JP6312292B2 - Β-amyloid removal system in blood - Google Patents

Description

  The present invention relates to a β-amyloid removal system in blood.

  Alzheimer's disease (AD) is a disease that is thought to become dementia due to the degeneration of nerve cells in the brain. Regarding the onset mechanism, the “amyloid hypothesis” starting from the accumulation of β-amyloid (hereinafter sometimes abbreviated as “Aβ”) in the brain is the most powerful, and soluble Aβ is a long-term enhancement of memory. It is known to strongly inhibit

It has been reported that administration of anti-Aβ antibody, which is an antibody against Aβ, and administration of Aβ vaccine, together with improvement of dementia symptoms, disappearance of brain Aβ deposition (Non-patent Document 1). Based on the amyloid hypothesis, Alzheimer's disease The possibility of treatment is suggested.
In addition, as a result of experiments using Alzheimer's disease model mice, when the Aβ in the brain decreases, the amount of Aβ in the blood increases significantly (Non-patent Document 2), and the Aβ-binding substance (Gelsolin) does not have an immunostimulatory function. And GM1 ganglioside) decrease in brain Aβ amount (Non-Patent Documents 3 and 4), and administration of anti-Aβ antibody Fab fragment having no immunostimulatory function decreases in brain Aβ amount (Non-Patent Document 5) and the like have been proposed, and a “pulling” hypothesis that brain Aβ is pulled into the blood as the amount of Aβ in the blood decreases is proposed. In connection with this hypothesis, it has been reported that the amount of Aβ in blood is reduced by artificial dialysis (Non-patent Documents 6 and 7).

It is known that Aβ is mainly composed of 40 amino acids Aβ _1-40 , 42 amino acids Aβ _1-42 , 38 amino acids Aβ _1-38 , 43 amino acids Aβ _1-43, and the like. Among them, Aβ _1-42 is considered to have strong aggregation ability and strong neurotoxicity.
In addition, it has been reported that Aβ oligomers are more neurotoxic than Aβ monomers (Non-patent Document 8). There is an equilibrium between Aβ monomers, Aβ oligomers, and soluble protofibrils, and as further aggregation proceeds, it deposits as fibrillar Aβ.

  Further, Patent Document 1 discloses a β-amyloid removal system that utilizes the fact that Aβ is adsorbed and removed from the hollow fiber surface.

JP 2012-16595 A

Bayer, A.A. et al. , Neurology, 2005, 64, 94-101. Lemere, C.I. A. et al. , Neurobiol. Dis. , 2003, 14, 10-18 Matsuoka, Y. et al. et al. , J. et al. Neurosci. , 2003, 23, 29-33 Bergamaschini, L.M. et al. , J. et al. Neurosci. , 2004, 24, 4148-4186 Levites, Y.M. et al. , J. et al. Neurosci. , 2006, 26, 11923-11928 Kyoto Medical Journal, Vol. 53, No. 1, June 2006, pp. 113-120 Isabel, R.M. et al. , Journal of Alzheimer's Disease, 2006, 10, 439-443. Walsh, DM. et al. , Nature, 2002; 416: 535-9

In light of the “withdrawal” hypothesis, the present inventors have found that efficient removal of Aβ in the blood of Alzheimer's disease patients is an effective means for treating or preventing Alzheimer's disease by reducing the accumulation of Aβ in the brain. Thought.
Accordingly, an object of the present invention is to provide a system that can efficiently remove Aβ in blood.

  As a result of intensive studies to solve the above problems, the present inventors have found that Aβ oligomers cannot be sufficiently removed by adsorption removal with a hollow fiber membrane. However, Aβ oligomers can be removed by passing blood across the hollow fiber wall and filtering in the thickness direction of the hollow fiber, and by filtering in the thickness direction, The present inventors have found that Aβ monomer can also be removed stably and completed the present invention.

That is, the present invention provides the following system for reducing the β-amyloid concentration in blood.
[1]
A housing with a blood inlet and outlet;
A hollow fiber membrane accommodated in the housing such that blood from the inlet flows into the hollow fiber lumen;
A blood circuit for bleeding blood outside the body and connecting it to the inlet;
A blood circuit that mixes blood that has been filtered out from the hollow fiber lumen after flowing in and blood that has flowed through the hollow fiber lumen after flowing in;
A blood circuit for returning the mixed blood,
A system for removing β-amyloid in blood, in which a part of blood is filtered across the hollow fiber lumen to the outside of the hollow fiber to remove β-amyloid with the hollow fiber.
[2]
The blood β-amyloid removal system according to [1], wherein the housing includes a filtered blood outlet and a passed blood outlet.
[3]
The blood β-amyloid removal system according to [1] or [2], further comprising a blood pump for allowing a part of blood to pass across the hollow fiber wall.
[4]
The blood β-amyloid removal system according to [3], comprising one blood pump that controls the flow rate of blood (QB) and the amount of filtered blood (QF).
[5]
The blood β-amyloid removal system according to [3], comprising a blood pump that controls the flow rate of blood (QB) to be introduced and a blood pump that controls the amount of blood filtration (QF).
[6]
The blood β-amyloid removal system according to any one of [1] to [5], further comprising a pressure loss structure that controls blood filtration rate (QF).
[7]
The β-amyloid removal system in blood according to [6], wherein the pressure loss structure is an orifice.
[8]
The blood β-amyloid removal system according to any one of [1] to [7], wherein the blood flow rate (QB) to be introduced is 20 to 200 mL / min.
[9]
The blood β-amyloid removal system according to any one of [1] to [8], wherein a ratio of (flowing blood flow) / (blood filtration amount) (QB / QF) is 2 to 100.
[10]
The β-amyloid removal system in blood according to any one of [1] to [9], wherein the housing in which the hollow fiber membrane is accommodated is a hemodialysis column.
[11]
Hollow fiber is polysulfone (PSf), polymethyl methacrylate (PMMA), polyethersulfone polyarylate polymer alloy (PEPA), polyethersulfone (PES), polyacrylonitrile (PAN), cellulose triacetate (CTA), modified cellulose (BEM) and the β-amyloid removal system in blood according to any one of [1] to [10], comprising at least one polymer selected from the group consisting of ethylene vinyl alcohol copolymer (EVAL).
[12]
The hollow fiber contains at least one polymer selected from the group consisting of polysulfone (PSf), polymethyl methacrylate (PMMA), polyethersulfone polyarylate polymer alloy (PEPA), and polyethersulfone (PES). The blood β-amyloid removal system according to any one of [1] to [11].
[13]
The β-amyloid removal system in blood according to [11] or [12], wherein the hollow fiber further contains polyvinylpyrrolidone.

