CN112402359A

CN112402359A - Polymer microneedle for inhibiting cell inflammatory factors to treat acute gout attack and preparation method thereof

Info

Publication number: CN112402359A
Application number: CN202011216538.8A
Authority: CN
Inventors: 霍亮; 向雄根; 黎永祥; 沈芝萍
Original assignee: Shenzhen Qianhai Yinggang Biotechnology Co ltd
Current assignee: Shenzhen Qianhai Yinggang Biotechnology Co ltd
Priority date: 2020-11-04
Filing date: 2020-11-04
Publication date: 2021-02-26
Also published as: CN113855621A

Abstract

The application discloses a polymer microneedle for inhibiting inflammatory response and treating acute gout attack and a preparation method thereof. The needle tip of the polymer microneedle is embedded with a medicinal preparation which is wrapped by a nano material and contains an effective dose of canamab; the pharmaceutical preparation contains 0.005-0.05 weight parts of kanamycin, 0.05-0.15 weight parts of sodium phosphate, 0.0025-0.025 weight parts of mannitol and 0.005-0.025 weight parts of sodium chloride. The microneedle patch is directly attached to a disease sign part, and is simple and convenient to use; the medicament can fully play a role by wrapping the nano material for slow release, has better fixed-point and targeted treatment effects and higher medicament utilization rate; not only solves the problem that the systemic circulation of the kanamycin antibody is difficult to effectively act on the pathological part for a long time, but also avoids various problems caused by excessive use of the kanamycin antibody in a systemic way.

Description

Polymer microneedle for inhibiting cell inflammatory factors to treat acute gout attack and preparation method thereof

Technical Field

The application relates to the technical field of gout treatment, in particular to a polymer microneedle for inhibiting inflammatory response and treating gout acute attack and a preparation method thereof.

Background

Gout is a common disease in the world, and according to epidemiological statistics, the prevalence rate of hyperuricemia in Europe and America in the 90 th of the 20 th century is reported to be 2% -24%, the prevalence rate of hyperuricemia reported in the United states in 1993 is 10.1%, and the total prevalence rate of hyperuricemia in China in 2008 is 13.2-21.04%. The total gout incidence in all parts of the world is between 0.13 and 15.3 percent, and the annual incidence in the world is between 0.20 and 0.35 percent. The incidence of gout and hyperuricemia in European and American countries is obviously higher than that in other countries, and the trend is increasing year by year. Wherein, the prevalence rate of gout patients in the United states is about 3.9%, and according to the estimation, about 920 ten thousand patients exist in the United states; gout patients in the United states and Europe exceed 800 million, and the number of Japanese adult male hyperuricemia is 25% -30%; in 2017, about 1.7 hundred million hyperuricemia patients in China are counted, and more than 3500 million patients with gout disease in China are conservatively estimated.

Gout (gout), a crystal-related arthropathy resulting from deposition of monosodium urate (MSU), is directly associated with hyperuricemia resulting from purine metabolic disorders and/or decreased uric acid excretion, and falls into the category of metabolic rheumatism. Gout is particularly characterized by acute characteristic arthritis and chronic tophus disease, can be complicated by nephropathy, and serious patients can have joint destruction, renal function impairment and other groups often accompanied by metabolic syndrome, such as abdominal obesity, hyperlipidemia, hypertension, type 2 diabetes and cardiovascular diseases. Gout is no longer considered to be only a disease of the joints, and the incidence of complications in patients with hyperuricemia is statistically very high, such as cardiovascular disease, type 2 diabetes, metabolic syndrome, chronic kidney disease, cancer, and premature aging. Whether elevated uric acid is associated with the pathogenesis of gout complications is still a controversial issue, but increasing epidemiological studies have found that hyperuricemia is an independent risk factor for gout complications. After the gout attack is relieved, the patient enters an asymptomatic period, which is also called an interval period, an intermission period or an attack interval; however, most patients relapse, and as the frequency of episodes increases, symptoms may persist more severely and the asymptomatic phase may shorten. Patients with recurrent episodes are characterized primarily by hyperuricemia, recurrent episodes of acute gouty arthritis, tophus deposition, chronic gouty arthritis and joint deformities, renal parenchymal lesions, and urate stone formation.

Acute attacks of gout cause severe pain. When pain occurs, the patient wakes up in a deep sleep in the middle of the night, and the patient describes the pain feeling as if the big toe is burned. The most commonly occurring joint is the big toe, the first metatarsal; however, the affected joints are not limited thereto, and are often found in joints of hands, knees, elbows, and the like. The affected joints are red and swollen and inflamed finally, tissues are soft after edema, activities are limited, over time, gout is damaged due to the frequently-attacked joints, joint ends of bones are eroded, and tophus deposits cause the joints to be in chronic inflammation, joint deformity and functional disorder, so that the disability rate is extremely high, and the daily life is influenced finally. Repeated attack patients and patients who develop chronic arthritis can use uric acid reduction drugs for long-term prevention to prevent gout from relapse or developing chronic tophus diseases.

Gout arthritis has not been treated completely to date, and the main goal of treatment is to alleviate the condition. Thus, the goal of treatment for acute gout attacks is to provide rapid and safe relief from pain and disability, including traditional therapies with nonsteroidal anti-inflammatory drugs, traditional disease-modifying glucocorticoids, representative drugs are acetaminophen, indomethacin, naproxen, ibuprofen, hydrocortisone, dexamethasone, prednisone, and the like. Even without treatment, episodes often are completely alleviated within days to weeks, especially in the early stages of the disease. Although early disease can be left untreated and can be completely alleviated within weeks; however, anti-inflammatory drugs can accelerate symptom relief and have a positive effect on treatment. However, although traditional drugs are more effective in treatment, some side effects limit their application, and some patients have poor curative effect in application, corticosteroids tend to destroy the immune system, and colchicine has higher toxicity; these drugs only temporarily eliminate pain and reduce inflammation.

