JPS597695B2 - Peptide preparations with immunoregulatory effects - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an immunoregulatory agent whose main component is a peptide having an immunoregulatory effect.

The discovery of the presence of immunosuppressive agents in serum was made in 1959.
Year, B. B. Kamri (B, B, Kamri
n) made by CB. B. Camryn: Proceedings of the Society for Experimental Biology and Medicine 7 (
Proc, of the Soc, for E
Experimental Biology & Medicine
ine) Volume 100, Page 58, 1959).

This report reports that the active ingredient is contained in the α2-globulin-rich Cohn fraction, and that the effective factor in human serum is also effective against rats, and that it acts across species distinctions. has been done.

Later, this thing was released in 1973 as Oktino (Occh
[The Journal of Immunology] was isolated from the human serum Cohn EV-1 by et al. The
Journal of Immunology
) Volume 110, No. 3, 1973].

The peptide is called IRA (Immunoreg).
Cooperband et al. of Boston University called it ulatory α-globulin).

This peptide is also herein abbreviated as IRA.

The method used by Octino et al. uses the rv-i fraction of Cohn, which contains 45% of the activity of IRA contained in the Cohn fraction, as a starting material, and the In this method, IRA is adsorbed onto a strongly basic anionic cellulose such as DEAE-cellulose, eluted with an aqueous sodium acetate solution having an ionic strength of 0.3, the eluate is then dialyzed in acidic conditions, and IRA is collected in the dialysate.

According to this method, the degree of purification of IRA is good, but the yield rate is low, and the recovery rate from fraction ① is only 0.4%.

The object of the present invention is to extract I from human blood with a good industrial recovery rate.
To provide an RA preparation.

Another purpose is to provide IRA injections.

The above purpose is to treat an aqueous solution of human blood or an α2-globulin-containing fraction thereof with an acid at a pH of 5.0 or less,
By ultrafiltering the treatment solution, the molecular weight is 1300.
Peptides smaller than 0 are collected in a filtrate, impurities contained in the filtrate are removed by adsorption on a cation exchanger, then IRA in the filtrate is adsorbed on an inorganic adsorbent, eluted, and injected. A preparation of the present invention of IRA which can be obtained is obtained.

The raw human blood used in the present invention includes not only normal blood but also hemolyzed blood, such as placental blood.

Although blood can be used as it is, it is preferable that
A fraction containing 2-glopurin is collected in advance and used.

In this way, the above-mentioned blood plasma fraction containing concentrated α2-globulin, and more preferably Cohn's fraction (2), which is usually discarded when collecting valuable serum components such as albumin, can be obtained. <<■-1 fraction of corn is suitable as a raw material for the present invention.

When using blood itself, it is preferable to dilute it.

For example, a blood extract obtained by extracting placental blood with water or saline can be used as is in the present invention.

The aqueous solution containing both blood and α2-globulin, which serve as raw materials, is treated with an acid at a pH of 5.0 or lower.

The acid used is a mineral acid such as hydrochloric acid, phosphoric acid, or sulfuric acid, or an organic acid such as acetic acid or formic acid, and the raw material aqueous solution is adjusted to pH 5.0 or less, preferably pH 1.5 by adding these acids or a buffer. -5.0, more preferably 2.0-3
．． Keep it at 5.

A treatment temperature of room temperature is sufficient, and a treatment time of 30 minutes to 24 hours.

By the above treatment, the non-covalent bond between IRA and glopurin in the raw material aqueous solution is broken and the IRA becomes free.

In order to separate the liberated IRA, both fractions having a molecular weight of less than 13,000 are selectively filtered out using ultrafiltration.

The preferred method for this is to use suitable hollow fibers, which allow for rapid operation and
and effective.

That is, Diafiber JDi manufactured by Amicon, USA
afiber) (for filtration of molecules with a molecular weight smaller than 10,000), Asahi Kasei Ultrafiltration ■
(for filtering substances with a molecular weight of less than 13,000), etc., are used.

This ultrafiltration removes proteins in the raw material.

The protein-free solution obtained as a filtrate is treated with a cation exchanger, such as CM-cellulose (carboxymethylcellulose, sold by Seikagaku Corporation), CM-Sephatex (carboxymethyl crosslinked dextrin, sold by Seikagaku Corporation). , and an acidic group-containing polysaccharide ion exchanger such as P-cellulose (phosphocellulose, sold by Seikagaku Corporation), and the highly antigenic contaminants contained therein are adsorbed and removed.

This step also removes hypotensive substances and minimizes subsequent production of new substances.

