JPH06145067A - Medicine for prevention and improvement of carinii infectious disease - Google Patents

Abstract

PURPOSE:To provide a medicine containing WF11243 substance (salt) exhibiting specified physical properties as the active component, excellent in safety and phylaxis effect and useful for preventing and improving carinii pneumon: a, etc. CONSTITUTION:The medicine contains WF11243 substance represented by the formula or its salt as the active component. In addition, hydrochloride of WF11243 substance exhibits, e.g. following physical properties; color and state of substance: white powder; melting point: 182 to 187 deg.C; specific rotatory power: [alpha]<23>D is +29 deg. (C=1.5, methanol); molecular formula: C71H115N14O23.HCl; elemental analysis: calculated value (wt.%) (as C71H115N14O23.HCl.8H2O) C: 49.74, H: 7.82 and N: 11-44, experimental value (wt.%): C: 49.65, H: 7.72 and N: 11.40; solvent solubility: readily soluble in methanol or water, sparlingly soluble in acetone and insoluble in n-hexane; color reaction: Iodine vapor reaction, cerium sulfate reaction and ninhydrin reaction are positive and Molisch reaction and Ehrlich reaction are negative; The dosage is preferably 0.5 to 100mg/kg per day on active component base.

Description

Detailed Description of the Invention

[0001] The present invention relates to a prophylactic / therapeutic agent for Pneumocystis carinii infection containing a WF11243 substance or a salt thereof as an active ingredient.

[0002]

2. Description of the Related Art Recently, diseases caused by protozoa,
Especially Pneumocystis carin
Various diseases caused by ii), such as carinii pneumonia, are highlighted, and there is a strong demand in the art for the development of preventive and therapeutic methods.

[0003]

[Means for Solving the Problems] In the present invention, molds isolated in Ayabe City, Kyoto Prefecture. No. WF produced by 11243 shares
It was confirmed that the 11243 substance or a salt thereof was effective for the preventive treatment of carinic pneumonia.

The present invention relates to a prophylactic / therapeutic agent for Pneumocystis carinii infection, which comprises as an active ingredient a WF11243 substance whose hydrochloride salt has the physical properties shown in Tables 5 to 8 below or a salt thereof.

[0005]

[Table 5]

[0006]

[Table 6]

[0007]

[Table 7]

[0008]

[Table 8]

Further, the WF11243 substance according to the present invention shows the properties of the polypeptide in view of the above-mentioned physicochemical properties. As a result of attempting the structure determination, the substance succeeded and the formula [1] I] was obtained.

[0010]

[Chemical 1]

The WF11243 substance according to the present invention is, for example, a microorganism No. 1 newly isolated from the deciduous leaf sample collected by the present inventors in Ayabe City, Kyoto Prefecture. In addition to being produced by the 11243 strain, it can be produced by a chemical synthesis method such as peptide synthesis.

This No. The 11243 strain spreads suppressively on various media, forming a pale orange colony. No. The bacteriological properties of strain 11243 are shown.

The properties of the culture on various media are shown in Table 1 shown in Tables 9 and 10 below. When inoculated in the center of malt extraction agar medium and cultured at 25 ° C. for 14 days, the growth was extremely inhibitory and spread to a diameter of 0.5 to 1.0 cm. The surface of the village was raised and had a light orange color. It also produced a colorless viscous leachate. The back of the village was grayish orange. Gave rise to anamorph. When the same culture was carried out on potato dextrose agar, the growth was extremely inhibitory (0.5 to 1.0 cm). The surface of the community was bulged, with radial grooves and was pale orange.
It also produced a colorless viscous leachate. The back of the village was pale yellow. Gave rise to anamorph.

[0014]

[Table 9]

[0015]

[Table 10]

No. The 11243 strain can grow at 7 to 29 ° C, and the optimum growth temperature is 22 to 26 ° C. (potato·
(Measured on dextrose agar).

