JP2001278895A - Peptide for reinforcing action of interferon and interferon action-reinforcing composition containing the peptide and the interferon - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a peptide which has an action for reinforcing the activity of interferon, and can enhance the efficacy of the interferon and reduce the use of the expensive interferon, by using the peptide together with the interferon. SOLUTION: The peptide containing an amino acid sequence of Ala-Lys-Pro- Ala-Pro-Pro-Lys-Pro at the N terminal. The composition containing the peptide and an interferon, or the peptide, the interferon and a flavin adenine dinucleotide.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a peptide that enhances the action of interferon, a composition containing the peptide and interferon with enhanced interferon action, and a composition containing the peptide, interferon and flavin adenine dinucleotide. A composition having enhanced interferon action.

[0002]

2. Description of the Related Art Interferon (IFN) is a generic term for glycoproteins produced and secreted by animal cells upon viral infection and the like, and stimulates fibroblasts in the same manner as α-type induced in leukocytes by viruses and nucleic acids. And γ-type, which is induced by stimulating lymphocytes with specific antigens and mitogens and is also called immune interferon. In addition, IFNs include those extracted from natural materials and those produced using animal recombination technology in animal cells or Escherichia coli, etc., such as `` feron '', `` smiferon '', `` campferon '',
It has been marketed under trademarks such as "Roferon" and "Intron".

[0003] Human IFN has been used in the treatment of tumors, but also in the treatment of chronic hepatitis B and C virus. However, the efficacy is low for chronic hepatitis B, and the effective rate for hepatitis C is only about 30%. Furthermore, due to the extremely high drug price of human IFN, re-administration after the completion of the administration schedule is not permitted in the treatment of chronic hepatitis C. On the other hand, the applicant company
The composition containing 15 μl of pig liver extract and 10 mg of flavin adenine dinucleotide (FAD) in 1 l of “Adelabin 9
The product is marketed as a remedy for hepatitis under the trademark No. The present inventors have found that Adelabin 9 remarkably enhances the antiviral effect of IFN (Japanese Patent Application Laid-Open No. 7-188052, Arzneim.-
Forsch./Drug Res. 47, 968-974, 1997). However, at that time, it was not enough to consider the mechanism of action in detail.

[0004]

As described above, IFN has problems not only in terms of effectiveness but also in terms of cost-effectiveness due to high drug price. This problem is based on the above-mentioned findings found by the present inventors (Adelabin No. 9
Is found to remarkably enhance the antiviral action of the compound of the present invention) by clarifying the mechanism of action and clarifying the action body. In addition, administering the substance of action together with IFN makes it possible to increase the effectiveness of IFN and to improve the cost-effectiveness. Therefore, the basic problem of the present invention is to elucidate the main body of action of “Adelabin 9”, which is a composition containing pig liver extract and flavin adenine dinucleotide (FAD), to enhance the action of IFN, or to increase the action of IFN. IFN to bring out less
It is to provide an action enhancer. An additional object of the present invention is to provide a composition containing IFN and an action enhancer having enhanced interferon action.

[0005]

