JP2010173973A - Cyp2a6 inhibitor and method for screening substance having cyp2a6 inhibitory activity - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a CYP2A6 inhibitor and a method for screening a substance having the CYP2A6 inhibitory activity. <P>SOLUTION: This CYP2A6 inhibitor contains (+)-camphor, (-)-camphor, (+)-borneol, (-)-borneol, (+)-fenchone, (-)-fenchone and a mixture of them as active ingredients. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a screening method for a CYP2A6 inhibitor and a CYP2A6 inhibitory active substance.

  Cytochrome P450 (CYP) is an enzyme mainly contained in the liver, and is said to be involved in about 80% of human drug metabolic reactions. CYP increases the water solubility by oxidizing a fat-soluble substrate and plays a role in facilitating the discharge from the body.

  CYP2A6 is a major metabolic enzyme of nicotine, which is the main component of tobacco, and metabolizes nicotine into an intermediate iminium ion. This iminium ion is further metabolized to the final product cotinine by aldehyde oxidase. Loss of nicotine due to metabolism in smokers leads to withdrawal symptoms such as lack of concentration, anxiety, and insomnia, and attempts to maintain nicotine levels by repeating smoking to eliminate these symptoms.

  Therefore, paying attention to the function of this CYP2A6, by suppressing the expression or enzyme activity of CYP2A6, the nicotine is not metabolized in the body for a long time and attempts to reduce the number of smokers' smoke (reduction of the number of smokers) or carcinogenicity Attempts to reduce the risk of carcinogenesis by preventing the activation of substances have been reported (for example, Patent Document 1).

  However, the CYP2A6 inhibitors proposed here are methoxalene, tranylcypromine, psoralen, pilocarpine, coumarin, chromone, esculetin, phenelzine, paroxetine, selegiline, and pargyline, all of which are anti-tinea agents. Prescription administration instructions are necessary and cannot be performed on a daily basis.

  In addition, the same document describes a CYP2A6 inhibitor using a natural product or an extract of a natural product, such as an extract such as Hypericum, Cicholium intybus or Bougainvllra spectabillis, in particular esculetin, Although esculin or esculin monohydrate is disclosed, its inhibitory effect is not satisfactory and its acquisition is not easy.

  Patent Document 2 describes a CYP2A6 inhibitor containing a soybean saponin extract fraction and / or a licorice extract as an active ingredient.

JP 2001-524516 A JP 2006-169186 A

  An object of the present invention is to provide a screening method for a CYP2A6 inhibitor and a CYP2A6 inhibitory active substance.

  As a result of intensive studies to achieve the above-mentioned object, the present inventor has found that monoterpenes, particularly bicyclic monoterpenes, specifically camphor, borneol, fenchone and the like are CYP2A6. It was found to have inhibitory activity.

  Conventionally, ketamine and 3-hydroxycotinine have been used as internal standard substances in screening methods for CYP2A6 inhibitory action. However, ketamine has been designated as a narcotic and is difficult to obtain, and 3-hydroxycotinine is expensive and has similar properties to the metabolites of cotinine, making accurate evaluation difficult. Therefore, this time, by using readily available trans-4'-carboxycotinine ester as an internal standard substance, it is possible to evaluate cheaply, simply and accurately compared to conventional screening methods. I found it. This was further developed and the present invention was completed here.

  That is, the present invention relates to the following CYP2A6 inhibitor and a screening method for a CYP2A6 inhibitory active substance (ie, a metabolic inhibitor of nicotine).

  Item 1. A CYP2A6 inhibitor containing (+)-camphor, (-)-camphor, (+)-borneol, (-)-borneol, (+)-fencon, (-)-fencon, and mixtures thereof as active ingredients.

  Item 2. CYP2A6 containing, as an active ingredient, an essential oil containing (+)-camphor, (-)-camphor, (+)-borneol, (-)-borneol, (+)-fencon, (-)-fencon, and mixtures thereof Inhibitor.

  Item 3. Item 3. A smoking frequency reducing agent comprising the CYP2A6 inhibitor according to Item 1 or 2.

  Item 4. Item 3. A preparation comprising the CYP2A6 inhibitor according to Item 1 or 2 and nicotine.

