JP5982676B2 - Biotinylated peptide compound having anti-PAF activity - Google Patents

Description

  The present invention binds to platelet activating factor (PAF) and lyso-PAF in vivo, inhibits PAF-operating receptor binding of PAF, and prevents and treats various inflammatory diseases caused by PAF receptor binding. The present invention relates to a biotinylated peptide compound useful for the above, and an anti-inflammatory agent containing this compound.

  Platelet activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, platelet-activating factor: PAF) is produced from various cell types such as platelets, neutrophils, and eosinophils. It is a lipid mediator that elicits a strong inflammatory response by binding to a specific G protein receptor. The pathological conditions caused by the overproduction of PAF include rheumatoid arthritis (Non-patent document 1), bronchial asthma (Non-patent document 2), dermatitis (Non-patent document 3), nephritis (Non-patent document 4), and periodontitis. There are many inflammatory diseases such as (Non-Patent Document 5). Accordingly, attempts have been made to diagnose these diseases and evaluate their therapeutic effects by measuring the PAF concentration (for example, Patent Document 1). On the other hand, inactivation of PAF by suppressing overproduction of PAF or inhibiting binding to a receptor is extremely useful for the prevention, treatment, improvement, prevention of deterioration, and prevention of recurrence of these pathological conditions. However, anti-inflammatory agents resulting from PAF activity targeting PAF molecules or PAF receptors have not yet been put into practical use.

  As a substance (PAF receptor antagonist) that antagonistically inhibits the binding between PAF and its receptor, for example, WEB-2086 (thieno-triazolodiazepine) (Non-patent document 6, Patent document 2) and CV-3988 (patent) Document 2) is known, and these PAF receptor antagonists are in the clinical trial stage as antiallergic agents and antiasthma agents. However, since these PAF receptor antagonists are ligands for the PAF receptor and do not bind directly to the PAF molecule, they are effective when administered locally but insufficiently when administered systemically. Furthermore, lysophospholipid / oxidized phospholipid receptors other than the PAF receptor also act as ligands, and thus there is a problem that unexpected side effects may occur.

  On the other hand, the oxidized low density lipoprotein (LDL) involved in the formation of early lesions of arteriosclerosis contains a lot of PAF-like lipids, but the present inventors specifically bind to the oxidized LDL. We have developed multiple types of peptides, a method for detecting oxidized LDL using these peptides, and a diagnostic agent for arteriosclerosis (Patent Document 3).

Special Table 2004-527770 Japanese Patent Laid-Open No. 06-305981 Japanese Patent No. 4017326

Vergne, P. et al., Mediators Inflamm., 6: 241-242, 1997 Tsukioka et al., Allergy, 42: 167-171, 1993 Czarnetzki, B., Clin. Exp. Immunol., 54: 486-492, 1983 Denizot, Y. et al., Nephrol. Dial. Transplant., 15: 1344-1347, 2000 Garito, M.L., J. Dent. Res., 74: 1048-1056, 1995 Casals-Stenzel, J., The Journal of Pharmacology, 241: 974-981, 1987

  The present invention prevents, recovers, ameliorates or worsens various inflammatory diseases caused by PAF agonist receptor binding by binding PAF and lyso-PAF and specifically inhibiting PAF receptor binding. The objective is to provide an effective and safe means for preventing recurrence.

In order to solve the above-mentioned problems, the present inventors have earnestly studied the oxidized LDL-recognizing peptide described in Patent Document 3, and as a result, have obtained the following knowledge.
(A) The oxidized LDL-recognizing peptide exhibits binding properties depending on the dose of PAF.
(B) The peptide strongly suppresses the cytotoxicity-inducing activity by PAF in cultured vascular endothelial cells.
(C) The peptide is bound depending on the dose of lysoPAF (1-O-alkyl-sn-glycero-3-phosphocholine), which is a precursor and metabolite of PAF.
(D) A PAF-induced inflammation suppression test (in vivo test) of the above peptide was performed using rats, but the anti-inflammatory effect in vivo was insufficient, and satisfactory results were not obtained.

