Amikacin sulfate multivesicular liposome for local injection and preparation method thereof
Technical Field
The invention belongs to the technical field of pharmaceutical preparations, and particularly relates to an amikacin sulfate multivesicular liposome for local injection, and a preparation method and application thereof.
Background
With the wide use and abuse of antibiotics, the problem of antibiotic resistance has become a global public health safety problem, especially the problem of gram-negative bacteria resistance, and the clinical treatment method for some drug-resistant bacteria infection is very limited. Throughout recent years of the year report by CHINET (China bacterial resistance monitoring network), amikacin, which is a representative of aminoglycoside antibiotics, has a very low resistance rate to enterobacteriaceae and Pseudomonas aeruginosa, but antibiotic therapy in the conventional mode of administration has various problems: after the antibiotic is administrated, the antibiotic is rapidly metabolized and discharged from the body, only a small part of the drug reaches the infected part, the bioavailability is low, so that large-dose administration and long-period treatment are often needed in clinic, and obvious toxic and side effects can be caused; in addition, antibiotics have poor therapeutic effect on bacteria in biomembranes, are difficult to enrich in cells and kill live bacteria in cells, and cause chronic infection and recurrent infection frequently.
Amikacin Sulfate (Amikacin Sulfate) belongs to the third-generation semi-synthetic aminoglycoside antibiotics, which was first developed in 1972 by the company Bristol, japan, and was first researched and developed by the institute of antibiotic industry, sichuan, china, and the first chemical name thereof is O-3-amino-3-deoxy- α -D-glucopyranosyl- (1 → 4) -O- [ 6-amino-6-deoxy- α -D-glucopyranosyl- (1 → 6) ] -N3- (4-amino-2-hydroxy-1-oxobutyl) -2-deoxy-L-streptomycin Sulfate, and the molecular structural formula is as follows:
the mechanism of action of amikacin sulfate is to inhibit protein synthesis by binding tightly to the a site of the 30S ribosomal 16S rRNA. Unlike other aminoglycoside antibiotics, amikacin sulfate has the advantage of producing few resistant strains, which are still sensitive to the product, especially to gram-negative bacteria and staphylococci. However, the amikacin sulfate preparation applied to clinic in China at present is mainly the common amikacin sulfate for injection, and the administration route mainly comprises the intramuscular route, the intravenous route and the intrathecal route. The preparation has large dosage, the drug release is carried out according to first-order kinetics, the fluctuation of the drug concentration in blood is large, the phenomenon of peak valley is often generated, the blood drug concentration is possibly beyond the lowest toxic concentration, the effective blood drug concentration has short maintenance time, the effective treatment aim can be achieved by repeated drug administration for many times, the labor cost is increased, the repeated drug administration has larger stimulation to human bodies, the stress response is obvious, and the preparation is not beneficial to the health of human beings.
In addition, clinical cases of toxic side effects due to amikacin sulfate are frequent, and there is evidence that in the treatment of acute infections lasting for 5-7 days, approximately 20% of patients may suffer from hearing loss due to the use of aminoglycoside antibiotics, and about 15% may affect the balance, and in addition, neurotoxicity (muscle paralysis and apnea) and nephrotoxicity are observed, with drug toxicity being positively correlated with blood levels. In addition, amikacin sulfate belongs to concentration-dependent antibiotics, and the sterilization effect is enhanced along with the increase of the drug concentration, so that the toxicity is increased, and the defects limit the wide application of the amikacin sulfate.
