CN116898824B

CN116898824B - Histamine H1 receptor antagonist slow-release nanoparticle, preparation method thereof, injection and application

Info

Publication number: CN116898824B
Application number: CN202311181984.3A
Authority: CN
Inventors: 白晓春; 周东方; 赖锐军
Original assignee: Third Affiliated Hospital Of Southern Medical University (academy Of Orthopaedics Guangdong Province)
Current assignee: Third Affiliated Hospital Of Southern Medical University (academy Of Orthopaedics Guangdong Province)
Priority date: 2023-09-14
Filing date: 2023-09-14
Publication date: 2024-01-23
Anticipated expiration: 2043-09-14
Also published as: CN116898824A

Abstract

The application relates to histamine H1 receptor antagonist slow release nanoparticles which are prepared from materials comprising a pharmaceutical active ingredient, an emulsifying agent and a slow release material, wherein the pharmaceutical active ingredient is histamine H1 receptor antagonist, and the slow release material is a high molecular polymer with molecular weight selected from 10000 Da-100000 Da. The application also relates to a preparation method of the histamine H1 receptor antagonist slow-release nanoparticle, which comprises the steps of dissolving a medicine active ingredient and a slow-release material in a first organic solvent to obtain a first organic phase, dissolving an emulsifying agent in a second organic solvent to obtain a second organic phase, mixing the first organic phase and the second organic phase, carrying out ultrasonic treatment, removing the organic solvent, and filtering. The application also relates to an injection, which comprises the histamine H1 receptor antagonist slow release nanoparticle. The application also relates to application of the histamine H1 receptor antagonist slow-release nano-particles in preparing a medicament for treating bone joint diseases.

Description

Histamine H1 receptor antagonist slow-release nanoparticle, preparation method thereof, injection and application

Technical Field

The application relates to the technical field of drug sustained release preparations, in particular to histamine H1 receptor antagonist sustained release nanoparticles, a preparation method, an injection and application thereof.

Background

Histamine is an active substance that is widely present in human tissues and is mainly contained in mast cells and basophils. Histamine is released when tissue is damaged or an inflammatory or allergic reaction occurs, and binding of histamine to specific receptors (histamine receptors) on target cells can produce adverse biological effects. Thus, histamine receptor blockers are of great clinical value.

Histamine receptors include three types of H1, H2 and H3, wherein histamine H1 receptors are distributed in bronchi and gastrointestinal smooth muscle and other wide tissues and organs, and upon binding to histamine, produce adverse physiological effects including smooth muscle contractile cramps, telangiectasias, etc., which can severely cause blood pressure drop and even shock. Histamine H1 receptor antagonists compete with histamine for the histamine H1 receptor and bind to the H1 receptor and are commonly used clinically as antiallergic agents.

The development of histamine H1 receptor antagonists has been to the third generation, however, only the first generation histamine H1 receptor antagonist drugs have injection formulations, but the sedative side effects of the first generation histamine H1 receptor antagonists are significant, greatly limiting further applications. Furthermore, there has been no report on sustained release dosage forms of histamine H1 receptor antagonists.

Disclosure of Invention

In view of the above, the technical staff of the application develop a histamine H1 receptor antagonist slow release nanoparticle through a large number of searching and creative experiments, which has the advantages of small particle size, high drug loading and long in-vivo and in-vitro sustained release time, can be used for preparing intramuscular injection, and has small injection stimulation and good patient compliance. The histamine H1 receptor antagonist slow release nanoparticle is prepared from a material comprising a medicinal active ingredient, an emulsifying agent and a slow release material, wherein the medicinal active ingredient is a histamine H1 receptor antagonist; the emulsifier comprises one or more of polyoxyethylene polyoxypropylene ether block copolymer, polyvinyl alcohol, tween 80, PEG-100 stearate, glyceryl stearate, arachidyl alcohol, fatty alcohol polyoxyethylene ether, glyceryl behenate, cetostearyl glucoside and combinations thereof; the slow release material is a high molecular polymer with molecular weight of 10000 Da-100000 Da;

the high molecular polymer comprises one or more of sodium alginate, chitosan, polylactic acid-glycolic acid copolymer, polycaprolactone, polylactic acid, polytrimethylene carbonate, polyglycolic acid, polyhydroxybutyrate-hydroxyvalerate copolymer, polyorthoester, polyanhydride and combinations thereof;

the medicine active ingredient accounts for 5% -10% of the total mass of the histamine H1 receptor antagonist slow release nanoparticle;

the particle size of the histamine H1 receptor antagonist slow release nanoparticle is 50 nm-200 nm.

In some embodiments, the histamine H1 receptor antagonist is selected from one or more of the following compounds: astemizole, azatadine, azelastine, brompheniramine, chlorocyclizine, chlorpheniramine, chloromadine, clemizole, cyproheptadine, dimethyindedine, diphenhydramine, dibenzline, hydroxyzine, hydroxypropyl piperazine, ketotifen, loratadine, nipradine, oxamide, pheniramine, promethazine, mepyramine, desloratadine, dimenhydrinate, mizolastine, terfenadine, topiramate, cetirizine, triprolidine, mehailin, clopyralid hydrochloride, and emedastine.

