JP4830093B2 - Preventive or therapeutic agent for non-bacterial inflammatory diseases - Google Patents

Description

  The present invention relates to a drug useful as a preventive or therapeutic agent for non-bacterial inflammatory diseases.

  Inflammatory bowel disease is an intractable disease that is increasing in frequency year by year, and development of a new treatment method is desired. In particular, inflammatory bowel disease, one of the inflammatory bowel diseases, is a disease of unknown cause that presents lesions such as ulcers and erosions in the mucosa and submucosa of the large intestine. It is. Current treatment methods for inflammatory bowel disease include pharmacotherapy and leukocyte removal therapy using 5-aminosalicylic acid preparations, steroids, and immunosuppressants. However, there are many intractable cases and steroid dependent cases, and the development of new treatments is desired.

  On the other hand, adrenomedullin (sometimes abbreviated as “AM” in this specification) is a potent vasodilatory antihypertensive peptide isolated and identified from human pheochromocytoma. It has been reported that it is distributed in cells and has various physiological activities (Non-patent Documents 1 and 2). In the gastrointestinal tract, its expression has been widely confirmed in the gastrointestinal mucosa, but there are few reports on detailed physiological effects. In recent years, it has been reported that adrenomedullin has a gastric acid secretion inhibitory action and a blood flow improving action, and acts curatively on gastric mucosal disorder model animals. On the other hand, adrenomedullin's NO releasing action, apoptosis inhibiting action, and IL-6 production inhibiting action using synovial cells of rheumatoid arthritis patients have been reported. Further, focusing on the fact that adrenomedullin has an antibacterial action, Patent Document 1 describes the possibility that adrenomedullin can be used as a useful component of a prophylactic and therapeutic agent for inflammatory bowel disease involving specific enteric bacteria. There are no examples of whether adrenomedullin is effective in the treatment of inflammatory bowel disease that is not due to bacterial infection (ie non-bacterial).

JP 2004-244378 A Salomone S., Caruso A., Cutuli VM., Mangano NG., Prato A., Amico-Roxas M., Bianchi A., Clementi G., Effects of adrenomedullin on the contraction of gastric arteries during reserpine-induced gastric ulcer. , Peptides. 2003 Jan; 24 (1): 117-22. Clementi G., Caruso A., Cutuli VM., Mangano NG., Salomone S., Lempereur L., Prato A., Matera M., Amico-Roxas M., Gasctroprotective effect of adrenomedullin administered substaneously in the rat., Peptides 2002 Jun; 23 (6): 1149-53.

  An object of the present invention is to provide a preventive or therapeutic agent for non-bacterial inflammatory diseases, particularly non-bacterial inflammatory bowel diseases.

The present invention includes the following inventions.
(1) Adrenomedullin, a modified form thereof having non-bacterial inflammation suppressing activity, or a salt thereof having non-bacterial inflammation suppressing activity as an active ingredient A preventive or therapeutic agent for bacterial inflammatory diseases.
(2) The preventive or therapeutic agent for a nonbacterial inflammatory disease according to (1), wherein the disease is a nonbacterial inflammatory bowel disease.
(3) The preventive or therapeutic agent for non-bacterial inflammatory diseases according to (1) or (2), wherein adrenomedullin or a modified form thereof is a peptide according to any one of the following (a) to (j).

