KR101823405B1 - Novel cilostazol salt compound and use thereof - Google Patents

Abstract

The present invention relates to a novel star sol compound indeed an oral absorption rate is increased, the mesylate More specifically, the star sol chamber of weakly basic drug (p K a, -1.2) and besylate (p K a, 0.7) , Cilostazol mesylate and cilostazolyl acetate synthesized by adding an acid such as N-methyl-2-pyrrolidone were stable to humidity and light exposure, and after the oral administration, improvement in the in vivo dissolution rate and bioavailability was confirmed, the cilostazol mesylate And cilostazolylate compounds can be used as oral preparations having improved oral absorption rate by improving the low solubility and dissolution rate problems of conventional cilostazol.

Description

Novel cilostazol salt compounds and their use {Novel cilostazol salt compound and use thereof}

The present invention relates to novel cilostazol salt compounds with increased oral absorption.

Quinolinone-based material, ciloxazole (6- [4- (Cyclohexyl- 1H-tetrazol-5-yl) butoxy] -3,4-dihydro-2 (1H) -quinolinone], is a phosphodiesterase By inhibiting the phosphodiesterase type III, platelet aggregation is suppressed and blood vessels are expanded to prevent or treat blood coagulation, improve central blood circulation, antiinflammatory, anti-ulcer, blood pressure lowering, asthma and cerebral infarction, It is known to work.

This cilostazol is a weakly basic molecule (pKa, 11.8) which shows a low dissolution rate in methanol and ethanol but is insoluble in water. Its absorption rate in the gastrointestinal tract is very low, variable, and its stability is very low, to be.

In addition, it is mainly absorbed from the upper gastrointestinal tract and decreases in absorption toward the lower part of the intestinal tract. As a result, there is a restriction on the absorption time when the formulation is formulated as a sustained-release preparation. Therefore, currently available cilostazol preparations are in the form of immediate release tablets , Commercial cilostazol preparations cause a rapid rise in blood drug concentration upon oral administration and cause side effects such as headache. In order to maintain the sustained pharmacological activity, it is inconvenient to take 50 ~ 100 mg formulations twice a day .

Therefore, various studies are being conducted to solve the side effects of the cilostazol and to solve low solubility or dissolution rate in order to improve the oral absorption rate and the bioavailability.

Korea Patent Publication No. 2006-0116422 (published on November 15, 2006)

The present invention is to provide a novel cilostazol salt compound which improves the oral absorption rate by improving the low dissolution rate and the dissolution rate of cilostazol.

The present invention provides a cilostazol mesylate compound represented by the following formula (1).

[Chemical Formula 1]

The present invention provides a preparation for oral administration containing the cilostazol mesylate compound as an active ingredient.

The present invention provides a cilostazol besylate compound represented by the following formula (2).

(2)

The present invention also provides a preparation for oral administration comprising the cilostazolyl compound as an active ingredient.

According to the present invention, cilostazol mesylate and cilostazolyl acetate are stable to humidity and light exposure, and exhibit improved dissolution and bioavailability in vivo after oral administration. Therefore, a low dissolution rate of cilostazol When cilostazol mesylate and cilostazolyl acetate compound are used as an alternative compound for improving the dissolution rate problem, an oral preparation having improved oral absorption rate can be provided.

Fig. 1 shows the chemical structure of each cilostazol salt. Fig. 1A shows cilostazol, Fig. 1B shows cilostazol mesylate, and Fig. 1C shows cilostazolylate.
Figure 2 is a star sol salt ¹ H NMR results for each thread, Figure 1A is a ¹ H NMR spectrum of cilostazol, Fig. 1B is a chamber of the ¹ H NMR spectrum, and FIG. 1C is cilostazol besylate of star sol mesylate ^& Lt; ¹ > H NMR spectrum.
FIG. 3 shows the dissolution ratios of the respective cilostazol salts at various pH conditions. FIG. 1A shows the cilostazol-free base, cilostazol mesylate, and cilostazol mesylate, Figure 1B shows the dissolution profile profile of cilostazol besylate, cilostazol-free base, cilostazol mesylate and cilostazol besylate identified at pH 4.5. Figure 1C is the dissolution profile profile of cilostazol-free base, cilostazol mesylate and cilostazol besylate at pH 6.8.
FIG. 4 shows the results of oral administration of cilostazol-free base, cilostazol mesylate and cilostazol besylate to rats at a dose of 20 mg / kg, This is the result of confirming the concentration of starzol.

