ANTAGONISTS FOR NICOTINIC ACETYLCHOLINE RECEPTOR, WHICH ARE CONTAINING BORNEOL OR CAMPHOR AS AN ACTIVE INGREDIENT
Technical Field
The present invention relates to an antagonist for nicotinic acetylcholine receptor, which contains borneol or camphor as an active ingredient. More specifically, the present invention relates to a novel medical use of a composition containing borneol of formula (1) or camphor of formula (2), as defined below, as an active ingredient, which specifically inhibits nicotinic acetylcholine receptor, which plays an important role for neurological activities, to inhibit secretion of catecholamine and therefore, can be effectively used as an agent for treatment of CNS diseases such as Alzheimer's disease, Parkinson's disease, etc., local anesthetic agent, muscle relaxant, anti-stress agent, adjuvant for prohibition of smoking, an agent for treatment of withdrawal syndrome, etc.
Background Art
Borneol ([(lS)-endo]-l,7,7-trimethylbicyclo[2.2.1]heptan-2-ol) and camphor
([lS]-endo)-l,7,7-trimethylbicyclo[2.2.1]heptan-2-one) are bicyclic triterpenes having two rings and have been used as an anti-inflammatory agent (US 5,889,049), an agent for treatment of diseases related to nitric oxide (US 5,990,177), an additive for anti-dandruff agent to provide a feeling of refreshment (International Patent No. 9939638) and the like.
They have also been used for the purpose of anti-pruritus, anesthesia, feeling of
refreshment, etc. (International Patent No. 9956548).
Borneol and camphor together with humulene (2,6,6,9-tetramethyl-l,4,8-cyclo- undecatriene) are the major ingredients in borneolum as the material for Chinese medicine. In the field of Chinese medicine, borneolum is a medicinal substance, which has been widely used for controlling heatstroke, toxicosis, abdominal pain, etc., as an agent for providing a feeling of refreshment and known as stimulating central nervous system and having an antibacterial activity against Staphylococcus aureus, E. coli, hog cholera, etc. In Korea, borneolum has been generally used as a component of "Woowhang Chung Shim Won" (see Korean Patent Publication No. 87-6998) and also been used as an indoor aromatic due to its specific odor (Korean Laid-open Patent Publication No. 96-46125). However, it has never been disclosed so far that borneol and camphor act specifically upon nicotinic acetylcholine receptor so that they can be used as an agent for treatment of various diseases, that is, an agent for treatment of CNS-related diseases such as Alzheimer's disease, Parkinson's disease or Huntington's disease, local anesthetic agent, muscle relaxant, anti-stress agent, adjuvant for prohibition of smoking, an agent for treatment of withdrawal syndrome, etc.
Dopamine, norepinephrine and epinephrine are generally called catecholamine and produced from tyrosine or phenylalanine in cerebral cells, adrenal medulla cells, sympathetic cells, etc. Catecholamines have been known as playing an important role in stress and emotional behaviors (see Cooper JR, Bloom FE, Roth RH, 1991, The biochemical basis of neuropharmacology, pp. 220-284, Oxford University Press) and act upon both peripheral and central nervous systems. When external signal is transmitted to nerve cells, calcium level in cells is increased, which acts just as a signal for secretion of neurotransmitters through nervous interstice to induce physiological phenomena including
heart rate, respiration, muscular contraction, metabolic control via control of insulin secretion, etc. Therefore, it has been known that many kinds of drugs for treatment of psychosis and anesthetic drugs act upon the regions related to mechanism of catecholamine secretion (see Seeman P, Nan Tol HH, 1994, Trends Pharmacol. Sci., 15: 264-270).
The receptors for signal transmission, which play an important role in catecholamine secretion include acetylcholine receptors, which are classified into muscarinic acetylcholine receptors and nicotinic acetylcholine receptors depending on the kinds of ligands and the mechanisms for signal transmission in cells. Muscarinic acetylcholine receptors exhibit their functions by the action of muscarine, which is an alkaloid present in mushrooms, as well as acetylcholine, and can be divided into several subgroups. The subgroups of muscarinic acetylcholine receptors include M2, which can open the calcium channel by activating G, proteins, Ml, M3 and M5 which are linked to G proteins such as Gθ7 Gq, etc. to activate phospholipase C, and M4, which activates G, thereby activating adenyl cyclase. As reviewed above, although all muscarinic acetylcholine receptors are linked to G proteins, they exhibit different responses depending on the types of existing cells. Meanwhile, nicotinic acetylcholine receptors also combine with nicotine as well as acetylcholine, and mediate the stimulant responses which can last only for about 1/1000 second. The nicotinic acetylcholine receptors form calcium and sodium ion channels, which can be opened and blocked by the ligand binding. As stated above, although muscarinic acetylcholine receptors and nicotinic acetylcholine receptors are different from each other in view of their acting mechanisms, both of them are acetylcholine receptors, and therefore, generally exhibit biological activities for controlling numerous functions of central and peripheral nervous systems including control of neurotransmitter secretion, respiration, appetite, drug intoxication, emotion, muscular
movement, memory, recognition, concentration, etc., which are greatly different depending on the kinds of ligands for receptor.
