JP5249737B2 - System for removing viruses and cytokines from blood - Google Patents

Description

  The present invention relates to a system for removing viruses and cytokines from blood.

  A state in which a virus enters the bloodstream of an animal infected with the virus and moves to the whole body is called viremia. In this state, the virus grows more and more in the host cell of the organ having affinity with the virus. In the body of an infected animal, immunocompetent cells work to combat the virus, produce various cytokines, and start preparing to produce antibodies against the virus. However, if the virus grows quickly and the virus increases before the antibody effect is still effective, the cytokines that should work locally increase excessively in the blood, causing damage to the organ and causing multiple organ failure. is there. Cytokines are essentially substances that have a role to protect the living body as an immunity, but when the immune system works actively and cytokines increase excessively, they damage the blood vessels of animals and various organs. Gives severe symptoms such as airway obstruction, encephalopathy, multiple organ failure.

  As a method for removing viruses from blood, blood is brought into contact with an adsorbent having a property of adsorbing viruses to adsorb and remove viruses (for example, see Patent Document 1), from blood of hepatitis C patients. After collecting plasma using a plasma separation membrane, it is further filtered with a membrane with a pore size that does not permeate hepatitis C virus, and the filtered plasma is mixed with blood rich in blood and returned to the patient, so-called double filtration There is a method using plasma exchange (for example, see Patent Document 2).

  Moreover, although not from blood, as a method for removing viruses from plasma fractionated preparations and antibody drugs, a method of filtering viruses with a filtration membrane is also known (see, for example, Patent Document 3).

  Furthermore, as a method of inactivating viruses, there are methods of using drugs and making the pH extremely acidic, but problems such as drug residues and blood degeneration must be solved.

In the method of removing cytokines from blood, the method of adsorbing and removing cytokines by bringing blood into contact with the material that adsorbs cytokines (see, for example, Patent Document 4), separating and discarding the patient's plasma with a plasma separator, Infusion of fresh frozen plasma, albumin and other supplements to patients, so-called simple plasma exchange method, continuous hemodiafiltration (CHDF) for removal of cytokines from high cytokine plasma of septic patients caused by bacterial infection There is a method (for example, see Non-Patent Document 1).
JP 2003-190276 A JP 2005-230165 A Japanese Patent Laid-Open No. 04-371221 International Publication No. 2003/055545 Pamphlet Journal of Japanese Society of Apheresis, Vol.23, No.1: 7-14, 2004

  When combining technologies by setting a new challenge to remove both viruses and cytokines from the patient's blood at the same time, various methods can be considered, but using the principle of membrane separation that can be separated according to the size of the substance to be removed When the system is constructed, the configuration as shown in FIG. 2 is considered as an example. The blood derived from the patient is first sent to the cytokine remover 101, where a low molecular protein containing cytokine and a liquid containing a substance having a smaller molecular weight are removed. The blood is then sent to the plasma separator 102, where it is separated into blood-rich blood and plasma, and the plasma obtained as filtrate is sent to the virus remover 103. The plasma sent to the virus remover is now filtered to remove the virus. The virus-removed plasma obtained as a filtrate is joined to blood rich in blood cells and returned to the patient.

However, the system of FIG. 2 can simultaneously remove both viruses and cytokines, but has the following problems.
First, since filtration of whole blood is performed at a low blood flow rate by the cytokine remover 101, the filtration flow rate of cytokine filtration is small. This is due to the fact that the filtration pressure cannot be increased to prevent damage to the blood cells, the adhesion of blood cells to the membrane, etc., and the plasma filtration flow rate in the downstream plasma separator 102 cannot be reduced much. Therefore, there is a restriction that blood cells cannot be concentrated excessively. In any case, a low filtration flow rate is a problem for therapeutic purposes to reduce cytokines and viruses in patient blood as quickly as possible. The blood outflow rate from patients infected with the virus is overwhelmingly lower than the blood outflow rate (about 200 mL / min) from dialysis patients. In the case of veins, 40-60 mL / min. The rate is about 80 to 100 mL / min, and the above-described problem is easily manifested.

  Second, the blood cell component of blood sent to the plasma separator 102 is already concentrated. Since blood cells and polymer proteins are concentrated by the amount of the liquid removed by the cytokine remover 101, the flow rate of plasma filtered by the plasma separator 102 is reduced. In addition, since the high molecular protein is concentrated, the filtration of the virus remover 103 is also affected, and the filtration efficiency is deteriorated.

  Third, since the cytokine remover 101 and the plasma separator 102 are connected in series, the optimum filtration pressure of the cytokine remover 101 and the optimum filtration pressure of the plasma separator 102 are different, so that adjustment is difficult. There is.