As a result of comprehensively examining these properties, this No. No. 11243 strain was identified as belonging to mold and was classified into No. 11243 strain was named and deposited with the Institute of Microbial Science and Technology of the Ministry of International Trade and Industry (accession number FERM
BP-3373 Deposit Date: April 23, 1991).

It should be understood that the production of the WF11243 substance is not limited to the use of the particular microorganisms described herein, merely for illustrative purposes. This invention includes artificial mutants and natural mutants that can be obtained from the microorganisms described by X-ray irradiation, ultraviolet irradiation, N-methyl-N'-nitro-N-nitrosoguanidine, 2-aminopurine and the like. It also covers the use of all mutants capable of producing the WF11243 substance.

The WF11243 substance according to the present invention is inoculated into a nutrient medium containing carbon and nitrogen sources capable of assimilating the substance-producing bacterium belonging to mold (for example, No. 11243 strain),
It can be produced by culturing under aerobic conditions (eg, shaking culture, aeration and stirring culture). As the carbon source, glucose, sucrose, starch, modified starch, fructose, glycerin and other carbohydrates are preferably used.

As the nitrogen source, oatmeal, yeast extract, peptone, gluten meal, cottonseed flour, cottonseed oil meal, soybean flour, corn steep liquor, dried yeast, wheat germ,
Peanut flour, chicken bone meat meal and the like are preferably used, but ammonium salts (for example, ammonium nitrate, ammonium sulfate, ammonium phosphate, etc.), inorganic and organic nitrogen compounds such as urea and amino acids can also be advantageously used.

It is advantageous to use these carbon sources and nitrogen sources in combination, but it is not always necessary to use pure ones. This is because the impure substance may be advantageous in some cases because it may contain a growth factor or a trace element.

If necessary, the following inorganic salts may be added to the medium: sodium carbonate, potassium carbonate, sodium phosphate, potassium phosphate, sodium chloride, potassium chloride, sodium iodide, iodine. Potassium chloride, magnesium salt, copper salt, cobalt salt and the like.

In particular, if the medium strongly foams, liquid paraffin, animal oil, vegetable oil, mineral oil, silicone or the like may be added when necessary.

For large-scale industrial production of the target substance, it is preferable to carry out aeration stirring culture as in the case of other fermentation products. For small-volume production, shake culture using a flask is preferable.

When the culture is carried out in a large tank, W
In order to prevent the growth delay of the bacteria in the production process of the F11243 substance, it is preferable to first inoculate and culture the produced bacteria in a relatively small amount of medium, then transfer the culture to a large production tank and perform the production culture there. In this case, the composition of the medium used for the pre-culture and the composition of the medium used for the production culture may be the same or may be different if necessary.

The culture is preferably carried out under aeration and stirring conditions,
For example, known methods such as stirring with a propeller or other machine, rotation or shaking of a fermenter, pumping, blowing of air are appropriately used. Use sterilized air for ventilation.

The culturing temperature may be appropriately changed within the range in which the WF11243 substance-producing bacterium produces this substance, but it is usually 1 to 40 ° C., preferably 14 to 36 ° C. The culturing time varies depending on the culturing conditions and the culturing amount, but is usually about 1 day to 1 week.

After completion of the culture, the desired WF1 is obtained from the culture.
Collect 1243 material. That is, bacterial cells are directly extracted with water and / or an organic solvent, or disrupted mechanically or by a known means such as ultrasonic waves, and then extracted with water and / or an organic solvent, followed by a conventional method. According to
Purify. In the case of a culture solution, it may be directly recovered and purified by a conventional method.

As a method for recovery and purification, for example, water extraction, organic solvent, solvent extraction with a mixed solvent of these; chromatography; recrystallization from a single solvent or a mixed solvent, etc. may be appropriately used alone or in combination. it can.

