JPH10120562A - V-atpase uncoupling proton pump inhibitor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject agent capable of treating and preventing diseases such as osteoporosis by compounding a cycloprodigiosin. SOLUTION: This agent contains a cycloprodigiosin or its salt, preferably cycloprodigiosin hydrochloride as an active component. The compound has V-ATPase-decoupling H<+> -pump inhibiting activity and is useful as a chemical reagent, an osteoporosis treating agent, an arteriosclerosis treating agent, an antibiotic, an anticancer agent stimulator, a sedative, etc. The daily dose of this compound is, in the case of oral administration, 0.1-0.5/kg, and in the case of parenteral administration, especially of injection, 0.1-0.5mg/kg. This compound is produced without having contaminants of other prodigiosins by culturing the strain Pseudoalteromonas denitrificans AK-1 (FERM P-15771), a kind of marine bacteria.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a V-ATPase uncoupling H ⁺ pump inhibitor. V-ATPas of the present invention
e The uncoupled H ⁺ pump inhibitor is composed of V-ATPase (vacuolar ATPase, vacuolar H
⁺ -ATPase), and inhibits only the H ⁺ (proton) pump activity, and can treat or prevent osteoporosis and other various diseases caused by the activity.

[0002]

2. Description of the Related Art Organelles belonging to the central vacuolar system such as synaptic vesicles, Golgi apparatus, vacuoles, and lysosomes have ATP which transports a group of protons.
ase exists. This ATPase is named after its location, V-ATPase (vacuolar ATPase).
, A vacuolar-type ATPase). V-ATPase
Is different from F-ATPase, an ATP synthase present in mitochondria, etc., and P-ATPase present in the gastric mucosa and controlling the acidification of gastric juice in terms of both structure and sensitivity to various drugs. It is an enzyme. V
-ATPase functions in conjunction with ATP hydrolysis to
To transport ⁺ into the organelle and create an electrochemical potential difference between H ⁺ inside and outside, or to acidify the pH inside. The difference in electrochemical potential of H ⁺ formed and the acidic pH are closely linked to the specific functions of each organelle. Examples include degradation of foreign substances in lysosomes and concentration of neurotransmitters in synaptic vesicles. It is also said to be involved in receptor recycling and protein sorting.
Bowman et al. Found that bafilomain, a macrolide antibiotic, was a potent inhibitor of V-ATPase (Proc. Natl. Acad. Sci. USA., 85 , 7972-7976 (198
8)).

[0003] Bifilomycin was found to inhibit acid secretion from macrophages, indicating that V-ATPase present in the plasma membrane regulates pH inside and outside cells (J. Biol). .Chem., 265, 7645-7654 (1990))
. Furthermore, it was confirmed that V-ATPase is involved in acid secretion from osteoclasts (J. Cell Biol., 111, 1305).
-1311 (1990)). That, V-ATPase is not only the internal granular acidic, also present in the plasma membrane, H ⁺
Is released outside the cell to create a locally acidic field outside the cell. In particular, V-ATPase is associated with the wavy edge of osteoclasts (ruf
It is present at the plasma membrane, called the fled border, and releases H ⁺ into the house resorption site, the Howship fossa, bringing the pH of the site to approximately 4, and its acidic pH releases Ca2 ⁺ from bone tissue. It facilitates the degradation and promotes the degradation of collagen by activating the protease.