For patients with chronic arthritis, controlling the blood uric acid level of the human body at a target level is the most fundamental and critical measure for preventing and treating gout. Can be used for long-term prevention of gout recurrence or development into chronic tophus disease by using uric acid lowering medicine. Although there are some treatment options available, the therapeutic effect of gout is not ideal. Based on the mechanism of gout formation, gout attacks cannot be fundamentally remedied if uric acid lowering drugs cannot control all patients' blood uric acid to normal levels below 5 mg/dL. However, the existing clinical medicines, whether singly administered or jointly administered, can not lower the blood uric acid level of gout patients to below 5mg/dL clinically even if the dosage reaches the intolerable level.

Studies have shown that high levels of uric acid (abbreviated UA) deposit MSU in tissues such as joints, and the deposition of MSU in joint cavities activates inflammatory cytokines, inducing the accumulation of macrophages and neutrophils, thereby causing painful arthritis. Oxidative stress plays a major role in the pathogenesis of gout and leads to a series of inflammatory events, such as the production of interleukin IL-1 β.

Depending on the pathogenesis of gout, inhibiting the inflammatory response and lowering serum UA levels are considered effective therapeutic strategies. Colchicine (col), corticosteroids and non-steroidal anti-inflammatory drugs are commonly used for the treatment of gouty arthritis. Allopurinol and Febuxostat (FBX) are the main clinical drugs for the treatment of hyperuricemia. However, many adverse reactions have been reported, including liver injury, renal toxicity, bone marrow suppression, and body allergies. Therefore, it is important to find alternative drugs and effective treatment methods for hyperuricemia and gouty arthritis.

The gouty inflammation is caused by the induction of proinflammatory cytokines by leukocytes from MSU crystals. Among the numerous cytokines, IL-1 β may play a particular role in the inflammatory network, as MSU crystals stimulate NLRP3 of monocytes, synovial monocytes and macrophage inflammasome, releasing IL-1 β. Animal studies and human clinical trials have provided convincing evidence for the role of IL-1 β in gout-related pain and inflammation. The effectiveness of IL-1 inhibition in hereditary autoinflammatory syndrome with mutations in NALP3 protein suggests that inhibition of IL-1 is effective in alleviating the inflammatory manifestations of acute gout. The MSU crystals trigger the release of IL-1 β via the innate immune pathway and the "inflammasome" complex (NLRP3 inflamasome) that leads to IL-1 β activation. The innate immune pathway includes TLR-2 and TLR-4 on the surface of monocytes and macrophages. The inflammasome acts as an intracellular sensor of inflammatory stimuli, regulating the activation of caspase (caspase-1). MSU crystals release active IL-1 beta by directly activating inflammatory corpuscle NLRP3 of monocytes or macrophages, so that neutrophils recruit more proinflammatory cytokines and release of chemokines to generate cascade amplification reaction of intense inflammation. At the same time, the proinflammatory cytokines and chemokines also inhibit the apoptosis of inflammatory cells so as to promote the continuation of inflammatory response.

Interleukin-1 (IL-1) consists of a group of cytokines that activate the expression of several proinflammatory genes. The 11 members of the IL-1 gene family include IL-1 β, as well as the anti-inflammatory interleukin-1 receptor antagonist (IL-1Ra) which acts as a regulator of IL-1 β signaling. Numerous studies have shown that the severity of inflammation is influenced by the relative amounts of IL-1 and IL-1 Ra.

Normally IL-1 β is in an inactive dormant state in immune cells (pro-IL-1 β), such as macrophages, monocytes and dendritic cells, and when immune stimulated caspase (caspase-1) is activated from the inactive dormant state (pro-caspase-1) by the action of the inflammasome NLRP3, shearing inactive IL-1 β to active IL-1 β. This IL-1 β is released into the extracellular joint synovial fluid, and IL-1 β activates IL-1 receptors on joint endothelial cells and macrophages, leading to signal transduction and gene activation, and to the secretion of a range of pro-inflammatory cytokines and chemokines. These pro-inflammatory cytokines and chemokines in turn recruit and activate leukocytes into the joint, amplifying the local joint inflammatory cascade of MSU crystals. Extrinsic symptoms include fever, redness, swelling, pain, and the like.

Each step is finely regulated throughout the MSU deposition process that triggers the cellular inflammatory response pathway. MSU crystals are recognized by pattern recognition receptors of the innate immune system, such as TLRs, and are phagocytosed by macrophages. Intracellular MSU crystals are recognized by the NLRP3 inflammasome polyprotein complex, resulting in activation of NLRP3, which cleaves pro-caspase-1 to active caspase-1. Cathepsin B, ROS and K ion extracellular discharge and other factors are also involved in the activation of NLRP3 stimulated by MSU. After Caspase-1 is activated, inactive IL-1 beta is sheared to produce active IL-1 beta, which is released into extracellular joint fluid. IL-1 β activates IL-1 receptors on articular endothelial cells and macrophages, leading to signal transduction and gene activation, and to the secretion of a range of proinflammatory cytokines and chemokines. These in turn recruit and activate leukocytes to the joints, thereby amplifying the inflammatory cascade.

In conclusion, in the acute gout attack process, inflammatory cells are mutually coordinated, immune proteins are mutually restricted, and cytokines are mutually induced, so that the whole inflammation generation and development process is accompanied. Although the exact molecular details of the NLRP3 inflammasome pathway and its role in MSU crystal deposition in the immune mechanisms of gout flares are not fully understood, the importance of urate crystals to activate the inflammasome complex, induce the release of proinflammatory cytokines such as IL-1 β, IL-8 and TNF and the interaction of these inflammatory cytokines on gout pathology has now been demonstrated, providing an idea and pathway for the treatment of gout flares.

With the recent emergence of biological preparation medicines, acute gout attack inflammation can be really encountered as "jindich". The biological preparation is a medicament with definite targeting, and compared with the traditional abiotic altered disease anti-gout joint inflammation medicament, the largest bright spot is that the biological preparation medicament can not only effectively relieve the disease condition, but also can block the damage of the disease to the joint. The biological preparation has the advantages of quick response, definite curative effect, good safety and the like, and brings great gospel for treating patients with gout arthritis and rheumatoid immune diseases. At present, the biological preparation medicine can effectively relieve the clinical symptoms of moderate and severe active rheumatoid arthritis patients, promote the treatment to reach the standard and possibly inhibit joint injury and deformity. IL-1 receptor blockers or IL-1 receptor antagonists, abbreviated as IL-1Ra, block IL-1 from binding to receptors by specifically binding to IL-1 receptors, thereby blocking IL-1-mediated inflammatory responses and reducing tissue damage due to immune responses, infections, inflammatory responses, and the like.

