CN112426427B

CN112426427B - Application of midazolam nanocrystal in preparation of medicine for improving blood brain barrier permeability

Info

Publication number: CN112426427B
Application number: CN202010586753.0A
Authority: CN
Inventors: 郑爱萍; 张洵; 李蒙; 张慧; 高静; 刘楠; 王增明; 高翔
Original assignee: Academy of Military Medical Sciences AMMS of PLA
Current assignee: Academy of Military Medical Sciences AMMS of PLA
Priority date: 2020-06-24
Filing date: 2020-06-24
Publication date: 2022-07-12
Anticipated expiration: 2040-06-24
Also published as: CN112426427A

Abstract

The invention provides application of midazolam nanocrystal and a composition thereof in medicines for improving blood brain barrier permeability. The midazolam nanocrystal solves the problem of solubility, improves the bioavailability, solves the problem of poor treatment effect of tablets and injection used as anticonvulsant and antiepileptic medicaments, can effectively penetrate through a blood brain barrier, and is used for treating epilepsy. The invention also provides a technical platform, and the nanocrystalline preparation improves the blood brain barrier permeability of the brain drug in the presence of the space protective agent and the charge stabilizer.

Description

Application of midazolam nanocrystal in preparation of medicine for improving blood brain barrier permeability

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to application of midazolam nanocrystal in preparation of a medicine for improving blood brain barrier permeability.

Background

Midazolam [ 8-chloro-6- (2-fluorophenyl) -1-methyl-4H-imidazo [1, 5-alpha ] [1,4] benzodiazepine, the structure of which is shown in formula I, plays a role through a gamma-aminobutyric acid (GABA) receptor binding site, and reduces the release of excitatory transmitter glutamic acid by inhibiting the reverse transport of a human glial cell glutamate carrier, thereby effectively controlling the onset of convulsion. The midazolam maleate tablet is suitable for treating sleep disorder and insomnia, and is especially suitable for people with difficulty in falling asleep and for taking medicine before operation or diagnostic operation. Indications for midazolam injection are "premedication prior to anesthesia induction, anesthesia induction and maintenance, conscious sedation for diagnostic or therapeutic procedures". However, the tablets and the injection have poor curative effect when used as anticonvulsant and antiepileptic medicaments.

Midazolam is poorly soluble in water under neutral conditions and its solubility increases at pH < 3. Midazolam injection (pH 2.9-3.7) is used for anesthesia, and has the defects of high irritation and the like when the midazolam injection is used for resisting convulsion through intramuscular injection. The nanometer suspension is neutral and has less irritation, but the dissolution and dissolution of the midazolam become the rate-limiting steps of the absorption in vivo because the midazolam is a poorly soluble drug. Therefore, increasing the dissolution rate of the drug is an important means for increasing the bioavailability of the drug. How to improve the solubility, dissolution rate and bioavailability of midazolam, develop a midazolam preparation suitable for large-scale industrial production and a preparation method thereof, and obviously improve the effectiveness, safety and stability of midazolam, which is a technical problem to be solved in the field.

Disclosure of Invention

The invention aims to provide a midazolam nanocrystal composition, which contains midazolam, a dispersion medium and a pharmaceutically acceptable carrier, wherein the midazolam comprises the following components in parts by weight: the weight volume percentage of the dispersion medium is 0.5-45.0%, preferably the pharmaceutically acceptable carrier is selected from any one of space protective agent and charge stabilizer or the combination thereof, and the dispersion medium is selected from any one of water, oil, polyethylene glycol and glycerol or the combination thereof.

In a preferred embodiment of the present invention, the oil is selected from any one of soybean oil, corn oil, tea oil and cottonseed oil, or a combination thereof.

In the preferable technical scheme of the invention, the particle size of the midazolam nanocrystal is less than or equal to 600nm, preferably less than or equal to 500nm, more preferably less than or equal to 400nm, still preferably 100nm-350nm, and still preferably 150-300 nm.

In a preferred technical scheme of the invention, the absolute value of the potential of the midazolam nanocrystal composition is more than 10mV, preferably more than 15 mV.

In a preferred technical scheme of the invention, the molar ratio of midazolam in the nanocrystal composition is as follows: the weight volume percentage of the dispersion medium is 1.0% to 40.0%, preferably 5.0% to 25.0%, more preferably 8 to 20%.

In a preferred technical scheme of the invention, the space protective agent in the nanocrystal composition is as follows: the weight volume percentage of the dispersion medium is 0.2% to 10.0%, preferably 1.0% to 5.0%, more preferably 1.5% to 3.5%, still more preferably 2.0% to 3.0%.

In a preferred embodiment of the present invention, the steric protector is selected from any one of a nonionic surfactant and a high molecular polymer, or a combination thereof.

In a preferred technical scheme of the invention, the nonionic surfactant is selected from any one of or a combination of polysorbate, glyceryl monostearate, poloxamer, span, maize and beneze.

In a preferred embodiment of the present invention, the high molecular polymer is selected from any one of hydroxypropyl methylcellulose (Hypromellose, HPMC), polyvinylpyrrolidone (PVP), Polyvinyl alcohol (PVA), tween, glycerol, decyl glucoside, hydroxypropyl cellulose, sodium carboxymethylcellulose, and sodium alginate, or a combination thereof.

In a preferred embodiment of the present invention, the steric protector is selected from any one of HPMC E5, HPMC E3, HPMC E6, HPMC E4M, HPMC K4M, poloxamer 188, poloxamer 407, PVP K12, PVP K17, PVP K30, PVA, tween 80, tween 20, sodium carboxymethylcellulose, glycerol, decyl glucoside, or a combination thereof.

In a preferred technical scheme of the invention, the charge stabilizer in the nanocrystal composition is as follows: the weight volume percentage of the dispersion medium is 0.1% to 3.0%, preferably 0.3% to 2.0%, more preferably 0.5% to 1.5%.

In a preferred embodiment of the present invention, the charge stabilizer is selected from any one of a zwitterionic surfactant and an anionic surfactant, or a combination thereof.

In a preferred embodiment of the present invention, the zwitterionic surfactant is selected from any one of lecithin and soybean lecithin, or a combination thereof.

In a preferred embodiment of the present invention, the anionic surfactant is selected from any one of Sodium Dodecyl Sulfate (SDS), docusate sodium (DOSS), or a combination thereof.

In a preferred embodiment of the present invention, the charge stabilizer is selected from any one of sodium dodecyl sulfate, docusate sodium, lecithin, and soybean lecithin, or a combination thereof.

In a preferred embodiment of the present invention, the HLB value (hydrophilic-lipophilic balance) of the charge stabilizer is not less than 10, preferably not less than 20, and more preferably not less than 30.

In a preferred technical scheme of the invention, the molar ratio of midazolam in the nanocrystal composition is as follows: the weight volume percentage of the dispersion medium is 0.5-45.0%, and the space protective agent: the weight volume percentage of the dispersion medium is 0.2-10.0%, and the charge stabilizer: the weight volume percentage of the dispersion medium is 0.1-3.0%.

In a preferred technical scheme of the invention, the nanocrystal composition comprises midazolam: the weight volume percentage of the dispersion medium is 1.0-40.0%, and the space protective agent: 1.0-5.0% by weight volume of dispersion medium, charge stabilizer: the weight volume percentage of the dispersion medium is 0.3-2.0%.

In a preferred technical scheme of the invention, the nanocrystal composition comprises midazolam: the weight volume percentage of the dispersion medium is 5-25%, and the space protective agent: the weight volume percentage of the dispersion medium is 2.0-3.0%, and the charge stabilizer: the weight volume percentage of the dispersion medium is 0.5-1.5%.

