JPS6233205B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for purifying several types of pyrodiene with different exothermic activity and gelling activity by separating pyrodiene contained in a purified solution according to specific gravity. The liquid is layered in a centrifugal separation capsule with the liquid, and processed using the density gradient centrifugal equilibrium separation method, and each of the specific gravity liquids, such as cesium chloride containing pyrodiene, separated by specific gravity, is processed in a mixed bed ion exchange device. This is a characteristic feature. In recent years, research has reported that pyrogen, especially bacterial endotoxin, has the ability to produce interferon against proliferative tumors and advanced cancer symptoms, and suppresses the growth and progression of these symptoms. pyrogens are in demand. Conventional methods for purifying pyrodiene include the Bois-Vin method, which is obtained by treating cultured fission bacteria with trichloroacetic acid and treating the precipitate with ether, and the Westfahl method, which involves extraction with phenol.
Commercially available products obtained by these purification methods contain impurities such as proteins and phospholipids, and it is desired to further increase the degree of purification. On the other hand, methods for producing sterilized purified water include removing coarse impurities from groundwater, river water, seawater, and tap water, and removing remaining colloidal substances, electrolytes, and microorganisms using conventional water treatment methods, such as coagulation-sedimentation, filtration, Normally, pharmacopoeial purified water is produced by sterilization, desalination, precision filtration (using a membrane of 0.2μ to 0.45μ), etc., which is then heat sterilized at around 121°C to produce sterile purified water. However, in reality, mixed bacteria survive in this sterilized purified water, and when subcultured in an appropriate medium, the mixed bacteria multiply exponentially within a few days, producing pyrogen. There were problems with these sterile purified waters for producing. Therefore, we focused on this point and used conventional reverse osmosis equipment or excess equipment to collect pharmacopoeically purified water or sterilized water using acetyl cellulose, aromatic polyamide, polyvinyl alcohol, polyion bond system, polyfluoroethylene, etc. Concentration of the permeable membrane can be achieved by treating it with a modular permeable membrane based on a graft copolymer of ethylene-vinyl copolymer, aliphatic polyamide, polyester, or other high-molecular substances. An aqueous solution containing pyrodiene was obtained on the side, and water free of pyrodiene was obtained on the permeate side of the permeable membrane. In this way, the pyrodiene in the purified water was concentrated. Thereafter, low-temperature concentration under reduced pressure was performed to reach a concentration ratio of approximately 10 ^-7 compared to the original raw water, and a dehydrated solid was obtained by freeze-drying. The dehydrated solid substance is easily dissolved in distilled water for pharmacopoeial injections and physiological saline, and this solubility showed positive results in both pharmacopoeial pyrogen tests (hereinafter abbreviated as pharmacopoeia tests), and the evaluation has been established in recent years. The Limulus test, which is currently underway, also showed a significant positive result, indicating that the dehydrated solid was highly purified pyrodiene. The threshold value at this time is
It was 10 μg/ml, and 1 μg/ml in the Limulus test. Based on the above knowledge, we have been conducting research on the purification of pyrodiene, which is a multi-chain complex compound, and have separated pyrodiene in solution according to specific gravity, and have separated several types of pyrodiene with different exothermic activity and gelling activity. This led them to devise a unique method for purifying pyrodiene, that is, the present invention. The present invention provides that purified solutions, such as purified water, purified physiological saline, purified sugar solution, etc. obtained by purifying solutions such as water, physiological saline, and sugar solution by known methods, contain a considerable amount of pyrogen. Therefore, the purified solution containing pyrodiene is permeated as necessary to concentrate the pyrodiene, and then this is layered on top of each of several solutions with different specific gravities and subjected to density gradient centrifugation. The equilibrium separation method is used to separate pyrogienes with different exothermic activities and gelling activities into solutions with different specific gravities, and for this purpose, selection of an appropriate specific gravity liquid is an important issue. Sucrose and Ficoll, which are commonly used as specific gravity liquids for ultracentrifugation, are positive in the Limulus test, and are unsuitable because they interfere with the exothermic activity and gelling activity of pyrodiene. A high-strength specific gravity liquid is also unsuitable because it affects the formation of concentration polarization during density gradient centrifugal equilibrium separation. As a result of various studies, it was found that cesium chloride (CsCI) or cesium sulfate (Cs ₂ SC ₄ ) solution is optimal as the specific gravity liquid when carrying out density gradient centrifugal equilibrium separation of pyrodiene according to the present invention. Note that these are preferably dissolved in distilled water for injection. Normally, in density gradient centrifugal equilibrium separation, several capsules in an ultracentrifuge are filled with specific gravities of solutions such as cesium chloride with different specific gravities, and a purified solution containing pyrodiene is layered on top of the capsules, which are then rotated at a constant speed. Centrifugation treatment is often performed in several cases. Next, each of the specific gravity liquids containing pyrodiene separated by specific gravity is diluted 100 to 3000 times with a purified liquid, such as distilled water for injection, and each is sequentially transferred to a mixed bed ion exchanger, such as H-type strong acid. Other impurities such as cesium chloride in the specific gravity liquid are removed by passing the liquid through a mixture of a strong basic cation exchange resin and an OH type anion exchange resin at a specified ratio at a constant flow rate. The treated liquid becomes a purified liquid (pure water) containing each separated pyrogen and flows out to the outside. It can be determined that these purified liquids containing pyrogienes with different specific gravity contain several kinds of pyrogienes with different exothermic activities and gelling activities in a purified state. Therefore, if necessary, the purified liquid containing the pyrodiene described above can be concentrated and freeze-dried to concentrate and solidify the purified pyrodiene having different specific gravities. The above describes a method of purifying a solution such as water by a known method and further purifying the pyrodiene in the solution, but the present invention is not limited to this.
