CN114903916B - Oral fullerene structure and preparation and application thereof in preventing and treating rheumatoid arthritis - Google Patents

Abstract

The invention relates to application of a fullerene structure in preparing a pharmaceutical composition for preventing and/or treating rheumatoid arthritis. The fullerene pharmaceutical composition can inhibit the inflammatory degree of mice with rheumatoid arthritis and relieve the damage of articular cartilage and bones caused by the rheumatoid arthritis.

Description

Oral fullerene structure and preparation, and its application in preventing and treating rheumatoid arthritis

Technical field

The present invention relates to the field of biomedicine, specifically to oral fullerene structures and preparations, and their application in preventing and/or treating rheumatoid arthritis.

Background technique

Rheumatoid arthritis (RA) is a chronic autoimmune joint disease that affects approximately 1% of the world's population, mostly affecting women and the elderly. Patients with RA are also prone to other complications, such as heart disease. Vascular disease, etc. These complications can also make it more difficult to treat rheumatoid arthritis. Although rheumatoid arthritis is a disease caused by an abnormal autoimmune response, its underlying pathogenic mechanisms are not fully understood and are currently thought to be triggered by a complex interaction of genetic and environmental factors. The disease involves not only small joints of the hands and feet but is also common in larger joints and is characterized by synovitis-related synovial hyperplasia accompanied by neovascularization, infiltration of lymphocytes and macrophages into the synovial tissue, and It can also cause damage to articular cartilage and bone.

To date, a variety of drugs have been licensed for use in the treatment of rheumatoid arthritis. Rheumatoid arthritis is initially treated with disease-modifying antirheumatic drugs (DMARDs), such as nonsteroidal anti-inflammatory drugs and glucocorticoids. A better DMARD is methotrexate, which can be used in combination with other drugs of this type. But today's treatment effects are still unsatisfactory. When arthritis is severe, these drugs cannot provide good therapeutic effects, and DMARDs can also produce toxic effects. Therefore, there is still an urgent need to find new anti-rheumatoid arthritis drugs with good therapeutic effects and less side effects.

Fullerene spherical molecules are similar to a sphere. Common fullerenes include C60, C70, C84, etc. Fullerene has a highly unsaturated structure and excellent electron acceptor properties. Therefore, fullerene and its functionalized derivatives have been widely used as free radical scavengers. Fullerene and its derivatives have good antioxidant activity and can exert many excellent biological effects, including liver protection, reducing neuronal damage, extending lifespan, regulating immune cells, etc. Many current studies have shown that fullerene and its derivatives significantly inhibit the production of pro-inflammatory cytokines in vitro, and studies have demonstrated the therapeutic effect of fullerene and its derivatives on many inflammatory disease models. These results are It shows that fullerenes and their derivatives have significant application potential in the treatment of autoimmune diseases such as rheumatoid arthritis.

Contents of the invention

One object of the present invention is to provide an application in preparing a pharmaceutical composition for preventing and/or treating rheumatoid arthritis. The second object of the present invention is to provide a pharmaceutical composition and method for preventing and/or treating rheumatoid arthritis using the above-mentioned fullerene pharmaceutical composition. In particular, the pharmaceutical composition of the present invention is a preparation not suitable for topical administration. In particular, the present invention relates to an orally administered pharmaceutical composition for preventing and/or treating rheumatoid arthritis, wherein The fullerene structure is water-insoluble.

In order to solve the above technical problems, the present invention provides an application of a fullerene structure in the preparation of pharmaceutical compositions for preventing and/or treating rheumatoid arthritis.

Preferably, the pharmaceutical composition is an oral formulation.

In one embodiment, the fullerene structure is a water-insoluble fullerene.

In one embodiment, the fullerene structure includes one or more of prototype fullerene and functionalized modifications of fullerene.

In one embodiment, the prototype fullerene includes one or more of hollow fullerene and metallofullerene.

In one embodiment, the functionalized modification of fullerene is selected from one or more fullerenes from the following group:

(1) Fullerene with functionalized groups on the surface,

(2) Fullerenes wrapped by functionalized biological small molecules,

(3) Fullerene supported by biocompatible carrier material,

(4) Functionalized supramolecular system fullerene formed by self-assembly.