In a preferred technical scheme of the invention, the nanocrystal composition contains 5.0% of midazolam, 2.5% of HPMC and 1.0% of SDS in percentage by weight and volume.

In a preferred technical scheme of the invention, the nanocrystal composition contains 5.0 percent by weight of midazolam, 2.5 percent by weight of HPMC and 0.5 percent by weight of SDS.

In a preferred technical scheme of the invention, the nanocrystal composition contains 10.0% of midazolam, 2.5% of HPMC and 1.0% of SDS in percentage by weight and volume.

In a preferred technical scheme of the invention, the nanocrystal composition contains 10.0% of midazolam, 2.5% of HPMC and 0.5% of SDS in percentage by weight and volume.

In a preferred embodiment of the present invention, the pharmaceutically acceptable carrier further comprises any one or a combination of a pH adjuster, a preservative, a lyoprotectant, and a flavoring agent.

In a preferred embodiment of the present invention, the pH adjuster is selected from any one of hydrochloric acid, sulfuric acid, chloric acid, nitric acid, hydrobromic acid, hydrofluoric acid, phosphoric acid, sulfonic acid, malic acid, sorbic acid, fumaric acid, citric acid, carboxylic acid, hydroxy acid, keto acid, acetic acid, oxalic acid, citric acid, succinic acid, formic acid, acetic acid, propionic acid, butyric acid, malonic acid, succinic acid, adipic acid, pyruvic acid, glutamic acid, tartaric acid, lactic acid, itaconic acid, ascorbic acid, fumaric acid, alpha-ketoglutaric acid, tartaric acid, sodium hydroxide, potassium hydroxide, ammonium hydroxide, sodium carbonate, sodium bicarbonate, sodium phosphate, disodium hydrogen phosphate, sodium dihydrogen phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, diamine hydrogen phosphate, and ammonium dihydrogen phosphate, or a combination thereof.

In a preferred embodiment of the present invention, the pH adjuster is a buffer, preferably any one or a combination of citric acid, potassium citrate, sodium citrate, malic acid, sodium malate, potassium hydroxide, sodium bicarbonate, sodium hydroxide, potassium carbonate, sodium carbonate, phosphoric acid, disodium hydrogen phosphate, sodium dihydrogen phosphate, monoethanolamine, diethanolamine, triethanolamine, lactic acid, sodium lactate, potassium lactate, propionic acid, sodium propionate, potassium propionate, tartaric acid, sodium tartrate, sodium fumarate, potassium tartrate, potassium fumarate, and fumaric acid.

In a preferred embodiment of the present invention, the preservative is selected from any one of benzoic acid or a salt thereof, sorbic acid or a salt thereof, parabens, sodium metabisulfite, chlorhexidine, sodium citrate, Butylated Hydroxytoluene (BHT), Butylated Hydroxyanisole (BHA), tocopherol, ethylenediaminetetraacetic acid, propyl gallate, quaternary ammonium compounds, or a combination thereof.

In a preferred technical scheme of the invention, the flavoring agent is selected from any one of xylitol, stevioside, mogroside, glycyrrhizin, rubusoside, sucrose, saccharin sodium, glycerol, sorbitol, mannitol and maltose or a combination thereof.

In a preferred technical scheme of the invention, the freeze-drying protective agent is selected from any one of or the combination of sucrose, maltose, lactose, fructose, glucan, mannitol, trehalose, sorbitol, xylitol, maltitol, oligosaccharide alcohol, polyethylene glycol and glycerol.

In a preferred technical scheme of the invention, the preparation form of the nanocrystal composition is selected from any one of suspension, injection, freeze-dried powder injection, microemulsion, aerosol, cream, suppository, gel and foaming agent.

In the preferred technical scheme of the invention, the nanocrystal composition is frozen and dried to obtain the freeze-dried solid preparation.

In a preferred embodiment of the present invention, the administration form of the nanocrystal composition is selected from any one of injection administration, mucosal administration, and oral administration, or a combination thereof.

The invention aims to provide a preparation method of a midazolam nanocrystal composition, which contains midazolam, a dispersion medium and a pharmaceutically acceptable carrier, wherein the midazolam comprises the following components in percentage by weight: the weight volume percentage of the dispersion medium is 0.5% -45.0%, preferably the pharmaceutically acceptable carrier is selected from any one or the combination of a space protective agent and a charge stabilizing agent, and also preferably the dispersion medium is selected from any one or the combination of water, oil, polyethylene glycol and glycerol, and the preparation method comprises the following steps: weighing required amount of midazolam, dispersion medium and pharmaceutically acceptable carrier, uniformly mixing, and wet grinding or high-pressure homogenizing to obtain the midazolam-based dispersion.

In the preferred technical scheme of the invention, the required amount of the uniform mixture of midazolam, the space protective agent, the charge stabilizer, the dispersion medium and other optional pharmaceutically acceptable carriers is subjected to ultrasonic homogenization treatment and then wet grinding or high-pressure homogenization to obtain the midazolam-based dispersion.

In the preferred technical scheme of the invention, the rotation speed of the ultrasonic homogenization is 10000-28000rpm, preferably 13000-25000rpm, and more preferably 16000-22000 rpm.

In the preferred technical scheme of the invention, the initial grinding speed is 800-.

In the preferred technical solution of the invention, the increasing range of the polishing speed is 100-1000rpm/min, preferably 300-800rpm/min, and more preferably 500-700 rpm/min.

In the preferred technical scheme of the invention, the grinding speed is 1500-.

In the preferred technical scheme of the invention, the grinding time is 20min-72h, preferably 40min-180min, and more preferably 60min-150 min.

In a preferred technical scheme of the invention, the nanocrystal composition comprises midazolam: the weight volume percentage of the dispersion medium is 1.0% to 40.0%, preferably 5.0% to 25.0%, more preferably 8 to 20%.

In a preferred embodiment of the present invention, the steric protective agent is selected from any one of a nonionic surfactant and a high molecular polymer, or a combination thereof.

In a preferred embodiment of the present invention, the HLB value of the charge stabilizer is not less than 10, preferably not less than 20, and more preferably not less than 30.

In a preferred technical scheme of the invention, the nanocrystal composition comprises midazolam: the weight volume percentage of the dispersion medium is 0.5-45.0%, and the weight volume percentage of the space protective agent is as follows: the weight volume percentage of the dispersion medium is 0.2-10.0%, and the charge stabilizer: the weight volume percentage of the dispersion medium is 0.1-3.0%.

In a preferred technical scheme of the invention, the nanocrystal composition contains 5.0% of midazolam, 2.5% of HPMC and 1.0% of SDS in weight/volume percentage.

In a preferred technical scheme of the invention, the nanocrystal composition contains 5.0% of midazolam, 2.5% of HPMC and 0.5% of SDS in percentage by weight and volume.

In a preferred technical scheme of the invention, the nanocrystal composition contains 10.0 percent by weight of midazolam, 2.5 percent by weight of HPMC and 1.0 percent by weight of SDS.

In a preferable technical scheme of the invention, the pH regulator, the preservative, the freeze-drying protective agent and the flavoring agent are optionally added before or after grinding.

