WO1994020531A1 - Physiologically active peptide - Google Patents
Physiologically active peptide Download PDFInfo
- Publication number
- WO1994020531A1 WO1994020531A1 PCT/JP1994/000345 JP9400345W WO9420531A1 WO 1994020531 A1 WO1994020531 A1 WO 1994020531A1 JP 9400345 W JP9400345 W JP 9400345W WO 9420531 A1 WO9420531 A1 WO 9420531A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- peptide
- cys
- cys residue
- lys
- thr
- Prior art date
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/18—Carboxylic ester hydrolases (3.1.1)
- C12N9/20—Triglyceride splitting, e.g. by means of lipase
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- the present invention relates to a bioactive peptide. Furthermore the present invention in detail relates to peptidyl-de having the activity of inhibiting the activity or inflammatory sites derived phospholipase A 2, which inhibits binding of Paris down into inflammatory sites derived phospholipase A 2 Prefecture.
- Phospholipase A 2 is an enzyme that hydrolyzes the ester bond at the 2-position of cell membrane phospholipids, and it is known that there are two types of membrane-bound PLA 2 and zymogenic PLA 2 .
- membrane-bound PLA 2 releases arachidonic acid (AA) from phospholipids.
- AA arachidonic acid
- PLA 2 breaks down phospholipids, breaks cell membranes, and produces lysolecithin, which has strong cytotoxicity. Recently, the importance of eye inflammation due to the action of cell membrane damage, its severity and multiple organ damage has been emphasized.
- PLA 2 of membrane-bound in these diseases are involved. Therefore, by inhibiting PLA 2, since it is possible to suppress the release of AA is a precursor of various physiologically active substances, considered to be useful for the prevention and / or treatment of such a variety of inflammatory diseases and Arerugi disease It is. In addition, since it can suppress cell membrane damage, it is considered to be useful for prevention and / or treatment of otitis, its severity and multiple organ damage. Also, by inhibiting the activity of PLA 2 can be suppressed prostaglandin ⁇ beauty leuco preparative Lin both biosynthesis, less side effects compared to steroid-based drugs and non-steroid drugs, strong anti-inflammatory work And anti-allergic effects can be expected.
- Hevalin is D-N-acetylglucosamine and D-glucuronic acid
- type II phospholipase A 2 Hosuhoriba inflammation local human inflammatory diseases and inflammatory animal models - Ze A 2 (hereinafter referred to as type II phospholipase A 2) is purified, its sex shape revealed (Efuji Ichiro Keizo Inoue; Biochemistry, 1330-1344 (1992)). It is thought that this enzyme has a function to promote the inflammatory response, and therefore, drugs that inhibit the activity of this enzyme are expected to show anti-inflammatory effects. Indeed, the type II phospholipase A 2 has Paris emission and strong affinity, are secreted into the extracellular with the inflammation. Furthermore, in order for its physiological activity to work, it needs to be taken up into other tissues or cells as well as FGF. At this time, it has been clarified that heparin or heparan sulfate plays an important role ( Keizo Inoue et al., Experimental Medicine, 1460 (1993)).
- the W093 / 01215 that contains the phospholipase A 2 inhibitor comprising five ⁇ Mi Bruno acid in the amino acid sequence of the phospholipase A 2.
- the Cys residue of the peptide to enhance the physiological activity of the peptide. Disclosure of the invention
- the present inventors have type II Hosuhoripa Ichize A 2, inter alia of the amino acid sequence of human type II phospholipase A 2, found parts position with Paris down binding capacity to, Paris down binding ability to this inhibiting the binding of Paris down to peptidase Dogahi preparative II phospholipase a 2 and having, inhibit the enzymatic activity of human type II phospholipase a 2 in the presence of Paris down to petit de stated child In addition, it was found that the above-mentioned activity was dramatically enhanced by making a predetermined modification to the Cys residue of the peptide. In view of such findings, the present inventors have made intensive studies and have reached the present invention.
- A--X-V 1 -Z-W 2 -Y 1 -V 2- Y 2 -B (I) (where A represents a peptide consisting of one to six amino acid residues. Represents a peptide consisting of 1 to 3 amino acid residues,, W 2 each independently represents Ser or Thr, X represents Lys or Arg, and V 1 and V 2 each represent Ser represents Arg, Thr, Tyr, Phe, or Lys, Y 1, Upsilon 2 is Table Phe, Tyr, or Trp independently, Z is represented Leu, Val, He, Pro, Phe, or Ala. )
- a ring structure is formed by a disulfide bond between the two Cys residues.