At present, an IL-1 receptor blocker for inhibiting gout attack, Anakinra (Kineret, Anakinra) administration means is injection administration by injection, but injection transdermal administration is used, and because frequent injection is needed, adverse side reactions such as cutin hyperplasia, red swelling, induration and the like can be caused on the skin of a frequently applied part, and strong pain and discomfort can be brought to patients. Subcutaneous and intramuscular injection of IL-1 receptor blockers has been shown to be effective in reducing gout inflammation, but administration of IL-1 receptor blockers by these systemic routes requires relatively high doses to achieve effective concentrations at the site of the disease condition. These relatively high systemic doses of IL-1 receptor blockers can lead to non-specific immunosuppression, thereby increasing the rate of infection and the incidence of various adverse effects. These disadvantages limit the therapeutic efficacy and wide use of IL-1 receptor blockers.

Therapies currently in the european and american gout research stage include Anakinra (Anakinra) and Canakinumab (Canakinumab) among other therapeutic biologics. However, to date, there is a lack of randomized controlled trials to evaluate the efficacy of Canakinumab in the treatment of gout. Although the biological agents have great breakthrough in the treatment of immune inflammatory diseases such as gout arthritis, the wide application of the biological agents is influenced by the bottleneck to be broken through. Some open pilot studies used Canakinumab to inhibit IL-1 until the symptoms of gout attack improved, and as a result found this regimen to be effective in some patients. However, some patients receive this treatment with only partial remission and relapse frequently occurs within 1-6 weeks after discontinuation of the drug. Moreover, Canakinumab has a short biological half-life and requires daily subcutaneous administration, and is therefore less suitable for preventing seizures; the short half-life of Canakinumab in the systemic circulation greatly limits its maximum drug action in the target tissues.

Disclosure of Invention

The application aims to provide a novel polymer microneedle for inhibiting inflammatory response and treating acute gout attack and a preparation method thereof.

The following technical scheme is adopted in the application:

one aspect of the application discloses a polymer microneedle for inhibiting inflammatory response and treating gout acute attack, wherein a needle tip of the polymer microneedle is embedded with a medicinal preparation which is wrapped by a nano material and contains an effective dose of canazumab; the pharmaceutical preparation contains 0.005-0.05 weight parts of canamycin, 0.05-0.15 weight parts of sodium phosphate, 0.0025-0.025 weight parts of mannitol and 0.005-0.025 weight parts of sodium chloride.

It should be noted that, the present application creatively combines the canazumab and the microneedle array, and takes advantages of both into account. On one hand, the pain of the patient in the treatment medication stage can be relieved by utilizing the characteristics of tiny microneedle patch needle head, minimally invasive painlessness, convenient use, friendly patient use experience and the like in the medication mode; on the other hand, the nano-material coated canakinumab is used for prolonging the drug release time, so that the canakinumab can block the inflammatory reaction of local focus, the absorptivity of the canakinumab is improved, and adverse side effects caused by the fact that a large amount of external drugs enter a human body in a short time are avoided by slow release. In addition, the polymer microneedles of the present application are advantageous in that they can be used to modify drugs for intravenous drip or subcutaneous injection into subcutaneous external application. This switching of the administration route has two major advantages: firstly, the convenience of medication can be increased, the medication can be finished by a patient at home, and particularly, when the patient suffers from gout attack to cause severe pain and is difficult to walk, the patient needs to conveniently relieve pain in time; secondly, medical cost is greatly reduced because of no need of assistance of medical care personnel.

It is also noted that the present application inventively embeds the canazumab in the nanoparticles and makes them into polymer microneedles for administration through the microneedles; the polymer microneedle disclosed by the application is attached to a disease sign part, so that the acute attack inflammatory reaction of gout can be effectively treated; moreover, the poisoning phenomenon can be reduced to the maximum extent by microneedle administration, and the damage to the kidney, the liver, the spleen, the heart, the pancreas, the bone marrow and the like can be reduced. It can be understood that the polymer microneedle of the present application acts directly on a disease condition site, and compared with the existing mode of intravenous drip or subcutaneous injection, the polymer microneedle of the present application acts more directly and has better treatment effect.

It should be further noted that, in terms of controlled release of the drug, the polymer microneedle of the present application uses a biodegradable nanomaterial to encapsulate the drug preparation to achieve the effect of controlled release. The drug preparation is wrapped by the biodegradable nano material, and after the drug enters a human body, the nano particles can be degraded and release the drug after a certain time, so that the drug can fully act on a focus part for a long time, and the drug has better fixed-point and targeted treatment effects and higher bioabsorption rate and utilization rate.

In general, the polymer microneedle combines the nanoparticles and the microneedle patch for controlled release of the drug, and has the advantages of painless minimally invasive property, convenience and easiness in use, high drug utilization rate and the like; the kanamycin monoclonal antibody directly acts on the pathological part and can control slow release, thereby well solving the problem that the systemic circulation of the kanamycin monoclonal antibody is difficult to act on the pathological part for a long time; meanwhile, because the administration of the kanamycin through a systemic route is not needed, the using dosage of the kanamycin is greatly reduced, so that the medicine cost is saved, and the problems of nonspecific immune suppression, various adverse reactions and the like caused by excessive use of the kanamycin through the systemic route are avoided.

Preferably, the canazumab further comprises at least one of a variant of canazumab with the same activity and a fusion protein of canazumab with the same activity.

It should be noted that the canazumab used in the present application may be conventionally used canazumab already reported at present; or amino acid variants having at least 90%, 95%, 97% or 99% identity thereto, or a canamab variant having the same or equivalent activity as that of the existing canamab by mutation such as deletion, insertion, inversion and substitution based on the existing canamab. The fusion protein of canazumab with the same activity is a fusion protein formed by fusing with other proteins or polypeptides, and such fusion protein can be used in the present application as long as it has the same or equivalent activity as that of canazumab.