The liver extract, which is a component of adelabin, contains nucleic acids, amino acids, and several kinds of peptides having different molecular weights. Nucleic acids and amino acids could have a liver-protective effect, but could not be expected to play a significant role in enhancing the antiviral effect of IFN. Therefore, the present inventors focused on the peptides in the liver extract, separated those peptides, analyzed the amino acid sequence, and performed a homology search to examine existing peptides having high homology. As a result, as shown in FIG. 1, the N-terminal side has a very characteristic sequence rich in proline and lysine, and has a molecular weight of 3000 to 90.
00 peptides, which are aggregates of peptides cleaved at certain points from HMG-17, a non-histone protein in the chromosome, and which strongly enhance the antiviral effect of IFN I found that. Further, in order to identify the active form, a short peptide having this characteristic N-terminal sequence was synthesized and its activity was examined.
It is presumed that Ala-Lys-Pro-Ala-Pro-Pro-Lys-Pro at the N-terminus is an essential sequence, and it is determined that a peptide having the sequence at the N-terminus has an action of enhancing IFN activity. I came to.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION The peptide of the present invention has Al
It is a peptide containing the amino acid sequence of a-Lys-Pro-Ala-Pro-Pro-Lys-Pro, preferably a peptide having a molecular weight of 9000 or less. Among them, peptides having 9 to 20 amino acids are preferable, and more preferably 12 to 1 amino acids.
5 peptides. Certain of the peptides of the present invention are peptides containing the amino acid sequence of Ala-Lys-Pro-Ala-Pro-Pro-Lys-Pro-Glu at the N-terminus, among which those having 9 to 20 amino acids are preferred. preferable. Further, some of the peptides of the present invention have Ala-Lys-Pro-Ala-Pro-Pro-
It is a peptide containing the amino acid sequence of Lys-Pro-Glu-Pro-Lys-Pro, and among them, those having 12 to 20 amino acids are preferable. Further, as the peptide of the present invention, Ala-Lys-
Pro-Ala-Pro-Pro-Lys-Pro-Glu-Pro-Lys-Pro-Lys-Lys-Al
A peptide consisting of the amino acid sequence of a can be exemplified.

[0007] The peptide of the present invention is a peptide that enhances the effect of IFN. Here, the effects of IFN
Although the examples show antiviral activity, the present invention is not limited to the antiviral activity, but includes the entire effects and effects of IFN, including antitumor activity and the like. The peptide of the present invention can be administered alone before or after administration of IFN, or the peptide of the present invention and IFN can be administered together in the form of a composition. Further, the peptide of the present invention can be administered together with FAD before and after administration of IFN, and further, the peptide of the present invention can be administered together with IFN and FAD in the form of a composition. Here, the IFN may be any of α, β and γ types. As the administration route of the peptide of the present invention, intravenous administration is preferable, similarly to IFN and Adelabin-9. The frequency of administration is preferably once a day in accordance with that of IFN. One dose of the peptide of the present invention in human is 1 μg / body to 1000 μg / body.
And preferably around 5 μg / body. In contrast, the dose of IFN is the usual dose (3 million, 60
(100,000 or 9 million IU / body), but may be half or quarter of that amount. When FAD is used in combination, the single dose of FAD is 0 to 40 mg / body, preferably 2 to 40 mg / body.
It is around 0mg / body. The peptide of the present invention does not require any special formulation, and only requires the lyophilized product to be dissolved in physiological saline and administered intravenously.

[0008] When the peptide of the present invention is used as a composition with IFN and FAD, the peptide of the present invention and a freeze-dried product of IFN may be mixed, or a powder of FAD may be additionally mixed.
The same applies to a case where the composition of the present invention is composed of the peptide and FAD. When the component content when the above composition containing the peptide of the present invention is created is shown as a single dose, the peptide of the present invention is 1 μg to 1000 μg, preferably around 5 μg, and IFN is 300 μg.
10,000, 6 million, or 9 million IU, FAD 0-40 mg, preferably 20
It will be around mg. This amount of IFN is a usual dose for a single administration, and can be further reduced to about one-half or one-fourth. Further, the peptide of the present invention has no problem of side effects. It is because there is no problem of side effects in "Adelabin 9" containing pig liver extract, and HMG-17 is only one amino acid difference between human and pig. It is clear from the fact that it is part of the human HMG-17 sequence. The peptide of the present invention can be synthesized by a peptide synthesizer or can be obtained from liver extract by the method described in Test Example 1 below. In addition, the liver extract is, for example, a pig liver extract, and Arzneim.-Forsch./Drug Res. 47, 968-9
74, 1997.

[0009]

[Examples] Test Example 1 Analysis of peptide component of liver extract (1) Experimental method 1) Digestion of nucleic acid component DNaseI, 1 mM MgC at a final concentration of 10 U / ml was added to 100 μl of liver extract.
l ₂ , 10 mM Phenylmethylsulphonyl Fluoride (PMSF) was added, and the mixture was treated at 25 ° C. for 18 hours. Then, DNaseI is 95 ℃
Inactivated by treatment for 10 minutes. In addition, the liver extract is a pig liver extract, and Arzneim.-Forsch./Drug Res. 47, 968-974,
Those prepared by the method described in 1997 were used.

