion of the intrathecal catheter, a single intravenous (IV) dose of antibiotic will be administered as prophylaxis against infection. Subjects will be placed in the lateral decubitus position and the epidural catheter will be inserted into the intrathecal space at lumbar position L3-L4 or L4-L5 under sterile conditions. After successful insertion of the catheter, an initial baseline sample 908 of CSF (about 1 ml) will be collected for CSF safety analysis (to analyze the glucose and protein levels as well as cell count and differential). During the first 30 minutes following catheter insertion, subjects will be asked to take a CogState® (CogState, Melbourne, Australia) computerized cognition test that lasts approximately 15 minutes. Thirty minutes after catheter insertion, three aliquots of approximately 0.5 ml CSF will be collected every 15 minutes for a total of 3 samples to establish a baseline for CSF amyloid-beta values.

Operational Scheme for Cohort 1. The first cohort of subjects will undergo a total of two cycles of CSF exchange, with one exchange being a mock filtration and the other exchange passing through a filter. As shown in FIGURE 9B, baseline samples 908 will be taken by directing the CSF along flow pathway 920 from the catheter 902 through the peristaltic pump 906 to a fraction collector (not shown).

Mock Exchange. As shown in FIGURE 9B, to perform the mock CSF exchange, 15 ml of CSF will be withdrawn along flow pathway 922 and infused back into the intrathecal space along flow pathway 924 through the same epidural catheter 902 by means of reversing the flow on the syringe pump, but without passing the CSF through the filter 912. After the infusion has been completed, three samples of CSF (about 0.5 ml each) will be collected every 15 minutes by directing the CSF along flow pathway 920 through the peristaltic pump 906 to the fraction collector. Cognitive measurements may also be performed during this period.

CSF Pheresis. Referring still to FIGURE 9B, about 15 minutes after the completion of the last CSF sample collection following the mock exchange, a directional valve 904 (e.g., a 3-way valve) will be switched to direct the flow of the withdrawn CSF into the filter pathway 926 for pheresis. After passing through the filter 912, the CSF will be reinfused into the intrathecal space through the same epidural catheter 902 by reversing the flow on the syringe pump. Ten minutes after the infusion is completed, three aliquots of post-procedure samples of CSF (about 0.5 ml each) will be collected every 15 minutes by directing the CSF along flow pathway 920 through the peristaltic pump 906 to the fraction collector for a total of three samples. Cognitive measurement may also be performed during this period.

As shown in FIGURE 9C, the patients in cohort 2 will undergo the same procedure as described above for cohort 1, except that these subjects will undergo four cycles of CSF pheresis using a clean filter 912 each time by switching the stopcock 904. The volume of CSF passed through each filter 912 is 15 ml, with about 15 minutes between each cycle. The CSF will be directed to a new filter 912 for each cycle by switching the appropriate stopcock 904 in the filtration pathway 926. Ten minutes after the infusion is completed, three aliquots of post-procedure samples of CSF (about 0.5 ml each) will be collected every 15 minutes by directing the CSF along flow pathway 920 through the peristaltic pump 906 to the fraction collector for a total of three samples. Cognitive measurement may also be performed during this period.

For safety monitoring, approximately 1.0 ml of CSF will be collected to measure albumin, glucose, protein levels, CSF cell counts, and microbiology culture immediately after initial lumbar puncture and immediately prior to catheter removal. An infectious disease sub- specialist, a neurologist, and an anesthesiologist will be available during the period of CSF pheresis for consultation. Further, while the spinal catheter is in place, tympanic temperature, heart rate, blood pressure, and cognitive measurement will be performed before and after the entire procedure. Following the collection of the final CSF sample, the epidural catheter will be removed and the patient will be kept supine in bed for a minimum of 12 hours. Tympanic temperature will be assessed each hour. A neurological assessment will be conducted after removal of the catheter as a safety measure, including a brief neurological exam and questions regarding headaches or any other adverse events. Cognitive screens will again be conducted approximately two hours after the CSF pheresis sessions are completed. Subjects will be monitored until day 7 for any adverse events.

While the preferred embodiment of the invention has been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the invention. While illustrative embodiments have been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the invention.

