JP4188897B2 - Virus infection / proliferation inhibitor - Google Patents

Description

  The present invention relates to a peptidic viral infection / proliferation inhibitor with few side effects.

  Many efforts have been made to overcome diseases caused by viral infection. However, in recent years, there are those that still have no effective preventive or therapeutic methods, such as the human immunodeficiency virus (HIV), which is prevalent in the world, and the influenza virus that is prevalent every year. Chemotherapy with antiviral drugs is a treatment for diseases caused by viral infection. However, at present, there are few antiviral agents that are administered systemically and show a clear effect. The reason for this is that the virus grows inside the cell, and its function for growth is almost dependent on the cell. In other words, after infection, the virus makes use of the proliferation function of the constituent cells of the human body whose proliferation should be treated. Therefore, many chemical substances that suppress the growth of viruses also act on cells and are toxic. Problems with antiviral agents include several problems derived from the characteristics of viral infections other than the low selective toxicity of drugs as described above. Particularly in acute and systemic viral infections, since the time when symptoms appear after the peak of viral in vivo growth has passed, there is a need for substances that rather prevent viral infection and suppress the growth. There is a method of vaccination as a method to protect against such viral infections, but this method is often known to show allergic reactions and various side effects, and it cannot cope with viral mutations. Is the reality.

  Under such circumstances, the present inventors have found that proteins having iron-binding ability such as lactoferrin (hereinafter sometimes referred to as LF), transferrin, ovotransferrin, etc. are infected with influenza virus or cytomegalovirus (CMV). A patent application was filed (for example, refer to Patent Document 1). LF is an iron-binding protein secreted into milk and is known to have antibacterial activity. LF has been studied in detail for human and bovine origin, and the entire amino acid sequence of human LF and bovine LF has already been determined (see, for example, Non-Patent Documents 1 and 2). In addition, an antiviral agent containing milk protein such as LF as an active ingredient is disclosed, and it is described therein that LF is effective against enveloped viruses and non-enveloped viruses ( For example, see Patent Document 2.) Recently, the present inventors also confirmed the infection / proliferation inhibitory effect of these iron-binding proteins on HIV and filed a patent application for an HIV infection / proliferation inhibitor containing LF as an active ingredient (for example, patent literature 3).

Thus, iron binding proteins such as LF have gradually been found to have an effect of suppressing infection and proliferation of viruses, and their practical application as antiviral agents has been expected. In order for these proteins to be actually used as infection / proliferation inhibitors, it is necessary that (i) when administered in the body, there is no problem in antigenicity, and (ii) they can be supplied in large quantities. Among these iron-binding proteins, bovine LF, bovine transferrin, or ovotransferrin obtained from chicken eggs can be supplied in large quantities, but there is a problem that it exhibits antigenicity when administered into the human body. On the other hand, human LF has no problem regarding antigenicity, but is difficult to supply in large quantities.
In order to solve such problems, the present inventors have found that the following [Chemical 4] and [Chemical 5] among the LF-derived peptide fragments have antiviral activity and filed a patent application ( For example, see Patent Document 4.)

[Chemical 4]
Cys-Phe-Gln-Trp-Gln-Arg-Asn-Met-Arg-Lys-Val-Arg-Gly-Pro-Pro-Val-Ser-Cys
(However, in the formula, Cys may be a reduced form, or two Cys in a peptide may be -SS-bonded to form an oxidized form.)

[Chemical 5]
Cys-Arg-Arg-Trp-Gln-Trp-Arg-Met-Lys-Lys-Leu-Gly-Ala-Pro-Ser-Ile-Thr-Cys
(However, in the formula, Cys may be a reduced form, or two Cys in a peptide may be -SS-bonded to form an oxidized form.)

  These peptides correspond to 20-37 residues of human LF and 19-36 residues of bovine LF, respectively, and intramolecular S—S bonds were not essential for antiviral activity. Since these peptides have an extremely short chain length compared to natural LF, they can be easily supplied by chemical synthesis or synthesis by genetic manipulation. In addition, in the infection protection test using mice, the amount of peptide of [Chemical Formula 4] and [Chemical Formula 5] necessary for completely suppressing the CMV infection was 0.1 g / kg body weight.

