KR20130103209A - Drug absorption enhancer for oral administration - Google Patents

Abstract

PURPOSE: A drug absorption enhancer for oral administration is provided to effectively accumulate a drug in cells and to minimize discharge of the drug to the outside of the cells, thereby enabling easy uptake of the drug into the cell. CONSTITUTION: A drug absorption enhancer for oral administration contains a compound of chemical formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient. The compound of chemical formula 1 enhances bioavailability and absorption of a drug in a dose-dependent manner. The compound helps drug absorption in gastrointestinal tract and is completely discharged to the outside from body.

Description

Drug absorption enhancer for oral administration

The present invention relates to oral administration absorption enhancers of drugs.

After the development of a new drug, the drug may be the basis for developing the drug to be administered to the living body so that the optimal pharmacological effect can be expressed in the simplest and most effective aspect. In general, the drug is usually administered three times a day, which is closely related to the half-life of the drug. If the drug can be maintained continuously after it is administered to the body, the lower the frequency of drug administration, the higher the medication compliance.

According to Cochburnet et al. In 1987, the compliance rate of medications was only 38% when administered three times a day, but as the number of doses decreased twice a day and once a day, compliance with the medication was 69. %, Increased to 90%. Thus, various studies have been conducted on techniques for promoting the absorption of drugs and improving the bioavailability through the diversification of administration routes as a method for convenience in drug administration.

Drug absorption promotion technologies include absorption enhancers, prodrugs or complexes, gastrointestinal stabilization / absorption, technology for transporting bio-essential materials, Nano / micro particle absorption absorption technology, poorly soluble drug solubilization technology, protein drug absorption promotion technology, and new route of administration.

Among them, absorption accelerators (absorption ehancer) is applied in various ways to the body and in vitro. When drugs are administered orally, most drugs are absorbed throughout the gastrointestinal tract but with varying degrees of absorption. However, in some drugs, the absorption site is limited to a part of the gastrointestinal tract, mainly due to physicochemical and biochemical properties. If the solubility of the drug varies greatly with pH, the pH of each part of the gastrointestinal tract varies from 1 to 8 (gastric pH 1.2-3.5, small intestine pH 6.3-8.0, large intestine pH 7.9-8.0). In particular, the absorption of the drug in a certain part may be limited by the pH difference, and in some cases, the absorption is limited by the presence of a specific enzyme. In addition, the conventional absorption accelerator has a disadvantage in that it is difficult to use as an absorption accelerator for oral use because the acceleration action is diminished when dilution and damage to the intestine when excessive use.

To date, the inhibitors of drug absorption in the gastrointestinal tract are known as P-glycoprotein (P-gp) and cytochrome P450 enzymes, which are ATP-dependent transporters in small intestinal epithelial cells. Cytochrome P450 (CYP3A), well known as the primary metabolic enzyme of drugs, is present in large amounts in the stomach and small intestinal epidermis, but its number and activity decreases as it descends into the large intestine, and this irregular distribution of CYP3A becomes a substrate for CYP3A. For some drugs (ACE inhibitors, antibiotics), their absorption may vary somewhat depending on the location of the gastrointestinal tract. In addition, P-glycoprotein expressed in small intestinal epithelial cells is expressed in the capillary endothelium of the blood brain barrier, as well as in the cell membranes of small intestine, biliary epithelial cells and renal epithelial cells. P-glycoprotein is an efflux transporter that releases various drugs for therapeutic purposes as well as harmful toxins out of the body, and is used in various tissues in the body (small intestine, large intestine, kidney, liver, brain, testes, placenta). (Cordon-Cardo et al., Proc. Natl. Acad. Sci., 86, pp695-698, 1989; Sugawara et al., Cancer Res., 68, pp1926-1929, 1988; Thiebaut et al., Proc. Natl.Acad. Sci., 84, pp7735-7738, 1987), which distributes the drug and limits the absorption of the drug in the intestine or the entry of the drug into the central nervous system. In particular, P-glycoprotein located in small intestinal epithelial cells is an important cause of inhibition of drug absorption (Lin and Yamazaki, Drug Metab. Rev., 35, pp417-454, 2003).

Thus, inhibition of the function of P-glycoprotein may increase the oral bioavailability of the drug or may promote the influx of the drug into the central nervous system, thereby increasing the effect of the drug. Accordingly, efforts have been made to increase the absorption of drugs by administering P-glycoprotein substrate drugs together with substances that inhibit P-glycoprotein activity.

Substances that promote drug absorption by inhibiting P-glycoprotein activity include verapamil, nifedipine, calmoduline antagonist, trifluoroperazine, and alkaloid, which are calcium channel inhibitors. Vincristine, vinblastine, and cyclosporine A, an immunosuppressive agent, are known.