  In view of the above-described problems, the problem of the present invention is that both the virus and the cytokine can be simultaneously removed using the principle of membrane separation, and the plasma separator, virus remover, and cytokine remover having the same membrane area can be used. An object of the present invention is to provide a system for removing viruses and cytokines from blood with a high filtration flow rate and easy control of the filtration pressure.

  As a result of intensive studies by the present inventors in order to solve the above problems, blood from a patient is first introduced into a plasma separator to obtain plasma as a filtrate, and this plasma is filtered by a virus remover to remove the virus. The blood from which the virus has been removed is introduced into the cytokine remover, the liquid containing the cytokine is filtered on the filtrate side, and the blood from which the virus and cytokine have been removed is derived from the plasma separator and the blood is concentrated. , The blood cell component of blood sent to the plasma separator and the high molecular weight protein are not concentrated, so the plasma flow rate of the plasma separator does not decrease, The plasma protein that is sent to the virus remover is not concentrated, so the filtration efficiency of the virus remover does not deteriorate and whole blood is sent to the cytokine remover. Since it is plasma, there is no influence of adhesion of blood cells, and the filtration pressure can be increased, so the filtration flow rate of the cytokine remover can be increased, and the plasma separator, virus remover, cytokine remover, and filtration pressure can be set independently. As a result, it was found that the problems described above can be solved and the above-mentioned problems can be achieved.

That is, the present invention includes the following.
(1) a blood flow path in which a blood inlet, a first conduit, a membrane-type plasma separator that separates blood cells and plasma, a second conduit, and a blood outlet are connected in this order;
A membrane virus remover with a Japanese encephalitis virus LRV of 1 or more, and a first conduit comprising a third conduit with one end connected to the filtrate outlet of the plasma separator and the other end connected to the plasma inlet of the virus remover. Plasma channels of
One end is connected to the filtrate outlet of a membrane-type cytokine remover with a molecular weight of 30,000 and a sieve coefficient of 0.7 or more in the fraction curve prepared using dextran, and the other end of the plasma of the cytokine remover A second plasma flow path consisting of a fourth conduit connected to the inlet;
A fifth conduit having one end connected to the plasma outlet of the cytokine remover and the other end connected to the second conduit;
A sixth conduit for draining the filtrate connected to a position leading to the filtrate chamber of the cytokine remover;
A seventh conduit for fluid replacement connected to one or more of the first conduit, the second conduit, the third conduit, the fourth conduit, and the fifth conduit;
A system for removing viruses and cytokines from blood.

(2) The system for removing viruses and cytokines from blood according to (1), further comprising an eighth conduit for introduction of dialysate connected to a position leading to the filtrate chamber of the cytokine remover.

(3) The system for removing viruses and cytokines from blood according to (1) or (2), further comprising a plasma outlet at the end opposite to the plasma inlet of the virus remover.

  By using the system for removing viruses and cytokines from blood according to the present invention, it is possible to remove both viruses and cytokines simultaneously using the principle of membrane separation, and plasma separators, virus removers, cytokine removals of the same membrane area Even if a vessel is used, the filtration flow rate is high, and the filtration pressure of each can be easily controlled. By using the system for removing viruses and cytokines from the blood according to the present invention, both virus and cytokines from the blood of patients suffering from viral plasma caused by viral infection and the accompanying cytokine storm (hypercytokinemia) Can be removed at the same time.

  The virus targeted in the embodiment of the present invention is a virus that can infect mammals and cause viral plasma and hypercytokinemia, and has a relatively large particle size such as rabies virus, Spanish cold virus, and influenza virus. Although a virus can be illustrated, it is not this limitation. Since viruses are susceptible to mutation, not only known viruses but also mutated viruses such as mutant forms of highly virulent avian influenza viruses are included in the viruses targeted by the embodiments of the present invention. In addition, bacterial infection such as Staphylococcus aureus can result in hypercytokinemia, but in the embodiment of the present invention, bacteria are not targeted for removal.

  Cytokines in the embodiment of the present invention play an important role in ecological defense such as immune response, inflammatory reaction, hematopoietic system, and are physiologically active substances that also act on the endocrine system, nervous system, etc. It is an important factor for maintaining the higher-order structure of the living body. For example, interleukins (IL-1-7, IL-9-13), chemokines (IL-8, GRO-α-γ, MCP-1-4, etc.), hematopoietic factors (erythropoietin, etc.), colony stimulating factors ( GM-CSF, G-CSF, etc.), tumor necrosis factor (TNF), interferon (IFN) and the like. Among these, the cytokine to be removed in the embodiment of the present invention is a cytokine having a molecular weight of 30,000 or less.