- Cys residue contained in A or B of one peptide is A multimeric ring structure is formed by a plurality of disulfide bonds formed with Cys residues contained in A or B of the peptide;
- FIG. 1 shows the results of reversed-phase HPLC of the peptide (2) in Example 1.2).
- FIG. 2 shows the results of reversed-phase HPLC of the peptide (11) in Example 3.2).
- FIG. 3 shows the results of reversed-phase HPLC of the peptide (16) in Example 3.3).
- FIG. 4 shows the results of reversed-phase HPLC of the peptide (13) in Example 4.2).
- FIG. 5 shows the results of reversed-phase HPLC of the peptide (21) in Example 5.
- FIG. 6 shows the results of reversed-phase HPLC of the peptide (22) in Example 8.
- FIG. 7 shows the results of reversed-phase HPLC of the peptide (17) in Example 6.
- FIG. 8 shows the results of reversed-phase HPLC of peptides (16) and (17) overlaid in Example 6.
- Figure 9 shows the binding inhibition measurement of constant results of heparinate emissions and human type II PLA 2 in Example 11.
- the symbol “ ⁇ ” represents the peptide (20) of the present invention
- the symbol “cross” represents the case using the control (Gly-Ala-Gly-Ala).
- At least one of A and B contains at least one Cys residue.
- A represents a peptide consisting of 1 to 6 amino acid residues.
- the amino acids constituting A are, in principle, L-amino acids, among which L-amino acids which are relatively hydrophilic, such as Lys, Arg, Gly, Asn, Ala, Gin, Ser, Preferable is a peptide consisting of 5 or 6 amino acid residues selected from the group consisting of Thr, Asp, Glu and Cys.
- a particularly preferred peptide comprising the above-described amino acid 5 residues or 6 residues is, for example, the amino acid 5 represented by Lys-Arg-Gly-Cys-Gly- (SEQ ID NO: 1). Residue peptides may be mentioned.
- A further includes Lys-Arg-Arg-Ala-Gly- (SEQ ID NO: 2), Lys-Ala- Thr-Asp-Ala-Asn- (SEQ ID NO: 3), Lys-Ala-Thr-Asp-Cys-Asn- (SEQ ID NO: 4) and the like.
- B represents a peptide consisting of one or three amino acid residues.
- the amino acid constituting B is an L-amino acid, and among them, an L-amino acid having a hydroxyl group or a mercapto group in its side chain, for example, an amino acid selected from the group consisting of Ser, Thr and Cys.
- Peptides consisting of two residues of amino acids or one residue of amino acids are preferred.
- a particularly preferred peptide consisting of the above-described two amino acid residues for example, a peptide represented by -Ser-Cys can be mentioned.
- B consists only of Ser.
- a and B can be used, provided that at least one of them contains a Cys residue.
- W 1 and W 2 each independently represent Ser or Thr, and in combination with A and B, A is Lys-Arg-Gly-Cys-Gly- (SEQ ID NO: 1).
- B is -Ser-Cys (referred to as a preferred embodiment)
- W 1 is Thr and W 2 is Ser.
- Lys or Arg is represented.
- X is preferably Lys.
- V 1 and V 2 each independently represent Ser, Arg, Thr, Tyr, Phe or Lys, and in the case where A and B are in the above preferred embodiment in combination with A and B Is preferably Phe or Lys, particularly V 1 Phe, and more preferably V 2 is Lys.
- Y 1 and Y 2 are each independently Phe, Represents Tyr or Trp, and in combination with A and B, when A and B are in the above-mentioned preferred embodiment, Phe or Tyr is preferable, and particularly, Y 1 is Tyr, and Y 2 is preferably Phe. .
- Z represents Leu, Val, He, Pro, Phe or Ala, and when A and B are combined with A and B in the above preferred embodiment, Leu is preferred.
- a as described above is Lys-Arg-Gly-Cys-Gly- (SEQ ID NO: 1), and B is -Ser-Cys, W 1 and W 2 are Ser or Thr, X is Lys or Arg, especially Lys, V 1 and V 2 are Phe or Lys, Y 1 and Y 2 are Phe Or Tyr and a bioactive peptide wherein Z is Leu.
- the peptides of W 1 -X-V 1 -Z-W 2 -Y 1 -V 2 -Y 2 in the peptide of the present invention include various W 1 , X, V 1 , Z, W 2 , Y 1 , V 2 and Y 2 may be any of various combinations.
