JPS6043333B2 - Method for producing anti-inflammatory active substances - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing anti-inflammatory active substances for use in the treatment of inflammatory diseases.

A hypothesis has been proposed that as one of the self-defense functions of living organisms, there are self-substances that exhibit therapeutic effects against inflammation.

For example, this substance exists in blood plasma and is said to be a low-molecular substance with a molecular weight of several hundred (Japanese Patent Application Laid-open No. 82214-1982), but the substance has not been sufficiently purified and its physicochemical properties is still not clear. The inventors,
Anti-inflammatory substance precursors with molecular weights different from such low-molecular-weight anti-inflammatory substances (molecular weight 1000 to 100
The present invention has been completed by separating 00) and converting it into an anti-inflammatory active substance by polymerizing it, thereby facilitating its purification.

According to the present invention, the anti-inflammatory active substance is a low molecule obtained by ultrafiltering body fluids such as serum, plasma, inflammatory exudate, milk, and saliva of mammals to remove high molecular weight proteins and carbohydrates. By taking the ultrafiltrate and reducing its calcium concentration, the low-molecular anti-inflammatory active substance precursor in the same fraction is polymerized, and the polymerized anti-inflammatory active substance that is not filtered by ultrafiltration is recovered. The active fraction was obtained by gel filtration chromatography, and then subjected to weakly basic ion-exchange chromatography, gel filtration chromatography, and weakly acidic ion-exchange chromatography. It can be obtained electrophoretically as a single substance.

This substance is thought to be a glycoprotein mainly composed of carbohydrates and some amino acids, and exhibits strong anti-inflammatory activity against carrageenan edema when administered intravenously. In addition, in an in vivo leukocyte fugue test using the CMC (carboxymethyl cellulose) sac method in rats, intravenous administration showed a strong fugetaxis inhibitory activity;
Even in a fugue test using the same substance, white blood cell fugue is significantly inhibited by the coexistence of the same substance. In carrying out the present invention,
Starting materials include body fluids of mammals such as humans, cows, pigs, etc.
Blood from sheep, rats, guinea pigs, etc., exudate from experimentally inflamed sites in rats, or secretions from humans, cows, horses, or pigs, such as milk or saliva, are used, but consideration should be given to the availability of large quantities of raw materials. Blood and milk from large animals such as cows, horses, and pigs are then optimal.

Each step of the fractionation and purification method will be explained below.

[First step] The raw material is first subjected to low-temperature centrifugation to remove blood cells, lipids, etc., and then subjected to ultrafiltration in order to remove high-molecular proteins, etc.

The following operations are performed in a cold room at around 4°C as much as possible.
For ultrafiltration, for example, Amicon CO.
) diaflow device was used, and the filter was UM.
It is advantageous to use a material that allows the passage of -10 equivalent molecular weights of 10,000 or less. Further, it is more preferable to use a follow fiber device (Model DC-2, HIDP-10) also manufactured by Amicon, as it can process a large amount of raw material in a short time.
For example, when ultrafiltrating serum, first add 1110 molar volume of 1% sodium azide (NaN3) to prevent bacterial propagation, and immediately place it in the ultrafilter without performing any operations such as dialysis or dilution. Put it on. During this process, when the calcium concentration of the solution decreases due to dialysis or dilution (approximately 5TrLg
% or less), some of the anti-inflammatory substance precursors become polymerized and do not flow out of the ultrafilter, resulting in a reduced yield. In addition, since the speed of ultrafiltration decreases with time due to high viscosity due to increase in content concentration, it is preferable to finish when 112 to 1B capacity of the raw material has been passed. The filtered low molecular weight fraction can be preserved by freezing. [Step (II)] The anti-inflammatory active substance precursor in the ultrapolar-filtered low-molecular fraction is subjected to a polymerization operation by reducing coexisting calcium ions.

