Sustained-release injection composition for treating or preventing inflammatory diseases and preparation method thereof

Abstract

The present invention relates to a sustained-release injection composition for treating or preventing inflammatory diseases and a method for preparing the same, and more particularly, to a sustained-release injection composition for treating or preventing inflammatory diseases and a method for preparing the same, the sustained release of cannabidiol for one month or more using microparticles containing cannabidiol, thereby being able to exhibit a therapeutic or preventive effect on inflammatory diseases. Moreover, the sustained-release injection composition containing cannabidiol, which is non-toxic and free from side effects caused by long-term use and can be continuously used for a long period of time, can exhibit a therapeutic or prophylactic effect on inflammatory diseases for one month or more by one injection, thereby significantly improving ease of administration.

Description

Technical Field

[0001] The invention relates to a sustained-release injection composition for treating or preventing inflammatory diseases and a preparation method thereof. Background Art

[0002] Osteoarthritis is a degenerative disease caused by damage to the cartilage of the knee and abnormal joint lubrication. It refers to arthritis caused by degenerative changes in the cartilage and surrounding bones in the synovial joint. Cartilage is the smooth tissue that covers the ends of the bones in the joint. Healthy cartilage helps absorb the impact caused by the movement of bones against each other. In osteoarthritis, the upper layer of cartilage breaks down and wears away, which allows the bone under the cartilage to come into direct contact. This direct contact can cause pain, swelling, and movement injuries to the joint, and over time, the joint may lose its normal shape and may develop osteophytosis. In addition, fragments of bone or cartilage may fall off and become free in the joint space, which will cause greater pain and injury.

[0003] People with osteoarthritis typically experience joint pain and loss of movement. Unlike other types of arthritis, osteoarthritis affects only the joints and not the internal organs. Osteoarthritis is the most common type of arthritis, and the second most common type of arthritis is rheumatoid arthritis, which is caused by a malfunction of the autoimmune system.

[0004] At present, known treatments for osteoarthritis include drug therapy, exercise therapy or surgical therapy, etc. Among them, in drug therapy, commonly used drugs include acetaminophen, nonsteroidal anti-inflammatory drugs (NSAIDs), corticosteroids, etc.

[0005] However, most drugs mainly relieve pain by treating inflammation and cannot fundamentally inhibit the degenerative changes of cartilage. Instead, they may accelerate cartilage damage and have side effects in the cardiovascular, gastrointestinal, kidney, liver, etc.

[0006] Therefore, it is necessary to develop therapeutic agents for treating or improving osteoarthritis, especially products that can achieve long-term sustained drug release through a single injection, thereby improving the therapeutic or preventive effect of osteoarthritis.

[0007] Prior art literature

[0008] Patent Literature

[0009] KR 10-2020-0066281A1 Summary of the invention

[0010] Technical problem to be solved by the invention

[0011] The object of the present invention is to provide a sustained-release injection composition for treating or preventing inflammatory diseases and a preparation method thereof.

[0012] Another object of the present invention is to provide a sustained-release injection composition that utilizes microparticles containing cannabidiol to continuously release cannabidiol for more than one month, thereby being able to exhibit a therapeutic or preventive effect on inflammatory diseases.

[0013] Another object of the present invention is to provide a method for preparing a sustained-release injection composition, which contains cannabidiol, which is non-toxic and has no side effects caused by long-term use, and can be used continuously for a long time. A single injection can show a therapeutic or preventive effect on inflammatory diseases for more than one month, thereby significantly improving the convenience of medication.

[0014] Means for solving technical problems

[0015] To achieve the above objectives, the present invention provides a sustained-release injection composition for treating or preventing inflammatory diseases, which comprises microparticles that continuously release cannabidiol (CBD) for more than one month, and the microparticles may contain cannabidiol and biodegradable polymers.

[0016] The microparticles may contain cannabidiol and biodegradable polymer in a weight ratio of 1:3 to 1:10.

[0017] The biodegradable polymer can be selected from the group consisting of polylactic acid, polylactide, poly(lactic-co-glycolic acid), poly(lactide-co-glycolide) (PLGA), polyphosphazene, polyiminocarbonate, polyphosphate, polyanhydride, polyorthoester, polycaprolactone, polyhydroxyvalerate, polyhydroxybutyrate, polyamino acid and combinations thereof.

[0018] In the poly(lactide-co-glycolide), a monomer ratio of lactide:glycolide may be 60:40 to 90:10.

[0019] The inflammatory disease may be osteoarthritis.

[0020] The microparticles can release 35% to 80% of the total weight of CBD after 18 hours under the following release test conditions:

[0021] [Release test conditions]

[0022] The dissolution test solution was prepared as follows: Tween 20 was mixed into 1 L of 10 mM PBS buffer (pH 7.4) to a concentration of 0.05% (w / v) to prepare a mixed solution, and then 0.02% (w / v) sodium azide and 0.0625% (w / v) sodium ascorbate were mixed into the mixed solution.

[0023] 100 mL of the dissolution test solution was injected into the injection vial, and the microparticles were added. At this time, the total weight of CBD in the microparticles was 15 mg. After the injection vial was completely sealed, the test was carried out at 120 rpm while maintaining 50°C. 18 hours after the start of dissolution, the injection vial was taken out, left to stand for 3 minutes, and then 1 mL of the test solution was accurately extracted with a syringe, centrifuged at 3000 rpm for 3 minutes, and the supernatant was taken as the test solution to confirm the release of CBD.

[0024] According to another embodiment of the present invention, a method for preparing a sustained-release injection composition for treating or improving inflammatory diseases may include: an oil phase solution preparation step, dissolving cannabidiol (CBD) and a biodegradable polymer in an organic solvent to prepare an oil phase solution; an aqueous phase solution preparation step, dissolving a surfactant in water to prepare an aqueous phase solution; and an emulsion formation step, mixing the oil phase solution and the aqueous phase solution to form an emulsion.

[0025] The prepared emulsion can be further subjected to: a residual solvent removal step, in which the prepared emulsion is collected into an aqueous phase solution to remove the residual solvent; a microparticle preparation step, in which the emulsion from which the residual solvent has been removed is washed and freeze-dried to prepare microparticles; and a mixing step, in which the freeze-dried microparticles are mixed with water for injection.