In some embodiments of the present application, the emulsifier is model number Pluronic F68;

the slow release material is polylactic acid-glycolic acid copolymer.

The application also relates to a preparation method of the histamine H1 receptor antagonist slow release nanoparticle, which comprises the following steps:

dissolving the active pharmaceutical ingredient and the sustained-release material in an organic solvent to obtain an organic phase;

dissolving an emulsifier in a buffer salt solution to obtain a water phase, wherein the pH value of the buffer salt solution is 7.0-7.6;

adding the organic phase into the aqueous phase under a dispersion condition to prepare emulsion;

removing the organic solvent in the emulsion and filtering.

In some embodiments of the present application, in the preparation method, the mass-to-volume ratio of the slow release material to the organic solvent is (0.5 to 5): 100;

the mass volume ratio of the emulsifying agent to the buffer solution is (1-5): 100;

the volume ratio of the organic phase to the aqueous phase is 1: (5-100).

In some embodiments of the present application, the preparation method includes one or more of the following technical features:

(a) The dispersing mode comprises the following steps: ultrasonic treatment is carried out at 25-50 kHz;

(b) The way of removing the organic solvent in the emulsion comprises the following steps: spin-drying at 36-42 deg.C under vacuum; and

(c) The filtering mode comprises the following steps: filtration was carried out with a filter membrane having a pore size of 200nm.

In some embodiments of the present application, the organic solvent is a low boiling point organic solvent having a boiling point of < 50 ℃.

In some embodiments of the present application, in the preparation method, the buffer salt solution is an aqueous solution of 0.05mol/L to 0.2mol/L of phosphate.

The application also relates to an injection, which comprises the histamine H1 receptor antagonist nanometer slow-release agent or the histamine H1 receptor antagonist slow-release nanoparticle prepared by the preparation method.

The application also relates to application of the histamine H1 receptor antagonist slow-release nano-particles in preparing a medicament for treating bone joint diseases.

Compared with the traditional histamine H1 receptor antagonist slow-release particles, the histamine H1 receptor antagonist slow-release nanoparticles are prepared from the high-molecular biodegradable polymer and a proper emulsifier serving as raw materials, and the high-molecular polymer can play a good role in drug slow-release and controlled-release. The histamine H1 receptor antagonist slow release nanoparticle has high drug content, small use volume and obvious action effect, can effectively relieve and treat osteoarthritis, maintains longer-term treatment effect after once administration, reduces the cost of repeated administration, reduces the pain and irritation of patients, and reduces adverse reactions and safety accidents caused by organic solvents.

The preparation method of the histamine H1 receptor antagonist slow-release nanoparticle is simple to operate, convenient and quick, can prepare the nanoparticle with uniform particle size and good stability, and can be used for industrial production.

The histamine H1 receptor antagonist slow release nanoparticle can be used for preparing medicines for treating osteoarticular diseases, such as osteoarthritis and cartilage loss and injury caused by osteoarthritis.

Drawings

In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present application, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a transmission electron microscope image of histamine H1 receptor antagonist slow release nanoparticles prepared in examples 1-3 of the present application;

FIG. 2 is a graph showing the particle size distribution of histamine H1 receptor antagonist slow release nanoparticles prepared in examples 1-3 of the present application;

FIG. 3 is a graph showing comparison of drug release curves of histamine H1 receptor antagonist release nanoparticles prepared in examples 1-1, 1-3, 1-4 and 1-5 of the present application at 37℃under different pH environments;

FIG. 4 is a graph showing drug release curves of histamine H1 receptor antagonist release nanoparticles prepared in comparative examples 1-1, 1-2 and 1-3 of the present application at 37℃under different pH conditions;

FIG. 5 is a comparative graph of safranin O-fast green staining results for examples 1-3, comparative examples 1-3 of the present application;

FIG. 6 is a graph comparing the quantification results of safranin O-fast green staining of examples 1-3, comparative examples 1-3 of the present application.

Detailed Description

The present application is further described below in conjunction with the embodiments, examples and figures. It should be understood that these examples are illustrative only of the present application and are not intended to limit the scope of the present application. Furthermore, it is to be understood that various changes and modifications may be made by one skilled in the art after reading the teachings of this application, and such equivalents are intended to fall within the scope of the claims appended hereto.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the description of the application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

Terminology:

unless otherwise indicated or contradicted, terms or phrases used herein have the following meanings:

herein, "preferred", "better", etc. are merely embodiments or examples that describe better results, and it should be understood that they do not limit the scope of protection of the present application.

In this application, "further," "still further," "particularly," and the like are used for descriptive purposes and are not to be construed as limiting the scope of the present application.