  Based on these findings, the present inventors have examined peptide modifications that allow the peptide to exert an anti-inflammatory action even in vivo. As a result, the peptide is excellent in vivo by the addition of biotin (biotinylation modification). The present invention was completed by confirming that it had the same effect.

That is, this invention provides the following as what solves the said subject.
(1) A peptide having an amino acid sequence of Tyr-Lys-Asp-Gly (SEQ ID NO: 1), Tyr-Lys-Asp-Lys-Glu (SEQ ID NO: 11) or Tyr-Lys-Gly-Lys (SEQ ID NO: 12) Biotinylated peptide compound that has biotin added and binds to platelet activating factor (PAF) and lyso-PAF in vivo to inhibit PAF-activated receptor binding of PAF.
(2) The peptide having the amino acid sequence of Tyr-Lys-Asp-Gly (SEQ ID NO: 1) has at least one amino acid residue before and after the sequence of positions 12-15 in SEQ ID NO: 2 (Tyr-Lys-Asp-Gly). The peptide compound of claim 1 comprising:
(3) An anti-inflammatory agent comprising the biotinylated peptide compound according to claim 1 or 2 as an active ingredient.

  “Peptide” means a molecule composed of a plurality of amino acid residues linked to each other by amide bonds (peptide bonds) or unnatural residue linkages. As described above, the peptide of the present invention is a molecule consisting of at least 4 amino acid residues (SEQ ID NO: 1) and at most 29 amino acid residues (SEQ ID NO: 2).

  “Biotinylated” refers to a state in which biotin is bound to either the N-terminus or C-terminus of the peptide.

  Other terms and concepts in the present invention are defined in detail in the description of the embodiments of the invention and the examples. Various techniques used for carrying out the invention of the present application can be easily and reliably implemented by those skilled in the art based on well-known literatures and the like, except for the technique that clearly indicates the source. For example, the preparation of the drug (pharmaceutical composition) of the present invention is described in Remington's Pharmaceutical Sciences, 18th Edition, ed. A. Gennaro, Mack Publishing Co., Easton, PA, 1990, etc. and Maniatis, in Molecular Cloning-A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 1989; Ausubel, FM et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, 1995, etc. ing. Furthermore, the terms in the present invention are basically based on the IUPAC-IUB Commission on Biochemical Nomenclature, or based on the meanings of terms conventionally used in the field.

  According to the inventions (1) and (2), a biotinylated peptide compound that binds to PAF and lyso-PAF in vivo and specifically inhibits PAF-activated receptor binding of PAF is provided.

  In addition, according to the invention (3), an effective and safe drug for preventing, recovering, ameliorating, or preventing aggravation / recurrence of various inflammatory diseases caused by binding of PAF to a PAF agonist receptor is provided.

The sequences of N-terminal biotinylated P21 and N-terminal biotinylated P4 and the structure of biotin used in the examples are shown. The binding of N-terminal biotinylated P21 using time-resolved fluorescence to PAF (A) or lyso-PAF (B) was demonstrated. The horizontal axis represents the concentration (μM) of each lipid, and the vertical axis represents the change in fluorescence intensity due to the binding of each lipid and peptide as an average value ± SD (n = 3). FIG. 5 shows the results of testing the anti-PAF effect by subcutaneous administration of N-terminal biotinylated P21 in Example 2, and the paw edema volume after each treatment is shown as an average value ± SD (ml). FIG. 5 shows the results of testing the anti-PAF effect by intravenous administration or subcutaneous administration of N-terminal biotinylated P21 and P4 in Example 2, and the foot edema volume after each treatment is shown as mean value ± SD (ml). It is the result of examining the correlation between the dose of peptide or N-terminal biotinylated P21 and P4 and anti-PAF activity in Example 2, the horizontal axis represents the dose (nmol) of each peptide per individual, and the vertical axis Changes in paw edema volume after each treatment are shown as mean ± SD (ml). This is the basis for calculating the 50% inhibition amount (ID ₅₀ ) of PAF activity of N-terminal biotinylated peptide compounds. The horizontal axis shows the dose (nmol) of N-terminal biotinylated P21 and N-terminal biotinylated P4 in log logarithm respectively. On the vertical axis, the change in foot edema volume (%) at each dose when the change in foot edema volume in each control group was taken as 100% was shown as the mean ± SD (ml). FIG. 3 shows the results of comparing the anti-PAF action of N-terminal biotinylated P21 and a PAF receptor antagonist in Example 3, and the foot edema volume after each treatment is shown as an average value ± SD (ml). In Example 4, it is the result of having tested the anti-PAF effect by intravenous administration of N terminal biotinylated PET3 which consists of a YKDKE sequence peptide, and N terminal biotinylated PCD36 which consists of a YKGK sequence peptide. Values are shown as ± SD (ml).