The present invention provides a cilostazol mesylate compound represented by the following formula (1).

[Chemical Formula 1]

The cilostazol mesylate can be synthesized through acid addition reaction of cilostazol free base with mesylate.

The cilostazol mesylate may exhibit 85 to 100%, 20 to 35% and 11 to 20% dissolution rates at pH 1.2, pH 4.5 and pH 6.8, respectively.

The present invention can provide a preparation for oral administration containing the compound of formula (1) as an active ingredient.

The present invention also provides a cilostazolyl compound represented by the following formula (2).

(2)

The cilostazolylate can be synthesized by acid addition reaction of cilostazol free base with besylate.

The cilostazolylate may exhibit a dissolution rate of 93 to 100%, 20 to 32% and 10 to 15% at pH 1.2, pH 4.5 and pH 6.8, respectively.

The present invention can provide a preparation for oral administration containing the compound of formula (2) as an active ingredient.

According to one embodiment of the present invention, gastrointestinal conditions are imitated with HCl buffer (pH 1.2), acetate buffer (pH 4.5) and phosphate buffer (pH 6.8) to produce cilostazol free base, cilostazol mesylate and cilostazol As shown in FIG. 3, in the case of cilostazol free base, the drug elution percentage accumulated over 6 hours under the conditions of pH 1.2, 4.5, and 6.8 was 25.4% ± 3.56 , 8.54% ± 1.89%, and 2.74% ± 0.341%, respectively.

On the other hand, the dissolution ratios of cilostazol mesylate and cilostazolyl acetate in the pH 4.5 buffer were 25.6% ± 4.57% and 26.6% ± 4.54%, respectively, and 13.0% ± 6.4% 2.07% and 12.1% ± 1.65%, respectively.

From the above results, it was confirmed that the elution rate and drug releasing degree of cilostazol mesylate and cilostazolyl acetate were greatly increased in the pH range of 1.2-6.8, and that the pH value of each test buffer was not changed after the dissolution of the cilostazol salt I could.

Thus, it has been found that the dissolution rate and drug release level of the two cilostazoles are significantly improved over the cilostazol free base, so that the two cilostazol salts can exhibit improved bioavailability in both the stomach and intestines.

In addition, in order to confirm the relative bioavailabilities (F) of the two cilostazol salts, it has been shown that the oral administration of cilostazol free base, cilostazol mesylate and cilostazolyl acetate to rats, The concentration-time profile was confirmed.

As a result, plasma cilostazol concentrations in the experimental group treated with cilostazol mesylate and cilostazolyl acetate were significantly higher than those in the group treated with cilostazol free base at all observation times (P <0.05 ).

In addition, as shown in Table 2, in the experimental group treated with cilostazol mesylate and cilostazolyl acetate, the oral absorption rate and the short T _max value were significantly faster than the experimental group treated with cilostazol free base. There was no significant difference between the groups treated with cilostazol mesylate and cilostazolylate.

On the other hand, it was confirmed that the terminal half-life of cilostazol appeared to be similar in all of the experimental groups treated with each of the three kinds of salts, and thus the cilostazol free base And the bioavailability of the composition of the present invention was greatly improved.

The preparations according to the present invention can be formulated into oral preparations by mixing with conventional pharmaceutically acceptable excipients, disintegrants, lubricants, sweeteners and the like. The excipient is preferably microcrystalline cellulose, lactose, corn starch, mannitol, low-substituted hydroxypropylcellulose, magnesium metasilicate aluminate, colloidal silicon dioxide and the like, and the disintegrant is preferably sodium croscarmellose, Magnesium stearate, talc, sodium stearyl fumarate, and the like are preferable as the lubricant. As the agent for oral administration, tablets, capsules, granules, solid dispersions and the like may be used.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are intended to illustrate the contents of the present invention, but the scope of the present invention is not limited to the following examples. Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

< Reference Example  1> Substance

It is also possible to use cilostazol-free base, benzene sulfonic acid, besylate, methane sulfonic acid (mesylate), chlorpropamide, dimethylsulfoxide, formic acid formic acid, polyethylene glycol 400 and Tween 80 were purchased from Sigma-Aldrich (St Louis, MO, USA). All solvents were purchased from Burdick and Jackson Company (Morristown, NJ, USA) with a high-performance liquid chromatography (HPLC) grade and other compounds were used at the highest grade.