[0026] The oil phase solution and the water phase solution are injected into each microchannel to make them flow, and an emulsion containing cannabidiol and a sustained-release agent can be formed at the point where the flow of the oil phase solution and the flow of the water phase solution intersect each other.

[0027] The oil phase solution may further include butylated hydroxytoluene (BHT).

[0028] Effects of the Invention

[0029] The present invention utilizes microparticles containing cannabidiol to continuously release cannabidiol for more than one month, thereby being able to exhibit therapeutic or preventive effects on inflammatory diseases.

[0030] Furthermore, since cannabidiol has a wide safety range when exposed to the whole body, by using a sustained-release injection composition containing the cannabidiol, the side effects caused by long-term use are reduced, and a single injection can show a therapeutic or preventive effect on inflammatory diseases for more than one month, thereby significantly improving the convenience of medication. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 1 is a CBD release test result of microparticles containing CBD according to an embodiment of the present invention.

[0032] Figure 2 The pharmacokinetic experimental results of microparticles containing CBD in rats according to one embodiment of the present invention are shown in FIG.

[0033] Figure 3 1 is a measurement result of body weight change in an osteoarthritis model to which microparticles containing CBD were administered according to one embodiment of the present invention.

[0034] Figure 4 1 is a measurement result of lower limb weight load in an osteoarthritis model to which microparticles containing CBD were administered according to one embodiment of the present invention.

[0035] Figure 5 This is the result of a foot avoidance threshold test in an osteoarthritis model before administration of microparticles containing CBD according to one embodiment of the present invention.

[0036] Figure 6 The figure is the result of the foot avoidance threshold test in the osteoarthritis model 2 hours after administration of microparticles containing CBD according to one embodiment of the present invention.

[0037] Figure 7 This is the result of the foot avoidance threshold test in an osteoarthritis model 4 days after administration of microparticles containing CBD according to one embodiment of the present invention.

[0038] Figure 8 This is the result of the foot avoidance threshold test in an osteoarthritis model 7 days after administration of microparticles containing CBD according to one embodiment of the present invention.

[0039] Fig. 9 This is the result of the foot avoidance threshold test in an osteoarthritis model 11 days after administration of microparticles containing CBD according to one embodiment of the present invention.

[0040] Fig.10 This is the result of a foot avoidance threshold test in an osteoarthritis model 14 days after administration of microparticles containing CBD according to one embodiment of the present invention.

[0041] Fig.11This is the result of the foot avoidance threshold test in an osteoarthritis model 21 days after administration of microparticles containing CBD according to one embodiment of the present invention.

[0042] Fig.12 This is the result of the foot avoidance threshold test in an osteoarthritis model 28 days after administration of microparticles containing CBD according to one embodiment of the present invention.

[0043] Fig.13 is a stability evaluation result of microparticles containing CBD according to one embodiment of the present invention. DETAILED DESCRIPTION

[0044] The present invention relates to a sustained-release injection composition for treating or preventing inflammatory diseases, comprising microparticles for continuously releasing cannabidiol (CBD) for more than one month, wherein the microparticles contain cannabidiol and a biodegradable polymer. Specific embodiments

[0046] Hereinafter, in order to enable those skilled in the art to easily implement the present invention, embodiments of the present invention will be described in detail. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein.

[0047] The cannabidiol (CBD) in the present invention is a physiologically active substance isolated from cannabis, which can be directly extracted from cannabis, or extracted CBD can be used, or it can be prepared by chemical synthesis, and its salts are all included, not limited to the above examples.

[0048] Hemp, also known as hemp, has traditionally been used as clothing material. More specifically, hemp fibers are used for fabrics, mosquito nets, ropes, fishing nets, papermaking raw materials, etc. Hemp fruits are pressed for oil and can be eaten or used in lamp oil, soap, varnish, paint, etc. Hemp residue is used as feed and fertilizer.

[0049] One of the representative ingredients of cannabis is delta-9-tetrahydrocannabinol (THC), a psychoactive chemical derived from Cannabis sativa or Cannabis indica plants, commonly known as marijuana, which is designated as an illegal drug for management in some countries. In countries where it is listed as an illegal drug, custody and handling are considered illegal without special permission. Another representative ingredient is cannabidiol (CBD), which is considered to have no psychoactive effect, unlike THC. Therefore, in recent years, various studies have been conducted on its use in the fields of medicines and cosmetics.

[0050] The World Health Organization (WHO) released a report stating that cannabidiol (CBD) oil made from cannabis extract is effective in treating epilepsy, Alzheimer's disease (dementia) and other diseases.

[0051] The World Anti-Doping Agency released the 2018 International Standard for the Prohibited List and began to exclude CBD from the banned drugs. CBD is widely used by athletes to treat pain. Although other substances obtained from cannabis plants such as hashish and marijuana are prohibited, CBD oil for medical purposes is allowed.

[0052] As mentioned above, many countries have allowed the use of CBD for medical purposes.

[0053] However, as mentioned above, when CBD is used for medical purposes, it is usually used in the form of CBD oil, so in order to treat or prevent specific diseases, it needs to be taken continuously for a long time. At present, there is no dosage form that improves the convenience of taking.

[0054] Therefore, the present invention provides an injectable composition for use in treating or preventing inflammatory diseases using CBD, and is characterized in that it contains microparticles that can continuously release CBD for more than one month through a single injection.

[0055] The microparticles are characterized by containing CBD and a biodegradable polymer.

[0056] Therefore, when the microparticles of the present invention are used as injections, a sustained CBD administration effect can be exhibited by a single administration. Specifically, the CBD concentration in the blood can be maintained above the effective concentration for more than one month by a single administration, the CBD concentration in the blood can be maintained above the effective concentration for several months by a single administration, the CBD concentration in the blood can be maintained above the effective concentration for three months by a single administration, the CBD concentration in the blood can be maintained above the effective concentration for six months by a single administration, and the CBD concentration in the blood can be maintained above the effective concentration for twelve months by a single administration.

[0057] In other words, the sustained CBD release effect can be shown for more than one month, more than three months, more than six months, or more than twelve months.

[0058] The sustained CBD release effect as described above may be affected by the degradation rate of the sustained-release agent, i.e., the biodegradable polymer.