A commercially available pyrodiene may be mixed into a purification solution, such as distilled water for pharmacopoeial injection, and this may be subjected to purification by specific gravity. In any case, pyrodiene that has been fractionated and purified according to its specific gravity has improved activity compared to before purification, and has increased medical usefulness. Examples of the present invention will be described below. Example 1 The raw water used was tap water sampled from an elevated water tank in Bunkyo Ward, Tokyo, but this raw water was positive according to the pharmacopoeial test, and was strongly positive, ie "++", according to the Limulus test. It was hot. This raw water is treated with a mixed-bed ion exchange device to deionize it to a specific conductivity of 0.1μυ/ml (25℃), and this deionized water is passed through an aromatic polyamide reverse osmosis module with a permeation pressure of 15 to 25 kg. /cm ² and a permeation flow rate of 425/module/time, a concentrated solution of about 10% was obtained on the concentration side from Iton deionized water. This concentrated liquid was concentrated under reduced pressure at 50°C to obtain 1. A small amount of solid matter precipitated therein was removed using a 0.45 μ filter, and the liquid was further concentrated under reduced pressure at below 50°C to approximately 50 c.c. This was quenched with acetone and dry ice, frozen, and dried under ultravacuum. This dried product was a dehydrated solid with a light yellow color and weighing about 100 mg. This solid substance was dissolved in distilled water for pharmacopoeial injection, and a gelation activity test was performed using a pharmacopoeial test and a limulus test. The gelation activity was 1 μg/ml, total organic carbon 34.13%, organic nitrogen 2.94%, iron 0.21%, and ammonium ion 0.208%. As mentioned above, since the solid substance was apparently a pyrogen, that is, pyrodiene in water, this solid substance was further purified by density gradient centrifugal equilibrium separation, and fractions of pyrodiene by specific gravity (density) were obtained. In other words, we were able to obtain a pure product with improved pyrodiene activity. In this process, it is convenient to separately use standard pyrodiene as a target, so in this experiment, we used a commercially available Escherichia coli bacterial endotoxin manufactured by Difco Lab in the United States.
Dissolved in distilled water for pharmacopoeial injections, this and the pyrodiene from the water treatment system described above were placed in an ultracentrifuge (Spinco E model manufactured by Beckman), and the differences in concentration polarization due to changes in rotational speed were measured using a Schilleren optical system. I confirmed it. It is thought that the number of bees detected by the Schilleren optical system at this time is almost attributable to the origin of pyrodiene, and for concentrated pyrodiene in water treatment systems, there are two rotational speeds of 17,000 rpm and 44,000 rpm, and for E. coli bacterial endotoxin,
17000rpm, 22000rpm~25000rpm and 56000rpm
Detection peaks were confirmed at three positions. In addition, a preliminary experiment was conducted to determine the type and specific gravity of the specific gravity liquid that dissolves pyrodiene during ultracentrifugation. First, as shown in FIG.
E. as a standard pyrogen.
Purified solution P containing coli-based bacterial endotoxin was layered on the top layer, and ultracentrifugation was performed at a rotation speed of 44,000 rpm for several hours. At a rotational speed of 17,000 rpm, the density is not distributed to each part of the density gradient shown in FIG. This is because the concentration polarization component of pyrodiene at a rotation speed of 17,000 rpm, that is, the first detection peak component in the Schilleren optical system, has weak exothermicity and weak gelation activity by the Limulus test, and is composed mainly of impurities contained in pyrodiene, that is, phospholipid bodies. The specific gravity is assumed to be less than 1.02. From rotation speed 25000rpm or more
The concentration polarized component up to 56000 rpm is distributed to each part of the density gradient of 1.02 to 1.20 (specific gravity at 20°C) of ficoll and sucrose. That is, when each density gradient fraction was fractionated and examined by electrophoresis, it showed the same tendency as the previously determined electrophoretic characteristics of E. coli, making it possible to know the existence of the component. Sucrose and Ficoll themselves are both positive in the Limulus test, and if they are used as a specific gravity solution, they will affect the exothermic activity and gelling activity of the material pyrodiene itself, so it is difficult to find the threshold of the gel-forming reaction of pyrodiene. was inappropriate. Therefore, in this experiment, cesium chloride (CsCI) was used as the specific gravity liquid for density gradient centrifugation.
Dissolved in distilled water for injection, use
Three types of specific gravity liquids were prepared: d ₁ =1.15, d ₂ =1.10, and d ₃ =1.05. Then, as shown in Figure 2, each of the 5 ml ultracentrifugal capsules 4 to 6 is filled with one type of cesium chloride specific gravity solution with a different specific gravity, and then the water treatment system described above is filled. The purified solution RP containing concentrated and purified pyrodiene was layered on top of the purified solution RP, and density gradient centrifugal equilibrium separation was carried out at a rotation speed of 44,000 rpm for about 2 minutes.
I spent time. Then, a sample was taken from each part of each capsule as shown in Figure 2 with a pipette, and the gelation state was classified by the Limulus test.
The active coordination of pyrodiene at each site was confirmed in the categories shown in Table 1.