In this application, "first," "second," etc. are for non-exhaustive list description purposes only, and it should be understood that no closed limitation on the number is made.

In the present application, "plural" in "one or more" is allowed to be two or more.

In the present application, the technical features described in an open manner include a closed technical scheme composed of the listed features, and also include an open technical scheme including the listed features.

In this application, reference is made to a numerical interval (i.e., a numerical range), where the optional numerical distribution is considered continuous, and includes two numerical endpoints (i.e., a minimum value and a maximum value) of the numerical range, and each numerical value between the two numerical endpoints, unless otherwise indicated. When a numerical range merely points to integers within the numerical range, both end integers of the numerical range are included, as well as each integer between the two ends, unless expressly stated otherwise. Further, when a plurality of range description features or characteristics are provided, these ranges may be combined. In other words, unless otherwise indicated, the ranges disclosed herein are to be understood to include any and all subranges subsumed therein.

In the present application, the weight may be a mass unit known in the chemical industry such as mu g, mg, g, kg.

In this application, molecular weight refers to weight average molecular weight.

In the present application, the administration mode of the histamine H1 receptor antagonist slow release nanoparticle is not particularly limited. Representative modes of administration include, but are not limited to: oral, rectal, parenteral (intravenous, intramuscular or subcutaneous) injection, and topical administration, inhalation.

The composition for injection may comprise a physiologically acceptable sterile aqueous or anhydrous solution, dispersion, suspension or emulsion, as well as sterile powder for reconstitution into a sterile injectable solution or dispersion. Suitable aqueous or nonaqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

Histamine H1 receptor antagonists (H1 antagonists) are commonly used clinically to combat allergic reactions and have been developed to the third generation. First generation H1 antagonists such as diphenhydramine, promethazine, etc. have high affinity for H1 receptors, but bind reversibly to H1 receptors, have short half-lives and are administered at a higher frequency. The first generation H1 antagonist has small molecular weight and is fat-soluble, and is easy to penetrate through the blood brain barrier to enter brain tissue, thereby generating a sedative effect on the central nervous system. Second generation H1 antagonists such as loratadine, cetirizine, and the like have little or less central nervous system inhibiting effect, and have long half-life and long lasting effect, and currently have gradually replaced the first generation antagonists. The third generation such as desloratadine is a further development on the basis of the second generation, with fewer side effects, particularly with significantly reduced side effects on the heart.

There is no report of a histamine H1 receptor antagonist sustained release agent at present, which is probably because the half-life of the second generation antagonist basically meets the requirement (for example, the action intensity of the main metabolite of loratadine, namely, the dehydroxymethyl ethoxy loratadine, is 4 times that of loratadine, the elimination half-life is 7-24 hours, and the half-life of loratadine is 8-11 hours). In view of the previous study of the subject group, the intracavitary injection of histamine H1 receptor antagonist once a week can delay the progress of animal osteoarthritis and reduce animal pain and knee joint cartilage abrasion degeneration; meanwhile, mesenchymal stem cells can be induced to differentiate into cartilage or cartilage-like formation in an in vitro cell model; histamine H1 receptor antagonists are well expected in the field of treatment of osteoarthritis; however, there is currently no sustained release injection of histamine H1 receptor antagonists, and the need for high frequency intra-articular injection due to the short half-life of the drug clearly does not meet future therapeutic demands. Accordingly, it would be desirable to provide a sustained release injection of histamine H1 receptor antagonists, because of the prolonged release of histamine H1 receptor antagonists in the knee joint, which maintains a longer-term therapeutic effect, thereby reducing the number of administrations, patient compliance and pain. And the slow release of the medicine can slow down the clearance of the medicine, improve the utilization rate and the treatment efficiency of the medicine, and play a better role in treatment.

In a first aspect of the present application there is provided histamine H1 receptor antagonist slow release nanoparticles made from a material comprising a pharmaceutically active ingredient (histamine H1 receptor antagonist), an emulsifier and a slow release material (high molecular polymer). The histamine H1 receptor antagonist can delay the drug release rate of the histamine H1 receptor antagonist through the encapsulation of a slow release material, maintain a longer-term treatment effect after once administration, reduce the cost of multiple administrations and reduce the pain and irritation of patients. In the application, the slow-release material refers to a material capable of reducing the absorption rate of a drug into a body by delaying the drug release rate of the drug from the dosage form, thereby playing a better treatment effect.

The histamine H1 receptor antagonist slow-release nanoparticle is prepared from a high molecular biodegradable polymer and a proper emulsifier serving as raw materials, and the high molecular weight polymer can play a good role in drug slow-release and controlled-release.

