Use of amlexanox

Abstract

Disclosed is use of amlexanox or a salt thereof or a solvate thereof in preparation of a drug having an inhibitory action on the smooth muscle cells, in particular a drug for preventing and treating vascular restenosis. Further disclosed is a medical device, particularly a drug stent, the surface of which is distributed with amlexanox or the salt thereof or the pharmaceutical composition thereof. Amlexanox has an activity in inhibition of the proliferation of smooth muscle cells, has a low inhibitory property of the endothelial cell growth, is particularly suitable for applying on a medical device to prevent the incidence of vascular restenosis, while not delaying the repair of endothelium.

Claims

1. A drug stent, comprising a stent body and an active substance and a pharmaceutical composition distributed on a surface of the stent body, wherein the active substance or the pharmaceutical composition has an inhibitory action on smooth muscle cells without inhibiting a growth of endothelial cells, wherein the active substance is amlexanox or a salt thereof, and the pharmaceutical composition comprises rapamycin, wherein a drug-loading rate of each of amlexanox and rapamycin on the drug stent is from 1 μmol to 6 μmol and a drug-loading molar ratio of amlexanox to rapamycin is 1:1.

Description

DETAILED DESCRIPTION OF THE INVENTION

(1) The technical solution of the present invention is further illustrated below in conjunction with specific embodiments. It should be understood that the embodiments are only intended to illustrate the present invention, and are not intended to limit the scope of the present invention. The experimental methods without indicating specific conditions in the following embodiments are generally carried out according to conventional conditions or according to the conditions recommended by the manufacturer, and the reagents without indicating the source in the following embodiments are commercially available.

(2) The term “carrier” as used in the present invention, unless otherwise specified, refers to a pharmaceutically acceptable carrier which does not evanish the biological activity or property of the compound, and is relatively non-toxic, for example, giving a subject a certain substance would not cause undesired biological effects or detrimental interactions with any of the contained components.

(3) The words “subject” or “patient” are used interchangeably herein to refer to an animal (including a human) that is acceptable for the treatment with the compounds and/or methods. “Subject” or “patient” is used herein to encompass both male and female genders unless specifically stated otherwise. Therefore, “subject” or “patient” includes any mammal, including but not limited to, human, non-human primates, such as mammals, dogs, cats, horses, sheep, pigs, cattle, etc., which is benefit from treatment with the compound. Animals suitable for treatment with the compounds and/or methods of the present invention are preferably human. In general, the terms “patient” and “subject” are used interchangeably herein.

(4) As used herein, the “medical instrument” refers to an appliance, device or apparatus directly or indirectly used on a patient. The medical instrument involved in the present invention may be an instrument implanted in the body or an extracorporeal instrument. The instrument can be used temporarily or implanted permanently. The medical instrument involved in the present invention includes, but is not limited to, the following devices: stent, stent graft, synthetic patch, lead, electrode, needle, surgical instrument, angioplasty ball, wound drainage tube, shunt, tube, infusion sleeve, cannula, pellet, implant, blood oxygenation generator, pump, vascular graft, vascular access port, heart valve, annuloplasty ring, suture, surgical clip, surgical nail, pacemaker, nerve stimulator, orthopedic instrument, cerebrospinal fluid shunt, implantable drug pump, vertebral cage, artificial disc occluder, artificial blood vessel, drug-eluting balloon, and the like.

Embodiment 1: Cell Experiment

(5) A. Drug Preparation:

(6) (I) Preparation of Amlexanox: a) Before the experiment, the drug stent loaded with amlexanox is placed into a Dimethyl Sulfoxide (DMSO) solution, completely dissolved, and further diluted to form a solution having a drug concentration of 10.sup.−2 mmol/ml; b) The drug solution having the drug concentration of 10.sup.−2 mmol/ml is diluted with DMSO to drug solutions having concentrations of 10.sup.−3, 10.sup.−4, 10.sup.−5, 10.sup.−6, 10.sup.−7, 10.sup.−8, 10.sup.−9 mmol/ml, respectively; c) The prepared drug solutions with a series of concentrations are separately packaged, and then are stored at −20° C. for temporary storage; d) When in use, the stored 10.sup.−2-10.sup.−9 mmol/ml drug solutions diluted by the DMSO are taken out and restored to normal temperature. The drug solution of each concentration is diluted by 1,000 times with the corresponding complete cell culture medium for cell tests, that is, the final concentrations used are 10.sup.−5, 10.sup.−6, 10.sup.−7, 10.sup.−8, 10.sup.−9, 10.sup.−10, 10.sup.−11, 10.sup.−12 mmol/mL, respectively.

