JPH0873499A - New peptide and immunopotentiator - Google Patents

Abstract

PURPOSE: To obtain a new peptide, available from an enzymolytic product by carrying out the enzymolysis of lactoferrins with a protease, having a specific amino acid sequence, capable of manifesting the immunopotentiating and cytomegalovirus phylactic activities and useful for an immunopotentiator, etc. CONSTITUTION: This new peptide comprises a peptide containing an amino acid sequence represented by formula I, a peptide deficient in 1-3 amino acid residues at the N-terminal of the peptide represented by formula I and a peptide, etc., containing an amino acid sequence represented by formula II (S-S bond may be SH groups in a reduced state). The peptide has mitogen activities and is capable of manifesting immunopotentiating and cytomegalovirus phylactic activities and is useful as a medicine such as an immunopotentiator. The peptide is obtained by carrying out the enzymolysis of a human lactoferrin with a protease such as pepsin, treating the resultant enzymolytic product by high- performance liquid chromatography and fractionating the product.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel peptide and an immunostimulant containing the peptide as an active ingredient.

[0002]

2. Description of the Related Art Lactoferrin (LF) is known as an iron-binding protein present in milk. It is present in various secretory fluids other than milk, is also present in joint cavities, serum, etc., and exhibits antibacterial action by binding to iron and depriving iron ions in the solution. In addition to antibacterial activity, lactoferrin is known to have activity such as binding to viruses and antiaging effects. Further, it has been confirmed that a part of the amino acid sequence of lactoferrin has an antibacterial activity different from iron-binding property (JP-A-5-78392, JP-A-5-148).
296). Thus, lactoferrin has various activities. For lactoferrin, a method for preparing a large amount of milk from milk has been developed. For example, JP-A-63-
Japanese Patent No. 255300 discloses a method for recovering lactoferrin from milk using a sulfate ester of a cross-linked polysaccharide having an affinity for lactoferrin. By using the lactoferrin thus obtained, some new physiological actions and detailed structures have been revealed. The structure and physiological activity of lactoferrin are described in a review article by Shimazaki et al. (Keiichi Shimazaki et al., Bioscience and Industry, Vol.51, 25-27, 1993).
Further, JP-A-5-178759 describes that lactoferrin activates the phagocytic ability of peripheral blood, particularly neutrophils, and improves immunity. However, it is not known that a peptide derived from lactoferrin induces mitogenic activity of lymphocytes and activates immune function.

The present inventors have investigated the physiological activity of peptides present in the structure of lactoferrin by subjecting lactoferrin to an enzymatic degradation treatment. It was confirmed that a specific peptide present in the structure of lactoferrin acts on anti-HIV activity and anti-HTLV to suppress infection, and is disclosed in Japanese Patent Application No. 5-240284 and Japanese Patent Application No. 6-1585.
Patent application was filed as No. 1. Furthermore, as a result of detailed examination of the enzymatic degradation product of lactoferrin, it was found that a peptide containing a specific peptide structure present in the amino acid sequence of lactoferrin has a strong lymphocyte mitogenic activity, and its action was examined. As a result, the present invention has been completed.

[0004]

DISCLOSURE OF THE INVENTION The present inventors have studied the physiological activity of lactoferrin, and as a result, have found that the enzymatic degradation product of lactoferrin has a strong mitogenic activity and a strong antitumor activity. Lactoferrin was incubated with pepsin, trypsin, chymotrypsin, and papain (all manufactured by Sigma) at lactoferrin / enzyme = 100/1 at 37 ° C. for 1 hour. After incubation, the pepsin degradation product returned the reaction solution to neutrality, and the others were heated at 80 ° C. for 5 minutes to stop the reaction. The generated precipitate was removed by centrifugation, and the supernatant was freeze-dried to obtain a powder of the enzymatic decomposition product of LF. When this lysate was used as a sample to measure the blastogenic and antitumor activity of lymphocytes by the method described in the following examples, it was found to have a strong activity that has never been reported. The results of this activity measurement are shown in Tables 1 and 2 below.