[0059] The biodegradable polymer can be selected from the group consisting of polylactic acid, polylactide, poly(lactic acid-co-glycolic acid), poly(lactide-co-glycolide) (PLGA), polyphosphazene, polyiminocarbonate, polyphosphate, polyanhydride, polyorthoester, polycaprolactone, polyhydroxyvalerate, polyhydroxybutyrate, polyamino acid and combinations thereof.

[0060] As an example of the above, the molar ratio of glycolide to lactide in poly(lactide-co-glycolide) may be about 40:60 to about 90:10, about 40:60 to about 85:15, about 40:60 to about 80:20, about 40:60 to about 75:25, about 45:55 to about 90:10, about 45:55 to about 80:20, but it is not limited to the above examples. Preferably, the molar ratio of glycolide to lactide in poly(lactide-co-glycolide) may be about 75:25 or about 50:50. When poly(lactide-co-glycolide) having the above-mentioned molar ratio range of glycolide to lactide is used as a sustained-release agent, CBD can be continuously released in vivo for more than one month.

[0061] The biodegradable polymer may include more than one polylactide and more than one poly(lactide-co-glycolide). In the present invention, the biodegradable polymer may include, for example, a combination of two polylactides, one polylactide and one poly(lactide-co-glycolide), two poly(lactide-co-glycolide), three polylactides, two polylactides and one poly(lactide-co-glycolide), one polylactide and two poly(lactide-co-glycolide), and the like, and in particular, may include one polylactide and one poly(lactide-co-glycolide), or two poly(lactide-co-glycolide), but is not limited thereto.

[0062] The biodegradable polymer may include two or more poly(lactide-co-glycolide). The biodegradable polymer may include one poly(lactide-co-glycolide) and one polylactide, but is not limited to the above examples, and any combination that can continuously release CBD in vivo for more than one month can be used without restriction.

[0063] The microparticles may contain CBD and biodegradable polymers in a weight ratio of 1:3 to 1:10, in a weight ratio of 1:3.5 to 1:9.5, and in a weight ratio of 1:4 to 1:9. The microparticles contained in the above range can be used as a sustained-release injection for sustained release of CBD within an injectable dose range when used as an injection composition. In other words, if the CBD relative to the biodegradable polymer is included at a value lower than the above range, in order to meet the total dose requirement of CBD administered as an injection, the amount of the injection composition will increase, which will lead to the problem of exceeding the injection dose that can be administered to humans once. In addition, if the CBD relative to the biodegradable polymer is included at a value exceeding the above range, it is difficult to prepare microparticles in which CBD is uniformly distributed.

[0064] The inflammatory disease may be osteoarthritis. It can also be used for other inflammatory diseases, and any disease for which CBD has been shown to have a therapeutic or improving effect on inflammatory diseases can be applied without limitation, but it is preferably used for the purpose of treating or improving osteoarthritis.

[0065] The microparticles can release 35% to 80% of the total weight of CBD after 18 hours under the following release test conditions:

[0066] [Release test conditions]

[0067] The dissolution test solution was prepared as follows: Tween 20 was mixed in 1 L of 10 mM PBS buffer (pH 7.4) to a concentration of 0.05% (w / v) to prepare a mixed solution, and then 0.02% (w / v) of sodium azide and 0.0625% (w / v) of sodium ascorbate were mixed in the mixed solution.

[0068] 100 mL of the dissolution test solution was injected into the injection vial, and the microparticles were added. At this time, the total weight of CBD in the microparticles was 15 mg. After the injection vial was completely sealed, the test was carried out at 120 rpm while maintaining 50°C. 18 hours after the start of dissolution, the injection vial was taken out, left to stand for 3 minutes, and then 1 mL of the test solution was accurately extracted with a syringe, centrifuged at 3000 rpm for 3 minutes, and the supernatant was taken as the test solution to confirm the release of CBD.

[0069] The above-mentioned CBD release test is a test that uses a dissolution test solution to confirm the sustained release of CBD. As mentioned above, in order for the microparticles containing CBD to continuously release CBD for more than one month, under the above release test conditions, at a time point after 18 hours, 35% to 80% should be released, 40% to 78% should be released, and 50% to 75% should be released relative to the total weight of CBD. When CBD is released within the above range, the microparticles can show the effect of continuously releasing CBD for more than one month. In other words, if the release is below the above range value, the initial CBD release effect is too weak to show the treatment or improvement effect of CBD on inflammatory diseases; if the release exceeds the above range value, the amount of CBD released is too much, and the sustained CBD release effect for more than one month cannot be achieved.

[0070] Furthermore, the microparticles of the present invention may further contain butylated hydroxytoluene (BHT).

[0071] The above-mentioned CBD may be a compound represented by the following chemical formula.

[0072] [Chemical formula]

[0073]

[0074] The compound CBD represented by the above chemical formula is a stable compound that does not oxidize when in contact with oxygen in the air. However, when it is formulated into microparticles containing a biodegradable polymer as described in the present invention, there is a problem of CBD oxidation.

[0075] As mentioned above, when CBD oxidizes, its color may change to yellow as mentioned earlier, and it may become less effective due to changes in the compound's structure caused by oxidation.

[0076] The butylated hydroxytoluene (BHT) can be included in the microparticles to prevent oxidation of CBD. In other words, in the microparticles, BHT can replace CBD for oxidation, thereby preventing oxidation of cannabidiol.

[0077] The microparticles of the present invention are spherical microparticles as described below, and when the solvent is completely removed through the preparation process, cannabidiol (CBD), the sustained-release agent and butylated hydroxytoluene (BHT) can be evenly distributed.

[0078] At this time, in an environment where the microparticles are in contact with oxygen, CBD will undergo an oxidation reaction, but since the oxidation reaction of butylated hydroxytoluene (BHT) occurs preferentially compared to the CBD, the oxidation reaction of CBD can be prevented.

[0079] Although the oxidation reaction of CBD can be prevented by the action as described above, this antioxidant effect is not produced by antioxidants other than the butylated hydroxytoluene (BHT). Only when butylated hydroxytoluene (BHT) is included, butylated hydroxytoluene (BHT) is oxidized preferentially to CBD, thereby exhibiting the antioxidant effect of CBD.