< Experimental Example  1> Cilostazol  Determination of moisture contained in salt

Water contained in cilostazol mesylate and cilostazolyl acetate was confirmed using Karl Fischer Moisture Titrator MKA-520 (Kyoto Electronics Manufacturing Co., Ltd., Kyoto, Japan).

< Experimental Example  2> In the compound Cilostazol  Confirm

Cilostazol was identified using an HPLC system (Agilent 1260; Agilent Technologies) consisting of a zigzag detector (G1314B), four pumps (G1311B), an autosampler (G1367E) and a column compartment (G1316A).

The mobile phase consisted of distilled water and acetonitrile (45:55, v / v) with a flow rate of 1.0 mL / min. The wavelength of the ultraviolet detector was 257 nm. A reversed-phase column (Gemini C18 150 mm × 4.6 mm, 5 μm particle size; Phenomenex, Torrence, CA, USA) was used and the temperature of the column was maintained at 30 ° C.

< Example  1> Cilostazol Mesylate  or Besylate  Compound manufacturing

Cilostazol of Figure 1A has _a p K 11.8 of a weakly basic drug, the present invention cilostazol in order to increase the stability of salts, can be administered orally and has a biopharmaceutical advantage, mesylate usable medicinal (p K a, -1.2) and linen rate (p K a, 0.7) and to prepare a new type of cilostazol salt of a selection of the counter ion represent the same acid.

First, 1.2 mmol of methanesulfonic acid or benzosulfonic acid was added to 10 mL of a chloroform solution in which cilostazol (370 mg, 1.0 mmol) was dissolved, and the mixture was stirred at 25 DEG C and mixed. Lt; / RTI &gt;

Then, the solvent was removed under reduced pressure, 10 mL of diethyl ether was added to the residue, and the mixture was stirred at 25 ° C for 1 hour, filtered, and dried under reduced pressure at 40 ° C to obtain cilostazol salt in powder form.

Two kinds of cilostazol salts, cilostazol mesylate and cilostazolyl acetate, as shown in Figs. 1B and 1C, were obtained by the acid addition reaction in the above process.

¹ H NMR (nuclear magnetic resonance) spectra of the cilostazol salts obtained using AVANCE III (500 MHz; Bruker, Billerica, MA, USA) using an undigested solvent or tetramethylsilane as an internal control, Respectively.

As a result, as shown in Fig. 2B, in the ¹ H NMR of cilostazol mesylate, a methyl peak containing three hydrogens of mesylate was confirmed at 2.99 ppm.

From the above results, it was confirmed that the cilostazol mesylate salt was generated in a ratio of 1: 1 of cilostazol and methanesulfonic acid.

Also, the same ratio was confirmed in ¹ H NMR results of cilostazolyl acetate as shown in Fig. 2C.

Cilostazol mesylate and cilostazolyl acetate showed 69% and 98% yields respectively as white powders. Moisture content was determined using Karl Fischer moisture measurement method. As a result, cilostazol mesylate and cilostazolyl acetate Showed very low moisture contents of 0.17% and 0.14%, respectively.

From the above results, it was confirmed that the two cilostazol salts were a water-free anhydrous form.

< Example  2> hygroscopicity ( Hygroscopicity ) Confirm

Indeed, since the low hygroscopicity of the compound in the manufacture and storage of pharmaceutical products is a very important property required for the materials of pharmaceutical products, it is necessary to confirm the moisture content contained in the prepared cilostazol mesylate and cilostazolyl acetate Hygroscopicity was confirmed according to the published method (Callahan et al.).

The two cilostazol salts were homogeneously sprayed in a petri dish in a petri dish and sealed with a dacer to measure the humidity conditions of 22% relative humidity (RH) and 92.5% RH (potassium nitrate saturated solution) at 25 ° C &Lt; / RTI &gt;

Three samples of the RH conditions were prepared. After one week, the weight of the sample was measured, and the weight (g) of water adsorbed per 100 g of the dried solid was measured to confirm hygroscopicity (g / 100 g).

