WO2001057078A1

WO2001057078A1 - Process for producing dialytic amyloid fiber polymerization nuclei, method of screening remedies for dialytic amyloidosis and remedies for dialytic amyloidosis

Info

Publication number: WO2001057078A1
Application number: PCT/JP2000/009145
Authority: WO
Inventors: Itaru Yamaguchi; Hiroshi Suda; Katsuyuki Ishii; Yoshiaki Tanaka; Hironobu Naiki; Fumitake Gejyo
Original assignee: Zeria Pharmaceutical Co., Ltd.
Priority date: 2000-02-01
Filing date: 2000-12-22
Publication date: 2001-08-09
Also published as: JP2005231998A; AU2001222228A1

Abstract

A process for producing dialytic amyloid fiber polymerization nuclei which comprises reacting dialytic amyloid fiber polymerization nuclei with recombinant β2-microglobulin to thereby effect passage fragmentation; a method of screening remedies for dialytic amyloidosis by inhibiting the amyloid fiber extending reaction with the use of the above-described polymerization nuclei; and remedies for dialytic amyloidosis such as glycosaminoglycan, fucoidan, sulfated diazo-colorant compounds, etc. which are obtained with the use of the above-described screening method.

Description

Description: Method for producing dialysed amyloid fibril polymerization nucleus, screening method for dialysis amyloidosis therapeutic agent, and dialysis amyloidosis therapeutic agent

The present invention relates to a method for producing a dialysis amyloid fibril polymerization nucleus, a method for screening a therapeutic agent for dialysis amyloidosis, and a therapeutic agent for dialysis amyloidosis. Background art

Amyloidosis is a factor in Alzheimer's disease, nephropathy, rheumatism, inflammation, diabetes, and neurotoxic diseases, and suppresses the formation and deposition of fibrils caused by amyloidosis, thereby reducing the symptoms of the resulting diseases. Can be improved. Dialysis amyloidosis; 3 ₂ _ be caused by microglobulin fibrosis and deposition are known. Organ lesions in amyloid deposition include dialysis complications such as carpal tunnel syndrome, destructive spondyloarthropathy, and bone cysts. These lesions, as a screening method for creating a drug that inhibits dialysis amyloid one cis to improve symptoms) 3 ₂ - inhibition test of microglobulin fibrils extension reaction has been attempted.

i3 ₂ - The fiber elongation reaction by-microglobulin, a polymerization nucleus extracted from the dialysis amyloidosis from patients amyloidosis tissue, ₂ 3 purified from urine or blood - It is known that the reaction of micro-Glo purine (Amy loid: In. J. Exp. Clin. Invest., 4, 223-232, 1997).

Since the materials used for these reactions use biological samples such as human tissues, urine, and blood, there are problems such as the need to obtain them and to prepare an environment for biohazard. Conventional dialysis amyloid fibrils polymerization nuclei and 3 ₂ - elongation reaction with microglobulin are those polymerization nuclei were taken from the dialysis amyloid one cis patient sets ^!. Therefore, there is a problem that it is used for only one reaction and it is difficult to obtain it. Also,

/ 3 ₂ — The disadvantage is that microglobulin also needs to be purified from urine and blood. Therefore, it can be mass-produced, and 3 ₂ - microglobulin fibrils elongation reaction without using a human tissue that can be used for dialysis amyloid fibrils polymerization nucleus production Nozomu rare, yet 3 ₂ can be easily obtained - using a micro-Glo purine Realization of the fiber elongation reaction is desired.

Producing a large amount of dialysis amyloid fibril polymerization nuclei can contribute to screening for a therapeutic agent for dialysis amyloidosis. Disclosure of the invention

The present invention, dialysis amyloidosis patients from dialysis amyloid fibrils polymerization nuclei and Rico Nbinanto i3 ₂ - reacting the micro Glo purine to form amyloid fibrils, the amyloid fibrils obtained fragmented by sonication, resulting An object of the present invention is to provide a method for producing a dialyzed amyloid fibril polymer nucleus by repeatedly and successively repeating the same reaction and fragmentation as described above by using a part of the obtained fragment as a dialysate amyloid fibril polymer nucleus.

