JPH0247447B2 - KOHOTAIKANOHIKUIIGMSOSEIBUTSU - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to IgM compositions with low anti-complement titers and thus capable of intravenous injection.

It is well known that among human immunoglobulins (Ig), IgG in particular is widely used to prevent various diseases and has remarkable preventive and therapeutic effects.
By the way, the biological activities of immunoglobulins differ depending on their type, and IgM is (i) produced earlier than IgG upon antigen stimulation of living organisms.

(ii) The molecular structure is a pentapolymer, and the binding value with the antigen is 10
(IgG is 2), and therefore has strong agglutination activity against bacteria, especially gram-negative bacteria.

(iii) It has characteristics such as bacteriolytic activity and opsonic activity that are stronger than those of IgG. These properties make IgM useful for treating bacterial infections.
It is said that it may be more suitable than IgG. Because of this, IgM (rich)
The preparations have attracted attention and development studies have been conducted, and some IgM preparations for intramuscular injection have already entered the practical stage, with reliable therapeutic effects being obtained. However, since it is administered by intramuscular injection, rapid effects due to the rapid rise in blood concentration due to large doses cannot be expected.
Moreover, pain at the injection site, delayed absorption, loss due to enzymatic degradation within muscle tissue, etc. are unavoidable.

On the other hand, attempts have been made to inject IgM intravenously, and Teimner et al.
They reported that they manufactured an IgM (rich) preparation and administered it intravenously to patients suffering from bacterial sepsis, etc., and observed improvement in symptoms (KDTympner et al. Mshr.
Kinderheilk, 123, 400−401 (1975)).

However, when IgM is intravenously injected as is,
As in the case of IgG, aggregates (polymers) produced during the manufacturing process are thought to cause rapid activation of complement, causing anaphylaxis-like side effects. As a way to reduce such side effects
Methods known in the case of IgG, namely enzymatic treatment employed for reducing the anti-complement titer of IgG, S
- All alkylation treatments, sulfonation treatments, etc.
There is a problem in that the pentapolymer structure that expresses the strong aggregation activity of IgM is destroyed, and the antibody activity that IgM originally has is significantly reduced.

Because of these problems, there are definite advantages to intravenous administration, as shown by the fact that the development of intravenously injectable IgG preparations has dramatically increased the versatility of immunoglobulin therapy and its therapeutic efficacy. Nevertheless, the development of intravenously injectable IgM preparations has been difficult and has not been put into practical use to date.

As a result of intensive research aimed at developing a safe intravenously administerable IgM preparation, the present inventors found that an IgM preparation (aqueous solution) containing basic amino acids has anti-complementary properties against normal human serum (NHS). The present invention was achieved based on the discovery that the body price was reduced to 30% or less, making intravenous administration possible.

That is, the present invention is an IgM composition comprising IgM and a basic amino acid and having a low anti-complement value.

IgM used in the present invention can be obtained, for example, from a fraction (F-fraction) of the ethanol fraction of human blood corn by the following method.
The fraction is extracted with a solution such as sodium chloride, ammonium sulfate, etc. is added to the extract to precipitate the immunoglobulin fraction, and the crude IgM is extracted by centrifugation.
Collect as a fraction. This crude IgM fraction is subjected to, for example, sepharose gel chromatography.
Collect only the IgM fraction. After this, further purification operations such as precipitation with polyethylene glycol and sepharose gel chromatography may be combined.

Specific examples of basic amino acids used in the present invention include arginine, lysine, ornithine, and histidine. Arginine and lysine are preferred.

The composition of the present invention preferably contains a basic amino acid in a range of 0.1 to 10 parts by weight per 1 part by weight of IgM. Although the method for preparing the composition is not limited in any way, it is convenient to dissolve a basic amino acid to a predetermined concentration in a purified aqueous IgM solution, or to mix an aqueous solution of a basic amino acid. The aqueous solution thus obtained (usually a physiological saline solution) is
For example, IgM preparations for intravenous injection are manufactured by adding solubilizers, stabilizers, tonicity agents, etc., or by subsequent freeze-drying. Or, again,
If 20% by weight or more of the total protein in an IgM preparation is IgM, the characteristics of an IgM preparation will be fully demonstrated, so the remaining less than 80% by weight should be from immunoglobulin components other than IgM (IgG, IgA, etc.). An intravenous IgM (rich) preparation containing basic amino acids may be prepared.

