JP3312243B2 - Chloride ion secretion inhibitor - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a chloride ion secretion inhibitor, and more particularly to an agent which inhibits chloride ion secretion in humans or other animals, and more particularly to creosote or a phenol derivative as a component thereof. And a chloride ion secretion inhibitor comprising:

[0002]

BACKGROUND OF THE INVENTION Creosote has conventionally been used as an intestinal fungicide or an antidiarrheal based on intestinal sterilization, an antidiarrheal or an antidiarrheal based on intestinal motility suppression and an antispasmodic.

[0003] That is, creosote is listed as a fungicide in item 1 of the category of antidiarrheals in column V in the approval standards for gastrointestinal drug production on page 240 of the 1988 edition of the Pharmaceutical Manufacturing Guidelines (edited by the Japanese Official Standards Association). . Also, Hiroshi Ito, “Pharmacology”
(Published by Kyodo Corporation, revised 6th edition on January 5, 1983)
Page 416 also states that creosote is used for intestinal preservation and has an expectorant effect by inhalation adaptation.
The Japanese Pharmacopoeia also states that it is used for expectoration, abnormal intestinal fermentation, food poisoning, and the like. The United States
Dispensary (The United States Dispensator
y), 27th ed. (1973), p. 355, also states that creosote is used as a fungicide for external use and as an expectorant for internal use. In addition, Norio Ogata (N.
Ogata) Other Pharmacology, 46
Vol., (1993), p. 173, states that creosote has an antidiarrheal action based on intestinal motility suppression.

The present inventor studied the pharmacological action of intestinal bactericide or creosote, which has been used as an antidiarrheal or expectorant based on the intestinal bactericidal action as described above, It has been found that creosote has a chloride ion secretion inhibitory action, which is a pharmacological action unexpected from conventional knowledge. The present invention has been completed based on this finding.

[0005] chloride secretion inhibitor of the present invention, Cleo <br/> saw store Rui are those comprising a mixture containing it as an active ingredient.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION Wood creoso
te, beechwood creosote) is primarily obtained by fractionation of wood tar and is transparent and liquid at room temperature (The Merck Index, S.M.
Budavari, published by Merck, 1989). It has a peculiar odor and is a mixture mainly composed of aromatic chemicals such as guaiacol and cresol (Ogata et al., N. Ogata,
T. Baba, Res. Commun. Chem. Pathol. Pharmacol. 66,
411-423, 1989). Other components include cresol, phenol and xylenol. This "creosote" is often mixed because of the same name, but is completely different from the "coal tar creosote" obtained from coal tar.

[0007] Creosote itself has been known as described above, and has been known in the Japanese Pharmacopoeia (13th revision) and the US National Formulary.
y) It is also listed in XII. The creosote is obtained by distilling tree tar obtained from trees such as beech, oak, maple, and pine, particularly from hardwoods, and then distilling the tree tar to 200 to 23.
This is a so-called wood creosote obtained by collecting a fraction at about 0 ° C. (760 mmHg) and is clearly distinguished from creosote obtained from coal tar (Norio Ogata, Tatsuya Baba, Research Communications Inc.) Chemical Pathology and Pharmacology, 66, 411-423, 1989 (N.
Ogata and T. Baba, Res.Commun.Chem.Pathol.Phar
macol. 66, 411-423, 1989)).

[0008] As described in the above literatures, creosote, which is an active ingredient of the drug of the present invention, contains about 20 to 35% of guaiacol, about 15 to 25% of cresol, phenol, cresols, xylenol. A mixture containing various phenol derivatives such as phenols, and is a colorless to pale yellow liquid having a characteristic smoke odor and tongue-like taste, and having a specific gravity of 1.064 or more.

By using the chloride ion secretion inhibitor of the present invention, secretion of an electrolyte solution containing chloride ions such as pancreatic juice and sweat can be suppressed. That is, chloride ion secretion is mainly performed from the chloride ion channel. The chloride ion channel is present in exocrine gland epithelial cells such as bronchi, sweat glands, liver and salivary glands, and is involved in secretion of electrolyte solution containing chloride such as pancreatic juice and sweat. A disease in which chloride channels are deficient is the genetic disease Cystic Fibrosis (CF). It is a highly mortal disease that causes systemic exocrine gland dysfunction, respiratory infections, and the like. The gene responsible for this has been cloned and the protein it produces is referred to as CFTR (Cystic Fibrosis Transmembrane Conductanc).
e Regulator), but this CFTR is c
It is known to be an AMP-dependent chloride ion channel. Gene deletion in CF is 50% of CFTR
The deletion of the eighth phenylalanine residue (ΔF508) is well known.

[0010] The opening mechanism of the CFTR is considered as follows. That is, first, cAMP in a cell is increased, whereby cAMP-dependent protein kinase A is increased.
(PKA) is activated and the regulatory region of CFTR (Regulato
(ry domain, R domain) is phosphorylated by this PKA, thereby opening the channel.