Further, the present invention is a dialysis amyloid one cis inhibitory effect dialysis and drug screening amyloid fibrils polymerization nuclei and recombinant 3 ₂ - dialysis amyloid by reacting a microglobulin - a method of screening for cis therapeutic agents, polymerization nucleus The present invention also provides a method for screening a therapeutic agent for dialysis amyloidosis, wherein the method comprises using the dialyzed amyloid fibril polymerization nucleus obtained by the above method.

Furthermore, the present invention provides a therapeutic agent for dialysis amyloidosis obtained by the above screening method. Further, the present invention comprises, as an active ingredient, glycosaminoglycan or a salt thereof, fucoidan, a diazo dye compound having a sulfate group, or a polymer compound in which glycosaminoglycan or fucoidan has a diazo dye compound having a sulfate group bonded thereto. It is intended to provide a therapeutic agent for dialysis amyloidosis.

Furthermore, the present invention relates to dialysis amyloidosis of a glycosaminodalican or a salt thereof, fucoidan, a diazo dye compound having a sulfate group, or a polymer compound in which a daricosaminoglycan or fucoidan has a diazo dye compound having a sulfate group bonded thereto. It provides a use for the manufacture of a therapeutic agent.

Furthermore, the present invention provides a method for treating glycosaminodalican or a salt thereof, fucoidan, a diazo dye compound having a sulfate group, or a polymer compound in which glycosaminodalican or fucoidan has a diazo dye compound having a sulfate group bonded thereto. It is intended to provide a method for treating dialysis amyloidosis, which comprises administering to a patient in need thereof. BEST MODE FOR CARRYING OUT THE INVENTION

In the method for producing a polymerized dialysis amyloid fiber nucleus of the present invention, first, a dialysis polymerization amyloid fiber nucleus (SO ₂ ) derived from human tissue is reacted with recombinant i32-microglobulin to form amyloid fibril. The obtained amyloid fiber is fragmented by ultrasonic treatment, and a part of the obtained fragment is used as the amyloid fibril polymerization nucleus, so that the reaction and fragmentation operations are repeated in the same manner as described above. By repeating the above steps, the dialyzed amyloid fibril polymerization nucleus (S 1) can be produced. The subculture repetition manipulate X times (X is an integer of 1 or more.) Repeating amyloid fibrils polymerization nucleus obtained by (S x) is 3 ₂ - increased affinity to microglobulin, the extension reaction Initial speed increased. Therefore, an infinite number of successive repetitive operations (S∞) can be performed, thereby realizing mass production of polymerization nuclei.

The starting material, the dialysed amyloid fibril polymerization nucleus (SO), is derived from human tissue Yes, it can be obtained by extraction from amyloid-deposited tissue from dialysis amyloidosis patients by the method of Pras et al. (J. Exp. Med., 130, 777-791, 1969).

Extension is fibrous constituents | 3 ₂ - microglobulin, human urine, instead of those obtained from your blood, commercially commercial sold recombinant 3 ₂ - may be able to use a mini Kuroguropurin This is a feature of the present invention.

Dialysis Amyloid fibrils polymerization nuclei and recombinant / 3 ₂ - sonication to fragment the amyloid fibrils formed by the reaction of micro Gros purine can be performed by ultrasonic fracture frame machine.

The amount of the fragment obtained by the sonication used for the polymerization nucleus (S x) in the successive extension reaction is preferably 2.5 to 100 g / mL, particularly preferably 10 g / mL. The amount of the recombinant 32 ₂ -microglobulin used is preferably from 10 xg / mL to 10 mg / mL (1::! To 1: 1 000).

1 1 subculture repetition fragmented by resulting polymer nuclei (S 1 1) 3 ₂ - does not include molecules other than Miku b globulin. The S 1 1 and recombinant 3 ₂ - Affinity micro globulin is increased, the initial rate of the extension reaction was increased.