It has been continuously and extensively studied to develop materials useful for treatment of various diseases through materials acting upon the receptors for such neurotransmitters or hormones. By way of example, Zantac® and Tagamet® as an agent for inhibiting H2 histamine receptor have been developed as an inhibitor of gastric acid secretion, and further, Salaracin® as an agent for inhibiting angiotensin receptor has been developed as an antihypertensive agent.
Thus, the present inventors have studied in order to find the novel compound which specifically acts only upon nicotinic acetylcholine receptor. As a result, we have identified that borneol and camphor, which have been known only as a substance for providing refreshment feeling and as an aromatic substance, specifically act only upon nicotinic acetylcholine receptor without acting upon calcium channel, sodium channel and receptors linked to phospholipase C, and therefore, can be used as an agent for treating diseases related to the activity of nicotinic acetylcholine receptor with little side effect, and thus completed the present invention.
Disclosure of the Invention
The purpose of the present invention is to provide a compound, which specifically acts only upon nicotinic acetylcholine receptor to inhibit its activity and reduce the secretion of catecholamine and therefore, is useful as an antagonist for nicotinic acetylcholine receptor.
Another purpose of the present invention is to provide a compound, which can be used as an agent for treatment of diseases related to the activity of nicotinic acetylcholine receptor, and has little side effect and is very safe.
Brief Description of Drawings
For a thorough understanding of the nature and purposes of the present invention, reference should be made to the following detailed description taken in connection with the accompanying drawings in which: Figure 1 is a graph showing that catecholamine secretion through activation of nicotinic acetylcholine receptor is inhibited by borneolum.
Figure 2a is a graph showing that catecholamine secretion through activation of nicotinic acetylcholine receptor is inhibited by borneol;
Figure 2b is a graph showing that catecholamine secretion through activation of nicotinic acetylcholine receptor is inhibited by camphor;
Figure 3a is a graph showing that calcium ion level in cells through activation of nicotinic acetylcholine receptor is inhibited by borneol ;
Figure 3b is a graph showing that sodium ion level in cells through activation of nicotinic acetylcholine receptor is inhibited by borneol;
Figure 4a is a graph showing that calcium ion level in cells through activation of nicotinic acetylcholine receptor is inhibited by camphor;
Figure 4b is a graph showing that sodium ion level in cells through activation of
nicotinic acetylcholine receptor is inhibited by camphor; and
Figure 5 is a graph showing that an increase of calcium ion level in cells through activation of calcium ion channel (A), sodium ion channel (B) and bradykinin receptor linked to phospholipase C (C) is not influenced by borneol, wherein a solid line represents the case not treated with borneol and a dotted line represents the case treated with 100 μM borneol.
Best Mode for Carrying Out the Invention
In order to achieve the above-mentioned purposes, the present invention provides an antagonist for nicotinic acetylcholine receptor, which contains borneol represented by the following formula (1) or camphor represented by the following formula (2):
Further, compound [(lR)-endo]-(+)-l,7,7-trimethylbicyclo[2.2.1]heptan-2-ol
represented by the following formula (3), which is an enantiomer of borneol, exhibits the same pharmacological effect as borneol and camphor above:
The above compounds specifically act only upon nicotinic acetylcholine receptor to inhibit catecholamine secretion and reduce the level of calcium and sodium ions in cells to be increased when nicotinic receptor is activated.
Therefore, the above compounds can be effectively used as an agent for treatment of CNS diseases related to the activity of nicotinic acetylcholine receptor, local anesthetic agent, muscle relaxant, anti-stress agent, an antidote for nicotine toxication and adjuvant for prohibition of smoking, an agent for treatment of withdrawal syndrome, etc. The CNS diseases include Alzheimer's disease, Parkinson's disease and Huntington's disease, etc.
Although the above compounds as an agent for treatment of above-mentioned diseases can be prepared by any of the generally known chemical synthetic methods, they can preferably be obtained by extraction from borneolum (Dryobalanops aromatica Gaertner) as the material for Chinese medicine. The use of borneolum extract containing said compounds is more advantageous than the chemical synthesis in view of safety and manufacturing costs.