Verapamil and nifedipine, like other calcium channel blockers, act as a substrate for P-glycoprotein and inhibit it. In vitro, nifedipine significantly increases the influx of calvediol (P-glycoprotein substrate) into kidney cells. Verapamil is known to cause side effects of gastrointestinal disorders such as angina and arrhythmia. Clinical application has failed (Woo et al., Pharm. Res., 20, pp 24-30, 2003; Kuppens et al., Cancer Invest., 23, pp443-464, 2005; Robert, Eur. J. Clin Invest., 29, pp 536-545, 1999). Vincristine and vinblastine are also known to have a number of serious side effects. Second-generation drug absorption enhancers, which are similar in structure to conventional first-generation drug absorption enhancers but have been developed to lose their efficacy, are used in drugs other than P-glycoprotein. Interactions with transporters and drug metabolizing enzymes are problematic and have limited applicability (Kuppens et al., Cancer Invest., 23, pp443-464, 2005). Such side effects, such as verapamil, are inferred to be due to the fact that the drug is absorbed into the verapamo density body while expressing its own efficacy and side effects while enhancing drug absorption.

Thus, the inventors of the present invention minimize the side effects, while developing a drug that enhances the gastrointestinal absorption of the drug, the compound according to the present invention confirmed that the drug has an excellent effect of promoting gastrointestinal absorption, and completed the present invention.

It is an object of the present invention to provide an oral absorption absorption enhancer of a drug which can not be absorbed into the body itself, but can be excreted to minimize side effects and enhance gastrointestinal absorption of the drug.

In order to achieve the above object, the present invention provides an oral absorption absorption enhancer of a drug that can excrete the compound itself is not absorbed into the body to minimize side effects and enhance the gastrointestinal absorption of the drug.

The compound according to the present invention not only has a better effect of accumulating the drug into cells than the conventional drug absorption enhancer verapamil, but also minimizes the drug released into the cell to facilitate the influx of the drug into the cell and the dose absorption of the drug. Dependently increasing the absolute bioavailability of the drug, and the compound of the present invention enhances the absorption of the drug in the body, and then itself is not absorbed into the body and is completely excreted, thereby causing other unnecessary effects and toxicity in the body. Since it does not have the effect, it can be usefully used as an absorption enhancer for oral administration of drugs.

1 is a diagram showing a graph comparing the amount of donomycin accumulated in the cells according to the compound of the formula (1) of the present invention.
2 is a graph showing a comparison of the amount of donomycin released to the extracellular according to the compound of formula (1) of the present invention.
Figure 3 is a diagram showing the concentration-time profile in plasma when orally administered with the compound of formula 1 of paclitaxel of the present invention.
4 is a diagram showing the concentration-time profile in plasma when orally administered verapamil with paclitaxel used as a drug control in the present invention.

Hereinafter, the present invention will be described in detail.

The present invention provides an oral administration absorption enhancer of a drug containing a compound of Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient:

The present invention includes not only the compound of Formula 1, but also pharmaceutically acceptable salts thereof, and possible solvates, hydrates, racemates, or stereoisomers that may be prepared therefrom.

The compound of formula 1 of the present invention may be used in the form of a pharmaceutically acceptable salt, and as the salt, an acid addition salt formed by a pharmaceutically acceptable free acid is useful. Acid addition salts include those derived from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid or phosphorous acid, and aliphatic mono- and dicarboxylates, phenyl-substituted alkanoates, hydroxyalkanoates, Dioleate, aromatic acid, aliphatic and aromatic sulfonic acids. Such pharmaceutically innocuous salts include, but are not limited to, sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate chloride, bromide, Butyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, succinate, maleic anhydride, maleic anhydride, , Sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, Methoxybenzoate, phthalate, terephthalate, benzene sulfonate, toluene sulfonate, chlorobenzene sulfide Sulfonate, methanesulfonate, propanesulfonate, naphthalene-1-sulphonate, naphthalene-1-sulphonate, , Naphthalene-2-sulfonate or mandelate.

The acid addition salts according to the invention are dissolved in conventional methods, for example, by dissolving a compound of formula 1 in an excess of aqueous acid solution and using the water miscible organic solvent, such as methanol, ethanol, acetone or acetonitrile. It can be prepared by precipitation.

An equivalent amount of the compound of formula 1 and an aqueous acid solution or alcohol may be heated and then the mixture is evaporated to dryness or the precipitated salt may be produced by suction filtration.

In addition, bases can be used to make pharmaceutically acceptable metal salts. The alkali metal or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess amount of an alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the salt of the undissolved compound, and evaporating and drying the filtrate. At this time, it is preferable for the metal salt to produce sodium, potassium or calcium salt.

The composition comprising the compound of the present invention, preferably containing 0.1 to 50% by weight based on the total weight of the composition is not limited thereto.

Hereinafter, the preparation method of Chemical Formula 1 according to the present invention is provided.

As shown in Scheme 1 below,

Preparing a compound of Formula 1 by reacting a starting material of dimethylsuberosin of Formula 2 with a pyridine compound in the presence of a base.