[0005]

[Table 1]

[0006]

[Table 2]

As described above, it was confirmed that the enzymatic decomposition product of lactoferrin has a strong immunostimulatory action and a strong antitumor effect presumed to be due thereto. The present inventors have further investigated the peptide structure present in the amino acid sequence of lactoferrin, and as a result, have elucidated for the first time the structure essential for exerting mitogenic activity. An object of the present invention is to provide a novel peptide having a mitogenic activity which is present in such an amino acid sequence of lactoferrin. Another object of the present invention is to provide an immunoactive agent containing such a peptide as an active ingredient. Another object of the present invention is to provide a protective agent for cytomegalovirus infection containing this peptide as an active ingredient.

[0008]

The peptide having mitogenic activity of the present invention must have the following amino acid sequence in the peptide sequence. That is, in the case of a peptide derived from human lactoferrin, it is a peptide containing the following sequence [I].

Gly-Arg-Arg-Arg-Arg-Ser-Val-Gln-Trp-Cys-Ala-Val-Ser-Gln-Pro-Glu-Ala-Thr-Lys [I]

The entire amino acid sequence of human lactoferrin has already been determined (MW Rey, et al., Nucleic Acid Re
s., Vol. 18, 5288, 1990). This peptide corresponds to the 1-19th amino acid sequence in the amino acid sequence of human lactoferrin, and peptides containing this sequence are included in the present invention. Hereinafter, the amino acid sequence of the peptide derived from lactoferrin will be described by the amino acid sequence number, with Gly as the first position according to this reference. This peptide may have one to three residues deleted from the N-terminus. As an example of a peptide containing this amino acid sequence, the following sequence [I
The peptide I] can be exemplified.

[0011]

[Chemical 3]

This peptide is 1-of human lactoferrin.
It corresponds to the 52nd position. Further, the SS bond of this peptide may be an SH group in a reduced state. Sequences [I] and [II] may have N-terminal amino acid residues 1 to 3 deleted. That is, it may correspond to the amino acid sequence of human lactoferrin 2-19th, 3-19th, 4-19th, or human lactoferrin 2-52th, 3-52nd, 4-52th.

In the case of a peptide derived from bovine lactoferrin, it is a peptide containing the following sequence [III].

Ala-Pro-Arg-Lys-Asn-Val-Arg-Trp-Cys-Thr-Ile-Ser-Gln-Pro-Asp-Ser-Phe-Lys [III]

The entire amino acid sequence of bovine lactoferrin has already been determined similar to human lactoferrin (PE
Mead, et al., Nucleic Acid Res., Vol. 18, 7167, 1990).
This peptide is 1 of the amino acid sequence of bovine lactoferrin.
It corresponds to the -18th amino acid sequence, and a peptide containing this sequence is included in the present invention. The amino acid sequence of a peptide derived from bovine lactoferrin will be described below according to this reference, with Ala as the first amino acid sequence number. An example of the peptide containing this amino acid sequence is the peptide of the following sequence [IV].

[0016]

[Chemical 4]

This peptide is 1-of bovine lactoferrin.
It corresponds to the 51st. Further, the SS bond of this peptide may be an SH group in a reduced state. Sequence [II
In [I] and sequence [IV], N-terminal amino acids 1 to 3 residues may be deleted. That is, bovine lactoferrin 2-1
It may correspond to the 8th, 3-18th, 4-18th or bovine lactoferrin 2-51st, 3-51st, 4-51st amino acid sequence. These peptides are small molecules and have low antigenicity upon administration as compared with the original protein.

