JP2928589B2 - Adsorbent for modified LDL and apparatus for removing modified LDL using the same - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to an adsorbent for adsorbing and removing harmful components from a liquid, and a removing device using the same. More specifically, the present invention relates to an adsorbent for selectively removing denatured LDL from body fluid to reduce various symptoms of arteriosclerosis or to halt the progress thereof, and to an apparatus for removing denatured LDL using the same.

[Problems to be Solved by Conventional Techniques / Inventions] The most characteristic lesion at the onset of arteriosclerosis is the presence of atheromatous plaque, and its major cell component is called a vacuole cell These cells have accumulated a large amount of ester-type cholesterol. The origin of the alveolar cells is thought to be that monocyte-derived macrophages in the blood formed cholesterol. These alveolar cells collect to form a fatty streak and further form atheromatous plaque, which leads to stenosis and occlusion of blood vessels, and causes arteriosclerotic diseases such as myocardial infarction. The source of cholesterol in the alveolar cells is thought to be from blood LDL.

Macrophages usually recognize and take up LDL
LD with poorly developed receptor and degenerated instead
L, especially denatured LDL with increased negative charge, such as oxidized LD
It was thought that there was a scavenger receptor that took in L, acetylated LDL, etc. Recently, this receptor was purified and its amino acid sequence was reported.

Until now, there have been drug-based and surgical methods for treating arteriosclerosis, but all of these methods inhibit LDL metabolism or push stenotic blood vessels.

The present inventors have conducted intensive studies in view of such circumstances, and as a result, selectively denatured LDL without substantially losing the active ingredient in the body fluid and without removing unmodified LDL (hereinafter referred to as normal LDL). Find an adsorbent that can be adsorbed,
The present invention has been completed.

[Means for Solving the Problems] That is, the present invention provides: (1) a modified LDL characterized in that a peptide having a selective affinity for the modified LDL is immobilized on a water-insoluble porous carrier. (2) a container having an inlet and an outlet for a fluid; an adsorbent filled in the container, which selectively binds the modified LDL; and a fluid and the fluid. The present invention relates to an apparatus for removing denatured LDL, which comprises a filter that can pass components to be passed but cannot pass the adsorbent.

[Actions and Examples] In the present invention, body fluids include blood, plasma, serum, ascites,
Lymph fluid, intra-articular fluid and fraction components obtained therefrom, as well as other biologically derived humoral components.

In the present invention, the modified LDL refers to a protein having properties different from normal LDL in terms of surface charge, structure, and the like as a result of normal LDL being oxidized or undergoing some chemical change.
Representative examples of these proteins include acetylated LDL, maleylated LDL, oxidized LDL, and the like.

A method for measuring denatured LDL present in the body has not been established at present, but its existence is considered to be certain. Experimentally, denatured LDL prepared chemically, for example, acetylated LDL, maleylated LDL, and oxidized LDL can be measured by labeling with a radioisotope. In other words, the amount of chemically-modified radioisotope-labeled LDL can be measured by the intensity of radiation, and normal LDL
The ratio of modified LDL in the mixture of
(The amount obtained by combining the denatured LDL amount and the normal LDL amount) can be measured as the total cholesterol amount, and the denatured LDL amount can be determined from the radiation intensity.

In the present invention, the water-insoluble carrier refers to a substance having a property of being insoluble in water for immobilizing a peptide having an affinity for denatured LDL. The water-insoluble carrier used in the present invention preferably has large pores of continuous fine pores.
That is, since the modified LDL is a macromolecule having a molecular weight of 1,000,000 or more, it is necessary that the modified LDL can easily enter the porous body in order to adsorb it efficiently.

There are various methods for measuring the pore size, and the mercury intrusion method is most frequently used, but it is difficult to apply the method to a case of a hydrophilic porous material. The exclusion limit molecular weight is often used as a standard of the pore diameter instead, and can be applied to any of a hydrophilic porous body and a hydrophobic porous body. As described in a compendium (eg, Hiroyuki Hatano, Toshihiko Hanai, Experimental High Performance Liquid Chromatography, Chemical Doujinshi) etc., molecules that cannot enter (exclude) pores in gel permeation chromatography Of those having the smallest molecular weight.

The exclusion limit molecular weight is generally investigated for globular proteins, dextran, polyethylene glycol, etc., but in the case of the carrier used in the present invention, the value obtained using the globular protein which seems to be most similar to LDL It is appropriate to use

The preferred exclusion limit molecular weight of the water-insoluble porous substance used in the present invention is from 400,000 to 100 million.