[Reaction Scheme 1]

At this time, as a reaction solvent that can be used, an aprotic polar solvent such as dimethylformamide (DMF), dimethyl sulfoxide (DMSO) or the like which does not adversely affect the reaction can be used, and preferably DMF can be used.

As the base, sodium carbonate, potassium carbonate, calcium carbonate, sodium hydroxide, potassium hydroxide, calcium hydroxide and the like can be used.

At this time, the reaction temperature is not particularly limited, but may be carried out within the boiling point range of the room temperature to the solvent, the reaction time is 12 hours to 24 hours.

As a result of performing the accumulation and excretion experiment of the drug by administering the compound of the present invention and the drug in an in vitro experiment to determine the effect of the drug absorption for oral administration of the compound of Formula 1 according to the present invention, Verapamil, a drug absorption enhancer, has a better effect of accumulating drugs into cells and a smaller amount of drug released into cells, thereby increasing the cytotoxicity of drugs (see Example 1).

In addition, as a result of measuring the pharmacokinetic parameters in order to determine the effect on the in vivo of the compound of Formula 1 according to the present invention, when the drug and the compound of the present invention orally administered, the drug alone The time to reach the maximum blood drug concentration, the maximum blood drug concentration was increased, the half-life of the drug (drug residence time) was increased, and the absorption rate and absorption rate of the drug were significantly increased. It confirmed that the effect is shown.

Furthermore, the compound of formula 1 according to the present invention, when administered alone intravenously, pharmacokinetic parameters are obtained, while analysis from plasma samples obtained by oral administration, quantitatively within the quantitative limit of HPLC (25 ng / mL) Pharmacokinetic parameters could not be derived (see Example 2).

From this, the compound of formula 1 according to the present invention promotes the absorption of paclitaxel in the body, and is then completely excreted without being absorbed into the body, so that it does not affect other unnecessary drugs and toxicity in the body, and thus absorbs oral administration of the drug. It can be usefully used as an enhancer.

Therefore, the compound according to the present invention has an excellent effect of enhancing the cytotoxicity of the drug, and the effect of accumulating the drug into the cell is superior to the conventional drug absorption enhancer Verapamil, and the amount of the drug released into the cell is less In addition, the dose-dependent increase in the absorption of the drug increases the absolute bioavailability of the drug, and the compound of the present invention is not absorbed in the body other than to enhance the absorption of the drug in the body, so other unnecessary drug effects and toxicity in the body, etc. Since it does not affect the oral administration of the drug can be usefully used as an absorption enhancer.

In addition, the compound according to the present invention can be usefully used as an oral absorption absorption enhancer of the drug because it enhances the absorption of the drug for oral administration in the body and is then completely excreted in the body, thereby not affecting other unnecessary effects and toxicity in the body. have.

In addition, the drug may be a drug having a low absorption efficiency in the gastrointestinal tract. Preferably, daunorubicin, doxorubicin, mitoxantron, vincrisitne, vinblastine, etoposide (VP-16) Teniposide (VM-26), actinomycin D, mitomycin C, colchicine, emethin, puromycin, Acyclovir, digoxin, quinidin, cimetidine, cyclophosphamide, 5-FU, amsacrine, daunomycin, paclitaxel, docetaxel And paclitaxel or docetaxel.

Oral administration of the drug of the present invention, the absorption enhancer may further include suitable carriers, excipients and diluents commonly used in the manufacture of a medicament.

Oral administration of the drug according to the invention absorption enhancers, respectively, in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like oral formulations, external preparations, suppositories and sterile injectable solutions in accordance with conventional methods It can be formulated and used. Examples of carriers, excipients and diluents that can be included in the composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

 In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may be formulated into the compositions of the present invention with at least one excipient such as starch, calcium carbonate, (sucrose), lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. As a suppository base, witepsol, macrogol, tween 61, cacao paper, laurin, glycerol gelatin and the like can be used.

Oral Administration of Drugs of the Invention Absorption enhancers may be administered orally or parenterally. Any parenteral administration method may be used, and systemic or topical administration is possible, but systemic administration is most preferred, and oral administration is most preferred. desirable.

Oral Administration of Drugs of the Invention The preferred dosage of the absorption enhancer depends on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art. However, for the desired effect, oral administration of the drug of the present invention, the absorption enhancer is preferably administered at 0.0001 to 0.03 g / kg, preferably at 0.001 to 8 mg / kg. The administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way.

Hereinafter, the present invention will be described in detail with reference to Production Examples and Examples.

The following Preparation Examples and Examples are merely illustrative of the present invention, but are not limited thereto.