- A is any of the amino acid sequences represented by SEQ ID NOs: 1 to 4, and W 1 to Y 2 are SEQ ID NOs: 5 to 7 And any combination of amino acids wherein B is Ser-Cys or Ser.
- B is Ser-Cys or Ser.
- Specific examples thereof include, for example, the following: Can be Lys-Arg-Gly-Cys-Gly-Thr-Lys-Phe-Leu-Ser-Tyr-Lys-Phe-Ser-Cys (SEQ ID NO: 8)
- Lys_Arg-Gly-Cys-Gly-Thr- Lys- Thr-Va Ser-Tyr- Thr-Phe-Ser-Cys (SEQ ID NO: 10)
- bioactive peptide of the present invention represented by the above formula (I) may have one or more amino acid residues substituted, deleted, and / or inserted.
- amino acid sequence of type II PLA 2 such snake venom (Garel J. van den Birgh et al , J. Cellular Biochemistry 39, 379 ⁇ 390 ( 1980)), similar to the bioactive peptide defined by the above formula (I), for example, Lys-Ala-Thr-Asp-Cys-Asn-Pro-Lys-Thr-Va Ser-Tyr-Thr-Tyr-Ser (SEQ ID NO: 25) can be mentioned.
- the amino acid residues constituting the bioactive peptide represented by the above formula (I) of the present invention are respectively defined as described above, and such amino acid residues are as defined above.
- L is not limited to an amino acid, bioactive to the onset bright object, such as activity to inhibit the binding of Paris down to human type II phospholipase a 2 Prefecture, human type II phospholipase a 2 Inhibition As long as it has activity, it may contain D-amino acid or yS, y-amino acid other than hy-amino acid.
- the functional groups on the side chains of these amino acid residues or the amino groups and / or carboxyl groups at the N-terminal and Z- or C-terminals of the peptide represented by the above formula (I) are known protective groups.
- esterification for example, methyl group, ester group, ethyl ester group, n-propyl ester group, isopropyl ester group, n-butyl ester group, sec-butyl ester group, tert-butyl ester group, benzyl Alkylation, for example, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, t tert-butyl, benzyl, trityl, carboxymethyl, aminocarboxymethyl, etc .; acylation, eg acetyl, benzoyl, benzyl By silyloxycarbonyl, 9-fluorenylmethyloxy
- At least one Cys residue contained in A and / or B is derivatized.
- At least one Cys residue is one of the peptides. Modified at the SH group.
- lower alkylbenzyl group for example, 4-methoxybenzyl group (Mob), acetamidemethyl group (—CH 2 NHC0CH 3 ), 2-pyridylsulfenyl group (SPy), 3-nitro-2- Examples thereof include a pyridylsulfonyl group (Npys), and a 4-methoxybenzyl group is particularly preferable.
- a linear multimer is formed by a disulfide bond formed between the residue and a Cys residue contained in A or B of another peptide.
- the multimer is preferably a dimer to a trimer, particularly preferably a dimer. As an embodiment of the dimer,
- a peptide containing a Cys residue only in A (a) and a peptide containing a Cys residue only in B (b) Is linked by a disulfide bond formed by
- a disulfide formed by each Cys residue is composed of a peptide (a) containing a Cys residue only in B and a peptide (b) containing a Cys residue only in B.
- a peptide containing a Cys residue in both A and B (a) and a peptide containing a Cys residue in both A and B (b) are: Linked by a disulfide bond formed by the Cys residue in A of the peptide (a) and the Cys residue in B of the peptide (b); and
- amino acid sequences of a plurality of peptides may be the same or different, and are preferably the same.
- a multimeric ring structure is formed by a plurality of disulfide bonds formed between the residue and a Cys residue contained in A or B of another peptide.
- a dimer or a trimer is preferable, and a dimer is particularly preferable.
- a dimer is particularly preferable.
- the peptide (a) containing a Cys residue in A and B and the peptide (b) containing a Cys residue in A and B are in the A of the peptide (a).
- a disulfide bond formed by the Cys residue of the peptide (b) and the Cys residue in the peptide (b), and the Cys residue in the peptide (a) B and the disulfide bond in the peptide (b) B A dimer cyclized by a disulfide bond formed with a Cys residue;
- the peptide (a) containing a Cys residue in A and B and the peptide (b) containing a Cys residue in A and B are the Cys in A of the peptide (a). Residue and the Cys residue in B of peptide (b). Dimerization is formed by the disulfide bond formed by the disulfide bond formed by the Cys residue in B of the peptide (a) and the Cys residue in A of the peptide (b). If
- Examples of specific structures of the peptides of the present invention include those shown in Tables 1 and 2 below. In these tables, the peptide structure has the N-terminal on the left and the C-terminal on the right.
