JP3561519B2 - New peptides and immunostimulants - Google Patents

Description

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

  Lactoferrin (LF) is known as an iron-binding protein present in milk. It is also present in various secretions other than milk, in joint cavities and in serum, etc., and exhibits antibacterial action by binding to iron and depriving the solution of iron ions. In addition, lactoferrin is known to have, in addition to antibacterial activity, activities such as binding to viruses and antiaging effects. In addition, it has been confirmed that a part of the amino acid sequence of lactoferrin has an antibacterial activity different from that of iron binding (for example, see Patent Documents 1 and 2). Thus, lactoferrin has various activities. A method for preparing lactoferrin in large amounts from milk has been developed. For example, a method of recovering lactoferrin from milk using a sulfated cross-linked polysaccharide having an affinity for lactoferrin is disclosed (for example, see Patent Document 3). Several new physiological actions and detailed structures have been found by using the lactoferrin thus obtained. Shimazaki et al. Described the structure and physiological activity of lactoferrin in a review (for example, see Non-Patent Document 1). Further, it is disclosed that lactoferrin activates the phagocytic activity of peripheral blood, particularly neutrophils, and improves immunity (for example, see Patent Document 4). However, it is not known that a lactoferrin-derived peptide induces lymphocyte mitogen activity and activates an immune function.

The present inventors have studied the physiological activity of a peptide present in the structure of lactoferrin by subjecting lactoferrin to an enzymatic degradation treatment. The inventors confirmed that a specific peptide present in the structure of lactoferrin acts on anti-HIV activity and anti-HTLV to suppress infection, and filed a patent application (for example, see Patent Documents 5 and 6). Furthermore, as a result of a detailed study 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 strong lymphocyte mitogenic activity. As a result, the present invention has been completed.
JP-A-5-78392 JP-A-5-148296 JP-A-63-255300 JP-A-5-178759 JP-A-7-69915 JP-A-7-206701 Keiichi Shimazaki et al., Bioscience and Industry, Vol.51, 25-27,1993

  An object of the present invention is to provide a novel peptide which is present in the amino acid sequence of bovine lactoferrin and has mitogenic activity. Another object of the present invention is to provide an immunostimulant containing such a peptide as an active ingredient. Another object of the present invention is to provide a cytomegalovirus infection protective agent comprising this peptide as an active ingredient.

  The present inventors have studied the physiological activity of lactoferrin, and as a result, have found that enzymatically degraded lactoferrin has strong mitogenic activity and strong antitumor activity.

  Lactoferrin was incubated for 1 hour at 37 ° C. with lactoferrin / enzyme = 100/1 using pepsin, trypsin, chymotrypsin and papain (all manufactured by Sigma). After the incubation, the reaction solution of the pepsin degradation product was returned to neutral, and the other reaction was stopped by heating at 80 ° C. for 5 minutes. The resulting precipitate was removed by centrifugation, and the supernatant was freeze-dried to obtain a powder of an enzyme-decomposed product of lactoferrin. When the enzymatic degradation product was used as a sample and the lymphocyte blastogenesis and antitumor activity were measured by the methods described in the following Examples, they were found to have a strong activity that has not been reported before. The results of the activity measurement are shown in Tables 1 and 2 below.

このようにラクトフェリンの酵素分解物には強い免疫賦活作用とこれによると推測される強い抗腫瘍効果が存在することが確認できた。
本発明者らは、さらにラクトフェリンのアミノ酸配列中に存在するペプチド構造に関して検討を行った結果、マイトージェン活性を発揮するに必須の構造を初めて解明した。

Thus, it was confirmed that the enzymatically degraded product of lactoferrin has a strong immunostimulatory effect and a strong antitumor effect presumably due to this.
The present inventors have further studied 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 mitogen activity.

  According to the present invention, a novel peptide having mitogenic activity, a method for producing the same, an immunostimulant containing the peptide as an active ingredient, and a cytomegalovirus infection protective agent are provided.

