JPS6193111A - Removal of pyrogen - Google Patents

Abstract

PURPOSE:In the removal of pyrogenic substances which dissolve in injection solution or transfusion solutions and develop fever in men and animals, the solution is stirred in the presence of a surfactant with a steroidal skeleton to form a micelle and the micelle is separated from the solution whereby the solution free from pyrogens is obtained. CONSTITUTION:A solution containing pyrogens is stirred together with a steroidal surfactant such as apocholic acid, chenodeoxycholic acid, or cholic acid to form a micelle in which the pyrogens are clathrated in the surfactant to effect the removal of the pyrogens. Since the surfactants with steroidal skeleton almost occur in nature, it would be safe, even when it remains in the solution and enters to the living body, because it is metabolized, decomposed and excreted.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for removing pyrogenic substances using a surfactant having a steroid skeleton.

Substances dissolved in injection solutions that sometimes cause fever in humans and animals are called pyrogenic substances, and it is a well-known fact that one such substance is Noribopolysasoccaride derived from Gram 1 Reediform. Pyrogenic substances that enter the human body through injections, infusions, or other routes cause fever and severe shock symptoms, so many researchers have been researching ways to eliminate them for many years. , Method for separating pyrogenic substances from solutions containing pyrogens under physiological conditions due to their strong heat resistance and chemical stability, Physiology in solutions of physiologically active substances containing pyrogenic substances At present, no definitive method has yet been developed for deactivating only the pyrogenic substance without deactivating the active substance.

Conventional techniques include a removal method using an endotoxin adsorbent devised by Niwa et al. and a method using Triton In addition, in the latter method, the synthetic surfactant and the formulation are mixed, so if the synthetic surfactant is not 100% recovered and remains in the formulation, etc. However, it is thought that there are many problems in applying these preparations to the human body.

As a result of research on a simple method for removing pyrogenic substances, the inventor of the present application discovered that by mixing and stirring a surfactant having a steroid skeleton (separately listed in Table 1) and a solution containing a pyrogenic substance, an interface having a steroid skeleton can be removed. The present invention has been completed based on the discovery that a method of forming pyrogen-conjugated micelles with an activator and then separating the micelles from the solution is effective for removing pyrogens from pyrogen-containing solutions.

Although some surfactants with a steroid skeleton are partially synthetic, most of them are natural products, so even if a trace amount of the surfactant remains in the solution to be treated after mixing with and separating from a pyrogenic substance-containing solution, It is extremely safe as it is metabolized and decomposed within the body and excreted from the body.

Furthermore, it is known that it causes less destruction of protein higher-order structure than surfactants such as SDS, SLS, and STS.

Furthermore, among surfactants with a steroid skeleton, it is true that bile acid-derived surfactants have a hemolytic effect, but their hemolytic effect is significantly attenuated by the addition of serum, so their safety is extremely low. expensive.

Putting this invention into practice/7i! This will be explained in detail based on one example.
Table L (1) Apocholic acid chenodeoxycholic acid 3α,7α-dehydroxy-12-keto-5β-furic acid sodium fluoric acid cholate sodium salt Dehydrocholic acid dehydroynylic acid sodium salt Dehydrolidonylic acid deoxycholic acid deoxynylic acid Sodium salt Gly: sodium chenodeoxycholate Gly: cholic acid (crystal) Gly: sodium salt glycodeoxynylic acid glyfuritocholate sodium salt 3β-hydroxy-Δ5-: hyodeoxycholic acid: hyodeoxycholic acid Lidonylic acid Table 1. (2) Lidonylic acid-3-ammonium sulfate salt glycolidenylic acid-3-sulfate disodium taurochenodeoxy: -
Sodium taurocholate Sodium tauronylate Sodium taurodehydronylate Sodium taurodeoxycholate Sodium taururtudeoxycholate Tau urundeoxycholate; Sodium lithocholic acid-3-sulfate Ammonium Taurolide Sodium fluoride Siquitonin Licoursodeoxy Sodium cholate The present invention is not limited by these.

Example 1 100 ml of a protein preparation containing 97 ml of pyrogen 10 and precipitated with 35% saturated ammonium sulfate was prepared at a pH of 12
, 5 phosphate buffer (1142yK2HPO, 3H20
, 140 KO Lq) was adjusted to pH 9.3 kg, 0.05% (W/W) sodium thioxycholate was added, and stirred at room temperature for 4 hours to form pyrogen-conjugated micelles. Ta. Next, salting out with ammonium sulfate saturated with 2096, centrifugation at 8.00 Or, p, m for 30 minutes to precipitate the micelles formed by sodium deoxycholate and separate them from the supernatant (protein preparation). Add a phosphate buffer with a pH of 12.5 to pure water to adjust the pH to 9.3, and dialyze with a sterilized and filtered solution to completely remove sodium deoxycholate that could not be completely removed by centrifugation. When a Japanese Pharmacopoeia Rabbit Pyrogenic Substance Test was conducted using the dialysis fluid, the total body temperature increase of the three Kupugi chickens was 0.4°C, which passed the pyrogenic substance test.

(Centrifuge tubes, dialysis tubes used for operations after centrifugation,
All instruments such as beakers were Pyrogen free, and all operations were performed aseptically. ) Example 2 A 40% human serum albumin solution containing 20 ng/ml of pyrogen was diluted to pH 9 using Tris buffer.
After adjusting to 0, add 0.5% (W/W) cholic acid)
After adding IJ cum and reacting at room temperature for 4 hours, it was left in a cold room at 4°C overnight to precipitate sodium cholate, and then reacted at 12.00 Or, p, m using a refrigerated centrifuge. 30
After centrifugation for a minute, the supernatant (human serum albumin dissolved tL) was separated, and the following Example 1. The same operations as those performed after dialysis were performed to obtain a pyrogen-free human serum albumin solution.

Example 3: 100mj of Japanese Pharmacopoeia hevarin natrichum solution containing 200g/me of pyrogenic substance? was adjusted to pH 9.5 using a phosphate buffer with pH 12.5, and then 0.5Φ (~
V/W) of sodium chenodeoxycholate was added,
After reacting at room temperature for 4 hours, pyrogens were removed from the solution using an alkaline ethanol solution, and the anion exchange resin Amberlite IRA-400 was thoroughly washed with pure water that does not contain pyrogens.
As a result of the Kusaya pyrogenic substance test, the total exothermic temperature of the three birds was 0.6°C. A heparinato IJ cum solution containing no pyrogenic substances was obtained. (Operation literature using ion exchange resin

Claims (1)

[Claims] A method for removing a pyrogen comprising the following steps: (a) A pyrogen-containing solution and a surfactant having a steroid skeleton are mixed and stirred to form pyrogen-conjugated micelles. (b) A pyrogen-free solution is obtained by separating the surfactant having a steroid skeleton and the pyrogen-conjugated micelles it forms from the original solution.