JPS58154596A - Modification of interferon - Google Patents

Abstract

PURPOSE:To modify the titled substance useful as an antiviral agent, antimali- gnant-tumor agent, etc., by treating an interferon with a boron-based reducing agent, thereby changing the electrical charge, etc. without losing the activity, delaying the clearance in the living body, and increasing the drug activity. CONSTITUTION:An interferon is dissolved in a 0.1M phosphate buffer solution added with 10% glycerol, left to stand for one night under ice cooling, added with a boron-based reducing agent such as an aqueous solution of NaBH4, and left under ice cooling. After the completion of the reaction, the mixture is dialyzed with 0.1M phosphate buffer solution, etc. for one night under ice cooling. The liquid left after the dialysis is freeze-dried, and reconstituted to an aqueous solution by adding water thereto. The objective reduced interferon having modified physical properties such as electrical charge, etc. can be obtained by this process without losing the activity.

Description

[Detailed description of the invention] The present invention relates to a method for modifying interferon.

Interferon is a protein produced by animal cells upon stimulation with inducers such as various viruses and double-stranded RNA, and acts on other cells of the same species to suppress the proliferation of viruses within those cells. Interferon is also known to exhibit cell growth-inhibiting effects and immune response regulating effects. In recent years, the therapeutic effects of interferon on certain viral diseases and even malignant tumors have been recognized, and its potential as a medicine has attracted attention. It is believed that chemical modification of interferon would be extremely effective in further developing interferon as a drug. In other words, it is expected that the chemical modification of interferon will be of great practical significance, as it is expected to delay the clearance in the living body and enhance the effects of interferon.

When modifying interferon, it would be of no practical value unless it could be chemically modified while retaining biological activities such as antiviral activity, but no such modification method has been found to date. There wasn't. Furthermore, it goes without saying that no modification method has been found to increase the efficacy of interferon, such as by delaying its clearance in vivo. The inventors of the present invention conducted intensive research to solve this situation, and as a result, they arrived at the following new points and completed the present invention.

The present inventors have discovered that when interferon itself is treated with a boron-based reducing agent, its charge and other physicochemical properties change without losing its antiviral activity.

In other words, when interferon is treated with a boron-based reducing agent, some of the multiple disulfide bonds are selectively reduced and cleaved, resulting in changes in other physicochemical properties such as charge. It seems that this will occur. Furthermore, there are multiple free amine groups derived from intermediate lysine residues in the protein portion of interferon, and the present inventors added a carbonyl compound, which will be explained later, to these free amine groups to form a Schiff base. It was discovered that the antiviral activity of interferon was not lost even if the Schiff base complex was treated with a boron-based reducing agent such as sodium borohydride to stabilize the bond between interferon and carbonyl compound. Ta. The present inventors have discovered that the interferon derivatives obtained by these modification methods all have different charges from the starting material interferon, and also have different in-vivo clearance.

That is, the present invention relates to a method for modifying interferon, which is characterized by treating interferon with a boron-based reducing agent.

The interferon used in the present invention can be produced by known methods in animal cells such as humans, mice, and rabbits, or can be produced in prokaryotic cells such as bacteria and yeast using interferon genes obtained from the above cells. Examples include standard products produced by a method and purified by a combination of known methods such as salting out, ion exchange chromatography, affinity chromatography such as antibody columns, or gel filtration. The interferons contained in these preparations all have disulfide bonds and also have free amino groups at the N-terminus and intermediate lysine residues of the protein portion. By using an interferon preparation with a specific activity exceeding 108 international units/Ig protein, it is also possible to obtain specifically labeled interferon using a radioactive isotope by the method of the present invention.
Even if an interferon preparation with a specific activity lower than this is used, interferon derivatives having antiviral activity and having different charge and other physicochemical properties can be obtained.

In the present invention, it is more preferable that the above-mentioned cinterferon is reacted with a carbonyl compound and then treated with a boron-based reducing agent.

The carbonyl compound in this case refers to the general formula and ketones. Examples include compounds such as formaldehyde, acetaldehyde, benzaldehyde, pyridoxal, pyridoxal-57-phosphate, 77t, fural, valeric aldehyde, and glyceraldehyde. The aldehydes and ketones mentioned herein also include sugars having a free carbonyl group. In the present invention, it is desirable to select a carbonyl compound that increases the efficacy of interferon as much as possible, such as by delaying its clearance in vivo. The amount of carbonyl compound to be used is about 2 to 500 times the molar amount of the protein contained in the interferon preparation.