As a previous finding regarding the LF fragment, a report that the 1-50 residue portion of human LF is the receptor binding region of the LF molecule (see, for example, Non-Patent Document 3), Human LF 4 to 52 A report that the corresponding peptide inhibited the binding of LF and lymphocytes (see, for example, Non-Patent Document 4), a peptide corresponding to residues 1 to 47 of human LF, and residues 17 to 41 of bovine LF There are reports that peptides have antibacterial activity (see, for example, Non-Patent Document 5), but there is no description of antiviral activity. In addition, regarding this antibacterial activity, a peptide containing 19 to 29 residues of human LF or a portion of 18 to 28 residues of bovine LF (see, for example, Patent Document 5), 21 to 26 residues of human LF or 20 to 24 of bovine LF. Peptides containing a residue part (for example, see Patent Document 6), peptides containing 21 to 25 residues of bovine LF or bovine LF 25 to 29 residue parts (see, for example, Patent Document 7), 20 to 20 of bovine LF Peptides containing a 24-residue part (for example, refer to Patent Document 8) are described, but each is different from a site essential for the antiviral effect of the present invention.
JP-A-2-233619 JP-A-1-233226 Japanese Patent Application No. 4-262035 Japanese Patent Application No. 5-069210 JP-A-5-078392 JP-A-5-148295 JP-A-5-148296 JP-A-5-148297 MW Rey et al., NeucleicAcid Res., Vol.18, 5288,1990 PE Mead et al., Neucleic Acid Res., Vol. 18, 7167, 1990 BF Anderson et al., J. Mol. Biol., 209, 711, 1989 D. Legrand et al., Biochemistry, 31,9243,1992 W. Bellamy et al., Biochem. Biophys. Acta, 1121, 130, 1992

  An object of the present invention is to provide a virus infection / proliferation inhibitor having a strong antiviral activity, which comprises an LF fragment which can be supplied in a large amount without any problem in antigenicity when administered into the body. .

  In order to obtain an LF fragment having a stronger antiviral activity than the conventional LF fragment, the present inventors chemically synthesized several LF fragments and examined their antiviral activity, The essential part regarding the antiviral activity of LF was searched. As a result, in bovine LF, the 24-39 residue part represented by the following [Chemical Formula 6] was found to be essential for antiviral activity. In addition, the number assigned to the amino acid sequence of natural bovine lactoferrin is used as the residue number of the LF peptide thereafter.

[Chemical 6]
Trp-Arg-Met-Lys-Lys-Leu-Gly-Ala-Pro-Ser-Ile-Thr-Cys-Val-Arg-Arg

For the purpose of developing a highly active antiviral peptide containing this active moiety, the present inventors have extended the peptide chain to the N-terminal side and the C-terminal side with the peptide shown in [Chem. 6] as the center. , at a significantly higher activity than LF derived peptide corresponding parts have been known heretofore to the part and 24-51 residues corresponding to the bovine LF 1 to 5 2 residues [Formula 7] [Chemical 8] I found out.

[Chemical 7]
Ala-Pro-Arg-Lys-Asn-Val-Arg-Trp-Cys-Thr-Ile-Ser-Gln-Pro-Asp-Ser-Phe-Lys-Cys-Arg-Arg-Trp-Gln-Trp-Arg- Met-Lys-Lys-Leu-Gly-Ala-Pro-Ser-Ile-Thr-Cys-Val-Arg-Arg-Ala-Phe-Ala-Leu-Glu-Cys-Ile-Arg-Ala-Ile-Ala- Glu-Lys

[Chemical 8]
Trp-Arg-Met-Lys-Lys-Leu-Gly-Ala-Pro-Ser-Ile-Thr-Cys-Val-Arg-Arg-Ala-Phe-Ala-Leu-Glu-Cys-Ile-Arg-Ala- Ile-Ala-Glu

Of these peptide fragments, [Chemical Formula 7] can have two SS bonds in the molecule, but this SS bond is not essential for the expression of viral activity. May or may not exist.
Therefore, Cys in [Chemical Formula 7] may be in a reduced form or may be in an oxidized form by S—S bond with each other.

