JPH10194985A - Substance for treating iron overload disease and therapeutic agent for iron overload disease - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject substance and the subject therapeutic agent more useful than that in the past by formulating a peptide as an active ingredient. SOLUTION: This therapeutic agent contains a substance for treating iron overload disease consisting of a peptide of the formula in an amount of 10<-7> -10wt.% and is formulated as oral administrating agents, injections, suppositories and preparations for external use. The dose is 10ng/kg-10mg/kg a day as the above peptide and the above preparation composition may be dividedly administrated 1-4 times/day. The above peptide is obtained by culturing, extracting and then recrystallizing the peptide-producing strain belonging to the genus Streptomyces It is useful for treating diseases such as serious thalassemia, iron refractory anemia, Diamond Blackfan anemia, aplasia anemia, sickle cell anemia, hemolytic anemia and iron toxic disease.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a therapeutic substance for an iron overload disease comprising a peptide represented by the following formula [1] and a therapeutic agent for an iron overload disease comprising the peptide as an active ingredient.

[0002]

[0002] Iron overload diseases include severe Mediterranean anemia, iron refractory anemia, Blackfan Diamond anemia, aplastic anemia, sickle cell anemia, other hemolytic anemia and iron intoxication. . In addition, iron overload may occur in patients who have transfused many times for the purpose of treating various diseases and the like. It is known that iron deposition due to iron overload is accompanied by tissue damage and causes heart diseases such as cardiac hypertrophy and heart failure, and liver diseases such as cirrhosis and hepatocellular carcinoma (Merck Manual, 16th edition, Japanese edition first edition, 100
6).

[0003] In the treatment of these iron overload diseases, administration of an iron chelating agent which has an affinity for iron ions and which is chelated and excreted is considered to be effective. Conventionally, iron chelating agents used for this treatment include deferoxamine mesylate [Prelog et al., Hel.
v. Chem. Acta 45, 31 (1962)].
By the way, in removing iron excessively present in a tissue, it is necessary to permeate a cell membrane. Therefore, it is generally considered that the higher the lipid solubility of a drug to be used, the more effective it is.
However, deferoxamine mesylate has low fat solubility,
There is a need for more effective drugs. In addition, deferoxamine mesylate has another problem that it has strong side effects such as audiovisual impairment, neuropathy, and lung impairment.

[0004]

DISCLOSURE OF THE INVENTION An object of the present invention is to provide a substance for treating iron overload disease and a therapeutic agent for iron overload disease which are more useful in view of the above problems.

[0005]

The substance for treating an iron overload disease of the present invention has the following formula [1]:

Embedded image Consists of a peptide represented by

[0006] The therapeutic agent for iron overload disease of the present invention is a therapeutic agent for iron overload disease containing the above peptide as an active ingredient.

[0007] The above-mentioned peptide is obtained by culturing the peptide-producing strain belonging to the genus Streptomyces, for example, Streptomyces nobilis (Streptomyces nobilis, hereinafter abbreviated as "S. nobilis"). Alternatively, it can be obtained by subjecting an extract extracted from a dried product of the same solution or a cultured cell with an organic solvent to various column chromatography, and recrystallizing a column chromatography fraction containing the target product.

[0008] Actinomyces S. cerevisiae producing the above peptides Nobilis is available from public preservation institutions, such as the RIKEN archival strain (JCM4274), which is ATCC 19252 in the United States and CBS in the Netherlands.
198.65).

[0009] The above-mentioned peptide can be obtained by the method described in WO 96/12732, which was previously filed by the present inventors in an international application.

[0010] The above peptides have higher lipophilicity than deferoxamine mesylate.

In order to obtain the therapeutic agent for iron overload disease of the present invention by formulating the above-mentioned peptides, they are usually formed into a pharmaceutical composition together with a pharmaceutical carrier. As a carrier, a filler, a disintegrant, a bulking agent, a binder, a coloring agent, a flavoring agent, a pH adjusting agent, a solubilizing agent, a suspending agent, which are generally used for preparing a drug according to a dosage form, Buffers, stabilizers, preservatives,
Excipients, surfactants, lubricants and excipients are exemplified. By selecting an appropriate solvent, the peptide can be used as it is as a liquid.

[0012] Dosage unit forms of the therapeutic agent for iron overload disease prepared using the above peptide include tablets, pills, drinking liquids, powders, suspensions, emulsions, granules, in addition to the above liquids. , Extracts, fine granules, syrups, infusions, decoctions, eye drops, troches, cataplasms, liniments, lotions, eye ointments, plasters, capsules, suppositories, enemas, injections (liquids) , Suspensions), patches, ointments,
Examples include jellies, pastas, inhalants, creams, sprays, nasal drops and the like.

The amount of the peptide to be contained in the therapeutic agent for iron overload disease is not particularly limited, and may be appropriately selected from a wide range. In the therapeutic agent for iron overload disease, the amount of the peptide is preferably 10 ^-7. In the range of 10 to 10% by weight.

[0014] The therapeutic agent for iron overload disease obtained from the therapeutic substance for iron overload disease of the present invention is administered by a method according to various forms when used. For example, injections are administered intravenously, intramuscularly, intradermally, subcutaneously, intraarticularly or intraperitoneally, and tablets, pills, drinking solutions, suspensions, emulsions, granules and capsules are administered. It is administered orally, in the case of suppositories it is administered rectally, and in the case of an external preparation it is sprayed, affixed or applied directly to the required site such as skin or mucous membrane.