[0004] V-ATP such as bafilomycin
It has been reported that ase inhibitors inhibit cholesterol ester accumulation (Eur. J. Biochem., 207, 383-
389 (1992)), and a substance having an activity of inhibiting acidification of acidic organelles by V-ATPase is expected to be used as a drug for treating or preventing arteriosclerosis. Also,
It is known that cancer cells become resistant to a plurality of anticancer agents at the same time. This is because P-glycoprotein (multi
It is thought that a drug efflux pump called drug resistant factor (MDR) exists and V-ATPase is involved in this drug efflux mechanism (Biochem. Pharmaco.
l. 36 , 2879-2887 (1988)). That is, daunomycin, an anticancer drug that serves as a substrate for P-glycoprotein, is concentrated in acidic organelles, which are considered to be lysosomes of drug-resistant cell lines, and the addition of bafilomycin makes it difficult to excrete daunomycin outside the cells. Increase the sensitivity of the cells to (J. Natl, Cancer Inst, 83 , 10
98-1102 (1991)). Therefore, in view of this, V-AT
The acidification inhibitory activity of acidified organelles by a Pase inhibitor has an anticancer drug-enhancing effect and is expected to be used as a medicine. Further, diphtheria toxin is considered to be activated by being cleaved by proteases in endosomes, to enter the cytoplasm and to exhibit toxicity. V-ATP
Addition of ase-inhibiting bafilomycin to cultured cells effectively inhibits the proteolysis of the endomembrane system involved in endocytosis, resulting in fragmentation and consequent loss of diphtheria toxin toxicity (J. B
iol. Chem. 265 , 21940-21945 (1990)). Viruses such as vesicular stomatitis virus and influenza virus are known to bind to receptors on animal cell surfaces and then enter the cells as endosomes.
It is clear that V-ATPase is involved
(J. Gen. Virol. 75 , 2595-2606 (1994); J. Virol. 70 , 5
76-579 (1996)). Therefore, it is considered that the V-ATPase inhibitor is used as an effective antiviral agent. Also,
Malaria is a disease caused by infection with malaria parasites, and it has recently been revealed that intracellular acidification is involved in the process of invasion and proliferation of malaria parasites into host hepatocytes and erythrocytes. Furthermore, it has been pointed out that V-ATPase is also present in the cell membrane of malaria parasite, and that this activity may be involved in infection. These things are appropriate V-ATPa
The use of inhibitors of se could protect against malaria infection. Synaptic vesicles are small organelles with a diameter of about 50 nm, which are often present in nerve endings, and are important for nerve chemical transmission. That is, a high concentration of neurotransmitter contained in the vesicle is released into the synaptic cleft by stimulation. V-ATPase accounts for about 10% or more of the total protein of synaptic vesicles, and contains H ⁺
(J. Exp. Biol. 172, 92-99 (1992)). The difference in electrochemical potential of the formed H ⁺ drives transport of mediators present in the vesicle membrane (monoamine transport, glutamate transport, γ-aminobutyric acid, acetylcholine transport)
(Biochem. Biophys. Res. Commun. 173, 443-448 (1991)). V
-Inhibition of ATPase also inhibits the transport system (ibid.). Therefore, inhibiting H ⁺ pumps V-ATPase would be expected as a sedative.

The present inventors have studied the V-ATPase inhibitory effect using various compounds in order to develop a novel substance having a V-ATPase inhibitory activity such as bafilomycin. Species of Pseudoalteromonas s denitri
ficans ) The red substance cycloprodigiosin hydrochloride produced by the AK-1 strain (FERM P-15771) is used for the uncoupling of V-ATPase H ⁺
It was found to have pump inhibitory activity. The use of this compound as an active ingredient of a V-ATPase uncoupled H ⁺ pump inhibitor was examined. In addition, they have found that free cycloprodigiosin obtained by removing hydrogen chloride from this compound has the same effect. Accordingly, the present invention provides a therapeutic agent for osteoporosis, a therapeutic agent for arteriosclerosis, an antibiotic, an anticancer agent, a sedative agent, etc., utilizing such a V-ATPase uncoupling H ⁺ pump inhibitory activity of cycloprodigiosin or salts. The present invention relates to a drug or a reagent for inducing organelles to be neutral. Conventionally, cycloprodigiosin has been known to have antibacterial and antifungal activities (Tetrahedron Letters 30 (13) 1725
(1989)), nothing is known about the above-mentioned inhibitory activities of cycloprodigiosin and its hydrochloride.

[0006]

SUMMARY OF THE INVENTION In view of the above,
An object of the present invention is to provide a novel V-ATPase uncoupling H ⁺ pump inhibitor. A further object of the present invention is to provide a novel use of cycloprodigiosin and salts thereof.