Preferably, the nanomaterial is a polylactic acid-glycolic acid copolymer.

Preferably, the polymeric microneedles are made from sodium carboxymethylcellulose.

The application also discloses application of the polymer microneedle in preparing a medicament for treating gout or rheumatoid arthritis.

Yet another aspect of the present application discloses a method of preparing a polymer microneedle of the present application, comprising the steps of:

preparing a pharmaceutical preparation, which comprises mixing the canazumab and the phosphate buffer solution according to a ratio, adding a polylactic acid-glycolic acid copolymer solution, uniformly dispersing, and preparing into first nanoparticles by adopting a spray dryer; mixing mannitol and sodium chloride, adding polylactic acid-glycolic acid copolymer solution, dispersing uniformly, and preparing second nanoparticles by using a spray dryer; uniformly mixing the first nanoparticles and the second nanoparticles in proportion to obtain a pharmaceutical preparation;

a drug preparation packaging step, which comprises dissolving polylactic acid-glycolic acid copolymer in organic solvent to prepare packaging material solution; uniformly dispersing the prepared nano particles of the pharmaceutical preparation into the prepared wrapping material solution, and adding a vitamin E polyethylene glycol succinate solution to form nano particles, so as to obtain the pharmaceutical preparation wrapped by the nano material;

preparing polymer microneedles, namely adding the pharmaceutical preparations wrapped by the nanomaterials into a sodium carboxymethylcellulose solution according to the dosage of each polymer microneedle to prepare a mixed solution, pouring the mixed solution into a microneedle patch mould, and then carrying out centrifugal treatment in a centrifugal machine to ensure that the mixed solution enters a pinhole cavity of the microneedle patch mould and most of the pharmaceutical preparations wrapped by the nanomaterials are deposited at a needle point; and then curing, forming and demolding to obtain the polymer microneedle of the application.

The demolding can refer to the existing polymer microneedle preparation method, for example, a substrate of a polymer microneedle patch is adopted and fixed on the substrate through adhesive bonding to perform demolding; or adopting sodium carboxymethyl cellulose solution without the medicine preparation coated by the nano material, and demoulding after solidification. The method is not particularly limited, and is specifically determined according to product design or production conditions.

Preferably, the organic solvent used in the pharmaceutical formulation encapsulation step is ethyl acetate.

Preferably, the nanoparticles are prepared at a temperature of about 0 ℃ and about 3 ℃.

Preferably, the centrifugal treatment conditions for the preparation of the polymer microneedles are 5000-6000rpm centrifugation for 3-6 min.

The polymer microneedle is prepared by centrifugal treatment, and on one hand, the mixed solution can be filled into a pinhole cavity of a microneedle patch die by centrifugal force; on the other hand, most of the medicinal preparation wrapped by the nano material can be deposited on the needle point, and the use efficiency of the polymer microneedle is improved.

The beneficial effect of this application lies in:

the polymer microneedle patch is directly attached to a disease sign part, and is simple and convenient to use; the medicament can fully play a role by wrapping the nano material for slow release, has better fixed-point and targeted treatment effects and higher medicament utilization rate; not only solves the problem that the systemic circulation of the kanamycin antibody is difficult to effectively act on the disease symptom part for a long time, but also avoids the problems of nonspecific immunosuppression, various adverse reactions and the like caused by excessive use of the kanamycin antibody in a systemic way. The polymer microneedle creatively combines the canazumab with the microneedle drug delivery system, and provides a more efficient and safe treatment scheme for clinically treating gout or IL-1 related diseases.

Drawings

FIG. 1 is a graph of the results of HPLC analysis of a nanomaterial-encapsulated pharmaceutical formulation of the examples of the present application;

FIG. 2 shows the results of the degree of degradation test of nanoparticles of the nanomaterial-encapsulated pharmaceutical formulation of the present application after being left for 1 day at room temperature;

FIG. 3 shows the results of the degree of degradation of nanoparticles of the nanomaterial-encapsulated pharmaceutical formulation of the present application after being left for 7 days at room temperature;

FIG. 4 shows the results of a degree of degradation test of nanoparticles of the nanomaterial-encapsulated pharmaceutical formulation of the present application after being left for 14 days at room temperature;

fig. 5 is a schematic view of a polymer microneedle patch in use in an embodiment of the present application;

fig. 6 is a schematic view of a polymer microneedle patch of an embodiment of the present application releasing a drug after application to the skin;

FIG. 7 is a confocal fluorescence microscope observation of polymer microneedles in an example of the present application;

FIG. 8 shows the experimental results of the in vitro transdermal delivery of polymer microneedles in the examples of the present application;

FIG. 9 shows the results of detecting the gouty arthritis inflammatory factor Il-1. beta. in each of the test group and the control group after the treatment with the polymer microneedles according to the examples of the present application;

FIG. 10 shows the results of detecting the gouty arthritis inflammatory factor Il-6 in each of the test group and the control group after the treatment with the polymer microneedles according to the examples of the present application;

FIG. 11 shows the test results of TNF- α after the polymer microneedles of the present application were used for the test groups and the control group.

Detailed Description

According to the influence of the interaction of inflammatory cytokines on gout, which is proved by the existing research, the application aims at the NLRP3 inflammasome and IL-1 beta signal blocking strategy, and achieves the purposes of disease alleviation and chronic disease complication avoidance by effectively inhibiting uncontrolled systemic and local inflammatory reaction for patients.

At present, a great deal of research is carried out on the utilization of a minimally invasive painless soluble microneedle patch as a novel drug delivery means, and related research results can be applied to the fields of vaccination, diabetes treatment, cancer treatment and the like. In the aspect of drug controlled release, the microneedle drug delivery system can also wrap the drug by using biodegradable nanoparticles to achieve the effect of controlled slow release. However, microneedles for gout treatment have been recently studied and reported.