As for the porous structure of the water-insoluble carrier, it is preferable that the pore volume is 20% or more from the viewpoint that total porosity is more preferable than surface porosity and that the adsorption capacity is large. As the shape of the water-insoluble porous carrier, any shape such as a granular shape, a spherical shape, a fibrous shape, a membrane shape, and a hollow fiber shape can be selected.

When a particulate water-insoluble porous substance is used, if the particle diameter is less than 1 μm, the adsorption capacity is small. Therefore, the particle size is preferably 1 μm or more and 5000 μm or less.

The water-insoluble porous carrier used in the present invention may be either organic or inorganic. However, it is preferable that the carrier has little adsorption (so-called non-specific adsorption) of a body fluid component other than the intended modified LDL. The water-insoluble porous substance is more preferably hydrophilic than hydrophobic because the non-specific adsorption is less when the substance is hydrophilic.

Further, it is convenient that a functional group which can be used for an immobilization reaction of a peptide used as a ligand is present on the surface of the carrier. Representative examples of these functional groups include a hydroxyl group,
Examples include an amino group, an aldehyde group, a carboxyl group, a thiol group, a silanol group, an amide group, an epoxy group, a halogen group, a succinylimide group, an acid anhydride group, and a tresyl group.

Representative examples of the water-insoluble porous carrier used in the present invention include soft porous materials such as agarose, dextran, and polyacrylamide, porous glass, inorganic porous materials such as porous silica gel, polymethyl methacrylate, polyvinyl alcohol, and styrene. And a porous polymer hard gel made from a synthetic polymer such as divinylbenzene copolymer and / or a natural polymer such as cellulose as a raw material, but is not limited thereto.

When the adsorbent of the present invention is used for extracorporeal circulation therapy, it is necessary to flow a high-viscosity fluid such as blood or plasma at a high speed, so that a hard water-insoluble porous substance having sufficient mechanical strength that does not cause consolidation It is preferable to use That is, as shown in Reference Examples below, a hard porous body is uniformly filled with a water-insoluble porous carrier in a cylindrical column, and the relationship between the pressure loss and the flow rate when an aqueous fluid flows is at least 0.3 kg / cm ² Up to a linear relationship.

The peptide used for the adsorbent of the present invention contains any of the following 42 amino acid sequences in its amino acid sequence, and any peptide can be used as long as the total number of amino acid sequences is 100 or less.

Examples of the peptide containing the 42 amino acid sequence include those containing the following 78 amino acid sequences.

In the present specification, the symbols constituting the amino acid sequence indicate the following amino acid residues, respectively.

G: glycine residue P: L-proline residue E: L-glutamic acid residue K: L-lysine residue D: L-aspartic acid residue R: L-arginine residue Q: L-glutamine residue N: L-asparagine residue I: L-isoleucine residue F: L-phenylalanine residue L: L-leucine residue T: L-threonine residue S: L-serine residue V: L-valine residue M: L -Methionine residue Also, the amino acid sequence is described such that the N-terminal amino acid is located on the left side and the C-terminal amino acid is located on the right side.

Examples of a method for synthesizing a peptide having such an amino acid sequence include a solid phase synthesis method, a liquid phase synthesis method, a method by genetic recombination, and the like. Peptides can be synthesized.

The peptide immobilized on the adsorbent of the present invention and having an affinity for denatured LDL may be one kind or two or more kinds.

The peptide having an affinity for denatured LDL, which is immobilized on the adsorbent of the present invention, contains the above-mentioned 42 amino acid sequences, and preferably has a molecular weight of about 10,000 or less. This corresponds to an amino acid sequence of about 100 amino acid residues or less as an amino acid sequence. When using a peptide with a molecular weight of about 10,000 or more (about 100 or more amino acid residues), it is safe to sterilize the adsorbent on which this is immobilized. It is conceivable that the problem in terms of sex will increase.

The adsorbent of the present invention is characterized in that a peptide having an affinity for denatured LDL is immobilized on a water-insoluble carrier. As a method for immobilizing the peptide on the water-insoluble porous carrier, various known methods can be used without any particular limitation. That is, there are a method by physical adsorption, a method by ionic bonding, a method of immobilization by covalent bonding, and the like. However, it is important for the preservation and stability of the adsorbent that the peptide is not desorbed and eluted, so it is immobilized by a covalent bond method that allows strong immobilization to minimize the desorption and elution of the peptide from the carrier. Is desirable.

Typical introduction methods include (1) a method in which a peptide is immobilized on a water-insoluble porous carrier by directly reacting an activated water-insoluble porous carrier with the peptide, and (2) a method in which the peptide and the water-insoluble porous carrier are used. And (3) a method in which a peptide and a water-insoluble porous carrier are bonded using a condensing agent.