< Manufacturing example  1> 6- (3- methyl - Boot -2- Enil ) -7- (pyridine-2- Ylmethoxy ) - Kromen Preparation of 2-one ( Chemical  One)

A 2-bromoethyl-pyridine (0.20 mmol) solution of dimethyl suverosine (30 mg, 0.19 mmol) and potassium carbonate (55 mg, 0.39 mmol) in DMF (3 ml) was prepared at room temperature with stirring. Was added. The reaction mixture was stirred for 18 hours at room temperature. When the reaction was completed, water was added and the mixture was extracted twice with ethyl acetate. The ethyl acetate extract was washed with water and brine, dried over anhydrous magnesium sulfate and filtered. The filtrate was concentrated under reduced pressure, and the residue was purified by flash column chromatography (ethyl acetate: n-hexane = 1: 2) to obtain 24 mg (yield 79%) of the title compound.

¹ H NMR (CDCl ₃ , 300 MHz) δ 8.65 (d, J = 4.2 Hz, 1H), 7.84 (t, J = 7.5 Hz, 1H), 7.63-7.56 (m, 2H), 7.36 (s, 1H) , 7.23 (s, 1H), 6.84 (s, 1H), 6.24 (d, J = 9.6 Hz, 1H), 5.36-5.29 (m, 3H), 3.42 (d, J = 3.43 Hz, 2H), 1.78 ( s, 3H), 1.70 (s, 3H);

MS (ESI) m / z 344 (M + Na) ⁺ , 322 (M + H) ⁺

In order to confirm the drug absorption enhancing effect of the compound of Formula 1 according to the present invention, the following experiments were performed in Examples 1-2.

Reagents and devices

RPMI 1640, trypsin-EDTA (0.25% trypsin-1mM EDTA) and antibiotic-antimycotic reagents were purchased from Invitrogen (Calsbad, USA), and fetal bovine serum serum, FBS) was purchased from Hyclone (South Logan, USA). Donomycin (DNM), verapamil, DMSO, sulforhodamine B (SRB), sodium bicarbonate, L-glutamine, trichloroacetic acid (TCA), cremophor ^® EL, Anhydrous ethanol, NACl, KCl, MgCl ₂ was purchased from Sigma-Aldrich, St. Louis, USA. CaCl ₂ and phosphoric acid were purchased from Showa chemical, Tokyo, Japan, and acetic acid was purchased from Daejung, Siheung, Korea. HEPES (N- (2-hydroxyethyl) piperazine-N'-2-ethanesulfonic acid), Triton X-100 and Tris base were purchased from USB (Cleveland, USA), Microscint TM40, scintillation cocktail was purchased from Packard Instrument Co. Inc., Meriden, USA, and [ ³ H] -DNM (1-5 Ci / mmol) was Perkin-Elmer Inc., Wellesley. , USA). 4-hydroxybenzoic acid n-hexyl ester was purchased from Tokyo Kassei Kogyo Co, Ltd., Tokyo, Japan, and paclitaxel was purchased from Samyang Genex (Daejeon, Korea). Acetonitrile for HPLC grade was purchased from Merck (Darmstadt, Germany), diethyl ether was purchased from Deoksan (Ansan, Korea), and heparin sodium salt from Shinpung (Seoul, Korea).

The Omnioire 0.45 μm membrane filter is available from Millford (MA, USA), Intramedic polyethylene tubing (PE60) is from Parsippamy, NJ, USA, 2 ml, 5 ml, and 10 ml of syringes were applied to a Korean rat (Ansam, Korea), a laboratory animal, Sprague-Dawauria male rats Purchased from Orient Bios Co. (Seongnam, Korea).

6 well plates and 96 well plates used for cell experiments were purchased from BD Biosciences (Bedford, USA). (Orbital shaker, SLOS-20, SLB, Seoul, Korea) was used as a device for measuring the number of cells in the culture medium (3158, Forma Scientific Inc., Marietta, USA), a radioactivity meter (Topcount NXT, Packard Instrument Co. Inc., Meriden USA) Korea), an inverted microscope (Axiovert 200, Carl Zeiss, Oberkochen, Germany) and an ELISA reader (3550, Bio-Rad, Hercules, USA). Analytical instruments include the Agilent HP1100 Series System (Hewlett-Packrad LP, Palo Alto, CA, USA), Micro-12 Centrifuse and Union 32R Centrifuse (Hanmil Science Industrial Co. Ltd., Anyang, Korea), Vortex- Jenny 2 (Scientific industries Inc., Bohemia, NY, USA), Ultrasonic Cleaner 3210-DTH (Branson μltrasonic Co., Danbury, CT, USA), Welch Pressure / Vacuum Pump 2522C (Thomas Industries, Inc., Skokie, IL, USA).

< Example  1> in vitro ( in vitro ) drug of  Accumulation and Discharge Experiment

In order to confirm the effects of the compounds of the present invention on the drug, cytotoxicity, anticancer drug accumulation and excretion experiments were performed in human breast cancer cells (MCF-7 / ADR) resistant to various anticancer drugs (Skehan et al., J.). Natl. Cancer.Inst., 82, pp 1107-1112, 1990; Critchfield et al., Biochem.Pharmacol., 48, pp1437-1445, 1994; Harker et al., Cancer Res., 45, pp4091-4096, 1985; Yeh et al., Cancer Res., 52, pp6692-6695, 1992; Zhang et al., J. 21, pp565-569, 2007; Chung et al., Phytother Res, 23, pp472-476, 2009) .