To prepare the peptide of the present invention, a conventional peptide synthesis method can be adopted. A solid-phase synthesis method is generally used as a peptide synthesis method. This solid-phase synthesis method is disclosed in "Izumitani et al., Fundamentals and Experiments of Peptide Synthesis (1985, Maruzen), pages 194 to 233". Can be mentioned. Also, other methods may be used.
Alternatively, human lactoferrin or bovine lactoferrin can be enzymatically decomposed with a protease and fractionated by chromatography. Lactoferrin for enzymatic degradation can be easily recovered from human or bovine milk. For example, the above-mentioned Japanese Patent Laid-Open No. 63-255
The sulfate ester of cross-linked polysaccharide having an affinity for lactoferrin disclosed in Japanese Patent Publication No. 300 can be used to recover from milk. As the enzyme used for the enzymatic decomposition of lactoferrin, any enzyme can be used as long as it is an enzyme normally used for the enzymatic decomposition of proteins. Examples of such an enzyme include pepsin, trypsin, chymotrypsin, papain and the like. Also, enzymes other than these can be used. These peptides can be collected from the thus obtained enzymatic degradation product by chromatographic treatment by a conventional method. Alternatively, it may be produced according to a conventional peptide production method.

The peptide of the present invention induces lymphocyte blastogenesis and exhibits an antiviral effect, particularly an effect of preventing infection against cytomegalovirus. The peptide of the present invention can be administered alone, or can be formulated using additives such as stabilizers and excipients used for formulation. The peptide of the present invention can be added to foods and livestock feeds for administration, and can also be used for applications such as pharmaceuticals and cosmetics. When used as a medicine, it can be used in a dosage form such as oral, injection, suppository, etc. Normally, by administering about 0.1 to 5 g per day for an adult,
It can be expected to have an immunostimulatory action and a virus infection defense effect. Further, the peptide of the present invention is a safe substance which does not show toxicity upon oral administration, and does not cause death of animals even at the maximum administrable dose even when administered intravenously.

The present invention will be described in more detail below with reference to examples.

Example 1 Preparation of enzymatic degradation product of lactoferrin (hereinafter referred to as LF)
Pepsin LF prepared from milk by the method described in isolation JP 63-255300 discloses the mitogen-activated peptide as a starting material was (Sigma) enzymatic degradation. Incubation was carried out at 37 ° C. for 1 hour at a ratio of LF / enzyme = 100/1. After the incubation, the pepsin degradation product returns the reaction solution to neutral, and the others are 80
The reaction was stopped by heating at 0 ° C for 5 minutes. The generated precipitate was removed by centrifugation, and the supernatant was freeze-dried to obtain a powder of the enzymatic decomposition product of LF. An HPLC equipped with a column in which two TSK gel G300SW columns (21.5 mm × 300 mm: manufactured by Tosoh) are connected in series to each other in the decomposition composition.
And separated. Elution is 0.015M NaCl
Absorption at 214 nm was measured using a 1 mM phosphate buffer solution (pH 7.4) containing 1% as an eluent. This gel filtration pattern is shown in FIG.

The lymphocyte blastogenic activity of each separated fraction was measured by the following method. C3H / HeN mouse spleen cells were collected, washed, and then suspended in RPMI1640 medium containing 10% fetal bovine serum. 5x spleen cells
Dispense into a 96-well microplate at 10 ⁵ / well, and the final concentration of each sample is 1 μg / ml,
It was added at 10 μg / ml and 100 μg / ml. Concanavalin A (final concentration 1 μg) in control wells
/ Ml), lipopolysaccharide (final concentration 100 μg
/ Ml) was added and the cells were cultured at 37 ° C. for 48 hours under 5% CO ₂ conditions. After culturing, 10 μl of 3- (4,5-dimethyl-2-thiazolyl) 2,5-diphenyl-2H tetrazolium bromide (hereinafter abbreviated as MTT) solution was added, and after culturing for 3 hours, the resulting MTT formazan was added to an ELISA reader. The absorbance was measured at 562-595 nm (the above method was based on the method disclosed in Med. Immunol, 12, 411 (1986)). The result was the average value of 10 wells, and the mitogen activity ratio (SI) was calculated based on the following formula.

S. I. = (Average absorbance of sample well) /
(Average absorbance of control well) x 100

The S. I. The values are shown in Table 3.

[Table 3]

Of the respective fractions, the elution position of the particularly highly active fraction 7 was measured again by HPLC and compared with the synthetic peptide shown below. As a result, the elution time of this fraction was consistent with that of bovine LF1-18. . Further, analysis of the amino acid sequence of this fraction confirmed that it had the sequence of bovine LF1-18.