That is, polymerization occurs by dilution with water, dialysis, or addition of a chelating agent that removes calcium, such as EDTA (ethylenediaminetetraacetic acid), GBDTA (glycolethylenediaminetetraacetic acid), etc.
M-1 Maki)? It will not come out in the liquid. Polymerization sufficiently occurs at a calcium ion concentration of 5m9%, and taking serum (containing about 10m9% calcium) as an example, it is only necessary to dilute the ultra-P filtrate twice with water. However, in order to ensure polymerization, it is more effective to use 3- to 4-fold dilution.
This low-calcification fraction was subjected to ultrafiltration (UM-10,
．． filter (such as UM-2), and collect the remaining high molecular fraction without being filtered. The target anti-inflammatory active substance can be significantly purified through this polymerization process by low calcium conversion and the subsequent ultrafiltration concentration process. One of the features of the present invention lies in this process. [Step II] The concentrated solution from Step II containing the anti-inflammatory active substance is subjected to gel permeation chromatography.

Sepha Dex G-75 etc. (Sepha
dexlPharmaciaCO. ) can be effectively used. Also, as a developing solution, 0.
A salt solution close to physiological conditions, such as 9% saline, can be used. The eluent from the chromatography column is collected in fixed amounts,
When a chromatogram is drawn using the ultraviolet absorption around 275 mμ as a guide, it can be seen that the anti-inflammatory active substance exists near the first peak and elutes almost immediately after the interstitial volume (VOidvOIume). This first peak and the tail portion flowing to the rear thereof are broadly sampled and subjected to ultrafiltration using a filter such as UM-2 to concentrate the active substance. [Step ■] The concentrated liquid from Step ■ is treated with a weakly basic ion exchanger such as DEA.
E-cellulose (diethylaminoethyl cellulose,
SefvaCO. The sample is subjected to ion exchange chromatography using a commercially available product such as

This ion exchanger is preliminarily mixed with a weakly acidic buffer of low ionic strength, for example, 0.05M, . The column is buffered with an acetate buffer having a pH of 5.8 or the like, and the concentrated solution of the second stage is adsorbed onto the chromatography column. In this case, the salt concentration after concentration is 0.
．． Adjust the solution to 1M saline. After adsorption, 0.2M acetate buffer (PH5.8) was thoroughly poured.
757T1. After removing as much of the eluting material as possible using μ ultraviolet absorption as an indicator, the buffer is exchanged with the same 1M buffer and the fractions to be concentrated and eluted are collected. Anti-inflammatory active substances are present in this fraction. [Stage ■] Stage ■? The 1M elution fraction of 100% is gel-filtered.

As for gel filtering, it is effective to use gels with a higher degree of cross-linking and a larger network structure than those used in the second stage, such as Sephadex G-200. Gel filtration chromatography is performed in the same manner as in step (2), and the first peak from which the anti-inflammatory active substance flows is collected. After concentration by ultrafiltration (UM-10, ■-2, etc.), it is thoroughly dialyzed against water to desalt it, and the dialyzed solution is freeze-dried and stored at low temperature. Although the anti-inflammatory active substance is purified to a very high degree through the purification up to step (2), one more step of purification may be performed. That is, weakly acidic ion exchangers such as CM-
Cephadex (carboxymethyl-cephadex)
This is the second stage where the liquid is passed through a column such as [Step (1)] The first peak obtained in the step (2) is dialyzed against water and subjected to ion exchange chromatography using CM-Sephadex.

CM-Sephadex is buffered in advance with a low-concentration buffer such as 0.02M acetate buffer (PH5.8), and after placing the active fraction on top, 0.02r. 4. Flow the same buffer solution in step 4 and collect the non-adsorbed elute. This contains anti-inflammatory active substances, and is it limitless? The desired active substance can be obtained by concentrating with filtration, thoroughly dialyzing with water, and freeze-drying. Next, the method of the present invention will be explained in more detail with reference to Examples.

Example 1 Fractionation and Purification of Anti-inflammatory Active Substances from Bovine Blood Bovine blood 180e was allowed to stand still to coagulate, left to stand at a low temperature of around 5°C to sufficiently separate serum, and then blood clots were removed.