- peptides (4), (17), (2), and (16) are preferred, and among these, peptides (4), (17), (2), and (16) can be particularly preferred.
- bioactive peptides of the present invention are described, for example, in “Basics and Experiments of Peptide Synthesis” (Nobuo Izumiya, Tetsuo Kato, Higashihiko Aoyagi, Michinori Waki: Maruzen Co., Ltd.) It can be obtained by purifying after synthesizing by the usual peptide synthesis method described in RC Sheppard "Solid phase peptide synthesis, a practical approach” (LRL press) and the like.
- the chemically modified amino acid is used.
- Conventionally known methods such as peptide synthesis using amino acids or chemical modification after peptide synthesis (“Basic and experimental methods for peptide synthesis” (Nobuo Izumiya, Tetsuo Kato, Higashihiko Aoyagi, Author: Maruzen, “Chemical Modification of Proteins” ⁇ top> ⁇ bottom> (Motonori Ohno, Yu Kanaoka, Fumio Sakiyama, Hiroshi Maeda); can be manufactured by Gakkai Shuppan Center.
- the peptide having an intermolecular disulfide bond of the present invention can be prepared by a conventional method, for example, “Basic and Experimental Peptide Synthesis” (Nobuo Izumiya, Tetsuo Kato, Tohiko Aoyagi, Michinori Waki: Maruzen Co., Ltd.) ) Or E. Atherton, RC Sheppard "Solid phase peptide synthesis, a practical approach (LRL press)".
- the peptide (2) of the present invention can be synthesized by a peptide synthesizer in a usual manner using the amino acid constituting the peptide (1) containing Cys already protected by Mob as a raw material, Subsequently, the obtained peptide (1) is dissolved in 10 mM ammonium acetate buffer at pH 8.0, and stirring is continued at room temperature for 3 days. If necessary, an oxidizing agent such as potassium ferricyanide or aqueous hydrogen peroxide can be added to promote the reaction. Thereafter, the peptide (2) can be obtained by purification by reverse phase HPLC.
- the peptide (7) is dissolved in 10 mM ammonium acetate buffer of PH 8.0, and the mixture is stirred at room temperature for 3 days, and then purified by reverse-phase HPLC to obtain the desired peptide (16).
- peptide (4) can be obtained from peptide (3).
- peptide (8) can be obtained from the peptide (4) in the same manner as the method for obtaining the peptide (7) from the peptide (2).
- the peptide (16) can be obtained from the peptide (8).
- Peptides (1) and (3) obtained in Examples 1 and 2 were dissolved in 10 mM ammonium acetate buffer at pH 8.0 to a concentration of 1 mgZml, and the solution was added at room temperature. Continue stirring for days. If necessary, an oxidizing agent such as lithium ferricyanide or aqueous hydrogen peroxide may be added to promote the reaction. Thereafter, the target peptide (5) can be obtained by purification by reverse phase HPLC.
- the peptide (9) is dissolved in a 10 mM ammonium acetate buffer solution (pH 8.0), and stirring is continued at room temperature for 3 days. If necessary, an oxidizing agent such as potassium fluoride or aqueous hydrogen peroxide can be added to promote the reaction. Then, purification by reverse phase HPLC yields the peptide (17).
- a 10 mM ammonium acetate buffer solution pH 8.0
- the peptide (10) and the peptide (12) obtained in Examples 3 and 4 were each dissolved in ammonium acetate buffer at pH 8.0 to a concentration of 1 mgZml, and the mixture was allowed to stand at room temperature. Continue stirring for 3 days. Alternatively, 2,2′-dithiobispyridininoacetic acid aqueous solution is added to the peptide (10) or the peptide (12), and the mixture is stirred for 30 minutes, and then the peptide (18) or the peptide (12) is added. 1 9) is obtained.
- the bioactive peptide of the present invention represented by the above formula (1) is converted into a corresponding acid addition salt or a hydrate thereof by a known method.
- Non-toxic, water-soluble salts are preferred.
- Suitable acid addition salts include hydrochloride.
- Inorganic acid salts such as hydrobromide, sulfate, phosphate, nitrate, or acetate, trifluoroacetate, lactate, tartrate, Oxalate, fumarate, maleate, citrate, benzoate, methansulfonate, ethanesulfonate, benzenesulfonate, toluenesulfonate, isethionate, glucuronate
- organic hydrochloric acid such as gluconate.