(A) a peptide consisting of the amino acid sequence of SEQ ID NO: 1, or a peptide consisting of the amino acid sequence of SEQ ID NO: 1 in which Cys at position 16 and Cys at position 21 are disulfide-bonded (b) a peptide consisting of the amino acid sequence of SEQ ID NO: 3, Or a peptide comprising the amino acid sequence of SEQ ID NO: 3 and a Cys at position 16 and a Cys at position 21 being disulfide-bonded (c) a peptide consisting of the amino acid sequence of SEQ ID NO: 5, or the amino acid sequence of SEQ ID NO: 5 (D) a peptide comprising the amino acid sequence of SEQ ID NO: 7 or a peptide comprising the amino acid sequence of SEQ ID NO: 7 and having a disulfide bond between Cys at position 16 and Cys at position 21 (e) ) A peptide consisting of the amino acid sequence of SEQ ID NO: 9, or an amino acid sequence of SEQ ID NO: 9. Peptide in which Cys at position 14 and Cys at position 19 are disulfide-bonded (f) Peptide having the amino acid sequence of SEQ ID NO: 11, or Cys at position 14 and Cys at position 14 consisting of the amino acid sequence of SEQ ID NO: 11 The peptide (g) according to any one of (a) to (f), wherein the disulfide bond is substituted with a —CH ₂ —CH ₂ — bond. A peptide having 1-15 amino acids deleted, substituted or added in the described peptide and having an activity of suppressing non-bacterial inflammation (i) The C-terminus is amidated (a) to (h) (J) The peptide according to any one of (a) to (h), wherein Gly is added to the C-terminus.

  The present invention provides a drug useful as a preventive or therapeutic agent for non-bacterial inflammatory diseases, particularly non-bacterial inflammatory bowel diseases.

  Hereinafter, the present invention will be described in detail.

  The drug according to the present invention is useful as a preventive or therapeutic agent for nonbacterial inflammatory diseases in the digestive tract such as organs, particularly the large intestine. Examples of such diseases include non-bacterial inflammatory bowel disease, particularly non-bacterial ulcerative colitis. Specifically, the drug according to the present invention is effective in reducing the ulcer area, and improving edema and inflammatory cell infiltration.

  The adrenomedullin used in the present invention may be derived from humans or other warm-blooded animals (for example, pigs, dogs, cows, rats, mice, etc.). A modified form of adrenomedullin having an activity of suppressing non-bacterial inflammation can also be used in the present invention.

The adrenomedullin or a modified form thereof used in the present invention is typically (a) a peptide consisting of the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence of SEQ ID NO: 1, wherein Cys at position 16 and Cys at position 21 are disulfide bonds. (B) a peptide consisting of the amino acid sequence of SEQ ID NO: 3, or a peptide consisting of the amino acid sequence of SEQ ID NO: 3 in which Cys at position 16 and Cys at position 21 are disulfide-bonded, (c) amino acid sequence of SEQ ID NO: 5 A peptide consisting of the amino acid sequence of SEQ ID NO: 5, a peptide in which Cys at position 16 and Cys at position 21 are disulfide-bonded, (d) a peptide consisting of the amino acid sequence of SEQ ID NO: 7, or the amino acid sequence of SEQ ID NO: 7 A peptide having a disulfide bond between Cys at position 16 and Cys at position 21, (e) SEQ ID NO: A peptide consisting of the amino acid sequence of 9, or a peptide consisting of the amino acid sequence of SEQ ID NO: 9 in which Cys at position 14 and Cys at position 19 are disulfide-bonded; (f) a peptide consisting of the amino acid sequence of SEQ ID NO: 11, or SEQ ID NO: 11 (G) any one of (a) to (f), wherein the Cys at position 14 and the Cys at position 19 are disulfide-bonded, and (g) the disulfide bond is replaced with a —CH ₂ —CH ₂ — bond. In the peptide according to (h), (a) to (g), one or more, for example, one or several, specifically 1 to 15, preferably 1 to 12 , More preferably 1-10, more preferably 1-8, more preferably 1-5, most preferably 1-3 amino acids deleted, substituted or added (I) the peptide according to any one of (a) to (h) in which the C-terminus is amidated, or (j) the Gly at the C-terminus. Is a peptide according to any one of (a) to (h). “C-terminal amidation” refers to one of peptide modification reactions, and the COOH group of the C-terminal amino acid of the peptide is in the form of CONH ₂ . Many physiologically active peptides that operate in vivo are initially biosynthesized as precursor proteins having a higher molecular weight, and in the process of intracellular translocation, they undergo a modification reaction such as C-terminal amidation and mature. Amidation is performed by the C-terminal amidating enzyme acting on the precursor protein. In the precursor protein, there is always a Gly residue on the C-terminal side of the residue to be amidated, followed by a basic amino acid sequence pair such as Lys-Arg or Arg-Arg on the C-terminal side. (Mizuno, Biochemistry Vol. 61, No. 12, pages 1435 to 1461 (1989)).