Blood cells in the serum fraction from which blood clots have been removed are removed by centrifugation.
In this case, a Dravall type continuous centrifuge, such as Birches (
Barces) Using a centrifuge, etc., increase the rotation speed to 5000 r.
．． p. If carried out at m, approximately 60' of clear serum can be obtained efficiently. Add sodium azide to the serum as a bactericidal agent.
．． 0.01%, and perform ultrafiltration operation. Using Amicon's high-flow cell type p-filtration machine (or Bio Engineering Co., Ltd.'s Diafilter MC-6 model machine),
A filter with a diameter of 150T0t (7) UM-10 is used, and the test is carried out under nitrogen gas pressure (4k91cf1). UM
What passed through the -10 filter had a molecular weight of about 10,000 or less, and a molecular weight of about 48' was obtained. Dilute this fluid by 4 times with water. During this dilution process, the low-molecular anti-inflammatory active substance precursor becomes a polymer and no longer passes through the filter during the next ultrafiltration. At this stage, the throughput is large, about 200 e, so it is efficient to use Amicon's follow fiber (DC-2 type, HIDP-10). In this way, those that pass are removed, and the polymer substances that do not pass are concentrated to a volume of 30 ml. In this case, it is convenient to concentrate using a small diaflow ultrafilter. This concentrated fraction is subjected to gel filtration.

First, Sephadex G-75 was suspended in 0.9% saline and loaded into a 5 x 95 cm chromatography column (Excel column, SB-501 Nitrous KK). Place 30ml of concentrated liquid on top of this, and the development is 0.9
% saline solution, and each eluting fraction is fractionated using the ultraviolet absorption of 2757n, μ as an index. The anti-inflammatory active substance is present in the first peak immediately adjacent to the interstitial volume (VOidVOlLlnle). The first peak fractions are combined and concentrated by ultrafiltration (filter UM-10) to approximately 30 mt. The concentrate is dialyzed overnight against water and then subjected to ion exchange column chromatography using weakly basic DEM-cellulose. DEAE-cellulose was buffered in advance with 0.01M acetate buffer at pH 5.8, and then placed on a 26×40C7Tt column (E
xcel). After placing the above concentrated solution on top of this and adsorbing it, 0.2M. ．． Thoroughly wash out the eluting material with an acetate buffer of pH=5.8, and after confirming that there is no more eluting material due to absorption of 2757T1,P, change to the same buffer of 1M. The active substance is concentrated by this 1M buffer and eluted through the column. Next, gel filtration is performed using Sephadex G-200.

G-2 in 0.9% saline as in the case of G-75 above.
Pack 00 into a column (4x95C!rl) and add DE to the top.
A 1M eluate (approximately 20 ml) from AE-cellulose chromatography was loaded and expanded with 0.9% saline. In this case too, the active substance is present in the first peak immediately after the interstitial volume. This first peak portion was filtered through ultrafiltration (UM-1
0 or UM-2), thoroughly dialyzed against water, and freeze-dried. At this stage, 26m9 of active substance was obtained. This substance is highly purified and exhibits strong anti-inflammatory activity. In addition, as a next step, ion exchange chromatography using weakly acidic CM-Sephadex is performed for further purification, but the anti-inflammatory activity may be partially reduced. The first peak of the freeze-dried sample or Sephadex G-200 chromatography was added to 0.01M acetate buffer (PH5.8).
After dialysis using a CM-Sephadex column (2.6 x 4
0 cr! Chromatography is performed using a gradient system in which sodium chloride is added to the same buffer solution. In the chromatographic fractionation, the portion eluted in the salt concentration range of 0.02 to 0.2M was collected and concentrated by ultrafiltration (UM-
10 or UM-2) and lyophilized after dialysis with water, 1
0 m9 of active preparation was obtained. Various properties of this substance are listed below.

[Physicochemical properties]

The freeze-dried sample is a white powder, and when dissolved in water, the pH =
4.3 (17y191m1).

The ultraviolet absorption spectrum of this product is 28 as shown in Figure 1.
It has a shoulder near 0wLμ. Moreover, the infrared absorption spectrum is as shown in FIG. The molecular weight is estimated to be 200,000 or more according to the gel filtration method. The elemental analysis values are as follows.

The composition of this product is as follows.

According to sugar analysis, it contains a lot of glucose, galactose, and fucose, and also contains trace amounts of rhamnose and mannose.

Amino acid analysis reveals aspartic acid, glutamic acid, glutamine, serine, glycine, alanine, valine, and leucine.

Note that sialic groups, glucuronic acid, and galactosamine are not recognized.