Item 5. A method for screening a CYP2A6 inhibitory substance,
(1) A step of adding a sample to a mixed solution containing CYP2A6, cytosol, NADPH generation system, nicotine, and potassium phosphate buffer, and reacting the mixture,
(2) adding a trans-4′-carboxycotinine ester as an internal standard substance and a reaction terminator to the reaction solution obtained in (1) above to stop the reaction; and
(3) After centrifuging the reaction solution obtained in (2) above, taking the supernatant, quantifying the amount of cotinine produced by HPLC,
A method comprising the steps of:

  The CYP2A6 inhibitor containing the specific bicyclic monoterpene of the present invention as an active ingredient has a high CYP2A6 inhibitory action. Therefore, using a CYP2A6 inhibitor, the metabolism of nicotine in the body can be delayed to maintain the nicotine concentration for a long time, and as a result, the amount of smoking can be reduced. The CYP2A6 inhibitor can be administered in various forms such as oral, transdermal, transpulmonary, and inhalation.

  Moreover, the screening method for CYP2A6 inhibitory substances of the present invention can be evaluated inexpensively, simply and accurately compared to screening methods in which ketamine or 3-hydroxycotinine is used as a conventional internal standard substance.

It is a graph which shows CYP2A6 inhibitory activity of various bicyclic monoterpenes.

The CYP2A6 inhibitor of the present invention includes monoterpenes (C ₁₀ ), bicyclic monoterpenes, specifically (+)-camphor ((+)-camphor), (−)-camphor ((−)-camphor). ), (+)-Borneol ((+)-borneol), (-)-borneol ((-)-borneol), (+)-fencon ((+)-fenchone), (-)-fencon ((-) -fenchone), and a mixture thereof as an active ingredient. Among these, (+)-camphor, (+)-Fencon, (-)-Fencon and the like are preferable because of their high CYP2A6 inhibitory activity.

  Moreover, since these terpenes are known as essential oil components of various plants, they are safe and easily available. The CYP2A6 inhibitor of the present invention includes essential oils containing the above-mentioned terpenes. For example, camphor, borneol and foncon can be isolated from plants such as camphor, fennel, dragon brain, and lavender.

  Essential oils from plants containing the above terpenes can be extracted using known methods. As for the extraction method, the corresponding plant can be subjected to extraction as it is, but it is preferable to subject the plant to extraction after further pulverization.

  The extraction solvent is not particularly limited. For example, alcohols such as methanol, ethanol, propanol and butanol; glycols such as 1,3-butylene glycol, glycerin and propylene glycol; esters such as ethyl acetate and butyl acetate Hydrocarbons such as hexane, cyclohexane and petroleum ether; water and the like can be used. These extraction solvents can be used singly or in combination of two or more. In particular, the use of at least one selected from the group consisting of alcohols such as methanol and ethanol and esters such as ethyl acetate and butyl acetate is easy to handle and provides an extract having excellent activity. It is preferable in that it can be performed.

  After obtaining the extract by the above-described method, the extract can be obtained by solid-liquid separation from the residue by a conventional method such as filtration or centrifugation, if necessary. In the present invention, the obtained extract can be used as it is as a CYP2A6 inhibitor. However, since the activity may be low, it may be used in the form of an extract or powder by appropriately concentrating or distilling off the solvent. it can.

  The CYP2A6 inhibitor of the present invention has a function of reducing the number of smokers smoking by suppressing the expression or enzyme activity of CYP2A so that nicotine is not metabolized in the body for a long time. Therefore, it is useful as a smoking frequency reducing agent.

  The CYP2A6 inhibitor is prepared by conventional methods such as tablets, powders, fine granules, granules, coated tablets, capsules, syrups, elixirs, troches, inhalants, suppositories, injections, ointments, It can be formulated into eye ointments, eye drops, nasal drops, ear drops, poultices, lotions and the like. Excipients, binders, lubricants, colorants, flavoring agents, and if necessary stabilizers, emulsifiers, absorption promoters, surfactants, pH adjusters, preservatives, Antioxidants and the like can be used. In general, ingredients and blending amounts that are generally used as raw materials for pharmaceutical preparations are appropriately selected to prepare a preparation by a conventional method.

  In the case of administering the preparation of the present invention, the administration form is not particularly limited and may be any method that is usually used, and may be any of oral, transdermal, pulmonary, inhalation, topical, rectal, and the like. Preferable examples include chewing gum, nasal spray, mouth spray, mouthwash, transdermal patch and the like. In particular, it is preferable to use in the form of a transdermal skin patch, aerosol administration with a nebulizer or the like, spraying into a space with a diffuser, or the like.