One of the peptides constituting the biotinylated peptide compound of the present invention is basically an oxidized LDL recognition peptide disclosed in Patent Document 3, and at least the amino acid sequence of SEQ ID NO: 1 (Tyr-Lys-Asp-Gly: Hereinafter, it may be described as “YKDG sequence”). Patent Document 3 describes the following as peptides that specifically recognize and bind to oxidized LDL containing PAF-like lipids (underlined portion is YKDG sequence).
P29: IKNASLSWGKW YKDG DKDAEITSEDVQQK (SEQ ID NO: 2)
P24: LSWGKW YKDG DKDAEITSEDVQQK (SEQ ID NO: 3)
P21: IKNASLSWGKW YKDG DKDAEI (SEQ ID NO: 4)
P16: LSWGKW YKDG DKDAEI (SEQ ID NO: 5)
P11: WGKW YKDG DKD (SEQ ID NO: 6)
P9: WGKW YKDG D (SEQ ID NO: 7)
P6: W YKDG D (SEQ ID NO: 8)
P4: YKDG (SEQ ID NO: 1)
On the other hand, according to Patent Document 3, the following peptides (P15, P7) do not have recognition / binding ability for oxidized LDL.
P15: IKNASLSWGKW YKDG (SEQ ID NO: 9)
P7: YKDG DKD (SEQ ID NO: 10)
That is, these peptides P15 and P7 are peptides having other amino acid residues only on either the N-terminal side or the C-terminal side of the YKDG sequence, whereas peptides P29 having the ability to recognize and bind to oxidized LDL, P24, P16, P11, P9, P6, and P4 are peptides containing at least one amino acid residue before and after the YKDG sequence alone or the YKDG sequence in SEQ ID NO: 2. Therefore, the peptide used in the present invention is the peptide consisting of the YKDG sequence at the shortest (P4) or the peptide consisting of the longest 29 amino acid residues including at least one amino acid residue before and after the YKDG sequence in SEQ ID NO: 2 (P29). It is.

  Another peptide constituting the biotinylated peptide compound of the present invention includes the amino acid sequence of SEQ ID NO: 11 (Tyr-Lys-Asp-Lys-Glu: hereinafter sometimes referred to as “YKDKE sequence”) and the amino acid of SEQ ID NO: 12. A peptide having a sequence (Tyr-Lys-Gly-Lys: hereinafter sometimes referred to as “YKGK sequence”). That is, the peptide having the YKDG sequence is a partial peptide of Asp-hemolysin, which is a protein toxin of Aspergillus fumigatus, whereas the YKDKE sequence and the YKGK sequence are partial peptides of a human protein. As a result of analyzing the amino acid sequences of many human physiologically active proteins, the present inventors found sequences similar to the partial peptide YKDG of Asp-hemolysin (the YKDKE sequence of endothelin-3 and the YKGK sequence of scavenger receptor CD36), It was confirmed that these biotinylated peptide compounds bind to PAF and lyso-PAF in vivo and inhibit PAF-binding of PAF. Hereinafter, the YKDKE sequence peptide of endothelin-3 (ET3) may be referred to as “PET3”, and the YKGK sequence peptide of CD36 may be referred to as “PCD36”.