The screening method of dialysis amyloidosis therapeutic agent of the present invention, the test agent is dissolved in acidic buffer, dialysis amyloid fibrils polymerization nuclei. 25 to 100 g / mL and Rico Nbinanto) 3 ₂ _ microglobulin 5-100 additives It is preferable to carry out an amyloid fiber elongation reaction. Uchiki et al.'S thioflavin T

(ThT) Measured by the fluorescence method (Amyloid: Int. J. Exp. Clin. Invest., 4, 223-232, 1997), and the increase in fluorescence intensity is used as an indicator of amyloid fibril elongation. Alternatively, the reaction mixture was centrifuged, the protein content in the centrifuged supernatant (i3 ₂ - microglobulin amount) is measured and a decrease in the protein content as an indicator of amyloid fibril elongation. In the ThT fluorescence method, since the increase in fluorescence intensity is reduced by suppressing the formation of amyloid fibrils, a drug with a smaller increase in fluorescence intensity suppresses amyloid sis, and as a therapeutic agent for dialysis amyloid cis. Are better. Moreover, the protein content in the centrifuged supernatant (3 ₂ - Mikuroguro In the determination of the amount of (brin), the decrease in the amount of protein in the centrifugal supernatant is suppressed by suppressing the formation of amyloid fibrils, so that a drug with a smaller decrease in the amount of protein in the centrifugal supernatant suppresses amyloidosis In other words, it is excellent as a therapeutic agent for dialysis amyloidosis.

The present invention includes the therapeutic agent for dialysis amyloidosis obtained by the above screening method. When a therapeutic agent for dialysis amyloidosis was searched for by the above screening method, it was found that daricosaminodalican or a salt thereof, which is a main component of cell connective tissue, had an excellent dialysis amyloidosis inhibitory action. Glycosaminodalican is preferably chondroitin sulfate C, dermatan sulfate, heparan sulfate, heparin or hyaluronic acid, and the salts thereof are physiologically acceptable, such as sodium salt, potassium salt and calcium salt. Are mentioned.

In addition, they found that fucoidan has an excellent inhibitory effect on dialysis amyloidosis. Fucoidan includes Gagomefukoidan, Mozukufukoidan, Futomozkofukoidan, Nebarimovkoidan, Ishigefukoidan, and Yirolovkoidan.

Furthermore, when a therapeutic agent for dialysis amyloidosis was searched for by the screening method of the present invention, a diazo dye compound having a sulfate group and a polymer compound having a diazo dye compound having a sulfate group bound to glycosaminoglycan or fucoidan were found. Have excellent dialysis amyloidosis inhibitory action. Diazo dye compounds having a sulfate group include Congo red, Direct Red 75, Direct Red 80, Direct Red 81 and Direct Red 81. Lianto Yellow (Brilliant Yellow), Brilliant Crocein MOO (Brilliant Crocein MOO) or Acid Violet 5 (Acid Violet 5).

The therapeutic agent for dialysis amyloidosis of the present invention is useful for treating dialysis amyloidosis patients. It is effective and its dosage varies depending on symptoms, administration method, administration period, etc., but it is usually 100-10000 mg / day for oral administration, preferably 900-1800 mg / day. Days, and is preferably administered in the range of 1 to 3 times a day. It can also be directly injected into the affected area or administered intravenously as a 1-100 g / mL solution for injection. These therapeutic agents are used at doses that do not cause toxicity or side effects when administered to the human body.

The active ingredient, such as glycosaminoglycan or a salt thereof, fucoidan, etc., is formulated into a formulation for oral administration or a formulation for parenteral administration by mixing pharmaceutically acceptable and commonly used carriers and additives. Can be. Preparations for oral administration include tablets, powders, granules and capsules, and preparations for parenteral administration include injections. Example

Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited thereto.

Example 1 (Method for producing dialyzed amyloid fibril polymerization nucleus)

(1) Formation of the base polymerized nucleus of amyloid fibrils (S 1)

• Preparation of human dialysed amyloid fibril polymerization nucleus (so)

After crude extraction from the synovium of a carpal tunnel syndrome patient by the method of Pras et al. (Supra), the crude extract was purified by sucrose density gradient ultracentrifugation, suspended in a 0.05% aqueous sodium azide solution, and used. Stored at 4 ° C until time.