In addition, the present invention provides the following method for reducing β-amyloid concentration in blood, and the following method for treating or preventing Alzheimer's disease.
[14]
A method for reducing the concentration of β-amyloid in blood,
A step of bleeding blood outside the body, a step of passing the removed blood through the hollow fiber lumen, a step of returning the passed blood to the body, and a part of the blood from the hollow fiber lumen to the hollow fiber A method of reducing the concentration of β-amyloid in blood, wherein the β-amyloid is removed by hollow fibers after being filtered through the outside.
[15]
The method according to [14], wherein the flow rate (QB) of blood to be introduced is 20 to 200 mL / min.
[16]
The method according to [14] or [15], wherein a flow rate (QB) of blood to be introduced is 40 to 60 mL / min.
[17]
The method according to any one of [14] to [16], wherein a ratio of (flowing blood flow) / (blood filtration amount) (QB / QF) is 2 to 100.
[18]
The method according to any one of [14] to [17], wherein a ratio of (flowing blood flow) / (blood filtration amount) (QB / QF) is 5 to 10.
[19]
The method to reduce the beta-amyloid density | concentration in the blood using the beta-amyloid removal system in blood in any one of [1]-[13].
[20]
The method for reducing the β-amyloid concentration in blood according to any one of [14] to [19], wherein the β-amyloid removal system in blood according to any one of [1] to [13] is used.
[21]
A method of treating or preventing Alzheimer's disease by reducing the concentration of β-amyloid in the blood,
Removing the patient's blood outside the body,
A step of passing the removed blood through the hollow fiber,
Returning the circulated blood to the patient's body,
A method in which a part of blood is filtered from the hollow fiber lumen to the outside of the hollow fiber to remove β-amyloid with the hollow fiber.
[22]
The method according to [21], wherein a flow rate (QB) of blood to be introduced is 20 to 200 mL / min.
[23]
The method according to [21] or [22], wherein the flow rate (QB) of blood to be introduced is 40 to 60 mL / min.
[24]
The method according to any one of [21] to [23], wherein a ratio of (flowing blood flow) / (blood filtration amount) (QB / QF) is 2 to 100.
[25]
The method according to any one of [21] to [24], wherein a ratio of (flowing blood flow) / (blood filtration amount) (QB / QF) is 5 to 10.
[26]
A method for treating or preventing Alzheimer's disease by reducing the β-amyloid concentration in blood using the β-amyloid removal system in blood according to any one of [1] to [13].
[27]
The method for reducing the β-amyloid concentration in blood according to any one of [14] to [20], wherein the β-amyloid removal system in blood according to any one of [1] to [13] is used. A method of treating or preventing a disease.

  ADVANTAGE OF THE INVENTION According to this invention, the system which can remove A (beta) in blood efficiently can be provided.

The schematic diagram of the Aβ removal system in blood in this embodiment is shown. The schematic using the product dialyzer of Example 1 is shown. The result of the removal rate of Aβ _1-40 in the pool liquid in Example 1 is shown. The relationship between the removal rate of Aβ _{1-40 in} the pool liquid 15 minutes after the start of circulation in Example 1 and QF is shown. The schematic of the circuit using the mini-module of Example 2 is shown. The result of Western Blot of the removal of the high molecular weight Aβ _1-42 oligomer, the low molecular weight Aβ _1-42 oligomer, and the Aβ _1-42 monomer in the pool solution over time in Example 2 is shown. The schematic using the product dialyzer of Example 3 is shown. The result of the Aβ _1-42 removal rate in the pool liquid in Example 3 is shown. The result of Aβ _1-42 oligomer removal rate in the pool solution in Example 4 is shown. The result of Western Blot in Example 4 is shown. The result of the removal rate of Aβ _1-42 monomer in Reference Example 1 is shown. The result of the removal rate of Aβ _1-42 oligomer in Reference Example 1 is shown.

  Hereinafter, modes for carrying out the present invention (hereinafter referred to as “the present embodiment”) will be described in detail below. In addition, this invention is not limited to the following embodiment, It can implement by changing variously within the range of the summary.