[0007]

DISCLOSURE OF THE INVENTION The present invention has been made to solve the above-mentioned problems, and is directed to cycloprodigiosin (formula I) or cycloprodigiosin hydrochloride (formula
II) V-ATPas containing other salts as active ingredients
e For uncoupled H ⁺ pump inhibitors.

[0008]

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[0009]

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Examples of such compounds include cycloprodigiosin or cycloprodigiosin hydrochloride, phosphate, sulfate, perchlorate, carboxylate (eg, acetate, citrate, oxalate) Etc.), iodate, bromate, picrate, sulfonate, tetrafluoroborate and the like.

[0011] cyclo Puroji Gio Singh, of marine bacteria Alteromonas rubra, (Alteromonas rubra) (Tetrahedron
Letters 24 (26), 2701-2704 (1983)) or Benekea gazogenes (ibid. 24 (27), 2)
797-2798 (1983)).
It is also known to synthesize 2-methylcyclohexanone as a starting material through the following steps (Tetrahedron Lett
ers 25 , (13) 1387-1388 (1984)).

[0012]

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The above compounds such as cycloprodigiosin hydrochloride can be obtained by converting cycloprodigiosin thus obtained into a salt form. As a means for forming a salt, a usual salt forming means can be used.
However, since cycloprodigiosin obtained from these marine bacteria produces several types of prodigiosins in the form of exudates, it is complicated to isolate cycloprodigiosin alone from the extract. Required a great isolation operation. In addition, the method for producing cycloprodigiosin by the chemical synthesis requires complicated steps over many steps.

The present inventors have been studying metabolites of marine bacteria and found that Pseudoalteromonas denitrificans (Pseudoalteromonas denitrificans).
ans) The AK-1 strain (FERM P-15771) was found to produce cycloprodigiosin hydrochloride without the inclusion of other prodigiosins. Therefore, when the cells are cultured, a large amount of cycloprodigiosin hydrochloride is produced and accumulated in the cells and in the medium, so that it can be produced in large amounts with high purity. This cycloprodigiosin hydrochloride can easily remove hydrogen chloride by column chromatography to obtain free cycloprodigiosin without hydrochloride. Then, the free cycloprodigiosin (referred to as a compound represented by the formula (I) in which hydrogen chloride has been removed from cycloprodigiosin hydrochloride) is treated with an acid such as phosphoric acid or sulfuric acid to obtain the above-mentioned salts. be able to. The cycloprodigiosin hydrochloride and other salts used in the present invention are soluble in water and can be used after being dissolved in distilled water or the like. Therefore, it can be used as an injection, a drink and the like.

According to the present invention, cycloprodigiosin or cycloprodigiosin hydrochloride and other salts are V-ATPas
e Uncoupling It has been found based on this finding that it has H ⁺ pump inhibitory activity.

The following experiment confirmed that cycloprodigiosin or a salt thereof according to the present invention has a V-ATPase uncoupling H ⁺ pump inhibitory action. [Experiment 1] PC12 cells (rat adrenal medulla cells) with or without differentiation induction by nerve growth factor (NGF) were cultured in 10% HS 5% FBS-DMEM medium. Cycloprodigiosin hydrochloride (50 nM) was added to the cells, and the cells were cultured for 1 hour. Then, the change in pH in the granules of the cells was observed with a fluorescence microscope (FIGS. 1 and 2) and a phase contrast microscope (FIGS. 3 and 4) using the accumulation of acridine orange as an index. The results are shown in FIGS. That is, FIGS. 1 and 3 show the state of cells in which PC12 cells were first induced to differentiate by NGF. 2 and 4 show the state of the cells that have not been induced to differentiate. The states of the cells treated with the cycloprodigiosin hydrochloride are shown as B and D. A and C are controls not treated with cycloprodigiosin hydrochloride. In the control, as shown by the arrow, the differentiated nerve terminal-like projections are stained with acridine orange. This is considered to be a synaptic vesicle, which is an acidic vesicle. In addition, around the cell nucleus, granules that seem to be relatively large lysosomes are stained. On the other hand, when treated with the above-mentioned cycloprodigiosin hydrochloride, the acridine orange staining of the protruding portion disappeared, indicating that the acidic environment of this portion disappeared.