As the processing conditions for this step, it is preferable to use an ion exchanger equilibrated with a buffer solution having a pH of 2 to 7, preferably 4 to 5.5, and a salt concentration of 0.1 to 0.5M.

When the IRA solution from which impurities have been removed is brought into contact with an inorganic adsorbent, IRA is adsorbed by the adsorbent.

The acquisition of IRA by an inorganic adsorbent is a novel discovery by the inventors, and is extremely inexpensive and more effective than the use of an anion exchanger by Octino et al.
This method is easy to process and extremely effective as an industrial manufacturing method.

Examples of the inorganic adsorbent used include silica gel, bentonite acid clay, and activated carbon. Among these, the amount of inorganic adsorbent used is not limited, but is usually 1 to 5 volumes per 100 volumes of IRA solution. used.

At that time, the pH of the solution is adjusted to 5.5 to 9.0, preferably << is p
Good results can be obtained by adjusting H to 7.0 to 8.0.

By stirring the mixture of IRA solution and adsorbent at room temperature for 30 minutes, about 75-95% of the IRA is adsorbed onto the adsorbent.

The contaminants remain in the aqueous solution and the IRA is recovered by eluting them from the inorganic adsorbent.

Elution of IRA from this inorganic adsorbent is carried out under acidic conditions.

The preferred pH of the eluate is 2 to 4, and its adjustment includes:
Inorganic or organic acids such as hydrochloric acid, phosphoric acid, sulfuric acid, formic acid, and acetic acid are used.

After neutralizing the eluted IRA solution if necessary, it is dried under reduced pressure or freeze-dried according to a conventional method to obtain powdered IRA.

In the case of the present invention, the recovery rate from corn fraction ① is approximately 1.2
%.

In addition, its purification can be performed in about one day, compared to the conventional three-way purification.
It is extremely efficient compared to a ~4 day process.

The activity of the obtained IRA is approximately 100-1000γ (
(as nitrogen), PHA method C Cooperva/de S,R.
(Cooperband, S.R.)
(The Journal Op Immunology, Vol. 109, No. 1, p. 154, 1972)] 70
Shows ~100% immunosuppressive effect.

This result shows that the activity is comparable to that of conventionally provided IRA.

The properties of this IRA are stable against heat treatment at 56°C for 30 minutes, and β-
A band is observed in the globulin region.

This substance is a peptide with a molecular weight of approximately 5000 as measured by Sephadex gel filtration. Since the IRA produced in this manner uses human body-derived materials such as plasma placental blood as a starting material, it can be said to be an extremely safe drug with no concerns about antigenicity.

It also shows little or no cytotoxicity to human lymphocytes in the presence of complement, and E. Rosette test [Menzoian et al.
l) The Journal of Immunology No. 113
Vol. 266 (1974)) prevents aggregation of immune cells.

If IRA with even higher purity is desired, the eluate from the inorganic adsorbent can be purified with an organic solvent such as a chlorinated hydrocarbon such as chloroform, or an ether such as diethyl ether.
RA can be extracted and purified.

Purity is significantly improved by this method.

This extraction process can also be combined with a step of eluting IRA from the inorganic adsorbent.

That is, elution can be performed by mixing the organic solvent and the eluent.

In such operations, a solvent that mixes with water, such as acetone or alcohol, can also be used as the solvent to be mixed.

Furthermore, the IRA of the present invention contains almost no antihypertensive substances and is safe for injection.

Although the activity of hepatitis viruses is usually not observed, it is possible that some raw materials may be contaminated with hepatitis viruses.

Therefore, the present invention also provides IRA that has been subjected to hepatitis virus (HBV) inactivation treatment.

Although the IRA according to the present invention is quite stable to heat in its aqueous solution as described above, its activity remains low when heated for 10 hours at a temperature of 60°C, which is known as HBV inactivation treatment of plasma protein components. decreases.

The present inventors have demonstrated that the IRA aqueous solution is stabilized against heat in the presence of neutral amino acids, monosaccharides, disaccharides, or sugar alcohols, and that
It was found that the activity was not significantly lost even when heated at ℃ for 10 hours.

In the present invention, the above thermal stabilization heat treatment can be applied to the IRA aqueous solution at any step during production.

However, it is preferred that this treatment be incorporated in a step prior to acid treatment of the IRA aqueous solution.

Examples of stabilizers are neutral amino acids (monoaminomonocarboxylic acids) such as glycine, alanine, parine, leucine, isoleucine, glucose, mannose, galactose,
These include monosaccharides such as fructose, disaccharides such as sucrose, maltose, and lactose, and sugar alcohols such as mannitol, sorbitol, and xylitol.