In one embodiment, the fullerene whose surface is modified with functionalized groups includes amino-, hydroxyl-, and carboxyl-modified fullerenes.

In one embodiment, the hollow fullerene is one or more cage structures composed of carbon atoms with a general formula of C _2m , 30≤m≤60, preferably, 30≤m≤45; more preferably Land, m is 30, 35 or 42.

In one embodiment, the metallofullerenes include M@C _2n , M ₂ @C _2n , MA@C _2n , M ₃ N@C _2n , M ₂ C ₂ @C _2n , M ₂ S@C _2n , one or more of M ₂ O@C _2n and M _x A _3-x N@C _2n , where: M and A both represent metal elements and M and A are selected from Sc, Y and lanthanide metal elements Any one of them, 30≤n≤60, 0≤x≤3; preferably, the metal fullerene is Gd@C ₈₂ .

In one embodiment, the fullerene is a prototype fullerene.

In one embodiment, the fullerene is _C60 .

In one embodiment, the oral formulation includes an oral solid formulation or an oral liquid formulation.

In one embodiment, the pharmaceutical composition is an oral tablet, oral capsule, oral granule, oral pill and/or oral suspension.

In one embodiment, the content of fullerene in the pharmaceutical composition of the oral liquid preparation is 4 mg/mL.

In one embodiment, the average particle size of the fullerene structure is 1-1000 nm, optionally, the average particle size is 1-200 nm.

In one embodiment, the method includes the step of orally administering an effective dose of any one of the aforementioned pharmaceutical compositions to an organism in need of prevention and/or treatment of rheumatoid joints.

In one embodiment, the pharmaceutical composition is in the form of a suspension, and the concentration range for use in vivo is 500 ppm-5000 ppm.

Beneficial effects:

(1) The inventor found through a large number of experiments that the fullerene structure not only has a strong ability to scavenge free radicals, but also can be rapidly metabolized in the body, has good biocompatibility, and has little toxic and side effects on organisms.

(2) The drug can be taken orally, which is convenient and can be taken for a long time. It is filtered and absorbed by the digestive system, reduces body damage, has little side effects and has significant curative effect.

(3) This drug can be metabolized outside the body. Due to its efficient free radical scavenging effect, it has an excellent effect in preventing and treating rheumatoid arthritis in the body, and can significantly improve joint redness, swelling and joint inflammation caused by rheumatoid arthritis. , cartilage and bone damage.

(4) For the prevention and treatment of rheumatoid arthritis in the present invention, the fullerene structure can be used as a preventive effect before the rheumatoid arthritis model matures; at the same time, its use after the onset of rheumatoid arthritis can eliminate excessive free radicals in the body. to prevent, treat and repair damaged joint function and other organ damage in rheumatoid arthritis

Description of drawings

Figure 1 is a photo of the hind limbs of mice with rheumatoid arthritis treated with C ₆₀ suspension and C ₆₀ -Ala;

Figure 2 shows the changes in joint scores of mice with rheumatoid arthritis treated with C ₆₀ suspension;

Figure 3 is a graph showing changes in the average cross-sectional area of the four paws of mice with rheumatoid arthritis treated with C ₆₀ suspension;

Figure 4 is a hematoxylin-eosin staining image of hindlimb knee joint sections of mice with rheumatoid arthritis treated with C ₆₀ suspension;

Figure 5 is a safranin-fast green stained image of hindlimb knee joint sections of mice with rheumatoid arthritis treated with C ₆₀ suspension;

Figure 6 is a picture of tartrate-resistant acid phosphatase staining of sections of hindlimb knee joints of mice with rheumatoid arthritis treated with C ₆₀ suspension.

Detailed ways

According to the above content of the present disclosure, various other forms of modifications, replacements or changes can be made according to the common technical knowledge and common means in the field without departing from the above basic technical ideas of the present disclosure.

I.Definition

Unless expressly stated otherwise, throughout the specification and claims, the term "comprises" or its variations such as "comprises" or "including" will be understood to include the stated elements or components and not Exclude other components or other components.