As a result, the hygroscopicity of cilostazol mesylate was 0.32% and 4.82% at 22% RH and 92.5% RH, respectively, and the hygroscopicity of cilostazolyl acetate was found to be 0.26% and 1.37%.

From the above results, it was confirmed that the two cilostazol salts were non-hygroscopic compounds when the increase in water content was a <20% (w / w) under conditions of 90% RH or more for 1 week.

< Example  3> Confirm stability

In order to confirm the stress stability of the two cilostazol salts, the effect on the salt form was ascertained for 6 weeks under temperature increasing conditions such as 40 ° C ± 2 ° C / 75% ± 5% RH and 60 ° C ± 2 ° C, Respectively.

Each of the prepared cilostazol salts (2 mg) was placed in a brown glass bottle and placed in a stability chamber (Labcare Pvt. Ltd., Mumbai, Japan) at 40 ° C ± 2 ° C / 75% ± 5% RH and 60 ° C ± 2 ° C for 6 weeks. India) and after 6 weeks sample purity was determined by the same method as "Cilostazol determination in salts".

In addition, two cilostazol salts were exposed to light, and the stability after light irradiation was confirmed for 24 hours.

2 mg of each cilostazol salt prepared above was sprayed into a clear glass bottle and placed in a light irradiation chamber (SUNTEST CPS +, Atlas Material Testing Technology, LLC, Chicago, Ill., USA) Respectively.

The light source was irradiated with 300.800 nm wavelength at 200 W / m ² for 24 hours using light from a xenon arc lamp filtered through a window glass to mimic indoor daylight.

The control group was protected with aluminum foil and irradiated with light under the same conditions in the light irradiation chamber at the same time as the experimental group, and all the samples were analyzed by the same method as "Cilostazol determination in salts".

As a result, as shown in Table 1, all of the test samples remained in a solid state without color loss, and showed good thermal stability even after storage of both cilostazol mesylate and cilostazolyl acetate. In each test group, 98.6% or more of the initial amount Recovered.

From the above results, it was confirmed that the two cilostazol salts exhibited stability without any photodegradation even after light exposure.

< Example  4> In vitro dissolution rate confirmation

Cilostazol free base, cilostazol mesylate and cilostazolyl acetate were prepared in vitro by mimicking gastrointestinal conditions with HCl buffer (pH 1.2), acetate buffer (pH 4.5) and phosphate buffer (pH 6.8) The elution effect was confirmed.

First, in order to confirm the dissolution rate of cilostazol salt cilostazol mesylate and cilostazolylate, the dissolution rate was measured at 37 ° C ± 10 ° C using a dissolution tester (708-DS; Agilent Technologies, Santa Clara, CA, USA) The dissolution rate was confirmed by repeating three times at a temperature of 0.5 캜.

Each sample weigh accurately weighed to 20.0 mg cilostazol in each of the samples was filled into hard gelatin capsules of size "5" (Capsugel, Morristown, NJ, USA) and placed in 900 mL of pH 1.2, 4.5, and 6.8 lysis buffer The dissolution medium and the baskets were spun at 100 rpm.

Samples of 1 mL of each dissolution medium were taken at 0.25, 0.5, 1, 2, 4, and 6 hour intervals and fresh media was added in equal amounts.

The collected sample was filtered with a polytetrafluoroethylene syringe filter (0.45 μm), appropriately diluted with filtered methanol, and the amount of cilostazol eluted by HPLC was analyzed.

The dissolution experiment was repeated three times and the results were expressed as mean values.

As a result, as shown in Fig. 3, the percentage of drug elution accumulated over 6 hours under the conditions of pH 1.2, 4.5, and 6.8 in the case of cilostazol free base was 25.4% ± 3.56%, 8.54% ± 1.89%, and 2.74% 0.341%.

From the above results, cilostazol showed high dissolution at low pH condition, while very low drug dissolution rate at high pH was confirmed because of the basic nature of cilostazol. From the above results, cilostazol was eluted with pH- .

