JPH088928B2 - Device for producing plasma from which low-density lipoprotein has been removed - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an apparatus for producing plasma from which low density lipoprotein, which is considered to have a close relationship with various diseases caused by an increase in plasma lipid, is selectively removed. <Br / > Ru.

[0002]

2. Description of the Related Art As is well known, it is considered that the increase of lipids in blood, particularly low density lipoprotein, is closely related to the cause or progression of arteriosclerosis. When arteriosclerosis progresses, there is an extremely high possibility of causing serious symptoms of the circulatory system such as myocardial infarction and cerebral infarction, and the mortality rate is high. So blood,
It has been expected that the selective adsorption and removal of low-density lipoprotein from body fluid components such as plasma will prevent the progression of the above-mentioned diseases, reduce the symptoms, and accelerate the healing.

The existing technology that can be used for the above purpose is adsorption by an adsorbent in which heparin is immobilized on agarose gel (Lupien, PJ, et.al .: A new approach to the man.
agement of familial hypercholesternlemia. Removal
of plasma-cholesterol based on the principle of af
finity chromatography. Lancet, 2: 1261 ~ 1264,197
6.) and chromatography using glass powder or glass beads (Carlson, LA: Chromatogra
phic separation of serum Lipoprotein on glass powd
er colums.Description of the method and some appl
ications. Clin. Chim. Acta, 5: 528 ~ 538, 1960.)
There is.

However, the adsorbent having heparin immobilized on agarose has a selective adsorption capacity for low-density lipoprotein, but its adsorption capacity is not sufficient, and since agarose is used as a carrier, its mechanical strength is poor. Sufficient and easy to handle
The operability was poor, clogging was likely to occur when body fluid was poured, and the pores were destroyed by the sterilization operation, making it extremely difficult to use. Further, the method using glass powder or glass beads is not practical because it has a low adsorption capacity and a low adsorption selectivity.

[0005]

The object of the present invention is generally applicable in view of the problems of the adsorption operation based on the above-mentioned conventional techniques, and selectively adsorbs low-density lipoprotein with high efficiency. However, it is an object of the present invention to provide an apparatus for producing purified plasma suitable for purification or regeneration of plasma, which has less non-selective adsorption, is safe, and can be easily sterilized.

[0006]

Means for Solving the Problems The inventors of the present invention have made extensive studies in accordance with the above-mentioned object, and as a result, have a chain polyanion having a large number of functional groups having a negative charge in the molecule and having a relatively large molecular weight on the surface. An adsorbent having a negative charge density within a specific range has a surprisingly high efficiency of adsorbing a low-density lipoprotein, and non-selective adsorption is small, and blood coagulation, a fibrinolytic system, The inventors have found that the complement system is rarely activated, and completed the present invention.

That is, the present invention comprises a chain polyanion having one or more functional groups having a negative charge in plasma per molecular weight of 200 and having a molecular weight of 5000 or more bound to the surface of a water-insoluble carrier. Charge density is 1μeq / m
An adsorbent having 1 to 1 meq / ml of 1 or more and 1 meq / ml or less is packed in a column having a plasma inlet and an outlet with 50 to 400 ml of space between two adsorbent leakage prevention filters, and a low specific gravity This is an apparatus for producing plasma in which low-density lipoprotein is selectively removed by intermittently passing plasma containing lipoprotein.

The adsorbent to be used in the present invention is a low-density lipoprotein. More specifically, it has a molecular weight of 2.2 × 10 ⁶ to 3.5 × 10 ⁶ and a hydration density of 1.0.
03 to 1.034 (g / ml), levitation coefficient (1.06
3) is 0 to 20 × 10 ^-13 cm ・ sec ^-1・ dyn ^-1・
g ^-1 , lipoprotein (SCA with a diameter of 20.0 to 30.0 nm)
NU, AM: plasma lipoproteins: an introduction.
"The Biochemistry of Atherosclerosis" ed. By SC
ANU AM, 1979, P.3-8). Lipoprotein with a smaller specific gravity than this, that is, the levitation coefficient (1.06
Lipoproteins having 3) larger than 20 × 10 ⁻¹³ cm · sec ⁻¹ · dyn ⁻¹ · g ⁻¹ may be adsorbed, but high specific gravity lipoproteins having high specific gravity are preferably not adsorbed.