As used in this article, the term "fullerene" is a series of spherical cluster molecules composed of an even number of carbon atoms, with 12 five-membered rings and the remaining six-membered rings. It is a cage-like structure composed of carbon atoms. Fullerenes include hollow fullerenes and metallofullerenes. The hollow fullerenes are cage-like structures composed of carbon atoms alone.

The term "metallofullerene" refers to the inclusion of various atoms, ions or atomic clusters within the carbon cage structure of fullerene to form a class of compounds with special structures and properties. Such compounds are often called endometallofullerenes. Fullerene is generally expressed in the form of M@C _2n , where M represents a metal element.

The term "water-soluble fullerene" or "water-soluble fullerene modification" refers to the water-soluble modification of fullerene particles such as hollow fullerene and metallofullerene particles. The modified fullerene particles are externally modified with multiple water-soluble functional groups. These chemical functional groups contain one or more hydrophilic groups such as hydroxyl, carboxyl, sulfhydryl or amino groups or a combination thereof, making the fullerene particles soluble in water, or directly using small hydrophilic biological molecules such as amino acids and peptide chains. Such modified metallofullerenes or their derivatives can also be loaded with biocompatible carrier materials, such as liposomes, cell membrane carriers, etc., or they can be self-assembled to form water-soluble metallofullerenes or their derivatives. Supramolecular systems, etc. The above modification methods can all be modified according to methods disclosed in the prior art.

The disclosure of all ranges in this disclosure should be considered a disclosure of all subranges and all point values within the range. For example: the disclosure of 1-1000 should be deemed to also disclose the ranges of 1-200, 200-300, etc., and also disclose the point values of 200, 300, 400, 500, 600, 700, 800, 900 and 100.

The term "treating" includes inhibiting, alleviating, preventing, or eliminating one or more symptoms or side effects associated with the disease, condition, or disorder being treated.

The use of the terms "reduce," "inhibit," "mitigate," or "reduce" is relative to a control. One skilled in the art will readily determine the appropriate controls for each experiment. For example, a reduced response in a subject or cell treated with a compound is compared to a response in a subject or cell not treated with the compound.

As used herein, the term "effective amount" or "therapeutically effective amount" means an amount sufficient to treat, inhibit, or alleviate one or more symptoms of the disease state being treated or otherwise provide the desired pharmacological and/or physiological effect. dose. The precise dosage will vary based on a variety of factors, such as subject-dependent variables (e.g., age, immune system health, etc.), disease or disease, and the treatment administered. An effective amount can be compared to a control. These controls are known in the art and discussed herein, and may be, for example, the subject's condition before or without administration of the drug or drug combination, or in the case of a drug combination, the combined effect may be Compare the effects with administering just one drug.

The term "pharmaceutical composition" means a composition comprising fullerene and, depending on the mode of administration and nature of the dosage form, at least one pharmaceutically acceptable ingredient selected from the following, including but not limited to: carriers, diluents, adjuvants Agents, excipients, preservatives, fillers, disintegrants, wetting agents, emulsifiers, suspending agents, sweeteners, flavoring agents, fragrances, antibacterial agents, antifungal agents, lubricants, dispersants, Temperature-sensitive materials, temperature regulators, adhesives, stabilizers, suspending agents, etc.

The above contents of the present disclosure will be further described in detail below through specific implementation methods in the form of examples. However, this should not be understood to mean that the scope of the above subject matter of the present disclosure is limited to the following examples. All technologies implemented based on the above contents of this disclosure belong to the scope of this disclosure.

II.Examples

The present disclosure is further explained below with reference to examples. Specific exemplary embodiments of the present disclosure have been described for purposes of illustration and illustration. These descriptions are not intended to limit the disclosure to the precise forms disclosed, and obviously many modifications and variations are possible in light of the teachings of this specification. The exemplary embodiments were chosen and described in order to explain certain principles of the disclosure and its practical application, thereby enabling others skilled in the art to make and utilize various exemplary embodiments of the disclosure and various different applications. Choice and change.