In some embodiments, the emulsifier comprises one or more of polyoxyethylene polyoxypropylene ether block copolymer, polyvinyl alcohol, tween 80, PEG-100 stearate, glycerol stearate, arachidyl alcohol, fatty alcohol polyoxyethylene ether, glyceryl behenate, cetostearyl glucoside, and combinations thereof;

in some embodiments, the emulsifier is selected from poloxamers, commercially available under the name of pramipexole (Pluronic), which is a novel class of polymeric nonionic surfactants, which are polyoxyethylene polyoxypropylene ether block copolymers, and in some embodiments, the emulsifier is Pluronic F68.

Further, the slow release material is a high molecular polymer with molecular weight selected from 10000Da to 100000 Da. In this application, the molecular weight of the slow release material may vary depending on whether it is a palliative treatment or a therapeutic treatment. In palliative applications, a relatively low dose is administered at relatively low frequency intervals over a long period of time, i.e. a longer period of sustained release of the histamine H1 receptor antagonist is required, at which time a sustained release material of greater molecular weight may be selected. In therapeutic applications, it is sometimes desirable to administer relatively high doses at relatively short intervals until the progression of the disease is delayed or stopped, i.e. a relatively short period of sustained release of the histamine H1 receptor antagonist is required, at which time a lower molecular weight sustained release material may be selected.

In some preferred embodiments, the weight average molecular weight of the polymeric slow release material is 10000-100000 daltons. For example, it may be, but is not limited to 10000 dalton, 20000 dalton, 30000 dalton, 40000 dalton, 50000 dalton, 60000 dalton, 70000 dalton, 80000 dalton, 90000 dalton or 100000 dalton, preferably 10000 to 80000 dalton, more preferably 10000 to 50000 dalton.

Further, the high molecular polymer comprises one or more of sodium alginate, chitosan, polylactic acid-glycolic acid copolymer, polycaprolactone, polylactic acid, polytrimethylene carbonate, polyglycolic acid, polyhydroxybutyrate-hydroxyvalerate copolymer, polyorthoester, polyanhydride, and combinations thereof.

In some embodiments, the high molecular polymer may be a polylactic acid-glycolic acid copolymer. Polylactic acid-glycolic acid copolymer (PLGA) is formed by random polymerization of monomer lactic acid and monomer glycolic acid, is a degradable functional polymer organic compound, and has good biocompatibility, no toxicity and good vesicle forming and film forming performances. The sustained release dosage form of the histamine H1 receptor antagonist with the polylactic acid-glycolic acid copolymer as a sustained release material has better stability and better drug release property.

In some embodiments, the emulsifier is Pluronic F68 and the slow release material is a polylactic acid-glycolic acid copolymer.

In some embodiments, the pharmaceutically active ingredient comprises 30% -40% of the total mass of the histamine H1 receptor antagonist slow release nanoparticle.

The particle size of the histamine H1 receptor antagonist slow release nanoparticle is 50 nm-200 nm. FIG. 2 is a graph showing the particle size distribution of histamine H1 receptor antagonist slow release nanoparticles in one embodiment, wherein the particle size of the nanoparticles is more than 90% between 80 and 120nm, and further more, the particle size of the nanoparticles is more than 99% between 90 and 100 nm.

In some embodiments, the histamine H1 receptor antagonist and the slow release material are present in a mass ratio of 1: (5-20), such as 1:5, 1:7.5, 1:10/1:12.5, 1:15, 1:17.5, 1:20, etc. When the mass ratio of the histamine H1 receptor antagonist main body to the sustained-release material is in the range of 1:5-20, the obtained histamine H1 receptor antagonist sustained-release nanoparticle has good stability, long in-vivo residence time, good drug release and high bioavailability.

In some preferred embodiments, the mass ratio of histamine H1 receptor antagonist to slow release material is 1 (5-20), and further may be 1 (5-10). When the mass ratio of the histamine H1 receptor antagonist to the sustained release material is 1:10, the obtained histamine H1 receptor antagonist sustained release dosage form has better stability, longer in vivo residence time, better drug release and higher bioavailability.

In some embodiments, the pharmaceutically active ingredient is at least one of a histamine H1 receptor antagonist or a histamine H1 receptor antagonist derivative or a histamine H1 receptor antagonist analog.

The application also relates to a preparation method of the histamine H1 receptor antagonist slow-release nanoparticle. At present, the core-shell structure nanoparticle is prepared by a plurality of modes including an O/W emulsification method, an O1/O2 emulsification method, a multiple emulsion-in-liquid drying method and the like, and the principle of the O/W emulsification method is that microspheres (nanoparticles) with proper particle sizes can be prepared by emulsifying an organic phase in which a drug and a polymer are dissolved in an aqueous phase of an emulsifier. In the traditional preparation, the O/W emulsification method has the defects of poor stability, uncontrollable particle size and the like. In the preparation method, the stability of the histamine H1 receptor antagonist slow release nano-particles in water is improved by using a high molecular nonionic surfactant such as Pluronic F68 and the like, and the preparation of the nano-scale histamine H1 receptor antagonist slow release nano-particles is realized by a high-frequency ultrasonic technology, so that the defects are overcome. Suitable emulsifiers constitute the intermolecular surface tension that results in a homogeneous stable dispersion or emulsion. The emulsifying agent is used for emulsifying the medicine active ingredient and the sustained-release material, so that the medicine active ingredient can be directly injected, and adverse reaction and safety accidents caused by the organic solvent are reduced.