[Biological properties]

Anti-carrageenan edema effect The anti-carrageenan edema effect in rats was carried out using Winter's method (1).If this freeze-dried sample solution was intravenously injected 3 hours before carrageenan administration, Significant suppression of foot edema is seen at the time of evaluation.

under. From the suppression rate shown in the table, the 50% suppression amount is calculated as D.
= 0.88μy/BOdy. Note that even if the protein portion of this substance is decomposed with pronase (PrOnase), the anti-carrageenan activity is not affected.

Inhibition of leukocyte migration using the CMC bag method (3): CMC (carboxymethyl cellulose) is subcutaneously administered to the back of rats and the number of leukocytes migrating to that area is measured.
The MC capsule method is used.

The standard solution is injected intravenously twice, at the time of CMC administration and 3 hours later. When looking at the local leukocyte count at 6 hours, a remarkable inhibition of migration was observed.

As shown in the table, leukocyte migration was inhibited by 72% with just two administrations of 0.5 μFllbOdy. (3) Yoshio Iizuka et al.; 24th Japanese Pharmacology Abstracts, p. 305 (1
967) Hiroshi Ishikawa, et al.; Pharmaceutical Journal, ? 1472 (1968) Leukocyte migration inhibition effect using the BOyden Chamber method (4): BOydenChem? divided into two chambers with a microbore filter? Guinea pig leukocytes in the upper chamber of r [polymorphonuclear leukocytes (PMNs in the peritoneal cavity)
-1euc0cyte)], its migratory factor (guinea pig serum + thymosan (ZymOsan)) is placed in the lower chamber, and the movement of leukocytes within the filter toward the factor is measured.

When the preparation is placed on the migration factor side, it does not have much effect on leukocyte migration, but when the preparation is placed on the leukocyte side (4.4pfI
m1), significantly inhibits leukocyte migration (86%).
This inhibitory effect is not due to the cytotoxicity of the preparation to leukocytes, but is due to migration inhibition. (4) S. Boyden
;J. Exptl. Med. , Kawa-, 453 (1962
) Example 2 A method using exudate from a rat's experimentally inflamed area as a raw material A sponge (for example, polyurethane maltbren can be used) is aseptically implanted on the back of a rat and removed after 4 days.

Normally, diameter 3, thickness 1C77! For a sponge with a diameter of 2
~3 pieces can be transferred to one animal, and about 10 nt of exudate from the sponge can be collected. This exudate is pooled and used as raw material. The exudate was centrifuged (10,000 R.p.m.
12) After removing the contaminated blood cells, enter the ultra-thigh temperature operation. The following procedure is the same as the method of Example 1. The anti-inflammatory active substance purified in this way also exhibits almost the same properties as the substance in bovine blood. . Example 3 Method using milk as raw material Fresh milk was centrifuged (one way R.p.
．． After removing floating lipids, perform centrifugation again to thoroughly remove lipids.

Defatted milk with a molecular weight of 10,000 or less is collected by ultrafiltration (UM-10, etc.). The difference between milk and serum is that it contains many times more calcium.
Dilution with a large amount of water is required to polymerize anti-inflammatory substance precursors due to low calcium content. However, if the volume increases, it will be difficult to carry out the next operation, so it is better to remove calcium by dialysis.In other words, put the fraction with a molecular weight of 10,000 or less extracted by ultrapolar filtration into a dialysis tube, and add water to the dialysis tube. The dialyzed fluid is thoroughly dialyzed against the dialysis fluid, except for the one that passes through Amicon's Ferro fiber, and the remaining polymers are concentrated, and then subjected to chromatography using Sephadex G-75.The following is an example. The procedure is carried out in the same manner as in 1. The substance thus extracted also exhibits almost the same properties as the anti-inflammatory substance in bovine serum.

[Brief explanation of the drawing]

Figure 1 shows the ultraviolet absorption spectrum of the anti-inflammatory active substance fractionated and purified from bovine serum according to Example 1.
Measurements were made by dissolving mg of the standard in 3 ml of water.

Claims (1)

[Claims] 1 After ultrafiltrating mammalian body fluids and subjecting the filtrate to polymerization by lowering the calcium concentration, ultrafiltration is performed again to fractionate the unfiltered polymer substances, and the resulting polymer A method for producing an anti-inflammatory active substance, which comprises purifying a molecular fraction.