  Examples of the peptide belonging to (h) above include peptides having the amino acid sequence of SEQ ID NO: 1, 3, 5, 7, 9 or 11 (having intramolecular bonds shown in (a) to (g) above. From 1 to 15 positions, 1 to 12 positions, 1 to 10 positions, 1 to 8 positions, 1 to 5 positions, or 1 to 3 position amino acids. . Among them, a peptide in which amino acids at positions 1 to 12 are deleted from a peptide having the amino acid sequence of SEQ ID NOs: 1, 3, 5 and 7 (which may have the above intramolecular bond), and SEQ ID NO: Peptides in which the amino acids at positions 1 to 10 are deleted from peptides having amino acid sequences of 9 and 11 (which may have the above intramolecular bond) are preferred. A peptide having an activity of suppressing non-bacterial inflammation, wherein one or several (for example, 1 to 5, 1 to 3, 1 or 2) amino acids have been deleted, substituted or added in these peptides. Can also be used in the present invention. Furthermore, a peptide in which the C-terminal of the peptide described in the paragraph of the present invention is amidated or a peptide in which Gly is added to the C-terminal can also be used in the present invention.

  Among the peptides described in the above (a) to (j), the peptide described in (a) to (f) having a C-terminal amidated and having a disulfide bond in the molecule is included in (i). “What has” is adrenomedullin mainly present in the living body, and the other peptides should be referred to as modified forms of adrenomedullin.

  Examples of the modified adrenomedullin other than those described above that can be used in the present invention include those in which a part of the amino acid residues constituting the peptides (a) to (j) above are amidated or esterified, and are non-bacterial And those having an activity of suppressing inflammation. Examples of the ester include those in which the C-terminal carboxyl group is esterified in the peptides (a) to (h) or (j).

  Adrenomedullin or a modified form thereof may be provided as a precursor or prodrug compound that is converted to adrenomedullin or a modified form thereof by metabolism in vivo. Such forms are also included in the scope of the present invention. As an example of the precursor, there is a peptide comprising the amino acid sequence of No. 1 to No. 185 encoded by the nucleic acid sequence of SEQ ID No. 2 or a partial sequence thereof and comprising amino acids of No. 95 to No. 146 Can be mentioned. Since the peptide described in (j) above is considered to be converted into a mature peptide by amidation of its C-terminal in vivo after administration, the peptide can also be included in adrenomedullin or a precursor of its modification.

  A salt of adrenomedullin or a modified product thereof having an activity of suppressing non-bacterial inflammation can also be used in the present invention. The salt may be any salt as long as it is a pharmaceutically acceptable salt with a base (for example, alkali metal) or an acid (organic acid, inorganic acid), for example, hydrochloride, hydrobromic acid, etc. Inorganic acid salts such as salts, hydroiodide, sulfate, nitrate, phosphate, carbonate, bicarbonate, perchlorate; formate, acetate, trifluoroacetate, propionate, shu , Glycolate, succinate, lactate, maleate, hydroxy maleate, methyl maleate, fumarate, adipate, tartrate, malate, citrate, benzoate , Cinnamate, ascorbate, salicylate, 2-acetoxybenzoate, nicotinate, isonicotinate and other organic acid salts; methanesulfonate, ethanesulfonate, isethionate, benzenesulfone acid , P- toluenesulfonate, sulfonic acid salts such as naphthalene sulfonate; sodium salt, alkali metal salts such as potassium salts; magnesium salts, alkaline earth metal salts such as calcium salts.

  Adrenomedullin or a modified form thereof can be produced by a method for purifying a peptide from human or warm-blooded animal tissue or cells, or can be produced according to a general peptide synthesis method. It can also be produced by culturing a transformant containing DNA encoding adrenomedullin.