(7) (II) Preparation of Rapamycin: a) Before the experiment, the drug stent loaded with rapamycin is placed into the DMSO solution, completely dissolved, and further diluted to form a solution having a drug concentration of 10.sup.−2 mmol/ml; b) The drug solution having the drug concentration of 10.sup.−2 mmol/ml is diluted with DMSO to drug solutions having the concentrations of 10.sup.−3, 10.sup.−4, 10.sup.−5, 10.sup.−6, 10.sup.−7, 10.sup.−8, 10.sup.−9 mmol/ml, respectively; c) The prepared drug solutions with a series of concentrations are separately packaged, and then are stored at −20° C. for temporary storage. d) When in use, the stored 10.sup.−2-10.sup.−9 mmol/ml drug solutions diluted by the DMSO are taken out and restored to normal temperature. The drug solution of each concentration is diluted by 1,000 times with the corresponding complete cell culture medium for cell tests, that is, the final concentrations used are 10.sup.−5, 10.sup.−6, 10.sup.−7, 10.sup.−8, 10.sup.−9, 10.sup.−10, 10.sup.−11, 10.sup.−12 mmol/mL, respectively.

(8) (III) Preparation of Combined Drug: a) Before the experiment, the drug stent loaded with rapamycin and amlexanox (the molar ratio is 1:1) is placed into the DMSO solution, completely dissolved, and further diluted to form a solution having an amlexanox or rapamycin concentration of 10.sup.−2 mmol/ml; b) The drug solution having the drug concentration of 10.sup.−2 mmol/ml is diluted with DMSO to drug solutions having the concentrations of 10.sup.−3, 10.sup.−4, 10.sup.−5, 10.sup.−6, 10.sup.−7, 10.sup.−8, 10.sup.−9 mmol/ml, respectively; c) The prepared drug solutions of with a series of concentrations are separately packaged, and then are stored at −20° C. for temporary storage; d) When in use, the stored 10.sup.−2-10.sup.−9 mmol/ml drug solutions diluted by the DMSO are taken out and restored to normal temperature. The drug solution of each concentration is diluted by 1,000 times with the corresponding complete cell culture medium for cell tests, that is, the final concentrations used are 10.sup.−5, 10.sup.−6, 10.sup.−7, 10.sup.−8, 10.sup.−9, 10.sup.−10, 10.sup.−11, 10.sup.−12 mmol/mL, respectively.
B. In Vitro Cell Proliferation and Tests Thereof a) Selection of cell inoculation concentration: A 10 cells with good growing status and stable growth are selected. After the digestion, cell suspensions having the concentrations of 1×10.sup.4/ml, 2×10.sup.4/ml, 4×10.sup.4/ml, 5×10.sup.4/ml, and 10×10.sup.4/ml are respectively prepared. The cell suspensions of the above concentrations are inoculated into a 96-well cell culture plate and cultured using the MTT colorimetry. Finally, the solution having a cell inoculation concentration exhibiting absorbance of 0.6-1.5 measured by a microplate reader at the wavelength of 570 nm is selected as the cell inoculation concentration for tests; b) The suspension having the selected cell concentration is prepared to be inoculated into the 96-well cell culture plate; c) The cell culture medium is absorbed, and the cell culture medium having the final concentration of drug is added. A drug-free cell culture medium is used as a blank control group, and a DMSO cell culture solution having the concentration of 1/1000 is used as a background control group. The cell culture medium is cultured for 72 hours in a cell incubator. d) The MTT is added to culture for 4 hours, the cell culture medium on the culture plate is discarded, and the DMSO is added to dissolve for testing the absorbance.
C. Experimental Groups

(9) The Human Aortic Smooth Muscle Cells (HASMCs) and Human Aortic Endothelial Cells (HAECs) are selected for experiments, and grouping is carried out according to drugs, each group implements 6 times of tests and the average result is as shown in Table 1, specifically as follows:

(10) 1. Control group: no addition of drugs;

(11) 2. Rapamycin group: the culture medium contains 1 μmol or 10 μmol of rapamycin;

(12) 3. Amlexanox group: the culture medium contains 1 μmol or 10 μmol of amlexanox; and

(13) 4. Rapamycin and amlexanox group: the culture medium contains 1 μmol or 10 μmol of rapamycin and amlexanox, respectively.

(14) D. Experimental Results

(15) The inhibition rates of smooth muscle cells and endothelial cells in each group of experiments are shown in Table 1 below.

(16) TABLE-US-00001 TABLE 1 Smooth muscle cell Endothelial cell Experimental groups inhibition rate (%) inhibition rate (%) Control group — — Rapamycin (1 μmol) 9.1 7.7 Amlexanox (1 μmol) 11.6 0.9 Rapamycin (6 μmol) 18.9 16.0 Amlexanox (6 μmol) 19.0 2.8 Rapamycin (10 μmol) 22.5 21.8 Amlexanox (10 μmol) 23.4 3.5 Rapamycin (1 μmol) + 30.5 3.9 amlexanox (1 μmol) Rapamycin (6 μmol) + 35.4 5.7 amlexanox (6 μmol) Rapamycin (10 μmol) + 34.9 6.4 amlexanox (10 μmol)

(17) The inhibition rates of the smooth muscle cells and endothelial cells for the groups shown in Table 1 are based on the data of the control group.