In a preferred embodiment of the present invention, the pH adjuster is a buffer, preferably any one or a combination of citric acid, potassium citrate, sodium citrate, malic acid, sodium malate, potassium hydroxide, sodium bicarbonate, sodium hydroxide, potassium carbonate, sodium carbonate, phosphoric acid, disodium hydrogen phosphate, sodium dihydrogen phosphate, monoethanolamine, diethanolamine, triethanolamine, lactic acid, sodium lactate, potassium lactate, propionic acid, potassium propionate, sodium propionate, tartaric acid, sodium tartrate, sodium fumarate, potassium tartrate, potassium fumarate, and fumaric acid.

In a preferable technical scheme of the invention, the preparation form of the nanocrystalline composition is selected from any one of suspension, injection, freeze-dried powder injection, microemulsion, aerosol, cream, suppository, gel and foaming agent.

In the preferred technical scheme of the invention, the nanocrystal composition is freeze-dried to obtain the freeze-dried solid preparation.

Another objective of the present invention is to provide a midazolam nanocrystal suspension, which contains midazolam, a dispersion medium and a pharmaceutically acceptable carrier, wherein the midazolam: the weight volume percentage of the dispersion medium is 0.5-45.0%, preferably the pharmaceutically acceptable carrier is selected from any one of space protective agent and charge stabilizer or the combination thereof, and the dispersion medium is selected from any one of water, oil, polyethylene glycol and glycerol or the combination thereof.

In the preferable technical scheme of the invention, the potential absolute value of the midazolam nanocrystal suspension is more than 10mV, preferably more than 15 mV.

In a preferred technical scheme of the invention, the molar ratio of midazolam in the nanocrystal suspension is as follows: the weight volume percentage of the dispersion medium is 1.0% to 40.0%, preferably 5.0% to 25.0%, more preferably 8 to 20%.

In the preferred technical scheme of the invention, the space protective agent in the nanocrystal suspension comprises: the weight volume percentage of the dispersion medium is 0.2% to 10.0%, preferably 1.0% to 5.0%, more preferably 1.5% to 3.5%, still more preferably 2.0% to 3.0%.

In a preferred technical scheme of the invention, the charge stabilizer in the nanocrystal suspension comprises: the weight volume percentage of the dispersion medium is 0.1% to 3.0%, preferably 0.3% to 2.0%, more preferably 0.5% to 1.5%.

In a preferred embodiment of the present invention, the HLB value (hydrophilic lipophilic balance) of the charge stabilizer is not less than 10, preferably not less than 20, and more preferably not less than 30.

In a preferred embodiment of the present invention, the pharmaceutically acceptable carrier further comprises any one or a combination of a pH adjusting agent, a preservative, and a lyoprotectant.

In a preferred technical scheme of the invention, the nanocrystal composition comprises midazolam: the weight volume percentage of the dispersion medium is 0.5-45.0%, and the space protective agent: the weight volume percentage of the dispersion medium is 0.2-10.0%, and the charge stabilizer: the weight volume percentage of the dispersion medium is 0.1-3.0%.

In the preferable technical scheme of the invention, the nano-crystalline suspension contains midazolam: the weight volume percentage of the dispersion medium is 1.0-40.0%, and the space protective agent: 1.0-5.0% by weight volume of dispersion medium, charge stabilizer: the weight volume percentage of the dispersion medium is 0.3-2.0%.

In a preferred technical scheme of the invention, the molar ratio of midazolam in the nanocrystal suspension is as follows: the weight volume percentage of the dispersion medium is 5% -25%, and the weight volume percentage of the space protective agent is as follows: the weight volume percentage of the dispersion medium is 2.0-3.0%, the charge stabilizer: the weight volume percentage of the dispersion medium is 0.5-1.5%.

In the preferred technical scheme of the invention, the nanocrystal suspension comprises 5.0 percent of midazolam, 2.5 percent of HPMC, 1.0 percent of SDS and the balance of water by weight volume percentage.

In the preferred technical scheme of the invention, the nanocrystal suspension comprises 5.0 percent of midazolam, 2.5 percent of HPMC, 0.5 percent of SDS and the balance of water by weight volume percentage.

In the preferred technical scheme of the invention, the nanocrystal suspension contains 10.0 percent by weight volume of midazolam, 2.5 percent by weight volume of HPMC, 1.0 percent by weight of SDS and the balance of water.

In the preferred technical scheme of the invention, the nanocrystal suspension comprises 10.0 percent of midazolam, 2.5 percent of HPMC, 0.5 percent of SDS and the balance of water by weight volume percentage.

In the preferred technical scheme of the invention, the nanocrystalline suspension is frozen and dried to obtain the freeze-dried solid preparation.

In a preferred technical scheme of the invention, the administration form of the nanocrystalline suspension is any one or combination of injection administration, mucosa administration and oral administration.

The invention aims to provide a preparation method of midazolam nanocrystal suspension, which contains midazolam, a dispersion medium and a pharmaceutically acceptable carrier, wherein the midazolam comprises the following components in percentage by weight: the weight volume percentage of the dispersion medium is 0.5% -45.0%, preferably the pharmaceutically acceptable carrier is selected from any one or the combination of a space protective agent and a charge stabilizing agent, and also preferably the dispersion medium is selected from any one or the combination of water, oil, polyethylene glycol and glycerol, and the preparation method comprises the following steps: weighing required amount of midazolam, dispersion medium and pharmaceutically acceptable carrier, uniformly mixing, and wet grinding or high-pressure homogenizing to obtain the midazolam-based dispersion.

In the preferred technical scheme of the invention, the required amount of the uniform mixture of midazolam, the space protective agent, the charge stabilizer, the dispersion medium and optionally other pharmaceutically acceptable carriers is subjected to ultrasonic homogenization treatment and wet grinding or high-pressure homogenization to obtain the midazolam-based dispersion.

In a preferred technical scheme of the invention, the rotation speed of the ultrasonic homogenization is 10000-28000rpm, preferably 13000-25000rpm, and more preferably 16000-22000 rpm.

In the preferred technical solution of the present invention, the increase of the polishing speed is 100-.

In the preferred technical scheme of the invention, the grinding time is 20min-72h, preferably 40min-180min, and more preferably 60-150 min.

In a preferable technical scheme of the invention, the particle size of the midazolam nanocrystal is less than or equal to 600nm, preferably less than or equal to 500nm, more preferably less than or equal to 400nm, still preferably 100nm-350nm, and still preferably 150-300 nm.

In the preferable technical scheme of the invention, the nano-crystalline suspension contains midazolam: the weight volume percentage of the dispersion medium is 1.0% to 40.0%, preferably 5.0% to 25.0%, more preferably 8 to 20%.

In a preferred embodiment of the present invention, the pH adjuster, the preservative, and the lyoprotectant are optionally added before or after grinding.

In the preferable technical scheme of the invention, the nano-crystalline suspension contains midazolam: the weight volume percentage of the dispersion medium is 5-25%, and the space protective agent: the weight volume percentage of the dispersion medium is 2.0-3.0%, and the charge stabilizer: the weight volume percentage of the dispersion medium is 0.5-1.5%.

In the preferred technical scheme of the invention, the nanocrystal suspension comprises 10.0% of midazolam, 2.5% of HPMC, 1.0% of SDS and the balance of water by weight volume percentage.

In the preferable technical scheme of the invention, the nanocrystal suspension contains 10.0 percent of midazolam, 2.5 percent of HPMC, 0.5 percent of SDS and the balance of water by weight volume percentage.

The other purpose of the invention is to provide the midazolam nanocrystal solid composition, which contains midazolam nanocrystal particles and a pharmaceutically acceptable carrier, wherein the particle size of the midazolam nanocrystal is less than or equal to 600nm, preferably less than or equal to 500nm, more preferably less than or equal to 400nm, further preferably 100-350 nm, and further preferably 150-300 nm.