  The blood introduction port referred to in the embodiment of the present invention refers to a portion connected to a catheter that is pierced into a patient's vein or artery, and examples thereof include a male connector and a luer connector.

  The conduit in the embodiment of the present invention refers to a tube capable of feeding a liquid, and can have a mounting portion to a pump that can be mounted on a roller pump, peristaltic pump, ironing pump, or the like. A vinyl chloride tube, a silicon tube, etc. can be used.

  The plasma separator referred to in the embodiment of the present invention is a container filled with a plasma separation membrane capable of separating whole blood into blood cell-rich components and plasma by filtering whole blood, and using the plasma separation membrane as a partition wall The container is divided into two chambers. As the plasma separation membrane, a hydrophilic polyethylene porous membrane having a pore size of about 0.1 to 0.8 μm, more preferably about 0.1 to 0.4 μm, and most preferably about 0.2 to 0.3 μm, cellulose acetate A porous membrane, a polysulfone porous membrane, a polyethersulfone porous membrane, or the like is used. If the pore size of the plasma separation membrane is too small, the permeability of the virus is deteriorated, and if it is too large, hemolysis tends to occur. The plasma separation membrane is preferably a hollow fiber membrane rather than a flat membrane because it is difficult for blood concentration spots to occur and the membrane area can be increased compared to the size of the container. Fill the hollow fiber membrane bundle along the longitudinal direction of the cylindrical container, and bond the outside of the hollow fiber membrane and the inside of the cylindrical container at both ends of the hollow fiber membrane bundle, The space is a blood chamber, the space formed between the outside of the hollow fiber membrane and the inner wall of the cylindrical container is a filtrate chamber, a blood inlet is provided at one end of the hollow fiber membrane bundle, a blood outlet is provided at the other end, and the filtrate It is preferable to provide a filtrate outlet at a position leading to the chamber. Plasma separators are commercially available from several companies and can be used.

  The blood outlet in the embodiment of the present invention refers to a portion connected to a blood return catheter or injection needle punctured in a patient's vein, and examples include male connectors, luer connectors, and the like. it can.

  The virus remover referred to in the embodiment of the present invention is obtained by filtering the plasma filtered by the plasma separator to remove the virus in the plasma and filtering it to obtain the plasma from which the virus has been removed as a filtrate. The container is filled with a virus removal membrane, the virus removal membrane is used as a partition, and the container is separated into two chambers. As the virus removal membrane, a porous membrane such as ethylene vinyl alcohol having an average pore diameter of about 45 to 60 nm, cellulose acetate, polysulfone containing a hydrophilizing agent, polyether sulfone containing a hydrophilizing agent, and hydrophilic polyvinylidene fluoride is used. It is done. Since the virus removal membrane can have a larger membrane area than the size of the container, a hollow fiber membrane is preferable to a flat membrane. Fill the hollow fiber membrane bundle along the longitudinal direction of the cylindrical container, and bond the outside of the hollow fiber membrane and the inside of the cylindrical container at both ends of the hollow fiber membrane bundle, The space is the plasma inlet chamber, the space formed between the outside of the hollow fiber membrane and the inner wall of the cylindrical container is the filtrate chamber, the plasma inlet is provided at one end of the hollow fiber membrane bundle, and the filtrate outlet is at a position leading to the filtrate chamber It is preferable to provide it. As the virus remover in the embodiment of the present invention, a commercially available filter can be used as a plasma component fractionation filter. In addition, if a plasma outlet is provided at the end opposite to the plasma inlet of the virus remover and used as an air vent or a plasma flush outlet, operability is improved. The LRV (log reduction value) of the Japanese encephalitis virus (size: 45-60 nm) of the virus remover needs to be 1 or more. A larger value of LRV means that the virus removal performance is higher, which is preferable. However, if the pore size of the virus removal membrane is too small, the protein permeability is deteriorated, which is not preferable. The range of LRV is preferably in the range of 1 to 4, more preferably in the range of 1 to 3, and still more preferably in the range of 1 to 2.

Here, a method for measuring the average pore size and LRV of the virus removal membrane will be described.
The average pore diameter is calculated by the following method. Ten hollow fiber membranes are bundled, and a module is prepared so that the effective length of each is 16 cm. One end of the module is closed, 200 mmHg pressure is applied from the other end, and water at a temperature of 37 ° C. is passed. At this time, the amount of water coming out through the hollow fiber membrane module is measured as the water permeability. Then, the average pore diameter 2r is calculated from the equation (1).