The experimental methods used in the following examples are conventional methods unless otherwise specified.

Materials, reagents, etc. used in the following examples can all be obtained from commercial sources unless otherwise specified.

Example 1: Preparation of prototype fullerene suspension (C ₆₀ suspension)

Mix fullerene C ₆₀ solid powder (purchased from Xiamen Funa New Material Technology Co., Ltd.), microcrystalline cellulose, copovidone, glyceryl behenate and other excipients evenly, and press into tablets with a tablet press; Before animal experiments, the tablets were dissolved in ultrapure water into a C ₆₀ suspension with a concentration of 4 mg/ml (5.6 mM) for use in subsequent examples.

Example 2: Preparation of amino acid modified fullerene (C ₆₀ -Ala)

Add 100 mg of C ₆₀ solid powder (purchased from Xiamen Funa New Material Technology Co., Ltd.) to 80 ml of toluene solution, dissolve with ultrasound, and transfer to a 250 ml single-necked flask.

Add 3g β-alanine and 6g sodium hydroxide to 9ml of aqueous solution, then add 18ml of ethanol, sonicate evenly, add dropwise to the above-mentioned single-neck flask in an oil bath, heat to 80°C, and react for 24 hours;

After the reaction, use a M.W.=3500 dialysis bag to remove small molecules, use a conductivity meter to monitor until the dialysis is completed, and concentrate the product to obtain water-soluble aminoacidized fullerene (C60-Ala), which is measured by DLS in the aqueous solution. The average particle size is 90-100nm, and the particle size distribution is uniform.

Example 3: C ₆₀ suspension and C ₆₀ -Ala inhibit the swelling of limbs in mice with rheumatoid arthritis

Animal model: DBA/1 male mice aged 7-8 weeks were selected and randomly divided into 3 groups, corresponding to the normal group, model group, C ₆₀ -Ala treatment group and C ₆₀ suspension treatment group.

Preparation of collagen adjuvant emulsion: Under sterile conditions, use a 1ml syringe to draw bovine type II collagen with a concentration of 2mg/ml, use another 1ml syringe to draw an equal amount of adjuvant, and use two syringes to push and pull back and forth to make the bovine type II collagen Type II collagen and adjuvant are mixed thoroughly until the liquid becomes milky white.

Normal group: 200 μl of normal saline was injected into the base of the tail on the first day, and 200 μl of normal saline was administered daily from the 28th day until the end of the experiment on the 48th day.

Model group: 200 microliters of bovine type II collagen adjuvant emulsion was injected into the tail root on the first day, and 200 microliters of normal saline was administered daily from the 28th day until the end of the experiment on the 48th day.

C ₆₀ -Ala treatment group: 200 μl of bovine type II collagen adjuvant emulsion was injected into the base of the tail on day 1, and 200 μl of 4 mg/ml C ₆₀ -Ala was injected intraperitoneally every day from day 28 until the end of the experiment on day 48. .

C ₆₀ suspension treatment group: 200 microliters of bovine type II collagen adjuvant emulsion was injected into the base of the tail on the first day, and 200 microliters of C ₆₀ suspension of 4 mg/ml (5.6mM) was administered daily from the 28th day. The experiment ended on the 48th day.

The mice were first cultured for 1 week to adapt to the laboratory environment. After the mice were stabilized, they were divided into groups according to their weight. As the first day of the experiment, the mouse rheumatoid joint modeling and treatment were as described in the experimental grouping. , other operations remain the same for each group. On the day of collection, that is, the 48th day of the experiment, the joint swelling of the limbs of the mice was observed, and the hind limbs of mice in different groups were photographed for comparison.

The corresponding test results are shown in Figure 1. It can be seen that the hind limb swelling of the mice in the model group was obvious. The C ₆₀ -Ala treatment group did not significantly reduce the swelling of the hind limbs of the mice with rheumatoid arthritis, while the C ₆₀ suspension treatment group It can significantly reduce the degree of swelling in mice with rheumatoid arthritis, bringing it close to the level of normal mice.

