CN115844899A - Inner ear local combination medicine composition for treating sensorineural deafness and application thereof - Google Patents
Inner ear local combination medicine composition for treating sensorineural deafness and application thereof Download PDFInfo
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Images
Abstract
The invention discloses an inner ear local combination drug composition for treating sensorineural deafness. The composition includes the anti-inflammatory drug dexamethasone and the antioxidant lipoic acid. The invention adopts a recrystallization method to prepare the lipoic acid microcrystal by taking the bulk drug thioctic acid crystal as the raw material, the change of the Chinese medicine perilymph liquid is observed by locally feeding the dexamethasone microcrystal and the lipoic acid microcrystal in the inner ear of the guinea pig, and a cell model quantitative combined effect is established. The invention also proves that the dexamethasone microcrystal and the lipoic acid microcrystal can inhibit the cytotoxicity induced by the cisplatin by combined administration, and the dexamethasone microcrystal and the lipoic acid microcrystal have strong synergistic effect. The invention provides a new medication strategy for the local treatment of sensorineural deafness of the inner ear, and promotes the progress of local combined medication in the treatment of the inner ear diseases.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to an inner ear local combination medicine composition for treating sensorineural deafness and application thereof.
Background
Sensorineural deafness is a common disease worldwide, seriously affects the health of patients and becomes a burden for society. Aging, noise exposure, gene mutation and the use of ototoxic drugs are the main causes of sensorineural deafness. Due to the complexity of the inner ear anatomy and the uncertainty of the pathological mechanism, there is no effective treatment method for chronic sensorineural deafness except for the implantation of artificial cochlea in some cases. For acute onset deafness, including sudden deafness, systemic treatment with steroids is the most common treatment, and the mechanism is not clear, but it is speculated that its strong anti-inflammatory action may reduce cytotoxic immune responses and increase microvascular blood flow. However, not only does large dose systemic steroid therapy cause systemic adverse effects, but also after treatment, about 50% of patients have no or limited improvement in hearing. Therefore, there is a need to develop new strategies for the treatment of sensorineural deafness.
Multiple studies have shown that increased levels of oxidative stress and inflammation are critical to the development and progression of inner ear injury. Lipoic acid is a naturally occurring compound that acts as a cofactor in the mitochondrial respiratory enzymes involved in energy metabolism production, scavenging free radicals, chelating metal ions, and regenerating endogenous antioxidants. Lipoic acid has been used to treat a range of diseases including diabetes, polyneuropathy, cataract and kidney disease based on its powerful antioxidant effect. The efficacy of lipoic acid to treat acute hearing loss has been demonstrated by scavenging reactive oxygen free radicals and protecting mitochondrial function.
Currently, intratympanic injection is an important means for clinical treatment of inner ear diseases. However, rapid absorption of the drug in the middle ear mucosa, and susceptibility to loss through the eustachian tube, has to be frequently injected, resulting in poor patient compliance. Currently, gels, polymer particles, mini-implants and controlled release drug delivery devices are used primarily as local delivery systems to prolong the residence time of drugs in the middle ear. Wherein, the drug microcrystal has the advantages of large drug-loading rate, easy preparation, reduction of side effect generated by excipient and the like. Additionally, poorly soluble drug crystals of uniform size and regular shape exhibit better solubility and bioavailability than spherical drug crystals.
However, topical medications have been developed for the treatment of sensorineural deafness in a single form. In order to meet the treatment requirements of deafness patients as soon as possible, the development of a combined drug form and a drug delivery mode with clinical value is urgently needed.
Disclosure of Invention
The invention aims to provide an inner ear local combination drug composition for treating sensorineural deafness.
In order to achieve the above object, the present invention provides a combination drug composition.
The combination drug composition provided by the invention comprises an anti-inflammatory drug and an antioxidant.
Further, the anti-inflammatory drug may be a glucocorticoid-like anti-inflammatory drug, which may be specifically prednisolone, methylprednisolone, dexamethasone, or the like.
The antioxidant may be vitamin C, vitamin E, lipoic acid, etc.
Further, the anti-inflammatory agent is dexamethasone or a pharmaceutically acceptable salt thereof.
The antioxidant is lipoic acid or a pharmaceutically acceptable salt thereof.
In a particular embodiment of the invention, the dexamethasone can be dexamethasone microcrystalline. The apparent shape of the dexamethasone crystallites may be quadrilateral, such as square or rhombus. The side length of the dexamethasone microcrystal can be 4.43-12.86 μm.