[0080] Relative to the total weight of cannabidiol and biodegradable polymer, the content of butylated hydroxytoluene (BHT) can be 0.11% to 9.9% by weight, 0.2% to 9% by weight, 0.3% to 8% by weight, or 0.5% to 5% by weight. Within the above range, spherical microparticles can be prepared, maintaining an appropriate content range of CBD, so as to be used as a sustained-release preparation, and stability can be improved by the antioxidant effect of CBD based on butylated hydroxytoluene (BHT).

[0081] According to another embodiment of the present invention, a sustained-release injection composition containing cannabidiol may include microparticles and a suspending agent, wherein the microparticles contain the cannabidiol.

[0082] The composition for injection may include a suspending solvent, and the suspending solvent includes an isotonic agent, a suspending agent and a solvent.

[0083] More specifically, the isotonic agent can be selected from the group consisting of D-mannitol, maltitol, sorbitol, lactitol, xylitol, sodium chloride and a mixture thereof, preferably D-mannitol, but not limited to the above examples.

[0084] The suspending agent may be selected from the group consisting of sodium carboxymethylcellulose, polysorbate 80, starch, starch derivatives, polyols, chitosan, chitosan derivatives, cellulose, cellulose derivatives, collagen, gelatin, hyaluronic acid (HA), alginic acid, algin, pectin, carrageenan, chondroitin, chondroitin sulfate, dextran, dextran sulfate, polylysine, titin, fibrin, agarose, fluran, xanthan gum, and a mixture thereof, preferably sodium carboxymethylcellulose and polysorbate 80, but not limited to the above examples.

[0085] The solvent may be injection water, and any solvent that can be used as injection water may be used without limitation.

[0086] According to another embodiment of the present invention, a method for preparing microparticles containing cannabidiol (CBD) may include: an oil phase solution preparation step, dissolving CBD and a biodegradable polymer in an organic solvent to prepare an oil phase solution; an aqueous phase solution preparation step, dissolving a surfactant in water to prepare an aqueous phase solution; an emulsion forming step, and mixing the oil phase solution and the aqueous phase solution to form an emulsion.

[0087] The preparation method can form an emulsion using an oil phase solution and an aqueous phase solution, wherein the oil phase solution is prepared by dissolving CBD, a sustained-release agent, and an antioxidant in an organic solvent, and the aqueous phase solution contains a surfactant.

[0088] As described below, the present invention prepares the emulsion by a microfluidic method using a microchannel, but is not limited to the preparation method, and microparticle preparation methods such as solvent evaporation method and membrane emulsification method can be applied.

[0089] The organic solvent can use a solvent that can completely dissolve CBD and biodegradable polymers. Specifically, any one of the group consisting of chloroform, ethyl chloride, dichloroethane, dichloromethane, trichloroethane, methylene chloride, methanol and mixtures thereof can be selected, preferably, the group consisting of methylene chloride, methanol and mixtures thereof can be selected. The organic solvent is not limited to the above examples, and any organic solvent that can completely dissolve CBD and biodegradable polymers that can be easily selected by those skilled in the art can be used.

[0090] The CBD and the biodegradable polymer in the organic solvent may be contained in a weight ratio of 1:3 to 1:10, a weight ratio of 1:3.5 to 1:9.5, or a weight ratio of 1:4 to 1:9.

[0091] The content of the biodegradable polymer in the oil phase solution is 10 to 20% by weight, preferably 12 to 18% by weight, and more preferably 15% by weight, but is not limited to the above examples. If the content is lower than the above range, the emulsion cannot be prepared; if the content exceeds the above range, the viscosity of the oil phase solution is too high, making it difficult to prepare the emulsion.

[0092] The biodegradable polymer can be selected from polylactic acid, polylactide, poly(lactic acid-co-glycolic acid), poly(lactide-co-glycolide) (PLGA), polyphosphazene, polyiminocarbonate, polyphosphate, polyanhydride, polyorthoester, polycaprolactone, polyhydroxyvalerate, polyhydroxybutyrate, polyamino acid and combinations thereof, preferably poly(lactide-co-glycolide) (PLGA) and / or polylactide (PLA), but is not limited to the above examples.

[0093] The oil phase solution may further contain butylated hydroxytoluene (BHT). As mentioned above, the butylated hydroxytoluene (BHT) may be contained in the oil phase solution, and thus contained in the microparticles, thereby preventing CBD from being oxidized. Relative to the total weight of CBD and the biodegradable polymer, the butylated hydroxytoluene (BHT) may be contained in an amount of 0.11% to 9.9% by weight, 0.2% to 9% by weight, 0.3% to 8% by weight, and 0.5% to 5% by weight.

[0094] The aqueous phase solution may contain the surfactant in an amount of 0.1 to 1.0 wt %, 0.2 to 0.5 wt %, or 0.25 wt %, and the rest may be water.

[0095] The surfactant may be selected from methylcellulose, polyvinyl pyrrolidone, lecithin, gelatin, polyvinyl alcohol, sorbitan monooleate (e.g., Span 80). TM80), polyoxyethylene sorbitan fatty acid esters (e.g., Tween 80 (Tween TM 80), etc.), any one of the group consisting of polyoxyethylene castor oil derivatives, sodium lauryl sulfate, sodium stearate, esteramines, linear diamines, fatty amines and mixtures thereof, preferably, polyvinyl alcohol, but not limited to the above examples, any surfactant that can prepare a completely spherical emulsion can be used.

[0096] As mentioned above, after the oil phase solution and the water phase solution are prepared separately, there is no limitation on the method of using them to prepare the emulsion.

[0097] However, in the present invention, the method of preparing microparticles by microfluidics will be emphasized.

[0098] The microchip used for the microfluidic method can be formed on a wafer or a glass substrate. A microchannel is formed on the microchip, and more specifically, the microchannel includes a channel for the flow of an oil phase solution, a channel for the flow of an aqueous phase solution, and a transport channel. The channel for the flow of the oil phase solution and the channel for the flow of the aqueous phase solution are formed in a manner that they intersect each other at one point, and one end of the transport channel can be connected to the portion where the two channels are joined.

[0099] The channel for the oil phase solution to flow and the channel for the water phase solution to flow may be connected to injection parts for injecting the oil phase solution and the water phase solution respectively, and one end of the transport channel may be connected to a recovery part for recovering the solution containing the emulsion.