The amount of stabilizer used is 5% (W/V) based on the aqueous solution.
Although it is above, preferably it is 15 to 20%.

Heating to inactivate the hepatitis virus is performed by a conventional method, and in the most common heat treatment at 60°C for 10 hours, the activity of IRA is still 95% by adding an appropriate amount of stabilizer.
Retained.

The stabilizer remaining in the aqueous solution after HBV inactivation can be separated by adsorbing IRA to the above-mentioned adsorbent, or it can be removed by dialysis.

Thus, the present invention provides an injectable human blood-derived IRA preparation that has been subjected to HBV inactivation treatment and has a minimal content of hypotensive substances.

Since this preparation suppresses transplant rejection, it can be effectively used to prevent and treat rejection during transplant surgery and maintain good conditions after surgery, and can also be used to treat cell-mediated immune diseases.

When this IRA is used as a medicine for treatment, the most preferred form is an injection, and intravenous injection is optimal.

It is considered that a dosage of 5 to 40 μg/kg per day is sufficient.

In a rat skin graft experiment, 20η/kg of IRA was administered per day.
As a result of continuous intravenous injection for 20 days, 85% engraftment was observed on the 20th day.

Furthermore, this IRA acute toxicity experiment was conducted using mice for 50 days.
0m9/kg, 1000■/ky were administered, but
No deaths were observed after 48 hours of observation.

Example 1 Using the PHA method obtained by Kohn's alcohol fractionation method, 4r
Cohn's IV- with 70% inhibitory activity at n9/ml
Pace for 1 fraction} 1 kg was suspended in 4 liters of cold distilled water and heated at 4°C.
Stir for 2 hours.

Centrifugation is performed to remove insoluble matter, and glacial acetic acid is added to the resulting supernatant to adjust the pH to 3.5, followed by gentle stirring for 1 hour.

This liquid is filtered through a hollow fiber (manufactured by Asahi Kasei Corporation) to collect a low molecular weight portion having a molecular weight of 13,000 or less.

1% by volume of CM-cellulose (manufactured by Seikagaku Kogyo Co., Ltd.) is added to this low molecular weight portion and stirred for 30 minutes to adsorb inactive contaminants and obtain the IRA active portion as a filtrate.

Activated carbon was added to this filtrate at a ratio of 4% by volume, and the pH was adjusted to 8.
0 to adsorb the active portion onto activated carbon.

IM acetic acid is used to elute active components.

The eluate was lyophilized to purify IRA6? I got it.

In cellulose acetate membrane electrophoresis, a band was observed in the β-globulin region, and its molecular weight was approximately 5,000.

Example 2 IRA-adsorbed activated carbon obtained by operating in the same manner as in Example 1 was mixed with the same volume of chloroform, stirred, and then the chloroform layer was separated, and this was dried under reduced pressure to volatilize the chloroform. , purified IRA5. I got l'.

This IRA2Or/ml is 60%, 50% by PHA method.
Almost 100% inhibitory activity was shown at 0.17 ml.

Example 3 100 human placentas are cut into pieces, suspended in 10 liters of 0.1M sodium chloride solution and stirred briefly.

Centrifugation is performed to obtain a clear extract of about 20 liters.

The extract was collected and adjusted to pH 2.0 using 2N hydrochloric acid.
Stir gently for 2 hours at room temperature.

A low molecular weight portion having a molecular weight of less than about 10,000 is collected using a hollow fiber (manufactured by Amicon, USA).

CM-Sephadex was added to the resulting aqueous solution at a ratio of 1% by volume, stirred for 30 minutes, and then filtered to obtain an IRA aqueous solution.

5% by volume of silica gel was added to this aqueous solution, and the pH was adjusted to 9.0 with IN sodium hydroxide.

After stirring for about 30 minutes, filter and collect the silica gel used for adsorption using Toyo Roshi A2.

0. After elution with 1 M formic acid, the product was dried under reduced pressure to obtain purified IRA900.

This IRA500r/ml showed 85% inhibitory activity by PHA method.

It was confirmed to be IRA by electrophoretic and molecular analysis.

Example 4 Silica gel adsorbed with IRA obtained by the same operation as in Example 3 was mixed with about % volume of acetone, stirred to elute IRA, and the eluate was dried under reduced pressure to obtain purified IR.
A750m9 was obtained.

This IRA is 20r/I71l, approximately 65% by PHA method.
, showed almost complete inhibitory activity at 500γ/1'ILl.