Inventive attempts of the present application combine canazumab with microneedle delivery systems and develop pharmaceutical formulations of canazumab suitable for use with polymer microneedles. Specifically, the kanamycin monoclonal antibody is wrapped by biodegradable nanoparticles and then prepared into polymer microneedles; after the medicine enters a human body, the nano particles can degrade and release the medicine after a certain time, so that the medicine can fully act with the inflammation focus part initiated by the joint urate crystallization to obtain better fixed point, accurately block the inflammation reaction of the local focus, and has high biological absorption rate. Therefore, the nano particles and the microneedle patch are combined to carry out drug controlled release, and the microneedle is soluble, painless and minimally invasive, convenient and easy to use, high in drug utilization rate and the like, so that the nano particles and the microneedle patch can be used for effectively and locally delivering the anti-canazumab in the skin and locally treating acute attack inflammation of gout, and have strong market competitiveness and wide application prospect.

It should be noted that the inventors of the present application actually developed a series of new drug microneedle delivery systems for new generation of therapeutic biological products, which have been proved to be capable of rapidly and effectively relieving the acute attack symptoms of gout without side effects and delaying the time interval of the second attack of gout with very good preventive effect in small clinical studies. The microneedle local administration is applied to quickly inhibit local cell inflammatory reaction of acute gout attack, and is convenient for repeated administration. Particularly, the polymer microneedle, the needle tip of which is embedded with the nano material-coated pharmaceutical preparation containing the effective dose of the canazumab, not only solves the problem that the general circulation of the existing canazumab is difficult to act on a disease symptom part for a long time, but also avoids the problems of nonspecific immunosuppression, various adverse reactions and the like caused by excessive use of the canazumab in a systemic way.

The present application will be described in detail with reference to specific examples. The following examples are intended to be illustrative of the present application and should not be construed as limiting the present application.

Examples

Preparation of pharmaceutical preparation

The pharmaceutical preparation of the embodiment comprises 0.005-0.05 weight parts of canazumab, 0.05-0.15 weight parts of sodium phosphate, 0.0025-0.025 weight parts of mannitol and 0.005-0.025 weight parts of sodium chloride. In this example, the drug formulation was prepared into nanoparticles and then used in subsequent experiments, and the specific preparation method is as follows:

weighing 0.05g of the canakinumab, dispersing the canakinumab in a sodium phosphate buffer solution with the pH value of 7.1-9.0, adding a small amount of polylactic acid-glycolic acid copolymer solution, wherein the dosage of the polylactic acid-glycolic acid copolymer is about 20-70% of the weight of the canakinumab, and preparing the first nano-particles by adopting a spray dryer. The sodium phosphate buffer contained 0.15g of sodium phosphate.

Weighing 0.025g of sodium chloride and 0.025g of mannitol, uniformly mixing, adding the mixture into the polylactic acid-glycolic acid copolymer solution, uniformly dispersing, wherein the dosage of the polylactic acid-glycolic acid copolymer is about 20-70% of the weight of the canadensin, and preparing the second nano-particles by adopting a spray dryer.

The first nanoparticles and the second nanoparticles are mixed uniformly to obtain the pharmaceutical preparation of the present example.

Second, nano-encapsulation and detection of pharmaceutical preparation

1. Nanoparticle preparation

In this example, polylactic-co-glycolic acid (abbreviated as PLGA) was used as the coating material, and the specific coating method was as follows:

(1) weighing 6g of PLGA, and dissolving the PLGA in 60mL of organic solvent ethyl acetate to prepare a wrapping material solution;

(2) preparing 2.7L vitamin E polyethylene glycol succinate solution with concentration of 0.3% w/v for later use;

(3) adding the medicinal preparation obtained by the step one and the step one of medicinal preparation into the wrapping material solution, and uniformly dispersing; then, dripping the wrapping material solution containing the medicinal preparation into a vitamin E polyethylene glycol succinate solution with twice volume by using a Pasteur pipette, and carrying out high-speed oscillation while dripping until the addition is finished, and then continuing to oscillate for 15s to obtain an emulsion; transferring the emulsion into an ultrasonic generator, keeping the container immersed in ice water, and ultrasonically treating for four times, 8s each time, in this example, an 800W ultrasonic instrument with an amplitude of 35% and a probe tip size of 1/8, pausing between each 8s of ultrasonic treatment to cool the solution and continuing, and moving the probe up and down to ensure uniform ultrasonic treatment;

(4) transferring all the products subjected to ultrasonic treatment in the step (3) into the rest vitamin E polyethylene glycol succinate solution, and continuously stirring for three hours to harden the nanoparticles to obtain the medicinal preparation wrapped by the nanomaterials;

(5) and (3) averagely dividing the product obtained in the step (4) into two centrifuge tubes, centrifuging for 30min at 17000g, removing supernatant, adding 30mL of deionized water into each centrifuge tube, washing at least twice, and finally adopting 10mL of deionized water for resuspension, wherein the obtained suspension, namely the nano-material-coated pharmaceutical preparation (nanoparticles), can be used for subsequent experiments.

2. Wrap rate detection

Accurately weighing 5mg of the prepared nano material-coated pharmaceutical preparation, and dissolving the nano material-coated pharmaceutical preparation in 4mL of acetonitrile to obtain a detection solution; the detection solution was filtered through a 0.22 μm teflon syringe filter and then analyzed by High Pressure Liquid Chromatography (HPLC).

This example uses a Waters 2695separationModule system (Waters Corp., Milford, MA, USA), TSKgel UP-SW Columns (4.6X 30cm), and is kept at room temperature; the detection wavelength is 280nm, and the flow rate is 1 mL/min; the mobile phase is a mixture of methanol 70 and water 30; the injection amount is 20 mL; the amount of active ingredient in each use was calculated using the calibration line.

The measurement results are shown in fig. 1, and the results show that almost all the drug in the nanomaterial-encapsulated pharmaceutical preparation prepared in this example is encapsulated in the PLGA nanoparticles, and the encapsulation rate reaches over 90%.