On the other hand, lysosomes were not significantly affected by this concentration of cycloprodigiosin hydrochloride, but the staining of acridine orange in the lysosome was completely abolished by the concentration of cycloprodigiosin hydrochloride of 1 μM. . This indicates that cycloprodigiosin hydrochloride neutralizes the inside of synaptic vesicles, which are acidic organelles, and also the inside of lysosomes at 1 μM.

[Experiment 2] (1) Using cycloprodigiosin hydrochloride at the concentration shown in FIG. 5, a phospholipid planar membrane method (July 10, 1992, issued by Tokyo Chemical Dojin, Japan) The Biochemical Society of Japan, New Biochemistry Laboratory Lecture, “Biomembrane and Membrane Transport (1)”, pp. 181-187). Thus, a specific resistance value was obtained. An increase in the specific resistance value indicates that the membrane is directly damaged. As a control, carbonyl cyanide-P-trifluoromethoxyphenylhydrazone was used. The results are shown in FIG. As can be seen in FIG. 5, the specific resistance of carbonyl cyanide-P-trifluoromethoxyphenylhydrazone increases significantly with increasing concentration, whereas the specific resistance of cycloprodigiosin hydrochloride increases. Very little direct damage to the membrane. This also suggests that cycloprodigiosin hydrochloride does not show ionophore activity and has very low toxicity.

(2) The medulla part was isolated from bovine adrenal gland, crushed with a Waring blender, and the crushed liquid was filtered through four layers of gauze. The filtrate was centrifuged and the chromaffin granule fraction was subjected to sucrose density gradient centrifugation. Prepared. The obtained chromaffin granule fraction was subjected to hypotonic treatment and centrifuged to obtain a chromaffin granule membrane fraction.

The V-ATPase of the chromaffin granule membrane thus obtained was stimulated with cycloprodigiosin hydrochloride at the concentration shown in FIG. 6 for 10 minutes, and the proton pump activity was measured using the accumulation of acridine orange as an index. did. On the other hand, ATPase activity was determined by quantifying the amount of inorganic phosphorus released from ATP. The result is shown in FIG. As shown in FIG. 6, the proton pumping activity is completely inhibited by 10 nM cycloprodigiosin hydrochloride. In contrast, V-ATPase activity was not inhibited at all even when cycloprodigiosin hydrochloride was used in a 1000-fold amount.

Furthermore, the effect of cycloprodigiosin hydrochloride on the bone resorption activity of osteoclasts based on the V-ATPase uncoupling H ⁺ pump activity inhibitory effect was examined. In other words, on November 10, 1994, published by Kyoritsu Shuppan Co., Ltd., edited by Tadao Ohno, “A Manual for Alternative Methods to Animal Experiments, Theory and Application Using Cultured Cells,” p. 102 Measurement method (J. Bona Miner. Ris., 8, 953-960 (199
According to 3)), the effect of cycloprodigiosin hydrochloride on the ability to form osteoclasts was examined.

That is, mouse bone marrow cells and osteoblasts were co-cultured on collagen gel in the presence of active vitamin D ₃ 10 ^-8 M. On the sixth day of the culture, the culture plate was treated with collagenase, and the formed osteoclast fraction was collected. The osteoclast fraction was spread on ivory sections (diameter: 4 mm) and cultured for 8 to 24 hours in the presence of a solution of cycloprodigiosin hydrochloride (0.1 to 100 nM) (n = 4). After completion of the culture, the cells on the sections were removed, and the resorption pits formed by the osteoclasts were stained with Mayer's hematoxylin staining solution. The resorption pits formed by osteoclasts are stained red. Therefore, the area of the resorption cavity was measured, and the area of the resorption cavity not treated with cycloprodigiosin hydrochloride was set to 100, and the resorption cavity area ratio of osteoclasts was determined.
The results are shown in Table 1.