The polyanion portion referred to in the present invention is a functional group having a molecular weight of 5,000 or more and having a negative charge in one molecule, that is, a functional group having a negative charge in plasma such as a sulfonic acid group and a sulfate group. It has a lot of groups. For example, polyvinyl sulfonic acid, vinyl synthetic polyanion such as polyvinyl phosphoric acid, polystyrene sulfonic acid, styrene polyanion such as polystyrene phosphoric acid, polyanion of sulfate each sugar such as dextran sulfate, peptide polyanion such as polytaurine, RNA, Examples thereof include nucleic acid-based polyanions such as DNA and polyanions such as polyphosphoric acid, polyphosphate ester, and poly-α-methylstyrene sulfonic acid.

Among them, the polyanion obtained by synthesizing is excellent in its chemical stability and can be sterilized by high pressure steam,
It is superior to natural products and can be recommended because it is easy to obtain a stable product against γ-ray sterilization, ethylene oxide sterilization, and the like, and the molecular weight can be adjusted relatively easily. Further, in the case of a polyanion obtained by synthesis, a polyanion which hardly causes activation of complement as seen in natural polysaccharides is easily obtained, which is preferable. Further, a vinyl anion that can be directly graft-polymerized to a carrier gives more preferable results in that a polyanion having a large molecular weight can be immobilized on the carrier with a high retention amount.

Further, since the low specific gravity lipoprotein, which is a target substance for adsorption, is a huge lipoprotein having a diameter of about 200Å, it is preferable that the structure of the polyanion be a chain structure, which extends long from the surface of the adsorbent. Is preferred. Further, the negative charge density in the chain polyanion has a molecular weight of 200
There is preferably at least one per hit. More preferably, it is desirable to have one in a unit having a molecular weight of 70 to 150. The molecular weight mentioned here includes the molecular weight of a functional group having a negative charge. When the molecular weight of the chain polyanion becomes small, the low specific gravity lipoprotein is not adsorbed so much.
000 or more is preferable, and the range of 25,000 to 1000000 is more preferable. It is considered that the large number of negatively charged functional groups possessed by the chain polyanion recognizes many points of the low-density lipoprotein to bind the low-density lipoprotein with strong Coulomb force.

The density of the negative charges is in the range of 1 μeq to 1 meq per 1 ml of the adsorbent, and the adsorption performance of the low-density lipoprotein is good,
It is in an appropriate range with good adsorption selectivity and little influence on coagulation / fibrinolysis system and complement system. If the negative charge density is lower than 1 μeq / ml, the adsorption capacity of low-density lipoprotein is less than the practical performance, and if it exceeds 1 meq, non-selective adsorption increases, which adversely affects the coagulation fibrinolytic system and complement system. More preferable range is 5μ
eq / ml to 700μeq / ml, more preferably 10μ
eq / ml to 500 μeq / ml, more preferably 20 μeq / ml
ml to 300 μeq / ml. The negative charge density can be measured according to the usual method for measuring the ion exchange capacity of a cation exchange resin.

The method for producing the adsorbent used in the present invention is as follows:
For example, a method of activating a carrier to covalently bond a chain synthetic polyanion at one end thereof, a method of graft-polymerizing an anion monomer on the carrier to form a graft chain of a polyanion, and the like can be mentioned. The carrier is a polyanion having a molecular weight of 5000 or more at a negative charge density of 1 μeq / ml.
From 1 to 1 meq / ml, both hydrophilic and hydrophobic carriers can be used. However, when a hydrophobic carrier is used, nonspecific adsorption of albumin to the carrier sometimes occurs, resulting in hydrophilicity. The carrier gives better results.