Claims

CLAIMSThe embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A composition comprising a biocompatible material that binds to amyloid- beta peptides present in endogenous bodily fluid, wherein in operation the composition is oriented such that endogenous bodily fluid contacts a surface of the biocompatible material under unidirectional flow conditions, and wherein the composition depletes at least a portion of the amyloid- beta peptides present in endogenous bodily fluid.

2. The composition of Claim 1, wherein the bodily fluid is cerebrospinal fluid.

3. The composition of Claim 2, wherein the biocompatible material depletes at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% of the amyloid-beta peptides present in the cerebrospinal fluid that contacts the biocompatible material.

4. The composition of Claim 1, wherein the biocompatible material comprises reactive groups that bind to amyloid-beta peptides.

5. The composition of Claim 1, wherein the biocompatible material further comprises a plurality of binding agents attached thereto that each selectively bind to amyloid-beta peptides.

6. The composition of claim 1, wherein the biocompatible material is at least one of tubing, filter(s), membrane(s), glass, beads, microspheres, nanoparticles, or a polymer.

7. The composition of claim 1, wherein the biocompatible material is sterilizable.

8. The composition of claim 5, wherein the binding agent is selected from the group consisting of an antibody, antibody fragment(s), diabody, aptamers, enzyme and peptide.

9. The composition of claim 1, wherein the biocompatible material is a polymer, and wherein a surface of the polymer comprises a plurality of binding agents attached thereto.

10. The composition of claim 9, wherein the polymer is selected from the group consisting of PTFE, polypropylene, PES, and polystyrene.

11. The composition of claim 5, wherein the binding agents are antibodies or antibody fragments that specifically bind amyloid-beta peptides.

12. The composition of claim 5, wherein the binding agent is an enzyme selected from the group consisting of the peptidases insulysin and neprilysin.

13. The composition of claim 6, wherein the biocompatible material is a filter or membrane, wherein a surface of the filter or membrane comprises a plurality of binding agents attached thereto.

14. The composition of claim 13, wherein the binding agents are antibodies or antibody fragments that specifically bind amyloid-beta peptides.

15. The composition of claim 6, wherein the biocompatible material is a plurality of beads or microspheres.

16. The composition of claim 15, wherein the surface of the beads or microspheres further comprises at least one of streptavidin, biotin, dextran, lectin, amine or carboxyl groups attached to the binding agent.

17. The composition of claim 1, wherein the composition is included in an implantable medical device.

18. A medical device for depleting a target substance from endogenous bodily fluid of a living subject, the medical device comprising:

(a) a composition comprising a biocompatible material that binds to a target substance present in the endogenous bodily fluid of a living subject;

(b) a housing enclosing the composition, the housing comprising a first end with an inlet for receiving bodily fluid comprising the target substance from a living subject, and a second end with an outlet for returning the bodily fluid depleted of at least a portion of the target substance to the living subject, wherein the composition is configured within the housing such that the endogenous bodily fluid directly contacts the composition.

19. The device of Claim 18, wherein the target substance is selected from the group consisting of amyloid-beta, tau, prion proteins, beta-2-microglobulin, bacteria, viruses, harmful metabolites, inflammatory mediators and cells.

20. The device of Claim 18, wherein the biocompatible material comprises reactive groups that bind to the target substance.

21. The device of Claim 18, wherein the biocompatible material comprises a plurality of binding agents attached thereto that each selectively bind to the target substance.

22. The device of claim 18, wherein the device is implantable into a living subject and the housing comprises the biocompatible material.

23. The device of claim 22, wherein the housing further comprises a plurality of inlets along the sides to receive bodily fluid comprising the target substance from a living subject.

24. The device of claim 18, wherein the device further comprises at least one check valve to maintain unidirectional flow of the bodily fluid of a living subject through the device and returned to the living subject.

25. The device of claim 18, wherein the device further comprises a sensor, wherein the sensor measures the binding capacity of the binding agent for the target substance and provides an output to a user regarding the status of the binding capacity.

26. The device of claim 18, wherein the composition is a replaceable cartridge within the housing.

27. The device of Claim 18, wherein the endogenous bodily fluid is selected from the group consisting of cerebrospinal fluid, blood, plasma, peritoneal fluid, and lymph.

28. The device of Claim 27, wherein the endogenous bodily fluid is cerebrospinal fluid.

29. The device of claim 28, wherein the composition is configured within the housing such that substantially all the cerebrospinal fluid that enters the inlet contacts the composition.