On the other hand, the dissolution rate and drug release rate of the two cilostazol salts were compared with cilostazol free base for 6 hours in all test lysis buffers.

As a result, in the HCl buffer (pH 1.2), cilostazol free base exhibited 25.4% ± 3.56% drug release over 6 hours, while cilostazol mesylate and cilostazolyl acetate showed 93.5% ± (P> 0.05). However, there was no significant difference in dissolution rate between cilostazol mesylate and cilostazol acetate (P> 0.05).

3B and 3C, the drug released from the cilostazol free base for 6 hours in the pH 4.5 and 6.8 lysis buffer was 8.54% ± 1.89% and 2.74% ± 0.341%, respectively, and the lower dissolution rate Was found to be due to the high pH solution.

On the other hand, cilostazol mesylate and cilostazolyl acetate showed a dissolution rate of 25.6% ± 4.57% and 26.6% ± 4.54% in the pH 4.5 buffer under the same time conditions, respectively, and 13.0% ± 2.07 % And 12.1% ± 1.65%, respectively.

From the above results, it was confirmed that both of the cilostazol mesylate and cilostazolyl acetate were greatly increased in the elution rate and the drug release degree in the pH range of 1.2-6.8, so that the two cilostazol salts were effectively used It was confirmed to be possible.

In addition, it was confirmed that the pH value of each test buffer did not change after elution of the cilostazol salt. From the results, it was confirmed that the dissolution rate and drug release level of the two cilostazol salts were significantly improved.

< Example  5> Pharmacokinetics ( Pharmacokinetic ) Confirm

1. Animal experiments

Male Sprague Dawley rats (8 wks, 250-270 g) were purchased from Orient Bio (Sungnam, Gyeonggi-do, South Korea) and protocols for animal experiments approved by the Animal Experimental Ethics and Use Committee of Catholic University Sacred Heart Campus Approval No 2014-027; Bucheon, Gyeonggi-do, South Korea).

All rats were given free access to water and food for 12 hours before drug administration, and a polyethylene tube (Clay Adams, Franklin Lakes, NJ, USA) was inserted into the carotid artery of the rat for blood sampling.

The rats were divided into three groups of 8 rats per group and cilostazol-free base (20 mg / kg), cilostazol mesylate or equivalent 20 mg / kg of cilostazol- free base) was orally administered.

For the oral administration of the drug, the free salt and the two cilostazol salts were dispersed in a solution of 5% Tween 80: polyethylene glycol 200: distilled water = 50: 10: 40 (v / v / v) 5 mL / kg of suspension was injected into the rats by oral gavage.

After injecting the drug, ~ 0.12 mL of blood was taken from the carotid arteries of the rats at 0 (control), 5, 15, 30, 45, 60, 90, 120, 240, 360, 480, 600 and 720 min and blood coagulation For prevention, the cannula was washed immediately with heparinized 0.9% NaCl injection solution (20 units / mL, 0.3 mL) immediately after collecting the blood sample.

The blood samples were centrifuged at 13,000 rpm for 10 minutes at 4 ° C immediately after collection, and then 50 μl of the plasma samples were stored at -80 ° C until the double mass analysis (LC-MS / MS) connected to the liquid chromatography.

2. Plasma Cilostazol  Confirm

Blood samples of each cilostazol-injected animal were analyzed by the modified LC-MS / MS analysis method (23) and the plasma cilostazol concentration was simultaneously ascertained by the above method.

The LC-MS / MS system consisted of Shimadzu Nexera HPLC coupled with a Shimadzu LCMS-8050 triple quadrupole mass spectrometer (Shimadzu Corporation, Kyoto, Japan).

Briefly, cilostazol and internal control (chlorpropamide, 500 ng / mL) were extracted from the plasma of the rats using 1 mL of tetra-butyl methyl ether, and analyzed on an ACE 3 C8 column (50 mm × 4.6 mm, 3 μm particle size ; Hichrom, Reading, Berkshire, UK) and acetonitrile containing distilled water and 0.1% formic acid at 25:75 (v / v) was used to perform chromatographic separation at a flow rate of 0.7 mL / min . The total run time per sample was 5.0 minutes.