The shape of the insoluble carrier may be any known shape such as a particle shape, a fiber shape, a hollow fiber shape, a film shape, etc.
From the viewpoint of the amount of chain polyanion having a molecular weight of 00 or more and the handling property as an adsorbent, the particulate or fibrous form is preferable.

The particulate carrier preferably has an average particle size of 25 μm to 2500 μm. The average particle size is classified in running water using a sieve specified in JIS-Z-8801, and then the intermediate value between the upper limit particle size and the adjusted particle size of each class is taken as the particle size of each class, and the weight average is the average. Calculate the particle size. The particle shape is preferably spherical, but is not particularly limited. When the average particle size is 2500 μm or more, the adsorption amount and adsorption rate of low-density lipoprotein decrease, and when the average particle size is 25 μm or less, coagulation system activation and blood cell adhesion are likely to occur. Examples of the particulate carrier that can be used include agarose-based, dextran-based, cellulose-based, polyacrylamide-based, glass-based, and silica-based activated carbon-based carriers, and hydrophilic carriers having a gel structure give good results. In addition, a commonly used carrier for immobilized enzyme and affinity chromatography can be used without particular limitation. Here, the carrier exclusion molecular weight of the carrier needs to be 2,000,000 or more, preferably 2.5 million to 10 million, and more preferably 3 million to 7 million. If this is shown by the average pore diameter, it is 20
It is in the range of 0Å to 3000Å, more preferably in the range of 250Å to 700Å, and preferably in the range of 300 to 650Å.

Porous particles, especially porous polymers, can also be used as the particulate carrier. The porous polymer particles used in the present invention are capable of immobilizing a polyanion having a molecular weight of 5000 or more at a negative charge density in the range of 1 μeq / ml to 1 meq / ml, and the polymer composition is polyamide type, polyester type or polyester type. Known polymers that can have a porous structure such as polymer, polyurethane, and vinyl compound polymers can be used, but vinyl compound porous polymer particles hydrophilized with a hydrophilic monomer give preferable results. .

It is necessary that the adsorbent used in the present invention does not cause clogging when flowing blood. Therefore, the carrier used in the present invention is preferably a hard carrier, and a synthetic polymer carrier, an inorganic carrier or the like is preferably used. The hard carrier referred to here is one in which the physical properties of the carrier are maintained at a certain value or more when an external force is applied. Specifically, the gel is filled in a container having a diameter of 10 mm and a length of 50 mm, When water is applied, the gel volume reduction rate is 10% when the difference between the inlet pressure and the outlet pressure of the column is 200 mmHg.
The following is preferable.

The porous structure preferably has an average pore size in the range of 200Å to 3000Å. If the average pore size is too small, the amount of low specific gravity lipoprotein adsorbed is too small, and if it is too large, It is not practical because the strength of polymer particles decreases and the surface area decreases. The surface area is preferably at least 10 m ² / g or more, more preferably 55 m ² / g or more. It is preferably 100 m ² / g or more.

The average pore size is measured by a mercury porosimetry porosimeter. This method is a method in which mercury is press-fitted into a porous material, the pore amount is determined from the amount of mercury that has entered, and the pore size is determined from the pressure required for press-fitting, and pores of 40 Å or more can be measured. The hole of the present invention is defined as a communicating hole from the surface having a hole diameter of 40 Å or more. The average pore diameter is dv, where r is the pore diameter and V is the cumulative porosity measured by a porosimeter.
The value of r is the maximum value of / dlogr.

When a fibrous carrier is used, the fiber diameter is 1 μm to 50 μm, more preferably 5 μm to 2 μm.
It is preferably in the range of 5 μm. If the fiber diameter is too large, the amount and rate of adsorption of low-density lipoprotein will decrease, and if it is too small, coagulation system activation, blood cell adhesion, and clogging will occur easily. Fibrous carriers that can be used include regenerated cellulosic fibers, nylon, acrylic,
Known fibers such as polyester can be generally used.