In one embodiment of the present invention, the lipoic acid may be lipoic acid microcrystals. The apparent shape of the lipoic acid crystallites may be quadrilateral, such as rectangular. The lipoic acid crystallites may have a length of 53.56-73.12 μm and a width of 29.31-46.07. Mu.m.
In the composition, the drug combination composition comprises dexamethasone microcrystal and lipoic acid microcrystal; when the dexamethasone microcrystal and the lipoic acid microcrystal are used in combination, the administration dosage ratio of the dexamethasone microcrystal to the lipoic acid microcrystal is 20 (100-150), and specifically can be 2.
In practical application, the drug combination composition can be used in the form of a compound preparation of the dexamethasone microcrystal and the lipoic acid microcrystal, namely, the dexamethasone microcrystal and the lipoic acid microcrystal are mixed together for use; it is also possible to use the dexamethasone crystallite in the form of a separate preparation and the lipoic acid crystallite in the form of a separate preparation simultaneously.
Specifically, the subject to be treated with the combination may be a human or a mammal (e.g., guinea pig, etc.).
In particular, the combination composition may be administered by local injection.
In one embodiment of the invention, the effective dose of dexamethasone microcrystals is 2 mg/ear and the effective dose of lipoic acid microcrystals is 15 mg/ear by in-ear injection into 7-8 week-old pure-white-red-order male guinea pigs (weighing 250-350 g).
In order to achieve the above object, the present invention also provides a lipoic acid microcrystal.
The appearance shape of the thioctic acid microcrystal provided by the invention is quadrilateral.
Further, the quadrangle may be a rectangle.
Further, the lipoic acid microcrystals may have a length of 53.56-73.12 μm and a width of 29.31-46.07. Mu.m.
The preparation method of the lipoic acid microcrystal also belongs to the protection scope of the invention. The preparation method comprises the following steps: recrystallizing the raw material lipoic acid crystal to obtain the lipoic acid microcrystal.
Further, the recrystallization method comprises the following steps: dripping anhydrous ethanol solution of the thioctic acid crystal of the raw material medicine into polyvinyl alcohol aqueous solution which is dispersed at high speed, placing the mixture into a paddle type stirrer after the dispersion is finished, continuously stirring the mixture to obtain suspension, standing the suspension overnight (volatilizing the anhydrous ethanol in the solution), centrifuging the suspension, and removing supernatant (removing the polyvinyl alcohol) to obtain the thioctic acid microcrystal.
Furthermore, the concentration of the absolute ethanol solution of the thioctic acid crystal of the bulk drug can be 20-30mg/mL or 20-25mg/mL or 25-30mg/mL or 20mg/mL or 25mg/mL or 30mg/mL, and specifically can be 25mg/mL.
In the dropping step, the dropping rate can be 0.5-2mL/min, or 0.5-1mL/min, or 1-2mL/min, or 0.5mL/min, or 1mL/min, or 2mL/min, specifically 1mL/min.
The concentration of the polyvinyl alcohol aqueous solution can be 1-10g/L, 1-5g/L, 5-10g/L, 1g/L, 5g/L or 10g/L, and specifically can be 10g/L.
The volume ratio of the absolute ethyl alcohol solution of the lipoic acid crystals to the polyvinyl alcohol aqueous solution can be 1: (10-20), specifically 1:10.
the rotation speed of the high-speed dispersion can be 4000-10800rpm, and specifically can be 8000rpm.
The stirring condition can be 400-1000rpm for 1h, and specifically can be 800rpm for 1h.
The temperature for standing overnight may be 4-25 deg.C, specifically 4 deg.C.
The centrifugation times can be multiple times, specifically 3 times, and the rotating speed of each centrifugation can be 5000rpm.
In order to achieve the above object, the present invention finally provides the use of the above composition or the above lipoic acid crystallites or lipoic acid crystallites prepared according to the above method in any one of the following (a 1) to (a 4):
(a1) Preparing a product for treating sensorineural deafness;
(a2) Preparing a product that inhibits ototoxic drug-induced cytotoxicity;
(a3) Treating sensorineural deafness;
(a4) Inhibiting ototoxic drug-induced cytotoxicity.
In the above application, the ototoxic drug may be cisplatin.
In the above application, the cell may be a HEI-OC1 cell.
In the above application, the sensorineural deafness includes presbycusis, sudden deafness, drug-induced deafness or noise-induced deafness.
In the above applications, the product may be a medicament or a pharmaceutical preparation or agent.