The blood β-amyloid removal system of the present embodiment,
A housing with a blood inlet and outlet;
A hollow fiber membrane accommodated in the housing such that blood from the inlet flows into the hollow fiber lumen;
A blood circuit for bleeding blood outside the body and connecting it to the inlet;
A blood circuit that mixes blood that has been filtered out from the hollow fiber lumen after flowing in and blood that has flowed through the hollow fiber lumen after flowing in;
A blood circuit for returning the mixed blood,
This is a β-amyloid removal system in blood in which a part of blood is filtered across the hollow fiber lumen to the outside of the hollow fiber to remove β-amyloid with the hollow fiber.

  In this embodiment, by guiding a part of blood in the film thickness direction of the hollow fiber membrane, the Aβ monomer is adsorbed not only on the hollow fiber lumen surface or the hollow fiber outer surface but also on the surface in the film thickness direction. , Aβ monomer concentration in blood decreases. In addition, it is considered that the Aβ oligomer concentration is decreased by filtering a part of the blood from the hollow fiber lumen to the outside of the hollow fiber and filtering the blood, so that the Aβ oligomer is removed by filtration on the surface of the hollow fiber wall.

  In this embodiment, since Aβ monomer and Aβ oligomer in blood can be efficiently removed, Aβ in patient blood can be efficiently removed. Thereby, it is considered that the accumulation of Aβ in the brain is reduced in the patient, which is an effective means for treating or preventing Alzheimer's disease.

  In this embodiment, a patient is not specifically limited, The patient who has developed Alzheimer's disease may be sufficient, and the patient before developing Alzheimer's disease may be sufficient.

  In this embodiment, by passing blood across the hollow fiber in the film thickness direction, the hollow fiber membrane surface (including the hollow fiber lumen surface and hollow fiber outer surface) and the hollow fiber membrane wall fine By filtering through the pores, removal of Aβ monomer, high molecular weight Aβ oligomer and Aβ protofibril in equilibrium with low molecular weight Aβ oligomer can be performed simultaneously.

In this embodiment, by guiding a part of blood in the film thickness direction of the hollow fiber membrane, the Aβ monomer is adsorbed not only on the hollow fiber lumen surface but also on the surface in the film thickness direction. Decreases. Further, it is considered that by filtering a part of blood from the hollow fiber lumen to the outside of the hollow fiber, the Aβ oligomer concentration is lowered because the Aβ oligomer is filtered and removed on the surface of the hollow fiber lumen or the hollow fiber membrane wall.
Then, by mixing the blood passing through the hollow fiber lumen and the blood coming out of the hollow fiber lumen to the outside of the hollow fiber to obtain blood for return, the blood Aβ monomer and the blood Aβ oligomer are obtained. At the same time, Aβ in the blood can be efficiently removed.

A schematic diagram of the Aβ removal system in blood in the present embodiment is shown in FIG. In FIG. 1, blood is passed through the hollow fiber lumen, and Aβ is adsorbed on the surface of the hollow fiber lumen and on the thickness part of the hollow fiber (the porous part between the inner surface and the outer surface of the hollow fiber membrane). . Further, when the blood passes from the hollow fiber lumen to the outside of the hollow fiber, the blood is filtered and Aβ contained in the blood is removed. It is considered that Aβ oligomers are mainly removed by filtration through the hollow fiber wall, and Aβ monomers are mainly adsorbed and removed by pores on the hollow fiber lumen surface and the hollow fiber wall surface.
In the filtered blood, Aβ monomer and Aβ oligomer are removed, and in the blood passed through the hollow fiber lumen, the Aβ monomer is removed, and the filtered blood and the hollow fiber lumen are passed through. It is considered that Aβ is removed by mixing the liquefied blood. The mixed blood from which Aβ has been removed is returned from the blood circuit.

The hollow fiber membrane used in the present embodiment is not particularly limited as long as it is a hollow fiber membrane capable of passing and filtering blood. For example, in artificial dialysis, blood filtration, and plasma separation, etc. The hollow fiber membrane used is mentioned, You may use a commercially available hollow fiber membrane.
Examples of the hollow fiber membrane include cellulose-based hollow fiber membranes such as cellulose acetate, modified cellulose and copper ammonia cellulose, polyacrylonitrile, polymethyl methacrylate, polyethylene, polyvinylidene fluoride, polyethersulfone polyarylate polymer alloy, polyvinyl Examples thereof include synthetic polymer-based hollow fiber membranes such as alcohol, polysulfone, polyamide, polyimide, polyphenyl ether, polyether sulfone, and ethylene vinyl alcohol copolymer. Among them, since an electrostatic bond and / or a hydrophobic bond is used, it is preferable to use a hydrophobic polymer, such as polysulfone, polymethyl methacrylate, polyethersulfone polyarylate polymer alloy, polyethersulfone, A hollow fiber membrane formed from a polymer such as polyacrylonitrile is preferred.

The material of the hollow fiber membrane preferably further contains polyvinyl pyrrolidone, polyethylene glycol, ethylene vinyl alcohol copolymer, polyhydroxyethyl methacrylate, etc., including blood compatibility. Examples thereof include hollow fiber membranes produced from a molecular mixture.
The polyvinyl pyrrolidone and the like may be spun by mixing with a polymer such as polysulfone, but may be used to coat a hollow fiber membrane obtained by spinning using a polymer such as polysulfone. .

  Although the hollow fiber membrane used in this embodiment can be manufactured by a well-known method, Preferably the thickness of a hollow fiber membrane is 0.01-1 mm.