In the preferable technical scheme of the invention, the midazolam nanocrystal particles are prepared by drying any one of the midazolam nanocrystal composition and the midazolam nanocrystal suspension.

In a preferred embodiment of the present invention, the drying is selected from any one or a combination of freeze drying, spray drying, vacuum drying and drying under reduced pressure.

In a preferred technical scheme of the invention, the midazolam nanocrystal particles contain 10% -95% of midazolam and 5% -90% of pharmaceutically acceptable carriers, and preferably the pharmaceutically acceptable carriers are selected from any one or combination of space protective agents, charge stabilizing agents and dispersion media.

In a preferred embodiment of the present invention, the pharmaceutically acceptable carrier is selected from any one of or a combination of a binder, a filler, a lubricant, a flavoring agent, a buffering agent, a wetting agent, a disintegrating agent, an effervescent agent, and other excipients.

In a preferred technical scheme of the invention, the nanocrystalline solid composition comprises the following components: 60-180 parts of midazolam nanocrystal particles, 100-200 parts of disintegrating agent, 35-70 parts of filler, 35-65 parts of adhesive and 2-15 parts of lubricant.

In a preferred technical scheme of the invention, the binder is selected from any one of or the combination of povidone, hydroxypropyl cellulose, methyl cellulose, hydroxypropyl methylcellulose, sodium carboxymethyl cellulose, polyvinyl alcohol, arabic gum and dextrin.

In a preferred embodiment of the present invention, the filler is selected from any one or a combination of lactose, powdered sugar, dextrin, starch or a derivative thereof, cellulose or a derivative thereof, an inorganic calcium salt, sorbitol, and glycine.

In a preferred technical scheme of the invention, the inorganic calcium salt is selected from any one of calcium chloride, calcium sulfate, calcium phosphate, calcium hydrophosphate and precipitated calcium carbonate or a combination thereof.

In a preferred technical scheme of the invention, the cellulose derivative is selected from any one or combination of microcrystalline cellulose, sodium carboxymethyl cellulose, ethyl cellulose and hydroxypropyl methyl cellulose, and preferably, the starch derivative is selected from any one or combination of sodium carboxymethyl starch, sodium starch glycolate, pregelatinized starch, modified starch, hydroxypropyl starch and corn starch.

In a preferred technical scheme of the invention, the lubricant is selected from any one of or a combination of sodium stearyl fumarate, stearic acid, magnesium stearate, calcium stearate, talcum powder and sucrose fatty acid ester.

In a preferred embodiment of the present invention, the wetting agent is selected from any one of sodium lauryl sulfate, water, ethanol, and isopropanol, or a combination thereof.

In a preferred technical scheme of the invention, the disintegrating agent is selected from any one of or the combination of carboxymethyl cellulose, carboxymethyl cellulose calcium, carboxymethyl starch sodium, cross-linked povidone and partially pregelatinized starch.

In a preferred technical scheme of the invention, the preparation form of the nanocrystalline solid composition is selected from any one of tablets, capsules, granules, powder and pills.

Another object of the present invention is to provide a method for preparing a midazolam nanocrystal solid composition, comprising the following steps: weighing required amount of midazolam nanocrystalline particles and a pharmaceutically acceptable carrier, and uniformly mixing to obtain the midazolam nanocrystalline particle.

In a preferred technical scheme of the invention, the preparation method of the tablet is selected from any one of dry granulation and powder direct compression.

In a preferred technical scheme of the invention, the dry granulation method comprises the following steps:

(a) material pretreatment: sieving the midazolam nanocrystal particles, the disintegrating agent, the filler, the lubricant and the adhesive for later use;

(b) mixing: weighing and uniformly mixing the midazolam nanocrystal particles, the disintegrating agent, the filler, the lubricant and the adhesive in the formula amount;

(c) putting the mixture in the step (b) into a dry granulating machine for granulation;

(d) and (3) total mixing: uniformly mixing the dry granules prepared in the step (c) with a disintegrating agent, a lubricating agent and an adhesive in a prescription amount;

(e) pressing plain tablets: and (d) pressing the granules prepared in the step (d) in a high-speed rotary tablet press to obtain the compound. In the preferable technical scheme of the invention, the coating is carried out after the plain tablets are pressed.

In a preferred technical scheme of the invention, the powder direct pressing method comprises the following steps:

(a) material pretreatment: sieving midazolam nanocrystal particles, a disintegrating agent, a filler, a lubricant and an adhesive for later use;

(b) mixing: weighing and uniformly mixing the midazolam nanocrystalline particles, the disintegrant, the filler, the lubricant and the adhesive in a prescription amount;

(c) pressing plain tablets: and (c) putting the uniformly mixed sample in the step (b) into a high-speed rotary tablet press for pressing to obtain the finished product.

In the preferable technical scheme of the invention, the coating is carried out after the plain tablets are pressed.

The invention also aims to provide the application of any one of the midazolam nanocrystal composition, the midazolam nanocrystal suspension and the midazolam nanocrystal solid composition or the combination thereof in preparing any one of anti-epilepsy and anti-convulsion medicines.

In a preferred embodiment of the present invention, the convulsion is selected from convulsion caused by any one or a combination of causes of fever, toxic substances, nerve damage, immature brain development, intracranial infection, electrolyte imbalance, metabolic disorder, environmental factor, genetic factor, and is preferably any one of neonatal convulsion, childhood convulsion, or a complication thereof.

In the preferred technical scheme of the invention, the fever can be exogenous intracranial fever.

In a preferred technical scheme of the invention, the cranial exogenous fever is fever caused by any one or a combination of reasons of varicella, influenza, gastroenteritis, otitis media, respiratory tract infection, tonsillitis and vaccination.

In a preferred embodiment of the present invention, the epilepsy is selected from any one of generalized seizures, partial/focal seizures, epileptic spasms, reflex seizures, or complications thereof.

In a preferred embodiment of the present invention, the generalized seizures are selected from any one of generalized tonic-clonic seizures, absence seizures, tonic seizures, clonic seizures, myoclonic seizures, dystonic seizures, or complications thereof.

In a preferred embodiment of the present invention, the partial epileptic seizure is selected from any one of simple partial seizures, complex partial seizures, secondary generalized seizures, or a complication thereof.

The invention also aims to provide a pharmaceutical composition containing the midazolam nanocrystal, which is prepared by combining any one of the midazolam nanocrystal composition, the midazolam nanocrystal solid composition and the midazolam nanocrystal suspension with any one of other antiepileptic drugs and other anticonvulsant drugs.

In a preferred embodiment of the present invention, the other antiepileptic drug is selected from any one of a sodium channel blocker, a drug for reducing neurotransmitter release, a drug for increasing gamma-aminobutyric acid (GABA) -mediated excitatory inhibition, an N-methyl-D-aspartate (NMDA) receptor blocker, an alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist, or a combination thereof.

In a preferred embodiment of the present invention, the sodium channel blocker is selected from any one of or a combination of phenytoin sodium, carbamazepine, oxcarbazepine, lamotrigine and lacosamide.

In a preferred embodiment of the present invention, the drug for reducing the release of the neurotransmitter is any one or a combination of ethosuximide, sodium valproate, lamotrigine, topiramate, levetiracetam, gabapentin and pregabalin.

In a preferred embodiment of the present invention, the drug for improving gaba-mediated excitability inhibition is any one or a combination of phenobarbital, phenytoin, benzodiazepines, felbamate, sodium valproate, topiramate, tiagabine and glycine.

In a preferable technical scheme of the invention, the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor antagonist drug is any one or combination of perampanel and zonisamide.