• Recombinant 3 ₂ — Preparation of microglobulin

Recombinant 3 ₂ - microglobulin (manufactured by Oriental Yeast Co., Ltd.) was desalted and dialyzed in refining water and lyophilized. Then, it was dissolved in a 5 OmM aqueous sodium chloride solution at a concentration of 300 to 500 iM, and stored at -80 ° C until use.

S 0 polymerized nucleus 1 0 g / mL, 25 M recombinant | 3 ₂ - microglobulin, 1 mL of a reaction solution containing 5 OmM citrate buffer (pH 2.5) and 100 mM sodium chloride was reacted at 37 ° C for 24 hours. The amyloid fibrils formed by this reaction were centrifuged at 15000 卬 m and 4 ° C for 2 hours to obtain a precipitate. This sediment was washed lightly with 0.5 mL of a 0.05% aqueous sodium azide solution, suspended in 300 L of a 0.05% aqueous sodium azide solution, and then sonicated (Ultrasonic Disruptor, TOMY UD-1201). ) To obtain 100 crushed fragments. This was used as the S 1 polymerization nucleus.

(2) Continuous formation of dialysis amyloid fibril polymerization nuclei

Using the S 1 polymerization nucleus obtained above as a primary polymerization nucleus, a successive polymerization nucleus (S 2 to S x) (X represents an integer of 1 or more) is formed by repeating the following method. Was.

The reaction solution, 50 Kuen acid buffer (p H 2. 5) and 100 polymerization nuclei in a mixture of chloride Natoriumu water solution (Sx) and Recombinant] 3 ₂ - microglobulin their respective final concentration 10 zg / mL And 25 were used. After preparing the reaction solution, dispense 30 L each of the reaction solution into an oil-free PCR tube (for 0.5 mL) under ice-cooling, set in a DNA thermal cycler (PJ480, Perkin ElmerCetus), and set at 4 ° C. The temperature was increased at the fastest speed to 37 ° C. 10 minutes after the start of the reaction, the reaction was stopped under ice cooling, and the fluorescence intensity was measured by the ThT method.

Add 5 L of the reaction solution for each elapsed time to 1 mL of 5 OmM glycine Z sodium hydroxide buffer (pH 8.5) containing 5 M ThT, mix with stirring, and immediately mix with a spectrofluorometer (Hitachi F-3010 ) Were measured at an excitation wavelength of 455 nm and an emission wavelength of 485 nm. Fluorescence measurements were performed three times on the same sample.

The initial reaction rates of the polymerization nuclei (S 0 to S 10) are as shown in Table 1. Table 1 First anti-J: IS degree

(S0-S10) (increase in fluorescence intensity / min)

S 0 0.28 ± 0.12

S 1 0.42 ± 0.08

S 3 0.6 7 ± 0.02

S 5 0.6.5 ± 0.15

S 7 0.92 ± 0.07

S10.1.14 ± 0.18 As is clear from Table 1, the initial reaction speed increased with each passage, and the initial reaction speed of S10 was four times that of S0.

Example 2 (Screening of dialysis amyloidosis using glycosaminodalican. Inhibitory effect of amyloid fibril elongation reaction)

Examples of glycosaminodalican include chondroitin sulfate C sodium salt (derived from shark joint, manufactured by Sigma), dermatan sulfate sodium salt (derived from bu-intestinal mucosa, Sigma), heparan sulfate sodium salt (derived from bu-intestinal mucosa) , Sigma), heparin sodium salt (derived from mucosal mucosa, manufactured by Sigma), or sodium hyaluronate (derived from human umbilical cord, manufactured by Nacalai Tesque, Inc.).

The above glycosaminodalican was dissolved in purified water to a concentration of 1 mg / mL, and diluted to a concentration of 300 g / mL.

To 75 / L of purified water, 15 M of 0.5 M citrate buffer (pH 2.5) was added and 15 L of 950 DIM of sodium chloride aqueous solution was added. Then, 3 0 0 / zg / the glycosaminoglycans (heparin sodium to the 1 mg / mL) Dali cans aqueous solution made adjusted to mL was added, further, 2 5 0 M recombinant of 3 ₂ - microglobulin (Oriental Yeast 15 and the 100 Aig / mL obtained in Example 1. 15 L of myloid fibril polymerization nucleus (S11) was added. The above operation was performed under ice cooling.