  In the present embodiment, the shape of the hollow fiber membrane in the housing is not particularly limited as long as the blood to be passed can be filtered from the hollow fiber lumen to the outside of the hollow fiber. The hollow fiber membrane module housed in the housing can be used.

As a form in which the hollow fiber membrane is provided in the housing, it is stored as a hollow fiber membrane bundle made of a plurality of hollow fiber membranes in a cylindrical housing as a hollow fiber membrane type module like a hollow fiber membrane column. May be.
In this case, both ends of the hollow fiber membrane bundle may be bonded and fixed (sealed) by a resin layer portion as a sealing portion formed by filling a resin composition such as polyurethane.
In the present embodiment, the housing is provided with a blood inlet and outlet for allowing blood to flow through and discharge from the housing. The blood may be mixed after exiting the housing outlet, or the mixed blood may exit the housing outlet.

  As a module having such a hollow fiber membrane, it is described in a module (dialysis column) used as a so-called dialyzer, Japanese Patent Application Laid-Open No. 2005-52716, International Publication No. 2006/024902, International Publication No. 2004/094047, and the like. And can be suitably used as a housing in which the hollow fiber membrane in this embodiment is accommodated. As the housing, a hemodialysis column or a commercially available hemodialysis column may be used.

In this embodiment, the blood sent from the blood circuit for blood removal is passed through the hollow fiber lumen of the hollow fiber membrane, but the blood flow rate (QB) flowing into the hollow fiber lumen is Preferably it is 20-200 mL / min, More preferably, it is 30-70 mL / min, More preferably, it is 40-60 mL / min.
In the present embodiment, the blood fed from the blood circuit for blood removal is passed through the hollow fiber lumen of the hollow fiber membrane, and a part of the blood goes out of the hollow fiber lumen to the outside of the hollow fiber. The ratio of (flow rate of blood flowing into the hollow fiber lumen) / (blood filtration amount from the hollow fiber lumen to the outside of the hollow fiber) (QB / QF) is preferably 2 to 100. Preferably it is 3-50, More preferably, it is 5-10.
In the present embodiment, an actual module may be designed in consideration of the Aβ removal rate and the blood throughput per unit time.

As a device for maintaining the blood flow rate, a blood pump may be provided in the blood Aβ removal system.
The blood pump is not particularly limited as long as it can continuously supply blood to the hollow fiber membrane, and examples thereof include a pump for a blood purification device, a pump for artificial dialysis, and a perista pump (roller pump).
In addition, when blood is usually passed through a central membrane module or the like, in order to prevent blood coagulation, an anticoagulant is contained in the blood so that the blood is passed. It may be provided.
In addition, a pressure monitor for measuring the pressure of the blood to be circulated and a monitor for confirming bubbles in the returned blood may be provided so that blood can be sampled. Also good.

With such a blood pump, it is preferable to pass a part of blood from the hollow fiber lumen to the outside of the hollow fiber, and the blood flow rate (QB) flowing into the hollow fiber lumen by one blood pump and the hollow fiber lumen The blood filtration amount (QF) from the hollow fiber to the outside of the hollow fiber may be controlled, the blood pump for controlling the blood flow rate (QB) flowing into the hollow fiber lumen, and the blood filtration from the hollow fiber lumen to the outside of the hollow fiber You may provide the blood pump which controls quantity (QF) separately.
By adjusting the blood throughput appropriately with a single blood pump, it is possible to adjust the pressure of the liquid to be fed and pass a part of the blood from the hollow fiber lumen to the outside of the hollow fiber. The blood pump may be provided on the inlet side or may be provided on the outlet side.

  In the blood Aβ removal system of the present embodiment, a replacement fluid pump that supplies replacement fluid, a replacement fluid tank that stores the replacement fluid, and the like may be provided.

In the Aβ removal system in blood of this embodiment, β-amyloid is removed by adsorption and filtration on the surface and thickness of the hollow fiber membrane, so that the blood is hollowed in order to increase the removal rate of β-amyloid. In order to efficiently contact the inside of the yarn lumen, the outside of the hollow fiber, and the inside of the film thickness, it is preferable to prevent reverse filtration even partially.
In the case of using a dialyzer-type hollow fiber membrane, since the reverse filtration can be prevented by setting the pressure on the dialysate side low, the flow rate on the dialysate side is not particularly limited. 100 mL / min or less is preferable.

  As a method of preventing such reverse filtration, the hollow fiber filling rate is made lower than the dialyzer for hemodialysis, or the ratio L / D of the length L and the diameter D of the container of the effective hollow fiber part is set as follows: For example, it is lower than the dialyzer for hemodialysis.

In the blood Aβ removal system of this embodiment, a portion of the blood is passed from the hollow fiber lumen to the outside of the hollow fiber to mix the blood filtered by the hollow fiber with the blood that has passed through the hollow fiber lumen. A blood circuit.
In such a blood circuit, a blood pump may be provided, or a pressure loss structure for controlling the blood flow rate (QB) flowing into the hollow fiber lumen may be provided. Moreover, you may provide the pressure loss structure which controls the blood flow rate which flows in into the outside of a hollow fiber.
The pressure loss structure is not particularly limited as long as a part of the blood flowing into the hollow fiber lumen can pass from the hollow fiber lumen to the outside of the hollow fiber. For example, Aβ in blood having an orifice It is good also as a removal system.