Cilostazol was detected and quantitated with a mass spectrometer in a reaction-monitoring mode with m / z 370.45 → 288.1 and chlorpropamide with m / z 277.05 → 111.05.

The optimized mass spectrum data was obtained under the following conditions.

Nitrogen was fed at a flow rate of 3.0 L / min and 10.0 L / min in the form of an inhaled gas and a dry gas, respectively, and the connector voltage was 4.5 kV; The desolvation line temperature is 250 DEG C; The heat block temperature was 400 ° C.

The collision energy was 18 V for cilostazol and 20 V for chlorpropamide.

Argon gas was used as collision-induced dissociation gas at a pressure of 270 kPa, and detector voltage was 1.72 kV.

Cilostazol was measured at a linear range of 5-2,000 ng / mL and a minimum value of 5 ng / mL.

The coefficient of variation for the precision and accuracy of all analyzes met the acceptance criteria for bioanalysis and the internal and daily precision of the analysis (n = 5) ranged from 5.91% to 10.1% And the daily accuracy (n = 5) was 88.9% to 105.4%.

Cilostazol was stable at various storage and conditioning conditions without associated interference or substrate effects.

3. Pharmacological and statistical analysis

The pharmacokinetic parameters were calculated using a non-treatment model with WinNonlin software (version 5.2; Pharsight Corporation, Mountain View, Calif., USA) and the peak plasma concentration (C _max ) (T _max ).

The area (AUC _t ) of the concentration-time curve from time 0 to the last observation time point was calculated by the linear trapezoidal formula.

The relative bioavailability (F) of the two cilostazol salts was calculated by the following equation.

Statistical analysis was performed using empirical analysis of variance among three methods of Duncan's multiple range test (Version 22; SPSS Inc., Chicago, IL, USA) to obtain independent data.

All results were expressed as mean ± SD except the median Tmax, and P <0.05 was considered significant for all analyzes.

The plasma concentration-time profile of cilostazol was confirmed after oral administration of cilostazol free base, cilostazol mesylate and cilostazolyl acetate to the rat.

As a result, plasma cilostazol concentrations in the experimental group treated with cilostazol mesylate and cilostazolyl acetate were significantly higher than those in the group treated with cilostazol free base at all observation times (P <0.05 ).

In addition, as shown in Table 2, the AUC _t of cilostazol mesylate and cilostazolyl acetate Values increased 2.87 times and 3.65 times, respectively, and C _max Values were increased by 2.94 and 3.88 times, respectively, while plasma concentration profiles of cilostazol mesylate and plasma concentration profiles of cilostazolylate were compared, indicating that AUC _t and C _max There was no significant difference between the two groups.

In addition, in the experimental group treated with cilostazol mesylate and cilostazolyl acetate, the oral absorption rate and the short T _max value were significantly faster than in the experimental group treated with cilostazol free base, and all the results showed that cilostazol mesyl There was no significant difference between the treated and cilostazolesylate treated groups.

On the other hand, it was confirmed that the terminal half-life of cilostazol appeared to be similar in all of the experimental groups treated with each of the three kinds of salts, and thus the cilostazol free base The oral absorption rate and the bioavailability of the composition of the present invention were greatly improved, and the results were similar to those of the in vitro dissolution.

From the above results, it was confirmed that the improved oral absorption rate of the cilostazol free base was due to the increase of solubility according to the sulfonic acid salt form.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Claims (8)

A cilostazol mesylate compound represented by the following formula (1) and synthesized through an acid addition reaction of cilostazol free base with mesylate.
[Chemical Formula 1]

delete The compound according to claim 1, wherein the cilostazol mesylate exhibits 85 to 100%, 20 to 35% and 11 to 20% dissolution rates at pH 1.2, pH 4.5 and pH 6.8, respectively. An agent for oral administration comprising the compound according to claim 1 as an active ingredient. A cilostazol besylate compound represented by the following formula (2), which is synthesized through an acid addition reaction of cilostazol free base with besylate.
(2)

delete The compound according to claim 5, wherein the cilostazolylate exhibits a dissolution rate of 93 to 100%, 20 to 32% and 10 to 15% at pH 1.2, pH 4.5 and pH 6.8, respectively. An agent for oral administration comprising the compound according to claim 5 as an active ingredient.

Images