The method of (1), that is, the method of immobilizing a peptide on a water-insoluble porous carrier by directly reacting the activated water-insoluble porous carrier with the peptide, comprises adding a hydroxyl-containing carrier to an epoxy solvent with epichlorohydrin in a basic solvent. There is a method in which the amino group or the carboxyl group of the peptide is reacted with the peptide and immobilized, or a method in which a commercially available activated carrier (tresylation carrier, CNBr activated carrier, etc.) is reacted with the peptide and immobilized. However, it is not limited to this. The method of (2), that is, the method of bonding and immobilizing the peptide and the water-insoluble porous carrier using a bifunctional crosslinking agent includes adding glutaraldehyde to a carrier having an amino group as a functional group and the peptide. There is a method of cross-linking the peptide with the carrier by using, for example, but not limited thereto. The method (3), that is, the method of bonding the peptide to the water-insoluble porous carrier using a condensing agent includes condensing a carboxyl group or an amino group of the peptide with an amino group or a carboxyl group of the carrier in the presence of dicyclohexylcarbodiimide. There is, but not limited to, a method of causing this to occur.

There are various methods for using the adsorbent of the present invention for treatment. The simplest method is to draw the patient's blood out of the body, store it in the blood bag, mix the adsorbent of the present invention with this, remove the denatured LDL, remove the adsorbent through a filter, and return the blood to the patient There is a way. Although this method does not require a complicated device, it has a drawback that the amount of processing performed at one time is small, the treatment is time-consuming, and the operation is complicated.

Another method is to pack the adsorbent of the present invention into a column, incorporate the adsorbent into an extracorporeal circulation circuit, and perform on-line adsorption removal. A container having an inlet and an outlet for the fluid,
The adsorbent filled in the container, and a method of passing a bodily fluid through a denatured LDL removing device comprising a filter that can pass a fluid and components contained in the fluid but cannot pass the adsorbent, is preferable. .

FIG. 1 shows a schematic sectional view of one embodiment of the modified LDL removal apparatus of the present invention. In FIG. 1, (1) is a container for filling the adsorbent (8) of the present invention, the container being a tubular body (2), a lid member (4) having an inlet (6), and an outlet ( 7) a cover member (4) having a cover member (4).
Is detachably attached to the body (2) with a packing (5) for preventing liquid leakage. Cylindrical body (2)
A filter or a mesh (3), which can pass a fluid and components contained in the fluid but cannot pass through the adsorbent (8) of the present invention, is mounted inside the lid members at both ends. The installation of the filter on the inflow side can be omitted.

The body fluid containing denatured LDL flows from the inlet (6) to the container (1).
Inside, passes through the filter (3) and comes into contact with the adsorbent (8). Here, only the denatured LDL is adsorbed by the adsorbent (8), and the body fluid from which the denatured LDL has been removed passes through the filter (3) on the outlet side and exits the vessel (1) from the outlet (7).

Hereinafter, the present invention will be described in more detail by way of examples, but the present invention is not limited to only these examples.

[Reference example] Biogel A5m (trade name, manufactured by Bio-Rad, particle size 50 to 100 mesh) on a glass column (inner diameter 9 mm, column length 150 mm) equipped with a filter with a pore size of 15 μm at both ends, from synthetic polymer Toyopearl HW65 (trade name, manufactured by Toyo Soda Kogyo Co., Ltd., particle size 50)
100100 μm), or Cellulofine GC700m, a porous cellulose gel (trade name, manufactured by Chisso Corporation, particle size 45-
(100 μm) were uniformly filled, water was circulated through the column by a peristaltic pump, and the relationship between the flow rate and the pressure loss ΔP was determined.

The result is shown in FIG. In FIG. 2, the flow rate (cm / min) is used to represent the flow rate. As is clear from the figure, the agarose gel, which is a soft gel, consolidates above a certain flow rate, and the flow rate does not increase even if the pressure is increased, whereas the hard gels such as Toyopearl and Cellulofine increase the pressure. The flow rate increases almost in proportion to. Therefore, when using the adsorption device of the present invention, it is preferable to use a hard gel as a carrier.

Production Example 1 CK Gel A-3 which is a porous cellulose gel (trade name,
Chisso Co., Ltd., globular protein exclusion limit molecular weight 50 million,
Particle size 45-105μm) 20ml NaOH 40g, heptane 120g in 100ml
And 10 drops of nonionic surfactant Tween 20 (trade name, manufactured by Kao Atlas Co., Ltd.). After stirring at 40 ° C. for 2 hours, 50 g of epichlorohydrin was added and the mixture was stirred for 2 hours, and the gel was washed with water and filtered to obtain a cellulose gel into which an epoxy group was introduced (hereinafter, referred to as an epoxidized gel).