In a preferred embodiment of the present invention, the anticonvulsant is any one or a combination of phenobarbital sodium, diazepam, phenytoin sodium, lidocaine, pregabalin, fosphenytoin, levetiracetam, topiramate, lorazepam, sodium valproate, and vigabatrin.

The invention also aims to provide application of any one of the midazolam nanocrystal composition, the midazolam nanocrystal solid composition and the midazolam nanocrystal suspension or the combination thereof in preparation of drugs for anesthesia and sedation or any one of the midazolam nanocrystal suspensions and the combination thereof.

In a preferred technical scheme of the invention, the sedation is preoperative sedation.

In the preferable technical scheme of the invention, the sedation is conscious sedation before diagnosis or endoscopic operation.

In a preferred technical scheme of the invention, the sedation is intraoperative sedation of anesthesia induction, general anesthesia or epidural anesthesia.

The invention also aims to provide application of any one or combination of the midazolam nanocrystal composition, the midazolam nanocrystal suspension and the midazolam nanocrystal solid composition in preparation of a medicine for improving blood brain barrier permeability.

In a preferred technical scheme of the invention, the midazolam nanocrystal composition, the midazolam nanocrystal solid composition and the midazolam nanocrystal suspension contain midazolam and a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier is preferably selected from any one or a combination of a space protective agent, a charge stabilizer and a dispersion medium.

In a preferred technical scheme of the invention, the nanocrystal composition contains midazolam, a dispersion medium and a pharmaceutically acceptable carrier, wherein the weight ratio of midazolam: the weight volume percentage of the dispersion medium is 0.5-45.0%, preferably the pharmaceutically acceptable carrier is selected from any one of space protective agent and charge stabilizer or the combination thereof, and the dispersion medium is selected from any one of water, oil, polyethylene glycol and glycerol or the combination thereof.

In a preferred technical scheme of the invention, the nanocrystal suspension contains midazolam, a dispersion medium and a pharmaceutically acceptable carrier, wherein the weight ratio of midazolam: the weight volume percentage of the dispersion medium is 0.5-45.0%, preferably the pharmaceutically acceptable carrier is selected from any one of space protective agent and charge stabilizer or the combination thereof, and the dispersion medium is selected from any one of water, oil, polyethylene glycol and glycerol or the combination thereof.

In a preferred technical scheme of the invention, the nanocrystalline solid composition contains midazolam nanocrystalline particles and a pharmaceutically acceptable carrier, wherein the midazolam nanocrystalline particles are prepared by any one of drying the midazolam nanocrystalline composition and the midazolam nanocrystalline suspension, preferably drying any one or combination of freeze drying, spray drying, vacuum drying and decompression drying.

In the preferable technical scheme of the invention, the midazolam nanocrystal particles contain 10-95% of midazolam and 5-90% of pharmaceutically acceptable carriers.

In a preferred technical scheme of the invention, the medicine for improving blood brain barrier permeability is selected from brain medicines.

In a preferred technical scheme of the invention, the brain drug is any one or combination of drugs for treating brain tumor, brain nervous system lesion, Parkinson's disease and cerebrovascular disease.

In a preferred technical scheme of the invention, the brain drug is selected from any one of temozolomide, 6-benzyl guanine, adriamycin, lexiscan, methotrexate, bevacizumab, rituximab, gemcitabine, manidipine, regorafenib, artemisinin, paclitaxel anticancer drugs, temozolomide, vinca alkaloids, 5-fluorouracil, anthracyclines anticancer drugs, pemetrexed, platinum anticancer drugs, camptothecin and derivatives thereof, cyclophosphamide, topotecan, lomustine and procarbazine or a combination thereof.

The dispersion medium of the present invention is a mixture of microparticles (molecules, ions, or molecular aggregates, etc.) of one (or more) substances distributed in another substance. Such as solutions, colloids, suspensions, emulsions, and the like. In a dispersion, a substance dispersed as fine particles is referred to as "dispersoid", also referred to as "dispersed phase"; the substance in which the fine particles can be dispersed is called a "dispersant", also called a "dispersion medium".

The dissolution instrument of the invention adopts a dissolution instrument of model ZRS-8G of Tiandatianfa science and technology limited company.

The nanometer particle size measurement, the potential measurement and the polydispersion coefficient measurement of the invention adopt a nanometer particle size measuring instrument with the model number of Nano-ES90 of Malvern company in UK.

The emulsifying homogenizer of the invention adopts an ultrasonic emulsifying homogenizer with the model C25 of Shanghai Hengchuan machinery Co.

The wet mill of the invention is a model research lab wet mill from walbao, switzerland.

Unless otherwise indicated, when the present invention relates to percentages between liquids, said percentages are volume/volume percentages; when the invention relates to percentages between liquid and solid, said percentages are volume/weight percentages; the invention relates to the percentages between solid and liquid, said percentages being weight/volume percentages; the balance being weight/weight percent.

Compared with the prior art, the invention has the following beneficial technical effects:

1. the midazolam nanocrystal composition has better quality and curative effect, and the nanocrystals have the advantages of uniform particle size distribution, no fine particle aggregation and stratification phenomenon, good stability, contribution to improving the solubility of midazolam, reduction of toxic and side effects of medicines, low irritation, improvement of bioavailability and the like.

2. The preparation method is simple and convenient to operate, remarkably shortens the production period, further remarkably reduces the production cost, and is suitable for large-scale industrial production.

Drawings

Figure 1 release curves for midazolam nanocrystal suspensions, midazolam drug substances, midazolam and adjuvant physical mixtures;

FIG. 2 comparison of muscle stimulation for different prescriptions;

figure 3 comparison of anticonvulsant and antiepileptic effects of midazolam nanocrystal suspensions, midazolam solutions, diazepam solutions;

figure 4 comparison of neurotoxicity of intramuscular administration of different drugs.

Detailed Description

The following description of the embodiments of the present invention is not intended to limit the invention, and those skilled in the art may make various changes and modifications without departing from the spirit of the invention, which is defined in the appended claims.

Example 1Mida (Chinese character of 'Mida')Preparation of zolam nanocrystalline suspension

Adding 2.5g HPMC E5 into 100ml water, heating to 50 ℃, stirring to completely dissolve, adding 1g SDS and 5g midazolam, and ultrasonically homogenizing for 5min to fully wet and uniformly suspend midazolam. Pouring the primary mixture into a wet grinder, wherein the initial rotating speed is 1500rpm, increasing the rotating speed by 500rpm every 5min until the rotating speed reaches 3000rpm, and continuously grinding for 1 h. And (3) diluting the ground nano suspension by 200 times, and detecting that the particle size of the prepared nano crystal is 286.6nm, the polydispersity index (PDI) is 0.124 and the Zeta potential is-23.4 mV.

Test example 1In vitro release experiments of midazolam nanocrystal suspensions

1. Solution preparation

(1) Mobile phase: phosphate buffer (pH3.5, 0.1 mol/L): the methanol content was 35:65 (V/V). The phosphate buffer solution was prepared by adding 9.8g of phosphoric acid and 4.2mL of triethylamine to 1L of water and adjusting the pH to 3.5 with 1mol/L of sodium hydroxide solution.

(2) Dissolution medium: 40.8g of potassium dihydrogen phosphate was weighed, placed in 6000mL of water, and after dissolution by stirring, the pH was adjusted to 7.4 with 2.0mol/L sodium hydroxide solution.