[0100] The microchannel may be formed on a material selected from the group consisting of a glass substrate, a silicon wafer, or a polymer film, but the material is not limited to the above examples, and any material that can form a microchannel may be used.

[0101] The polymer film may be selected from the group consisting of polyimide, polyethylene, fluorinated ethylene propylene, polypropylene, polyethylene terephthalate, polyethylene naphthalate, polysulfone and a mixture thereof, but is not limited to the above examples.

[0102] As an example, aluminum is deposited on a silicon wafer using an e-beam evaporator, and a photoresist is patterned on the aluminum using photolithography. Subsequently, the aluminum is etched using the photoresist as a mask, and after removing the photoresist, the silicon is etched using deep reactive ion etching (DRIE) using the aluminum as a mask. After removing the aluminum, a glass anode is bonded to the wafer and sealed to prepare the above-mentioned microchannel.

[0103] The average diameter of the microchannel is 60 to 150 μm, preferably 80 to 100 μm, but not limited to the example. If a microchannel with an average diameter smaller than the above range is used, an emulsion with a diameter too small may be prepared, thereby affecting the effective release of the drug and the absorption in vivo.

[0104] Furthermore, if the average diameter of the microchannel exceeds the above range, the average size of the prepared microparticles will exceed 120 μm, which may increase the foreign body sensation and pain when administered as an injection, and as the diameter of the microchannel increases, the particle size distribution of the prepared particles will also increase, making it difficult to prepare microparticles of uniform size.

[0105] Furthermore, the average diameter of the microchannel is closely related to the average diameter of the particles, and is also closely related to the flow ratio (μl / min) of the oil phase solution and the water phase solution.

[0106] Moreover, the cross-sectional width (w) and cross-sectional height (d) of the microchannel are closely related to the average diameter (d') of the prepared microparticles. The ratio of the width (w) of the microchannel cross section to the average diameter (d') of the microparticles is in the range of 0.7 to 1.3, and the ratio of the height (d) of the microchannel cross section to the average diameter (d') of the microparticles is in the range of 0.7 to 1.3.

[0107] In other words, after the average diameter (d') of the microparticles to be prepared is determined, based on this, the length of the width (w) and height (d) of the microchannel cross section is set to a ratio range of 0.7 to 1.3 of d', so that microparticles of the desired size can be prepared.

[0108] To prepare microparticles using the microchip, an oil phase solution may be injected into the channel where the oil phase solution flows, and an aqueous phase solution may be injected into the channel where the aqueous phase solution flows, thereby forming an emulsion at the junction of the two channels.

[0109] When the oil phase solution and the water phase solution are respectively injected into the microchannel, the flow ratio of the oil phase solution to the water phase solution can be 1:10 to 1:50, 1:15 to 1:40, 1:15 to 1:30, or 1:15 to 1:25. As described above, microparticles with uniform diameters can be prepared by adjusting the flow ratio of the oil phase solution to the water phase solution.

[0110] The emulsion formed in the above-mentioned joined part can be collected by the previously prepared aqueous phase solution, that is, a mixed solution of surfactant and water. The prepared aqueous phase solution can not only be injected into the microchannel for forming the emulsion, but also filled in the water tank for preventing the collected emulsion from agglomerating.

[0111] The emulsion collected in the water tank can remove the residual organic solvent. The step of removing the residual organic solvent can be carried out by stirring under certain temperature conditions and stirring speeds, thereby evaporating and removing the residual organic solvent present in the emulsion. At this time, the stirring conditions are: a first stirring at 15 to 20° C. for 50 to 70 minutes; a second stirring at 20 to 40° C. for 50 to 70 minutes; and a third stirring at 40 to 60° C. for 1 to 3 hours.

[0112] The stirring speed is kept consistent from the first to the third stirring stages, and the stirring speed is 300 to 500 rpm, and can be 400 rpm.

[0113] As described above, the stirring step is characterized in that the temperature is gradually increased as the stirring step proceeds, and the evaporation rate of the organic solvent in the emulsion can be adjusted by increasing the temperature stepwise.

[0114] The temperature of the oil phase solution and the water phase solution when flowing through the microchannel is also 5 to 20°C, preferably 10°C. In other words, after flowing through the microchannel and forming a junction to generate an emulsion, the temperature of the collected emulsion is maintained at a low temperature of 5 to 20°C until the first stirring. Only by maintaining a low temperature during the emulsion preparation process can spherical particles be prepared and maintained. In other words, if it is not under low temperature conditions, it is difficult to prepare uniform spherical particles.

[0115] Subsequently, in the second stirring process, the temperature is gradually increased and the stirring time is extended, so that the organic solvent present in the emulsion is slowly moved to the surface, so that as the organic solvent evaporates on the surface, the influence on the emulsion properties can be minimized. In other words, if the organic solvent evaporates too fast, the surface of the microparticles finally prepared may be not smooth and form pores. In order to avoid this problem, as described above, by gradually increasing the temperature and increasing the stirring process time, the evaporation rate of the organic solvent can be adjusted, and the surface properties of the microparticles prepared can be controlled.

[0116] The third stirring step is to raise the temperature of the external water phase to a temperature close to the boiling point of the organic solvent and stir it after the organic solvent in the emulsion is extracted by the external water phase, thereby removing the saturated organic solvent from the water phase and making it easier to remove the residual organic solvent in the emulsion.

[0117] Finally, the microparticles from which the residual organic solvent has been removed are subjected to a step of washing and drying, that is, the microparticles from which the surface organic solvent has been removed by stirring are washed multiple times with sterile filtered pure water to remove the residual surfactant in the microparticles, and then freeze-dried.

[0118] The finally prepared microparticles have the property that CBD is uniformly distributed in the microparticles composed of spherical biodegradable polymers, and may contain CBD and the biodegradable polymer in a weight ratio of 1:3 to 1:10.

[0119] Furthermore, as mentioned above, in addition to CBD, butylated hydroxytoluene (BHT) is evenly distributed in the finally prepared microparticles. When in contact with oxygen in the air, BHT will be preferentially oxidized, thereby preventing the oxidation of CBD.

[0120] The weight ratio of CBD and biodegradable polymer contained in the microparticles is the same as the weight ratio in the oil phase solution when preparing the emulsion. This is because after forming the emulsion through the microchannel and removing all organic solvents in the emulsion, microparticles containing CBD and biodegradable polymer in the same weight ratio as in the oil phase solution can be prepared.