Aiming at the problems existing in the traditional antibiotic administration mode, the novel lipid administration system is utilized to deliver the antibiotic, so that the application prospect is good. Multivesicular liposomes (MVL) are aggregates of closely packed non-concentric lipid bimolecular vesicles developed by Kim S in 1983, a novel liposome for drug delivery. As a drug carrier, the multivesicular liposome can be biodegraded, has no toxicity and immunogenicity, can form a drug reservoir by locally injecting multivesicular liposome suspension into a focus part, and the drug release needs to penetrate the layer-by-layer obstruction of lipid and phospholipid bilayers to generate good slow release action, thereby not only reducing the drug dose, but also improving and prolonging the drug curative effect, and the drug has no burst release phenomenon when being released under the therapeutic dose, and can also avoid the fluctuation or toxicity level in the body and the absorption by a reticuloendothelial system; because the multivesicular liposome contains a large amount of water-phase encapsulated antibiotic drugs with good water solubility, high encapsulation efficiency and large drug-loading rate can be obtained, and the lipid carrier can also improve the pharmacokinetics and biological distribution of the drugs and reduce the drug toxicity; has targeting selectivity to focus parts, enhances the antibacterial activity of the medicine to intracellular and extracellular pathogens, and reduces the occurrence of drug resistance. Multivesicular liposome formulations currently on the market and approved by FDA in the United states are developed by SkyePharma for the prevention and treatment of meningeal leukemia and lymphoma meningitis
(cytarabine multicapsule liposome injection) and morphine sulfate multicapsule liposome injection applied to acute and chronic pain and clinical anesthesia treatment
And Pacira corporation developed non-opioid topical long-acting analgesics for postoperative analgesia at the surgical site
(bupivacaine liposome injection suspension) officially prepared by NMPA drug evaluation center at 2021 and 3 months domesticallyThe green leaf pharmaceutical ropivacaine hydrochloride liposome suspension injection (LY09606) was admitted to clinical stage. The drugs that have been studied and encapsulated in multivesicular liposomes relate to anti-tumor, analgesic, antibacterial, antiviral and biomacromolecule drugs such as liraglutide. In addition, there is a foreign review that companies are currently developing liposomal suspensions of amikacin multivesicules (DePoAmikacin)
TM) And the phase I clinical study stage is entered in 2005, but relevant contents in the preparation process study are not obtained through further search, and no product is on the market.
Therefore, there is a need for an amikacin sulfate multivesicular liposome and related injection preparations, which can solve the above problems of systemic action of the traditional amikacin sulfate for injection. How to provide the amikacin sulfate multivesicular liposome, the local administration on a bacterial infection focus is realized, the drug dosage is reduced, the multivesicular liposome is used for encapsulating drugs to realize a slow release effect, the administration frequency is reduced, the toxic and side effects are reduced, and the compliance of patients is improved, which becomes a technical problem to be solved urgently.
Disclosure of Invention
The invention aims to solve the technical problems, provides an amikacin sulfate multivesicular liposome for local injection and a preparation method thereof, and also provides a specific application of the amikacin sulfate multivesicular liposome. The amikacin sulfate multivesicular liposome provided by the invention has the characteristics of high encapsulation efficiency, large drug loading rate and uniform particle size, and can be prepared into powder injection for injection, so that the sustained-release effect is better, the bioavailability of the drug is favorably improved, and the curative effect is obviously improved.
The invention aims to provide an amikacin sulfate multivesicular liposome for local injection, which is characterized by comprising the following raw materials in parts by weight:
35-180 parts of amikacin sulfate;
35-300 parts of a lipid material;
195-410 parts of an osmotic pressure regulator;
0.50-35.50 parts of auxiliary emulsifier material;
wherein the lipid material is prepared from phospholipid, cholesterol, triglyceride and a membrane stabilizer according to the mass ratio of (5-7.5): (3-5.5): (3-6): (1.0-3.0).
The amikacin sulfate multivesicular liposome is a light milky and uniform suspension after redissolving, has uniform particle size, high encapsulation efficiency and large drug loading capacity, can solve the problem of large using amount of amikacin sulfate for injection, further reduces the occurrence of toxic and side effects, is encapsulated in a lipid material, is locally injected to an infected part sensitive to the amikacin sulfate, can be accumulated in a large amount in an injection part or a drug delivery cavity, and slowly releases the drug without burst release.
Further, the phospholipid is selected from at least one of Soybean Phospholipid (SPC), Hydrogenated Soybean Phospholipid (HSPC), egg yolk lecithin (EPC) or dipalmitoyl phosphatidylcholine (DPPC); the triglyceride is at least one selected from triolein, tricaprylin and soybean oil. Unlike the multivesicular liposome injection products on the market, the preferred natural phospholipid of the invention has better biocompatibility, low price and easy obtainment, and simultaneously has the advantages of low phase transition temperature and relatively mild preparation conditions.