  PET3 and PCD36 may be peptides having a total length of about 29 amino acid sequences with at least one amino acid residue added before and after the YKDKE sequence and YKGK sequence, respectively. That is, the entire amino acid sequence of human endothelin-3 is known (for example, GenBank: AAP35748), and the YKDKE sequence is located at positions 102 to 106 of the entire amino acid sequence. In the case of human CD36, the YKGK sequence is present at positions 230 to 233 of the entire known amino acid sequence (for example, GenBank: EAW7700). According to these known amino acid sequences, amino acid residues can be added before and after PET3 peptide and CD36 peptide.

  These peptides can be prepared as recombinant peptides using gene recombination techniques, but preferably chemically synthesized using a commercially available peptide synthesizer (for example, Applied Biosystems A431 or A433). Can do. The synthesis is carried out according to a known method, for example, from the C-terminus of the peptide using an amino acid derivative. As the amino acid derivative, it is preferable to use an amino acid derivative in which an amino terminal group required for bonding is derivatized with a fluorenylmethyloxycarbonyl (Fmoc) group. The reactive side group of the amino acid used is a protecting group that can be easily cleaved after completion of peptide synthesis, such as triphenylmethyl (Trt), t-butyl ether (tBu), t-butyl ester (0 tBu), t-butoxy. It contains carbonyl (Boc) or 2,2,5,7,8-penta-methylchroman-6-sulfonyl (Pmc). In addition to 20 natural amino acids, these peptides include non-natural amino acids and amino acid analogs (for example, Hunt, The Non-Protein Amino Acids: Chemistry and Biochemistry of the Amino Acids, Barrett, Chapman and Hall, 1985). May be included.

  The synthesized peptide is biotinyl by a known method using a biotin-introducing reagent such as NHS-biotin and NHS-SS-biotin (sulfosuccinimidyl 2- (biotinamido) ethyl-1,3-dithiopropinate). Turn into. The biotin addition may be at the N-terminus or C-terminus of the peptide.

  The anti-inflammatory agent of the present invention contains the biotinylated peptide compound as an active ingredient. Diseases targeted by this drug are various inflammatory diseases in which PAF is involved as a causative agent such as rheumatoid arthritis, bronchial asthma, dermatitis, nephritis, periodontitis, etc. that develops due to receptor binding of PAF. The agent of the present invention effectively acts to prevent, alleviate, recover, or separately protect against these diseases.

  This anti-inflammatory agent can be formulated with the aforementioned biotinylated peptide compound itself, but is usually mixed with one or more pharmacologically acceptable carriers, and is used in the technical field of pharmaceutics. It is desirable to administer it as a pharmaceutical formulation produced by any method well known in the art.

  It is desirable to use the most effective route for treatment, and oral administration or parenteral administration such as buccal, respiratory tract, rectal, subcutaneous, intramuscular and intravenous is desirable. Can be given intravenously.

  Suitable formulations for oral administration include emulsions, syrups, capsules, tablets, powders, granules and the like. Liquid preparations such as emulsions and syrups include sugars such as water, sucrose, sorbitol and fructose, glycols such as polyethylene glycol and propylene glycol, oils such as sesame oil, olive oil and soybean oil, p-hydroxybenzoic acid Preservatives such as esters, and flavors such as strawberry flavor and peppermint can be used as additives. For capsules, tablets, powders, granules, etc., excipients such as lactose, glucose, sucrose, mannitol, disintegrants such as starch and sodium alginate, lubricants such as magnesium stearate and talc, polyvinyl alcohol, hydroxy A binder such as propylcellulose and gelatin, a surfactant such as fatty acid ester, and a plasticizer such as glycerin can be used as additives.