[0025]

[Example 2] Preparation of human LF enzymatic degradation product and mitogenic activity pepti
Isolation of human LF was carried out in the same manner as in Example 1 to prepare an enzymatic degradation product of human LF. I. Active peptides were identified by retention time by HPLC and amino acid sequence. As a result, S. I. It was confirmed that the active peptide was human LF1-19.

[0026]

Example 3 Chemical Synthesis of Active Peptide The peptides identified in Examples 1 and 2 and peptides containing the amino acid sequences thereof were synthesized. In this example, human L
The synthetic examples of F1-19, human LF1-52, bovine LF1-18, and bovine LF1-51 are shown. Other peptides described herein were also synthesized according to this example. (1) By a synthetic peptide synthesizer 431A (ABI ) of human LF1-19 ,
A linear protected peptide was synthesized on a 0.25 mmol scale using para-hydroxymethylphenoxymethyl polystyrene (HMP) resin with a 9-fluorenylmethyloxycarbonyl (Fmoc) group as the amino-terminal protecting group. Obtained HMP resin-bound protected peptide 1455 mg
In the presence of phenol, 1,2-ethanedithiol and thioanisole, the peptide was cleaved from the HMP resin by trifluoroacetic acid (TFA) and the protecting group was removed at the same time. 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 500
mg is HPLC [column: octadecyl 4PW (2
1.5 × 150 mm, (Tosoh Corporation), elution: 0.1% T
Gradient elution with water-acetonitrile containing FA]
Was purified by to obtain 410 mg of the linear purified peptide. The purity of the obtained purified peptide was 93% as a result of analysis by HPLC.

(2) Human LF1-52 [ ²⁰ Cys (Acm), ³⁷ C
ys (Acm)], human LF1-52 and [ ¹⁰ CysSH, ²⁰ Cys
(Acm), ³⁷ Cys (Acm), ⁴⁶ CysSH] Human LF1-52 synthetic peptide synthesizer 431A (ABI)
Parahydroxymethylphenoxymethyl polystyrene (HMP) resin was used, and 9-fluorenylmethyloxycarbonyl (Fmoc) group was used as the amino-terminal protecting group,
The SH group of ²⁰ Cys and ³⁷ Cys was converted to acetamidomethyl (A
cm) group to synthesize a linear protected peptide on a 0.25 mmol scale. The obtained HMP resin-bound protected peptide 2337 mg was treated with trifluoroacetic acid (T) in the presence of phenol, 1,2-ethanedithiol and thioanisole.
FA) simultaneously cleaved the peptide from the HMP resin and removed the protecting group. After removing TFA by concentration under reduced pressure, the crude peptide was crystallized with ethyl ether,
This was dissolved in 5% acetic acid and freeze-dried. The obtained linear crude peptide (970 mg) was purified by HPLC (column: octadecyl 4PW (21.5 × 150 mm, Tosoh Corp.), elution: gradient elution with water-acetonitrile containing 0.1% TFA) and linear purification. Peptide [ ¹⁰ CysS
H, ²⁰ Cys (Acm), ³⁷ Cys (Acm), ⁴⁶ CysSH] human LF (1-5
2) 607 mg was obtained. The purity of the obtained purified peptide was 96% as a result of analysis by HPLC. By air oxidation of this peptide in the presence of potassium ferricyanide
By forming an SS bond in ¹⁰ Cys and ⁴⁶ Cys and further purifying by HPLC, [ ²⁰ Cys (Acm), ³⁷ Cys (A
cm)] Human LF (1-52) 450 mg 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 120 mg of human LF (1-52). As a result of analysis by HPLC, the purity of this peptide was 89%.

(3) Synthesis of bovine LF1-18 A peptide having the amino acid sequence of bovine LF1-18 was synthesized in the same manner as in Example 2 to obtain 365 mg of a chain peptide having a purity of 98%.