2. Conditions of the experiment

(1) Midazolam nanocrystal suspension: taking 50mg/mL nanocrystal suspension prepared in example 1, adding water, diluting to 10mg/mL, taking 1mL, and adding into a dissolution instrument;

(2) midazolam raw material medicine: putting 10mg of midazolam raw material medicine into 1mL of water, and adding the midazolam raw material medicine into a dissolution instrument;

(3) physical mixture: 10mg of midazolam +5mg of HPMC E5+2mg of SDS were taken and placed in 1ml of water and added to a dissolution apparatus.

(4) The three groups are dissolved out by adopting a paddle method at the rotating speed of 75rpm at 37 ℃, the dissolution medium is a potassium dihydrogen phosphate solution with the pH value of 7.4, 2mL samples are respectively taken at 5min, 10min, 20min, 30min, 45min, 60min, 2h, 4h and 8h, the samples are filtered, the sample injection detection is carried out, the cumulative release degree is calculated, and the release curve is drawn.

HPLC chromatographic conditions: octadecylsilane chemically bonded silica is used as a filler, and a mobile phase is phosphate buffer (0.1mol/L, pH3.5) -methanol (35:65, v/v); detection wavelength: 220 nm; the column temperature is 40 ℃; the flow rate is 1.0 ml/min; sample introduction amount: 10 μ L. The results are shown in Table 1 and FIG. 1.

TABLE 1

Test example 2Irritation test of midazolam nanocrystal suspension

1. Laboratory animal

Male new zealand big ear white rabbits 4, weighing about 2.5-3.5kg, about 3-4 months old, normal grade.

2. Experimental methods

(1) Administration of test and control

The administration route is as follows: intramuscular injection administration;

administration volume: 5mg/3kg (3 kg body weight per rabbit, 0.5ml per administration);

frequency and time of administration: dosing was 1 time per day, 8 am: 30-11: 30, of a nitrogen-containing gas; the administration is continued for 5 days, and the administration volume is adjusted according to the body weight change.

(2) Solution preparation

Blank control (negative control): 0.9% sterilized normal saline is prepared.

1.7% acetic acid positive control: 1.7g of acetic acid solution was added to 98.3g of water and mixed well.

SDS + HPMC E5 solution: 1g SDS and 2.5g HPMC E5 were added to 100ml water and mixed well.

Midazolam nanocrystal suspensions (MDZ/ncs (sds)): 5% midazolam, 1% SDS, 2.5% HPMC E5, prepared as in example 1.

TABLE 2

At 24 hours after administration, each group of animals was sacrificed by anesthesia and exsanguination, and the presence or absence of redness, congestion, exudation, denaturation or necrosis at the injection site was visually observed and recorded. Further histopathological examination of the injection site was performed simultaneously. Histopathological sections were scanned after HE staining.

The muscle stimulation response grading criteria are shown in table 3.

TABLE 3

The results show that the blank control: no obvious abnormality and inflammation are seen, and the reaction grade is 0. 1.7% acetic acid positive control: a large amount of myofibers are necrosed and dissolved in tissues, inflammatory cells are more, muscle cell gaps are filled, muscle bundle interstitium generates edema, myofibers generate the conditions of degeneration, atrophy, necrosis and the like, and the reaction level is 5. SDS + HPMC E5 solution: the tissue staining is uniform, the stroma has no obvious abnormality and inflammation, and the reaction grade is 0. Midazolam nanocrystal suspension (MDZ/ncs (sds)): the morphological structure of the muscle fiber is not abnormal, the arrangement is neat, the boundary is obvious, the obvious abnormality and inflammation are not seen in the interstitium, and the reaction level is 0. The results are shown in Table 4 and FIG. 2.

TABLE 4 histomorphological examination

Example 2Preparation of midazolam nanocrystal suspension

Adding 2.5g HPMC E5 into 100ml water, heating to 55 deg.C, stirring to dissolve completely, adding 0.5g SDS and 5g midazolam, and ultrasonic homogenizing for 5min to wet midazolam thoroughly and suspend uniformly. And pouring the primary mixture into a wet grinder, wherein the initial rotating speed is 1500rpm, increasing the rotating speed by 500rpm every 5min until the rotating speed reaches 3000rpm, and continuously grinding for 1h to obtain the product. And (3) diluting the ground nano suspension by 200 times, and detecting that the particle size of the prepared midazolam nano crystal is 267.3nm, the polydispersity index (PDI) is 0.132 and the Zeta potential is-28.1 mV.

Test example 3Midazolam nanocrystal suspension stability study

The nanocrystal suspension prepared in example 2 was placed at 4 ℃, 25 ℃ (room temperature) and 40 ℃ (accelerated) respectively, sampled at 0, 1, 2, 3, 4, 5, 6, 7 and 8 months, diluted 200 times, and the particle size was measured. The results are shown in Table 5.

TABLE 5

Test example 4Anticonvulsant and antiepileptic effect research of midazolam nanocrystal suspension

1. Anticonvulsant and antiepileptic effects

1) Preparation of a pentaerythrityl solution of 6.9 mg/mL: 276.00mg of pentaerythrityl nitrogen were weighed, placed in a 50mL centrifuge tube, diluted to 40mL with physiological saline solution, and mixed well for use.

2) Preparing 1mg/mL midazolam nanocrystal suspension: precisely transferring 1mL of midazolam nanocrystal (50 mg/mL) suspension, placing the midazolam nanocrystal suspension into a 50mL volumetric flask, adding a physiological saline solution, diluting the midazolam nanocrystal suspension to a constant volume to a scale, and fully and uniformly mixing the midazolam nanocrystal suspension for later use.

Diluting 1mg/mL midazolam nanocrystal suspension to 0.5mg/mL, 0.25mg/mL, 0.125mg/mL and 0.0625mg/mL respectively by using a physiological saline solution for later use.

3) Preparing 1mg/mL midazolam solution: 50.00mg of midazolam is precisely weighed and placed in a 50mL volumetric flask, diluted with a physiological saline solution with the pH value of 3.5 to a constant volume to be scaled, and fully and uniformly mixed for later use.

1mg/mL midazolam solution was diluted to 0.5mg/mL, 0.25mg/mL, 0.125mg/mL, and 0.0625mg/mL, respectively, with a physiological saline solution having a pH of 3.5.

4) Preparation of diazepam solution 1 mg/mL: accurately weighing 50.00mg diazepam, placing the diazepam in a 50mL volumetric flask, dissolving the diazepam in ethanol, diluting to 50mL, and fully mixing for later use.

The 1mg/mL diazepam solution was diluted with ethanol to 0.5mg/mL, 0.25mg/mL, 0.125mg/mL, 0.0625mg/mL, respectively, for use.

Model group: SD rats 10, each weighing 250 + -20 g, male and female halves, were given a 6.9mg/mL solution of pentaerythrine and the number of convulsions recorded in the rats.

Midazolam nanocrystal suspension test groups: SD rats 60 were divided into 6 groups of 10 rats each weighing 250 + -20 g, each male and female half, administered intramuscularly with midazolam nanocrystalline suspensions at a dose of 0.0625mg/kg, 0.125mg/kg, 0.25mg/kg, 0.5mg/kg, 1.0mg/kg, 2.0mg/kg, respectively, and after 2min, 6.9mg/mL of a solution of pentylenetetrazol, and the number of convulsions in the rats was recorded.

Anticonvulsant and antiepileptic effects of midazolam and diazepam solutions: the experimental methods such as animal grouping, administration modes and the like are the same as the midazolam nanocrystal suspension.