By carrying out the present invention, a virus infection / growth inhibitor is provided.
The effects of the composition having a virus infection / growth inhibitory action according to the present invention are summarized as follows.
(1) It is possible to prevent virus infection, and for an already infected person, it is possible to prevent the virus from further proliferating in the body and increase the number of infected cells.
(2) Since it is a composition having an active ingredient as an ingredient that is usually ingested as a food, there is little concern about side effects due to administration.
(3) Since it is a small molecule, it can be prepared relatively easily and in a large amount by a chemical synthesis method or the like as compared with binding proteins such as LF. Therefore, the use is not limited to treatment of a specific patient, and it is possible to widely prevent infection and proliferation of viruses.
The virus infection / growth inhibitor of the present invention is useful for the prevention or treatment of influenza or AIDS and the protection against cytomegalovirus infection during organ transplantation.

The present invention relates to a viral infection and proliferation inhibitor containing, as an active ingredient, represented Lupe peptide by the following formula 9.
The peptide may be in the form of a pharmacologically acceptable salt.

[Chemical 9]
T rp-Arg-Met-Lys-Lys-Leu-Gly-Ala-Pro-Ser-Ile-Thr-Cys-Val-Arg-Ar g

Further, a peptide obtained by extending the peptide chain to the N-terminal side and C-terminal side based on the sequence shown in [Chemical 9] can be used as an active ingredient. Examples of such peptides include the peptides shown in [Chemical Formula 7] and [Chemical Formula 8] described above. Furthermore, pharmacologically acceptable salts thereof such as hydrochloride and acetate can also be used. The peptide represented by [Chemical 9] is a sequence corresponding to residues 24 to 39 of bovine LF, and is a region essential for antiviral activity in the present invention as described above. Hereinafter, a peptide containing an amino acid sequence derived from LF as an active ingredient in the virus infection / growth inhibitor of the present invention is described by the number of the amino acid sequence in LF, such as bovine LF (24-51).
In order to obtain the peptide of the present invention, any of ordinary chemical synthesis, isolation from LF protease degradation products, genetic recombination, etc. may be used. Furthermore, after obtaining a linear peptide using a commercially available synthesis apparatus such as a peptide synthesizer, an SS bond may be formed by a usual method, or an SS bond may not be formed.

  These peptides or peptide derivatives can be formulated singly or by adding an excipient or stabilizer. The viral infection / growth inhibitor of the present invention can be administered orally, as an injection, or as a suppository. Usually, the effect is shown by administering 0.1 to 1.0 g per day for an adult. Moreover, since these peptides are part of proteins conventionally used as food materials, there is no problem in terms of safety. In particular, bovine-derived peptide fragments are preferred in practice because of their low antigenicity.

  The virus infection / growth inhibitor according to the present invention will be described in detail below with reference to examples. The synthesis was carried out by solid phase synthesis according to the method described in Izumiya et al., “Basics and Experiments of Peptide Synthesis” (1985 Maruzen) p194-233.

Synthesis of bovine LF (24-39) Using peptide synthesizer 431A (ABI), parahydroxymethylphenoxymethyl polystyrene (HMP) resin was used, and the 9-fluorenylmethyloxycarbonyl (Fmoc) group was used as the amino-terminal protecting group. A linear protected peptide was synthesized on a 0.25 mmol scale by extending the sequential peptide chain from the C-terminal side. In the presence of phenol, 1,2-ethanedithiol, and thioanisole, 1340 mg of the obtained HMP resin-bound protected peptide was simultaneously cleaved from the HMP resin and removed with a trifluoroacetic acid (TFA). After removing TFA by concentration under reduced pressure, the crude peptide was crystallized with ethyl ether, dissolved in 5% acetic acid, and lyophilized. 543 mg of the obtained linear crude peptide was purified by HPLC [column: octadecyl 4PW (21.5 × 150 mm, Tosoh Corp.), elution: gradient elution with water-acetonitrile containing 0.1% TFA]. 367 mg of peptide was obtained. The purity of the obtained purified peptide was 90% as a result of analysis by HPLC.