In the present invention, adrenomedullin may be provided as a nucleic acid molecule (eg, DNA or RNA) comprising a gene sequence encoding adrenomedullin. That is, the drug according to the present invention includes a so-called gene therapy form. Examples of DNA encoding adrenomedullin include (1) DNA containing the nucleotide sequence of SEQ ID NO: 2, 4, 6, 8, 10 or 12 or nucleotide sequence of SEQ ID NO: 2, 4, 6, 8, 10 or 12 DNA comprising a partial sequence partially containing a region encoding adrenomedullin, (2) DNA comprising a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 2, 4, 6, 8, 10 or 12, or a sequence Mammals that hybridize under stringent conditions with DNA comprising a base sequence complementary to a partial sequence partially containing the region encoding adrenomedullin among the base sequences of numbers 2, 4, 6, 8, 10 or 12 DNA encoding a peptide or precursor thereof having an activity of suppressing non-bacterial inflammation, and (3) due to degeneracy of the genetic code (1 And (2) is not arranged hybridize which is determined, a DNA encoding a polypeptide having the same amino acid sequence is used. Hybridization can be performed according to a known method or a method analogous thereto. The stringent conditions include, for example, 42 ° C., 50% formamide, 4 × SSPE (1 × SSPE = 150 mM NaCl, 10 mM NaH ₂ PO ₄ .H ₂ O, 1 mM EDTA pH 7.4), 5 × Denhardt's solution, 0 .1% SDS.

  The above-mentioned “partial sequence partially including a region encoding adrenomedullin in the base sequence of SEQ ID NO: 2, 4, 6, 8, 10 or 12” will be described with an example. In SEQ ID NO: 2, the region from 439th T to 594th C encodes adrenomedullin, so that a partial sequence of SEQ ID NO: 2 containing this region, for example, from 157th A to 711th T Can be used in the present invention.

  When the gene comprising the DNA shown in (1) to (3) above is transcribed and translated in vivo, a precursor protein having a molecular weight larger than that of adrenomedullin is usually generated. It undergoes modification reactions such as amidation and matures into active adrenomedullin.

  Use of adrenomedullin, a modified form thereof or a salt thereof, or a gene encoding adrenomedullin as a preventive or therapeutic agent for non-bacterial inflammatory diseases can be performed according to a conventional method. For example, aseptic tablets or capsules, elixirs, microcapsules or the like, or aseptic solutions with water or other pharmaceutically acceptable liquids, as needed Alternatively, it can be used parenterally in the form of an injection such as a suspension. Any administration method such as local direct administration can be used. For example, adrenomedullin, modified forms thereof, salts thereof, etc. are required for generally accepted pharmaceutical practice together with pharmaceutically acceptable carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders, etc. It can be manufactured by mixing in unit dosage form. The amount of active ingredient in these preparations is such that an appropriate volume within the indicated range can be obtained. It can also be used in combination with other components useful as a medicine.

  Additives that can be mixed into tablets, capsules and the like include binders such as gelatin, corn starch, gum tragacanth and gum arabic, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid and the like A swelling agent, a lubricant such as magnesium stearate, a sweetening agent such as sucrose, lactose or saccharin, a flavoring agent such as peppermint, red oil, or cherry are used. When the dispensing unit form is a capsule, a liquid carrier such as fats and oils can be further contained in the above-mentioned type of material. Sterile compositions for injection can be formulated according to conventional pharmaceutical practice, such as dissolving or suspending active substances, naturally occurring vegetable oils such as sesame oil, coconut oil etc. in a vehicle such as water for injection. . Examples of aqueous solutions for injection include isotonic solutions (eg, D-sorbitol, D-mannitol, sodium chloride, etc.) containing physiological saline, glucose and other adjuvants, and suitable solubilizing agents. For example, you may use together with alcohol (for example, ethanol), polyalcohol (for example, propylene glycol, polyethylene glycol), a nonionic surfactant (for example, polysorbate 80 (TM), HCO-50), etc. Examples of the oily liquid include sesame oil and soybean oil. You may use together with a benzyl benzoate, benzyl alcohol, etc. as a solubilizing agent. Buffers (eg, phosphate buffer, sodium acetate buffer), soothing agents (eg, benzalkonium chloride, procaine, etc.), stabilizers (eg, human serum albumin, polyethylene glycol, etc.), storage You may mix | blend with an agent (for example, benzyl alcohol, phenol, etc.), antioxidant, etc. The prepared injection solution is usually filled in a suitable ampoule. Since the preparation thus obtained is safe and has low toxicity, for example, humans and mammals (eg, mice, rats, guinea pigs, rabbits, chickens, sheep, pigs, cows, cats, dogs, monkeys, baboons, chimpanzees, etc. ).