The method for immobilizing the chain polyanion having a molecular weight of 5000 or more on the surface of the insoluble carrier is a covalent bond,
Any known method such as ionic bond, physical adsorption, embedding or precipitation insolubilization on the polymer surface can be used, but in view of the elution property of the chain polyanion, it can be fixed and insolubilized by covalent bonding. preferable. Therefore, it is possible to use an immobilized enzyme, a known method for activating a carrier generally used in affinity chromatography, a method for binding to a ligand, and a method for graft polymerization using a carrier as a trunk polymer and a chain polyanion as a branch.

Examples of the activation method include a cyanogen halide method, an epichlorohydrin method, a bisepoxide method, a halogenated triazine method, a bromoacetyl bromide method, an ethyl chloroformate method, and a 1,1′-carbonyldiimidazole method. be able to. The activation method of the present invention is not limited to the above examples, as long as it can perform a substitution and / or addition reaction with a nucleophilic reactive group having an active hydrogen such as an amino group, a hydroxyl group and a thiol group of a ligand. Considering the thermal stability and thermal stability, the method using an epoxide is preferable, and the epichlorohydrin method is particularly recommended. Further, for a carrier having a silanol group such as a silica type or a glass type, various silane coupling agents are preferably used.

Examples of the graft polymerization method include a chain transfer method, an emulsion polymerization method, a cerium salt, a persulfate-lithium halide, a hydrogen peroxide-metal salt, and the like. A mercapto group, a graft polymerization method with a polymer having a functional group such as a diazo group, a graft polymerization method by air or ozone oxidation, a radiation graft method, and the like, among them, a hydroxyl group, a thiol,
A method of graft-polymerizing an anion monomer onto a carrier having a reducing group such as aldehyde or amine with an initiator such as cerium salt or iron salt is simple and recommended.
The graft polymerization system is preferably used because it can fix a polyanion having a relatively large molecular weight to the inside of the carrier.

Two or more kinds of chain polyanions having a molecular weight of 5,000 or more may be bound to the carrier. The method for binding the polyanion having a molecular weight of at least 5000 to the carrier at a negative charge density of 1 μeq / ml to 1 meq / ml has been described in detail above by exemplifying the method for producing the adsorbent used in the present invention. The adsorbent of the invention is not limited to this.

For example, a method of polymerizing (copolymerizing) using a polymerizable monomer having a chain polyanion having a molecular weight of 5000 or more, a method of activating a polyanion having a molecular weight of 5000 or more, and then binding it to a carrier are also adopted. can do. The low-density lipoprotein adsorbent used in the present invention is used by being filled and held in a container having a blood inlet / outlet port.

In the drawings, reference numeral 1 shows an example of an adsorption device containing a low specific gravity lipoprotein adsorbent used in the present invention. An adsorbent leakage prevention filter 3 is provided inside an opening of one end of a cylinder 2. A cap 6 having a blood introduction port is screw-fitted through a tight packing 4, and a blood outlet port 7 is provided through a packing 4'where an adsorbent leakage prevention filter 3'is tightly fitted inside the other end opening of the cylinder 2. The cap 8 having the above is screw-fitted to form a container, and the adsorbent layer 9 is formed by filling and holding the adsorbent between the filters 3 and 3 '.

The adsorbent layer 9 may be filled with the aforementioned low-density lipoprotein adsorbent alone, or may be mixed or laminated with another adsorbent. As another adsorbent, for example, activated carbon having a wide adsorption capacity can be used. As a result, a broader clinical effect due to the synergistic effect of the adsorbent can be expected. When used for extracorporeal circulation, the volume of the adsorbent layer 9 is preferably about 50 to 400 ml. When the apparatus of the present invention is used in the form of extracorporeal circulation, it can be roughly used in the following two ways. One is that the blood taken out of the body is separated into a blood plasma component and a blood cell component by using a centrifuge, and then only the blood plasma component is passed through the device to be purified and then combined with the blood cell component in the body. Is a way to get it back,
The other is a method in which blood taken out from the body is directly passed through the device for purification.