[Table] However, the above-mentioned cesium chloride specific gravity liquids of each specific gravity of d ₁ , d ₂ , and d ₃ dissolve pyrodiene in water at a constant rate at 20°C. Table 2 shows the degree determined by Limulus reaction. Therefore, the actual distribution concentration by pyrodiene specific gravity is
The concentration of pyrodiene in each specific gravity liquid in Table 1 is minus the concentration of intrinsically soluble pyrodiene in Table 2. From the above experimental results, the practical threshold of gelling activity in the limulus _test is 5 × 10 ^-3 μg/
ml, 1 × 10 ^-2 μg/ml in d ₂ , 1 in d ₃
×10 ^-1 μg/ml.

[Table] Next, the specific gravity liquids of d ₁ , d ₂ , and d ₃ containing concentration-polarized pyrodiene were separated by specific gravity, respectively.
Collect the sample in a container that has been dry-heat sterilized at 250°C for 30 minutes, dilute it several thousand times with a diluent, such as distilled water for injection, and sequentially transfer each diluted solution to a mixed-bed ion exchanger, such as a strong acid Liquid passing rate SV10 ~ through an ion exchange resin cylinder in which H type cation exchange resin and OH type strongly basic anion exchange resin are evenly mixed in a 1:1 ratio.
Run the liquid at 60°C. Cesium chloride in the specific gravity liquid is almost quantitatively adsorbed and removed, and pyrodiene passes through the ion exchange resin layer and is eluted into pure water as a treatment liquid. After concentrating the pure water containing pyrodiene, it is freeze-dried to form a solid substance, d ₁ , d ₂ ,
Purified pyrodiene was obtained by gravity fractionation, that is, concentration polarization, in each specific gravity solution of _d3 . These three types of dehydrated solid pyrodiene were redissolved in a pure water solution and subjected to a pharmacopoeia test and a limulus test, and the results were compared with the pyrodiene concentrated from the water treatment system before the treatment of the present invention. are shown in Table 3.