The preparation method is simple, convenient and quick to operate, and the prepared histamine H1 receptor antagonist slow-release nanoparticles are good in stability, long in-vivo residence time, good in drug release and high in bioavailability, and are beneficial to popularization and application of industrial production.

In some embodiments, the preparation of histamine H1 receptor antagonist slow release nanoparticles comprises the steps of:

s100: providing a pharmaceutically active ingredient and a sustained release material;

s200: dissolving the active pharmaceutical ingredient and the sustained-release material in an organic solvent to obtain an organic phase;

s300: dissolving an emulsifier in a buffer salt solution to obtain a water phase;

s400: adding the organic phase into the aqueous phase under a dispersion condition to prepare emulsion;

s500: removing the organic solvent in the emulsion and filtering.

In some embodiments, the boiling point of the organic solvent in the step S100 is not higher than 50 ℃, so that the step S500 can remove the organic solvent more conveniently, and the preparation process is simplified, for example, the purification and impurity removal operations such as distillation, extraction and the like are not required by evaporation at normal temperature or micro-heat. In some embodiments, the organic solvent is selected from one or more of acetone, chloroform, methylamine, dimethylamine, diethyl ether, pentane, methylene chloride, carbon disulfide, and combinations thereof, more preferably from methylene chloride. Dichloromethane has good volatility and low toxicity. And dichloromethane is selected as an organic solvent, so that toxic and side effects can be reduced to the greatest extent.

In some embodiments, in step S200, the pharmaceutically active ingredient and the sustained release material are dissolved in an organic solvent to obtain an organic phase. Further, the pharmaceutically active ingredient and the sustained-release material are dissolved in an organic solvent under stirring or ultrasonic conditions.

In some embodiments, the mass to volume ratio of the slow release material to the organic solvent is (0.5-5): 100.

in some embodiments, in step S200, every 5-10 mg of the pharmaceutically active ingredient and 10-50 mg of the slow release material are dissolved in 0.5-2 ml of the organic solvent. The sustained release material may be, for example, but not limited to, 10mg, 15mg, 20mg, 25mg or 30mg, the pharmaceutically active ingredient may be, for example, but not limited to, 5mg, 6mg, 7mg, 8mg, 9mg or 10mg, and the organic solvent may be, for example, but not limited to, 0.5mL, 1mL, 1.5mL or 2mL.

In some embodiments, in step S300, the emulsifier is dissolved in a buffer salt solution to obtain an aqueous phase. Further, the emulsifier is dissolved in the buffer salt solution under stirring or ultrasonic conditions.

In some embodiments, the pH of the buffer salt solution is 7.0 to 7.6, further selected from phosphate solution, tris solution, or a combination thereof. In some embodiments, the buffer salt solution is an aqueous solution of 0.05mol/L to 0.2mol/L phosphate.

In some embodiments, the mass to volume ratio of the emulsifier to the buffer salt solution is (1-5): 100.

in some embodiments, in step S300, 0.1 to 2mL of emulsifier is dissolved per 1 to 10mL of buffer salt solution and mixed. The emulsifier may be, for example, but not limited to, 0.1mL, 0.2mL, 0.3mL, 0.4mL, 0.5mL, 0.6mL, 0.7mL, 0.8mL, 0.9mL, or 1mL.

In some embodiments, in step S400, the dispersing means comprises ultrasound. Further, ultrasonic waves are carried out at 25-50 kHz.

In some embodiments, in step S400, the volume ratio of organic phase to the aqueous phase is 1: (5-100).

In some embodiments, the means for removing the organic solvent from the emulsion comprises: spin-drying at 36-42 deg.C under vacuum.

In some embodiments, the manner of filtering includes: filtration was carried out with a filter membrane having a pore size of 200nm.

The application also relates to an injection, which comprises the histamine H1 receptor antagonist slow release nanoparticle. Currently, only the first generation histamine H1 receptor antagonist drugs (chlorpheniramine maleate, promethazine, diphenhydramine and derivatives thereof) are in injection form, but the common side effect of the first generation histamine H1 receptor antagonists is that the drugs can pass through the blood brain barrier, causing somnolence. The second generation and the third generation drugs can not pass through the blood brain barrier, thus reducing the occurrence of side effects, but no injection formulation exists in the market. In addition, the half-life period of the second generation and the third generation medicaments is short, and the repeated injection is required to be performed for a plurality of times, so that the joint cavity of the patient is damaged.