The dose of the therapeutic agent for iron overload disease of the present invention is as follows:
It is appropriately selected depending on the purpose of use, symptoms, etc.
10 ng / kg to 10 mg of the above peptide per day
/ Kg range. In addition, the above-mentioned pharmaceutical composition is used for 1 to 4
Of course, it is also possible to administer the drug in divided doses / day.

[0016]

BEST MODE FOR CARRYING OUT THE INVENTION

Example 1 Actinomyces S. aureus obtained from RIKEN Nobilis (JCM
4274) was subjected to shaking culture (pre-preculture) in 100 ml of a starch / ammonium medium supplemented with 0.2% (w / v) of yeast extract. Subsequently, 3 ml of the same medium was inoculated with 60 ml of the pre-precultured culture solution and shake-cultured (seed culture) for 25 hours.
did. In addition, a starch / ammonium medium (distilled water 100m
1 g of soluble starch and 0.1 g of dipotassium hydrogen phosphate in 1 l.
285 liters of the seed culture was inoculated into 285 liters (containing 0.05 g of ammonium chloride and 0.05 g of ammonium chloride), and cultured with shaking at about 30 ° C for 8 days.

This culture solution is centrifuged, and the resulting cells 2
To 15 g (wet weight), 2.15 liter of dichloromethane was added, and after sonication for 30 minutes, 8.6 liter of dichloromethane was further added, and the whole was stirred at room temperature for 1 hour. After filtering off the cells, the obtained filtrate was concentrated to dryness. After the dried product of the extract was dissolved in a small amount of hexane, the hexane phase was extracted three times with methanol. The hexane insolubles and the methanol extract were redissolved in methanol.

Next, the solvent extract obtained as described above is
ODS (octadecyldimethylchlorosilane) carrier 28
g. Then, about 30 g of the extract-adsorbing carrier was charged on the 3.2-cm-diameter column packed with 275 g of the ODS carrier to prepare an ODS column. Purification was performed using the ODS column under the following conditions. As an elution solvent, a) methanol: acetonitrile: water = 1.5 liter of 7: 7: 6, b) methanol: acetonitrile: water = 1.2 liter of 8: 8: 4, c) methanol: acetonitrile: water = 9 : 9: 2
150 ml, d) methanol: acetonitrile: water =
1 liter of 19: 19: 2 and e) 1 liter of methanol were flowed in this order at a flow rate of 10.5 ml / min. The fractionation was carried out every time the solvent composition was changed. In particular, the fraction eluted with methanol: acetonitrile: water = 19: 19: 2 was fractionated little by little (by 2 minutes) using an appropriate fraction collector. For each eluted fraction, ODS-80T
M, HPLC using a Tosoh column having an inner diameter of 4.6 mm × length of 25.0 cm (Hitachi, Pump L-600)
0, L-6200, detector L-3000, column oven 655A-52) under the conditions of a detection wavelength of 210 nm, a column temperature of 40 ° C., and a flow rate of 1 ml / min, water: acetonitrile: methanol = 6: 7: 7 (0
Min) to 0: 1: 1 (30 min). Of the above fractions, only those containing a peak with a retention time of 18 to 20 minutes were collected and treated as the same fraction (840 m
g), repeated recrystallization using methanol-water,
330 mg of needle crystals were obtained.

The structure of this substance was determined to be the formula [I] from the following instrumental analysis data.

Structural analysis data Instrument analysis data of the substance obtained above is shown below.

1. MS · ESI-MS: m / z = 913.6 (M + H-H 2 O) +, 931.6 (M + H) +, 953.6 (M + Na) + · HRFAB-MS Found: m / z = 913.5079 (M + H−H ₂ O) ⁺ , m / z = 913, 953, 931 (913 is main, 931 is very small) Calcd for: C ₄₅ H ₆₉ N ₈ O ₁₂ m / z = 913.5053

[0022]

3. Amino acid analysis D-serine, L-alanine and D-
N-methyl-phenylalanine was observed.

Test Example 1 The peptide of the formula [I] obtained above was dissolved in methanol to give 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM
M, a 0.5 mM solution was prepared. Further, ferric chloride was dissolved in methanol to prepare a 0.1 mM solution.

In 1 ml of the above ferric chloride methanol solution,
1 ml of a methanol solution of the peptide of the formula [I] or methanol alone at each concentration was added and mixed. The absorbance at a wavelength of 530 nm of each mixture was measured using a spectrophotometer (manufactured by Shimadzu Corporation, UV-26) using methanol as a blank.
0). The results are shown in Table 1.

[0026]

[Table 1]

As can be seen from Table 1, the above peptide is
A chelate was formed at a molar ratio of 3: 1 with trivalent iron ion (Fe ³⁺ ). From this, it was confirmed that the above-mentioned peptide is useful as a therapeutic agent for the above-mentioned iron overload disease.

[0028]

The constitution of the therapeutic substance for iron overload disease of the present invention is as described above, and the present invention provides a therapeutic substance for iron overload disease having higher utility.

The composition of the therapeutic agent for iron overload disease of the present invention is as described above. Since the present invention comprises the peptide of formula [1] as an active ingredient, the therapeutic agent for iron overload disease has higher utility. I will provide a.

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Claims (2)

[Claims] (1) The following formula (1): A therapeutic substance for an iron overload disease comprising the peptide represented by the formula: 2. A therapeutic agent for iron overload disease comprising the peptide according to claim 1 as an active ingredient.