The reaction solution was dispensed in 50 L aliquots into PCR tubes (for 0.5 mL) and heated to 37 ° C to initiate the amyloid fibril elongation reaction. Amyloid fiber elongation was measured by the ThT method, and the increase in fluorescence intensity 2 hours or 4 hours after the start of the reaction was used as an index.

In the control group, purified water was added in place of the aqueous glycosaminoglycan solution.

The measurement method of the T h T method was performed as follows.

5 OmM glycine Z sodium hydroxide buffer containing 5 M ThT (pH

8.5) To 1 mL, 5 xL of the reaction solution was added and mixed. Immediately after that, the fluorescence intensity (excitation wavelength 455ηιη, fluorescence wavelength 485nm) was measured. This operation was performed three times, and the average value of the three fluorescence intensities was calculated.

Table 2 shows the suppression effect of glycosaminoglycan on the fluorescence intensity.

Table 2

As is evident from Table 2, glycosaminoglycan strongly suppresses dialysis amyloid fibril elongation reaction and is useful as a therapeutic agent for dialysis amyloidosis.

Example 3 (Screening for dialysis amyloidosis using fucoidan. Inhibitory effect on the elongation reaction of sulfide fibers)

Fucoidan collected the following seaweed using the method of Furukawa et al. (Hijiyama University Junior College bulletin)

Bul. Hiyama Univ. Jun. Coi., 34, 81-87, 1999).

Gagome (Kjell bandit iella, a commercial product from Hokkaido)

Mosque (Nemacystus decipiens)

Futo Mosque (Tinocladia crassa)

Nebarimo (Leathesia di f formis)

Isshige (Is ige okamurai)

Iroge (Ishige fol iacea)

The purified fucoidan was dissolved in purified water to a concentration of lmg / ml.

75 Add 0.5 M citrate buffer (pH 2.5) to 15 L of purified water, and add 15 L

An aqueous solution of 95 OmM sodium chloride was added 15 times. Next, 1 mg / mL of added the fucosyltransferase Idan aqueous 1 5 Ji, further, 250 M of recombinant i3 ₂ - obtained in Miku b globulin (manufactured by Oriental Yeast Co., Ltd.) 1 5 and Example 1

15 L of 100 g / mL amyloid fibril polymerization nucleus (S11) was added. The above operation was performed under ice cooling.

The reaction solution was dispensed in 50 L portions into a PCR tube (for 0.5 mL) and heated to 37 ° C to initiate the amyloid fibril extension reaction. Amyloid fiber elongation was measured by the ThT method, and the increase in fluorescence intensity 4 hours after the start of the reaction was used as an index.

In the control group, purified water was added in place of the fucoidan aqueous solution.

Table 3 shows the effect of suppressing the fluorescence intensity of each fucoidan. Table 3

As is evident from Table 3, each fucoidan potently inhibits the dialysis amyloid fibril elongation reaction and is therefore useful as a therapeutic agent for dialysis amyloidosis.

Example 4 (Screening for dialysis amyloidosis using Congo Red. Inhibitory effect of amyloid fibril elongation reaction)

Congo Red (Sigma) was dissolved in purified water to a concentration of 1 OmM and diluted to a concentration of 300.

75 Add 0.5 M citrate buffer (pH 2.5)

95 OmM aqueous sodium chloride solution 15 was added. Next, the 300 M Congo - a-intensity aqueous 1 5添力[], further 250 M recombinant / 3 ₂ - obtained in Miku b globulin (manufactured by Oriental Yeast Co., Ltd.) 1 5 and Example 1

1 00

15 L of myloid fibril polymerization nucleus (S11) was added. The above operation was performed under ice cooling.

The reaction solution was dispensed in 50 L aliquots into PCR tubes (for 0.5 mL) and heated to 37 ° C to initiate the amyloid fibril elongation reaction. Amyloid fiber elongation was measured by the amount of centrifugal supernatant protein, and the decrease in the amount of centrifugation supernatant protein before the start of the reaction and 4 hours after the reaction was used as an indicator of amyloid fiber elongation. The control group was prepared by adding purified water instead of the above Congolese. Quantification of the protein in the centrifuged supernatant was performed as follows.