The recovery and purification of the WF11243 substance is carried out by appropriately utilizing the known method as described above, but it may be carried out as follows, for example. The culture was extracted with acetone water, adsorbed on a neutral neutral adsorption resin [eg HP-20 (manufactured by Mitsubishi Kasei)], eluted with acidified acetone water, concentrated, and washed with ethyl acetate. , Butanol, and if necessary, repeated desorption with a neutral adsorption resin for purification. W
The F11243 substance is an amphoteric substance and can react with a base or an acid to form a salt. The WF11243 substance can be recovered and purified even in a free state (WF11243 substance itself), and also as a salt thereof,
Can be purified. Moreover, these can also be mutually converted by a conventional method.

Examples of the salt of WF11243 substance with a base include alkali metal salts such as sodium salt and potassium salt, alkaline earth metal salts such as calcium salt and magnesium salt, salts with inorganic base such as ammonium salt, methylamine salt and ethylamine. Salt, propylamine salt, isopropylamine salt, butylamine salt, t-butylamine salt, dimethylamine salt, diethylamine salt, trimethylamine salt, triethylamine salt, pyridine salt, picoline salt, dicyclohexylamine salt, N, N'-dibenzylethylenediamine salt Examples thereof include salts with organic bases such as organic amine salts such as arginine salts, asparagine salts, and amino acid salts such as glutamate. Examples of salts with acids include hydrochloride, hydrobromide and hydroiodide. Inorganic acid addition salts such as sulfates, nitrates, and phosphates Organic acid addition salts such as acetate, trifluoroacetate, maleate, tartrate, methanesulfonate, benzenesulfonate, formate and toluenesulfonate, amino acid addition salts such as aspartate and glutamate Acid addition salts such as

The preparation of the present invention contains WF1 as an active ingredient.
Rectal administration of 1243 substance and / or its salts, transpulmonary (nasal or buccal inhalation), nasal drop, eye drop, external (topical),
Oral or parenteral (including subcutaneous, intravenous and intramuscular)
Can be used in the form of a solid, semi-solid or liquid preparation containing an organic or inorganic carrier or excipient suitable for administration or inhalation. The active ingredient is used, for example, in tablets, pellets, troches, capsules, suppositories, creams, ointments, aerosols, inhalable powders, solutions, emulsions, suspensions and other dosage forms suitable for use. It can be formulated with conventional non-toxic pharmaceutically acceptable carriers. In addition, auxiliary, stabilizing, thickening, coloring and perfuming agents can be used if desired. W
The F11243 substance and / or its salt may be contained in the formulation in an amount sufficient to produce the desired therapeutic effect on the course or condition of the disease.

When this formulation is applied to humans,
Intramuscular or oral administration is preferred. The therapeutically effective amount of the active ingredient varies depending on the age and condition of each patient to be treated, but in general, in the case of intravenous administration, the daily dose of the active ingredient is 0.01 to 100 mg per 1 kg of human body weight,
In the case of intramuscular administration, the daily dose is 0.1 per 1 kg of human body weight.
˜100 mg, and in the case of oral administration, a daily dose of 0.5 to 100 mg per 1 kg of human body weight can be administered for the treatment or prevention of infectious diseases.

The present invention uses the WF11243 substance and / or a salt thereof for the prevention and / or treatment of Pneumocystis carinii infection. In treating or preventing Pneumocystis carinii infection, the following points are particularly mentioned. It should be noted.

For administration by inhalation, the compounds of the present invention are conveniently delivered in the form of an aerosol spray from pressure vessel or nebulizer. The compounds can also be supplied as powders which can be formulated and the powder composition can be inhaled by an insufflation powder inhaler. The preferred delivery system for inhalation is a metered dose inhalation aerosol, which can be formulated as a suspension or solution of the compound in a suitable propellant such as a fluorocarbon or hydrocarbon.

Aerosol administration is the preferred method of administration because it is desirable to treat the lungs and bronchi directly. Venting is also a desirable method, especially when the infection spreads to the ears and other body cavities.

Parenteral administration can also be used by intravenous infusion administration.

The present invention will be described in more detail with reference to Reference Examples and Examples.