Further, in the present embodiment, it is also possible to control the pores of the hollow fiber to generate a filtration pressure and allow a part of blood to pass from the hollow fiber lumen to the outside of the hollow fiber.
It is preferable that the pores of the hollow fiber in the present embodiment have such a small size that hardly allows albumin to pass therethrough.
Further, by controlling the pressure difference (TMP) between the inside and outside of the membrane, a part of blood can be passed from the hollow fiber lumen to the outside of the hollow fiber.

In the present embodiment, a method for reducing the β-amyloid concentration in blood can be performed.
The method of reducing the β-amyloid concentration in the blood of this embodiment is as follows:
A step of bleeding the blood outside the body, a step of passing the removed blood through the hollow fiber lumen, and a step of returning the passed blood into the body.
In this method, β-amyloid in the blood is reduced by filtering a part of blood from the hollow fiber lumen to the outside of the hollow fiber to remove β-amyloid.
In this embodiment, it is preferable to use the Aβ removal system in blood of this embodiment in order to filter a part of blood from the hollow fiber lumen to the outside of the hollow fiber.

The method of reducing the concentration of β-amyloid in the blood is performed by extracorporeal circulation in which the blood is removed from the body, the removed blood is passed through the hollow fiber membrane, and the passed blood is returned to the body. Can be implemented.
Such a series of steps can be implemented as a method similar to blood purification techniques such as so-called artificial dialysis and blood filtration. For example, a blood circuit comprising a flexible tube for circulating a patient's blood extracorporeally. Is used.
A blood circuit for performing extracorporeal circulation generally includes an arterial blood circuit in which an arterial puncture needle for collecting blood from a patient is attached to the tip, and a vein in which a venous puncture needle for returning blood to the patient is attached to the tip. A hollow fiber membrane is provided between the side blood circuit and the arterial side blood circuit and the venous side blood circuit.
The arterial blood circuit, hollow fiber membrane, and venous blood circuit are connected by a flexible tube, and blood removed from the patient is passed through the flexible tube and contacts the hollow fiber membrane. Be made.
That is, blood is removed from the patient's body through the artery side blood circuit, introduced into the blood circuit, passed through the flexible tube in the blood circuit, and passed through the hollow fiber membrane.
The β-amyloid in the blood passed through the hollow fiber membrane comes into contact with the hollow fiber membrane, so that β-amyloid is adsorbed and filtered by the hollow fiber membrane, and the β-amyloid in the blood after passing through the hollow fiber membrane The concentration decreases. In this embodiment, not only Aβ monomers but also low molecular weight Aβ oligomers, high molecular weight Aβ oligomers, Aβ protofibrils and the like can be efficiently removed, so that β-amyloid in blood can be efficiently removed. . Thereby, it is provided as a method of reducing the β-amyloid concentration in blood.
The blood that has flowed out from the hollow fiber membrane is returned to the body from outside the patient's body as blood with a decreased β-amyloid concentration in the blood from the venous blood circuit.

The method for reducing the β-amyloid concentration in blood of the present embodiment is applied to a patient, preferably an Alzheimer's disease patient, so that the β-amyloid concentration in the blood in the Alzheimer's disease patient can be reduced. it can. Patients with Alzheimer's disease who are not affected by Alzheimer's disease but who have a higher Aβ level in the blood than normal, or who have no symptoms of Alzheimer's disease. In patients who can be classified as, it may be possible to reduce the β-amyloid concentration in the blood.
The Alzheimer's disease patient is not particularly limited as long as it is a patient capable of extracorporeal circulation. In addition, the method for reducing the β-amyloid concentration in the blood of the present embodiment can also be used as a method for preventing the onset of Alzheimer's disease by applying it to patients who have not yet developed Alzheimer's disease.

  In this embodiment, β-amyloid is adsorbed and filtered on the hollow fiber membrane, thereby reducing the β-amyloid concentration in the blood. However, “β-amyloid is adsorbed on the hollow fiber membrane” A state where the thread membrane surface and β-amyloid are electrostatically and / or hydrophobically bonded. Further, “β-amyloid is filtered into the hollow fiber membrane” means a state in which β-amyloid is physically trapped in the pores having a thickness. The trapped β-amyloid may also be electrostatically and / or hydrophobically bonded to the hollow fiber membrane surface.

  In this embodiment, by passing the patient's blood through the hollow fiber membrane, β-amyloid is dissociated from the β-amyloid-albumin complex as a form in blood and adsorbed to the hollow fiber membrane. One of these albumins is free in the blood.

  The method for reducing the β-amyloid concentration in the blood according to the present embodiment preferably includes a β-amyloid-albumin complex bound to albumin in the blood, and the method removes blood from the body. A step of passing the drained blood through a hollow fiber membrane having a dialysis hollow fiber membrane into a housing having at least an inlet and an outlet, and a step of returning the passed blood to the body. The blood flow rate is 20 to 200 mL / min as the amount of blood flowing into the hollow fiber lumen, and the step of passing the liquid adsorbs β-amyloid from the β-amyloid-albumin complex to the hollow fiber membrane. It is also possible to reduce the β-amyloid concentration in the blood by releasing albumin and thus reducing the β-amyloid concentration in the blood.

  In the present embodiment, a method for treating or preventing Alzheimer's disease can also be provided by reducing the β-amyloid concentration in the blood. In the method for treating or preventing Alzheimer's disease, the blood Aβ removal system according to this embodiment may be used.