Example 5 Formic acid was added to an aqueous extract of corn IV-1 fraction paste obtained in the same manner as in Example 1, the pH was adjusted to 4.5, and the mixture was left for 2 hours.

The low molecular weight portion (molecular weight 13
000 or less) was collected and P-cellulose (manufactured by Seikagaku Corporation) was collected.
2% by volume and further adjusted to pH 7. with IN sodium hydroxide.
It was set to 0.

The P-cellulose adsorbed with impurities was filtered off, and 3% by weight of acid clay was added to the filtrate to adsorb IRA onto the acid clay.

After elution with 0.5N acetic acid, the concentrated IRA solution was further extracted with chloroform and dried under reduced pressure to obtain IRA of about 4.71.

The immunosuppressive effect of this product was measured together with PHA, and 500γ
showed an inhibitory activity of 85%.

It was confirmed to be IRA by electrophoretic and molecular analysis.

Example 6 Using 800 human placentas, they were finely ground to give 0.
Add 100 liters of 1M sodium chloride solution and stir briefly.

The extracted placental blood is centrifuged to produce a clear extract of 200ml.
I got l.

Total protein was obtained from this placental extract, which was precipitated by 50% saturation with ammonium sulfate.

Per 1 kg of this precipitate, 4 to 6 liters of distilled water was added to dissolve it, and a small amount of hydrochloric acid was added to this solution to adjust the pH to 5.0, followed by gentle stirring at room temperature for 2 hours.

Using hollow fiber (manufactured by Asahi Kasei), the molecular weight is approximately 1.
The low molecular weight fraction below 3000 was collected.

This collected solution was mixed with CM-cellulose at pH 5.0, 0.
It was developed using a column equilibrated with 15M phosphate buffer.

A sufficient amount of water was run through and the eluate was collected.

4% by weight of acid clay was added to this eluate to adsorb IRA.

The adsorbed acid clay was taken out, and the same procedure as in Example 5 was carried out. Purified IRA8 was eluted with IN hydrochloric acid and dried under reduced pressure.
I got a g.

This ■RA500γ/rIll showed 90% inhibitory activity by PHA method.

It was confirmed to be IRA by electrophoretic and molecular analysis.

Example 7 4 m by PHA method obtained by Cohn's alcohol fractionation method
1 kg of IV-1 fraction paste having 9/ml and 70% inhibitory activity was suspended in 4 liters of cold distilled water and stirred at 4°C for 2 hours.

After centrifugation to remove insoluble materials, a solution of glycine dissolved in the supernatant at a concentration of 15% (W/V) was heated at 60° C. for 10 hours.

After heating, the mixture was dialyzed against distilled water, and the resulting precipitate was removed by centrifugation to obtain a clear solution.

Glacial acetic acid was added to the solution to adjust the pH to 3.5.

This liquid was filtered through a Hollow Fihar (manufactured by Asahi Kasei Corporation) to collect a low molecular weight portion having a molecular weight of less than 13,000.

1% by weight of CM-cellulose (manufactured by Seikagaku Kogyo Co., Ltd.) was added to the low molecular weight portion and stirred to adsorb inactive contaminants, thereby obtaining the IRA active portion in the filtrate.

Activated carbon was added to this filtrate at a concentration of 4% by weight, and the pH was adjusted to 38%.
% ammonia solution to 8.0, and the active portion was adsorbed onto activated carbon.

For elution, 0. IN hydrochloric acid was used. The obtained eluate was dried under reduced pressure to obtain purified IRA10. Got the OP.

This I RA5 0 0 1 /rnl is 7 using the PHA method.
It showed an inhibitory activity of 4%.

In cellulose acetate membrane electrophoresis, a band was observed in the β-globulin region, the molecular weight of which was approximately 5000, and it was confirmed to be IRA.

In addition, HBV activity was measured using Ausria-125 (Au
sria-1 2 5 ■) (manufactured by Abbott 1) Nyol radioimmunoassay method (Japanese Patent Application Laid-Open No. 48-4991 9
The results were negative.

The blood pressure lowering substance was measured in adult dogs by the rate of decrease in average arterial blood pressure before administration of the drug solution, and the rate of decrease was approximately 75%, which was determined to be negative.

Example 8 Approximately % volume of ether was added to the eluate obtained in the same manner as in Example 7, and after stirring thoroughly, the ether layer was separated and dried under reduced pressure to obtain approximately 61 purified IRA.