3. Measurement of Release Rate of nanomaterial-Encapsulated pharmaceutical formulations

20mg of the prepared nanomaterial-encapsulated pharmaceutical formulation was added to a 10mL capped glass vial and added to 6mL of a 20mM PBS solution at pH 7.4. Wherein, the PBS solution contains 20% methanol by volume, and the effect of the methanol is to accelerate the drug release. The vial was shaken in a shaker at 50rpm and the temperature was set at 37 ℃. At each preset interval, the vial was removed from the shaker and allowed to stand for several minutes to allow the microspheres to settle to the bottom. Then, 1mL of the supernatant was removed and an equal amount of fresh methanol-containing PBS was added. The vial was placed back in the shaker. The removed solution samples were analyzed using an HPLC system and the methods described above.

The measurement result shows that the sustained-release time of the drug preparation wrapped by the nano material in the embodiment is about 5 days; in addition, the release speed of the drug can be controlled by controlling the dosage of the polylactic acid-glycolic acid copolymer, so that the sustained release effect is achieved, and the release time of the drug can be controlled to realize a better drug utilization effect.

4. Stability testing of nanomaterial-encapsulated pharmaceutical formulations

The degradation degree of the nanoparticles is judged by detecting the molecular weight change of the polylactic acid-glycolic acid copolymer serving as the wrapping material of the nanoparticles, so that the stability of the nanoparticles of the drug preparation wrapped by the nanoparticles is represented.

Specifically, this example tests the degree of molecular weight decrease of the polylactic acid-glycolic acid copolymer after the nanoparticles are left at room temperature (about 25 ℃) for 1 day, 7 days, and 14 days, respectively. The method for measuring the molecular weight comprises the following steps: determining the molecular weight of the original and degraded polymer samples by gel permeation chromatography; wherein the gel permeation color system consists of a Waters HPLC pump and a differential refraction detector; two gel permeation chromatography columns of polystyrene type cross-linked copolymer were connected together to cover a wider range of molecular weights, maintaining the temperature at 35 ℃; connecting a polystyrene type cross-linked copolymer protective column before two gel permeation chromatographic columns; chloroform was used as a mobile phase at a flow rate of 0.25mL/min and an injection volume of 100. mu.L; polystyrene standards were used to establish molecular weight calibration lines. The test results are shown in fig. 2 to 4, fig. 2 shows the test results after leaving for 1 day, fig. 3 shows the test results after leaving for 7 days, and fig. 4 shows the test results after leaving for 14 days.

The measurement results show that the molecular weight of the nanoparticles hardly decreases within 1 day in the nanomaterial-encapsulated pharmaceutical formulation of this example, the molecular weight decreases by only about 10% within 7 days, and the time for 50% decrease in molecular weight is longer than 14 days, indicating that the nanoparticles of this example have good stability.

Preparation and detection of polymer microneedle

1. Polymer microneedle preparation

Dissolving the medicinal preparation wrapped by the nano material prepared in the embodiment into a 10% sodium carboxymethyl cellulose solution to prepare a mixed solution; wherein, the amount of the medicinal preparation is 5 percent to 20 percent (W/W) of the sodium carboxymethyl cellulose, and the embodiment is 20 percent (W/W). Adding 75 mu L of the mixed solution into a microneedle patch mould, placing the mould in a centrifuge and centrifuging at 6000rpm for 4min to ensure that the mixed solution fully enters a pinhole cavity on the mould and most of the medicinal preparation wrapped by the nanomaterial is deposited on a needle point. After centrifugation, the mixed solution was removed from the cavity and allowed to air dry overnight to allow the mixed solution to solidify. Then, 300. mu.L of a 10% sodium carboxymethylcellulose solution was added to the mold, and the mixture was centrifuged at 4000rpm for 1 min. After centrifugation, the mold was allowed to stand and air dried overnight to form microneedle patches. After the patch was formed, the patch was peeled off from the mold, and the polymer microneedle patch of this example was obtained. The polymer microneedle patch of this example had a base area of 1.5cm by 1.5cm, a microneedle density of 100 needles per square centimeter, and a microneedle height of about 500 μm to 1 mm. It will be appreciated that the footprint of the patch and the density of microneedles are both dependent on the mould; the height of the microneedles depends on the mold on the one hand and on whether the mixed solution effectively enters the pinhole cavity of the mold on the other hand; although the mixed solution is fully introduced into the pinhole cavity by centrifugation, the mixed solution still cannot completely fill the pinhole cavity, so that the height of the microneedle is 500-1 mm, and the height range can meet the use requirement.

Wherein, after 50 μ L of the mixed solution is added into the microneedle patch mould, the centrifugation speed can be 5000-.

When the microneedle patch of the present embodiment is used, as shown in fig. 5, the microneedle patch 01 is directly applied to the affected part; the microneedles of the microneedle patch 01 penetrate the epidermis 11 and dermis 12, with the tips of the needles slightly contacting the subcutaneous tissue 13, releasing the drug directly to the location of the disease symptoms. In fig. 5, a is a schematic view of the microneedle patch applied to an affected part; b is a schematic diagram of a drug preparation wrapped by a nanomaterial in a microneedle, wherein 02 represents drug preparation nanoparticles; the C diagram is a diagram of the pathological part of the patient, and 14 in the A diagram and the C diagram shows the inflammation focus part caused by urate crystallization. The polymer microneedle is shown in fig. 6, the bottom of the polymer microneedle is a water-soluble sodium hydroxymethyl cellulose base 31, and the needle tip part contains a large amount of a drug preparation 32 coated by aqueous polylactic acid-glycolic acid copolymer; after the microneedle is inserted into the skin, its action process is shown in fig. 6, including penetration 33, implantation 34, rapid release 35, and sustained release 36; after the needle is penetrated 33, the needle point part stays in the skin, the medicinal preparation wrapped by the nano material is planted at the affected part, the medicinal preparation wrapped by the nano material is quickly released from the needle point part of the microneedle, and the medicament is continuously released in the biodegradation process of the nano wrapping material; the drug preparation wrapped by the nano material is gradually released by controlling the biodegradation of the micro needle; the degradation of the nano-coating material is controlled, and the medicinal preparation is slowly released, so that the medicinal preparation can be applied to the disease symptom part for a long time, the utilization rate and the action period of the medicinal preparation are improved, and the effect of effectively treating gout is achieved.