Further, the present invention includes a pharmaceutical composition containing one or more compounds according to the present invention together with a non-toxic, inert, pharmaceutically acceptable excipient, and a process for preparing said composition. For clinical purpose, the compound of the present invention can be administered via oral or parenteral route and can be prepared and used in any of conventional pharmaceutical formulations.
For practical administration in the clinical field, the compounds of the present invention can be administered in any form of various oral and parenteral formulations, which are prepared using conventionally used fillers, extenders, binders, wetting agents, disintegrants, diluents including surfactants or excipients.
The present invention also includes the pharmaceutical formulation in a dosage unit form. The formulation can be in individual dosage form, for example, tablets, coated tablets, capsules, pills, suppositories and ampules, wherein the content of the active ingredient in the unit dosage form corresponds to a fraction or a multiple of the individual dose. The unit dosage form can contain, for example, one, two, three or four times or 1/2, 1/3 or 1/4 times of the individual dose. The individual dose is preferably a single dose of the active ingredient, which corresponds conventionally to a total of, or 1/2, 1/3 or 1/4 times, the daily dosage.
The non-toxic, inert, pharmaceutically acceptable excipients include solid, semi-solid or liquid diluents, fillers or all types of formulation auxiliaries.
The preferable formulations include tablets, coated tablets, capsules, pills, granules, suppositories, solutions, suspensions and emulsions, pastes, ointments, gels, creams, lotions, powders and sprays, etc.
In the preferred embodiment according to the present invention, 50% inhibitory concentration (IC50) of borneol is about 70 μM and IC50 of camphor is about 55 μM. Thus, the effective dose of the compound according to the present invention is 1.5-13.2 mg kg, more preferably 1.5-3.1 mg/kg.
The present invention will be more specifically explained hereinafter.
In the preferred embodiment of the present invention, we have searched for the compounds having antagonistic activity against nicotinic acetylcholine receptor among numerous materials for Chinese medicine. About 50 kinds of materials for Chinese medicine were extracted with hot water to obtain respective hot water extracts, which were studied in cells of bovine adrenal medulla to find out a material capable of inhibiting catecholamine secretion due to activation of nicotinic acetylcholine receptor. As a result, we could find that borneolum exhibits a most potent activity.
Bovine adrenal medulla cells have been generally and widely used as the model cell for controlling catecholamine secretion since they have both the characteristics of nerve cells and endocrine cells and therefore, secret catecholamine under external stimulation (see Park TJ, Shin SY, Suh BC, Suh EK, Lee IS, Kim YS, Kim KT, 1998, Synapse, 29: 248-256). Therefore, the present invention used those bovine adrenal medulla cells as the model cell. The level of catecholamine secretion was measured by introducing [3H]-norepinephrine into the cells, treated the cells with the extract of borneolum and then measuring the radioactivity in the culture medium to determine the amount of [3H]-epinephrine secreted out of the cells.
As a result, catecholamine secretion due to activation of nicotinic acetylcholine receptor was inhibited to 50% by 6 μg/ml of borneolum and completely inhibited by 30
μg/ml of borneolum. Although the increase of calcium ion level in cells plays an important role in catecholamine secretion, the increase of calcium ion level in cells due to activation of nicotinic acetylcholine receptor was also inhibited by borneolum. Further, the activation of nicotinic acetylcholine receptor also induces an increase of sodium ion level in cells, which was also inhibited by borneolum.
Since the result derived from inhibition of an increase of calcium and sodium ion levels in cells as mentioned above is similar to the effect of inhibiting catecholamine secretion, it can be identified that catecholamine secretion is inhibited by acting the extract of borneolum directly upon nicotinic acetylcholine receptor to inhibit the activity of receptor.
The effect of borneol, which is a constituent of borneolum, on catecholamine secretion was also examined. As a result, it could be identified that borneol effectively inhibited catecholamine secretion with 50% inhibition at 70 μM concentration and 100% inhibition at 300 μM concentration. The increase of calcium ion and sodium ion levels in cells due to activation of nicotinic acetylcholine receptor was also inhibited by borneol to the similar extent. Therefore, it can be seen that borneol directly combines with nicotinic acetylcholine receptor to inhibit the activity of nicotinic acetylcholine receptor, thereby inhibiting catecholamine secretion.