[0039]

Reference Example 1 (1) Fermentative production of WF11243 substance Sucrose 4%, cottonseed oil cake 2%, dry yeast 1%, peptone 1%, KH ₂ PO ₄ 0.2%, CaCO ₃ 0.
A pre-culture medium consisting of 2% and Tween 80 0.1% was dispensed into a 500 ml Erlenmeyer flask by 160 ml and sterilized at 121 ° C. for 30 minutes.
No. 11243 shares (FERM BP
-3373) slant culture was inoculated with 1 platinum loop each, and
The cells were shaken at 4 ° C. for 4 days.

Next, modified starch 2%, glucose 0.5
%, Cottonseed oil cake 1%, gluten meal 1%, KH ₂ PO _{4
2%, Na 2 HPO 4 ·} 12H 2 O 1.5%, ZnSO 4
・ 7H ₂ O 0.001%, ADEKA NOL LG-10
9 (Asahi Denka Co., Ltd.) 0.025% and Silicon KM70 (Shin-Etsu Chemical Co., Ltd.) 0.025% were prepared in advance, and 20 liters of this main culture medium was injected into a 30-liter jar fermenter. . After sterilizing this at 121 ° C for 30 minutes, inoculate 2% of the preculture obtained above and
Cultured for a day. Agitation is 200 rpm, aeration is 20 l /
Went in minutes. The amount of WF11243 substance in the culture is HP
LC [column: Hibar LiChrospher 100RP18 (manufactured by Merck); solvent: 50% acetonitrile water containing 0.5% NH ₄ H ₂ PO ₄ ; detection: UV 210 nm; flow rate: 1 ml
/ Min]. The test sample was prepared by adding an equal amount of acetone to the culture solution, filtering the solution, and concentrating the solution to an appropriate amount.

(2) Extraction and purification of WF11243 substance

An equal amount of acetone was added to 75 l of the culture obtained by the above-mentioned culture method, and the mixture was allowed to stand overnight at room temperature with occasional stirring and then filtered to obtain a culture extract. 65 l of water was added to the extract and the pH was adjusted to 6 with NaOH 6N.
After correction to 5, it was applied to 6.5 l of HP-20 (manufactured by Mitsubishi Kasei Co.). After washing the column with 35 liters of water and 27 liters of 40% acetone water, the target substance was eluted with 48 liters of 80% acetone water containing 0.002N HCl as a final concentration.

The purification was carried out by antibacterial activity against Candida albicans, and HPLC (column: YMC Packed Column).
AM-303 (S-5, 120A, ODS) YMC Co., LTD]; Mobile layer: 0.
45% acetonitrile water containing 5% NH ₄ H ₂ PO ₄ ; detection: UV 210 nm; flow rate: 1 ml / min; retention time (RT): 10.9 minutes] was used as an index.

The eluate obtained above was concentrated under reduced pressure to 1.9 l, adjusted to pH 6.0 and washed with 2 volumes of ethyl acetate. Next, the active ingredient was extracted with 1.9 l of butanol, concentrated under reduced pressure, and replaced with 1 l of water. PH of the aqueous solution
Was adjusted to 3.0 with 1N HCl and then ethyl acetate 1 was added again.
The aqueous solution was washed with 1 l and then the active ingredient was extracted with 1 l of butanol. The organic solvent layer was washed with 1 L of 1% aqueous sodium hydrogen carbonate and 1 L of hydrochloric acid having a pH of 4, and then the organic solvent layer was concentrated under reduced pressure. The residue was then dissolved in 3.0 l of 50% acetonitrile water and applied to a 1.3 l HP-20 column. The column was 3.5 l of water and 3.5% of 50% methanol water.
1, 80% methanol water 3.5 l, methanol water 3.8
After washing with 1, the final concentration was 0.002N HCl.
The active ingredient was eluted with 2.7 l of 80% aqueous acetone containing Next, the eluate was concentrated under reduced pressure, the residue was dissolved in 5 l of 20% methanol water, and the reverse phase carrier (YMC ・ Gel, ODS-A
M, 120-S50, YMC Co., LTD) column (500 ml)
Attached to. The column was equilibrated with 0.5% NH ₄ H 20% aqueous acetonitrile containing ₂ PO _4, including after charging a solution containing a target substance, a _{0.5% NH 4 H 2 PO 4} 30
% Acetonitrile water 1 liter, same 35% acetonitrile water 1
l, washed with 1 l of the same 40% acetonitrile water, and the target substance was eluted with 2 l of the same 45% acetonitrile water.