  Hereinafter, the present embodiment will be described more specifically with reference to examples and comparative examples, but the present embodiment is not limited to only these examples.

In the following examples and reference examples, as Aβ, hydrochloride of human Aβ _1-40 or human Aβ _1-42 manufactured by Wako Pure Chemical Industries, Ltd. was used. For Aβ _1-40 or Aβ _1-42 , 100 μg / mL Aβ was stored frozen as pure water or dimethyl sulfoxide (DMSO) solution, and the stock solution was thawed immediately before use.

<Example 1> Removal of Aβ _{1-40 in} a pool solution using a product dialyzer A circuit using a polysulfone (PSf) dialyzer (APS13EA Asahi Kasei Medical Co., Ltd., Type V) and a hemodialysis circuit (Nikkiso Co., Ltd.) is shown in FIG. I assembled as follows.
A MASTER FLEX (7518-10) made by Cole Palmer instrument was used for the blood side and the filtration pump, and a PERISTA pump (SJ-1211) made by ATTO was used as a filtration pump only when QF = 1 mL / min.
Bovine serum albumin (BSA) adjusted to 5 mg / mL by priming the dialyzer hollow fiber lumen with 500 mL of calcium-free phosphate buffered saline (PBS) (Wako Pure Chemical Industries, Ltd., pH 7.2) at a flow rate of 200 mL / min 200 mL of PBS solution of Wako Pure Chemical Industries, Ltd. (for globulin-free biochemistry) was circulated for 30 minutes at a flow rate of 50 mL / min. Thereafter, 200 mL of 4 ng / mL Aβ _1-40 and 5 mg / mL BSA in PBS was passed through the dialyzer at a flow rate of 50 mL / min.
A PBS solution of 4 ng / mL Aβ _1-40 and 5 mg / mL BSA in 250 mL was used as a pool solution (corresponding to a human body) and circulated at QB = 50 mL / min, QF = 1, 5, 15, 25 mL / min. Pool liquid was collected after 5, 15, 30, 60 minutes. The concentration of Aβ _1-40 in the collected sample was measured by ELISA using Human βAmyloid (1-40) ELISA Kit, High-Sensitive (Wako Pure Chemical Industries, Ltd.), and Aβ ₁ in the pool solution after circulation for X minutes. _{The -40} removal rate was calculated by the following formula.
Pool liquid A [beta] _1-40 removal rate (X min) = 100 × {1- (pooled solution A [beta] _1-40 levels in the pool liquid A [beta] _1-40 levels / circulation starting after X minutes circulation)}
The results are shown in FIG. In the case of QB = 50 mL / min, Aβ _1-40 can be removed faster and more efficiently than when QF = 0 mL / min, even if filtration is performed in a small amount compared to QB of QF = 1 mL / min. In other words, if filtration is caused more than when there is no filtration rate (QF), the Aβ _1-40 solution flowing through the hollow fiber lumen obtains a flow path to the outside of the hollow fiber. It was presumed that Aβ _1-40 was adsorbed and filtered at the hollow fiber wall portion while moving out of the hollow fiber.
From the graph (Fig. 4) with the Aβ _1-40 removal rate in the pool liquid 15 minutes after the start of circulation on the vertical axis and QF on the horizontal axis (Figure 4), when the QB is 50 mL / min, the QF is 1 mL / min or more. It can be seen that the Aβ _1-40 removal rate is improved, and if the QF is 5 mL / min or more, a high Aβ _1-40 removal rate can be stably obtained. In FIG. 4, there is a significant difference in the removal rate between QF = 0 mL / min and QF = 5 mL / min (p = 0.031). Moreover, there is a significant difference in the removal rate between QF = 0 mL / min and QF = 25 mL / min (p = 0.037).

<Example 2> Removal of Aβ _{1-42 in} a pool solution using a mini module 80 hollow fibers of polysulfone (PSf) dialyzer (APS-SA Asahi Kasei Medical Co., Ltd., IV type) are taken out so that the effective length is 80 mm. A dialyzer type mini-module (membrane surface area 0.0037 m ² ) encapsulated with urethane was prepared.
Using a silicon tube with an inner diameter of 1 mm, the circuit was assembled as shown in FIG. 5 so that both the liquid that passed through the hollow fiber lumen of the mini module and the filtrate filtered through the hollow fiber wall returned to the pool liquid. Pool and filtrate were collected after 30 minutes, 1, 2, 18 and 24 hours.
Using two Atris PERISTA pumps (SJ-1211L), QB was 9 mL / hr (equivalent to QB = 50 mL / min when converted to product dialyzer size), and QF was 1.8 mL / hr (product dialyzer The flow rate was adjusted so that QF = 10 mL / min in terms of size).
An Aβ _1-42 oligomer was prepared by adjusting 50 μg / mL Aβ _1-42 and 2.5 mg / mL BSA in PBS as a pool solution.
In order to confirm Aβ _1-42 oligomers in Western Blot, experiments were conducted at Aβ _1-42 concentrations that were 1 million times higher than normal blood in consideration of the sensitivity of Western Blot.
Aβ _1-42 oligomer removal was examined by SDS-PAGE (electrophoresis) under reducing conditions using MES SDS Running Buffer and NuPAGE® Novex 4-12% Bis-Tris Gel (Invitrogen), then PVDF (electrophoresis) Polyvinylidene difluoride, Atto) transferred to a membrane, monoclonal antibody 6E10 against Aβ _1-16 (Covance 1: 2000) was used as the primary antibody, and the secondary antibody was anti-mouse IgG-HRP (Rockland, human, bovine, etc. (Pre-absorbed protein) was used at 1: 5000, and chemiluminescence was performed on Western Blot using ECL-Prime (GE Healthcare).
The results are shown in FIG. Both the high molecular weight Aβ _1-42 oligomer and the low molecular weight Aβ _1-42 oligomer in the pool solution were removed over time, and were hardly detected on Western Blot at 18 hours and 24 hours.
Further, in the filtrate that passed through the hollow fiber wall portion, bands of Aβ _1-42 oligomer and Aβ _1-42 monomer were hardly confirmed except that a very thin band of BSA was detected.