- the toxicity of the bioactive peptide of the present invention is extremely low, and it can be judged that it is sufficiently safe to use as a medicine.
- Bioactive base peptide of the present invention impair inhibitory binding of the type II PLA 2 and Bae valine, also has the effect of say equal to inhibit the enzymatic activity of type II PLA 2 in the presence of Paris down to. Therefore, these peptides inhibit various types of inflammatory diseases (eg, rheumatoid arthritis, organ inflammation) by inhibiting the secreted type II PLA 2 from migrating to other tissues or cells and exerting its activity.
- inflammatory diseases eg, rheumatoid arthritis, organ inflammation
- Asthma allergic disease, generalized intravascular coagulation (DIC), ischemic vascular disorders (myocardial infarction, cerebral infarction), restenosis (restenosis), ulcer, sepsis, ARDS, nephritis, spleenitis, its severe It is useful for prevention and / or treatment of hyperplasia and multiple organ disorders.
- DIC generalized intravascular coagulation
- ischemic vascular disorders myocardial infarction, cerebral infarction
- restenosis restenosis
- ulcer sepsis
- ARDS nephritis
- spleenitis its severe It is useful for prevention and / or treatment of hyperplasia and multiple organ disorders.
- Dosage varies depending on age, symptoms, therapeutic effect, administration method, treatment time, etc., but is usually in the range of 1 mg to l OOOmg per adult per day, once or several times a day.
- Administered or parenterally preferably intravenously once to several times daily, in lmg to 100mg per adult per day, or daily It is continuously administered intravenously for 1 to 24 hours.
- the dose varies depending on various conditions, so that a dose smaller than the above-mentioned dose may be sufficient, or may be required beyond the range.
- the bioactive peptide of the present invention When administering the bioactive peptide of the present invention, it is used as a solid composition, a liquid composition and other compositions for oral administration, an injection for parenteral administration, an external preparation, a suppository and the like. It is possible.
- Solid compositions for oral administration include tablets, pills, capsules, powders, granules and the like.
- Capsules include hard capsules and soft capsules.
- Liquid compositions for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, elixirs, etc., and commonly used inert diluents (water). , Ethanol, etc.).
- compositions for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.
- Other compositions for parenteral administration include one or more active substances and external preparations such as topical solutions, ointments and liniments formulated in a conventional manner, suppositories for rectal administration Agents and accessories.
- Examples of carriers and excipients for pharmaceuticals include solid or liquid non-toxic pharmaceutical substances. Examples of these include lactose, stearyl Examples include magnesium phosphate, starch, talc, gelatin, agar, pectin, gum arabic, olive oil, sesame oil, cocoa butter, ethylene glycol, and the like, and other commonly used ones.
- Human type II PLA 2 was prepared by genetic engineering according to the report of JJ Seilhamer et al. (Journal 'ob' Biological. Chemistry Vol. 264, p. 5335 (1989)).
- the recombinant virus was obtained by transfection into insect cell SF-9 together with the baculovirus (AcNPV) DNA of the above.
- the SF-9 culture supernatant on day 3 after infection with the recombinant virus was collected, passed through a heparin sepharose column or a sulfated cellophine column (manufactured by Seikagaku), and then subjected to reverse phase HPLC for human II. type and the (inflamed local origin) PLA 2 was made fine.
- Reference Example 1 human type II purification by (inflammatory sites derived) PLA 2 500 / g 5 mM of 2 ml sulfates O j Kushii succinimidyl 6 - (Kiso to Piochin'a Mi de - scan) of 0.1 M NaHCO 3 solution in 1 hour after the reaction at room temperature, the purified Paris Nsefu Arosukaramu to give Piochin arsenate preparative II type PLA 2.
- Reference example 3. Creating a heparin join plate
- PBS phosphate buffered saline
- BSA 30mgZml bovine serum albumin
- the Fmoc method was carried out using Applied Biosystems' 431 type peptide synthesizer.
- the peptide (1) was synthesized. Deprotection was performed by the TFA clear ledge method. Purification was by reversed-phase HPLC (column: YMC-Pack R & D ODS S-5 120A, 20 x 250 mm, flow rate: 15. OmlZmin, mobile phase: 0.1% trifluoroacetic acid, 0 to 50% acetonitril concentration gradient) , 60 minutes).