  Formulations suitable for parenteral administration include injections, suppositories, sprays and the like. The injection is prepared using a carrier made of a salt solution, a glucose solution, or a mixture of both. Suppositories are prepared using a carrier such as cacao butter, hydrogenated fat or carboxylic acid. The spray is prepared using a carrier that does not irritate normal tissues and organs such as the recipient's oral cavity and airway mucosa, and that facilitates absorption by dispersing the active ingredient as fine particles.

  Specific examples of the carrier include lactose and glycerin. Furthermore, preparations such as aerosols and dry powders are possible depending on the nature of the carrier used. In these parenteral preparations, the components exemplified as additives for oral preparations can also be added.

  The dose or frequency of administration varies depending on the intended therapeutic effect, administration method, treatment period, age, body weight, etc., but the effective dosage is, for example, about 1 μg to about 10 mg / kg body weight, preferably about 10 μg per day. ~ 5mg / kg body weight. The total daily dose is administered in a single dose or divided doses (for example, about 2 to 6 times).

  EXAMPLES Hereinafter, although an Example is shown and this invention is demonstrated further in detail and concretely, this invention is not limited to the following examples.

The materials used in the following examples are as follows.
N-terminal biotinylated peptide compounds:
A peptide compound (hereinafter referred to as N-terminal biotinylated P21, N-terminal biotinylated P4) in which the N-terminus of each of P21 peptide (SEQ ID NO: 4) and P4 peptide (SEQ ID NO: 1) is biotinylated is synthesized with a purity of 95% or more. Purified (confirmed by high performance liquid chromatography and mass spectrometry). FIG. 1 shows the sequences of N-terminal biotinylated P21 and N-terminal biotinylated P4 and the structure of biotin.

Further, peptide compounds obtained by biotinylating the N-terminus of PET3 (SEQ ID NO: 11) and PCD36 (SEQ ID NO: 12) (hereinafter, N-terminal biotinylated PET3 and N-terminal biotinylated CD36) were synthesized and purified in the same manner.
PAF and lyso PAF:
PAF uses 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (PAF C-16, Enzo Life Sciences (BIOMOL), L-100), and lyso PAF uses 1-O-hexadecyl-sn. -Glycero-3-phosphocholine (C-16, Cayman Chemical Company, 60906) was used.

Evaluation of N-terminal biotinylated P21 binding to PAF and lyso-PAF
Binding analysis of N-terminal biotinylated P21 to PAF and lyso-PAF was performed using time-resolved fluorescence. Specifically, 100 μl of each concentration of PAF or lysoPAF (0-30 μM each) was added to a 96-well microplate and allowed to stand overnight at 4 ° C., and then each well was washed with PBS at pH 7.4. after washing times, and blocked by PAF, SuperBlock each well of lyso PAF-immobilized plate ^R blocking Buffer (thermo Scientific). Further, each well was washed 3 times with PBS, 100 μl each of N-terminal biotinylated P21 (1 μM) was added, reacted at 37 ° C. for 30 minutes, and then europium-labeled streptavidin (Parkin) diluted 1,000 times with PBS. Elmer) 100 μl each was added and allowed to react for 30 minutes at room temperature. After washing 8 times with PBS, 100 μl of DELFIA enhancement solution (PerkinElmer) was added to each well, and time-resolved fluorescence of europium was measured using a Wallac ARVOsx 1420 Multilabel counter (PerkinElmer) (excitation wavelength 340). nm, measurement wavelength 615 nm).

  The results are as shown in FIG. 2, and the N-terminal biotinylated peptide P21 showed binding depending on the dose of PAF and lyso-PAF.

Effects of N-terminal biotinylated peptide compounds on PAF activity in vivo
In order to examine the effect of N-terminal biotinylated peptide compounds on PAF bioactivity in vivo, we used the rat paw edema model (Henriques et al., Br. J. Pharmacol., 106: 579-582, 1992) A PAF-induced inflammation suppression test was conducted.
1) Induction and quantification of foot edema
5-8 week old male Wistar rats (Claire Japan) weighing 150-200 grams were anesthetized by inhalation with diethyl ether. A 10 mM PAF solution dissolved in ethanol is diluted 500-fold with a solution of 150 mM sodium chloride, 10 mM tris (hydroxymethyl) aminomethane (hereinafter, Tris, pH 7.5) and 0.25% bovine serum albumin, and ultrasonicated for 5 minutes. A PAF solution was prepared by treatment. 50 μl of PAF solution (final concentration 20 μM, 1 nmol) was subcutaneously administered between the rat hind footpads, and the foot edema was measured and evaluated 1 hour after the maximum edema due to PAF.