Regarding the passage speed of blood or plasma, since the adsorbent has a very high adsorption efficiency, the particle size of the adsorbent can be made coarse and the packing degree can be lowered, so that the shape of the adsorbent layer can be reduced. A high passing speed can be given regardless of. Therefore, a large amount of body fluid can be treated. In the device of the present invention, blood containing low-density lipoprotein is intermittently passed through the adsorption device. The intermittent liquid flow has various advantages as illustrated below.

First, the adsorbent, which has reached a saturated adsorption amount and is deactivated, can be regenerated between the intermittent passages. In this case, two or more same adsorbing devices are prepared so that the blood to be treated is continuously processed, and the blood to be treated is always passed through one adsorbing device to regenerate other adsorbing devices or to prepare for regeneration. If it is placed in a state, efficient production of purified plasma can be realized.

[0030] Usually, the amount of low-density lipoprotein contained in the blood to be treated cannot be accurately grasped before the treatment. Therefore, the treatment capacity of the adsorption device, the amount of the blood to be treated, and It is difficult to know the balance with the amount of low-density lipoprotein of B. Therefore, the amount of blood that is far below the processing capacity of the adsorption device is discarded and the adsorption device is discarded, or conversely, the amount of blood to be processed that exceeds the processing capacity of the adsorption device is passed through the adsorption device. The material may not be able to adsorb the low-density lipoprotein contained in the blood to be treated.

In order to avoid such an inconvenience, the blood to be treated is intermittently passed through the adsorbing device, and the plasma which comes out from the blood outlet side of the adsorbing device is appropriately supplied between the intermittent liquid passing. By confirming the processing capacity of the adsorption device by checking whether or not low-density lipoprotein remains in the inside, it is possible to save the trouble of re-adsorbing the blood to be treated in the end,
Moreover, since the number of times of damaging precious blood to be contacted with the adsorbent is reduced and the adsorption device can be utilized without wasting its capacity, the plasma from which low-density lipoprotein has been removed can be used. It can be efficiently manufactured in a short time.

[0032]

As described above, the adsorbent used in the present invention is capable of selectively adsorbing and removing low-density lipoprotein in blood at a high rate, and an adsorbing device using the adsorbent is very compact. It is simple and safe. In addition, other components in plasma proteins are not adsorbed non-selectively, low-density lipoproteins can be adsorbed with high efficiency, and coagulation fibrinolysis and complement system are hardly activated. The device of the present invention
The device is applicable to purification and regeneration of blood such as hyperlipidemia, and is also effective for safe and reliable treatment of diseases caused by hyperlipidemia.

[0033]

The embodiments of the present invention will be described in more detail with reference to the following examples. Example 1 Vinyl acetate 100 g, triallyl isocyanurate 4
1.4 g (X = 0.40), ethyl acetate 100 g, heptane 100 g, polyvinyl acetate (polymerization degree 500) 7.5
g and 2,2'-azobisisobutyronitrile 3.8
g, and 1% by weight of polyvinyl alcohol, 0.05% by weight of sodium dihydrogen phosphate dihydrate, and 400 ml of water in which 1.5% by weight of disodium hydrogen phosphate dodecahydrate was dissolved. Was placed in a flask and sufficiently stirred, followed by heating and stirring at 65 ° C. for 18 hours and further at 75 ° C. for 5 hours to carry out suspension polymerization to obtain a granular copolymer. Filter water wash,
Then, after extraction with acetone, transesterification of the copolymer was carried out at 40 ° C. for 18 hours in a solution consisting of 46.5 g of sodium hydroxide and 2 liters of methanol. The obtained polyvinyl alcohol gel had an average particle size of 98 μm and an exclusion limit molecular weight of 4.8 million.