1. Cell culture

MCF-7 / ADR cells, multi-drug resistant human breast cancer cells, were treated with FBS 10%, 10 mM HEPES, 24 mM sodium bicarbonate, 2 mM L in a 37 ° C. cell incubator with 5% CO ₂ and 95% air. -Was cultured in RPMI 1640 medium containing glutamine and antibiotics.

2. Experiment

(1) effect on cytotoxicity

MCF-7 / ADR cells were added to a 96 well plate containing 5,000 revisions per well, and then incubated at 37 ° C. for 24 hours to allow cells to adhere to the plate. Dounmycin (DNM) in the concentration range of 1.0 × 10 ⁻⁶ M to 1.0 × 10 ⁻⁴ M, verapamil (drug control group) at a concentration of 50 μM, known as drug absorption enhancer, and a sample of the compound according to the present invention were prepared in each well. Was added.

In this case, a group in which only the same amount of DMSO solvent was added instead of coumarin derivative compounds was used as a control. After 2 hours, the culture medium (DNM ± compound according to the present invention, verapamil and DMSO) was removed and the cells washed twice with RPMI 1640 and then fresh media was added. After 3 days, the cytotoxicity was examined by SRB staining. The method is briefly described as follows.

Cells were fixed with 10% TCA for 1 hour, washed 4 times with water and dried. SRB reagent (0.4% w / v in 1% acetic acid) was added to each well and washed 4 times with 1% acetic acid after 30 minutes. After drying the plate, the dye bound to the protein was dissolved in 10 mM Tris base for 90 minutes, the absorbance at 530 nm was measured, and the IC ₅₀ value was determined using a table curve. In this case, the IC ₅₀ value means the concentration of B that can reduce the size of reaction A to 50% by using drug B, assuming that the size of any reaction A is 100%.

The results are shown in Table 1 below.

division Donomycin IC ₅₀ (μM) The compound of formula (1) 7.86 Solvent control 50.7 Drug control group (verapamil) 6.03

As shown in Table 1, the compound of Formula 1 according to the present invention reduces the IC ₅₀ value of doomycin to 7.86 μM in MCF-7 / ADR cells similarly to verapamil (6.03 μM) known as a representative drug absorption enhancer. It was confirmed.

(2) intracellular [ ³ H] - Donomycin  Impact on Accumulation

To study the effect of the compound according to the present invention on P-glycoprotein activity, MCF-7 / ADR cells were put in a 6 well plate to 150,000 / well and cultured for 72 hours. Each well was then treated with uptake buffer (137 mM NaCl, 5.4 mM KCl, 2.8) containing 50 μM concentration of verapamil or the compound of formula 1 according to the present invention at 0.05 μM concentration of [ ³ H] -DNM. 1 ml of mM CaCl ₂ , 1.2 mM MgCl ₂ , 10 mM HEPS, pH 7.4) was added and incubated for 2 hours. The absorption buffer of each well was then removed and ice cold stop solution (137 mM NaCl, 14 mM Tris, pH 7.4) was added to terminate [ ³ H] -DNM accumulation. Then 1 ml of lysis buffer (1% Triton X-100) was added to each well and shaken at 150 rpm for 90 minutes, then 100 μl of the 200 μl scintillation cocktail was obtained. And mixed overnight at 80 rpm and then radioactivity was measured with a radiometer (LSC). The results of intracellular accumulation in the control group and the intracellular accumulation results obtained after treating the cells with the compound according to the present invention were statistically treated by the student's t-test, and the p value was 0.05 or less (p <0.05). It was determined that the significance, the results are shown in Table 2 and FIG.

Intracellularly accumulated
Amount of donomycin (%) Compound of compound 1 270% Drug control
(Verapamil) 176% Solvent control 100%

As shown in Table 2 and FIG. 1, when the compound of Formula 1 according to the present invention was added, the amount of donomycin accumulated in the cells was 270%, about 1.5 times higher than that of verapamil (176%) used as a drug control. It was confirmed that the degree is excellent, and about 2.7 times better than the solvent control (100%) was confirmed that the effect.

(3) ³ H] - Donomycin  Impact on emissions

To confirm the effect of coumarin derivative compounds on the release of DNM from cells to reconfirm whether the increase in the accumulation of DNM in human breast cancer cells of the compounds according to the invention inhibited the release of DNM introduced into cells. The experiment was performed.

MCF-7 / ADR cells were placed in 6 well plates at 150,000 / well, followed by incubation for 72 hours, followed by 1 ml of absorption buffer containing 0.05 [mu] M [ ³ H] -DNM in each well 37 Incubate for 1 hour at ℃ accumulates DNM into cells. Then, the absorption buffer containing [ ³ H] -DNM was removed and the cells were washed, and then each well was added with 50 µM concentration of Verapamil and the absorption buffer containing the compound of Formula 1 according to the present invention for 1 hour. Incubated for 2 hours.