[0121] The average diameter of the microparticles may be 30 to 70 μm, 30 to 65 μm, or 30 to 60 μm. Furthermore, the standard deviation of the average diameter may be 1 to 30 μm, 1 to 20 μm, 1 to 10 μm, or 1 to 7 μm. Within the above diameter range, it can be confirmed that uniform particles can be prepared, and when the uniform particles are used as sustained-release injections, the foreign body sensation can be reduced to improve the convenience of medication, and the initial excessive release can be prevented during in vivo injection, showing a sustained CBD release effect.

[0122] Preparation Example

[0123] Preparation of microparticles

[0124] 1) Preparation of oil phase solution

[0125] Cannabidiol, a biodegradable polymer (one or more of a lactide-co-glycolide copolymer and a lactide copolymer) and butylated hydroxytoluene (BHT) are dissolved in methylene chloride to prepare an oil phase solution. At this time, the biodegradable polymer in the oil phase solution is contained at 15% by weight, and the weight ratio of cannabidiol to the biodegradable polymer is 1:3 to 1:10.

[0126] 2) Preparation of aqueous solution

[0127] Polyvinyl alcohol was dissolved in water to prepare an aqueous phase solution, wherein the content of polyvinyl alcohol in the aqueous phase solution was 0.25 wt %.

[0128] 3) Preparation of microparticles

[0129] The oil phase solution and the aqueous phase solution were injected into the microchannel formed on the silicon wafer to prepare microparticles. At this time, the flow ratio of the oil phase to the aqueous phase was 1:20, and the temperature condition was maintained at 10.0°C. The generated microparticles were collected in a water tank filled with the aqueous phase solution and stirred at 10.0°C for 1 hour, 30.0°C for 1 hour, and 45.0°C for 2 hours, with a stirring speed of 400rpm.

[0130] 4) Washing and collection

[0131] After the stirring is completed, the microparticles are washed with sterile filtered purified water and collected in powder form after freeze drying.

[0132] The microparticles were prepared according to the following method using the ingredients and contents listed in Table 1 below.

[0133] Table 1

[0134]

[0135] Experimental Example 1 CBD Release Experiment 1) Preparation of standard solution

[0136] Take about 15 mg of cannabidiol standard and put it into a 100 mL volumetric flask. Add about 50 mL of diluent (acetonitrile) and perform ultrasonic treatment for 10 minutes. After sufficient cooling, adjust to the scale line with the test solution and filter with a 0.45 μm filter (water-soluble PTFE) as a standard solution (concentration: 0.15 mg / mL).

[0137] 2) Preparation of dissolution test solution

[0138] Tween 20 was mixed into 1 L of 10 mM PBS buffer (pH 7.4) to a concentration of 0.05% (w / v), and then 0.02% (w / v) of sodium azide and 0.0625% (w / v) of sodium ascorbate were mixed into the mixed solution to prepare a dissolution test solution.

[0139] 3) Preparation of test solution

[0140] Weigh each sample accurately to a main component mass of 15 mg, place it in an injection vial, accurately add 100 mL of dissolution test solution and seal it. Perform the test at a constant temperature of about 50°C and oscillate at a speed of 120 rpm. Place the injection vial in a horizontal position. At 2 hours, 18 hours, and 72 hours after the start of dissolution, take out the injection vial containing the sample, let it stand for 3 minutes to allow the sample to settle, then accurately draw 1 mL of test solution with a syringe, centrifuge at 3000 rpm for 3 minutes, and take the supernatant as the test solution.

[0141] 4) HPLC conditions

[0142] -Detector: UV (220nm)

[0143] - Column: ODS column (4.6*150mm, 5μm)

[0144] -Flow rate: 1.0mL / min

[0145] -Mobile phase: methanol: purified water = 85:15 (Isocratic method)

[0146] - Calculation formula: main peak area of ​​test solution × amount of standard sample × purity of standard sample (%) × dilution factor / peak area of ​​main component in standard solution × corresponding amount of cannabidiol in test solution (mg)

[0147] The results of the CBD dissolution test under the above experimental conditions are as follows Figure 1 As shown in Table 2:

[0148] Table 2

[0149] 2 hours 18 hours 72 hours Comparative Example 1 44.5±0.3 82.6±2.4 97.9±0.2 Comparative Example 2 3.0±3.9 32.2±0.1 47.9±0.1 Example 1 31.2±6.5 74.8±0.1 88.6±0.5 Example 2 29.6±0.4 65.2±3.8 78.2±0.7 Example 3 22.9±10.6 56.4±2.5 77.4±1.8 Example 4 28.9±0.6 66.7±1.2 85.0±0.3 Example 5 12.1±8.2 57.8±1.0 72.3±0.2

[0150] According to the above experimental results, it can be confirmed that Comparative Example 1 releases 44.5% of CBD in 2 hours, 82.6% in 18 hours, and 97.9% in 72 hours, that is, almost completely released. This indicates that the initial CBD release is large and the long-term continuous CBD release effect cannot be achieved.

[0151] In addition, Comparative Example 2 released 47.9% at 72 hours. Although it can show the effect of long-term sustained release, it only released 3% of CBD at 2 hours. The initial release amount was too low, and there was a problem that CBD had little effect on the treatment and improvement of inflammatory diseases.

[0152] In contrast, in the case of Examples 1 to 5, an appropriate degree of CBD release effect was exhibited at 2 hours, and the release was not completely completed at 72 hours, thus confirming that a long-term sustained drug release effect was exhibited.

[0153] Experimental Example 2

[0154] Pharmacokinetic study in rats

[0155] The experimental conditions used to conduct the pharmacokinetic studies in rats were as follows:

[0156] Table 3

[0157]

[0158] As a control group, CBD isolate solution was used. The experimental animals were male SD rats aged 6 weeks after birth, purchased from KoaTech, South Korea, and the experiment was conducted after one week of adaptation. As experimental feed, ordinary feed for experimental animals was provided, and during the feeding process, the animals could freely take in water and feed.

[0159] General symptoms

[0160] During the experiment, all animals were observed twice a day (morning and afternoon) for symptom changes, and their body weights were measured at the time of introduction and grouping.