Synthesis of bovine LF (24-51) Synthesis was carried out in the same manner as in Example 1 to obtain 432 mg of a chain peptide of bovine LF (24-51) having a purity of 93%.

Synthesis of N-acetyl-bovine LF (24-39) -amide (Ac-bovine LF (24-39) -NH ₂ ) Using benzhydrylamine resin with peptide synthesizer 431A (ABI), tert-butyloxycarbonyl (t Synthesis was performed on a 0.25 mmol scale by sequentially extending the peptide chain from the C-terminal side using the -Boc) group as a protective group at the amino terminal. After all the condensation reactions were completed, the amino terminal t-Boc group was removed, and this was acetylated with acetic anhydride. Cleavage of the peptide from the resin and removal of the protecting group were performed simultaneously by HF treatment. Reduced pressure to remove the HF to give the crude Ac- bovine LF (24-39) -NH ₂ purposes Concentration. This crude peptide was purified by HPLC in the same manner as in Example 1 to obtain 42 mg of a purified peptide having a purity of 91%.

Bovine LF (1-5 2 ), [ ¹⁹ Cys (Acm), ³⁶ Cys (Acm)] Bovine LF (1-5 2 ) and [ ⁹ CysSH, ¹⁹ Cys (Acm), ³⁶ Cys (Acm), ⁴⁵ CysSH] Synthesis of bovine LF (1-5 2 ) Using peptide synthesizer 431A (ABI), parahydroxymethylphenoxymethyl polystyrene (HMP) resin was used, and the 9-fluorenylmethyloxycarbonyl (Fmoc) group was protected at the amino terminal. A linear protected peptide was synthesized on a 0.25 mmol scale by protecting the ¹⁹ Cys and ³⁶ Cys SH groups with an acetamidomethyl (Acm) group and extending the sequential peptide chain from the C-terminal side. In the presence of phenol, 1,2-ethanedithiol and thioanisole, 2767 mg of the obtained HMP resin-bound protected peptide was simultaneously cleaved from the HMP resin and removed from the protecting group with trifluoroacetic acid (TFA). After removing TFA by concentration under reduced pressure, the crude peptide was crystallized with ethyl ether, dissolved in 5% acetic acid, and lyophilized. The obtained linear crude peptide (865 mg) was purified by HPLC [column: octadecyl 4PW (21.5 × 150 mm, Tosoh Corp.), elution: gradient elution with water-acetonitrile containing 0.1% TFA]. purified peptide ^{[9 CysSH, 19 Cys (Acm)} , 36 Cys (Acm), 45 CysSH ] to give the bovine LF (1-5 2) 589mg. The purity of the obtained purified peptide was 95% as a result of analysis by HPLC.
This peptide was oxidized by air in the presence of potassium ferricyanide to form an S—S bond at ⁹ Cys, ⁴⁵ Cys, and further purified by HPLC to obtain a purity of 88% [ ¹⁹ Cys (Acm), ³⁶ Cys (Acm)] 467 mg of bovine LF (1-5 2 ) was obtained.
Further, this peptide was treated with iodine to simultaneously remove the Acm group and form an S—S bond, and purified by HPLC to obtain 101 mg of bovine LF (1-5 2 ). As a result of analysis by HPLC, the purity of this peptide was 87%.

Production of virus infection / growth inhibitor An example of producing a peptide injection preparation obtained by the methods of Examples 1 to 4 is shown.
(1) Bovine LF (24-39) 4g
Tween 80 2mg
Glycine 2g
The above composition was dissolved in physiological saline for injection, and the total volume was adjusted to 20 ml. After sterilization, 2 ml was dispensed into vials, and freeze-dried and sealed.
(2) Bovine LF (24-51) 4g
Tween 80 1mg
Sorbitol 2g
Glycine 1g
The above composition was dissolved in physiological saline for injection, and the total volume was adjusted to 20 ml. After sterilization, 2 ml was dispensed into vials, and freeze-dried and sealed.