Styrene sulfonic acid (one sulfonic acid group per 184 molecular weight) per 1 volume of this gel
Was added with 10 volumes of a methanol solution in which 5 w / v% was dissolved,
The adsorbent was obtained by irradiating 25 kGy of γ-rays and radiation-polymerizing polystyrene sulfonic acid on the surface of the polyvinyl alcohol gel. The negative charge density of the obtained adsorbent was 58 μeq / ml,
The molecular weight of polystyrene sulfonic acid was about 5,500.

Cholesterol adsorption capacity was measured as follows using the obtained adsorbent. Add 1 volume of adsorbent to 6 volumes of plasma with total cholesterol concentration of 235 mg / dl,
And reacted for 2 hours. The adsorption capacity of the adsorbent calculated from the amount of total cholesterol reduction before and after the reaction is 1 per 1 ml of adsorbent.
It was 4.2 mg. 200 pp of the adsorbent thus obtained
An adsorber was experimentally manufactured by filling the same container having an inlet and an outlet for plasma as shown in the drawing together with m 2 of sodium pyrosulfite buffer solution and performing high-pressure steam sterilization. The amount of adsorbent that could be filled in the adsorber was 10 ml.

Total cholesterol concentration of 235 mg in the adsorber
20 ml of plasma / dl was perfused at 0.5 ml / min. At this time, the total cholesterol concentration in the plasma discharged from the adsorber was 2.0 mg /
It was dl, and the adsorptivity of cholesterol was very high.
Thereafter, the perfusion of plasma was stopped, the adsorber was washed with 30 ml of physiological saline (pharmacy method), and then stored at 4 ° C. for 2 days. After storage, total cholesterol level 2
20 ml of 35 mg / dl plasma was perfused. At this time, the total cholesterol concentration in the plasma discharged from the adsorber was 4.6 mg / dl, which showed the same high cholesterol adsorbing capacity as before storage. As described above, the adsorbability of cholesterol was stably high before and after storage, and the adsorbent could be used without wasting the adsorbing ability of the adsorbent.

Example 2 An adsorption device similar to the adsorption device used in Example 1 was used. Total cholesterol concentration of 250.7 mg / dl in the adsorber
100 ml of plasma was perfused at 0.5 ml / min. The total cholesterol concentration in the plasma discharged from the adsorber at the beginning of perfusion was 0.2 mg /
Although it exhibited high adsorption performance, the total cholesterol in the plasma flowing out from the adsorber gradually increased from the time when approximately 50 ml of plasma was perfused, and when 100 ml of plasma was perfused, the total cholesterol before and after the adsorbent was No reduction in cholesterol was seen.

Next, the perfusion of plasma was stopped and the physiological saline solution 5 was added.
0 ml of this adsorber was perfused at 0.5 ml / min, and then 10 ml of a 5% sodium chloride aqueous solution was perfused at 0.5 ml / min to release LDL cholesterol adsorbed on the adsorbent. After release, 50 ml of physiological saline was perfused into the adsorber at 0.5 ml / min, and again 50 ml of plasma having a total cholesterol concentration of 250.7 mg / dl was perfused at 0.5 ml / min. At this time, the total cholesterol concentration in the plasma discharged from the adsorber was 0.4 mg / dl,
It exhibited high adsorption performance again.

[Brief description of drawings]

FIG. 1 is a cross-sectional view showing an example of an adsorption device for selectively adsorbing low-density lipoprotein incorporated in the device of the present invention.

Claims (1)

[Claims] A chain polyanion having at least one functional group having a negative charge in plasma per molecular weight of 200 and having a molecular weight of 5000 or more is bound to the surface of a water-insoluble carrier, and the negative charge density thereof is 1 μeq / ml or more, 1 meq / 4 or less of the adsorbent, and 50 to 4 between two adsorbent leakage prevention filters separated in a column having a plasma inlet and an outlet.
The adsorption device formed by 00ml filled, the plasma containing low density lipoprotein intermittently passed through, to produce a plasma for selectively removing a low density lipoprotein device.