Neurotoxicity experiments of midazolam nanocrystal suspensions: SD rats 50 were divided into 5 groups of 10 rats each, each half of the males and females, and given intramuscular injections of midazolam nanocrystal suspensions at doses of 0.625mg/kg, 1.25mg/kg, 2.5mg/kg, 3.0mg/kg, 4.0mg/kg, respectively, the rats were placed on horizontal bars and the number of drops per group was recorded.

Neurotoxicity experiments of midazolam and diazepam solutions: the experimental methods such as animal grouping, administration modes and the like are the same as the midazolam nanocrystal suspension.

Results were statistically analyzed using SPSS 19.0 software using one-way analysis of variance, p<Statistical differences at 0.05 and calculation of ED using Probit analysis₅₀。

The result shows that when the dosage of the midazolam nanocrystal suspension is 0.25mg/kg, the convulsion control rate can reach 100%. Under the same dosage, the convulsion control rate of the diazepam solution is 50%, and the convulsion control rate of the midazolam solution is 90%. ED calculated by Probit₅₀. The results are shown in Table 6, Table 7 and FIG. 3.

TABLE 6 anticonvulsant and antiepileptic effects by intramuscular injection

Note: with 0.0625mg/kg^-1Midazolam nanocrystal group comparison of p<0.05; and 0.125mg kg^-1Comparison of midazolam nanocrystals<0.01; compared with the midazolam nanocrystal, the diazepam solution,^△p<0.05.

TABLE 7

2. Midazolam nanocrystal suspension neurotoxicity study

Recording the drop number of each group of mice, and carrying out Probit analysis and calculation according to the unbalance rate to obtain TD₅₀. The results are shown in Table 8, Table 9 and FIG. 4.

TABLE 8 comparison of neurotoxicity of intramuscular administration

TABLE 9 median poisoned dose comparison

The results show that the effect of the midazolam nanocrystal suspension on controlling epileptic seizure is superior to that of a midazolam solution and a diazepam solution. And the therapeutic index (TI,112.58) of the midazolam nanocrystal suspension is far greater than that of diazepam (13.09) and a midazolam solution (35.33), so that the midazolam nanocrystal suspension has higher therapeutic safety.

TABLE 10 therapeutic index comparison

Test example 5Blood brain barrier permeability research of midazolam nanocrystal

1. Laboratory animal

10 male SD rats, 2 weeks old, available from Beijing Wittingle laboratory animal technology Co., Ltd, certificate number SCXK (Jing) 2016-.

2. Experimental method

The solution and mode adopted for preparing the midazolam nanocrystal suspension and the midazolam solution are referred to the experimental example 4.

(1) Isolation and primary culture of brain microvascular endothelial cells

SD rats of 2 weeks were taken, cultured for 1 day and then sacrificed, the head was clipped off with hemostatic forceps, soaked in 75% alcohol for 2min, and placed in D-Hank's solution for soaking, and brain tissue was taken out and placed in a culture dish in ice to obtain the cerebral cortex.

1) D-Hank's brain cortex was washed 3 times, transferred to serum free DMEM and minced, 4mL of 0.1% collagenase type II (containing 120. mu. DNasel) was added, digested in a water bath at 37 ℃ for 1.5h, and the tubes were shaken every 20 min.

2) Centrifuging at 1000rpm for 8min, discarding supernatant, adding 20% BSA for resuspension, centrifuging at 14000rpm at 4 deg.C for 20min, removing middle layer tissue and large blood vessel, and collecting bottom precipitate.

3) 2mL of 0.1% collagenase/dispase and 40. mu.DNase I were added, digested for 1h, centrifuged at 1000rpm for 8min, and the supernatant was discarded.

4) 3mL of rat endothelial cell isolate 1 was added to the centrifuge tube, 1mL of isolate 2 was slowly added to the upper layer, and finally 2mL of DMEM-resuspended cell sap without FBS was added to the uppermost layer to ensure that the liquid levels in each layer were well-separated, at 4 ℃ 1000 × g, and centrifuged for 20 min.

5) The liquid level of the centrifugal tube after centrifugation is divided into three layers, middle layer endothelial cells are absorbed and put in DMEM without FBS, centrifugation is carried out for 5min at 1000rpm, washing is carried out for 2 times, and the supernatant is discarded.

6) ECM was added and resuspended in culture flasks for 30min before transferring to FN-coated culture flasks and puromycin (4. mu.g/mL) was added.

7) At 37 ℃ 5% CO₂Culturing in a cell culture box until the culture bottle is full.

(2) Passage of BMECs

1) The culture medium was aspirated and washed 3 times with PBS.

2) Adding 0.25% trypsin for digestion for 1.5min, observing the digestion under microscope, and adding culture medium to stop digestion when the degree is appropriate.

3) Transferring the digested cells to a centrifuge tube, centrifuging at 1000rpm for 5min, and discarding the supernatant after centrifugation.

4) Resuspending with ECM specific culture medium, adjusting cell density to 1 × 105/mL, and inoculating to FN-coated 25cm²A culture flask.

5) At 37 ℃ 5% CO₂After the culture in the cell culture box is completed to 70 percent of growth, the liquid is changed every day.

6) And (5) carrying out passage again after the cells grow into a full bottle.

(3) Blood brain barrier model established by brain microvascular endothelial cells

1) The medium was aspirated off and washed 3 times with PBS.

2) Adding 0.25% trypsin for digestion for 1-2min, observing digestion under microscope, and adding culture medium to stop digestion when appropriate degree is reached.

3) The cells were placed in a 15mL centrifuge tube, centrifuged at 1000rpm for 5min, and the supernatant was discarded. The ECM culture was resuspended and plated at a density of 1 × 106/well in the upper chamber of a Transwell 24-well plate (FN incubation).

4) At 37 deg.C, 5% CO₂After culturing in a cell incubator and observing cell fusion after about 2 to 3 days, TEER experiments were performed.

(4) TEER experiment

Rat brain microvascular endothelial cells were seeded into Transwell cells on 24-well plates at a density of 1 × 106/well, the resistance value was measured from the first day of seeding, and the culture and monitoring were continued for 7 days with TEER values exceeding 200 Ω · cm²And the in vitro Blood Brain Barrier (BBB) model is successfully constructed.

(5) Method for detecting cytotoxicity of midazolam nanocrystal suspension by CCK-8 method

Cells were seeded in 96-well plates at a density of 5 × 104 cells per well, and 100 μ L of ECM was added per well for culture. After the cells are attached to the wall, the midazolam nanocrystal suspension and the midazolam solution with the concentrations of 10 mug/mL, 20 mug/mL, 40 mug/mL, 60 mug/mL and 80 mug/mL are respectively added, and sampling detection is respectively carried out for 24h, 48h and 72 h. And measuring the absorbance value of the supernatant at the wavelength of 450nm to evaluate the influence of the midazolam nanocrystal suspension and the midazolam solution on the activity of the cells. The cell viability was calculated as: cell survival (%) ═ a experimental group/a control group × 100%. For each concentration, 6 replicate wells were set and their mean was counted.

The results show that the cytotoxicity of the midazolam nanocrystal suspension and the midazolam solution is increased and the survival rate is gradually reduced with the increase of the administration concentration. The cytotoxicity of the midazolam solution after 24h, 48h and 72h of cell culture is higher than that of the midazolam nanocrystal suspension, and the midazolam nanocrystal suspension has better biological safety. The results are shown in Table 11.

TABLE 11 cytotoxicity of midazolam nanocrystal suspensions in BMECs

(6) Blood brain barrier permeability study of midazolam nanocrystal suspension

1) Taking a culture flask full of brain microvascular epithelial cells, washing with PBS for 3 times, digesting with 0.25% trypsin for 2min, and immediately adding 10% FBS-containing ECM culture medium to terminate digestion after observing cell suspension under a microscope.