<Example 3> Removal of Aβ _1-42 oligomer in pool liquid using product dialyzer Polysulfone (PSf) dialyzer (APS13EA Asahi Kasei Medical, type V) and hemodialysis circuit (Nikkiso Co., Ltd.) Assemble as shown in FIG.
A MASTER FLEX (7518-10) made by Cole Palmer instrument was used for the blood side and the filtration pump, and a PERISTA pump (SJ-1211H) made by ATTO was used as a filtration pump only when QF = 1 mL / min.
The dialyzer hollow fiber lumen was primed with 500 mL of PBS at a flow rate of 200 mL / min, and 200 mL of BSA adjusted to 5 mg / mL was circulated at a flow rate of 50 mL / min for 30 minutes. Thereafter, 200 mL of 1.6 μg / mL Aβ _1-42 and 5 mg / mL BSA in PBS was passed through the dialyzer at a flow rate of 50 mL / min.
A PBS solution of 1.6 μg / mL Aβ _1-40 and 5 mg / mL BSA in 250 mL was used as a pool solution and circulated at QB = 50 mL / min and QF = 5 mL / min. Further, as a control, circulation was performed without performing filtration by setting QF = 0 mL / min.
The dialyzer inlet and outlet, filtrate (usually dialysate inlet), and pool were collected after 30,120 minutes. The concentration of Aβ _1-42 in the collected sample was measured by ELISA using Human βAmyloid (1-42) ELISA Kit, High-Sensitive (manufactured by Wako Pure Chemical Industries, Ltd.), and Aβ in the pool solution after circulation for X minutes. _1-42 removal rate was calculated by the following formula.
Pool liquid A [beta] _1-42 removal rate (X min) = 100 × {1- (pooled solution A [beta] _1-42 levels in the pool liquid A [beta] _1-42 levels / circulation starting after X minutes circulation)}
Further, the Aβ _1-42 removal rate before and after the dialyzer after circulation for X minutes (removal rate before and after D) was calculated by the following equation.
A β removal rate before and after D = 100 × {1− (dialyzer outlet Aβ _1-42 concentration at X minute circulation point / dialyzer inlet Aβ _1-42 concentration at X minute circulation point)}
Aβ _1-42 oligomers were measured using an Amyloid β Oligomers (82E1 specific) ELISA manufactured by IBL.
The results are shown in Table 1. By setting the QF to 5 mL / min, a decrease in the oligomer concentration of the pool solution was observed. The Aβ oligomer concentration in the filtrate at QF = 5 mL / min was 1% or less of the feed solution (dialyzer inlet concentration) flowing into the dialyzer.
As for the Aβ _1-42 monomer, as in Example 1, removal of QF = 5 mL / min was faster and more efficient than QF = 0 mL / min (FIG. 8). Further, Aβ _1-42 monomer was not detected in the filtrate.

<Example 4> Removal of Aβ _1-42 oligomer in pool liquid using minimodule 80 hollow fibers of polysulfone (PSf) dialyzer (APS-SA Asahi Kasei Medical Co., Ltd., IV type) have an effective length of 80 mm. Using a dialyzer-type minimodule (membrane surface area 0.0037 m ² ) encapsulated with urethane, it was examined whether Aβ oligomers would be removed by filtration into hollow fibers.
Quantitative evaluation of Aβ oligomers was carried out in an amyloid β Oligomers (82E1 specific) ELISA (Immuno-Biological Laboratories (IBL)). The qualitative examination of the molecular weight of the Aβ oligomer was conducted by Western Blot as in Example 2.
The following Aβ _1-42 solution was prepared and filtered.
Experiment 1: 5 μg / mL Aβ _1-42 in PBS.
Experiment 2: PBS solution of 50 μg / mL Aβ _1-42 .
Experiment 3: PBS solution of 5 μg / mL Aβ _1-42 and 2.5 mg / mL BSA.
Experiment 4: PBS solution of 50 μg / mL Aβ _1-42 and 2.5 mg / mL BSA.
Connect a silicone tube to the hollow fiber lumen outlet (V side) of the mini module and close it with forceps. Of the dialysate ports of the mini module, close the Aβ _1-42 solution inflow side with a cap, and the hollow fiber lumen outlet side. The dialysate port was opened and the total filtrate was sampled from here.
The Aβ _1-42 solution was fed at 20 μL / min to the hollow fiber lumen inlet (A side) of the mini module using a Terumo syringe pump. In Experiments 1 and 2, immediately before the experiment, the circuit and the minimodule were washed with PBS in a total filtration mode at 200 μL / min for 30 minutes. In Experiments 3 and 4, immediately before the experiment, the circuit and the minimodule were primed with a PBS solution of 2.5 mg / mL BSA at 200 μL / min for 10 minutes in the total filtration mode.
The results are shown in FIG. Moreover, the result of Western Blot is shown in FIG. Feed solution other A [beta] _1-42 monomers, dimers, trimers, low molecular weight A [beta] _1-42 oligomers of tetramers, etc., and, although a molecular weight of more high molecular weight A [beta] _1-42 oligomers 50kDa was present in a large amount, after filtration In the solution, Aβ _1-42 oligomer and Aβ _1-42 monomer were not detected over the entire molecular weight region. The Aβ _1-42 oligomer was efficiently removed by filtration through a hollow fiber.
Bands such as dimer, trimer, and tetramer can be confirmed on SDS-PAGE, but larger aggregates are formed in the Aβ _1-42 solution, and the hollow fiber pore (11.8 kDa β2 microglobulin is 30% It was thought that it was not possible to pass. This aggregate formation is thought to occur even in the absence of albumin.
The band of the albumin region in the BSA-containing samples of Experiments 3 and 4 is the same as the bovine blood-derived protein contained in BSA and reagent BSA from the Western Blot analysis of the samples of Experiments 1 and 2 not containing Aβ _1-42. It has been shown that this is a cross-reaction with a secondary antibody.