(3) Bovine LF (1-5 2 ) 4 g
Tween 80 1mg
Sorbitol 2g
Glycine 1g
The above composition was dissolved in physiological saline for injection, and the total volume was adjusted to 20 ml. After sterilization, 2 ml was dispensed into vials, and freeze-dried and sealed.
(4) Ac- bovine LF (24-39) -NH ₂ 4g
Sorbitol 4g
Human serum albumin 50mg
The above composition was dissolved in 0.01 M PBS of pH 7.0, and the total volume was adjusted to 20 ml. After sterilization, 2 ml was dispensed into vials, and freeze-dried and sealed.

  Experimental examples are shown below, and the effects of the present invention will be described in more detail.

Experimental example 1

Infection / growth inhibitory effect of LF-derived peptide against cytomegalovirus (CMV) Method: LF-derived peptide was dissolved in MEM medium supplemented with 2% serum and sterilized by filtration to obtain a stock solution. This stock solution was diluted with MEM medium supplemented with 2% serum as necessary. Human fetal fibroblasts (HEL cells) were suspended in MEM medium containing 2% serum containing the sample and incubated for 10 minutes. The cells were removed by centrifugation, and further washed twice with MEM medium supplemented with 2% serum, and then suspended in MEM medium supplemented with 2% serum. Human CMV (TANAKA strain) was added thereto, cultured for 24 hours, and then fluorescently stained with human CMV positive serum to measure the ability of human CMV to be adsorbed to cells.

  Results: Table 1 shows the infection / growth inhibitory effect of human LF-derived peptides on CMV. From the sequences common to peptides having antiviral activity among the peptides shown in Table 1, it was speculated that the 25 to 40 residue portion contains a structure essential for activity. This corresponds to the 24-39 residue part in the case of bovine LF. Table 2 also shows the minimum concentration required for bovine LF-derived peptides to inhibit 90% of CMV infection / growth compared to controls. From this result, it was revealed that the peptide according to the present invention exhibits an antiviral effect at a lower concentration than natural LF and LF-derived peptides known so far. Of the peptides used here, those other than the peptides constituting the present invention were also chemically synthesized in the same manner as in the method shown in Example 1, and the purity thereof was determined as a result of analysis by HPLC. It is confirmed that it is 85% or more.

[Table 1]
Inhibitory growth inhibition effect of human LF-derived peptide on CMV
__________________________________________________________
Peptide ^{* 1} SS bond Infection inhibition rate (%)
__________________________________________________________
LF (1-52) ¹⁰ Cys- ⁴⁶ Cys, ²⁰ Cys- ³⁷ Cys 95
LF (1-52) ¹⁰ Cys- ⁴⁶ Cys, 95
LF (1-52) 97
LF (18-52) 24
LF (25-52) 91
LF (1-40) 97
LF (28-40) 0
LF (19-31) 2
LF (2-19) 14
LF (4-29) 0
LF (18-42) 97
__________________________________________________________
^{* 1} Peptide concentration is 0.5mg / ml

[Table 2]

Experimental example 2

Infection / Proliferation Inhibitory Effect of LF Derived Peptide on HIV Method: Culture supernatant of MOLT-4 / HTLV-III _B (hereinafter abbreviated as MT-4) cell which is a HTLV-III _B persistently infected strain of HIV Was used as a virus solution. The supernatant was stored at -80 ° C. Cells used for the assay were human T cell line MT-4. MT-4 was passaged using RPMI 1640 medium containing 10% fetal calf serum (FCS). The sample (LF-derived peptide) was dissolved in a medium (RPMI1640) and 1 ml was added to the cells as a target concentration. After incubation for 60 minutes, MT-4 cells were infected with HIV so that moi (cell / infection virus ratio) = 0.01, and 1 ml of a cell solution prepared to 3 × 10 ⁵ / ml was added. After culturing the cells for 3 days, HIV-infected cells were measured using the indirect fluorescent antibody method.
HIV-infected cells were measured by the indirect fluorescent antibody method using HIV-infected patient serum as a primary antibody. More than 500 cells were observed under a fluorescence microscope, and the ratio of fluorescently stained cells was calculated. In addition, HIV-infected MT-4 cells cultured without adding a sample as a positive control and cell culture without adding a virus solution as a negative control were simultaneously performed.