30. The device of claim 28, wherein the flow rate of cerebrospinal fluid through the device is from 5 mL/hour to 160 mL/hour.

31. The device of claim 18, wherein the biocompatible material further comprises binding agents that specifically bind amyloid-beta peptides.

32. The device of claim 31, wherein the binding agent is selected from the group consisting of an antibody, antibody fragment(s), diabody, aptamer, enzyme, and peptide.

33. The device of claim 32, wherein the binding agent is an enzyme selected from the group consisting of the peptidases insulysin and neprilysin.

34. The device of claim 18, wherein the biocompatible material is at least one of tubing, filter(s), membrane(s), glass, beads, microspheres, nanoparticles, or a polymer.

35. The device of claim 34, wherein the biocompatible material is a polymer.

36. A medical device for depleting at least a portion of amyloid-beta from endogenous cerebrospinal fluid in a living subject, the medical device comprising:

(a) a composition comprising a biocompatible material that binds to amyloid- beta present in endogenous CSF of a living subject; and

(b) a housing enclosing the composition, the housing comprising a first end with an inlet for receiving endogenous CSF comprising amyloid-beta from a living subject, and a second end with an outlet for returning the CSF depleted of at least a portion of amyloid-beta to the living subject, wherein the composition is configured within the housing such that the endogenous CSF directly contacts the composition.

37. A method of depleting amyloid- beta peptides from endogenous cerebrospinal fluid of a living subject, comprising:

(a) contacting endogenous cerebrospinal fluid of a living subject with a composition comprising a biocompatible material that binds to amyloid-beta peptides present in cerebrospinal fluid; and

(b) returning the cerebrospinal fluid depleted of at least a portion of amyloid- beta peptides to the living subject.

38. The method of Claim 37, wherein the biocompatible material comprises reactive groups that bind to amyloid-beta peptides.

39. The method of Claim 37, wherein the biocompatible material comprises a plurality of binding agents attached thereto that each selectively bind to amyloid-beta peptides.

40. The method of Claim 37, wherein the biocompatible material depletes at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% of the amyloid-beta peptides present in the cerebrospinal fluid that contacts the biocompatible material.

41. The method of claim 37, wherein the cerebrospinal fluid is contacted with the composition exterior to the living subject.

42. The method of claim 37, wherein the cerebrospinal fluid is contacted with the composition implanted within the living subject.

43. The method of claim 42, wherein the implanted composition is positioned in the central nervous system in the living subject in such a manner that the endogenous physiological flow of the cerebrospinal fluid results in the contacting of the fluid with the composition and the returning of the cerebrospinal fluid depleted of at least a portion of amyloid-beta peptides to the subject.

44. A system for depleting a target substance from endogenous bodily fluid of a living subject, the system comprising:

(a) a medical device that includes:

(i) an inlet port for receiving endogenous bodily fluid from a living subject;

(ii) a composition comprising a biocompatible material that binds to a target substance present in the bodily fluid of a living subject;

(iii) an outlet port for returning the bodily fluid depleted of at least a portion of the target substance to the living subject;

(b) a pump that is fluidically connected to the medical device of (a); and

(c) a computer comprising a memory, an analog and digital interface, and a user interface for controlling fluid movement through the system.

45. The system of claim 44, wherein the target substance is selected from the group consisting of amyloid-beta, tau, prion proteins, beta-2-microglobulin, bacteria, viruses, harmful metabolites, inflammatory mediators and cells.

46. The system of claim 44, wherein the endogenous bodily fluid is selected from the group consisting of cerebrospinal fluid, blood, plasma, peritoneal fluid, and lymph.

47. The system of claim 44, wherein the biocompatible material comprises reactive groups that bind to the target substance.

48. The system of claim 44, wherein the biocompatible material further comprises a plurality of binding agents that each selectively bind to the target substance.

49. The system of claim 44, further comprising at least one sensor for detecting the amount of the target substance in the bodily fluid.

50. The system of claim 45, wherein the target substance is amyloid-beta.

51. The system of 50, wherein the endogenous bodily fluid is CSF.

52. The system of claim 51, wherein the system depletes at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% of the amyloid-beta peptides present in the CSF that contacts the composition.

53. The system of claim 51, wherein the flow rate of CSF through the medical device is from about 5 mL/hour to about 160 mL/hour.