(18) As shown in Table 1, compared with rapamycin, the smooth muscle cell inhibition rate of amlexanox is slightly higher, and the endothelial cell inhibition rate of amlexanox is greatly reduced, showing a particularly prominent of the inhibitory selectivity. In addition, in the case of the combined use of amlexanox and rapamycin, a smooth muscle cell inhibition rate of more than 30% is able to be obtained at a low drug content, and the endothelial cell inhibition rate is greatly reduced compared to the inhibition rate of the single use of rapamycin. Moreover, the endothelial cell inhibition rate of the combination of amlexanox and rapamycin is not obviously increased, with the drug content increases.

Embodiment 2 Drug Eluting Stent

(19) 300 mg of amlexanox (drug) and 300 mg of PLGA (drug carrier) are mixed in 20 ml of chloroform. After the solute is completely dissolved, the solution is uniformly sprayed on the surface of the L605 cobalt-chromium alloy metal stent by ultrasonic atomization until the drug-loading rate reaches 50 μg/cm.sup.2. The drug eluting stent is obtained after the solvent is completely volatilized at room temperature.

Embodiment 3 Nano-Microporous Preloading Type

(20) Fine marks are formed on the surface of a stainless steel stent body through friction processing; the micronized amlexanox and the stent body are placed into a high-pressure sealing device; the device is activated to make the micronized drug particles embedded in the fine marks of the stent body; and the drug eluting stent is obtained after the drug-loading rate reaches 50 μg/cm.sup.2.

Embodiment 4 Preparation Method of Grooved Drug Eluting Stent

(21) First, a stent body is provided, and a groove is formed on the surface of the stent body for subsequent loading of the drug.

(22) Next, amlexanox and a solvent are mixed to obtain a solution, where the solvent is selected from the group consisting of paraffins, olefins, alcohols, aldehydes, amines, esters, ethers, ketones, aromatic hydrocarbons, hydrogenated hydrocarbons, terpene hydrocarbons, halogenated hydrocarbons, heterocyclic compounds, nitrogen-containing compounds, and sulfur-containing compounds.

(23) Then, the solution is loaded onto the stent body. Specifically, the solution is loaded in the groove on the surface of the stent body. The solution is loaded by: dripping or coating, and the coating includes one or more of ultrasonic atomization spraying, chemical vapor deposition, physical vapor deposition, ion beam spraying, dip coating, micro-spraying and brush coating.

(24) Finally, the drug-loaded stent is obtained after the solvent is volatilized.

Embodiment 5 Animal Experiment

(25) A. Research Subject:

(26) Small male pigs aged 1-2 months are selected and fed with induced formula feed. Pigs with hyperglycemia, hyperinsulinemia, and early diabetic nephropathy such as microalbuminuria, urine sugar, and nephritis are selected as subjects.

(27) B. Grouping

(28) 1. Rapamycin stent group: animals are implanted with the stent containing rapamycin (the rapamycin loading rate of the stent is 140 μg/cm.sup.2);

(29) 2. Amlexanox stent group: animals are implanted with the stent containing amlexanox (the amlexanox loading rate of the stent is 140 μg/cm.sup.2);

(30) 3. Rapamycin+amlexanox stent group 1: animals are implanted with the stent containing both rapamycin and amlexanox (the rapamycin loading rate and the amlexanox loading rate of the stent are 140 μg/cm.sup.2 and 20 μg/cm.sup.2, respectively);

(31) 4. Rapamycin+amlexanox stent group 2: animals are implanted with the stent containing both rapamycin and amlexanox (the rapamycin loading rate and the amlexanox loading rate of the stent are 140 μg/cm.sup.2 and 70 μg/cm.sup.2, respectively); and

(32) 5. Rapamycin+amlexanox stent group 3: animals are implanted with the stent containing both rapamycin and amlexanox (the rapamycin loading rate and the amlexanox loading rate of the stent are 140 μg/cm.sup.2 and 140 μg/cm.sup.2, respectively).

(33) For the preparation of the above stent groups, please refer to Embodiment 4.