Production Example 2 Was synthesized by a solid phase synthesis method using an automatic peptide synthesizer (Model 431A manufactured by Applied Biosystems). The peptide obtained by the automatic synthesis was treated with trifluoromethanesulfonic acid according to a conventional method to obtain 1.0 g of a crude peptide a.

The obtained crude product was fractionated by liquid chromatography and purified to obtain 80 mg of the desired purified product of peptide a.

Production Example 3 Is synthesized in the same manner as in Production Example 2;
0.8 g of crude product of peptide b was obtained.

The obtained crude product was fractionated by liquid chromatography and purified to obtain 50 mg of a purified product of the desired peptide b.

Example 1 Production Example 2 was added to 1 ml of the epoxidized gel obtained in Production Example 1.
A solution prepared by dissolving 10 mg of the obtained peptide a in 2 ml of water and adjusting the pH to 10 was added thereto, followed by shaking at room temperature for 24 hours to obtain a cellulose gel on which peptide a was immobilized.

Example 2 Preparation Example 3 was added to 1 ml of the epoxidized gel obtained in Preparation Example 1.
A solution prepared by dissolving 10 mg of the obtained peptide b in 2 ml of water and adjusting the pH to 10 was added, and the mixture was shaken at room temperature for 24 hours to obtain a cellulose gel on which peptide b was immobilized.

Example 3 Peptide a and peptide b obtained in Examples 1 and 2
After washing the adsorbent having immobilized thereon and the carrier (for comparison) used in Production Example 1 with physiological saline, each adsorbent and 1.0 ml of the carrier were placed in a polypropylene microtube (7 ml dissolved amount), and denatured. Add 4.0 ml of normal LDL saline solution or normal LDL saline solution at 37 ° C.
Shake for hours. After this adsorption operation, the gel was sedimented by centrifugation, and the amount of denatured LDL in the collected supernatant or normal LDL was measured.
The change in the amount was measured using a total cholesterol amount measurement kit (WAKO). Table 1 shows the results.

 The rate of change in Table 1 is shown by the following equation.

Denatured LDL concentration change rate (%) = (total cholesterol concentration after adsorption operation / total cholesterol concentration of stock solution) x 100 Normal LDL concentration change rate (%) = (total cholesterol concentration after adsorption operation / total cholesterol of stock solution) Table 1 shows that the adsorbent obtained by immobilizing peptide a and peptide b on the water-insoluble porous carrier adsorbs modified LDL. A small change in the amount of normal LDL indicates that the modified LDL is selectively adsorbed.

[Advantage of the Invention] Since the adsorbent in which the peptide having the specific amino acid sequence of the present invention is immobilized on a carrier selectively adsorbs modified LDL, it is possible to remove the modified LDL without substantially impairing other components in the body fluid. it can.

[Brief description of the drawings]

FIG. 1 is a schematic cross-sectional view showing one embodiment of the modified LDL removal apparatus of the present invention, and FIG. 2 is a flow rate and pressure drop when three types of carriers are packed in a column and water is passed through the column. Δ
It is a graph which shows the relationship with P. (Main symbols in the drawings) (1): Container (3): Filter (6): Inlet (7): Outlet (8): Adsorbent

Continuation of the front page (58) Field surveyed (Int. Cl. ⁶ , DB name) C07K 17/04, 14/47 B01J 20/26 CA (STN) REGISTRY (STN)

Claims (3)

(57) [Claims] An adsorption method for selectively binding denatured LDL, wherein a peptide comprising the following 42 amino acid sequences and the total number of amino acid sequences of 100 or less is immobilized on a water-insoluble porous carrier. body. (Here, the symbols constituting the above amino acid sequence indicate the following amino acid residues, respectively: G: glycine residue P: L-proline residue E: L-glutamic acid residue K: L-lysine residue D: L- Aspartic acid residue R: L-arginine residue Q: L-glutamine residue N: L-asparagine residue I: L-isoleucine residue F: L-phenylalanine residue L: L-leucine residue T: L- Threonine residue S: L-serine residue V: L-valine residue M: L-methionine residue Also, the amino acid sequence is such that the N-terminal amino acid is located on the left and the C-terminal amino acid is located on the right. Write.) 2. The modified peptide according to claim 1, wherein said peptide comprises the following 78 amino acid sequences:
An adsorbent that selectively binds LDL. 3. A container having a fluid inlet and a fluid outlet,
3. The modification according to claim 1, wherein the container is filled in the container.
An apparatus for removing denatured LDL, comprising an adsorbent that selectively binds LDL, and a fluid that can pass through the fluid and components contained in the fluid but cannot pass through the adsorbent.