On the other hand, drug-free absorption buffer containing no these substances was added to the control group. After 1 hour, the cells were washed twice with an ice-cold stop solution. Then, 1 ml of lysis buffer was added to each well, shaken at 150 rpm for 90 minutes, and 100 µl was taken. 200 μl of the fluorescent mixture was added and mixed overnight at 80 rpm, followed by radioactivity (LSC).

At this time, since the measured value is the amount of DNM remaining in the cell, the cells were cultured with 0.05 μM concentration of [ ³ H] -DNM for 1 hour immediately after incubation with the coumarin derivative compound at the total amount of DNM introduced into the cell for 1 hour. After culturing the cells, the amount of [ ³ H] -DNM released from the cells was calculated for 1 hour by subtracting the remaining amount in the cells. The results are shown in Table 3 and FIG. 2.

Exocytosed
Amount of donomycin (%) Compound of compound 1 40.7 Drug control
(Verapamil) 48.8 Solvent control 64.5

As shown in Table 3 and Figure 2, when the compound of Formula 1 according to the present invention is added, the amount of donomycin released to the extracellular is 40.7%, about 10% than Verapamil (48.8%) used as a drug control group It was measured in a small amount, it was confirmed that the effect is about 37% superior to the solvent control (64.5%).

Therefore, the compound of formula 1 according to the present invention is excellent in the effect of increasing the cytotoxicity of the drug donomycin, and excellent in the effect of accumulating the drug into the cell than the conventional drug absorption enhancer Verapamil, and is discharged to the outside of the cell It can be seen that it reduces the amount of drug.

< Example  2> in vivo in vivo ) Measurement of oral absorption rate enhancement effect of drug

In order to examine the effect of the compound of the present invention on the oral absorption of the anticancer agent, the change in the oral absorption rate of paclitaxel was observed by the combination administration of the anticancer agent paclitaxel and the compound of the present invention (Malingre et al., Invest. New. Drugs., 19, pp 155-162, 2001). The results were compared using Verapamil as a drug control (Perez-Tomas R., Curr. Med. Chem., 13, pp1859-1876, 2006).

(1) Animal experiment

A healthy sprague-dauris male rat (female) was fed with normal feed and water in a constant temperature, humidity and light controlled environment (23 ± 2 ° C, 50 ± 5% 6-7 weeks old, 260-280 g). Animals were stabilized in the above environment for at least 1 week and then carotid arteries were anesthetized with PE-60 tubes one day prior to drug treatment. At this time, diethyl ether was used to lightly inhale and anesthetize, and the mice were allowed to move freely by putting one rat in each cage to recover from the surgery. Feed and water were also freely ingested. Drug administration was carried out the morning after surgery.

(2) Drug administration

Paclitaxel was prepared from the currently available Taxol ^占 formulation (anhydrous ethanol: Crmpo EL: saline = 1: 1: 4) and the compound of formula 1 according to the present invention as a test substance and verapamil Respectively.

Paclitaxel was administered intravenously 2 mg / kg, orally 25 mg / kg, and verapamil and the compound of formula 1 according to the present invention were orally administered 0.5, 2 and 5 mg / kg paclitaxel 25 mg / kg, Respectively. When the drug was administered, the final concentration of paclitaxel was 2 mg / ml, and verapamil and the compound of formula (1) according to the present invention were also prepared and used at the same concentration. However, when the dose of verapamil and the test substance was 0.5 mg / kg, the final concentration was set to 1 mg / ml in consideration of errors that may occur when a small amount is administered. At this time, the proportions of the respective solvents were the same as above.

Blood samples were collected in 200 μl of each blood sampling point using a 1 ml syringe coated with heparin (100 IU / ml) diluted with 0.9% physiological saline. Blood samples were taken and replaced with equivalent amounts of physiological saline containing heparin. Blood samples were obtained by centrifugation at 11,000 rpm for 15 minutes and stored at -70 ° C until analysis. The time of sampling was 0, 0.25, 0.5, 1, 2, 3, and 4 hours after oral administration, and the time of sampling was 0, 0.033, 0.083, 0.25, 0.5, 1, 2, 3, 4, 6, 4, 6, 8, 10, and 24 hours.

(3) HPLC  Plasma sample preparation for analysis

5 μl of n-hexyl p-hydroxybenzoic acid (3 μg / ml) was added to 50 μl of the plasma sample as an internal standard, and 45 μl of acetonitrile as an extract was added thereto. The sample was vortexed for 2 minutes, And centrifuged. Then, supernatant was taken and 40 μl was injected into HPLC.