(4) Bovine LF (1-51), [ ¹⁹ Cys (Ac
m), ³⁶ Cys (Acm)] bovine LF (1-51) and [ ⁹ CysS
H, ¹⁹ Cys (Acm), ³⁶ Cys (Acm), ⁴⁵ CysSH] Bovine LF (1-5
Synthesis of 1) Synthesis was carried out in the same manner as in Example 3, and 576 mg of 88% pure bovine LF (1-51) and 90% pure [ ¹⁹ Cys
(Acm), ³⁶ Cys (Acm)] Bovine LF (1-51) 390m
g, 82% pure [ ⁹ CysSH, ¹⁹ Cys (Acm), ³⁶ Cys (Acm), ⁴⁵
CysSH] bovine LF (1-51) was obtained.

[0030]

[Example 4] Measurement of lymphocyte blastogenic activity of synthetic peptide The lymphocyte blastogenic activity of the synthetic peptide obtained in Example 3 was measured. The measurement was according to the method shown in Example 1. The measurement results are shown in Table 4 below. Each synthetic peptide has a strong S. I. Had activity.

[0031]

[Table 4]

[0032]

Example 5 Measurement of Antitumor Effect of Synthetic Peptide In order to confirm the immunostimulatory effect of the synthetic peptide obtained in Example 3, an experiment was carried out by using the growth inhibitory effect of ascites tumor as an index, and changes in immune markers were confirmed. I observed. BALB / c
Male mice (10 mice per group) were intraperitoneally transplanted with 10 ⁵ to 10 ⁶ ascites tumor cells (Meth A cells). From the day of tumor implantation, the synthetic peptide was intraperitoneally administered every other day five times. Further, as a positive control, muramyl dipeptide (MDP) was similarly administered at 3 mg / kg, and as a negative control, carrageenan was similarly administered. Table 5 shows the survival rate of the mice 20 days after the tumor transplantation. 0.005 g / body weight k for samples
A tumor growth inhibitory effect was observed at a dose of g or more, and in the synthetic peptide administration group, death of mice was not observed at 0.05 g / kg body weight. It was also confirmed that the NK cells of the peptide-administered animal were activated. The measurement of NK cell activation was carried out by using mouse spleen cells as effector cells and target cells with ⁵¹ Cr-labeled tumor cells (YAC-1) mixed at a ratio of 100: 1 to release the amount of ⁵¹ Cr. The NK activity value was measured.
The NK activity value of the animals administered with the substance of the present invention was significantly higher than that of the control.

[0033]

[Table 5]

[0034]

Example 6 Cytomegalovirus infection protective effect of synthetic peptide Various synthetic peptides of human and bovine LF were prepared, and the cytomegalovirus infection protective effect of this peptide was confirmed. As experimental animals, SPF-BALB / cA males, 4
Weekly age was used as 5 animals per group. This mouse was infected with a mouse cytomegalovirus (MCMV) Smith strain that passed through the mouse salivary gland 10 times or more, and the survival rate was determined to make a determination. Virus infection is 1 × 10
⁶ PFU / mouse was inoculated into the abdominal cavity by intraperitoneal injection, and PBS solution of each peptide was intraperitoneally 0.2
The dose was 5, 0.125, 0.025, 0.005 g / kg (body weight). The surviving animals were dissected, the spleen was removed, and the NK cell activity of splenocytes was measured. The results are shown in Table 6.
It was shown to.

[0035]

[Table 6]

It was confirmed that the peptide of the present invention has activity on the N-terminal side of human or bovine LF. It was also confirmed that this activity had no effect even if 3 residues from the N-terminus were deleted. Furthermore, the NK cell activity was increased in all the animals administered with the peptide of the present invention. This activity was also in agreement with the cytomegalovirus infection protective activity.

[0037]

Example 7 This example shows an example in which the peptide of the present invention was formulated. (1) Tablets 170 g of lactose, 5 g of potato starch, 20 g of bovine LF1-18 synthesized in Example 3 and 5 g of talc stearate were mixed and tableted by a conventional method to produce 200 1 g tablets containing 100 mg of peptide. did. (2) Injection 5 g of mannitol in 100 ml, 100 mg of human LF1-19 synthesized in Example 3, human serum albumin 1
An aqueous solution containing 00 mg and 2 mg of sodium caprylate was aseptically prepared, dispensed in 1 ml aliquots, lyophilized and sealed.