After 280 ml of the eluted active fraction was diluted with an equivalent amount of water, it was again applied to a column (180 ml) of a reverse phase carrier (YMC.Gel, ODS-AM, 120-S50) and 0.5% NH ₄ was added. H ₂ PO ₄
Containing 30% acetonitrile water 0.4 l, 35% acetonitrile water 0.4 l, 40% acetonitrile water 0.4.
After washing the column with 4 l, the column was developed with the same 43% acetonitrile water to elute the target substance. 55 ml of this elution active fraction
Was diluted with an equivalent amount of water and applied to a 40 ml HP-20 column. The column was diluted with 180 ml of water with an equivalent amount of water and applied to a 40 ml HP-20 column. After washing this column with 180 ml of water, the final concentration was 0.002
The target substance was eluted with 100 ml of 80% acetone water containing N HCl. The eluate was concentrated under reduced pressure to remove acetone and then freeze-dried to obtain a white powder of hydrochloride of WF11243 substance (referred to as FR901469 substance).
2 mg was obtained.

(3) Physicochemical properties of FR901469 substance

FR901469 thus obtained
The physicochemical properties of the substances are as shown in Tables 1 to 4. The amino acid analysis was performed as follows. First, 1 mg of FR901469 substance was added to 6N HCl (1
ml) was used for hydrolysis for 20 hours at 110 ° C. in a sealed tube. After completion of the hydrolysis, the reaction mixture was evaporated to dryness, which was then analyzed by an amino acid analyzer (Hitachi 835 Automatic Amino-Acid A
nalyzer). The result is Thr
(4), Gly (1), Ala (1), Val (1),
They were Tyr (1) and Orn (1). Then, its chemical structure was determined as shown in formula (I) below.

[0048]

[Chemical 2]

[0049]

[Reference Example 2] Biological properties of FR901469 substance (1) Antibacterial activity

The antibacterial action of the FR901469 substance was measured by the following microbroth dilution conventional method using a 96-well multi-tray.

A test bacterial suspension (viable cell count: 2 × 10 5 cells / ml) was prepared from yeast cultured in a slant medium in a yeast nitrogen base dextrose (YNBD) medium.
A serial 2-fold dilution series of FR901469 substance in YNBD was made and 100 μl was added to each well. After adding 100 μl of the test bacterial suspension to each well, the wells were incubated at 37 ° C for 24
Time (Candida and Aspergillus genera) or 48 hours (Cryp
tococcus) cultured. After the completion of the culture, the turbidity of each well was measured, and the drug concentration in the well showing half the turbidity of the drug-free well was expressed as the 50% growth inhibitory concentration (IC ₅₀ ). The results are shown in Table 2 below in Table 11.

[0052]

[Table 11]

(3) Candida al in experimental mouse infection
Infection protection effect of FR901469 substance on bicans

The effectiveness of FR901469 substance in the body as an antifungal agent was clarified by the following method.

The test animals were 4 weeks old and weighed 18 to 21.
g of ICR female mouse is used. One group consists of 5 animals. Candida albicans FP633 suspended in physiological saline was infected by injecting into the lateral tail vein of the mouse so that the viable cell count was 2 × 10 ⁶ per mouse. 1 hour after infection,
A physiological saline solution of FR901469 substance was administered to mice by subcutaneous injection. This subcutaneous injection was further repeated once a day from the day after infection for 3 days. The number of surviving mice was observed 14 days after the infection, and the obtained results are shown in Table 3 shown in Table 12 below.