However, the most effective modes of administration for osteoarticular diseases include injection, such as intravenous injection, intramuscular injection, and intra-articular injection, and the like, and the number of administrations should be reduced as much as possible, and the pain of the patient is alleviated, and it is also required to avoid toxic and side effects while prolonging the half life and retention time of the drug.

The application also relates to application of the histamine H1 receptor antagonist slow-release nano-particles in preparing a medicament for treating bone joint diseases. The osteoarthropathy includes degenerative arthritis, bursitis, synovitis, cervical spondylosis, lumbar spondylosis, scapulohumeral periarthritis, hyperosteogeny, rheumatic arthritis, rheumatoid arthritis, femoral head necrosis and the like, and is common in middle-aged and elderly people. Among them, degenerative arthritis (osteoarthritis, OA) is one of the most common arthropathy, manifested by joint pain, stiffness, and severe disability.

Under normal physiological conditions, both articular cartilage and subchondral bone are in dynamic anabolic and catabolic balance, and maintenance of this balance is an important guarantee for the joint to exert its physiological functions. While the physiological dynamics of articular cartilage and subchondral bone of patients with osteoarticular diseases are in an unbalanced state, the synthesis of extracellular matrix of articular cartilage is reduced, the decomposition is increased, the subchondral bone generation and bone resorption are uncoupled, and the joint is subjected to degenerative change. Researchers in this application have found through extensive literature research that histamine H1 receptor antagonists may be of positive interest in the treatment of bone joint disorders.

In some embodiments, the osteoarticular disease includes osteoarthritis, cartilage degradation, and cartilage loss.

The following are some specific examples.

The experimental parameters not specified in the following specific examples are preferentially referred to the guidelines given in the application document, and may also be referred to the experimental manuals in the art or other experimental methods known in the art, or to the experimental conditions recommended by the manufacturer.

The starting materials and reagents referred to in the following specific examples may be obtained commercially or may be prepared by known means by those skilled in the art.

EXAMPLE 1 preparation of histamine H1 receptor antagonist sustained release nanoparticles

1.1. Weighing the raw materials according to the following table (Table 1)

TABLE 1

In Table 1, the slow release material is polylactic acid-glycolic acid copolymer (molecular weight: 50000 Da), the pharmaceutically active material is chlorpheniramine, the organic solvent is dichloromethane, the emulsifier is Pluronic F68, and the phosphate buffer solution is an aqueous solution of phosphate (pH 7.4, mass concentration: 0.05 mol/L)

1.2. According to the formula of Table 1, the weighed raw materials are respectively prepared into histamine H1 receptor antagonist slow release nanoparticles, and the specific operation is as follows: mixing the slow release material and the organic solvent of the medicine active component to obtain an organic phase. The emulsifier is dissolved in a buffer salt solution to prepare an aqueous phase. And (3) dropwise adding the organic phase into the water phase at 25 kHz-50 kHz, and carrying out ultrasonic treatment for 30-40 minutes to obtain the cloud-like white emulsion. Setting the temperature of a rotary evaporator to be 36-42 ℃, vacuumizing an organic solvent in the spin-drying emulsion, and filtering residues by using a filter membrane with the aperture of 200 nanometers to obtain histamine H1 receptor antagonist slow-release nanoparticles.

Example 2 topography testing

2.1. Transmission electron microscope observation

The histamine H1 receptor antagonist slow release nanoparticles prepared in examples 1-3 were subjected to transmission electron microscopy. The results are shown in FIG. 1. As can be seen from fig. 1, the histamine H1 receptor antagonist slow release nanoparticles of the present application are spherical and uniformly dispersed.

2.2. Average particle size test

The histamine H1 receptor antagonist slow release nanoparticles prepared in examples 1-3 were subjected to particle size distribution determination by Dynamic Light Scattering (DLS) nanoparticle size analyzer (nanoBrook 90Plus Zeta). The results are shown in FIG. 2. As can be seen from fig. 2, the histamine H1 receptor antagonist slow release nanoparticle of the present application has an average particle size of 100 nm.

Example 3 sustained Release test

The histamine H1 receptor antagonist slow release nanoparticles prepared in each example and comparative example are taken as samples, and histamine H1 receptor antagonist release curves of the histamine H1 receptor antagonist slow release nanoparticles in PBS at different pH values (7.4, 6.5 or 5.0) are tested, and the specific operation is as follows:

3.1. sample processing

Dissolved in PBS (0.01 mol/L, 1 mL) of different pH (7.4, 6.5, 5.0) and packed in dialysis tubing (MWCO=3500). The tube was then immersed in 20mL of PBS (pH 7.4, 6.5 or 5.0) and continuously shaken at 80 rpm. 1mL of dialysate was withdrawn at different time intervals and replaced with fresh PBS solution.

3.2. Detection of

The content of histamine H1 receptor antagonist was determined by high performance liquid chromatography. The relative release rate of histamine H1 receptor antagonist was calculated as a function of time (37 ℃).