It has been known that calcium channel, sodium channel and receptors linked to phospholipase C as well as nicotinic acetylcholine receptor increase calcium ion level in cells and induce catecholamine secretion. Thus, in order to confirm whether or not the effect of borneol and camphor according to the present invention is originated from a specific inhibition of the activity of nicotinic acetylcholine receptor, a test was conducted
to examine the effect of borneol and camphor on the increase of ion levels in cells due to the activation of calcium channel, sodium channel or receptors linked to phospholipase C. As a result, it could be identified that borneolum and borneol have no effect on the increase of calcium ion levels in cells due to the activation of calcium channel, sodium channel or receptors linked to phospholipase C. Therefore, it can be seen that borneolum and its major ingredient, borneol specifically inhibit only the activity of nicotinic acetylcholine receptor and eventually inhibit catecholamine secretion.
The present invention is more specifically illustrated by the following examples. However, it should be understood that these examples are provided only for illustration of the present invention but not intended to limit the present invention in any manner.
Example 1
Inhibitory effect of borneolum on catecholamine secretion due to activation of nicotinic acetylcholine receptor
Borneolum was extracted in water bath for 3 hours or more to obtain the hot water extract. Aliquot of the extract was lyophilized to weigh its mass. Bovine adrenal medulla cells were separated into medullary cells and cortical cells by treatment with collagenase and then only medullary cellular tissues were collected and treated again with collagenase to separate the cells from tissues. Finally, the cells were passed through a fine nylon mesh to obtain only a single cell, which was then incubated for 2 to 3 days in DMEM/F12 medium (pH 7.4, Life Technologies, Inc.) for cell culture.
To determine catecholamine secretion 1 μCi/ml of radioactively labeled [ H]-norepinephrine and 0.1 M vitamin C were added to the medium of adrenal medulla
cells and introduced into cells by incubating at 37°C for one hour. Then, [3H]-norepinephrine which is not introduced into cells was washed out by Locke's solution (154 mM sodium chloride, 5.6 mM potassium chloride, 5.6 mM glucose, 1 mM calcium chloride, 1 mM magnesium chloride and 5 mM Hepes, pH 7.4). The cells were then treated with various concentrations of the hot water extract of borneolum for 10 minutes and the medium was collected. The radioactivity was measured by means of a liquid scintillation counter to determine the amount of [3H]-norepinephrine secreted out of the cells.
Figure 1 is the result showing that the secretion of [3H]-norepinephrine caused by treatment with DMPP (l,l-dimethyl-4-phenylpiperazineium iodide), which has been known to specifically activate nicotinic acetylcholine receptor, is inhibited depending on the concentration of borneolum. From this result, it can be seen that the secretion of [3H]-norepinephrine due to DMPP treatment is inhibited as the concentration of borneolum increases. Borneolum exhibited about 50% inhibitory effect at concentration of 6 μg/ml and 100% inhibitory effect at concentration of 30 μg/ml.
Example 2
Inhibitory effect of borneol on catecholamine secretion due to activation of nicotinic acetylcholine receptor
The test was conducted according to the same procedure as Example 1 except that borneol (Sigma, USA) was used instead of the extract of borneolum.
Figure 2a is the result showing that the secretion of [ H]-norepinephrine caused by DMPP treatment is inhibited depending on the concentration of borneol, which is the major
ingredient of borenolum. From this result, it can be seen that the secretion of [3H]-norepinephrine due to DMPP treatment is inhibited as the concentration of borneol increases. Borneol exhibited about 50% inhibitory effect at concentration of 70 μM and 100% inhibitory effect at concentration of 300 μM. Therefore, it may be regarded that the inhibitory effect of borneolum is originated from its major ingredient, borneol.
Example 3
Inhibitory effect of camphor on catecholamine secretion due to activation of nicotinic acetylcholine receptor
The test was conducted according to the same procedure as Example 1 except that camphor (Sigma, USA) was used instead of the extract of borneolum.
As a result, it can be seen that camphor exhibited 50% inhibitory effect at concentration of about 55 μM.
Example 4
Inhibitory effect of borneol on increase of calcium and sodium levels in cells due to activation of nicotinic acetylcholine receptor
1) Effect on calcium ion level
To determine a change of calcium level in cells, fura-2/AM (Molecular Probe Inc., USA), which is a fluorescent dye specifically binding to calcium, was introduced into the cells by adding 3 μM of the dye to the medium and then incubating bovine adrenal medulla cells at 37°C for 40 minutes. Then, fura-2/AM not introduced into the cells was washed
out. Ultraviolet ray was irradiated thereto using a spectrofluorometer in turns at 340 nm and 380 nm and then the fluorescence emitted at 500 nm was measured to obtain the rate to the increase of calcium level in cells. From the obtained value, calcium level was quantified by means of the following mathematical equation (1) (see Grynkiewicz G, Poenie M, Tsien RY, 1985, J. Biol. Chem., 260: 3440-3450):
Calcium level in cells = Kp[(R-Rmιn)/(Rmax-R)]/(S{2/Sb2) (1)
In the above equation (1), Kp denotes a dissociation constant of the complex of fura-2 and calcium; R denotes a ratio of fluorescences at 340 nm and 380 nm; Rmιn denotes a ratio of fluorescence when calcium level is 0; Rmax denotes a ratio of fluorescence when calcium level is in the state of saturation; S 2 denotes a fluorescent value when calcium level is 0; and Sb2 denotes a fluorescent value when calcium level is in the state of saturation.