[0056]

[Table 12]

(4) Toxicity test

F5 female mice, 5 weeks old, were given F
The R901469 substance was intraperitoneally injected once daily at a dose of 100 mg / kg for 3 consecutive days, and there were no deaths, and the weight gain was exactly the same as in the non-administered mouse group.
The high safety of the substance was confirmed.

[0059]

[Reference Example 3] Production of injectable preparation (1) FR901469 substance produced in Reference Example 1 5g (2) Dietary salt 9g (3) Sodium hydrogencarbonate 1g (1)-(4) All components were dissolved in distilled water 100ml After that, 1 ml each was dispensed into ampoules to manufacture 1000 injections.

[0060]

[Reference Example 4] Production of inhalation aerosol

2% by weight of the compound represented by the formula (I), 33% by weight of ethanol, and 65% by weight of propellant (a mixture of propane: isobutane = 70: 30) were filled in a valve-equipped pressure resistant container to produce an inhalation aerosol agent. did.

[0062]

Example 1 White powder of FR901469 substance produced in Reference Example 1 was administered to a nude mouse in which severe Pneumocystis carinii pneumonia was formed, and the therapeutic effect was determined by determining the number of cysts of Pneumocystis carinii in the lung. It was judged by examining.

[0063]

[Materials and methods] 10 ⁴ Pneumocystis carinii cysts of 4 4-week-old (female) BALB were used.
/ C strain nude mice were nasally inoculated, and 4 months later, one mouse was dissected to examine the number of cysts in the lung before administration of the FR901469 substance. At the same time, it was also confirmed that severe carini pneumonia was formed. FR901
FR901469 for 2 mice in the 469 substance administration group
The substance was dissolved in 0.1 ml of physiological saline so that the concentration of the substance became 10 mg / kg, and then the substance was continuously administered to mice until the end of the experiment.
Subcutaneous injection was carried out for a day. 0.1m for 2 mice in control group
l of physiological saline was subcutaneously injected in the same manner. FR901
After administration of 469 substances, all mice in both groups were dissected and examined for the number of cysts in the lung. The number of cysts in the lung was calculated by preparing a 10% lung emulsion with a phosphate buffer solution, smearing 25 μl of the emulsion on a slide glass, and staining with toluidine blue under a microscope.

[0064]

[Results and Discussion] When FR901469 substance was administered to mice in which severe carinii pneumonia was formed, the number of cysts in the lung was significantly reduced as shown in Table 4 shown in Table 13 below.

[0065]

[Table 13]

The reduction rate of cysts in the FR901469 substance-administered group mice was 99.98% as compared with the control group.
From the above results, it was revealed that the FR901469 substance has a significant therapeutic effect on carini pneumonia.

[0067]

INDUSTRIAL APPLICABILITY The present invention has found that the WF11243 substance or a salt thereof is an excellent prophylactic / therapeutic agent for carini pneumonia.

[0068]

[Brief description of drawings]

FIG. 1 is a drawing showing a ¹³ C nuclear magnetic resonance spectrum of FR901469 substance.

FIG. 2 is a drawing showing a ¹ H nuclear magnetic resonance spectrum of FR901469 substance.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification number Internal reference number FI technical display location C12R 1: 645) 7804-4B (72) Inventor Tomoko Nakanishi Minami Otodo, Tsuchiura City, Ibaraki Prefecture 3- 14-17 (72) Inventor Masaharu Hashimoto 2-15-3 Umezono, Tsukuba, Ibaraki Prefecture 2-15-3 (72) Masakuni Okuhara 2-14-10 Umezono, Tsukuba, Ibaraki Prefecture

Claims (1)

[Claims] 1. A prophylactic / therapeutic agent for Pneumocystis carinii infection, which comprises, as an active ingredient, a WF11243 substance whose hydrochloride salt has the physical properties shown in Tables 1 to 4 below or a salt thereof. [Table 1] [Table 2] [Table 3] [Table 4]