Edema was quantified by measuring rat paw volume immediately before administration of PAF and 1 hour after administration. For edema, the water volume that increases when the foot is immersed in water was measured in milliliters (ml). In addition, an oil pen was used to mark the boundary between the hairline of the hind legs and the heel so that the difference could be accurately compared by immersing the legs at each time point.
2) Administration routes of various peptides and N-terminal biotinylated peptide compounds Various peptides or N-terminal biotinylated peptide compounds are injected locally into the <1> rat hind footpad or <2> into the tail vein, respectively. Administered. Specifically, in <1>, 100 μl of a peptide solution prepared at various concentrations with PBS (pH 7.4) was subcutaneously administered between the rat's hind foot pads, and in <2>, PBS was used in the same manner as above from the tail vein. 100 μl of peptide solution prepared at each concentration was intravenously administered. In both <1> and <2>, the same amount of PBS was administered from each route in the control group.
3) Anti-PAF effect by subcutaneous administration of N-terminal biotinylated P21 Rats (n = 3 per group) were subcutaneously injected with 100 μl N-terminal biotinylated P21 (10 nmol) or carrier dissolved in PBS between the hind footpads 15 minutes later, 50 μl of PAF (1 nmol) or carrier solvent (150 mM sodium chloride, 10 mM Tris (pH 7.5) and 0.25% bovine serum albumin) was administered to the same site, and 1 hour later. Foot edema was measured and evaluated.

The results are as shown in FIG. 3. Each foot edema volume is 0.11 ± 0.035 ml with vehicle alone, 0.52 ± 0.14 ml with PAF alone, 0.12 ± 0.033 ml with N-terminal biotinylated P21 alone, and N-terminal biotinyl. The combined use of P21 and PAF resulted in 0.15 ± 0.12 ml, and N-terminal biotinylated P21 did not develop edema and markedly suppressed foot edema due to PAF.
4) Comparison of anti-PAF effect by intravenous and subcutaneous administration of N-terminal biotinylated peptide compound Rats (n = 3 per group) were treated with 100 μl N-terminal biotinylated P21 (10 nmol), N Terminal biotinylated P4 (10 nmol) or PBS was administered intravenously or subcutaneously, 15 minutes later, 50 μl of PAF solution (1 nmol) was subcutaneously administered to the same site, and foot edema was measured 1 hour later. evaluated.

The results are as shown in FIG. Each foot edema volume is 0.083 ± 0.047 ml with vehicle alone, 0.52 ± 0.14 ml with PAF alone, 0.16 ± 0.073 ml, 0.15 ± 0.12 ml by intravenous or subcutaneous administration of N-terminal biotinylated P21, respectively. Intravenous and subcutaneous administration of N-terminal biotinylated P4 resulted in 0.15 ± 0.038 ml and 0.13 ± 0.025 ml, respectively, and both biotinylated peptide compounds significantly inhibited paedema caused by PAF by intravenous administration and subcutaneous administration.
5) Correlation between dose of peptide or N-terminal biotinylated peptide compound and anti-PAF activity Dose of peptide (P21, P4) and various N-terminal biotinylated peptide compounds (N-terminal biotinylated P21, N-terminal biotinylated P4) And the correlation between anti-PAF activity was investigated. Specifically, rats (n = 3 per group) were intravenously administered with 100 μl of each molar amount of peptide (0.625, 1.25, 5, 10 or 20 nmol) or PBS dissolved in PBS, and 15 After 50 minutes, 50 μl of PAF (1 nmol) or carrier solvent was subcutaneously administered between the hind footpads, and changes in foot edema after 1 hour were measured and evaluated.