- Peptide (2) Dissolve 10 mg of the peptide (1) obtained in the above 1) in 10 ml of 10 mM ammonium acetate buffer at pH 8.0, and continue stirring at room temperature for 3 days. Purify by reverse phase HPLC as in (1), 5 mg of tide (2) was obtained. The results of this HPLC (absorbance 210 nm) are shown in FIG.
- Peptide (6) The peptide (6) was synthesized in the same manner as in 1) using the amino acid constituting the peptide (6) as a raw material. It was confirmed by the peptide sequencer that the obtained peptide had the desired amino acid sequence.
- the peptide (Fmoc) method was performed using Applied Biosystems' 431 type peptide synthesizer. 3) was synthesized. Deprotection was performed by the TFA clear ledge method. Purification was performed by reversed-phase HPLC (column: YMC-Fack R & D ODS S-5 120A, 20 x 250 mm, flow rate: 15. OmlZmin, mobile phase: 0.1% trifluoroacetic acid, 0 to 50% acetate nitrile concentration gradient) (60 minutes) to obtain 10 mg of peptide (3).
- Peptide (4) 10 mg of the peptide (3) obtained in 1) above at pH 8.0 was dissolved in 10 ml of 50 mM ammonium acetate buffer solution, and 0.3% hydrogen peroxide solution 60 / z 1 was added under ice-cooling. The mixture was stirred for 30 minutes and purified by reversed-phase HPLC in the same manner as for peptide (3). 4 mg of peptide (4) was obtained.
- Peptide (11) Dissolve 10 mg of the peptide (10) obtained in the above 1) in 10 ml of 10 mM ammonium acetate buffer at pH 8.0, and continue stirring at room temperature for 3 days. Purification by reverse phase HPLC was carried out in the same manner as in 1) to obtain 5 mg of the desired peptide (11). The result of this HPLC (absorbance 210 nm) is shown in FIG. 3) Peptide (16): After dissolving 5 mg of the peptide (11) obtained in 2) above in 50 ml of a 2N aqueous hydrochloric acid solution, add 5.3 ml of 1 mg / ml iodine // 35% acetic acid solution to this solution.
- Peptide (13) 10 mg of the peptide (12) obtained in 1) was dissolved in 10 ml of lOmM ammonium acetate buffer, and stirred at room temperature for 3 days, followed by purification by reversed-phase HPLC in the same manner as for peptide (1) to obtain 5 mg of the desired peptide (13).
- the result of this HPLC (absorbance 210 nm) is shown in FIG.
- the obtained peptide had the above amino acid sequence. Further, the obtained peptide (16) was confirmed to have a molecular weight of about 3446 by ion spray mass spectrometry.
- Peptide (21) was synthesized by the Fmoc method using Applied Biosystems' Model 431 peptide synthesizer. Deprotection was performed by the TMSBr clean pegging method. Purification was performed by reverse phase HPLC (column; Vydac Protein C4, 20 ⁇ 250 mm-flow rate 25 mlZ min, mobile phase; 0.1% trifluoroacetic acid, 5-55% acetonitrile gradient, 60 min). The results of HPLC (absorbance 210 nm) are shown in FIG. From Fig. 5, the target time was set to 25.2 to 25.4 minutes. It turns out that tide (21) was obtained. The reaction yield was 45%. The amino acid sequence of the obtained peptide (21) was confirmed by a gas-phase protein sequencer 477 of Applied Biosystems.
- This peptide (21) is converted to the bioactive peptide of the present invention by modification of the Cys residue in A and / or B and formation of a di- or disulfide bond.
- Peptide (17) 10 mg of the peptide (21) obtained in Example 5 was dissolved in 10 ml of 10 mM ammonium acetate buffer at pH 6.0, and the mixture was stirred at room temperature for 1 day, followed by reverse phase HPLC (column: YMCPack®). & D ODS S-5 120A, 20x250-Flow rate 15. OmlZmin., Mobile phase: 0.1% trifluoroacetic acid, 0-50% acetonitrile concentration gradient, 60 min. ) 5 mg were obtained. The result of this HPLC (absorbance 210 nm) is shown in FIG.
- Example 5 3 mg of the peptide obtained in Example 5 was dissolved in 1 liter of ammonium acetate buffer at pH 8.0, air-oxidized at room temperature, and then isolated by reversed-phase HPLC to obtain the desired peptide. (20) 2.5 mg were obtained.
- This peptide (23) is converted to the linear dimer of the present invention by modification of the Cys residue in B or formation of a disulfide bond between the two molecules.
- This peptide (24) is converted to a linear dimer by modification of the Cys residue in A or formation of a disulfide bond between the two molecules.