The invention discloses that the combined use of dexamethasone and lipoic acid can be used for treating sensorineural deafness for the first time, and the lipoic acid microcrystals can promote the absorption of the dexamethasone microcrystals through testing the pharmacokinetic change in a guinea pig body. Protection studies of cisplatin-induced HEI-OC1 cytotoxicity demonstrated that the combined administration of dexamethasone and lipoic acid microcrystals showed a stronger cytoprotective effect than the single administration, i.e., the combined administration could achieve a synergistic effect at the cellular level. The invention provides a new medication strategy for the local treatment of sensorineural deafness of the inner ear, and promotes the progress of local combined medication in the treatment of the inner ear diseases.
Drawings
FIG. 1 is a scanning electron microscope image of dexamethasone drug substance, dexamethasone microcrystal, lipoic acid drug substance and lipoic acid microcrystal. (a) is a scanning electron microscope image of dexamethasone bulk drug; (b) is a scanning electron micrograph of dexamethasone microcrystal; (c) is a scanning electron microscope picture of the lipoic acid bulk drug; and (d) is a scanning electron microscope image of the thioctic acid microcrystals.
FIG. 2 is a graph showing the behavior of a Chinese medicinal composition in the form of perilymph after tympanogram injection of dexamethasone microcrystal and thioctic acid microcrystal alone or in combination.
FIG. 3 is a CCK-8 method for determining the protective effect of dexamethasone crystallite and lipoic acid crystallite alone or in combination on cisplatin-induced HEI-OC1 cytotoxicity.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The dexamethasone drug substance (DEX) in the following examples is a product of Tianjin Tianyao drug industry, gmbH, having a product number NED190807.
The lipoic acid drug substance (LA) in the following examples is a product of Michelin Biotechnology Ltd of Shanghai, having a product number of A835604-25g.
Cisplatin in the following examples is a product of Shanghai Michelin Biochemical technology, inc., having a product number of D807330-5g.
The following examples of pure white-red male guinea pigs were obtained from the institute of radiology, institute of medical research, and Beijing coordination, the Chinese academy of medical sciences.
The HEI-OC1 cells in the examples described below are products of ATCC under the accession number CRL-7782.
Example 1 an inner ear topical combination composition for the treatment of sensorineural deafness and method of preparation thereof
The inner ear local combination drug composition for treating sensorineural deafness provided by the invention consists of dexamethasone microcrystal and lipoic acid microcrystal. The preparation method of the dexamethasone microcrystal and the lipoic acid microcrystal comprises the following steps:
1. preparation of dexamethasone crystallites (DEX MCs)
1) Mixing 100mg of dexamethasone raw material medicine with the absolute ethanol solution to ensure that the concentration of the dexamethasone raw material medicine is 10mg/mL to obtain the dexamethasone absolute ethanol solution.
2) Adding a dexamethasone absolute ethyl alcohol solution into a polyvinyl alcohol aqueous solution with the mass volume concentration of 1g/L at the dropping rate of 2mL/min, wherein the volume ratio of the dexamethasone absolute ethyl alcohol solution to the polyvinyl alcohol aqueous solution is 1.
3) The suspension was obtained by high speed stirring at 10800 rpm. Standing overnight at 4 deg.C, and volatilizing anhydrous ethanol to obtain microcrystalline suspension.
4) Centrifuging the microcrystalline suspension for 3 times (the rotation speed of each centrifugation is 5000 rpm), and removing supernatant (removing excessive polyvinyl alcohol) to obtain dexamethasone microcrystalline.
Scanning electron micrographs of dexamethasone drug substance and dexamethasone microcrystal are shown in fig. 1 (a) and (b). As can be seen from the figure, the dexamethasone crystallites are square or diamond-shaped, with the side length of the dexamethasone crystallites being 4.43-12.86 μm.
2. Preparation of lipoic acid microcrystals (LA MCs)
1) Dissolving 250mg of lipoic acid raw material medicine into the absolute ethyl alcohol solution to ensure that the concentration of the lipoic acid raw material medicine is 25mg/mL, thus obtaining the lipoic acid absolute ethyl alcohol solution.
2) Adding the lipoic acid absolute ethyl alcohol solution into a polyvinyl alcohol aqueous solution with the mass volume concentration of 10g/L, which is dispersed at a high speed (a high-speed dispersion machine, the rotating speed is 8000 rpm) at the dropping speed of 1mL/min, wherein the volume ratio of the lipoic acid absolute ethyl alcohol solution to the polyvinyl alcohol aqueous solution is 1:10.