  A gene (usually DNA) encoding adrenomedullin can be administered by a so-called gene therapy technique. For example, the DNA encoding adrenomedullin is expressed by (a) administering and expressing the DNA encoding adrenomedullin to the patient, or (b) inserting and expressing the DNA encoding adrenomedullin in a cell or the like. By transplanting into the patient, the amount of adrenomedullin in the cells of the patient can be increased, and the effect of adrenomedullin can be fully exerted. Administration of DNA encoding adrenomedullin can be carried out according to conventional means after inserting the DNA alone or into an appropriate vector such as a plasmid vector, retrovirus vector, adenovirus vector, adenovirus associated virus vector.

  The dose of adrenomedullin varies depending on symptoms, but is typically 0.01 to 10 mg / 60 Kg / day for intravenous administration or enteral administration for humans.

  EXAMPLES Hereinafter, although this invention is demonstrated based on an Example, this invention is not limited only to the following Example.

  The effect of adrenomedullin on non-bacterial inflammatory diseases in the large intestine was examined using acetic acid-induced colorectal ulcer model rats.

[Method]
Wistar male rats (6-7 weeks old) were used. According to a report by Kojima et al. (Kojima R, Hamamoto S, Moriwaki M, Iwadate K, Ohwaki T. The new experimental ulcerative colitis model in rats induced by subserosal injection of acetic acid. Folia Pharmacol J 2001; 118: 123-30.) An ulcer was created in the large intestine 5 cm from the anus using the subserosa acetic acid injection method. Immediately after the creation of the ulcer, 0.5 ml of a solution of human adrenomedullin (having the amino acid sequence of SEQ ID NO: 1) (0.25 to 1.0 μg / 0.5 ml physiological saline) was transanally administered for one day using a plastic tube. One dose was administered. The control group was similarly administered with 0.5 ml of physiological saline. The mice were sacrificed 3 days, 5 days and 10 days after the ulcer was created, and histopathological findings, ulcer area and tissue weight were examined. In addition, the large intestine 3 cm in length was excised centering on the ulcer, and cytokines (IL-6, IFN-γ) in the tissue were measured by ELISA method using the homogenized supernatant.

[Histopathological findings]
FIG. 1 (a) shows a macroscopic image of an ulcer after 5 days in the control group, and FIG. 1 (b) shows an AM 1.0 μg / day administration group. FIG. 1 (c) shows a loupe image of an ulcer 5 days after the control group, and FIG. 1 (d) shows an AM 1.0 μg / day administration group. Highly reproducible ulcer formation was observed by injecting acetic acid under the serosa of the large intestine (FIGS. 1 (a) and (c)). In addition, in the AM 1.0 μg / day administration group, reduction of the ulcer area, improvement of edema and inflammatory cell infiltration were observed as compared with the control group (FIGS. 1B and 1D).

[Ulcer area]
FIG. 2 shows the ulcer area after 5 days in each test section. There was a tendency to reduce the ulcer area in a dose-dependent manner from 0.25 to 1.0 μg / day (n = 5, P <0.05).

  FIG. 3 shows the time course of the ulcer area in the control group and the AM 1.0 μg / day administration group. The control group showed a spontaneous improvement trend at the peak on the fifth day, whereas the AM 1.0 μg / day administration group showed an improvement tendency after 3 days, and the ulcer almost disappeared on the 10th day ( n = 3, P <0.05).

[Tissue weight]
FIG. 4 shows the wet weight (mg) of the tissue after 5 days in the control group and AM 1.0 μg / day administration group. Compared to the control group, the tissue was significantly lighter in the AM 1.0 μg / day administration group (n = 5, P <0.05).