The drug of the present invention has a property of preventing the chloride ion channel from opening or a property of closing the open chloride ion channel, that is, the activity of the creosote or phenol derivative which is an active ingredient thereof, ie, a chloride ion secretion inhibitory activity. In humans and other animals, it is effective in suppressing gastric acid secretion, suppressing hyperhidrosis, suppressing GABAergic neurotransmission, suppressing runny nose, suppressing pollakiuria, and increasing abnormal chloride ion secretion.

[0012] Creosote, or a component thereof or a mixture containing the same, is used in a general form in medical drugs. Common forms include, for example, tablets, pills, powders, capsules (soft capsules, hard capsules), granules, oral liquids, and injections (intravascular, intramuscular, subcutaneous, skin) Internal administration) or suppositories. Tablets, granules, when preparing in the form of powder, conventionally known carriers can be widely used, for example, lactose, sucrose, glucose, starch, excipients such as crystalline cellulose, such as hydroxypropylcellulose, methylcellulose, Gelatin, tragacanth, gum arabic,
Binders such as sodium alginate, for example, starch, carboxymethyl cellulose, disintegrants such as calcium carbonate,
For example, lubricants such as magnesium stearate, talc, stearic acid and the like can be used. The tablet may be coated with a normal coating as needed, and may be, for example, a sugar-coated tablet, a film-coated tablet, or the like, and may be a two-layer tablet or a multilayer tablet. In addition, granules and powders can be coated with a normal coating.

For the purpose of suppressing chloride ion secretion,
For dosages of creosote or its components,
Since it depends on the type or sex of the target animal, gender, age, and degree of symptoms, it cannot be unconditionally determined. However, in the case of oral administration or rectal administration (suppository) in humans, it is approximately 1 kg / adult body weight per day. 1 to 500
mg, preferably 2 to 100 mg. Also,
In the case of administration as an injection, adult weight per day is 1
The amount is 0.2 to 300 mg, preferably 0.2 to 50 mg per kg. These daily doses may be administered in 2 to 4 divided doses.

[0014]

EXAMPLES Pharmacological tests performed on the chloride ion inhibitor of the present invention will be described below. Pharmacological test 1 [ Examination of chloride ion secretion inhibitory action using fluorescent indicator in cultured cells] In this experiment, cells were established from human pancreatic cancer in which a normal chloride ion channel (CFTR) is genetically deficient. CFPAC-1 (ATC
C (American Type Culture Collection) CRL-1
918, Address: 12301 ParklawnDrive Rockville, MD
20852 USA, Tel: (301) 881-2600, FAX: (301) 816-4365)
And MDCK cells (ATCC CCL-34), which contain normal CFTR in dog kidney-derived cells.

100% MDCK cells in a culture flask
Culture in culture medium (Iscove's modified Dulbecco's medium] 90%, fetal calf serum; 10%, 2 mM calcium chloride until confluence (3
After 7 ° C., 5% CO ₂ /95% air), 10 mM N-
(Ethoxycarbonylmethyl) -6-methoxy-quinolium bromide (10 mM N- (ethoxycarbonylmethyl) -6-m
ethoxy-quinolinium bromide, chloride ion fluorescent indicator,
The mixture was replaced with a culture solution containing MQAE), and culturing was performed overnight. Next, the cells were treated with buffer 1 (2.4 mM dipotassium hydrogen phosphate [K ₂ HPO ₄ ], 0.6 mM potassium dihydrogen phosphate [KH ₂ PO ₄ ], 10 mM Hepes [Hepe]
s], washed three times with 10 mM D-glucose, 1 mM magnesium sulfate, 130 mM sodium chloride, 1 mM calcium sulfate, pH 7.4) and then 0.25% (w / v)
Cells are detached with trypsin, 0.02% (w / v) ethylenediaminetetraacetic acid (EDTA) solution, and
It was suspended at a density of × 10 ⁶ cells / ml. Take 1 ml of this cell suspension and centrifuge (1000 rpm × 4
Min) and the supernatant was removed, and 3 ml of buffer 2 (2.4 mM dipotassium hydrogen phosphate [K ₂ HPO ₄ ], 0.6 mM potassium dihydrogen phosphate [KH ₂ PO ₄ ], 10 mM Hepes [H
epes], 10 mM D-glucose, 1 mM magnesium sulfate, 130 mM sodium nitrate, 1 mM calcium sulfate, pH 7.4), and after stirring, put into a fluorescence cell and measure fluorescence with a fluorescence spectrophotometer (excitation wavelength;
360 nm, fluorescence wavelength; 450 nm).