[0038]

EFFECTS OF THE INVENTION The present invention provides a novel peptide, a method for producing the same, an immunostimulant containing this peptide as an active ingredient, and a cytomegalovirus infection protective agent.

[0039]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 19 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type: N-terminal fragment Origin: Human lactoferrin Sequence characteristics Sequence: Gly Arg Arg Arg Arg Ser Val Gln Trp Cys Ala Val Ser Gln Pro Glu 1 5 10 Ala Thr Lys

SEQ ID NO: 2 Sequence length: 52 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type: N-terminal fragment Origin: Human lactoferrin Sequence characteristics Sequence: Gly Arg Arg Arg Arg Ser Val Gln Trp Cys Ala Val Ser Gln Pro Glu 1 5 10 15 Ala Thr Lys Cys Phe Gln Trp Gln Arg Asn Met Arg Lys Val Arg Gly 20 25 30 Pro Pro Val Ser Cys Ile Lys Arg Asp Ser Pro Ile Gln Cys Ile Gln 35 40 45 Ala Ile Ala Glu 50

SEQ ID NO: 3 Sequence length: 18 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type: N-terminal fragment Origin: Bovine lactoferrin Sequence characteristics Sequence: Ala Pro Arg Lys Asn Val Arg Trp Cys Thr Ile Ser Gln Pro Asp Ser 1 5 10 15 Phe Lys

SEQ ID NO: 4 Sequence length: 51 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type: N-terminal fragment Origin: Bovine lactoferrin Sequence characteristics Sequence: Ala Pro Arg Lys Asn Val Arg Trp Cys Thr Ile Ser Gln Pro Asp Ser 1 5 10 15 Phe Lys Cys Arg Arg Trp Gln Trp Arg Met Lys Lys Leu Gly Ala Pro 20 25 30 Ser Ile Thr Cys Val Arg Arg Ala Phe Ala Leu Glu Cys Ile Arg Ala 35 40 45 Ile Ala Glu 50

[Brief description of drawings]

FIG. 1 shows an HPLC pattern of an enzymatic degradation product of bovine lactoferrin with pepsin. The peptide of the present invention is present at the peak marked with ↓ in the figure.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display area C07K 7/08 ZNA 8318-4H C12P 21/06 9282-4B

Claims (9)

[Claims] 1. A peptide comprising an amino acid sequence represented by the following amino acid sequence [I]. Gly-Arg-Arg-Arg-Arg-Ser-Val-Gln-Trp-Cys-Ala-Val-Ser-Gln-Pro-Glu-Ala-Thr -Lys (I) 2. The peptide according to claim 1, wherein 1 to 3 amino acid residues at the N-terminal of amino acid sequence [I] are deleted. 3. A peptide containing the amino acid sequence represented by the following amino acid sequence [II]. Embedded image However, the S—S bond may be a reduced SH group. 4. A peptide comprising an amino acid sequence represented by the following amino acid sequence [III]. Ala-Pro-Arg-Lys-Asn-Val-Arg-Trp-Cys-Thr-Ile-Ser-Gln-Pro-Asp-Ser-Phe-Lys (III) 5. A peptide containing the amino acid sequence represented by the following amino acid sequence [IV]. Embedded image However, the S—S bond may be a reduced SH group. 6. An immunostimulant comprising the peptide according to claim 1 or a pharmacologically acceptable salt thereof as an active ingredient. 7. A cytomegalovirus infection protective agent comprising the peptide according to claim 1 or a pharmacologically acceptable salt thereof as an active ingredient. 8. A method for producing a peptide, which comprises enzymatically decomposing human lactoferrin with a protease, and collecting any of the peptides of claims 1 to 3 from the enzymatic decomposition product. 9. A method for producing a peptide, which comprises enzymatically decomposing bovine lactoferrin with a protease, and collecting the peptide of claim 4 from the enzymatic decomposition product.