  The CYP2A6 inhibitor of the present invention can be used together with nicotine to allow nicotine to remain in the body for a long time without being metabolized, thereby further reducing the smoking impulse of the smoker. For example, the above-mentioned CYP2A6 inhibitor preparation can be used in combination with known nicotine preparations (eg chewing gum, nasal spray, mouth spray, mouthwash, transdermal patch, etc.). Moreover, the CYP2A6 inhibitor and nicotine may be contained in the preparation.

  In order to reduce the number of smokers who smoke, the CYP2A6 inhibitor of the present invention can be contained in the filter of the cigarette with filter. Moreover, it is also possible to spray into space using a diffuser in a smoking room or the like.

In addition, the screening method for the CYP2A6 inhibitory active substance (ie, nicotine metabolism inhibitor) of the present invention comprises:
(1) A step of adding a sample to a mixed solution containing CYP2A6, cytosol, NADPH generating system, nicotine, and potassium phosphate buffer to prepare and react a plurality of reaction solutions having different sample concentrations;
(2) adding a trans-4′-carboxycotinine ester as an internal standard substance and a reaction terminator to the reaction solution obtained in (1) above to stop the reaction; and
(3) After centrifuging the reaction solution obtained in (2) above, taking the supernatant, quantifying the amount of cotinine produced by HPLC,
including.

  According to this screening method, in the reaction of step (1), for example, baculovirus recombinant CYP2A6 is used as CYP2A6, pooled human liver cytosol is used as cytosol, and 0.5 mM NADP +, 5 mM is used as NADPH production system. A system such as G-6-P or 0.5 units / mL G-6-P DH is used. As nicotine, commercially available products can be used. For example, products such as Tokyo Chemical Industry Co., Ltd., Kanto Chemical Co., Inc., Fluka, SIGMA, ALDRICH, etc. can be used. The potassium phosphate buffer can be adjusted to pH 7.0 to 8.0 (particularly 7.4). Examples of the solvent for dissolving the sample include dimethyl sulfoxide (DMSO). The sample concentration in the plurality of reaction solutions can be appropriately selected depending on the degree of CYP2A6 inhibitory activity. The reaction can usually be performed at about 37 ° C. for 30 to 120 minutes.

  In the step (2), trans-4'-carboxycotinine ester (particularly methyl ester) which is an internal standard substance is used. Moreover, as a reaction terminator, it is a reagent which can stop the reaction of an enzyme, Usually, acetonitrile / methanol mixed solution, acetone, etc. are used. The above-mentioned internal standard substance is prepared by converting readily available trans-4'-carboxycotinine to diazomethane, for example, by reacting with diazomethane. As trans-4'-carboxycotinine, commercially available products can be used. For example, products such as Tokyo Chemical Industry Co., Ltd. and ALDRICH can be used.

In step (3), the reaction solution stopped in step (2) is centrifuged at 4 to 10 ° C. and 1000 to 3000 rpm for 10 to 30 minutes, and the supernatant is sampled to produce cotinin by HPLC. Quantify the amount. The HPLC analysis conditions can be appropriately set. For example, an ODS column is used, acetonitrile / sodium phosphate buffer is used as the mobile phase, the flow rate is 0.1 to 1.5 mL / min, and the column temperature is 30 to 40 ° C. Can be set. For detection of cotinine, for example, a UV wavelength of 260 nm is used. An absolute calibration curve for cotinine can be created to calculate the amount of cotinine produced. Inhibitory activity is evaluated by determining the 50% inhibitory concentration (IC ₅₀ value) of each sample.

  According to this screening method, the remarkable effect that it can be evaluated cheaply, simply and accurately is exhibited as compared with the conventional screening method using ketamine or 3-hydroxycotinine as an internal standard substance.