- the peptide was synthesized and purified in the same manner as in Example 5 to obtain a peptide (25), and its amino acid sequence was confirmed.
- the peptide was synthesized and purified in the same manner as in Example 7 to obtain a peptide (28), and its amino acid sequence was confirmed.
- Table 3 also shows the results. From the table, the peptides (1), (2), (4), (6), (11), (13), (14), (16), (17), and (20) are excellent humans. Type
- Example 13 The present invention was applied to the mouthend toxin shock of the ⁇ ° C.
- the mouse endotoxin shock model was created with reference to the method of Galanos et al. (Pro Natl. Acad. Sci. USA 76, 5939-5943, 1979). That is, an 8-week-old C57BLZ6 mouse (female, weighing 16-20 g) was given 12 mg of galactosamine (manufactured by Wako), 0.1 g of LPS (E. coli ⁇ 0 ⁇ B12 strain, LIST Biological Laboratories In), 200 g / a zg or 800 g of physiological saline solution of peptide (2) of, and administered simultaneously intraperitoneally ((one group consisting of 8 animals).
- Raw food salt water 1/8 Table 6 shows that the peptide (2) of the present invention has an excellent life-prolonging effect in a mouse endotoxin shock model, and is therefore effective for DIC, especially for sepsis.
- Peptide of the present invention had activity in inhibiting the action of phospholipase A 2, anti-inflammatory agents, are expected to be effective as an antiallergic agent
Description
Claims
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU61562/94A AU6156294A (en) | 1993-03-03 | 1994-03-03 | Physiologically active peptide |
EP94908491A EP0687685A4 (en) | 1993-03-03 | 1994-03-03 | PHYSIOLOGICALLY ACTIVE PEPTIDES |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP4260893 | 1993-03-03 | ||
JP5/42608 | 1993-03-03 | ||
JP6/7662 | 1994-01-27 | ||
JP766294 | 1994-01-27 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1994020531A1 true WO1994020531A1 (en) | 1994-09-15 |
Family
ID=26341992
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/JP1994/000345 WO1994020531A1 (en) | 1993-03-03 | 1994-03-03 | Physiologically active peptide |
Country Status (3)
Country | Link |
---|---|
EP (1) | EP0687685A4 (ja) |
AU (1) | AU6156294A (ja) |
WO (1) | WO1994020531A1 (ja) |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5786324A (en) * | 1994-03-24 | 1998-07-28 | Regents Of The University Of Minnesota | Synthetic peptides with bactericidal activity and endotoxin neutralizing activity for gram negative bacteria and methods for their use |
WO2018236931A1 (en) * | 2017-06-19 | 2018-12-27 | Allegro Pharmaceuticals, Inc. | PEPTIDE COMPOSITIONS AND ASSOCIATED METHODS |
US10639347B2 (en) | 2009-11-10 | 2020-05-05 | Allegro Pharmaceuticals, LLC | Peptides useable for treatment of disorders of the eye |
US11673914B2 (en) | 2009-11-10 | 2023-06-13 | Allegro Pharmaceuticals, LLC | Peptide therapies for reduction of macular thickening |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1998055504A1 (en) * | 1997-06-05 | 1998-12-10 | Institut Pasteur | HsPLA2 GR II PEPTIDES EXHIBITING AN ANTICOAGULANT EFFECT |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5266562A (en) * | 1987-11-19 | 1993-11-30 | The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services | Anti-inflammatory agents |
JPH01199995A (ja) * | 1988-02-04 | 1989-08-11 | Kyowa Hakko Kogyo Co Ltd | ホスホリパーゼa↓2阻害ペプチド |
-
1994
- 1994-03-03 WO PCT/JP1994/000345 patent/WO1994020531A1/ja not_active Application Discontinuation
- 1994-03-03 EP EP94908491A patent/EP0687685A4/en not_active Withdrawn
- 1994-03-03 AU AU61562/94A patent/AU6156294A/en not_active Abandoned
Non-Patent Citations (3)
Title |
---|
J. Biol. Chem., Vol. 252, No. 14 (1977), ROBERT L. HEINRIKSON et al., "Amino Acid Sequence of Phospholipase A2-Alpha from the Venom of Crotalus Adamanteus. A New Classification of Phospholipases A2 Based Upon Structural Determinants", pp. 4913-4921. * |