3) The suspension was placed in a paddle stirrer and stirring was continued for 1h at 800 rpm. Standing overnight at 4 deg.C, and volatilizing anhydrous ethanol to obtain microcrystalline suspension.
4) Centrifuging the microcrystalline suspension for 3 times (the rotation speed of each centrifugation is 5000 rpm), and removing the supernatant (removing the excessive polyvinyl alcohol) to obtain the thioctic acid microcrystalline.
Scanning electron micrographs of the lipoic acid drug substance and the lipoic acid microcrystals are shown in FIGS. 1 (c) and (d). As can be seen from the figure, the lipoic acid crystallites are oblong with a length of 53.56-73.12 μm and a width of 29.31-46.07. Mu.m.
Example 2 pharmacokinetic study in guinea pigs with dexamethasone microcrystalline alone, lipoic acid microcrystalline alone or dexamethasone microcrystalline and lipoic acid microcrystalline in combination
1. Experiment grouping and experiment method
Grouping experiments:
healthy pure white-red male guinea pigs (7-8 weeks, 250-350g in body weight) were divided into three groups according to the administration: DEX MCs group (20 mg/mL), LA MCs group (150 mg/mL), DEX LA group/LA DEX Group (DEX MCs 20mg/mL + LA MCs 150 mg/mL). The administration to each group is as follows:
DEX MCs group (20 mg/mL): dexamethasone microcrystals (DEX MCs) were mixed with ultrapure water to give a suspension of DEX MCs at a concentration of 20 mg/mL.
LA MCs group (150 mg/mL): lipoic acid microcrystals (LA MCs) were mixed with ultrapure water to give a suspension of LA MCs at a concentration of 150 mg/mL.
DEX LA group/LA DEX Group (DEX MCs 20mg/mL + LA MCs 150 mg/mL): dexamethasone microcrystals (DEX MCs) and lipoic acid microcrystals (LA MCs) are mixed with ultrapure water to obtain a microcrystals suspension with the concentration of DEX MCs being 20mg/mL and the concentration of LA MCs being 150 mg/mL.
The experimental method comprises the following steps:
guinea pigs were anesthetized with abdominal cavity, the hair was removed from the back of the ear, and bilateral retroauricular incisions were made with local disinfection. The muscle fascia on the surface of the temporal bone is separated, a small hole with the diameter of 1mm is drilled by an electric drill, and 0.1mL of the medicines of each group are respectively injected. After the three groups of animals are administrated at different time points, cochlear perilymph fluid is extracted, the drug concentration is measured by high performance liquid chromatography, and pharmacokinetic parameters comprising drug peak time (Tmax), mean Retention Time (MRT), area under a blood concentration-time curve (AUC), maximum blood concentration (Cmax) and the like are measured by adopting DAS2.0 pharmacokinetic statistical software.
2. Results of the experiment
The results are shown in FIG. 2, in the DEX MCs group and DEX LA In the group, the dexamethasone concentration in the perilymph fluid peaked at 12h (112.94. + -. 9.77. Mu.g/mL) and 1h (138.23. + -. 8.97. Mu.g/mL), respectively, indicating that when dexamethasone and lipoic acid are administered in combination, the Tmax is significantly reduced and the Cmax is slightly increased compared to the individual administration. In addition, in DEX LA In the group, dexamethasone was not detected on day 7. These results indicate that LA promotes the absorption of DEX. However, lipoic acid showed similar pharmacokinetic profiles whether administered alone or in combination with a single dose of dexamethasone. And 1 hour after tympanogram injection in perilymph fluidLipoic acid levels reached a maximum, although there was a variation at some time points, e.g. 3h DEX The lipoic acid concentration of the group was significantly higher than that of the LA MCs group, but no significant difference was observed between the two groups at other time points. The above results indicate that the absorption of DEX is improved in the presence of LA, which is a simpler and more effective method of promoting drug absorption.
Example 3 protection study of dexamethasone microcrystalline alone, lipoic acid microcrystalline alone, or dexamethasone microcrystalline and lipoic acid microcrystalline combined administration on cisplatin-induced HEI-OC1 cytotoxicity
1. Experiment grouping and experiment method
Using DMEM medium containing 10% fetal bovine serum at 37 deg.C and 5% CO 2 And culturing the HEI-OC1 cells in an incubator with relative humidity of 90%. Dexamethasone microcrystals (0.01-100 mu M), lipoic acid microcrystals (0.25-4 mM) or cisplatin (5-120 mu M) with different concentrations are taken to be respectively co-cultured with HEI-OC1 cells for 24h, and the CCK-8 method is utilized to screen out the optimal concentration of the drug (the optimal protection concentration of the dexamethasone microcrystals or lipoic acid microcrystals: the selected drug, and the half inhibition concentration of cisplatin: the selected drug).