The results are as shown in FIG. 6. Although suppression of PAF-induced edema was observed for both P21 and P4, the effect was insufficient (up to about 50% suppression). In contrast, various biotinylated peptide compounds showed anti-PAF activity depending on the dose, and a stable inhibitory effect was observed.
6) Calculation of 50% inhibitory dose (ID ₅₀ ) of N-terminal biotinylated peptide compound
From the result of 5), the ID ₅₀ value of the N-terminal biotinylated peptide compound was calculated. The results are as shown in FIG. 6, and the ID ₅₀ values of N-terminal biotinylated P21 and N-terminal biotinylated P4 were calculated as 1.62 nmol and 2.00 nmol, respectively.

Comparison of anti-PAF activity of N-terminal biotinylated peptide compounds and PAF receptor antagonists
We compared the effects of N-terminal biotinylated peptide compounds and known PAF receptor antagonists on the anti-PAF activity in vivo. Each group of rats (n = 3) was pretreated with a known PAF receptor antagonist (CV-3988: Wako Pure Chemical 037-14381, Alprazolam: Wako Pure Chemical 016-17171) or N-terminal biotinylated P21 did. Specifically, for each PAF receptor antagonist, 2 mg each (CV-3988: 3.4 μmol, alprazolam: 6.5 μmol) was dissolved in 200 μl of ethanol and administered intraperitoneally. Was subcutaneously administered between the hind foot pads. On the other hand, for N-terminal biotinylated P21, 20 nmol (dissolved in 100 μl PBS) was intravenously administered per animal, and PAF was subcutaneously administered 15 minutes later. On the other hand, only PAF was administered to the control group of rats. In each treatment, foot edema was measured and evaluated 1 hour after PAF administration.

  The results are as shown in FIG. 7, and each foot edema volume is 0.083 ± 0.047 ml for vehicle alone, 0.52 ± 0.14 ml for PAF alone, 0.17 ± 0.029 ml for N-terminal biotinylated P21 treatment, CV-3988 treatment was 0.22 ± 0.076 ml, alprazolam treatment was 0.18 ± 0.077 ml, and N-terminal biotinylated P21 was a significant anti-antibody even at very low doses of 150-300 times each PAF receptor antagonist. PAF activity was shown.

Effect of N-terminal biotinylated PET3 and PCD36 on PAF activity Using the same rat paw edema model as in Example 2, N-terminal biotinylated PET3 and PCD36 were tested for inhibition of PAF-induced inflammation.

  Rats (n = 3-4 per group) were intravenously administered with 100 μl N-terminal biotinylated PET3 (20 nmol), N-terminal biotinylated PCD36 (20 nmol) or PBS alone in PBS via the tail vein. 15 minutes later, 50 μl of PAF (1 nmol) or carrier solvent (150 mM sodium chloride, 10 mM Tris (pH 7.5) and 0.25% bovine serum albumin) was subcutaneously administered between the hind footpads, and 1 hour later. Foot edema was measured and evaluated.

  The results are as shown in FIG. 8, and each foot edema volume is 0.083 ± 0.047 ml with vehicle alone, 0.35 ± 0.11 ml with PAF alone, 0.063 ± 0.025 ml with N-terminal biotinylated PET3 and PAF, The combined use of N-terminal biotinylated PCD36 and PAF resulted in 0.16 ± 0.024 ml, and all N-terminal biotinylated peptide compounds significantly suppressed paf edema due to PAF.

  Provided is a means for effective and safe prevention, recovery, improvement, prevention of deterioration, and prevention of recurrence against various inflammatory diseases caused by PAF agonist receptor binding of PAF.

Claims (1)

An anti-inflammatory agent comprising, as an active ingredient, a biotinylated peptide compound in which biotin is added to a peptide consisting of the amino acid sequence of SEQ ID NO: 1, 4 , 11 or 12.

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