The peptide having mitogenic activity of the present invention needs to have the following amino acid sequence in the peptide sequence.
That is, in the case of a bovine lactoferrin-derived peptide, it is a peptide containing the following sequence [I].
Ala-Pro-Arg-Lys-Asn-Val-Arg-Trp-Cys-Thr-Ile-Ser-Gln-Pro-Asp-Ser-Phe-Lys [I]

The entire amino acid sequence of bovine lactoferrin has already been determined (PE Mead et al., Nucleic Acid Res., Vol. 18, 7167, 1990). This peptide corresponds to the amino acid sequence of positions 1 to 18 in the amino acid sequence of bovine lactoferrin, and a peptide having this sequence is included in the present invention. Hereinafter, the amino acid sequence of the peptide derived from bovine lactoferrin is described by the amino acid sequence number, with Ala as the first, according to this document.
As an example of a peptide containing this amino acid sequence, a peptide having the following sequence [II] can be exemplified.

This peptide corresponds to positions 1-51 of bovine lactoferrin. Further, the SS bond of this peptide may be a reduced SH group.
In the sequence [I] and the sequence [II], the N-terminal amino acid 1 to 3 residues may be deleted. That is, it may correspond to the amino acid sequence of bovine lactoferrin 2-18, 3-18, 4-18, or bovine lactoferrin 2-51, 3-51, 4-51.
These peptides have the advantage that they are small molecules compared to the original protein and have low antigenicity upon administration.

To prepare the peptide of the present invention, a general peptide synthesis method can be employed. As a peptide synthesis method, a solid phase synthesis method is generally used, and this solid phase synthesis method is disclosed in, for example, "Izumiya et al., Basics and Experiments of Peptide Synthesis (Maruzen, 1985), pp. 194-233". Can be mentioned. Other methods may be used.
Further, bovine lactoferrin can be enzymatically decomposed with a protease and separated by chromatography. Lactoferrin to be subjected to enzymatic degradation can be easily recovered from bovine milk. For example, it can be recovered from milk using a sulfated ester of a cross-linked polysaccharide having an affinity for lactoferrin disclosed in JP-A-63-255300.
As the enzyme used for the enzymatic degradation of lactoferrin, any enzyme that is commonly used for the enzymatic degradation of proteins can be used. Examples of such an enzyme include pepsin, trypsin, chymotrypsin, and papain. Also, other enzymes can be used.
These peptides can be collected by subjecting the enzymatic degradation product thus obtained to a chromatographic treatment in a conventional manner.
Further, the peptide may be produced according to other ordinary peptide production methods.

The peptide of the present invention induces lymphocyte blastogenesis and exhibits an antiviral effect, particularly a protective effect against cytomegalovirus.
The peptide of the present invention can be administered alone, or can be formulated using additives usually used for the formulation such as stabilizers and excipients.
The peptide of the present invention can be administered after being added to food or livestock feed, and can also be used for applications such as pharmaceuticals and cosmetics. When used as a pharmaceutical, it can be used in dosage forms such as oral, injection, and suppository. Usually, by administering about 0.1 to 5 g per adult per day, an immunostimulatory effect and a protective effect against virus infection can be expected. Things.
The peptide of the present invention is a safe substance which does not show toxicity when administered orally and which does not produce dead animals at the maximum physically administrable dose even when administered intravenously.
Hereinafter, the present invention will be described in more detail with reference to Examples.

(Preparation of lactoferrin (hereinafter referred to as LF) enzymatic degradation product and isolation of mitogen-active peptide)
Pepsin (manufactured by Sigma) was subjected to enzymatic degradation using LF prepared from milk by the method described in JP-A-63-255300. The mixture was incubated at 37 ° C. for 1 hour at a ratio of LF / enzyme = 100/1. 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 resulting precipitate was removed by centrifugation, and the supernatant was freeze-dried to obtain a powder of an enzymatically degraded LF. This decomposed composition was subjected to HPLC equipped with a column in which two TSK gel G300SW columns (21.5 mm × 300 mm: manufactured by Tosoh) were connected in series, to perform separation. Elution was performed using a 1 mM phosphate buffer (pH 7.4) containing 0.015 M NaCl as an eluent, and the absorption at 214 nm was measured. This gel filtration pattern is shown in FIG.