3.3. Experimental results

FIG. 3 is a graph showing comparison of drug release curves of histamine H1 receptor antagonist release nanoparticles prepared in examples 1-1, 1-3, 1-4 and 1-5 under different pH environments. As can be seen from FIG. 3, in the physiological environment (joint cavity microenvironment pH) at pH 7.4, the histamine H1 receptor antagonist slow release nanoparticles of example 1-1 released 36% of histamine H1 receptor antagonist within 7 days, the histamine H1 receptor antagonist slow release nanoparticles of example 1-4 released 34% of histamine H1 receptor antagonist within 7 days, and the histamine H1 receptor antagonist slow release nanoparticles of example 1-5 released 24% of histamine H1 receptor antagonist within 7 days. Examples 1-3 histamine H1 receptor antagonist slow release nanoparticles exhibited higher stability with only 18% histamine H1 receptor antagonist released within 7 days.

The histamine H1 receptor antagonist in the histamine H1 receptor antagonist slow release nanoparticles of examples 1-1 released 71% and 85% or more, respectively, the histamine H1 receptor antagonist in the histamine H1 receptor antagonist slow release nanoparticles of examples 1-4 released 61% and 74% or more, respectively, within 48 hours, and the histamine H1 receptor antagonist in the histamine H1 receptor antagonist slow release nanoparticles of examples 1-4 released 50% and 69% or more, respectively, within 48 hours at pH 6.5 and pH 5.0. Examples 1-3 histamine H1 receptor antagonist slow release nanoparticles exhibited higher stability, with histamine H1 receptor antagonists released 42% and 70% or more, respectively, within 48 hours.

FIG. 4 is a graph showing drug release curves of histamine H1 receptor antagonist release nanoparticles prepared in comparative examples 1-1, 1-2 and 1-3 under different pH environments. As can be seen from FIG. 4, in the physiological environment (joint cavity microenvironment pH) at pH 7.4, 37% of histamine H1 receptor antagonist was released within 7 days from the histamine H1 receptor antagonist slow-release nanoparticles of comparative example 1-1, 29% of histamine H1 receptor antagonist was released within 7 days from the histamine H1 receptor antagonist slow-release nanoparticles of comparative example 1-2, and 41% of histamine H1 receptor antagonist was released within 7 days from the histamine H1 receptor antagonist slow-release nanoparticles of comparative example 1-3.

EXAMPLE 4 anti-osteoarthritis Effect test

Animal experiments were performed with the histamine H1 receptor antagonist slow release nanomaterials of examples 1-3 and comparative examples 1-3, and the specific procedures are as follows:

the histamine H1 receptor antagonist slow release nano-materials obtained in the above examples 1-3 and comparative examples 1-3 were dispersed and dissolved with physiological saline to prepare a drug suspension with a mass volume concentration of 100 mg/mL.

Cutting off the ligament of the meniscus at the inner side of the experimental side (right knee joint) by using a C57BL/6 mouse with the age of 12 weeks, cutting off part of the meniscus at the inner side, constructing a model (DMM model) of the meniscus at the inner side of the mouse, avoiding the loss of articular cartilage in the operation, and performing only false operation treatment on the contrast side (left knee joint);

the drug suspensions obtained in examples 1-3 and comparative examples 1-3 were used for injection into the intra-articular cavity of the knee joint of experimental side of DMM-molded mice, 100uL each time, once every 4 weeks, and the effect was evaluated 8 weeks after the operation. The evaluation was performed by imaging after safranin O-fast green staining and by OARSI scoring for osteoarthritis.

The safranin O-fast green coloring agent can intuitively reflect the structures of articular cartilage, subchondral bone and bone tissues after being dyed, and the principle is that basophilic cartilage and basic dye safranin O are combined to be red, and acidophilic bone and acid dye are combined to be green or blue, and the red cartilage is contrasted with the red cartilage, so that the cartilage tissues and the bone tissues are distinguished. The staining results are shown in FIG. 5.

FIG. 5A is safranin O-fast green staining of a comparative (left) knee joint without actual surgical modeling;

fig. 5B is safranin O-fast green staining of untreated knee joint on the experimental side (right);

FIG. 5C is safranin O-fast green staining of experimental (right) knee joints after treatment with comparative examples 1-3;

fig. 5D is safranin O-fast green staining of experimental (right) knee joints after treatment of examples 1-3.

Safranin O staining is approximately proportional to the concentration of anions, indirectly reflecting the proteoglycan content and distribution in the matrix. When cartilage is damaged, glycoproteins in the cartilage are released, causing uneven distribution of matrix components, resulting in light or no staining of safranin O. Safranin O-stained cartilage matrix can be quantitatively analyzed by the osteoarthritis OARSI scoring standard. The scoring criteria are as follows:

TABLE 2 OARSI scoring criteria for knee osteoarthritis in mice

OARSI scores for the staining results of fig. 5 are shown in fig. 6. In fig. 6, the ordinate represents the score, and the abscissa represents the correspondence: the "comparative side" corresponds to the scoring result of the staining chart of fig. 5 a; the "experimental side" corresponds to the scoring result of the staining chart of fig. 5B; "experimental side-comparative examples 1-3" corresponds to the scoring results of the staining chart of fig. 5C; "Experimental side-examples 1-3" corresponds to the scoring results of the staining pattern of D in FIG. 5.