Further, the membrane stabilizer is at least one of negatively charged phosphatidyl glycerol, fatty acid or stearylamine; the negatively charged phosphatidylglycerol comprises dipalmitoyl phosphatidylglycerol (DPPG), 1, 2-dicaprylyl-SN-glycero-3-phospho-RAC- (1-glycerol) (DEPG), 1, 2-dimyristoyl-SN-glycero-3-phospho-RAC- (1-glycerol) (DMPG), 1, 2-dioleoyl-SN-glycero-3-phospho-RAC- (1-glycerol) (DOPG); the fatty acid comprises oleic acid or stearic acid. It has been reported that most of the polycystic liposome preparations adopt negatively charged phospholipids as membrane stabilizers, but the membrane stabilizers are high in cost and not easy to obtain, the materials selected by the invention are easy to obtain and low in price, and particularly, the preferred stearylamine is used as the membrane stabilizer, so that the phospholipid membrane can be prevented from being combined and damaged by increasing electrostatic repulsion between phospholipid bimolecular layers. The addition of different amounts of stearylamine in the oil phase can obviously improve the formability of the multivesicular liposome, the formed multivesicular liposome has uniform particle size, and the generation of phospholipid fragments can be greatly reduced to eliminate the phenomenon of aggregation and precipitation.
Further, the osmotic pressure regulator is at least one selected from sucrose, glucose, behenyl fatty acid sorbitol or trehalose. The auxiliary emulsifier material is selected from one or more of L-Lysine (L-Lysine) and polyvinyl alcohol (PVA), the preferable auxiliary emulsifier material is polyvinyl alcohol, Lysine is selected as the auxiliary emulsifier in the conventional multivesicular liposome formula, and the invention discovers that higher encapsulation efficiency can be obtained by adopting the polyvinyl alcohol through comparison.
The invention also aims to provide a preparation method of the amikacin sulfate multivesicular liposome for local injection, which comprises the following steps:
(1) weighing lipid materials according to parts by weight, and dissolving the lipid materials in an organic solvent to be used as an oil phase; preparing amikacin sulfate solution containing osmotic pressure regulator as an internal water phase; preparing a coemulsifier solution containing an osmotic pressure regulator as an external water phase;
(2) dispersing the inner water phase into the oil phase, and mixing to form stable W/O type primary emulsion;
(3) refining the primary emulsion, and quickly injecting the refined primary emulsion into an external water phase to form multiple emulsion by vortex mixing;
(4) placing the multiple emulsion in a hot water bath to remove the organic solvent to obtain amikacin sulfate multivesicular liposome suspension A;
(5) and (4) purifying the product obtained in the step (4) to obtain amikacin sulfate multivesicular liposome suspension B, and freeze-drying to obtain the amikacin sulfate multivesicular liposome.
According to the preparation method of the amikacin sulfate multivesicular liposome, the W/O type colostrum which is milky white in appearance, good in fluidity and stable can be prepared in the step (2).
Further, the organic solvent in the step (1) is selected from one of chloroform and a mixed solution of chloroform and diethyl ether; the volume ratio of the internal water phase to the oil phase is 0.5-2: 2; the volume ratio of the colostrum to the external water phase is 1: 1 to 4.
Further, the mixing method in the step (2) comprises any one of a vortex method, a stirring method or an ultrasonic cell disruption method, wherein the ultrasonic power of an ultrasonic cell disruption instrument is preferably 150-500W, and the ultrasonic time is 3-10 min; the method for removing the organic solvent in the step (4) includes any one of room temperature evaporation, reduced pressure rotary evaporation or nitrogen blowing.
Further, the temperature of the hot water bath in the step (4) is 37-40 ℃ until the turbidity of the suspension is reduced and the suspension is free of organic solvent smell.
Further, the purification step in step (5) is: and selecting one or more of ultrapure water, 5% glucose solution and 0.9% sodium chloride solution by adopting a centrifugal method, washing and precipitating for 2-3 times, and re-suspending to obtain the amikacin sulfate multivesicular liposome suspension B.
The amikacin sulfate multivesicular liposome suspension B prepared by the invention is light milky and uniform suspension in appearance, uniform in particle size, high in encapsulation rate, large in drug loading capacity, good in biocompatibility and biodegradability, easy to suspend by light shaking and precipitation at the bottom after being stored for 20 days at 4 ℃, and low in drug leakage rate. The amikacin sulfate multivesicular liposome freeze-dried powder injection is prepared by freeze-drying the amikacin sulfate multivesicular liposome suspension B by adopting a freeze-drying technology, and can improve the stability and better transport and store.