2r: average pore diameter (nm)
Kw: constant (2.0)
V: Water permeability (mL / min)
d: Film thickness of hollow fiber membrane (μm)
μ: Viscosity of water (cp)
p: Pressure difference (mmHg)
A: membrane area (cm ² )
Pr: Porosity (%)
The porosity Pr is calculated by the following method. The apparent density ρ _a is obtained from the inner diameter, the film thickness, the length and the absolute dry weight of the hollow fiber membrane, and the porosity is obtained from the equation (2).
ρ _a = W _d / V _w
= 4W _d / π · l (D _o ² −D _i ² )
P _r = (1−ρ _a / ρ _p ) × 100 (2)
P _r ; porosity (%)
W _d : Absolute dry weight of hollow fiber membrane (g)
V _w : Volume of hollow fiber membrane (cm ³ )
l: Length of hollow fiber membrane (cm 2)
D _o : outer diameter of the hollow fiber membrane (cm 2)
D _i ; Inner diameter of hollow fiber membrane (cm 2)
ρ _p ; density of membrane material (g / cm ³ )

LRV is measured as follows. Japanese encephalitis virus was prepared from Perkin strain, and the titer before and after filtration was measured by TCID ₅₀ using BHK-21 cells. The titer of the original solution was 10 ^10.5 TCID ₅₀ (mL ⁻¹ ). Here, the TCID ₅₀ (50% infection end point) method is a method for measuring the amount of virus infection. First, the virus solution to be measured is diluted 10 times, and each diluted solution is inoculated into a certain number of cells, cultured for a certain period of time, and cells in which the cytopathic effect (CPE) due to the virus is recognized are positive and are not recognized. A thing is negative. The appearance rate of positive cells in each dilution is plotted logarithmically, and the dilution rate showing 50% positive is defined as TCID ₅₀ . For this calculation, the Reed-Muench method was used. LRV is calculated by the following formula (3). The reason for using the Japanese encephalitis virus as an indicator of virus removal performance is that it has an average size as a virus. When a large amount of plasma is totally filtered, the virus removal membrane also reduces the pore size as the virus gets smaller. This is because it is easy to cause clogging, but the virus is large enough to avoid clogging.
LRV = 1og ₁₀ (N _o / N _f ) (3)
N _o ; titer of virus in the original solution before filtration N _f ; titer of virus in the filtrate after filtration

  The cytokine remover referred to in the embodiment of the present invention means that a substance having a molecular weight of 30,000 or less is easily permeated and a substance having a molecular weight of albumin or higher is difficult to permeate. The container is divided into two chambers. Cytokine removal membranes include polysulfone or polyethersulfone containing ethylene vinyl alcohol, hydrophilic polymer, and sieving coefficient with molecular weight of 30,000 in fraction curves prepared using dextran of various molecular weights of 0.7 or more. Porous membranes such as those based on cellulose, cellulose acetate and polyacrylonitrile are used. For these porous membranes, a highly water-permeable membrane is used among membranes used for artificial kidneys, or, for example, a hollow fiber porous membrane for artificial kidneys with high water permeability is taken as an example. Further, a highly water-permeable porous membrane obtained by finely adjusting the composition ratio of (core liquid) can be used. The cytokine-removing membrane is preferably a hollow fiber membrane rather than a flat membrane because the membrane area can be increased compared to the size of the container. Fill the hollow fiber membrane bundle along the longitudinal direction of the cylindrical container, and bond the outside of the hollow fiber membrane and the inside of the cylindrical container at both ends of the hollow fiber membrane bundle, The space is the plasma chamber, the space formed between the outside of the hollow fiber membrane and the inner wall of the cylindrical container is the filtrate chamber, the plasma inlet is provided at one end of the hollow fiber membrane bundle, the plasma outlet is provided at the other end, and the filtrate It is preferable to provide a filtrate outlet at a position leading to the chamber. Larger sieving coefficient with molecular weight of 30,000 in fraction curves prepared using dextran of various molecular weights is suitable for the purpose of removing cytokines, but if the pore size of cytokine removal membrane is too large, useful protein The sieving coefficient of albumin is also increased, and albumin is lost. Although lost albumin can be supplemented with a replacement fluid, if possible, the loss of albumin should be kept low. The sieve coefficient having a molecular weight of 30,000 in the fraction curve prepared using dextran having various molecular weights is preferably in the range of 0.7 to 1.0, more preferably 0.8 to 0.98, and still more preferably 0.85. It is in the range of ~ 0.95.