[0161] Blood sampling

[0162] At 14 time points (0 time) before and 4 hours, 8 hours, 24 hours, 3 days, 5 days, 1 week, 2 weeks, 4 weeks, 6 weeks, 8 weeks, and 12 weeks after the test substance was administered to acclimated rats by subcutaneous injection (SC), 500 to 1000 μL of blood was collected from the jugular vein, and then immediately centrifuged at 1000 g for 10 minutes to separate the serum and store it in a frozen state at -70°C.

[0163] Through the above experiments, the pharmacokinetic (pK) determination results of the microparticles of Examples 1 to 5 are shown in Tables 4 and Figure 2 As shown:

[0164] Table 4

[0165]

[0166]

[0167] (Unit: μg / mL) Based on the above experimental results, it can be confirmed that Examples 1 to 5 of the present invention maintain the CBD concentration in the blood for at least 1 month. In other words, Example 1 was confirmed to be 0.036 μg / mL at 4 weeks, and Example 4 also showed 0.782 μg / mL.

[0168] Furthermore, in Examples 2 to 5, the CBD concentration in the blood could still be confirmed at 7 weeks, and thus it could be confirmed that a sustained CBD release effect was exhibited.

[0169] Experimental Example 3

[0170] Osteoarthritis (OA) Treatment Trials

[0171] After the rats were acclimated, they were divided into groups and the area around the left knee joint was cleanly depilated. The osteoarthritis-inducing substance monosodium iodoacetate (MIA, Sigma, USA) was adjusted to a concentration of 60 mg / mL with 0.9% sterile saline and stained with BD Ultra-Fine. TM II insulin syringe (Insulin Syringe) was used to administer 50 μL of each into the joint cavity. The right knee joint was administered with an equal amount of 0.9% sterile saline (saline). Two weeks after administration of monosodium iodoacetate (MIA), the test substances shown in Table 5 below were injected into the joint cavity, and the therapeutic effects were evaluated:

[0172] Table 5

[0173]

[0174]

[0175] Dosage

[0176] In the CBD solution and Example 2 to Example 4 groups, 14 days after the administration of MIA, the drug was administered into the left knee joint cavity, and the same amount of 0.9% sterile saline was administered only into the right knee joint. The same amount of 0.9% saline was also administered to the normal control group and the positive control group.

[0177] Dose level selection

[0178] - CBD solution: Based on existing literature, the effective concentration in the MIA-OA model was selected as 300 μg / joint.

[0179] -Example 2 to Example 3: Maximum feasible dose level (1-2 mg / joint) when considering the volume of microspheres and the amount that can be administered into the rat joint cavity (30 μL).

[0180] Weight measurement results

[0181] The body weight measurements during the trial were as follows: Figure 3 As shown. The positive control group had the lowest weight gain, while in the CBD solution and Examples 2 to 4 administration groups, there was a tendency for increased weight gain compared to the positive control group. In the Example 3 and Example 4 administration groups, the weight gain was statistically significantly increased compared to the positive control group. When osteoarthritis (OA) is induced, it is generally observed that the amount of feed intake is reduced due to pain and walking disorders, resulting in a tendency to lose weight. However, when CBD was administered, it was confirmed that the weight gain increased compared to the positive control group due to reduced pain and improved feed intake activity, and this tendency was more obvious in Examples 3 and 4, especially.

[0182] Measuring lower limb weight load

[0183] The weight load of the lower limbs was measured as follows: before administration of MIA (0 day), 6 days and 13 days after administration of MIA (before injection of the test drug), and 2 hours, 4 hours, 3 days, 6 days, 10 days, 13 days, 20 days and 27 days after administration of the test drug, the left and right feet were measured respectively using an incapacitance tester (Ugo Basile, Italy). The weight (g) of the two feet was measured respectively when the abdomen of the rat (Rat) was not in contact with the sensor of the instrument. The measurement results were analyzed by the weight ratio (ratio) (%, left / right) measured by the left and right feet.

[0184] The weight (g) of both sides of the lower limbs was measured on each measurement day. In the case of induced arthritis, the weight load on the induced side (left side) was reduced. The weight load value was calculated based on the following formula:

[0185] Weight load value = normal lower limb weight (right side) / induced arthritis lower limb weight (left side)

[0186] Relative pain ratio (%) = (average weight load value of each group / average weight load value of normal control group) × 100

[0187] The test results are as follows Figure 4 As shown:

[0188] The positive control group showed a significant change in weight bearing compared to the normal control group, confirming the induction of arthritis. Two hours after administration, the CBD solution and Example 2 to 4 administration groups showed a tendency to significantly improve weight bearing compared to the positive control group.

[0189] The CBD solution administration group showed no significant difference compared with the PC control group 2 hours after administration. The Example 4 administration group showed a tendency to improve the weight load compared with the PC control group 14 days after administration, and showed statistically significant improvement compared with the control group at 14 days and 28 days. The Example 3 administration group showed a tendency to reduce pain compared with the PC control group 14 days and 21 days after administration, and significance was confirmed at 14 days.

[0190] Paw withdrawal threshold test

[0191] The avoidance reaction test was performed before administration of MIA (0 day), 7 days and 14 days after administration of MIA (before administration of test drug injection), and the foot avoidance reaction was evaluated after mechanical stimulation using an electronic von-Frey aesthesiometer (IITC Life science, USA) 2 hours, 4 hours, 4 days, 7 days, 11 days, 14 days, 21 days and 28 days after administration of test drug. In order to measure the foot avoidance reaction, rats were placed in a transparent acrylic container on a wire mesh and adapted to the new environment for about 20 minutes. The determination method is as follows: an electronic von-Frey filament is applied vertically to the sole of the foot to measure the threshold (threshold) (g force) that triggers a sharp avoidance reaction. The measurement results are analyzed with the g force ratio (left / right) measured by the left and right feet.

[0192] Paw withdrawal threshold ratio = Left threshold (induced side) / Right threshold (normal side). When arthritis is relieved, the left and right ratios tend to be equal (ratio 1).

[0193] The test results are as follows Figures 5 to 12 shown.