[Cytokine evaluation]
FIG. 5 shows the amounts of IL-6 (FIG. 5 (a)) and IFN-γ (FIG. 5 (b)) in the tissue after 5 days in the control group and AM 1.0 μg / day administration group (weight unit is pg). ). The amount of IL-6 was significantly low in the AM 1.0 μg / day administration group (n = 5, P <0.05). There was no significant difference between the two groups regarding the amount of IFN-γ. This tendency suggests that the anti-ulcer action by AM may be mediated by a Th2-type immune response.

1A shows a macroscopic image of an ulcer after 5 days in a control group, and FIG. 1B shows an AM 1.0 μg / day administration group. FIG. 1 (c) shows a loupe image of an ulcer 5 days after the control group, and FIG. 1 (d) shows an AM 1.0 μg / day administration group, respectively. FIG. 2 shows the ulcer area after 5 days for each test group. FIG. 3 shows the time course of ulcer area in the control group and AM 1.0 μg / day administration group. FIG. 4 shows the wet weight (mg) of the tissue after 5 days in the control group and the AM 1.0 μg / day administration group. FIG. 5 shows the amounts of IL-6 (a) and IFN-γ (b) in the tissue after 5 days in the control group and AM 1.0 μg / day administration group.

Claims (2)

(1) Adrenomedullin,
(2) a modified product in which two Cys in the amino acid sequence of adrenomedullin are disulfide bonded,
(3) a modified product in which the disulfide bond is substituted with a —CH ₂ —CH ₂ — bond,
(4) A modified product in which 1 to 15 amino acids are deleted, substituted or added in the amino acid sequence of adrenomedullin or a modified product thereof of (1) to (3),
(5) In the amino acid sequence of adrenomedullin of (1) to (4) or a modified product thereof, a modified product in which the C-terminus is amidated, or (6) the adrenomedullin of (1) to (4) or a modified product thereof A modified form of adrenomedullin selected from a modified form in which Gly is added to the C-terminus in the amino acid sequence, which has an activity of suppressing non-bacterial inflammation, or a salt thereof, which is non-bacterial A preventive or therapeutic agent for non-bacterial inflammatory bowel disease, which contains as an active ingredient a substance having an activity of suppressing inflammation of. The agent for preventing or treating nonbacterial inflammatory bowel disease according to claim 1 , wherein adrenomedullin or a modified form thereof is the peptide according to any one of the following (a) to (j).
(A) a peptide consisting of the amino acid sequence of SEQ ID NO: 1, or a peptide consisting of the amino acid sequence of SEQ ID NO: 1 in which Cys at position 16 and Cys at position 21 are disulfide-bonded (b) a peptide consisting of the amino acid sequence of SEQ ID NO: 3, Or a peptide comprising the amino acid sequence of SEQ ID NO: 3 and a Cys at position 16 and a Cys at position 21 being disulfide-bonded (c) a peptide consisting of the amino acid sequence of SEQ ID NO: 5, or the amino acid sequence of SEQ ID NO: 5 (D) a peptide comprising the amino acid sequence of SEQ ID NO: 7 or a peptide comprising the amino acid sequence of SEQ ID NO: 7 and having a disulfide bond between Cys at position 16 and Cys at position 21 (e) ) A peptide consisting of the amino acid sequence of SEQ ID NO: 9, or an amino acid sequence of SEQ ID NO: 9. Peptide in which Cys at position 14 and Cys at position 19 are disulfide-bonded (f) Peptide having the amino acid sequence of SEQ ID NO: 11, or Cys at position 14 and Cys at position 14 consisting of the amino acid sequence of SEQ ID NO: 11 The peptide (g) according to any one of (a) to (f), wherein the disulfide bond is substituted with a —CH ₂ —CH ₂ — bond. A peptide having 1-15 amino acids deleted, substituted or added in the described peptide and having an activity of suppressing non-bacterial inflammation (i) The C-terminus is amidated (a) to (h) (J) The peptide according to any one of (a) to (h), wherein Gly is added to the C-terminus.

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