The lymphocyte blastogenic activity of each of the separated fractions was measured by the following method. After collecting and washing C3H / HeN mouse spleen cells, they were suspended in RPMI1640 medium containing 10% fetal calf serum. The spleen cells were dispensed into a 96-well microplate at 5 × 10 ⁵ / well, and the samples were added thereto to give final concentrations of 1 μg / ml, 10 μg / ml, and 100 μg / ml, respectively. Concanavalin A (final concentration 1 μg / ml) and lipopolysaccharide (final concentration 100 μg / ml) were added to control wells, 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 further culturing for 3 hours, the resulting MTT formazan was subjected to ELISA reader. Was used to measure the absorbance at 562-595 nm (the above method was based on the method disclosed in Med. Immunol, 12, 411 (1986)). The results were averaged in 10 wells, and the mitogen activity ratio (SI) was calculated based on the following equation.

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

  S. of each fraction I. The values are shown in Table 3.

各フラクションの内、特に活性の高かったフラクション７について再度ＨＰＬＣによりその溶出位置を測定し、以下に示した合成ペプチドと比較した結果、このフラクションの溶出時間はウシＬＦ１−１８と一致した。またこのフラクションのアミノ酸配列を分析したところウシＬＦ１−１８の配列を有することが確認できた。

The elution position of fraction 7, which had particularly high activity, among the fractions was measured again by HPLC, and compared with the synthetic peptide shown below. As a result, the elution time of this fraction coincided with that of bovine LF1-18. When the amino acid sequence of this fraction was analyzed, it was confirmed that it had the sequence of bovine LF1-18.

(Chemical synthesis of active peptide)
The peptide identified in Example 1 and the peptide containing the amino acid sequence thereof were synthesized. In this example, a synthesis example of bovine LF1-18 and bovine LF1-51 was shown. The synthesis of other peptides described in this specification was also performed according to this example.

(1) Synthesis of bovine LF1-18 A 9-fluorenylmethyloxycarbonyl (Fmoc) group was used as a protecting group at the amino terminus using a peptide synthesizer 431A (ABI) using parahydroxymethylphenoxymethyl polystyrene (HMP) resin. A linear protected peptide was synthesized on a 0.25 mmol scale. 1455 mg of the obtained HMP resin-bound protective peptide was simultaneously separated from the HMP resin with trifluoroacetic acid (TFA) in the presence of phenol, 1,2-ethanedithiol and thioanisole, and the protective group was removed. After removing TFA by concentration under reduced pressure, the crude peptide was crystallized with ethyl ether, dissolved in 5% acetic acid, and freeze-dried. 500 mg of the obtained crude crude peptide was purified by HPLC [column: octadecyl 4PW (21.5 × 150 mm, Tosoh Corporation), elution: gradient elution with water-acetonitrile containing 0.1% TFA] 365 mg of the purified peptide was obtained. The purity of the obtained purified peptide was 98% as a result of analysis by HPLC.

(2) Bovine LF (1-51), [ ¹⁹ Cys (Acm), ³⁶ Cys (Acm)] Bovine LF (1-51) and [ ⁹ CysSH, ¹⁹ Cys (Acm), ³⁶ Cys (Acm), ⁴⁵ CysSH Synthesis of bovine LF (1-51) A 9-fluorenylmethyloxycarbonyl (Fmoc) group was protected with an amino-terminal protecting group using a peptide synthesizer 431A (ABI) using a parahydroxymethylphenoxymethyl polystyrene (HMP) resin. Then, the SH group of ²⁰ Cys and ³⁷ Cys was protected with an acetamidomethyl (Acm) group to synthesize a linear protected peptide on a 0.25 mmol scale. In the presence of phenol, 1,2-ethanedithiol, and thioanisole, 2337 mg of the obtained HMP resin-bound protective peptide was simultaneously cleaved from the HMP resin with trifluoroacetic acid (TFA) and the protective group was removed. After removing TFA by concentration under reduced pressure, the crude peptide was crystallized with ethyl ether, 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 Corporation), elution: gradient elution with water-acetonitrile containing 0.1% TFA) and linear purification. The peptide [ ⁹ CysSH, ¹⁹ Cys (Acm), ³⁶ Cys (Acm), ⁴⁵ CysSH] bovine LF (1-51) was obtained. The purity of the obtained purified peptide was 82% as a result of analysis by HPLC. This peptide was subjected to air-oxidation in the presence of potassium ferricyanide to form an SS bond at ⁹ Cys and ⁴⁵ Cys, and further purified by HPLC to give 90% pure [ ¹⁹ Cys (Acm), ³⁶ Cys (Acm)]. 390 mg of bovine LF (1-51) was obtained. The peptide was further treated with iodine to remove the Acm group and simultaneously form an SS bond, followed by purification by HPLC to obtain 576 mg of bovine LF (1-51). As a result of analysis by HPLC, the purity of the peptide was 88%.