[Table] As is clear from Table 3 above, the specific gravity purified pyrodiene purified by the method of the present invention has a stronger exothermic activity than pyrodiene concentrated and purified from a normal water treatment system, and The gelation activity threshold was also improved. Therefore, by further separating the conventional purified pyrodiene by specific gravity, it is possible to classify it by each exothermic activity and gelling activity, so it is possible to obtain pyrodiene of higher purity depending on the purpose of use than the conventional purified pyrodiene. Therefore, it can be usefully used in various medical fields. Example 2 As pyrodiene, a type of endotoxin from Escherichia coli strain 055B5 was dissolved in phosphate buffer pH 6.98 to a concentration of 10,000 ppm to obtain a pyrodiene stock solution. Next, prepare a pyrogen-free specific gravity solution of cesium chloride (CsCI) so that the specific gravity is 1.15, 1.10, and 1.05 at 20℃, and place it in a capsule (inner volume 5 ml) for ultracentrifugation. 4 for each book
Dispense 5 ml each, distinguish d ₁ (specific gravity 1.15), d ₂ (specific gravity 1.10), and d ₃ (specific gravity 1.05) for each specific gravity liquid capsule, and add 0.5 ml of pyrodiene on top of each specific gravity liquid. Gently dispense the stock solution in layers. Afterwards, seal the top of the pyrodiene stock solution with liquid paraffin liquid and use the Beckmann SPINCO accessory.
Attach three capsules to the SW50E rotor,
The rotation speed was increased to 56,000 r.p.m., and after being maintained for about 3 hours, the density gradient centrifugal equilibrium separation was completed 8 hours after the initial operation by automatic stop without any control. Thereafter, the capsule was taken out, and the cesium chloride layer was carefully fractionated and drawn out from the upper part, and transferred to another container to obtain a pyrodiene distribution liquid by specific gravity fraction. This solution was diluted several thousand times with pure water that does not contain pyrodiene, and passed through a normal mixed bed type ion exchange resin cylinder at SV2 to 15. The ion exchange resin at this time is a normal H type strongly acidic cation exchange resin or an OH type strongly basic anion exchange resin. It was confirmed by checking for chloride ions using the mercuric nitrate method (JIS K0101) that the pure water containing pyrodiene flowing out from the ion exchange resin cylinder did not leak cesium chloride. In this way, a pyrodiene diluted pure water solution free of cesium chloride was obtained, and this pure water diluted solution was concentrated under reduced pressure and further freeze-dried to obtain a purified pyrodiene solid. When the threshold value was measured using the usual Limulus test (INVITRO) and Pharmacopoeia test (INVIVO), the results were as shown in Table 4 below. In other words, the components from which cesium chloride is finally removed by ion exchange treatment are roughly divided into the following three components, each of which exhibits a different pyrodiene activity than before the treatment according to the present invention, and the _d1 component in particular has a higher activity than before the treatment according to the present invention. I obtained a pure product that shows the following.

【table】 [Brief explanation of the drawing]

Figure 1 shows a preliminary experiment for selecting a specific gravity liquid for density gradient centrifugal equilibrium separation to be used in the present invention. An explanatory diagram showing a state in which a gradient multilayer solution is supplied and a standard pyrodiene solution P is superimposed on it. FIG. 2 is an embodiment of the present invention, and capsules 4, 5,
Cesium chloride specific gravity liquid d ₁ with different specific gravity,
d ₂ and d ₃ were respectively supplied, and refined sugar pyrodiene RP treated with a conventional well-known water treatment system was layered on top of them, and density gradient centrifugal equilibrium separation was performed. FIG. 1 to 6...Capsule, 4-0 to 4-5...Sample collection position, 5-0 to 5-4...Sample collection position,
6-0 to 6-5...Sample collection position, F...Ficoll, S...sucrose, P...standard pyrodiene, RP...
Purified pyrogen.

Claims (1)

[Claims] 1. A solution containing pyrodiene is layered with several types of specific gravity liquids such as cesium chloride with different specific gravities, density gradient centrifugal equilibrium separation is performed, pyrodiene is separated according to specific gravity in each specific gravity liquid, and each separated pyrodiene is further contained. After diluting a solution such as cesium chloride with a pyrodiene-free solution, each of the diluted solutions is sequentially passed through a mixed-bed ion exchange device to remove cesium chloride, etc., and each separated pyrodiene-containing solution is used as a treatment solution. A method for purifying several types of pyrogienes with different exothermic activity and gelation activity by obtaining