2) Taking 24-well plate, adding 1mL culture medium into lower layer, placing upper layer into transwell chamber, adding 200 μ L culture medium containing 1 × 106/mL, adding 200 μ L ECM culture medium into lower chamber, keeping liquid level flat, measuring resistance value after 2-4 hr, and calculating resistance value (Ω · cm)²) (R cell-R blank) x S membrane (cm)²)。

3) Sucking the ECM culture medium of the upper chamber and the lower chamber, adding 1200 mu L of D-Hanks into the lower chamber, adding 200 mu L of drug-containing culture medium into the upper chamber, and adding 10 mu g/mL of midazolam nanocrystal suspension and midazolam solution into the upper chamber respectively. Taking 200 μ L of the medicine from lower chamber at 5min, 10min, 20min, 30min, 45min, and 60min respectively, adding into sample vial, taking out, and replenishing solution.

4) The concentrations of midazolam that passed through the BBB were measured by HPLC. HPLC method is phosphate bufferedLiquid (0.1mol/L, formulation: 9.81g phosphoric acid in 1000mL water, 4mL triethylamine, pH adjusted to 3.5 with 2mol/L NaOH): methanol 35: 65; column temperature: 30 ℃; lambda [ alpha ]_max220 nm; flow rate: 1.0 mL/min; sample introduction amount: 10 μ L.

The result shows that the blood brain barrier cumulative transmittance of the midazolam nanocrystal is (18.59 +/-0.86)% which is 2.5 times of that of the midazolam solution (7.44 +/-0.14)%. The results are shown in Table 12.

Table 12 permeation rate of midazolam nanocrystal suspension in blood brain barrier model

Example 3Preparation of midazolam nanocrystal suspension

Adding 2.5g HPMC E5 into 100ml water, heating to 60 ℃, stirring for dissolving, then adding 0.5g SDS and 10g midazolam, and carrying out ultrasonic homogenization for 5min to fully wet the midazolam and uniformly suspend. And pouring the primary mixture into a wet grinder, taking 1500rpm as an initial rotating speed, increasing 500rpm every 5min until 3500rpm, and continuously grinding for 2h to obtain the final product. And diluting the ground nano suspension by 200 times, wherein the detected particle size is 223.6nm, the polydispersity index (PDI) is 0.124, and the Zeta potential is-25.8 mV.

Example 4Preparation of midazolam nanocrystal suspension

Adding 8g of HPMC E5 into 100ml of water, heating to 55 ℃, stirring until the HPMC E5 is completely dissolved, adding 2g of SDS and 45g of midazolam, and ultrasonically homogenizing for 20min to fully wet the midazolam and uniformly suspend the midazolam. And pouring the primary mixture into a wet grinder, wherein the initial rotating speed is 1500rpm, increasing the rotating speed by 500rpm every 10min until 3000rpm is reached, and continuously grinding for 4h to obtain the product. And (3) diluting the ground nanosuspension by 200 times, and detecting that the particle size of the prepared midazolam nanocrystal is 245.6nm, the polydispersity index (PDI) is 0.231, and the Zeta potential is-30.3 mV.

Example 5Preparation of midazolam nanocrystal suspension

Adding 0.25g of HPMC K4M into 100ml of water, heating to 55 ℃, stirring until the mixture is completely dissolved, adding 0.05g of SDS and 0.5g of midazolam, and ultrasonically homogenizing for 5min to fully wet the midazolam and uniformly suspend the midazolam. And pouring the primary mixture into a wet grinder, wherein the initial rotating speed is 1500rpm, increasing the rotating speed by 500rpm every 2min until 3000rpm is reached, and continuously grinding for 2h to obtain the final product. And (3) diluting the ground nano suspension by 200 times, and detecting that the particle size of the prepared midazolam nano crystal is 342.3nm, the polydispersity index (PDI) is 0.215, and the Zeta potential is-18.3 mV.

Example 6Preparation of midazolam nanocrystal suspension

Adding 2.5g HPMC E5 into 100ml water, heating to 50 ℃, stirring to completely dissolve, adding 0.5g DOSS and 5g midazolam, and ultrasonically homogenizing for 5min to fully wet and uniformly suspend midazolam. Pouring the primary mixture into a wet grinder, wherein the initial rotating speed is 1500rpm, increasing the rotating speed by 500rpm every 5min until the rotating speed reaches 3000rpm, and continuously grinding for 1 h. And diluting the ground nano suspension by 200 times, and detecting that the particle size of the prepared nano crystal is 293.4nm, the polydispersity index (PDI) is 0.154 and the Zeta potential is-25.8 mV.

Example 7Preparation of midazolam nanocrystal suspension

Adding 2.5g of CMC Na into 100ml of water, heating to 50 ℃, stirring until the CMC Na is completely dissolved, then adding 0.5g P188 and 5g of midazolam, and carrying out ultrasonic homogenization for 5min to fully wet and uniformly suspend the midazolam. Pouring the primary mixture into a wet grinder, wherein the initial rotating speed is 1500rpm, increasing the rotating speed by 500rpm every 5min until the rotating speed reaches 3000rpm, and continuously grinding for 1 h. And diluting the ground nano suspension by 200 times, and detecting that the particle size of the prepared nano crystal is 328.4nm, the polydispersity index (PDI) is 0.203 and the Zeta potential is-35.2 mV.

Example 8Midazolam nanocrystalline tablet and preparation method thereof

The midazolam nanocrystalline tablet comprises the following components in percentage by weight:

the preparation method of the midazolam nanocrystalline tablet comprises the following steps:

1. freeze-drying the midazolam nanocrystal suspension of example 3 to obtain freeze-dried particles;

2. sieving microcrystalline cellulose, crospovidone, hypromellose, and pulvis Talci;

3. weighing required amount of midazolam nanocrystal suspension freeze-dried particles, microcrystalline cellulose, crospovidone, hydroxypropyl methylcellulose and talcum powder, stirring, uniformly mixing, and placing the prepared uniform mixture into a high-speed rotary tablet press for tabletting to obtain the midazolam nanocrystal suspension.

The above description of the specific embodiments of the present invention is not intended to limit the present invention, and those skilled in the art may make various changes and modifications according to the present invention without departing from the spirit of the present invention, which is defined in the appended claims.

Claims

1. The preparation method of the midazolam nanocrystal suspension comprises the steps of adding 2.5g of HPMC E5 into 100ml of water, heating to 55 ℃, stirring until the HPMC E5 is completely dissolved, adding 0.5g of SDS and 5g of midazolam, carrying out ultrasonic homogenization for 5min to fully wet and uniformly suspend the midazolam, pouring a primary mixture into a wet grinding machine, increasing the initial rotation speed to 1500rpm every 5min until the rotation speed reaches 3000rpm, continuously grinding for 1h to obtain the midazolam nanocrystal suspension, diluting the ground nanocrystal suspension by 200 times, and detecting that the particle size of the prepared midazolam nanocrystal is 267.3nm, the polydispersity number ZePDI is 0.132, the potential is-28.1 mV, and the cumulative blood brain barrier transmittance of 60 minutes of the midazolam nanocrystal suspension is (18.59 +/-0.86)%; the medicine for improving the permeability of the blood brain barrier is selected from brain medicines, and the brain medicines are selected from any one or combination of medicines for treating brain tumor, brain nervous system lesion, Parkinson disease and cerebrovascular disease.