According to the invention, the drug loading is increased, the drug dosage is reduced, the amikacin sulfate multivesicular liposome suspension is locally injected to the focus part to form a drug reservoir, and the drug generates a good slow release effect through the layer-by-layer barrier of lipid and phospholipid bilayers, so that the bioavailability is improved, the preparation toxicity is further reduced, and a better antibacterial treatment effect is realized.
The invention also aims to provide application of the amikacin sulfate multivesicular liposome, which is used for preparing the prepared amikacin sulfate multivesicular liposome into a medicament for treating or preventing local soft tissue and closed body cavity infection and Foreign Body (FBs) infection caused by staphylococcus and gram-negative bacteria.
The invention has the following beneficial effects:
the invention provides an amikacin sulfate multivesicular liposome for local injection and a preparation method thereof. The injection can be locally injected to a focus part to form a drug reservoir, and the drug can generate a good slow release effect through layer-by-layer separation of lipid and phospholipid bilayers, so that the bioavailability is improved, the toxicity of the preparation is reduced, and a better antibacterial treatment effect is realized.
Drawings
FIG. 1 is a particle morphology of AMKX-MVLs (amikacin sulfate multivesicular liposomes) under a fluorescence inverted microscope, 200 (left) and 400 (right) according to example 3 of the present invention; FIG. 2 is a graph of the cumulative release profiles (n 6) of AMKX-MVLs (amikacin sulfate multivesicular liposomes) in different pH release media according to example 1 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more clearly understood, the present invention is described in detail below with reference to the following embodiments, and it should be noted that the following embodiments are only for explaining and illustrating the present invention and are not intended to limit the present invention. The invention is not limited to the embodiments described above, but rather, may be modified within the scope of the invention.
In the embodiment of the invention, the appearance of the amikacin sulfate multivesicular liposome is observed by adopting a fluorescence inverted microscope;
the method for calculating the entrapment rate and the drug loading capacity of the amikacin sulfate multivesicular liposome in the embodiment of the invention comprises the following steps:
the calculation method of the encapsulation efficiency comprises the following steps:
the calculation method of the drug loading rate comprises the following steps:
example 1
The amikacin sulfate multivesicular liposome comprises the following raw material formula in the following table 1: combining literature data and drug administration dosage, fixing the concentration of the main drug TO be 50mg/mL, wherein the mass ratio of SPC TO CH TO TO TO ODA in the lipid material is 4: 3: 2: 1, investigating the medicine-fat ratio as 3: 1,2: 1,1: 1 the appearance, particle size and encapsulation efficiency of the product varied.
TABLE 1
The preparation process comprises the following steps:
SPC, CH, TO and ODA with the formula amount are dissolved in 3ml of chloroform respectively for each group of samples TO be used as oil phases for later use; dissolving amikacin sulfate in 3ml of 7% sucrose solution to serve as an internal water phase for later use; 625mg of glucose is dissolved in 12.5ml of 0.5% PVA (polyvinyl alcohol) solution to be used as an external water phase for standby; dispersing the inner water phase into the oil phase, stirring at 10000r/min for 5min, and then breaking the ultrasonic cells for 4min (power 300W) under the ice bath condition to form stable W/O type primary emulsion; sucking primary emulsion with injector, injecting into 12.5ml external water phase at a certain speed, and vortex mixing for 10s to form multiple emulsion; transferring the multiple emulsion into an open glass bottle, placing in water bath at 37 + -0.2 deg.C, stirring at a certain speed while blowing nitrogen for 10-15min, and removing organic solvent until turbidity of the suspension is reduced and no organic solvent is smelled to obtain amikacin sulfate multivesicular liposome suspension A (AMKX-MVLs). The appearance of suspension a changed from a light milky translucent homogeneous to milky homogeneous with increasing lipid mass.
The encapsulation efficiencies of the samples 1 to 3 are respectively 21.05%, 29.70% and 51.98%, the average particle diameter D50 values are respectively 54.292 μm, 50.188 μm and 45.592 μm, the drug-loading rates are respectively 35.91%, 33.99% and 21.80%, and the encapsulation efficiencies after purification can reach 54.42%, 65.73% and 94.15%. The results of comparative experiments show that the encapsulation efficiency of the medicine can be improved by adjusting the medicine-fat ratio, but when the medicine-fat ratio reaches 1: 1, the formed colostrum becomes sticky, and the quality of lipid is increased continuously, which is not beneficial to the formation of multiple emulsion.