The reaction solution was centrifuged at 14500 rpm at 4 ° C for 20 minutes to precipitate amyloid fibrils, and the amount of protein in the supernatant was measured using a Micro BCA protein quantification kit (Pierce).

Table 4 shows the results.

Table 4

As is clear from Table 4, Congolese is useful as a therapeutic agent for dialysis amyloidosis because it strongly suppresses the elongation reaction of dialysis amyloid fibrils.

Example 5 (Preparation of tablet containing chondroitin sulfate C sodium salt)

20 g of chondroitin sulfate C sodium salt, lactose 100 g, corn starch 36 g, crystalline cellulose 30 g, carboxymethylcellulose calcium 10 g and magnesium stearate 4 g are uniformly mixed, and the diameter is 7.5 using a single tablet press. Each tablet was made into a tablet of 20 Omg with a punch.

Example 6 (Preparation of injection containing chondroitin sulfate C sodium salt)

Take 0.1 mg of chondroitin sulfate C sodium salt, dissolve in water for injection to make 100 mL, filter using a 0.2 m membrane filter, fill aseptically into a vial, and mix with the injection. did.

Industrial applicability

ADVANTAGE OF THE INVENTION According to this invention, the dialysis amyloid fiber polymerization nucleus can be manufactured in large quantities. Therefore, it is industrially useful, and the obtained dialyzed amyloid fibril polymerization nucleus can be used as it is for screening for a therapeutic agent for dialysate amyloidosis.

Claims

The scope of the claims

1. Reacting the dialyzed amyloid fibril nucleus derived from a dialysis amyloidosis patient with recombinant 32 ₂ -microglobulin to form amyloid fibrils, fragmenting the obtained amyloid fibrils by sonication, A method for producing a dialyzed amyloid fibril polymer nucleus by repeatedly performing the same reaction and fragmentation as described above for a portion of the obtained fragment as a dialyzed amyloid fibril polymer nucleus.

. 2 with an agent for screening dialysis amyloidosis inhibitory effect dialysis amyloid fibrils polymerization nuclei and recombinant / 3 ₂ - in the screening methods of dialysis amyloidosis therapeutic agent by reacting a micro Gros purine, according to claim 1 as a polymerization nucleus A method for screening a therapeutic agent for dialysis amyloidosis, which comprises using a dialyzed amyloid fibril polymerization nucleus obtained by the method.

3. A therapeutic agent for dialysis amyloidosis obtained by the screening method according to claim 2.

4. Dialysis using glycosaminodalican or a salt thereof, fucoidan, a diazo dye compound having a sulfate group, or a polymer compound in which glycosaminodalican or fucoidan is bonded with a diazo dye compound having a sulfate group as an active ingredient. Amyloidosis treatment.

5. The therapeutic agent for dialysis amyloidosis according to claim 4, wherein the glycosaminodalican is chondroitin sulfate (: dermatan sulfate, heparan sulfate, heparin or hyaluronic acid).

6. The therapeutic agent for dialysis amyloidosis according to claim 4, wherein the fucoidan is gagomefucoidan, mozukufucoidan, fen ', nevarimofucoitan.

7. Diazo dye compounds having sulfate group are Congo Red, Direct Red 75, Direct Red 80, Direct Red 81, Brilliant Yellow, 5. The therapeutic agent for diffusive amyloidosis according to claim 4, which is Priliantoclosenmo or Acid Violet 5.

8. Production of a therapeutic agent for dialysis amyloidosis of glycosaminodalican or a salt thereof, fucoidan, a diazo dye compound having a sulfate group, or a polymer compound in which glycosaminoglycan or a diazo dye compound having a sulfate group is bonded to fucoidan. Use for

9. Patients who need to be treated with glycosaminodalican or a salt thereof, fucoidan, a diazo pigment compound having a sulfate group, or a polymer compound in which glycosaminoglycan or a diazo pigment compound having a sulfate group is bonded to fucoidan. A method for treating dialysis amyloidosis, comprising administering to a patient.