2. Polymer microneedle drug distribution testing

In order to observe the distribution of the drugs in the polymer microneedle, the present example adds the fluorescent dye sulforhodamine B into the wrapping material of the drug preparation nano-microsphere, namely the polylactic acid-glycolic acid copolymer, and adds the fluorescent dye coumalin 314 into the sodium carboxymethylcellulose for preparing the polymer microneedle; preparing polymer microneedles according to the foregoing method; then observing the distribution of fluorescent molecules by using a confocal fluorescent microscope; and judging the distribution of the drug preparation nano-microspheres in the polymer microneedles according to the distribution conditions of the fluorescent dye sulforhodamine B and the fluorescent dye kumalin 314.

The result is shown in fig. 7, the left graph is an observation result graph of the fluorescent dye coumalin 314, the middle graph is an observation result graph of the fluorescent dye sulforhodamine B, and the right graph is a superimposed view of two fluorescence channels of the coumalin 314 and the sulforhodamine B. The results in fig. 7 show that most of the fluorescent dye is distributed at the tip of the microneedle, consistent with the expected results.

3. Polymer microneedle in vitro transdermal drug delivery experiment

To evaluate the time-dependent intradermal release rate of microneedle kanamycin, the present example uses Cy 3-labeled kanamycin microneedles, which are inserted into a pigskin sample, and then at different time intervals, the transfer efficiency and the drug release kinetics profile of Cy 3-labeled kanamycin are determined by measuring the change in Cy3 fluorescence in the pigskin sample.

Dividing the polymer microneedle patch prepared in the embodiment into 9 groups, and sequentially and respectively staying for 10s, 30s, lmin, 2min, 5min, l0min, 15min, 20min and 25min after each group is pricked into the pigskin; the polymer microneedle patch was then pulled out. And observing the appearance of the tip end of the micro needle of the pulled polymer micro needle patch and the entering and distribution conditions of the drugs in the pig skin by using a microscope, and detecting the content of the drugs in the micro needle patch before and after treatment by using a spectral fluorescence instrument.

Partial statistical results are shown in fig. 8, as the retention time increases, the microneedle patch absorbs tissue water in the pigskin and slowly dissolves to release tip drugs, and the fluorescence of the tip end of the microneedle slowly weakens; accordingly, the fluorescence intensity of the pig intradermal drug is gradually increased. In this example, about 50% of the kanamycin embedded microneedle tips were released onto the skin within 5 minutes, with peak release between 10-20 minutes and about 75% of the kanamycin released into the skin microenvironment.

4. Distribution test of drug released by polymer microneedle after skin penetration

Two groups of rats of 3 rats each under the same conditions were used in this example to perform a molecular distribution test after skin permeation of the drug released from the microneedle patch. Specifically, the microneedle patch of this example was applied to the dorsal skin of two groups of rats, respectively, and after 10 minutes, the microneedle patch was pulled out, and after 2 hours and 6 hours of treatment, the skin near the penetration site was dissected and prepared for imaging; the frozen sections were imaged by confocal microscopy (EVOS M7000) to determine the distribution of the released drug formulation.

The results show that after 2 hours of treatment, there is a deposition of fluorescent molecules in the dermis at a depth of about 100 μm; after 6 hours of treatment, continued diffusion of the fluorescent molecules was observed in a larger area within the dermis layer near the needle puncture site. It can be seen that the microneedle patch of this example can stably carry a drug and deliver drug molecules into the skin.

5. Polymeric microneedle skin irritation test

In this example, the prepared microneedle patch was applied to the same position of the back skin of the rat once a day for 3 consecutive days in such a manner that the microneedle patch was attached for 10 minutes and then pulled out. The safety of the polymer microneedle patch of this example was evaluated by observing whether there was visible skin irritation on the skin treated with the microneedle patch.

The results showed that no visible irritation was observed on the skin treated with the polymer microneedle patch of this example, compared to the skin without any treatment. Dissecting the skin around the microneedle puncture site, performing histological examination, observing whether skin inflammatory cell infiltration exists, and using untreated skin as a control group; the results show that no significant infiltration of skin inflammatory cells was observed on skin repeatedly inserted with microneedle patches compared to untreated skin. The above test results show that the polymer microneedle patch of this example does not induce a significant inflammatory reaction in the skin, and is highly safe.

6. Animal experiments with polymeric microneedles

The test mainly detects the influence of the kanamycin on the gout cell inflammatory factors, constructs an acute model of the gouty arthritis of a rat, and treats the gout cell inflammatory factors by using the polymer prepared in the example. The method comprises the following specific steps:

50 male Wistar rats of 8 weeks of age were used. All rats were placed in plastic cages at constant temperature 23 + -1 deg.C and relative humidity 55%, with a 12 hour light/dark cycle for a light time of 7:00-19:00, and were free to eat standard food and mineral water.

Sodium Urate (MSU) -induced gouty arthritis model in rats:

MSU (Sigma-Aldrich) suspensions were prepared in a biosafety cabinet with sterile water.

50 rats were randomly divided into 5 groups of 10 rats each. Day 5, 4:00 pm, except for CTRL rats injected with 0.9% saline, the remaining rats were injected intra-articularly to the right ankle (0.1mL) with 20mg/mL of MSU. Blood samples of 200 microliters were collected from the tail vein of rats prior to MSU injection. Blood samples of 200 microliters were collected from the tail vein of rats before they were euthanized on days 6, 7, and 8.