Adding HEI-OC1 cells in logarithmic growth phase into a 96-well plate, adding 100 mu L of dexamethasone microcrystal, lipoic acid microcrystal or combined drug (dexamethasone microcrystal and lipoic acid microcrystal) with optimal concentration into each well after culturing for 24h, respectively, adding cisplatin after 4h, continuing culturing for 24h, adding CCK-8, and measuring absorbance value at 450 nm. The treatment groups were divided into the following treatment groups according to the administration: the dose for each group is shown in figure 3 (d). Meanwhile, HEI-OC1 cells added with the culture medium are used as a negative control group, and only the culture medium is added as a blank control group, and the cell survival rate is calculated according to the following formula: cell viability = (OD dosing group-OD blank group)/(OD negative group-OD blank group), which reflects the protective effect of dexamethasone crystallite alone, lipoic acid crystallite alone or dexamethasone crystallite and lipoic acid crystallite dosing in combination on cisplatin-induced HEI-OC1 cytotoxicity by cell viability.
2. Results of the experiment
The results are shown in fig. 3, the HEI-OC1 cells are respectively treated by dexamethasone microcrystals with different concentrations or lipoic acid microcrystals with different concentrations, no obvious cytotoxicity is found, and 1 mu M dexamethasone microcrystals and 1mM lipoic acid microcrystals are selected for carrying out the cell protection experiment. In addition, 20 μ M cisplatin-treated cells were approximately 50% less viable than the control group. The cell viability of the cisplatin group, lipoic acid crystallite + cisplatin group, dexamethasone crystallite + lipoic acid crystallite + cisplatin group and dexamethasone crystallite + lipoic acid crystallite + cisplatin group were further determined to be 41.50 ± 3.73%, 48.72 ± 3.10%, 53.28 ± 1.44% and 60.21 ± 3.59%, respectively. The results indicate that pretreatment of cells with dexamethasone crystallite alone, lipoic acid crystallite alone and dexamethasone crystallite and lipoic acid crystallite administration in combination can protect HEI-OC1 cells from cisplatin-induced cell damage, wherein dexamethasone crystallite and lipoic acid crystallite administration in combination shows a stronger cytoprotective effect than dexamethasone crystallite and lipoic acid crystallite administration alone.
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.
Claims (10)
1. A combination composition comprises an anti-inflammatory agent and an antioxidant.
2. The composition of claim 1, wherein: the anti-inflammatory drug is dexamethasone or pharmaceutically acceptable salt thereof;
the antioxidant is lipoic acid or a pharmaceutically acceptable salt thereof.
3. The composition according to claim 1 or 2, characterized in that: the anti-inflammatory drug is dexamethasone microcrystal;
or the antioxidant is lipoic acid microcrystals.
4. The composition according to any one of claims 1 to 3, characterized in that: the combination composition comprises dexamethasone microcrystal and lipoic acid microcrystal; the dosage ratio of dexamethasone microcrystal and thioctic acid microcrystal in the combined administration is 20 (100-150).
5. Lipoic acid microcrystals, characterized in that: the appearance shape of the lipoic acid microcrystal is quadrilateral.
6. The lipoic acid crystallite of claim 5, characterized in that: the quadrangle is rectangular;
or the length of the lipoic acid microcrystal is 53.56-73.12 mu m, and the width of the lipoic acid microcrystal is 29.31-46.07 mu m.
7. A process for the preparation of the lipoic acid microcrystals of claim 5 or 6, comprising the following steps: recrystallizing the raw material lipoic acid crystal to obtain the lipoic acid microcrystal.
8. Use of a composition according to any one of claims 1 to 4 or of a lipoic acid crystallite according to claim 5 or 6 or of a lipoic acid crystallite prepared according to the process of claim 7 in any one of the following (a 1) to (a 4):
(a1) Preparing a product for treating sensorineural deafness;
(a2) Preparing a product that inhibits ototoxic drug-induced cytotoxicity;
(a3) Treating sensorineural deafness;
(a4) Inhibiting ototoxic drug-induced cytotoxicity.
9. Use according to claim 8, characterized in that: the ototoxic drug is cisplatin.
10. Use according to claim 8 or 9, characterized in that: the sensorineural deafness comprises presbycusis, sudden deafness, drug deafness or noise deafness.
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