The antibody titer of the IgM composition of the present invention is not substantially different from the antibody titer that IgM originally has.

In the present invention, the anti-complement titer is measured against normal human serum (NHS) using the method of Kabat and Mayer (Experimental).
Immunochemistry 225 (1961)). Anti-complement value of 2% solution of IgM composition is 30
% or less is particularly suitable for intravenous preparations with few side effects.

Hereinafter, the present invention will be explained in detail with reference to Examples. The percentages in the examples are by weight unless otherwise specified.

Example 1 (1) Extract the fraction obtained by Cohn's ethanol fractionation method with a 0.45% sodium chloride aqueous solution, add ammonium sulfate to the extract to make it a half-saturated ammonium sulfate aqueous solution, and centrifuge the precipitated globulin fraction. collected by
This was dissolved in a small amount of pure water to obtain a crude IgM fraction.

This crude IgM fraction was gel-filtered with Sepharose 4B, and the shaded fraction in the chromatogram shown in Figure 1 was collected.
The IgM fraction was collected, polyethylene glycol 4000 was added to it to give a concentration of 5 to 10%, and the precipitated fraction was collected by centrifugation. This was dissolved in a small amount of sodium phosphate buffered saline, gel-filtered through Sepharose 4B, the IgM fraction was collected in the same manner as above, and further concentrated by ammonium sulfate salting out.
Dialyzed against sodium phosphate buffered saline containing 0.5% polyethylene glycol 4000;
Purified IgM with a purity of about 90% was obtained.

(2) Dissolve L-arginine at a concentration of 2% in the 2% IgM obtained in (1) above, leave it at 4°C for 1 hour, and add 2% arginine to the Ig solution (2%).
was prepared.

(3) The anti-complement titer of the 2% arginine-added IgM solution prepared in (2) above against human standard complement was measured according to the Kabat and Mayer method. That is, 0.5 ml of IgM strength solution supplemented with 2% arginine was added to 0.5 ml of human standard complement, incubated at 37°C for 1 hour, cooled on ice, diluted 40 times with gelatin-added veronal buffer, and incubated. The anti-complement titer was determined by measuring the post-basis complement titer using the method of Kabat and Mayer.

As a result, the anti-complement value of IgM added with 2% arginine was 3.5%. The anti-complement value of IgM without arginine was 42%.

Example 2 L-arginine was dissolved in 2% IgM obtained by the method described in Example 1 (1) to give a concentration of 1% and 5%, and left at 4°C for 1 hour. % arginine added IgM and 5% arginine added IgM
were prepared respectively. The anti-complement value of each arginine-added IgM solution was measured by the method described in Example 1 (3). As a result, the anti-complement value of IgM added with 1% arginine was 15%, and the anti-complement value of IgM added with 5% arginine was 1%.

Example 3 L-lysine was dissolved to a 2% concentration of IgM obtained by the method described in Example 1 (1) to a concentration of 1%, 2%, and 5%, and 1% arginine was added to each. Added IgM, 2% lysine added IgM, and 5% lysine added IgM were prepared, and the anti-complement value was measured by the method described in Example 1 (3). As a result, the anti-complement value was 18% for IgM with 1% lysine, 5% for IgM with 2% lysine, 1.8% for IgM with 5% lysine, and 41% for IgM without lysine.

[Brief explanation of drawings]

FIG. 1 is a chromatogram of Sepharose 4B gel filtration of the crude IgM fraction.

Claims (1)

[Claims] 1. An IgM composition with a low anti-complement value comprising IgM and a basic amino acid. 2. The IgM composition with a low anti-complement value according to claim 1, wherein the basic amino acid is contained in an amount of 0.1 to 10 parts by weight per 1 part by weight of IgM. 3. The IgM composition with a low anti-complement value according to claim 1, wherein the basic amino acid is arginine and/or lysine.