(Measurement of lymphocyte blastogenesis activity of synthetic peptide)
The lymphocyte blastogenesis effect of the synthetic peptide obtained in Example 2 was measured.
The measurement was performed according to the method described in Example 1. The measurement results are shown in Table 4 below.
Each synthetic peptide has strong S. cerevisiae. I. Had activity.

(Measurement of antitumor effect of synthetic peptide)
In order to confirm the immunostimulatory effect of the synthetic peptide obtained in Example 2, an experiment was performed using the growth inhibitory effect of ascites tumor as an index, and changes in immune markers were observed.
BALB / c male mice (10 animals per group) to ¹⁰ 5 to 10 ⁶ ascites tumors cells (Meth A cells) were transplanted intraperitoneally. From the day of tumor implantation, the synthetic peptide was administered intraperitoneally five times every other day. In addition, muramyl dipeptide (MDP) 3 mg / kg was similarly administered as a positive control, and carrageenan was similarly administered as a negative control.
Table 5 shows the survival rates of the mice 20 days after the tumor implantation. Regarding the sample, a tumor growth inhibitory effect was observed at a dose of 0.005 g / kg or more, and no death of mice was observed at 0.05 g / kg of body weight in the synthetic peptide administration group. In addition, it was confirmed that NK cells of the peptide-administered animal were activated. NK cell activation was measured by using mouse spleen cells as effector cells, using ⁵¹ Cr-labeled tumor cells (YAC-1) as target cells, mixing at a ratio of 100: 1, and releasing ⁵¹ Cr. The NK activity value was measured from. The NK activity value of the animal to which the substance of the present invention was administered was significantly higher than that of the control.

(Protective effect of synthetic peptide on cytomegalovirus infection)
Various synthetic peptides of bovine LF were prepared, and the protective effect of this peptide on cytomegalovirus infection was confirmed.
As experimental animals, SPF-BALB / cA male, 4 weeks old, were used as 5 animals per group. The mouse was infected with a mouse cytomegalovirus (MCMV) Smith strain that had passed the mouse salivary gland 10 times or more, and the survival rate was determined to determine the survival rate.
Virus infection was performed by intraperitoneal inoculation at a concentration of 1 × 10 ⁶ PFU / mouse. Simultaneously with infection, PBS solutions of each peptide were intraperitoneally injected at 0.25, 0.125, 0.025, and 0.005 g / mouse. Kg (body weight) was administered. Surviving animals were dissected, their spleens were excised, and NK cell activity of spleen cells was measured.
The results are shown in Table 6.

  It was confirmed that the peptide of the present invention has activity on the N-terminal side of bovine LF. It was also confirmed that this activity had no effect even if three residues from the N-terminal were deleted. Furthermore, all animals to which the peptide of the present invention was administered had increased NK cell activity. This activity was also consistent with the cytomegalovirus protective activity.

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

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

Claims (6)

A peptide comprising the amino acid sequence represented by the following amino acid sequence [I].
Ala-Pro-Arg-Lys-Asn-Val-Arg-Trp-Cys-Thr-Ile-Ser-Gln-Pro-Asp-Ser-Phe-Lys (I)   2. The peptide according to claim 1, wherein the N-terminal amino acid residue of the amino acid sequence [I] is deleted by 1 to 3 residues. A peptide comprising the amino acid sequence represented by the following amino acid sequence [II]:

However, the SS bond may be a reduced SH group.   An immunostimulator comprising the peptide according to claim 1 or a pharmacologically acceptable salt thereof as an active ingredient.   A cytomegalovirus infection protective agent comprising the peptide according to claim 1 or a pharmacologically acceptable salt thereof as an active ingredient. A method for producing a peptide, which comprises subjecting bovine lactoferrin to enzymatic degradation with a protease and collecting the peptide according to any one of claims 1 to 3 from the enzymatic degradation product.

Images