Figure 3a is the result showing that the increase of calcium level in cells, which occurs by treating the cells with DMPP to specifically activate nicotinic acetylcholine receptor, is inhibited depending on the concentration of borneol. From this result, it can be seen that the increase of calcium level in cells due to DMPP treatment is inhibited as the concentration of borneol increases.
2) Effect on sodium ion level
Further, to determine a change of sodium level in cells, SBFI (sodium-binding benzofuran isophthalate, Molecular Probe Inc., USA), which is a fluorescent dye specifically binding to sodium, was introduced into the cells, and then SBFI not introduced
into the cells was washed out. Ultraviolet ray was irradiated thereto using a spectrofluorometer in turns at 340 nm and 380 nm and then the fluorescence emitted at 530 nm was measured to obtain the rate to the increase of sodium level in cells. From the obtained value, sodium ion level was quantified by means of the mathematical equation (1) as described in the above 1).
From the result as shown in Figure 3b, it can be seen that the increase of sodium level in cells, which occurs by treating the cells with DMPP to specifically activate nicotinic acetylcholine receptor, is inhibited depending on the concentration of borneol.
As shown above, in view of the fact that the inhibitory effect of borneol on the increase of calcium and sodium ion levels in cells due to activation of nicotinic acetylcholine receptor is similar to the inhibitory effect of borneol on catecholamine secretion, it can be regarded that borneol directly inhibits the activity of nicotinic acetylcholine receptor.
Example 5
Inhibitory effect of camphor on increase of calcium and sodium levels in cells due to activation of nicotinic acetylcholine receptor
The inhibitory effect of camphor on the increase of calcium ion and sodium ion levels in cells due to activation of nicotinic acetylcholine receptor was determined according to the same procedure as Example 4 except that camphor was used instead of borneol.
From the result as shown in Fig. 4, it could be seen that camphor inhibits the increase of calcium ion level at concentration of about 88 μM and further inhibits the
increase of sodium ion level by 50% at concentration of about 19 μM.
Example 6
Inhibitory effect of borneol on increase of calcium level in cells due to activation of calcium channel, sodium channel and receptors for phospholipase C
To identify whether or not the inhibition of increase of calcium and sodium ion levels in cells by borneol and camphor is derived from a specific inhibition of the activity of nicotinic acetylcholine receptor, the test was conducted to determine whether or not borneol can reduce the calcium level in cells due to activation of calcium channel, sodium channel and receptors for phospholipase C. To activate only calcium channel, the cells were treated with potassium chloride at concentration as high as 70 M to activate a potential-dependent calcium channel, sodium channel was activated by veratridine treatment, and bradykinin receptor for phospholipase C was activated by bradykinin treatememt. Borneol was added at concentration of 100 μM or not added (control group). The specific method for measuring a change of calcium level in cells is as shown in Example 4.
Figure 5A is the result showing that borneol has no effect on the increase of calcium level in cells, which occurs by treating the cells with high concentration of potassium chloride (KG) to activate a potential-dependent calcium channel; Figure 5B is the result showing that borneol has no effect on the increase of calcium level in cells, which occurs by treating the cells with veratridine, which has been known to activate a potential-dependent sodium channel; and Figure 5C is the result showing that borneol has no effect on the increase of calcium level in cells, which occurs by treating the cells with
bradykinin which activates bradykinin receptor linked to phospholipase C.
As a result, it can be seen that borneol has no effect on the activation of calcium channel, sodium channel and receptors linked to phospholipase C, all of which play an important role in signal transmission in nerve cells.
Industrial Applicability
According to the present invention, borneol and camphor specifically act only upon nicotinic acetylcholine receptor to inhibit catecholamine secretion, and therefore, can be effectively used as an agent for treatment of diseases related to nicotinic acetylcholine receptor, which has little possibility of occurring the side effect and is highly safe. Particularly, they can be effectively used as an agent for treatment of CNS diseases, local anesthetic agent, muscle relaxant, anti-stress agent, adjuvant for prohibition of smoking, an agent for treatment of withdrawal syndrome, etc.