  Results: The experimental results are shown in Table 3. The results show the concentration required to prevent HIV infection by 90%. From this result, it was revealed that the peptide according to the present invention exhibits an antiviral effect at a lower concentration than natural LF and LF-derived peptides known so far.

[Table 3]

Experimental example 3

Infection / Proliferation Inhibitory Effect of LF-Derived Peptide on Influenza Virus Method: The infection / proliferation-inhibitory effect of LF-derived peptide on influenza virus is described in General Research on Virus Experiments, National Institute of Preventive Health, edited by the Alumni Association, p. 113-129 According to Maruzen (1973), it was carried out by the incubation method of hatched chicken eggs. Human LF, bovine LF and the peptide prepared in Example 1-3 were dissolved in physiological saline and sterilized by filtration. Twenty-five eggs hatched on the 10th day of the ages were divided into 5 groups of 5; the control group was inoculated with physiological saline alone, and the other group was inoculated with 100 μl of the sample solution into the urine cavity. Three hours later, 100 µl of a virus solution of influenza virus A / PR / 8/34 was inoculated into each urine cavity of each hatched chicken egg. The amount of virus used here is the amount of virus that can show a hemagglutination (HA) reaction in a solution obtained by diluting the urine fluid collected after inoculation into the urine cavity 64 times. Two days later, each egg was left overnight in an ice chamber, and then urine was collected. The obtained urine was diluted stepwise with physiological saline, chick-stabilized red blood cells (Takeda Pharmaceutical) were added thereto, and virus was quantified by performing HA reaction.

  Results: The experimental results are shown in Table 4. The results indicate the concentration required to block 90% of influenza virus infection. From this result, it was revealed that the peptide according to the present invention exhibits an antiviral effect at a lower concentration than natural LF and LF-derived peptides known so far.

[Table 4]

Experimental Example 4

In Vivo CMV Infection Protection Test Method: 4-week-old male Balbc / AJcl mice (10 per group) were used. Samples were dissolved in phosphate buffered saline (PBS) and administered intraperitoneally to mice. Six hours later, 1 × 10 ⁶ PFU of mouse CMV was intraperitoneally administered, and the protection rate was evaluated based on the survival rate of the mice after 10 days.

  Results: Table 5 shows the experimental results. It was revealed that the peptide according to the present invention has an infection protective effect against mouse CMV when administered at a dose of 0.01 g / kg body weight or more.

[Table 5]

Claims (3)

A virus infection / growth inhibitor comprising a peptide represented by the following [Chemical Formula 1] or a pharmacologically acceptable salt thereof as an active ingredient.
[Chemical 1]
T rp-Arg-Met-Lys-Lys-Leu-Gly-Ala-Pro-Ser-Ile-Thr-Cys-Val-Arg-Ar g Viral infection / growth inhibitor comprising the peptide represented by the following [Chemical Formula 2] or a pharmacologically acceptable salt thereof as an active ingredient
[Chemical 2]
Ala-Pro-Arg-Lys-Asn-Val-Arg-Trp-Cys-Thr-Ile-Ser-Gln-Pro-Asp-Ser-Phe-Lys-Cys-Arg-Arg-Trp-Gln-Trp-Arg- Met-Lys-Lys-Leu-Gly-Ala-Pro-Ser-Ile-Thr-Cys-Val-Arg-Arg-Ala-Phe-Ala-Leu-Glu-Cys-Ile-Arg-Ala-Ile-Ala- Glu-Lys
(However, in the formula, Cys may be a reduced form, or may be an oxidized form in which two Cys in a peptide are bonded to form an -S-S- bond) Viral infection / growth inhibitor comprising the peptide represented by the following [Chemical Formula 3] or a pharmacologically acceptable salt thereof as an active ingredient
[Chemical 3]
Trp-Arg-Met-Lys-Lys-Leu-Gly-Ala-Pro-Ser-Ile-Thr-Cys-Val-Arg-Arg-Ala-Phe-Ala-Leu-Glu-Cys-Ile-Arg-Ala- Ile-Ala-Glu
(However, in the formula, Cys may be a reduced form, or may be an oxidized form in which two Cys in a peptide are bonded to form an -S-S- bond)