The sieve coefficient having a molecular weight of 30,000 in a fraction curve prepared using dextran having various molecular weights can be obtained as follows.
First, a mini module having an effective membrane area (area actually used for filtration) of the cytokine removal membrane of 0.01 m ² is prepared. To this, a 37 ° C solution obtained by dissolving dextran of various molecular weights in pure water so that the concentration of dextran of each molecular weight was 50 mg / dL was used as a mother liquor at a flow rate of 3 mL / min from the plasma inlet side to the plasma outlet side. Pour, bring the filtrate to a flow rate of 1.0 mL / min and return the filtrate to the mother liquor. After 20 minutes, the filtrate is collected for 5 minutes, and the filtrate and the mother liquor are each analyzed using liquid chromatography, the sieve coefficient is determined for each molecular weight, and the fraction curve using the sieve coefficient is drawn. And the sieve coefficient of molecular weight 30,000 is calculated | required from this fraction curve. Calculation of the dextran sieving coefficient of each molecular weight is based on the following formula (4).
Sieve coefficient = concentration of filtrate / concentration of mother liquor (4)

  The albumin sieving coefficient of the cytokine removal membrane used in the cytokine remover is preferably 0.2 or less, more preferably 0.1 or less, still more preferably 0.01 to 0.05, and preferably 0.005 to 0.01. .

The albumin sieve coefficient can be determined as follows.
First, as described above, a mini module having an effective membrane area of 0.01 m ² is prepared. Human serum prepared by adding physiological saline and adjusting the total protein concentration to 6.5 g / dL is heated to 37 ° C. and heated from the plasma inlet side to the plasma outlet side at a linear velocity of 0.4 cm / sec. The filtrate is collected by applying a pressure of 25 mmHg across the membrane, and the filtrate is returned to the mother liquor. After 60 minutes, the filtrate is collected for 5 minutes, and the concentration of albumin is determined by the EIA (enzyme immunoassay method) method and the BCG (bromcresol green coloring method) method, and the sieve coefficient is calculated from the following formula (5).
Sieve coefficient = (concentration of filtrate / concentration of mother liquor) (5)

  In the embodiment of the present invention, the seventh conduit is a conduit for replacement fluid, and is a conduit for replacing the electrolyte solution or the like according to the amount of filtrate in the cytokine remover. Albumin lost in the cytokine remover can also be replenished. When supplementing a protein having a high molecular weight such as albumin, the seventh conduit is preferably connected to the second conduit or the fifth conduit, which is a blood return circuit.

  The eighth conduit is a dialysate introduction circuit used when not only filtration but also dialysis is simultaneously performed in the cytokine remover. It can be used when low molecular weight substances are to be removed by dialysis.

Hereinafter, a method of using the system for removing viruses and cytokines from blood according to the embodiment of the present invention will be described.
FIG. 1 is a schematic diagram of a system for removing viruses and cytokines from blood according to an embodiment of the present invention. Blood from the patient is sent from the blood inlet 1 to the plasma separator 3 by a blood pump (not shown) attached on the first conduit 2 where it is separated into blood components rich in blood cells and plasma. The plasma separated by the plasma separator 3 is sent to the virus remover 6 through the third conduit 7 by a plasma pump (not shown) attached on the third conduit 7. The plasma is completely filtered through the virus removal membrane of the virus remover 6 to remove the virus. The plasma from which the virus has been removed is sent from the plasma outlet of the virus remover 6 through the fourth conduit 9 to the cytokine remover 8 from the plasma inlet of the cytokine remover 8. Plasma is aspirated by a cytokine removal pump (not shown) attached to a sixth conduit 11 connected to a position leading to the filtrate chamber of the cytokine remover 8 and passes through the sixth conduit 11 together with a low molecular substance containing cytokine. Discarded as filtrate. The remaining plasma is sent to the second conduit 4 through the plasma outlet of the cytokine remover 8, the fifth conduit 10, mixed with blood components rich in blood cells, and returned to the patient through the second conduit 4 and the blood outlet 5. The At this time, a replacement fluid corresponding to the amount of filtrate removed by the cytokine remover 8 is performed through the seventh conduit 12 by a replacement fluid pump (not shown) attached on the seventh conduit 12, and this is also returned to the patient. . In FIG. 1, it is described that the fluid is replaced in the fifth conduit 10 and returned through the second conduit 4 and the blood outlet 5. The conduit to be replenished by the seventh conduit 12 can be selected from the first conduit, the third conduit, and the fourth conduit in the case of conditions close to total filtration, depending on the flow rate of the filtrate in the cytokine remover 8. When the filtrate flow rate is smaller than the plasma flow rate from the plasma inlet, the second conduit and the fifth conduit can be used. If the filtrate flow rate is between these values, both conduits can be replenished simultaneously.

  The blood pump, plasma pump, and cytokine removal pump used in the embodiment of the present invention can be of a type in which each conduit can be externally mounted, for example, a roller pump, a finger pump, etc., and a roller pump is preferable.