[0194] The threshold value of the positive control group (Positive control, PC) was significantly increased compared with the normal control group (Normal control). Two hours after administration, the CBD solution and the groups administered with Examples 2 to 4 all showed a significant improvement response compared with the positive control group (Positive control). Specifically, the CBD solution (CBD solution) administration group showed a tendency to lower the threshold value compared with the PC control group 2 hours after administration, and there was no significant difference compared with the control group afterwards. The Example 2 administration group also showed a tendency to continue to improve 4 days after administration, and was statistically significantly improved at 4 days, 21 days, and 28 days. The Example 4 administration group also showed a tendency to continue to improve 4 days after administration, and was statistically significantly improved at 4 days and 11 days. Finally, the Example 3 administration group also showed a tendency to continue to improve 4 days after administration, and was statistically significantly improved at 11 days, 14 days, and 21 days.

[0195] According to the results of the lower limb heavy load measurement test and the foot avoidance threshold test, it can be confirmed that the CBD solution (CBDsolution) administration group is effective in the initial administration, but there is no significant difference compared with the PC control group afterwards. In contrast, Examples 2 to 4 were confirmed to show a long-term sustained therapeutic effect compared with the PC control group, thereby confirming through experiments that it not only shows a therapeutic and improvement effect in the osteoarthritis model, but also has a long-term sustained therapeutic effect.

[0196] Experimental Example 4

[0197] Stability evaluation

[0198] Microparticles were prepared in the same manner as in Example 1 with the same composition as in Example 1 except that BHT was not included.

[0199] About 100 mg of microparticles were weighed and placed in a 5 mL glass vial and stored in a stability test chamber with or without a cap (closed / open). To evaluate the stability, the test was conducted under long-term conditions (temperature: 25 ± 2 ° C, relative humidity: 60 ± 5%), and the daily changes in properties were observed to confirm whether yellowing occurred. On the other hand, the encapsulation rate test was conducted on the initial sample and the sample after 6 days of storage to confirm whether the content changed.

[0200] The experimental results are as follows Fig.13 shown.

[0201] Immediately after the preparation of the microparticles, no color change was observed. After 6 days, regardless of whether the glass vial was capped or not, in the absence of BHT, a color change to yellow was observed.

[0202] On the other hand, as shown in Example 1, when 1% by weight of BHT was contained, no color change occurred, and it was confirmed that excellent stability was exhibited.

[0203] The preferred embodiments of the present invention are described in detail above, but the scope of the present invention is not limited thereto. Various modifications and improvements made by those skilled in the art based on the basic concept of the present invention, as long as they are defined in the claims, belong to the scope of the present invention.

[0204] Industrial Applicability

[0205] The invention relates to a sustained-release injection composition for treating or preventing inflammatory diseases and a preparation method thereof.

Claims

1. A sustained-release injection composition for treating or preventing inflammatory diseases, characterized in that: The invention relates to microparticles that continuously release cannabidiol for more than one month, wherein the microparticles contain cannabidiol and a biodegradable polymer.

2. The sustained-release injection composition for treating or preventing inflammatory diseases according to claim 1, characterized in that: The microparticles contain cannabidiol and biodegradable polymer in a weight ratio of 1:3 to 1:

10.

3. The sustained-release injection composition for treating or preventing inflammatory diseases according to claim 1, characterized in that: The biodegradable polymer is selected from the group consisting of polylactic acid, polylactide, poly(lactic acid-co-glycolic acid), poly(lactide-co-glycolide), polyphosphazene, polyiminocarbonate, polyphosphate, polyanhydride, polyorthoester, polycaprolactone, polyhydroxyvalerate, polyhydroxybutyrate, polyamino acid and combinations thereof.

4. The sustained-release injection composition for treating or preventing inflammatory diseases according to claim 3, characterized in that: In the poly(lactide-co-glycolide), the monomer ratio of lactide:glycolide is 40:60 to 90:

10.

5. The sustained-release injection composition for treating or preventing inflammatory diseases according to claim 1, characterized in that: The inflammatory disease is osteoarthritis.

6. The sustained-release injection composition for treating or preventing inflammatory diseases according to claim 1, characterized in that: The microparticles release 35% to 80% of the total weight of cannabidiol after 18 hours under the following release test conditions: [Release test conditions] The dissolution test solution was prepared as follows: Tween 20 was mixed at a concentration of 0.05% (w / v) in 1 L of 10 mM PBS buffer (pH 7.4) to prepare a mixed solution, and then 0.02% (w / v) sodium azide and 0.0625% (w / v) sodium ascorbate were mixed in the mixed solution; 100 mL of the dissolution test solution was injected into an injection bottle, and the microparticles were added. At this time, the total weight of cannabidiol in the microparticles was 15 mg. After the injection bottle was completely sealed, the test was carried out at 50°C and oscillated at 120 rpm. 18 hours after the start of dissolution, the injection bottle was taken out and allowed to stand for 3 minutes. Then, 1 mL of the test solution was accurately extracted using a syringe, and the solution was centrifuged at 3000 rpm for 3 minutes. The supernatant was taken as the test solution to confirm the release of cannabidiol.

7. A method for preparing a sustained-release injection composition for treating or improving inflammatory diseases, characterized in that: include: an oil phase solution preparation step, dissolving cannabidiol and a biodegradable polymer in an organic solvent to prepare an oil phase solution; an aqueous solution preparation step, dissolving a surfactant in water to prepare an aqueous solution; as well as The emulsion forming step is to mix the oil phase solution and the water phase solution to form an emulsion.

8. The method for preparing a sustained-release injection composition for treating or improving inflammatory diseases according to claim 7, characterized in that: Also includes: a residual solvent removal step, collecting the prepared emulsion into an aqueous phase solution to remove the residual solvent; a microparticle preparation step, washing and freeze-drying the emulsion from which the residual solvent has been removed to prepare microparticles; and The mixing step is to mix the freeze-dried microparticles with water for injection.

9. The method for preparing a sustained-release injection composition for treating or improving inflammatory diseases according to claim 7, characterized in that: The oil phase solution and the water phase solution are injected into each microchannel to make them flow, and an emulsion containing cannabidiol and a sustained-release agent is formed at a point where the flow of the oil phase solution and the flow of the water phase solution intersect each other.

10. The method for preparing a sustained-release injection composition for treating or improving inflammatory diseases according to claim 7, characterized in that: The oil phase solution further comprises butylated hydroxytoluene.

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