[0023]

[Table 1] 面積 Cycloprodigiosin absorption area ratio Hydrochloride concentration (nM) ───────い な い Untreated 100.0 0.1 85.0 1 79.8 10 53.9 100 19.6 ────────────────── ───────

As shown in the table, cycloprodigiosin hydrochloride strongly suppressed the ability of osteoclasts to form resorption pits at 100 nM. Therefore, since cycloprodigiosin hydrochloride strongly inhibits the bone resorption function of osteoclasts, it can be used as a therapeutic drug for osteoporosis using this activity.

As described above, cycloprodigiosin hydrochloride has no ionophore activity in the phospholipid planar membrane method (1),
In the experiment using chromaffin granule membrane in (2), V-ATPase
Specifically inhibits only H ⁺ pump activity. Such a property is the same as that of conventional V-ATPas such as bafilomycin.
e It is not recognized as an activity inhibitor, and it is considered that the action of cycloprodigiosin is due to a completely different mode of action from conventional V-ATPase inhibitors. The cycloprodigiosin hydrochloride in the present invention specifically inhibits V-ATPase uncoupled H ⁺ pump activity without inhibiting V-ATPase activity. Therefore, it may be used for the following applications based on this activity inhibiting action.

(1) Reagents Used for Analysis of Phenomena Involving Acidified Organelles In the acidified organelles, as shown in Experiment 1, the effective concentration of neutralization differs depending on the organelles. It can be induced and can be used as a reagent to analyze the accompanying phenomenon.

(2) Drug for treating osteoporosis With the aging, the concentration of serum calcium is kept unchanged without change, but the secretion of calcium metabolizing hormone is greatly changed. Parathyroid hormone works to stimulate the mobilization of calcium into extracellular fluid from bones by mobilizing osteoclasts, but the blood level of this hormone increases, and its antagonist, calcitonin, reduces Hormones that suppress the action of cells and reduce calcium mobilization, on the contrary, decrease. It is also said that the blood concentration of active vitamin D decreases. Changes in these hormones
It is said that calcium is released from bone, and the calcium keeps serum calcium concentration constant and deposits on the inner wall of blood vessels to trigger arteriosclerosis. Therefore,
Neutralizing acidic pH suppresses the release of Ca ²⁺ , and is expected to act as a therapeutic agent for osteoporosis.

(3) Arteriosclerosis inhibitor Also, since it has been reported that a V-ATPase inhibitor inhibits the accumulation of cholesterol ester, an action as an arteriosclerosis inhibitor is also expected. (4) Anticancer drug effect enhancer It is known that cancer cells become resistant to a plurality of anticancer drugs, because a drug efflux pump called P-glycoprotein exists in the plasma membrane. It has been pointed out that V-ATPase may be involved in such a drug excretion mechanism. However, the addition of bafilomycin, which is known as a V-ATPase inhibitor, makes the anticancer drug daunomycin, which serves as a substrate for P-glycoprotein, become cell-free. It is hard to be discharged outside,
At the same time, there is a report that the sensitivity to daunomycin increases. That is, cycloprodigiosin hydrochloride, which is a V-ATPase uncoupled H ⁺ pump inhibitor, also inhibits drug excretion, and can be expected to have an anticancer drug-enhancing effect.

(5) Antibiotics It has been clarified that the process of expressing toxicity of diphtheria toxin and the process of infecting viruses with animal cells involves endosome acidification via endosome V-ATPase. Therefore, cycloprodigiosin hydrochloride, a V-ATPase uncoupled H ⁺ pump inhibitor, can also be expected as a new inhibitory antiviral agent. In addition, V-
Since it has been shown that ATPase may be involved, cycloprodigiosin hydrochloride can be expected as an effective antimalarial agent. (6) Sedatives By inhibiting the H ⁺ pump, the transport system of neurotransmitters is inhibited. Therefore, cycloprodigiosin hydrochloride can also be expected as a sedative by inhibiting this transport system.

When these medicaments are used in the present invention, they may be used alone or in combination with other medicaments. In particular, when used as a therapeutic agent for osteoporosis, it may be used in combination with calcitonin, vitamin D or an inorganic calcium salt. When used as an anticancer drug enhancer,
You may use together with an anticancer agent. Furthermore, when used as a reagent, it can be used in the form of a kit in combination with acridine orange or the like.