J. Biol. Chem., Vol. 264, No. 10 (1989), RUTH M. KRAMER et al., "Structure and Properties of a Human Non-Pancreatic Phospholipase A2", pp. 5768-5775. * |
See also references of EP0687685A4 * |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5786324A (en) * | 1994-03-24 | 1998-07-28 | Regents Of The University Of Minnesota | Synthetic peptides with bactericidal activity and endotoxin neutralizing activity for gram negative bacteria and methods for their use |
US10639347B2 (en) | 2009-11-10 | 2020-05-05 | Allegro Pharmaceuticals, LLC | Peptides useable for treatment of disorders of the eye |
US11666625B2 (en) | 2009-11-10 | 2023-06-06 | Allegro Pharmaceuticals, LLC | Pharmaceutical compositions and preparations for administration to the eye |
US11673914B2 (en) | 2009-11-10 | 2023-06-13 | Allegro Pharmaceuticals, LLC | Peptide therapies for reduction of macular thickening |
WO2018236931A1 (en) * | 2017-06-19 | 2018-12-27 | Allegro Pharmaceuticals, Inc. | PEPTIDE COMPOSITIONS AND ASSOCIATED METHODS |
Also Published As
Publication number | Publication date |
---|---|
EP0687685A4 (en) | 1998-07-01 |
EP0687685A1 (en) | 1995-12-20 |
AU6156294A (en) | 1994-09-26 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US11622998B2 (en) | Anti-inflammatory peptides and composition comprising the same | |
US11098294B2 (en) | Anti-inflammatory peptides and composition comprising same | |
KR102179392B1 (ko) | 글루카곤, glp-1 및 gip 수용체 모두에 활성을 갖는 삼중 활성체의 지속형 결합체 | |
US10174079B2 (en) | Compstatin analogs with improved pharmacokinetic properties | |
KR100208873B1 (ko) | 폴리펩타이드 및 그것으로 부터 제조된 항-hiv 제제 | |
US7718610B2 (en) | Retrocyclins: antiviral and antimicrobial peptides | |
DK3045183T3 (en) | PEGYLED APELIN AND APPLICATIONS THEREOF | |
KR20110099069A (ko) | 진균 및/또는 원생생물 감염의 치료방법 | |
WO1991011458A1 (en) | CYCLIC PEPTIDES CONTAINING Arg-Gly-Asp FLANKED BY PROLINE | |
WO1994020531A1 (en) | Physiologically active peptide | |
US5338725A (en) | Anti-aggregatory agents for platelets | |
JP4290564B2 (ja) | C5aアナフィラトキシンの突然変異タンパク質、このような突然変異タンパク質をコードする核酸分子ならびにC5aアナフィラトキシンの突然変異タンパク質の医薬的使用 | |
US9556235B2 (en) | F11 receptor (F11R) antagonists as therapeutic agents | |
US6797806B2 (en) | Fragments of CR1 and their use | |
JPH06263796A (ja) | 生理活性ペプチド | |
US20080299072A1 (en) | Chemokine Antagonists | |
JPWO2008050830A1 (ja) | 抗hiv剤 | |
JPH10182479A (ja) | ペプチド誘導体類からなる薬剤 | |
EP4011904A1 (en) | Masp inhibitory compounds and uses thereof | |
US20240010684A1 (en) | Masp inhibitory compounds and uses thereof | |
CA2001094A1 (en) | Chemical compounds | |
Bowerman et al. | Aromatic Versus Hydrophobic Contributions to Amyloid Peptide Self-Assembly | |
WO1992019644A1 (en) | Vasorelaxant peptide | |
CA2779710A1 (en) | F11 receptor (f11r) antagonists as therapeutic agents | |
CA2178927A1 (en) | Peptide inhibitors of cxc intercrine molecules |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AK | Designated states |
Kind code of ref document: A1 Designated state(s): AU CA JP KR US |
|
AL | Designated countries for regional patents |
Kind code of ref document: A1 Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE |
|
ENP | Entry into the national phase |
Ref country code: CA Ref document number: 2134449 Kind code of ref document: A Format of ref document f/p: F |
|
DFPE | Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101) | ||
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
WWE | Wipo information: entry into national phase |
Ref document number: 1994908491 Country of ref document: EP |
|
ENP | Entry into the national phase |
Ref country code: US Ref document number: 1995 513854 Date of ref document: 19950905 Kind code of ref document: A Format of ref document f/p: F |
|
WWP | Wipo information: published in national office |
Ref document number: 1994908491 Country of ref document: EP |
|
NENP | Non-entry into the national phase |
Ref country code: CA |
|
WWW | Wipo information: withdrawn in national office |
Ref document number: 1994908491 Country of ref document: EP |