According to fig. 6, it can be seen that the sustained-release nanomaterial provided in embodiments 1-3 of the present invention can maintain the effect of relieving and treating osteoarthritis on the basis of once injection every 4 weeks, is superior to the therapeutic effect of comparative examples 1-3, can reduce the injection frequency of 1 injection per week at present, can maintain the longer-term therapeutic effect in the middle period after 1 injection, can reduce the time and economic cost of multiple injections during injection, and reduces the multiple pain of clinical patients, thereby having higher value.

Therefore, the histamine H1 receptor antagonist slow release agent of the comparative example cannot realize long-acting stable release, has good stability, long release time and better application value, is suitable for preparing medicines for treating various bone joint diseases including osteoarthritis, can be injected, and can slowly release the active ingredients of the medicines for long time at affected parts.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. Unless otherwise conflict with the purpose and/or technical solution of the present application, the present application relates to the cited documents which are incorporated by reference in their entirety for all purposes. When reference is made to a cited document in this application, the definitions of the relevant technical features, terms, nouns, phrases, etc. in the cited document are also incorporated by reference. Examples of the relevant technical features and preferred modes to be cited in the present application when the cited documents are referred to in the present application are incorporated by reference in the present application, but are not limited to being able to implement the present application. It should be understood that when a reference is made to the description herein, it is intended to control or adapt the present application in light of the description herein.

The technical features of the above-described embodiments and examples may be combined in any suitable manner, and for brevity of description, all of the possible combinations of the technical features of the above-described embodiments and examples are not described, however, as long as there is no contradiction between the combinations of the technical features, they should be considered to be within the scope described in the present specification.

The above examples merely represent various embodiments of the present application and are not to be construed as limiting the claims. It should be noted that it would be apparent to those skilled in the art that various modifications and improvements could be made without departing from the spirit of the present application, which would be within the scope of the present application. Further, it will be understood that various changes or modifications may be made to the present application by those skilled in the art after reading the foregoing teachings, and equivalents thereof will be within the scope of the present application. It should also be understood that those skilled in the art, based on the technical solutions provided in the present application, can obtain technical solutions through logical analysis, reasoning or limited experiments, all fall within the protection scope of the claims attached to the present application. The scope of the patent application is therefore intended to be covered by the appended claims, which description and drawings should be construed in view of the appended claims.

Claims

1. The histamine H1 receptor antagonist slow release nanoparticle is characterized by being prepared from materials comprising a pharmaceutical active ingredient, an emulsifying agent and a slow release material;

the medicine active ingredient is histamine H1 receptor antagonist, and the model of the emulsifier is Pluronic F68; the slow release material is polylactic acid-glycolic acid copolymer;

the particle size of the histamine H1 receptor antagonist slow release nanoparticle is 50 nm-200 nm;

the preparation method of the histamine H1 receptor antagonist slow-release nanoparticle comprises the following steps: 7.5mg of the pharmaceutically active ingredient and 50mg of the slow release material were dissolved in 1mL of methylene chloride to obtain an organic phase,

dissolving the emulsifier accounting for 1 percent of the total weight in 50mL of phosphate buffer salt solution to obtain a water phase, wherein the pH value of the phosphate buffer salt solution is 7.0-7.6,

adding the organic phase into the aqueous phase under the ultrasonic condition of 25kHz to prepare emulsion;

rotary evaporating and vacuumizing, spin-drying the solvent in the emulsion, and filtering the residue with a filter membrane with the aperture of 200nm;

the histamine H1 receptor antagonist is selected from one or more of the following compounds: brompheniramine, chlorpheniramine, diphenhydramine, pheniramine, mepyramine, tripiramine and clopyralid hydrochloride.

2. The histamine H1 receptor antagonist slow release nanoparticle according to claim 1, wherein the vacuum is applied by rotary evaporation at 36-42 ℃.

3. The histamine H1 receptor antagonist slow release nanoparticle according to claim 1 or 2, wherein said phosphate buffer salt solution is an aqueous solution of 0.05mol/L to 0.2mol/L phosphate.

4. The histamine H1 receptor antagonist slow release nanoparticle according to claim 1 or 2, wherein the time of ultrasound is 30-40 minutes.

5. An injection comprising histamine H1 receptor antagonist slow release nanoparticles according to any one of claims 1 to 4.

6. Use of histamine H1 receptor antagonist slow release nanoparticles according to any one of claims 1-4 in the manufacture of a medicament for the treatment of osteoarticular diseases.