When these are used as medicaments, cycloprodigiosin, cycloprodigiosin hydrochloride or other salts can be used as such or in combination with other drugs. Usually, it is administered orally or parenterally in the form of a suitable formulation together with excipients and other auxiliaries. Oral preparations include powders, granules, tablets, sugar coatings, pills, capsules, drinks and the like.
Parenteral preparations include suppositories, cataplasms, injections and the like. The injection may be in the form of intramuscular, arterial or intravenous injection. As these excipients and other auxiliaries, lactose, sucrose, various starches, glucose, cellulose or derivatives thereof, magnesium stearate, talc and the like are used. Further, a solvent such as distilled water can be used. These can be made into oral or parenteral preparations by means commonly known as preparation means. The dose varies depending on symptoms, age, gender, etc., but in the case of oral administration, cycloprodigiosin or its hydrochloride or other salt is 0.1 to 0.5 / kg body weight per day,
In the case of parenteral administration, it is desirable to administer 0.1 to 0.5 mg / kg of body weight per day by injection.

[0032] For the toxicity of these compounds, cyclo Puroji Gio thin hydrochloride ICR female mice (5 weeks old, body weight 25~29g) LD ₅₀ value was administered intraperitoneally was at least 10 mg. Further, when the specific resistance of cycloprodigiosin hydrochloride was measured by the phospholipid flat membrane method as described above, no ionophore activity was observed, and thus it was judged that the toxicity was extremely low.

A production example of the cycloprodigiosin hydrochloride of the present invention will be described. Bacto peptone (Difco) 2.5g, Bacto yeast extract (Difco) 0.5g and iron (III) phosphate n-hydrate (Wako Pure Chemical Industries) 0.1g are dissolved in 1L of natural seawater, sterilized and 3ml Inside, Pseudoalteromonas denitrificans AK
-1 strain (FERM P-15771) was inoculated, and cultured with shaking at 15 to 20 ° C for 1 day. The obtained culture was cultured in a medium 6 having the above composition.
Transferred to 00 ml and cultured for 1 day.

The medium cultured in this way was 22,000 r.
After centrifugation at pm for 20 minutes, the cells were collected. 5-10 cells
g was recovered. A mixed solution of acetone-diethyl ether (4: 1) was added to the cells, and a dye (cycloprodigiosin hydrochloride) was extracted at 200 shaking / min. Extract at 12,000 rpm for 10
After centrifugation for minutes, a supernatant was obtained. Anhydrous magnesium sulfate was added to the supernatant to adsorb and remove water, and the magnesium sulfate was removed by filtration. The filtrate was concentrated to dryness and analyzed by thin layer chromatography (K
ieselgel 60 F ₂₅₄ ; developing solvent benzene: ether = 1:
1). A single spot was obtained at Rf 0.20. This concentrate was dissolved in 5 ml of methylene chloride, and recrystallized from this solution by utilizing a temperature difference. Wash the generated crystals with pentane,
5 mg of crystals were collected by suction filtration. The crystals are needle-like crystals with a red metallic luster, have no melting point, and
Decomposed at 30 ° C. The crystal was subjected to elemental analysis and its M
ass spectrum, UV spectrum, IR spectrum, H-NMR
Analysis of the spectrum showed that it was consistent with that of cycloprodigiosin hydrochloride (Chem. II) (Tetrahedron Letters,
24 (26), 2701-2704 (1983)).

5 mg of cycloprodigiosin hydrochloride thus obtained was dissolved in 10 ml of methylene chloride and passed through a silica gel column (diameter 1.5 cm, length 12 cm, flow rate 1.2 ml / min), and the solvent was distilled off. Free cycloprodigiosin 4mg
I got The fact that this substance was free cycloprodigiosin was confirmed by data such as N-NMR.

Next, formulation examples are shown as examples.