(4) HPLC  analysis

HPLC was used as a device for analyzing plasma samples. The analysis conditions were as follows. The composition of the mobile phase was 0.1% phosphoric acid: acetonitrile = 50:50 (v / v), the flow rate was 0.5 ml / min and the running time was 30 minutes. The column was a Capcell Pak C18 MG120 column (3 mm × 250 mm, 5 μM, Shiseido, Tokyo, Japan) and the wavelength of the UV detector was set at 227 nm.

(5) The compound of formula Paclitaxel  Effect on gastrointestinal absorption

In order to investigate the effect of the compound of the present invention on the gastrointestinal tract absorption of the anticancer drug paclitaxel, paclitaxel (25 mg / kg) and verapamil (0.5, 2, 5 mg / kg) 2, 5 mg / kg) was orally administered to experimental animals simultaneously. The plasma samples obtained were analyzed using HPLC, and PK parameters were derived using the WinNonlin program.

In this case, the initial concentration after intravenous administration (C _0), the peak plasma concentration (C _max), time (T _max) to reach maximum plasma concentration, in vivo elimination half-life (t _1/2), elimination rate constant (ke), (AUC _last ), plasma drug concentration to infinity (AUC _inf ), whole body clearance (Cl or Cl / F) and apparent distribution volume (Vd or Vd / F) was derived.

When only paclitaxel was administered intravenously (see Table 4), when only compound of formula 1 was administered intravenously (see Table 5), when the compound of formula 1 according to the present invention was orally administered with paclitaxel (see Tables 6 and 3). ) And plasma concentration-time profiles of paclitaxel when orally administered paclitaxel and verapamil (see Table 7 and FIG. 4).

Paclitaxel  In the case of single intravenous administration Pharmacokinetics  parameter PK parameter PTX 2 mg / kg (IV) C ₀ (μg / ml) 3.89 ± 0.25 AUC _last (μg · h / ml) 1.53 ± 0.83 AUC _INF (μg · h / ml) 1.83 ± 0.99 t _1/2 (h) 11.9 ± 1.48 ke (1 / h) 0.059 ± 0.007 Vz (ml) 5166 ± 842 Cl (ml / h) 301 ± 32.2

Intravenous administration of the compound of Formula 1 alone Pharmacokinetics  parameter PK parameter Mg / kg (IV) C ₀ (μg / ml) 4.33 ± 1.24 AUC _last (μg · h / ml) 0.83 ± 0.12 AUC _INF (μg · h / ml) 0.85 ± 0.12 t _1/2 (h) 1.31 ± 0.74 ke (1 / h) 0.67 ± 0.35 Vz (ml) 2800 ± 1268 Cl (ml / h) 1549 ± 250

Paclitaxel  Exclusive; And Paclitaxel  When the compound of formula (I) is administered orally at the same time Pharmacokinetics  parameter PK parameter PTX (25 mg / kg)
(n = 5) Dose of Compound of Formula 1 0.5 mg / kg (n = 5) 2 mg / kg (n = 5) 5 mg / kg (n = 4) C _max (μg / mL) 0.19 ± 0.04 0.33 ± 0.01 0.34 + 0.04 1.16 ± 0.20 T _max (h) 2.0 ± 0.0 2.7 ± 0.4 3.0 ± 0.0 0.5 ± 0.0 AUC _last (μg · h / ml) 0.70 + 0.17 2.65 ± 0.40 2.58 ± 0.29 6.46 ± 1.38 AUC _INF (μg · h / ml) 0.77 ± 0.16 2.78 ± 0.38 3.08 ± 0.39 7.43 ± 2.13 t _1/2 (h) 2.85 ± 0.89 5.29 ± 1.04 9.56 ± 0.99 11.13 ± 7.75 ke (1 / h) 0.26 + 0.07 0.14 ± 0.03 0.07 ± 0.01 0.09 ± 0.05 Vz / F (ml) 35142 ± 16840 18859 ± 6669 30639 ± 4850 13557 ± 6062 Cl / F (ml / h) 8224 ± 1835 2419 ± 376 2222 ± 278 971 ± 253 Absolute BA (%) 3.68 12.1 13.4 32.4 Relative BA (%) 360 398 961

Paclitaxel  Exclusive; And Paclitaxel Verapamil  At the same time, Pharmacokinetics  parameter PK parameter PTX (25 mg / kg)
(n = 5) The dose of verapamil co-administered with PTX (25 mg / kg) 0.5 mg / kg (n = 5) 2 mg / kg (n = 4) 5 mg / kg (n = 4) C _max (μg / mL) 0.19 ± 0.04 0.23 ± 0.05 0.16 ± 0.06 0.21 ± 0.05 T _max (h) 2.0 ± 0.0 2.2 ± 0.4 2.4 ± 0.5 2.6 ± 0.9 AUC _last (μg · h / ml) 0.70 + 0.17 1.50 + - 0.38 1.18 0.35 2.04 ± 0.75 AUC _INF (μg · h / ml) 0.77 ± 0.16 1.82 0.36 1.36 0.31 2.28 ± 0.84 t _1/2 (h) 2.85 ± 0.89 10.29 ± 5.30 8.77 ± 3.00 7.13 + - 0.50 ke (1 / h) 0.26 + 0.07 0.09 ± 0.05 0.09 0.03 0.10 ± 0.01 Vz / F (ml) 35142 ± 16840 56313 ± 29339 70085 ± 31691 36674 ± 15240 Cl / F (ml / h) 8224 ± 1835 3803 ± 29339 5375 ± 1279 3584 ± 1588 Absolute BA (%) 3.68 7.9 5.9 9.9 Relative BA (%) 235 175 294