Example 2
Combining literature reports with pre-experimental results, this example details the effect of stearylamine as a film stabilizer, whether or not it is added to the formulation and the amount of it, on the appearance, particle size and encapsulation efficiency of the product.
The amikacin sulfate multivesicular liposome comprises the following raw material formula of 2:
TABLE 2
The preparation process comprises the following steps:
SPC, CH, TO and ODA with the formula amount are dissolved in 3ml of chloroform respectively for each group of samples TO be used as oil phases for later use; dissolving amikacin sulfate in 3ml of 7% sucrose solution to serve as an internal water phase for later use; dissolving 1.0006g of glucose in 20ml of 0.5% PVA solution to serve as an external water phase for later use; dispersing the inner water phase into the oil phase, stirring at 10000r/min for 5min, and breaking the ultrasonic cells for 4min (power 300W) under ice bath condition to form stable W/O type primary emulsion; sucking primary emulsion with injector, injecting into 12.5ml external water phase at a certain speed, and vortex mixing for 10s to form multiple emulsion; transferring the multiple emulsion into an open glass bottle, placing in water bath at 37 + -0.2 deg.C, stirring at a certain speed while blowing nitrogen for 10-15min, and removing organic solvent until turbidity of the suspension is reduced and no organic solvent is smelled to obtain amikacin sulfate multivesicular liposome suspension A (AMKX-MVLs).
Wherein, sample 4 is not added with stearylamine, and the phenomena of particle adhesion, aggregation and precipitation appear in the process of removing the solvent; samples 5-7, which were in the form of a pale milky translucent homogeneous suspension, were stored at 4 ℃ and allowed to stand for 0.5-1h, after which the liposomes began to precipitate and were resuspended by gentle shaking with mean particle sizes D50 of 48.152 μm, 45.592 μm and 43.435 μm, encapsulation efficiencies of 49.47%, 51.98% and 54.27% and drug loadings of 25.38%, 21.80% and 20.29%, respectively. After purification (amikacin sulfate multivesicular liposome suspension B), the entrapment rate can reach 93.37%, 94.15% and 94.24%.
Example 3
The amikacin sulfate multivesicular liposome comprises the following raw material formula of 3:
TABLE 3
The preparation process comprises the following steps:
in the two groups of samples, SPC, CH, TO and ODA with the formula amounts are respectively dissolved in 3ml of chloroform-ether mixed solution (v/v, 1: 1) TO be used as oil phases for later use; dissolving amikacin sulfate in 3ml of 7% sucrose solution to serve as an internal water phase for later use; 1g of glucose was dissolved in 20ml of a 0.5% PVA solution and used as an external aqueous phase. Dispersing the inner water phase into the oil phase, stirring for 9min at 10000r/min, sucking primary emulsion by a syringe, injecting the primary emulsion into 12.5ml of outer water phase at a certain speed, carrying out vortex mixing for 10s to form multiple emulsion, transferring the multiple emulsion into an open glass bottle, placing the open glass bottle in a water bath at 37 +/-0.2 ℃, carrying out nitrogen blowing for 10-15min at the same time of stirring at a certain speed, removing the organic solvent until the turbidity of the suspension is reduced and the taste of the organic solvent is eliminated, and obtaining amikacin sulfate multivesicular liposome suspension A (AMKX-MVLs). Sample 9 disperses the inner water phase into the oil phase, ultrasonic cell disruption is carried out for 4min (power 300W) under ice bath condition after 10000r/min stirring for 5min to form stable W/O type primary emulsion, and the subsequent steps are the same as sample 8.
The appearance of the sample 8 is milky and uniform, the appearance of the sample 9 is light milky and semitransparent and uniform, the average particle size D50 values of the samples measured by a laser particle sizer are 46.272 mu m and 28.513 mu m respectively, the encapsulation efficiency is 50.44 percent and 62.48 percent respectively, and the drug loading rate is 27.38 percent and 21.81 percent respectively. The encapsulation efficiency can reach 85.01 percent and 94.79 percent after purification. Preferably, the stirring and ultrasonic cell disruption method is combined with the measurement results of various indexes of particle size, encapsulation efficiency and drug-loading rate as the preparation process of the W/O type colostrum.