Example 1 Cycloprodigiosin hydrochloride 0.5 g Lactose 400 g Potato starch 100 g Microcrystalline cellulose 30 g Light anhydrous silicic acid 30 g Cycloprodigiosin 0.5 g, Lactose 400 mg, Bayrecio starch 100 mg, Crystalline cellulose 30 mg and Light anhydrous 30 mg of silicic acid was mixed, and a solution of 20 mg of hydroxypropylcellulose in methanol (10% by weight of hydroxypropylcellulose) was added, kneaded, and granulated. Next, this is
The mixture was extruded through a 0.8 mm screen to form granules, dried, added with 10 mg of magnesium stearate, and tableted in 200 mg increments to give tablets.

[0037]

Example 2 Cycloprodigiosin hydrochloride 20 mg Tween 80 0.4 g Distilled water 200 ml Cycloprodigiosin hydrochloride 20 mg was dissolved in distilled water 200 ml together with Tween 80 0.4 g, and filled in a 20 ml ampoule.
The solution was sterilized by heating to prepare an intravenous injection.

[0038]

According to the present invention, the cycloprodigiosin or the hydrochloride salt thereof has a V-
These compounds can be effectively used as chemical reagents, osteoporosis drugs, arteriosclerosis drugs, antibiotics, anticancer drug enhancers, sedatives, etc. by utilizing the ATPase uncoupling H ⁺ pump inhibitory action. When the obtained agent is used as a medicine, it can be expected to have a therapeutic effect on these diseases by administering orally or parenterally. When used as a reagent, it may be used as a kit.

[Brief description of the drawings]

FIG. 1 shows fluorescence micrographs (400 × magnification) of PC12 cells treated or not treated with cycloprodigiosin hydrochloride in Experiment 1.

[Explanation of symbols]

A: PC12 cells induced to differentiate with NGF and not treated with cycloprodigiosin hydrochloride B: PC12 cells induced to differentiate and treated with cycloprodigiosin hydrochloride

FIG. 2 shows fluorescence micrographs of PC12 cells treated with cycloprodigiosin hydrochloride or untreated PC12 cells in Experiment 1 (400 × magnification).

[Explanation of symbols]

C: PC12 cells not induced to differentiate and not treated with cycloprodigiosin hydrochloride D: PC12 cells not induced to differentiate and treated with cycloprodigiosin hydrochloride

FIG. 3 shows phase contrast micrographs of PC12 cells treated with cycloprodigiosin hydrochloride or untreated PC12 cells in Experiment 1 (400 × magnification).

[Explanation of symbols]

A: PC12 cells induced to differentiate with NGF and not treated with cycloprodigiosin hydrochloride B: PC12 cells induced to differentiate and treated with cycloprodigiosin hydrochloride

FIG. 4 shows phase contrast micrographs of PC12 cells treated with cycloprodigiosin hydrochloride or untreated PC12 cells in Experiment 1 (400-fold magnification).

[Description of References] C: PC12 cells not induced to differentiate and not treated with cycloprodigiosin hydrochloride D: PC12 cells not induced to differentiate and treated with cycloprodigiosin hydrochloride

FIG. 5 shows the specific resistance of the phospholipid flat membrane of cycloprodigiosin hydrochloride in Experiment 2 (1).

FIG. 6: V of cycloprodigiosin hydrochloride from experiment 2 (2)
-The effect on ATPase activity and V-ATPase activity is shown.

[Explanation of symbols]

-○-V-ATPase conjugated H ⁺ pump activity-×-V-ATPase activity

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI A61K 31/40 AED A61K 31/40 AED AGA AGA // C07D 403/14 207 C07D 403/14 207 (72) Inventor Shinichi Nakatsuji Hyogo 1479-1 Kaneji, Kamiguno-cho, Ako-gun, Japan Himeji Institute of Technology Inside the Company (72) Inventor Keiko Kawauchi 329 Sakagoshi, Ako City, Hyogo Prefecture Inside the Ako Kasei Co., Ltd.

Claims (3)

[Claims] 1. V-ATPase (vacuolar ATP) containing cycloprodigiosin or a salt thereof as an active ingredient.
ase) Uncoupled H ⁺ pump inhibitor. 2. The inhibitor according to claim 1, wherein the active ingredient is cycloprodigiosin. 3. The inhibitor according to claim 1, wherein the active ingredient is cycloprodigiosin hydrochloride.