2) SDS-PAGE and transcription to PVDF membrane Liver extract digested with nucleic acid components is used for peptide peptide
(SHM-601, Mori Washina) to separate peptide components by SDS-PAGE, and a part of the gel was Coomassie Brilliant Blue
(CBB) Stained with R-250. Peptides in the remaining gel was transferred to PVDF membrane using a transblot SD (Bio Rad) (Immobilon P ^SQ, Millipore). This membrane was used for amino acid analysis after confirming the peptide band with CBB.

3) Amino acid analysis The amino acid sequence of the peptide is determined by ABI Model 476A Micro Seq
Analysis was performed using the uence Analysis System (Applied Biosystems) up to 20 or 15 residues from the N-terminus. The deduced amino acid sequence was determined from the chromatogram based on the result calculated by Model 610A Data Analysis System. 4) Search for homology with known proteins The deduced amino acid sequence is available from NCBI (National Center for Biotechnology Inf
ormation; http://www.ncbi.nlm.nih.gov) was searched for homology with proteins registered in the swissprot database and the like using the BLASTp program.

(2) Results and discussion The peptide in the liver extract was found to be 3 kDa to 9 kDa by SDS-PAGE.
A molecular weight peak leading to Da was shown. A band that can be clearly confirmed was cut out, and the amino acid sequence from its N-terminus was determined. From the sequence, the protein whose homology was recognized in the protein database was HMG-17, a human non-histone chromosome protein. FIG. 1 shows the partial amino acid sequence of HMG-17, the sequence of the liver extract peptide determined, and the sequence considered to be an essential sequence. Most of the N-terminal amino acid sequences of the peptides present in the liver extract were found to be such sequences.

Test Example 2 Effect of Liver Extract Peptide Fraction on Herpes Simplex Type 1 Virus (HSV-1) The liver extract peptide fraction was separated, and its enhancing effect on the antiviral activity of IFN was examined. (1) Experimental method 1) Separation of liver extract peptide fraction The liver extract prepared in the same manner as in Test Example 1 was fractionated on a monoQ column, and Fr1 was used as an unadsorbed fraction, and Fr2 and Fr3 were used as elution fractions of peptide components. I got

2) Effect of Combination of Liver Extract Peptide Fraction with IFN After separating Fr1-Fr3, the solution amount was adjusted to the amount equivalent to the liver extract before fractionation and used. HeLa cells cultured in a monolayer on a 96-well culture plate were added drug and 50TCID ₅₀ HSV.
-1 was added, and the cells were cultured in a 5% CO ₂ incubator at 37 ° C. for 2 days. The drug addition group was IFN-α2a “campferon” alone group (6.25, 12.5, 25, 50, 100 IU / ml), IFN-α
2a and liver extract peptide fraction (2.5 and 5 respectively for Fr1 to Fr3)
μl / ml). After completion of the culture, the cells were fixed with 5% formalin, stained with 0.05% crystal violet, and observed under a microscope for cell damage (CPE) caused by HSV-1. The IFN activity enhancing effect of the liver extract peptide fraction was IFN-
The IC ₅₀ values of the anti-HSV-1 activity of a2a, was determined by comparing the presence or absence of liver extract peptide fraction combination.

(2) Results and Discussion Fr1 is a portion that does not adsorb to the monoQ column, and no peptide component was detected. Fr2 and Fr3 were eluted fractions of the monoQ column and were found to consist of peptide components. The effect of the liver extract fractionation is shown in Table 1. IFN-
The IC50 of α2a was ₄₄ IU / ml. On the other hand, Fr1 containing no peptide component shows no change to its value,
There was no FN activity enhancing effect. On the other hand, when combined liver extract peptide fraction Fr2 or Fr3 is at any concentration of 2.5 and 5 [mu] l / ml, decreased IC ₅₀ values of IFN-a2a to about 10 IU / ml, the activity of IFN is enhanced Was done. From the above,
The peptide fraction of liver extract was found to enhance the antiviral effect of IFN.