Example 4
The amikacin sulfate multivesicular liposome comprises the following raw material formula of 4:
the preparation process comprises the following steps:
weighing EPC, CH, TO and ODA with the formula amount in each group of samples, dissolving the EPC, CH, TO and ODA in 3ml of chloroform, and taking the EPC, CH, TO and ODA as oil phases for later use; dissolving amikacin sulfate in 3ml of 8% sucrose solution to serve as an internal water phase for later use; samples 10, 11 and 12 respectively select a 20mmol/L L-lysine-4% glucose solution, a 40mmol/L L-lysine-4% glucose solution and a 60mmol/L L-lysine-4% glucose solution as external water phases for standby. Dispersing the water phase in each sample into oil phase, stirring for 5min at 10000r/min, breaking ultrasonic wave cells for 4min (power 300W) under ice bath condition to form stable W/O type colostrum, sucking the colostrum with a syringe, injecting into 12.5ml of external water phase at a certain speed, mixing by vortex for 10s to form multiple emulsion, transferring the multiple emulsion into an open glass bottle, placing in water bath at 37 +/-0.2 ℃, stirring at a certain speed and blowing nitrogen for 10-15min, removing the organic solvent until the turbidity of the suspension is reduced and no organic solvent is smelled, and obtaining amikacin sulfate multivesicular liposome suspension A (AMKX-MVLs).
The samples 10 and 12 have the phenomenon of particle adhesion, aggregation and sedimentation when organic solvents are removed for 10min, the sample 11 is in a milky uniform appearance, the average particle size D50 value is 50.378 mu m when the sample is measured by a laser particle sizer, the encapsulation rate is 48.54%, the drug loading rate is 12.78%, and the encapsulation rate can reach 80.17% after purification.
Example 5
The amikacin sulfate multivesicular liposome suspension B (purified A) prepared in example 2 of the present invention was stored at 4 ℃ and the appearance was observed and leakage was examined, the results are shown in Table 5. The result shows that the amikacin sulfate multivesicular liposome suspension B can be stored for 21 days at the temperature of 4 ℃, the appearance of the amikacin sulfate multivesicular liposome suspension B has no obvious change, but the leakage rate is increased, which is probably related to the particle size of the amikacin multivesicular liposome suspension B being in micron-sized size, so the amikacin sulfate multivesicular liposome suspension B is considered to be prepared into a freeze-dried product preparation.
TABLE 5
Example 6
The amikacin sulfate multivesicular liposome suspension B (A) prepared in the embodiment 2 of the invention is taken and placed in a vacuum freeze dryer, the freeze-drying process (prefreezing-sublimation drying-desorption drying) is set to prepare a freeze-dried product, the freeze-dried product is sealed by an aluminum-plastic combined cover and stored for 30 days at the temperature of 25 ℃, the appearance of the freeze-dried product is observed to be white cake-shaped solid, the surface is flat and smooth, the powder texture is fine, a proper amount of 0.9 percent sodium chloride is added for slight shaking, the freeze-dried product can be completely redissolved in a short time and is milky uniform suspension, and the leakage rate is also reduced.
Example 7
Amikacin sulfate multivesicular liposome suspension B3ml prepared in example 3 of the present invention was put into a clean dialysis bag (Mw 14000), air bubbles were removed from the dialysis bag and both ends were tied, and the dialysis bag was placed in a centrifuge tube containing phosphate buffer solution with pH 5.5 and pH 7.4. Placing the centrifuge tube in a shaker at constant temperature of 37 deg.C, setting shaker rotation speed of 50r/min, sampling 2ml in 0.5, 1,2, 4, 10, 24, 48, and 72h respectively, supplementing fresh release medium with the same temperature and volume, determining drug concentration of the sample and calculating drug cumulative release percentage according to the established determination method of amikacin sulfate content, and drawing a release curve with time as abscissa and drug cumulative release percentage as ordinate as figure 2. According to the in vitro release result, the amikacin sulfate multivesicular liposome suspension can realize the sustained and slow release of the medicament in vitro in release media with different pH values, has no burst release effect, and is released more quickly in the release media with the pH value of 5.5.