(34) C. Stent Implantation

(35) Aspirin and clopidogrel are administered daily starting three days before surgery. The animals are anesthetized before surgery, and are fixed with their backs on the operating table, then a venous access is established, a cannula is inserted in the trachea, and a ventilator is provided for assisting in breathing. The right femoral artery is punctured after coronary angiogram and local disinfection, a guide wire is delivered through a puncture needle, a 6F femoral artery sheath is delivered along the guide wire, and 150 Ukg of heparin is administered through the sheath. A 6F right coronary guiding catheter is delivered through the sheath to perform left and right coronary angiography. The target vessel selection avoids large vascular branches as much as possible. A balloon is swelled with a pressure pump in vitro to release the stent, and the balloon is retrieved after the stent is completely adhered to the wall and causes injury. The angiography is rechecked after surgery. The catheter is retrieved, the femoral artery sheath is drawn out, and the surgical site is locally pressurized to stop bleeding. The pigs are sent back to the pigpen after awake for feeding.

(36) D. Experimental Results

(37) The pigs are continuously fed for 45 days after the stent is implanted. The vascular intimal hyperplasia at the injury site is examined 45 days later. The thicknesses of the intima of the bleeding vessel implanted with the stent and media of the bleeding vessel implanted with the stent are assayed to calculate the FM ratio, and the results are shown in Table 2 below.

(38) TABLE-US-00002 TABLE 2 No. Experimental groups I/M 1 Rapamycin stent group 0.553 ± 0.301 2 Amlexanox stent group 0.349 ± 0.160 3 Rapamycin + amlexanox stent group 1 0.190 ± 0.104 4 Rapamycin + amlexanox stent group 2 0.225 ± 0.133 5 Rapamycin + amlexanox stent group 3 0.207 ± 0.089

(39) It can be seen from the above results that the I/M of the combination of rapamycin and amlexanox is smaller than that of the single rapamycin or single amoxicillin, which indicates that the combination of rapamycin and amlexanox has a lower inhibition ratio over the intima in animals, and thus it is more conducive to rapid endothelialization of blood vessels.

Embodiment 6 Pharmaceutical Composition Eluting Stent

(40) Amlexanox, paclitaxel and poly(styrene-b-isobutylene-b-styrene) triblock copolymer (SIBS) are respectively weighed in a weight ratio of 1:1:10 and mixed, and then added with tetrahydrofuran until a solid content percentage reaches 1%. A stent body having a groove is prepared, and the prepared solution is then injected into the groove by micro-spraying. The drug-loaded stent is obtained after being dried in vacuum for six hours to remove the tetrahydrofuran.

Embodiment 7 Drug-Loading Balloon

(41) A drug balloon for treating vascular stenosis is prepared using a nylon balloon.

(42) (1) The surface of the nylon balloon is pretreated for 30 minutes by low temperature plasma under nitrogen at −20° C. with the output power of 2,000 W, the frequency of 25 Hz, and the air pressure of 1 Pa;

(43) (2) Polylysine is dissolved in ethanol to obtain a 60 mg/ml solution, the balloon flap obtained in step (1) is wound. The solution is dropwise coated to the balloon using a Hamilton MOD710SYR 100 μl NR syringe (DL Naturegene Life Sciences, Inc., China), and is dried naturally, so that a modified balloon is obtained;

(44) (3) Amlexanox is dissolved in a mixed solution of DMSO/water (with the volume ratio of 70:30) to prepare a 15 mg/ml amlexanox solution. 400 mg of lysine is added to 10 ml of the above amlexanox solution and dissolved by stirring at 10 rpm. The solution is placed at −5° C. for 24 hours, and filtered to obtain a solid. The solid is dried at 40° C. for 60 minutes to obtain a drug crystal; and

(45) (4) The drug crystal obtained in step (3) is brushed onto the surface of the modified balloon obtained in step (2). The modified balloon is weighed and repeatedly brushed five times, dried at 60° C. under 1,000 Pa vacuum, flapped and wound, packaged and sterilized, so that the drug balloon is obtained.

(46) It is assayed that the content of amlexanox in the prepared drug balloon is 345 μg.

Embodiment 8 Drug-Loading Balloon

(47) 500 mg of adhesive (such as PVP) is accurately weighed and dissolved in 10 mL of anhydrous ethanol, and stirred for 15 min for future use; 75 mg of amlexanox is dissolved in 10 ml of 50% ethanol solution and stirred for 15 min for future use. The adhesive solution is placed in a sample tube, and the balloon is immersed in the adhesive solution at a speed of 5 mm/s, and the balloon is pulled out at the rate of 2 mm/s after staying for 15 s. Then, the amlexanox solution is placed in another sample tube, and the balloon coated with the adhesive is treated five times in the same coating manner to obtain a drug loading balloon. The drug-loading rate is 200 μg/cm.sup.2.

(48) The technical solution provided by the present invention is able to inhibit the biological activity of proliferation of vascular smooth muscle cells without inhibiting the growth of the endothelial cell, namely providing a selective inhibitory effect. The technical solution provided by the present invention is able to prevent the incidence of vascular restenosis without delaying endothelial repair. Therefore, it is able to shorten the time for patients to take antithrombotic and platelet drugs after surgery, and thus greatly improves the long-term efficacy.