This IRA 2 0 1 /ml is approximately 65% by PHA method.
%, showed almost complete inhibitory activity at 500 r/ml.

IRA was confirmed by electrophoretic and molecular weight analysis, and HBV and hypotensive substances were negative.

Example 9 100 human placentas are cut into pieces, suspended in 10 liters of 0.1M sodium chloride solution and stirred briefly.

A clear extract of about 20% was obtained by centrifugation.

The extracts were collected, mannitol was added to a concentration of about 15% W/V, and heat-treated at 60° C. for 10 hours.

The heated solution was dialyzed against a saline solution, and the resulting precipitate was removed by centrifugation to obtain a clear solution.

The solution was adjusted to pH 2.0 using 2N hydrochloric acid and gently stirred at room temperature for 2 hours.

Approximately 1000 using hollow fiber (manufactured by Amicon)
Collect the low molecular weight parts of 0 or less and CM- according to Example 1.
Sephadex treatment was performed.

Next, 5% by weight of silica gel was administered, and 5% by weight of silica gel was administered.
The pH was adjusted to 9.0 with Jum.

After stirring for about 30 minutes, the silica gel used for adsorption was collected by filtration using Toyo Roshi A2.

After elution with a mixture of diethyl ether and methanol in the same volume as the silica gel, sterilization filtration was performed, and the filtrate was dried under reduced pressure to obtain 900 mg of purified IRA.

This IRA500γ/Tll showed 100% inhibitory activity by PHA method.

It was confirmed to be IRA by electrophoretic and molecular analysis.

Furthermore, HBV activity was negative, and the presence of hypotensive substances was also negative.

Test 1 Test Example 1 (Confirmation of drug implantation effect and safety) An animal test was conducted using IRA derived from plasma and placenta obtained in Examples 8 and 9, respectively.

Using 15 mice, 5 mice received placental blood-derived IRA and the other 5 mice received plasma-derived IRA at 20η/kg/each.
After continuous intravenous administration for 20 days, the effect on IRA skin grafting experiments was examined.

IRA was the IRA powder obtained in Examples 8 and 9 as it was,
It was dissolved in raw saline to prepare a 10% by weight IRA aqueous solution.

20rrI9/k in advance 24 hours before the start of the transplant experiment.
g and then once a day for 20 days.

Surface observation after 20 days showed that both IRA-administered groups had a survival rate of about 85-100%, compared to 0% in the control.

This suggests the effectiveness of the drug-embedding effect of the present IRA injection as an immunosuppressant.

Test Example 2 (Acute Toxicity) An acute toxicity test was conducted using the IRA obtained in Examples 8 and 9.

Using 20 mice, a solution containing 500 m97 kg of IRA and 10 mice of 1000 drops/kyo IRA was administered intravenously to 10 mice and observed for 48 hours, but there were no deaths in either case. I was not able to admit.

Claims (1)

[Scope of Claims] 1. An immunosuppressive α-globulin preparation derived from human blood, in which hepatitis viruses have been inactivated and hypotensive substances have been removed. 2 Immunosuppressive α-globulin at 100-10001/v
A formulation according to claim 1, which contains tl and has an inhibitory activity of 70 to 100% as determined by the PHA method. 3. The preparation according to claim 1 or 2, wherein the immunosuppressive α-globulin is derived from placental blood. 4. The preparation according to claim 1 or 2, wherein the immunosuppressive α-globulin is derived from plasma. 5. The preparation according to claim 1, wherein the immunosuppressive α-globulin is an injectable preparation. 6 A claim consisting of inactivating a hepatitis virus by heat treatment in the presence of a stabilizer selected from the group consisting of neutral amino acids, soluble sugar alcohols, and monosaccharides and disaccharides, and then removing the stabilizer. Preparations according to paragraph 1. 7. The formulation according to claim 6, wherein the heat treatment is performed at 60° C. for 10 hours. 8. The preparation according to claim 6, wherein the neutral amino acids are glycine, alanine, parine, leucine, and isoleucine. 9. The preparation according to claim 6, wherein the monosaccharide is glucose, mannose, galactose, or fructose. 10 The preparation according to claim 6, which is a disaccharide, sucrose, maltose, or lactose. The preparation according to claim 6, which is a 11-soluble sugar alcohol, mannitol, sorbitol, xylitol. 12. A formulation according to claim 6, in which the stabilizer is removed by dialysis. 13 The amount of stabilizer added is at least 5% (W/V)
A formulation according to claim 6. 14. The formulation according to claim 13, wherein the amount of stabilizer added is 15 to 20% (W/V).