When performing extracorporeal circulation treatment of blood using the system for removing viruses and cytokines from blood according to an embodiment of the present invention, a blood anticoagulant is used to prevent blood coagulation. Actually, a branch circuit for injecting the anticoagulant into the first conduit 2 is provided, and the anticoagulant is injected by a metering pump. As the anticoagulant used, heparin, low molecular weight heparin, nafamostat mesylate, gabexate mesylate, citric acid, and the like can be used.
In addition, plasma dialysis of plasma can be performed by connecting the eighth conduit 13 to a position leading to the filtrate chamber of the cytokine remover and supplying dialysate.

  Further, if a plasma outlet 14 is provided at the end of the virus remover 6 opposite to the plasma inlet, air removal during priming of the entire system or concentration of high molecular protein occurs when the virus removal membrane becomes clogged. This is useful for so-called flushing, where the layers are washed away.

  Hereinafter, embodiments of the present invention will be described in more detail by way of examples. The following examples and comparative examples were performed assuming a clinical scale of 1/10.

(Plasma separator)
A polyethylene hollow fiber hydrophilized with ethylene vinyl alcohol having an inner diameter of 330 μm, a film thickness of 50 μm and an average pore diameter of 0.3 μm was used as a plasma separation membrane. The hollow fiber membrane bundle is filled along the longitudinal direction of the cylindrical container, and the outer side of the hollow fiber membrane and the inner side of the cylindrical container are bonded with urethane at both ends of the hollow fiber membrane bundle. The inner space is the blood chamber, the space between the outside of the hollow fiber membrane and the inner wall of the cylindrical container is the filtrate chamber, the blood inlet is provided on one end of the hollow fiber membrane bundle, and the blood outlet is provided on the other end A filtrate outlet was provided at a position leading to the filtrate chamber. The effective membrane area was 0.05 m ² .

(Virus remover)
As the virus removal membrane, a cellulose hollow fiber membrane having an inner diameter of 330 μm, a film thickness of 35 μm, and an average pore diameter of 50 nm was used. Fill the hollow fiber membrane bundle along the longitudinal direction of the cylindrical container, and bond the outside of the hollow fiber membrane and the inside of the cylindrical container at both ends of the hollow fiber membrane bundle, The space is the plasma chamber, the space formed between the outside of the hollow fiber membrane and the inner wall of the cylindrical container is the filtrate chamber, the plasma inlet is provided at one end of the hollow fiber membrane bundle, and the filtrate outlet is provided at a position leading to the filtrate chamber It was. The membrane area was 0.2 m ² . The LRV of Japanese encephalitis virus in the hollow fiber membrane used here was 2.1 (99.2% removal rate) as measured by the method described above.

(Cytokine remover)
As the cytokine removal membrane, a blend hollow fiber membrane of polysulfone and polyvinylpyrrolidone having an inner diameter of 200 μm and a thickness of 45 μm was used. Fill the hollow fiber membrane bundle along the longitudinal direction of the cylindrical container, and bond the outside of the hollow fiber membrane and the inside of the cylindrical container at both ends of the hollow fiber membrane bundle, The space is the plasma chamber, the space formed between the outside of the hollow fiber membrane and the inner wall of the cylindrical container is the filtrate chamber, the plasma inlet is provided at one end of the hollow fiber membrane bundle, the plasma outlet is provided at the other end, and the filtrate A filtrate outlet was provided at a position leading to the chamber. The membrane area was 0.15 m ² . The sieve coefficient with a molecular weight of 30,000 in the fraction curve prepared using dextran, measured by the method described above, was 0.9, and the albumin sieve coefficient was 0.05.

(Blood filtration)
Bovine whole blood (500 mL) to which heparin was added at a rate of 10 units / mL as a blood anticoagulant was kept at 37 ° C., and this was used as a mother liquor for blood perfusion. The blood hematocrit was 37% and the total protein was 6.5 g / dL. The system shown in FIG. 1 was used, and a silicon tube was used for each conduit, and a peristaltic pump was used for each pump. The blood flow rate was 6 mL / min, the filtration flow rate in the plasma separator was 3 mL / min, the filtration flow rate in the virus remover was also 3 mL / min, and the filtration flow rate of the cytokine removal filter was 2 mL / min. As a replacement fluid, physiological saline was injected into the fifth conduit at a flow rate of 2 mL / min. Then, blood perfusion was performed until 500 mL of blood was completed.

  As a result, abnormal filtration pressure due to clogging of the plasma separator, virus remover, and cytokine remover was not observed, and the plasma filtration volume of the virus remover was 250 mL, and the filtrate in the cytokine remover was 167 mL. In the embodiment of the present invention, the abnormal filtration pressure means that the transmembrane pressure difference (TMP) is 200 mmHg or more.