Examples of the boron-based reducing agent used in the present invention include sodium borohydride and sodium cyanoborohydride. The amount of reducing agent to be used is approximately 2 to 20 oO times the amount of protein contained in the interferon preparation. The reaction between the interferon preparation and the carbonyl compound was 0.5 at pH 7-10 and temperature o~10°C.
It is preferable to carry out the treatment for up to 24 hours. If the reaction between the dicarbonyl compound and interferon is carried out in the presence of a reducing agent, the entire amount of the reducing agent may be added at the start of the reaction, or the reducing agent may be added in small amounts during the course of the reaction. The above reaction conditions are also preferred when treating an interferon preparation with only a reducing agent. After the reaction is completed, the reaction product is purified by dialysis, chromatography, etc. to obtain the desired interferon derivative. If a radioactive isotope-labeled aldehyde is used as the carbonyl compound, it is possible to know how much interferon has been modified without purification.

Examples of the present invention will be described below, but the present invention is not limited thereto.

Example 1゜The Journal of General Vi
volume 55, pages 225-266 (1976
Mouse interferon (molecular weight s6.ooo) with a specific activity of 1×108 international units/my protein purified according to the method of Yamamoto et al.
The solution was dissolved in 300 μt of 0.1 M phosphate buffer (pH 8,0) containing 10 tiglycerin so as to give ug. 100 of them
100 μt of the above phosphate buffer was added to μt to prepare reaction system 1. For the other 100μt, pyridoxal 5■/WLe
A reaction system 2 was prepared by adding 100 µt of a hexapyric acid buffer solution containing the above. To the remaining 100 .mu.t, 11 .mu.t of the above phosphate buffer containing 5 .mu./me of pyridoxal-57-phosphate was added to prepare reaction system 6. After leaving it under water cooling overnight, reaction system 1,
2 and filtration were added four times each under ice-cooling. After further standing for one hour under water cooling, reaction systems 1, 2 and 6 were dialyzed against the above phosphate buffer overnight under water cooling. After the dialysis fluid was freeze-dried, 100 μt was added to each solution to reconstitute it into an aqueous solution. The titers of interferon obtained after the reactions in reaction systems 1, 2 and 3 were as shown in Table 1.

Ampholine (pH 3,5-8
) Isoelectric focusing was performed on a 25 mg column at 4° C. and 310 V for 5 hours. 1#! The interferonka titer was measured in 7 fractions. The results are shown in Figures 1 to 4 together with the migration pattern of unreacted interferon, and each reaction system showed a different migration pattern. In addition, in Figs. 1 to 4, the fractionation was performed on the acidic side.

Example 2゜Example 1. The interferon preparation obtained in
d Mice were injected into the tail vein in groups of 6 mice. After a certain period of time, blood was collected by cardiac puncture, and the interferon titer in the serum was measured. Figure 5 was obtained by calculating how much of the given interferon was recovered into the bloodstream, with 1/12 of the mouse's body weight being taken as the mouse's blood volume. Delayed clearance was observed in all reaction systems compared to unreacted interferon.

[Brief explanation of the drawing]

FIG. 1 shows Example 1. The experimental results of isoelectric focusing of raw material (unreacted) interferon are shown. Similarly, FIGS. 2 to 4 show Example 1. The experimental results of isoelectric focusing of interferons obtained in reaction systems 1 to 3 shown in FIG. FIG. 5 shows Example 2. The recovery rate of interferon titer in mouse serum is shown. 1 to 6 in the figure represent Example 1, respectively. The values related to the interferon obtained in reaction systems 1 to 6 are shown, and 4 shows the value related to the raw material (unreacted) interferon. Patent applicant Toshi Co., Ltd. Foundation Tokyo Metropolitan Institute of Clinical Medicine ↑ [Figure 9 2 Figure 9 ? Figure 113'. Figure 4 o
++t& fraction 060 time (4F)

Claims (1)

[Claims] (1) A method for modifying interferon, which comprises treating interferon with a boron-based reducing agent.