In order to open the CFTR of MDCK cells, 250 μM of 8- (4-chlorophenylthio) -cyclic AMP (8- (4-chlorophenylthienyl) was added two and three minutes after the start of measurement.
o) -cyclic AMP, CPT-cAMP, and membrane-permeable cAMP) are shown in FIG. 1 (a). One minute after the start of measurement, 30 μm
g / ml creosote (wood creosote) was added, and 1.3 minutes later, 250 μM CPT-cAMP was added. The results are also shown in FIG. 1 (b). In addition, 30 μg / ml
FIG. 1 also shows creosote alone (c) and the result (d) without any addition. In addition, CPT-cAMP
Is added to time 0 minutes.

As a result, it was found that creosote inhibited cAMP-dependent chloride ion secretion observed only in MDCK cells.

The fact that chloride ion secretion from MDCK cells is from a cAMP-dependent chloride ion channel (CFTR) was confirmed using CFPAC-1 cells in CPT-c.
For the case where AMP was added and the case where nothing was added,
The same experiment as in MDCK cells was performed and confirmed. The result is shown in FIG.

Pharmacological test 2 [ Examination of chloride ion secretion inhibitory effect using ³⁶ Cl in cultured cells] A test was performed using MDCK cells used in pharmacological test 1. A culture solution of MDCK cells up to 100% confluence (Eagle's E.M.
s MEM medium]) After culturing with 90% and 10% fetal calf serum (the culture conditions were the same as in pharmacological test 1), the culture solution was 1.1 μC
The culture solution was replaced with a culture solution to which labeled sodium chloride (Na ³⁶ Cl) was added so as to be i / ml, and the culture was further performed for 20 hours. Next, 5 ml of buffer 3 (10 mM Hepes, 138
mM sodium chloride, 5 mM potassium chloride, 2.5 mM
Sodium dihydrogen phosphate, 1.8 mM calcium chloride,
1 mM magnesium sulfate, 10 mM D-glucose,
The cell layer was washed three times with pH 7.2) and 0.25% (w /
v) Cells were detached with trypsin, 0.02% (w / v) EDTA solution, and suspended in buffer solution 3. The cell suspension was centrifuged (1000 rpm × 4 minutes), and the supernatant was removed.
(10 ml Hepes, 138 mM sodium nitrate, 5 mM
Potassium nitrate, 2.5 mM sodium dihydrogen phosphate,
1.8 mM calcium nitrate, 1 mM magnesium sulfate,
10 mM D-glucose, was suspended in pH 7.2), and centrifuged under the same conditions, the supernatant was removed, with buffer 4 1 × 10 ⁶
Cells / ml.

Next, CPT-cAMP or creosote was added as described below, and the mixture was allowed to stand for 8 minutes with stirring so as not to precipitate cells. Then, the mixture was centrifuged (under the same conditions as above), 100 μl of the supernatant was put in a ready cap (manufactured by Beckman) which was a solid scintillator, dried, and then measured with a liquid scintillation counter. When 250 μM CPT-cAMP was added to 1 ml of this cell suspension, 250 μM CPT-cAMP and 30 μg were added.
FIG. 3 shows the results obtained when / ml creosote was added, when nothing was added (blank), and when only 30 μg / ml creosote was added.

The results shown in FIG. 3 indicate that creosote inhibits chloride ion secretion from the cAMP-dependent chloride channel (CFTR).

Pharmacological Test 3 [ Examination of Concentration Dependence in Creosort's Chloride Channel Inhibiting Effect] The creosote concentration was determined to be 0, 1, 3, 10, 15, 30, 30 in the same manner as in the aforementioned pharmacological test 2.
The effect of 250 μM CPT-cAMP on chloride ion secretion was tested by changing to μg / ml. FIG. 4 shows the results.

As can be seen from the figure, the concentration dependence of creosote's chloride ion channel inhibitory action was confirmed.

[0024]

According to the present invention, a novel chloride ion secretion inhibitor can be provided. By using the chloride ion secretion inhibitor of the present invention, secretion of an electrolyte solution containing chloride ions such as pancreatic juice and sweat can be suppressed.

[Brief description of the drawings]

FIG. 1 is a graph showing the results of pharmacological test 1.

FIG. 2 is a graph showing other results in pharmacological test 1.

FIG. 3 is a graph showing the results of pharmacological test 3.

FIG. 4 is a graph showing the results of pharmacological test 4.

──────────────────────────────────────────────────の Continued on the front page (58) Field surveyed (Int. Cl. ⁷ , DB name) A61K 35/78 A61K 31/05 BIOSIS (DIALOG) CA (STN) MEDLINE (STN)

Claims (4)

(57) [Claims] 1. A inhibits chloride secretion inhibitor secretion of chloride ions, Kureoso store Rui chloride secretion inhibitor characterized from consisting that mixtures containing it as an active ingredient. 2. The chloride ion secretion inhibitor according to claim 1, which is used for treating hyperhidrosis. 3. The chloride ion secretion inhibitor according to claim 1, which is used for treating nasal hypersecretion. 4. The chloride ion secretion inhibitor according to claim 1, which is used for treating gastric hyperacidity.