<Reference Example 1>
A polysulfone (PSf) hollow fiber dialyzer (APS-13EA Asahi Kasei Medical) was disassembled, and the hollow fiber was taken out and cut into a length of about 2 mm. The hollow fiber pieces were packed into two kinds of mini columns (4-2 column: 0.629 cm × 2.01 cm, 5-1 column: 0.350 cm × 62.14 cm) composed of a silicon tube and a polypropylene joint.
The hollow fiber fragments in the mini-column were primed with a PBS solution of 10 mg / mL BSA at 60 mL / hr for 20 minutes with a PERISTA pump, and then fed with a PBS solution of 40 ng / mL Aβ _1-42 and 10 mg / mL BSA at 15 mL / hr. did.
Aβ _1-42 oligomer was measured using IBL's amyloid β Oligomers (82E1 specific) ELISA, and Aβ _1-42 monomer was measured using Wako Pure Chemical Industries' human β amyloid (1-42) ELISA kit Wako. did.
The Aβ _1-42 removal rate before and after the dialyzer after circulation for X minutes (removal rate before and after D) was calculated by the following equation.
Aβ removal rate before and after D = 100 × {1− (Aβ _1-42 concentration after dialyzer / Aβ _1-42 concentration before dialyzer)}
The removal rate of Aβ _1-42 monomer is shown in FIG. 11, and the removal rate of Aβ _1-42 oligomer is shown in FIG. Only adsorption removal occurs in the hollow fiber fragment, but the Aβ _1-42 monomer is efficiently removed only by the adsorption removal. On the other hand, Aβ _1-42 oligomer can be removed to some extent by removing only adsorption, but a large amount of hollow fiber fragment (5-1 column) was used.

  The blood Aβ removal system of the present invention may reduce the amount of Aβ in the brain, and may be effective in the treatment or prevention of Alzheimer's disease.

Claims (12)

A housing with a blood inlet and outlet;
A hollow fiber membrane accommodated in the housing such that blood from the inlet flows into the hollow fiber lumen;
A blood circuit for bleeding blood outside the body and connecting it to the inlet;
A blood circuit that mixes blood that has been filtered out from the hollow fiber lumen after flowing in and blood that has flowed through the hollow fiber lumen after flowing in;
A blood circuit for returning the mixed blood,
A part of blood is filtered from the hollow fiber lumen to the outside of the hollow fiber , the Aβ monomer is adsorbed , and β-amyloid is removed by the hollow fiber.
The hollow fiber contains at least one polymer selected from the group consisting of polysulfone (PSf), polymethyl methacrylate (PMMA), polyethersulfone polyarylate polymer alloy (PEPA), and polyethersulfone (PES). , Β-amyloid removal system in blood.   The blood β-amyloid removal system according to claim 1, wherein the housing includes a filtered blood outlet and a passed blood outlet.   The blood β-amyloid removal system according to claim 1, further comprising a blood pump that filters a part of the blood across the hollow fiber wall.   The blood β-amyloid removal system according to claim 3, comprising one blood pump for controlling a blood flow rate (QB) and a blood filtration rate (QF) to be introduced.   The blood β-amyloid removal system according to claim 3, comprising a blood pump for controlling a blood flow rate (QB) to be introduced and a blood pump for controlling a blood filtration rate (QF).   The blood β-amyloid removal system according to any one of claims 1 to 5, further comprising a pressure loss structure that controls a blood filtration rate (QF).   The blood β-amyloid removal system according to claim 6, wherein the pressure loss structure is an orifice.   The blood β-amyloid removal system according to any one of claims 1 to 7, wherein a blood flow rate (QB) to be introduced is 20 to 200 mL / min.   The (beta) -amyloid removal system in blood of any one of Claims 1-8 whose (flow blood flow to flow in) / (blood filtration amount) (QB / QF) ratio is 2-100.   The β-amyloid removal system in blood according to any one of claims 1 to 9, wherein the housing in which the hollow fiber membrane is accommodated is a hemodialysis column. The β-amyloid removal system in blood according to any one of claims 1 to 10, wherein Aβ oligomer is filtered to remove β-amyloid with a hollow fiber. The β-amyloid removal system in blood according to any one of claims 1 to 11 , wherein the hollow fiber further contains polyvinylpyrrolidone.