  That is, 79% of the total plasma volume of 315 mL (500 mL × (1−0.37)) can be filtered with a virus remover using a virus removal membrane with Japanese encephalitis virus LRV of 2.1, and a molecular weight of 30,000 dextran sieve. A cytokine remover using a cytokine removal membrane with a coefficient of 0.9 and an albumin sieve coefficient of 0.05 was able to remove 53% of the total plasma volume. The time required was 83 minutes.

(Comparative Example 1)
The same plasma separator, virus remover, and cytokine remover as those in Example 1 were used. The blood was obtained from the same individual as in Example 1, and 500 mL of whole blood was kept at 37 ° C. as a mother liquor in the same manner as in Example 1. The same system as shown in FIG. 2 was used as the system, and the tubes, pumps, and the like were the same as in Example 1. The blood flow rate was 6 mL / min, the filtrate flow rate of the plasma separator and the filtrate flow rate of the virus removal filter were 1.8 mL / min, and the filtrate flow rate of the cytokine removal filter was 1.2 mL / min. As the replacement fluid, physiological saline was injected into the blood outlet of the plasma separator at a flow rate of 1.2 mL / min. The reason for setting such a flow rate is to set the flow rate of plasma filtered from whole blood to one half of the blood flow rate, as in the first embodiment.

  As a result, abnormal filtration pressure due to clogging of the plasma separator, virus remover, and cytokine remover was not observed, but the amount of plasma filtration of the virus removal filter was 150 mL, and the filtrate in the cytokine removal filter was 100 mL. It was only 60% of the amount.

  That is, only 48% of the total plasma volume of 315 mL can be filtered with a virus remover using a virus removal membrane with a Japanese encephalitis virus LRV of 2.1, a dextran sieve coefficient with a molecular weight of 30,000 is 0.9, and an albumin sieve coefficient is Only 32% of the total plasma volume could be removed with a cytokine remover using a 0.05 cytokine removal membrane. The required time was 83 minutes as in Example 1.

  Blood filtration was performed under the same conditions as in Example 1 except that a polysulfone hollow fiber membrane containing polyvinylpyrrolidone having an inner diameter of 230 μm, a film thickness of 45 μm, and an average pore diameter of 60 nm was used as the virus removal membrane. The LRV of the Japanese encephalitis virus of the virus removal membrane used here was 1.1 (removal rate: 92.1%) as measured by the method described above.

  As a result, abnormal filtration pressure due to clogging of the plasma separator, virus remover, and cytokine remover was not observed, and the plasma filtration volume of the virus remover was 250 mL, and the filtrate in the cytokine remover was 167 mL.

  That is, 79% of the total plasma volume of 315 mL can be filtered with a virus remover using a virus removal membrane with a Japanese encephalitis virus LRV of 1.1, a dextran sieve coefficient with a molecular weight of 30,000 is 0.9, and an albumin sieve coefficient is 0. A cytokine remover using a .05 cytokine removal membrane was able to remove 53% of the total plasma volume. The time required was 83 minutes.

  Blood filtration was performed under the same conditions as in Example 1 except that an ethylene vinyl alcohol hollow fiber membrane having an inner diameter of 175 μm, a film thickness of 40 μm, and an average pore diameter of 45 nm was used as the virus removal membrane. The LRV of the Japanese encephalitis virus in the virus removal membrane used here was 2.7 (99.8% removal rate) as measured by the method described above.

  As a result, abnormal filtration pressure due to clogging of the plasma separator, virus remover, and cytokine remover was not observed, and the plasma filtration volume of the virus remover was 250 mL, and the filtrate in the cytokine remover was 167 mL.

  That is, 79% of the total plasma volume of 315 mL can be filtered with a virus remover using a virus removal membrane with a Japanese encephalitis virus LRV of 2.7, a dextran sieve coefficient with a molecular weight of 30,000 is 0.9, and an albumin sieve coefficient is 0. A cytokine remover using a .05 cytokine removal membrane was able to remove 53% of the total plasma volume. The time required was 83 minutes.

  Blood filtration was performed under the same conditions as in Example 1 except that a polyacrylonitrile hollow fiber membrane having an inner diameter of 200 μm and a film thickness of 35 μm was used as the cytokine removal membrane. The sieving coefficient with a molecular weight of 30,000 was 0.7 and the albumin sieving coefficient was 0.1 in the fractionation curve prepared using dextran measured by the above-described method of the cytokine removal membrane.

  As a result, abnormal filtration pressure due to clogging of the plasma separator, virus remover, and cytokine remover was not observed, and the plasma filtration volume of the virus remover was 250 mL, and the filtrate in the cytokine remover was 167 mL.