Example 4: Effect of C ₆₀ suspension on inhibiting the onset of rheumatoid arthritis in mice

Animal model: DBA/1 male mice aged 7-8 weeks were selected and randomly divided into 3 groups, corresponding to the normal group, model group and C ₆₀ suspension treatment group.

Preparation of collagen adjuvant emulsion: Under sterile conditions, use a 1ml syringe to draw bovine type II collagen with a concentration of 2mg/ml, use another 1ml syringe to draw an equal amount of adjuvant, and use two syringes to push and pull back and forth to make the bovine type II collagen Type II collagen and adjuvant are mixed thoroughly until the liquid becomes milky white.

Normal group: 200 μl of normal saline was injected into the base of the tail on the first day, and 200 μl of normal saline was administered daily from the 28th day until the end of the experiment on the 48th day.

Model group: 200 microliters of bovine type II collagen adjuvant emulsion was injected into the tail root on the first day, and 200 microliters of normal saline was administered daily from the 28th day until the end of the experiment on the 48th day.

C ₆₀ suspension treatment group: 200 microliters of bovine type II collagen adjuvant emulsion was injected into the base of the tail on the first day, and 200 microliters of 4 mg/ml C60 suspension was administered daily from the 28th day until the 48th day of the experiment. Finish.

The mice were first cultured for 1 week to adapt to the laboratory environment. After the mice were stabilized, they were divided into groups according to their weight. As the first day of the experiment, the mouse rheumatoid joint modeling and treatment were as described in the experimental grouping. , other operations remain the same for each group. The joints of the mice were scored every 2-3 days after administration, and the width and thickness of the four paws of the mice were measured with vernier calipers to calculate the average cross-sectional area of the four paws of the mice until the end of the experiment.

The corresponding test results are shown in Figures 2 and 3. It can be seen from Figure 2 that compared with the model group, the C ₆₀ suspension treatment group can reduce the joint scores of mice with rheumatoid arthritis. It can be seen from Figure 3 that the C ₆₀ suspension treatment group can reduce the joint scores of mice with rheumatoid arthritis. The suspension treatment group can significantly alleviate the swelling of the paws of mice with rheumatoid arthritis, proving that C ₆₀ suspension treatment can reduce the joint swelling of mice and inhibit the onset of rheumatoid arthritis.

Example 5: C ₆₀ suspension inhibits the inflammatory effect of rheumatoid arthritis mouse joints

Animal model: DBA/1 male mice aged 7-8 weeks were selected and randomly divided into 3 groups, corresponding to the normal group, model group and C ₆₀ suspension treatment group.

Preparation of collagen adjuvant emulsion: Under sterile conditions, use a 1ml syringe to draw bovine type II collagen with a concentration of 2 mg/ml, use another 1ml syringe to draw an equal amount of adjuvant, and use two syringes to push and pull back and forth to make the bovine type II collagen Type II collagen and adjuvant are mixed thoroughly until the liquid becomes milky white.

Normal group: 200 μl of normal saline was injected into the base of the tail on the first day, and 200 μl of normal saline was administered daily from the 28th day until the end of the experiment on the 48th day.

Model group: 200 microliters of bovine type II collagen adjuvant emulsion was injected into the tail root on the first day, and 200 microliters of normal saline was administered daily from the 28th day until the end of the experiment on the 48th day.

C ₆₀ suspension treatment group: 200 microliters of bovine type II collagen adjuvant emulsion was injected into the base of the tail on the first day, and 200 microliters of 4 mg/ml C60 suspension was administered daily from the 28th day until the 48th day of the experiment. Finish.

The mice were first cultured for 1 week to adapt to the laboratory environment. After the mice were stabilized, they were divided into groups according to their weight. As the first day of the experiment, the mouse rheumatoid joint modeling and treatment were as described in the experimental grouping. , other operations remain the same for each group. After harvesting the materials, the mouse hindlimb knee joints were embedded in paraffin and sectioned, and hematoxylin-eosin (HE) staining was performed to observe the inflammation in the joints.