The 50 rats were randomly divided into 5 groups (n: 10/group), each group being designed as follows:

1) a CTRL control group, after MSU is injected on the fifth day, a nano microneedle patch without the kanamycin antibody is attached to the right ankle of the rat, and the nano microneedle patch is replaced every day for 3 days; 2) the method comprises the following steps of (1) sticking a gouty arthritis rat to the right ankle of the rat by using a nano microneedle patch without kanamycin, and replacing the nano microneedle patch every day for 3 days; 3) Gouty arthritis rats-canazumab-low dose group containing canazumab 75mg/kg nano microneedle patch (canamab from Sigma-Aldrich, USA) replaced the nano microneedle patch daily for 3 days; 4) gouty arthritis rat-canamumab-high dose group (canamumab from Sigma-Aldrich, USA) containing 200mg/kg of canamumab nano microneedle patch, changing the nano microneedle patch every day for 3 days; 5) gouty arthritis rats were given daily intramuscular injections of 40mg/kg Triamcinolone acetonide (Triamcinolone acetonide) for 3 days. And analyzing the influence of the Carnacumab nano microneedle patch on the cell inflammatory factors of the gouty arthritis model rat by using the CTRL control group and the gouty arthritis rat as a negative control group and a model control group respectively.

In the acute gouty arthritis rat model, changes in serum IL-1 β, IL-6 and tumor necrosis factor (TNF- α) levels at different times after MSU injection were determined using R & D Systems, Minneapolis, MN, USA ltd ELISA kits, and the detection procedure was performed according to the manufacturer's instructions. The test results are shown in table 1 and fig. 9 to 11. Wherein, fig. 9 is a detection result of the gouty arthritis inflammatory factor IL-1 β of each test group and the control group after being treated by the polymer microneedle, fig. 10 is a detection result of the gouty arthritis inflammatory factor IL-6 of each test group and the control group after being treated by the polymer microneedle, and fig. 11 is a detection result of the gouty arthritis tumor necrosis factor TNF- α of each test group and the control group after being treated by the polymer microneedle.

TABLE 1 Effect of the microneedle patch of this example on the inflammatory cytokines in rats with acute gouty arthritis

The results of the tests in table 1 are mean ± Sd, where the mean is the mean of n ═ 10 for each group and the standard deviation is from 3 replicates, i.e. three replicates are run for one sample.

The results in Table 1, FIGS. 9 to 11 show that hyperuricemia and the levels of proinflammatory cytokines IL-1 and IL-6 are elevated in rats in the gouty arthritis model group, (P < 0.05). Compared with the experimental control group of rats, the level of Il-1 beta of the kanamycin microneedle treated group of rats is reduced by 34.2 percent (P <0.05), the level of Il-6 is reduced by 27.3 percent (P <0.05) and the level of tumor necrosis factor-alpha (TNF-alpha) is reduced by 13.2 percent (P < 0.05).

The tests show that the polymer microneedle prepared by the method can be directly attached to the disease sign part, has a good treatment effect on gout arthritis, solves the problem that the systemic circulation of the kanamycin antibody is difficult to act on the disease sign part for a long time, and avoids the problems of nonspecific immunosuppression, various adverse reactions and the like caused by excessive use of the kanamycin antibody in the systemic way. The polymer microneedle of the embodiment provides a more efficient and safe treatment scheme for clinically treating gout or IL-1 related diseases.

The foregoing is a detailed description of the present application in connection with specific embodiments thereof, and implementations of the present application are not to be considered limited to those descriptions. It will be apparent to those skilled in the art from this disclosure that many more simple derivations or substitutions can be made without departing from the basic inventive concepts herein.

Claims

1. A polymer microneedle for inhibiting inflammatory response and treating acute gout attack, which is characterized in that: the needle tip of the polymer microneedle is embedded with a medicinal preparation which is wrapped by a nano material and contains an effective dose of canazumab;

the pharmaceutical preparation contains 0.005-0.05 weight parts of canazumab, 0.05-0.15 weight parts of sodium phosphate, 0.0025-0.025 weight parts of mannitol and 0.005-0.025 weight parts of sodium chloride.

2. A polymeric microneedle according to claim 1, characterized in that: the canazumab also includes a variant of canazumab with the same activity and/or a fusion protein of canazumab with the same activity.

3. A polymeric microneedle according to claim 1 or 2, characterized in that: the nano material is polylactic acid-glycolic acid copolymer.

4. A polymeric microneedle according to claim 1 or 2, characterized in that: the polymeric microneedles are prepared from sodium carboxymethylcellulose.

5. Use of the polymer microneedle according to any one of claims 1 to 4 for the preparation of a medicament for treating gout or rheumatoid arthritis.

6. A method of manufacturing polymeric microneedles in any one of claims 1-4, wherein: comprises the following steps of (a) carrying out,

preparing a pharmaceutical preparation, which comprises mixing the canazumab and a phosphate buffer solution according to a ratio, adding a polylactic acid-glycolic acid copolymer solution, uniformly dispersing, and preparing into first nanoparticles by adopting a spray dryer; mixing mannitol and sodium chloride, adding polylactic acid-glycolic acid copolymer solution, dispersing uniformly, and preparing second nanoparticles by using a spray dryer; uniformly mixing the first nanoparticles and the second nanoparticles in proportion to obtain a pharmaceutical preparation;

a drug preparation packaging step, which comprises dissolving polylactic acid-glycolic acid copolymer in organic solvent to prepare packaging material solution; uniformly dispersing the prepared nano particles of the medicinal preparation into the prepared wrapping material solution, and adding a vitamin E polyethylene glycol succinate solution to form nano particles, so as to obtain the medicinal preparation wrapped by the nano material;

preparing polymer microneedles, namely adding the pharmaceutical preparations wrapped by the nanomaterials into a sodium carboxymethylcellulose solution according to the dosage of each polymer microneedle to prepare a mixed solution, pouring the mixed solution into a microneedle patch mould, and then carrying out centrifugal treatment in a centrifugal machine to ensure that the mixed solution enters a pinhole cavity of the microneedle patch mould and most of the pharmaceutical preparations wrapped by the nanomaterials are deposited at a needle point; and then curing, forming and demolding to obtain the polymer microneedle.

7. The method of claim 6, wherein: in the step of coating the pharmaceutical preparation, the organic solvent is ethyl acetate.

8. The method of claim 6, wherein: in the step of coating the pharmaceutical preparation, the nanoparticles are prepared at a temperature of about 0 ℃ and about 3 ℃.

9. The production method according to any one of claims 6 to 8, characterized in that: in the preparation of the polymer microneedle, the centrifugation treatment condition is 5000-6000rpm centrifugation for 3-6 min.