Next, the present invention will be specifically described, but the present invention is not limited thereto.
[HPLC analysis conditions]
L-column ODS (4.6 × 150 mm, manufactured by CERI) was used, the mobile phase was 10% acetonitrile / 25 mM sodium phosphate buffer, the flow rate was 1.0 mL / min, and the column temperature was 40 ° C. A UV wavelength of 260 nm was used for detection. The amount of cotinine produced was calculated from an absolute calibration curve for cotinine. The inhibition was evaluated by determining the IC ₅₀ value, which is the 50% inhibitory concentration of each.
[Preparation method of internal standard substance]
30 mg of trans-4′-carboxycotinine (Tokyo Kasei Kogyo Co., Ltd.) was dissolved in 20 mL of acetone, 2 mL of diazomethane was added and reacted at room temperature for 20 minutes. After the reaction, the solvent was distilled off to obtain 39 mg of trans-4′-carboxycotinine methyl ester which is a methyl ester form.

Example 1 (Analysis method of nicotine metabolism using HPLC)
Six types of bicyclic monoterpenes of (+)-camphor, (−)-camphor, (+)-borneol, (−)-borneol, (+)-fencon, (−)-fencon were used as samples, The following nicotine metabolism analysis was performed.

  5 μL baculovirus recombinant CYP2A6 (20 nmol / mL, manufactured by BD), 15 μL pooled human liver cytosol (0.3 mg / mL, manufactured by BD), 52.5 μL NADPH generating system (0.5 mM NADP +, 5 mM G-6− P, 0.5 units / mL G-6-PDH), 10 μL of nicotine (50 mM), and 50 mM potassium phosphate buffer (pH 7.4) were mixed to make a total volume of 250 μL. This mixture was reacted at 37 ° C. for 30 minutes. Monoterpene, an inhibitor, was dissolved in DMSO and prepared to 200, 100, 50, and 25 μM in a total reaction volume of 250 μL.

  After the reaction, 25 μL of trans-4′-carboxycotinine methyl ester (25 μM) as an internal standard substance and 250 μL of a mixed solution of acetonitrile / methanol (1: 1, v / v) as a reaction terminator were added. After stopping the reaction, the mixture was centrifuged at 4 ° C. and 3,000 rpm for 20 minutes, and the supernatant was taken and the amount of cotinine produced was quantified by HPLC. The result is shown in FIG.

FIG. 1 shows inhibitory activity in all bicyclic monoterpenes. In particular, (+)-camphor, (+)-fencon, and (-)-fencon showed the strongest inhibitory effect, and all had an IC ₅₀ value of 22 μM. Moreover, it was suggested that the inhibitory effect tends to be higher in the ketone body than in the alcohol body.

Example 2 (formulation example)
Using the camphor and foncon analyzed in Example 1, preparations of Production Examples 1 to 5 below can be prepared.

  Formulation Example 1 (Patch formulation adhesive gel)

  Formulation Example 2 (Patch formulation adhesive gel)

  Formulation Example 3 (Spray formulation)

  Formulation Example 4 (Spray formulation)

Formulation Example 5 (chewing gum formulation)
The following composition A is prepared.

  The above composition A is mixed with the following gum base and fragrance to produce a chewing gum.

  Formulation Example 6 (Patch formulation adhesive gel)

  Formulation Example 7 (Patch formulation adhesive gel)

  Formulation Example 8 (Spray formulation)

  Formulation Example 9 (spray formulation)

Formulation Example 10 (chewing gum formulation)
The following composition B is prepared.

  The above composition B is mixed with the following gum base and fragrance to produce a chewing gum.

Claims (5)

A CYP2A6 inhibitor containing (+)-camphor, (-)-camphor, (+)-borneol, (-)-borneol, (+)-fencon, (-)-fencon, and mixtures thereof as active ingredients. CYP2A6 containing, as an active ingredient, an essential oil containing (+)-camphor, (-)-camphor, (+)-borneol, (-)-borneol, (+)-fencon, (-)-fencon, and mixtures thereof Inhibitor. A smoking frequency reducing agent comprising the CYP2A6 inhibitor according to claim 1 or 2. A preparation comprising the CYP2A6 inhibitor according to claim 1 or 2 and nicotine. A method for screening a CYP2A6 inhibitory substance,
(1) A step of adding a sample to a mixed solution containing CYP2A6, cytosol, NADPH generation system, nicotine, and potassium phosphate buffer, and reacting the mixture,
(2) adding a trans-4′-carboxycotinine ester as an internal standard substance and a reaction terminator to the reaction solution obtained in (1) above to stop the reaction; and
(3) After centrifuging the reaction solution obtained in (2) above, taking the supernatant, quantifying the amount of cotinine produced by HPLC,
A method comprising the steps of:

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