[0016]

[Table 1]

Test Example 3 Action of Liver Extract Synthetic Peptide on Herpes Simplex Type 1 Virus (HSV-1) Nine of the amino acid sequences characteristic of liver extract peptide
Peptides consisting of 12 and 15 amino acids were synthesized, and the enhancing effect of IFN on antiviral activity was examined.

(1) Experimental method HSV-1 MIYAMA was added to HeLa cells cultured in a monolayer on a 24-well plate.
The strain was added at 500 PFU / 0.4 ml and infected for 1 hour at 37 ° C. in a 5% CO ₂ incubator. After washing the cell layer, a drug was added and the cells were cultured for 2 days. Drug addition group, IFN-α2a `` Campferon '' 6.3 IU / ml alone group, IFN-α2a 6.3 IU / ml
And liver extract synthetic peptide 1 (peptide 1) or liver extract synthetic peptide 2 (peptide 2) as a mixed addition group, and the concentrations of these peptides were 0.01, 0.1, and 1 μg / ml. Two days after the culture, the amount of virus in the culture supernatant was measured by the plaque method. Next, liver extract synthetic peptide 2 was prepared in the same manner.
(Peptide 2) and liver extract synthetic peptide 3 (peptide 3) were tested. Further, as a FAD combination test, a test similar to the above was performed, except that only the drug addition group was changed as follows. Here, the drug addition group was IFN-α2a 6.3 IU
/ ml alone group, IFN-α2a 6.3 IU / ml and FAD 25 μg / ml (adderabine 9 2.5 μl / ml equivalent) mixed addition group, IFN-α2a 6.3I
U / ml, FAD 25 μg / ml and peptide 1 or 2 were mixed and added, and the peptide concentration was the same as above.

As a control peptide, BAM-12P having 12 amino acids and a completely different sequence was used.
A similar test was performed in combination with α2a. In addition, for the peptides 1, 2, and 3, the antiviral activity was evaluated alone by the same test. Peptide 1 is a peptide having 9 amino acids, Peptide 2 is a peptide having 12 amino acids, and Peptide 3 is a peptide having 15 amino acids. The amino acid sequences are shown below together with the control peptide. Peptide 1: Ala-Lys-Pro-Ala-Pro-Pro-Lys-Pro-Glu Peptide 2: Ala-Lys-Pro-Ala-Pro-Pro-Lys-Pro-Glu-Pro-Lys-Pro Peptide 3: Ala -Lys-Pro-Ala-Pro-Pro-Lys-Pro-Glu-Pro-Lys-Pro-Lys-Lys-Ala BAM-12P: Thr-Gly-Gly-Phe-Met-Arg-Arg-Val-Gly- Arg-Pro-Glu

(2) Experimental Results and Discussion Peptide 1, Peptide 2 and Peptide 3 are
HSV-1 activity was not observed. On the other hand, IFN-α-2a 6.3IU
The growth of virus at the time of addition of 1 μg / ml was 70
% And peptide 2 significantly suppressed to 48% (FIG. 2).
In addition, the effect of peptide 3 was equal to or higher than that of peptide 2 (FIG. 3). Since peptide 3 has the strongest effect of enhancing the anti-HSV-1 activity of IFN-α-2a, it was found that the length of the peptide chain was also involved in the enhancing effect. In addition, since the control peptide BAM-12P was ineffective, the characteristic amino acid sequence at the N-terminus of the liver extract peptide was required for the effect of enhancing the anti-HSV-1 activity of IFN-α-2a. There was found.

In addition, IFN of these liver extract synthetic peptides
The activity-enhancing effect was further enhanced in the presence of FAD, and at a concentration of 1 μg / ml, peptide 1 significantly suppressed virus growth to 53% and peptide 2 to 38% (FIG. 4).
From the above, when the sequence consisting of 9 amino acids of peptide 1 has the effect of enhancing the antiviral activity of IFN, and when the sequence comprises 12 or 15 amino acids, the activity of enhancing the antiviral activity of IFN is further improved. Is strengthened,
It was also found that the IFN activity enhancing action of these liver extract synthetic peptides was further enhanced by FAD.