  In other words, 79% of the total plasma volume of 315 mL can be filtered with a virus remover using a virus removal membrane with a Japanese encephalitis virus LRV of 2.7, a dextran sieve coefficient with a molecular weight of 30,000 is 0.7, and an albumin sieve coefficient is 0. A cytokine remover using a cytokine removal membrane of .1 could remove 53% of the total plasma volume. The time required was 83 minutes.

  Blood filtration was performed under the same conditions as in Example 1 except that physiological saline was injected into the fourth conduit 9 at a flow rate of 3 mL / min and the filtration flow rate of the cytokine removal filter was changed to 5 mL / min.

  As a result, abnormal filtration pressure due to clogging of the plasma separator, virus remover, and cytokine remover was not observed, the plasma filtration volume of the virus remover was 250 mL, and the filtrate in the cytokine remover was 415 mL (diluted by half). Therefore, the filtered plasma volume was 207.5 mL).

  That is, 79% of the total plasma volume of 315 mL can be filtered with a virus remover using a virus removal membrane with a Japanese encephalitis virus LRV of 2.1, a dextran sieve coefficient with a molecular weight of 30,000 is 0.9, and an albumin sieve coefficient is 0. 66% of the total plasma volume could be removed with a cytokine remover using a .05 cytokine removal membrane. The time required was 83 minutes.

(Reference example)
The cytokine removal performance of the cytokine remover used in Example 1 and Example 4 was measured. A solution obtained by adding interleukin-6 (IL-6, molecular weight 22-28 kD) to 315 mL of bovine serum was used as a mother liquor, and was passed through the cytokine remover at a flow rate of 3 mL / min, and the serum that passed through the cytokine remover was returned to the mother liquor. . The filtrate flow rate was 2 mL / min, and physiological saline was added to the mother liquor at the same flow rate as the filtrate flow rate. Serum was perfused and filtered under the above conditions for 83 minutes, and the IL-6 concentration in the mother liquor before perfusion and the IL-6 concentration in the mother liquor after perfusion were measured by enzyme immunoassay (ELISA method). As a result, in the cytokine remover of Example 1, the IL-6 concentration was lowered to 47%, and in the cytokine remover of Example 4, it was lowered to 59%.

  By using the system for removing viruses and cytokines from the blood according to the present invention, both virus and cytokines from the blood of patients suffering from viral plasma caused by viral infection and the accompanying cytokine storm (hypercytokinemia) Can be removed at the same time, and the virus and cytokine removal performance is good and the operability is good. Therefore, it is expected to be useful for the treatment of the above patients in the medical field.

It is a schematic diagram which shows the system which removes a virus and cytokine from the blood which concerns on embodiment of this invention. It is a schematic diagram which shows the system which set the new subject that removes both a virus and cytokine from a patient's blood simultaneously, and combined the membrane separation technique.

Explanation of symbols

1 blood inlet 2 first conduit 3 plasma separator 4 second conduit 5 blood outlet 6 virus remover 7 third conduit 8 cytokine remover 9 fourth conduit 10 fifth conduit 11 sixth conduit 12 seventh conduit 13 eighth Conduit 14 Plasma outlet 101 Cytokine remover 102 Plasma separator 103 Virus remover

Claims (3)

A blood flow path in which a blood inlet, a first conduit, a membrane-type plasma separator that separates blood cells and plasma, a second conduit, and a blood outlet are connected in this order;
A membrane type virus remover having an LRV of Japanese encephalitis virus of 1 or more, and a third conduit having one end connected to the filtrate outlet of the plasma separator and the other end connected to the plasma inlet of the virus remover A first plasma channel;
A membrane type cytokine remover having a molecular weight of 30,000 in a fraction curve prepared using dextran and having a sieve coefficient of 0.7 or more, and one end connected to the filtrate outlet of the virus remover, and the other end of the cytokine remover A second plasma flow path comprising a fourth conduit connected to the plasma inlet of
A fifth conduit having one end connected to the plasma outlet of the cytokine remover and the other end connected to the second conduit;
A sixth conduit for draining the filtrate connected to a position leading to the filtrate chamber of the cytokine remover;
A seventh conduit for injecting replacement fluid connected to any one or more of the first conduit, the second conduit, the third conduit, the fourth conduit, and the fifth conduit;
A system for removing viruses and cytokines from blood.   The system for removing viruses and cytokines from blood according to claim 1, further comprising an eighth conduit for introducing dialysate connected to a position leading to the filtrate chamber of the cytokine remover.   The system for removing viruses and cytokines from blood according to claim 1 or 2, further comprising a plasma outlet at an end opposite to the plasma inlet of the virus remover.

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