As shown in Table 5, when the compound of formula 1 of the present invention was administered intravenously, pharmacokinetic parameters were obtained, whereas the results of analysis from plasma samples obtained by oral administration showed the limit of HPLC (25 ng / ml). Pharmacokinetic parameters could not be derived because they were not quantified within.

Referring to Table 6 or FIG. 3, when paclitaxel and the compound of the present invention are orally administered, the maximum blood drug concentration attainment time is increased depending on the dose, and the maximum blood drug concentration is increased depending on the dose compared to the case of paclitaxel alone. It was found that the half-life of the drug (drug residence time in the blood) was increased, and the drug had an effect of significantly increasing the absorption rate and absorption rate of the drug.

Meanwhile, referring to Table 7 and FIG. 4, in the case of paclitaxel administered orally with verapamil, verapamil has a low blood concentration and an area under a low plasma concentration-time curve compared to the case where the compound of Formula 1 is administered at all doses. It can be seen that (AUC) is indicated. In particular, when administration of verapamil 5.0 mg / kg, the maximum blood concentration (C _max ) is 0.21 ± 0.05 μg / ㎖ is only one fifth of the level compared to the same dose of the compound of Formula 1 have. In addition, the maximum blood concentration reaching time (T _max ) was confirmed to be delayed by 5 times compared to the case of administration of the same dose of the compound of Formula 1 in the case of verapamil 5.0 mg / kg.

From these results, the compound of formula 1 according to the present invention increases the absorption of paclitaxel more effectively than verapamil so that the absolute bioavailability and relative bioavailability of paclitaxel when compared to the same doses between verapamil and the compound of formula 1 It can be seen that the increase by the compound of 1.

From this, the compound of formula 1 according to the present invention promotes the absorption of paclitaxel in the body, and is then completely excreted without being absorbed into the body, so that it does not affect other unnecessary effects and toxicity in the body, thus absorbing oral administration of the drug. It can be usefully used as an enhancer.

On the other hand, the compound of Formula 1 according to the present invention can be formulated in various forms according to the purpose. The following are some examples of formulation methods containing the compound represented by Formula 1 according to the present invention as an active ingredient, but the present invention is not limited thereto.

< Formulation example  1> Manufacture of Pharmaceuticals

One. Sanje  Produce

500 ng of compound of formula 1

Lactose 1 g

The above components were mixed and packed in airtight bags to prepare powders.

2. Preparation of tablets

500 ng of compound of formula 1

Corn starch 100 mg

Lactose 100 mg

2 mg of magnesium stearate

After mixing the above components, tablets were prepared by tableting according to a conventional method for producing tablets.

3. Preparation of Capsule

500 ng of compound of formula 1

Corn starch 100 mg

Lactose 100 mg

2 mg of magnesium stearate

After mixing the above components, the capsules were filled in gelatin capsules according to the conventional preparation method of capsules.

4. Preparation of injections

500 ng of compound of formula 1

Mannitol 180 mg

Na ₂ HPO ₄ 2H ₂ O 26 mg

Distilled water 2974 mg

According to a conventional method for preparing an injection, an injection was prepared by containing the above components in the contents shown.

Claims (5)

Oral administration absorption enhancer of a drug containing the compound of formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient:
[Formula 1]

.
According to claim 1, wherein the compound of formula 1 oral absorption absorption enhancer of the drug, characterized in that to enhance the bioavailability of the drug.
The method of claim 1, wherein the compound of formula 1 orally administered absorption enhancer of the drug, characterized in that the dose-dependent increase in the absorption of the drug.
According to claim 1, wherein the compound of formula (1) after promoting the absorption of the drug in the gastrointestinal tract, the compound itself is not absorbed into the body, orally administered absorption enhancer of the drug, characterized in that completely excreted in the body.
According to claim 1, wherein the drug is daunorubicin, doxorubicin, mitoxantron, mitcristine, vincrisitne, vinblastine, etoposide (VP-16) (etoposide) (VP-16)), teniposide (VM-26), actinomycin D, mitomycin C, colchicine, emetin, Puromycin, acyclovir, digoxin, quinidin, cimetidine, cyclophosphamide, 5-FU, amsacrine, donomycin daunomycin), paclitaxel and docetaxel is one kind selected from the group consisting of oral administration absorption enhancers.

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