Test Example 4 Action of Alanine-Substituted Peptide The N-terminal sequence of the liver extract peptide is a very characteristic sequence mainly composed of proline and lysine.
It is considered to be the main body of activity enhancing action. Therefore, among the liver extract synthetic peptides consisting of 12 amino acid sequences, a peptide in which proline was substituted with alanine was prepared, and IFN-α2a
And the antiviral effect was examined in combination with FAD.

(1) Experimental method In the same manner as in Test Example 3, after infecting HeLa cells cultured in a monolayer with HSV-1, liver extract was obtained under the conditions of IFN-α2a6.3 IU / ml and FAD 25 μg / ml. Synthetic peptide 2 (peptide 2), and
An alanine-substituted peptide (substituted peptide) obtained by substituting alanine for the proline at the 3rd, 5th and 8th positions from the N-terminus was added at a concentration of 0.1 and 1 μg / ml, respectively. The viral load was measured by the plaque method. Peptide 2: Ala-Lys-Pro-Ala-Pro-Pro-Lys-Pro-Glu-Pro-Lys-Pro Substituted peptide: Ala-Lys- Ala- Ala - Ala -Pro-Lys- Ala- Glu-Pro-Lys -Pro

(2) Experimental Results and Discussion As shown in FIG. 5, peptide 2 has an effect of enhancing the activity of IFN, but proline at positions 3, 5, and 8 from the N-terminus. Did not show the effect of enhancing IFN activity. From the above, Ala-Lys-Pro-Ala-Pro-Pro-Lys-
It is presumed that the Pro sequence is an important sequence for expressing the effect of enhancing IFN activity.

[0025]

The peptide of the present invention has the effect of enhancing the activity of IFN. By using the peptide of the present invention in combination with IFN, the effectiveness of IFN can be increased or the amount of expensive IFN used can be reduced. be able to.

[Brief description of the drawings]

FIG. 1 is a partial amino acid sequence of a liver extract peptide and HMG-17, and an essential amino acid sequence having an action of enhancing IFN activity.

FIG. 2 shows that liver extract synthetic peptides 1 and 2 are IFN anti-HSV-1
Graph showing potentiating activity.

[FIG. 3] Liver extract synthetic peptides 2 and 3 are IFN anti-HSV-1
Graph showing potentiating activity.

[FIG. 4] Synthetic peptide of liver extract and anti-HSV-IFD of IFN.
1 Graph showing that activity is enhanced.

FIG. 5 is a graph showing that the activity disappears when proline at positions 3, 5, and 8 from the N-terminal of a liver extract synthetic peptide is substituted with alanine.

 ──────────────────────────────────────────────────続 き Continuing from the front page (72) Inventor Hidetane Saito 3-3-7-301 F-term, 3-3-7-301 Sakuradai, Nerima-ku, Tokyo 4C084 BA01 BA17 BA18 BA23 BA44 CA33 CA59 DA21 NA05 ZB011 ZB032 ZC012 ZC751 ZC752 4C086 AA01 AA02 AA03 EA18 MA03 MA04 NA05 ZB01 ZB03 ZC01 ZC75 4H045 AA10 AA30 BA10 BA15 BA16 BA17 CA40 DA15 EA28 EA29 FA10 FA71 HA02 HA03 HA04

Claims (6)

[Claims] 1. Ala-Lys-Pro-Ala-Pro-Pro-Lys-N-terminal
A peptide containing the amino acid sequence of Pro. 2. Ala-Lys-Pro-Ala-Pro-Pro-Lys- at the N-terminus
The peptide according to claim 1, which comprises an amino acid sequence of Pro-Glu-Pro-Lys-Pro. 3. Ala-Lys-Pro-Ala-Pro-Pro-Lys-Pro-Glu-
A peptide having the amino acid sequence of Pro-Lys-Pro-Lys-Lys-Ala. 4. An interferon action enhancer comprising the peptide according to claim 1, 2 or 3 as an active ingredient. 5. A composition comprising the peptide according to claim 1, 2 or 3, and an interferon. 6. A composition comprising the peptide according to claim 1, 2 or 3, interferon and flavin adenine dinucleotide.