The corresponding test results are shown in Figure 4. It can be seen from the figure that a large number of inflammatory cells gathered in the joints of the mice in the model group, and the inflammatory infiltration was serious, while the C ₆₀ suspension treatment group could significantly alleviate the inflammation in the mice with rheumatoid arthritis. Cell infiltration, inhibiting the degree of inflammatory infiltration.

Example 6: Effect of C ₆₀ suspension on alleviating joint cartilage and bone damage in mice with rheumatoid arthritis

Animal model: DBA/1 male mice aged 7-8 weeks were selected and randomly divided into 3 groups, corresponding to the normal group, model group and C60 suspension treatment group.

Preparation of collagen adjuvant emulsion: Under sterile conditions, use a 1ml syringe to draw bovine type II collagen with a concentration of 2mg/ml, use another 1ml syringe to draw an equal amount of adjuvant, and use two syringes to push and pull back and forth to make the bovine type II collagen Type II collagen and adjuvant are mixed thoroughly until the liquid becomes milky white.

Normal group: 200 μl of normal saline was injected into the base of the tail on the first day, and 200 μl of normal saline was administered daily from the 28th day until the end of the experiment on the 48th day.

Model group: 200 microliters of bovine type II collagen adjuvant emulsion was injected into the tail root on the first day, and 200 microliters of normal saline was administered daily from the 28th day until the end of the experiment on the 48th day.

C ₆₀ suspension treatment group: 200 microliters of bovine type II collagen adjuvant emulsion was injected into the base of the tail on the first day, and 200 microliters of 4 mg/ml C60 suspension was administered daily from the 28th day until the 48th day of the experiment. Finish.

The mice were first cultured for 1 week to adapt to the laboratory environment. After the mice were stabilized, they were divided into groups according to their weight. As the first day of the experiment, the mouse rheumatoid joint modeling and treatment were as described in the experimental grouping. , other operations remain the same for each group. After collecting the materials, the mouse hind limb knee joints were embedded in paraffin and sectioned and stained with safranin-fast green. The basophilic cartilage tissue was combined with the basic dye safranin to appear red, and the eosinophilic bone tissue was combined with the acidic dye fast green to appear red. Colored green or blue to observe the destruction of cartilage and bone in mouse joints. In addition, tartrate-resistant acid phosphatase (Trap) staining was performed to observe the number of osteoclasts in the joints. Trap staining makes the osteoclasts red to show the distribution and number of osteoclasts.

The corresponding test results are shown in Figures 5 and 6. It can be seen from Figure 5 that the articular cartilage and bone of the mice in the model group were destroyed in large areas, and the bone was seriously eroded. The C ₆₀ suspension treatment group can significantly reduce Loss of articular cartilage and bone destruction in mice with rheumatoid arthritis. It can be seen from Figure 6 that the joints of mice in the model group contain a large number of osteoclasts, and the C ₆₀ suspension treatment group can significantly reduce the number of osteoclasts in the joints of mice with rheumatoid arthritis and inhibit the growth of osteoclasts. production and aggregation, thereby reducing damage to cartilage and bone.

The foregoing descriptions of specific exemplary embodiments of the present invention have been presented for purposes of illustration and illustration. These descriptions are not intended to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teachings. The exemplary embodiments were chosen and described in order to explain certain principles of the invention and its practical applications, thereby enabling others skilled in the art to make and utilize various exemplary embodiments of the invention and various different applications. Choice and change. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims (3)

1. The application of the fullerene structure in the preparation of a pharmaceutical composition for the treatment of rheumatoid arthritis, characterized in that the pharmaceutical composition is an oral suspension, The fullerene structure is water-insoluble fullerene, The fullerene is C ₆₀ , The oral suspension is made by mixing fullerene C ₆₀ solid powder and auxiliary materials including microcrystalline cellulose, copovidone, and glyceryl behenate evenly, and pressing them into tablets with a tablet press; Dissolve in pure water into a C ₆₀ suspension with a concentration of 4mg/ml. 2. The application according to claim 1, wherein the average particle size of the fullerene structure is 1~1000nm. 3. The application according to claim 2, wherein the average particle size of the fullerene structure is 1 to 200 nm.