CN114949242A - Application of selenium-containing compound in preparation of osteoclast differentiation inhibitor - Google Patents

Application of selenium-containing compound in preparation of osteoclast differentiation inhibitor Download PDF

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CN114949242A
CN114949242A CN202210098486.1A CN202210098486A CN114949242A CN 114949242 A CN114949242 A CN 114949242A CN 202210098486 A CN202210098486 A CN 202210098486A CN 114949242 A CN114949242 A CN 114949242A
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osteoclast differentiation
differentiation inhibitor
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陈填烽
邹斌华
贺利贞
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Jinan University
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Abstract

The invention discloses application of a selenium-containing compound in preparing an osteoclast differentiation inhibitor. The inventor of the invention discovers for the first time that selenides such as selenadiazole, nano-selenium modified by chitosan, nano-selenium modified by lentinan, nano-selenium modified by polyethylene glycol, sodium selenite, selenocystine and the like have the characteristic of inhibiting the generation of osteoclasts, so that the selenides can be used as osteoclast differentiation inhibitor medicines for treating diseases treated by the osteoclast differentiation inhibitor; particularly selenocysteine, has the advantages of small toxicity and remarkable pharmacological action.

Description

Application of selenium-containing compound in preparation of osteoclast differentiation inhibitor
Technical Field
The invention relates to the field of medicines, in particular to application of a selenium-containing compound in preparation of an osteoclast differentiation inhibitor.
Background
Stabilization of bone metabolism requires a dynamic balance and coupling between Osteoclast (OCs) -mediated bone resorption and Osteoblast (OBs) -mediated bone formation, which in healthy individuals maintain a functional balance that, once broken, may lead to the development of skeletal disease.
Osteoclasts are the only cells in the body responsible for bone resorption. It can be activated by osteoclast precursor cells to differentiate into mature osteoclast cells with functions. Since the differentiated osteoclasts can absorb bone, over-activation of osteoclasts can cause bone loss, osteoporosis, fracture pain, secondary reactive bone hyperplasia and even disability. The diseases related to the overactivation of osteoclasts are many, the classic is postmenopausal osteoporosis, and in addition, tumor metastasis bone destruction, inflammation-related bone destruction (rheumatoid arthritis, septic arthritis, periodontitis and the like) and the like are more important. Osteoclast activation is associated with a huge number of patients with bone destruction, with billions of patients worldwide with primary osteoporosis alone (including mainly postmenopausal osteoporosis, etc.).
The bone destruction diseases related to the overactivation of osteoclast are various in types, the etiology is also very diversified, even one disease has various pathogenesis-related factors, and some of the diseases belong to intractable diseases with uncertain etiology or diversified etiology. However, the medicine which directly aims at osteoclast and inhibits the differentiation and formation of osteoclast can directly improve related bone destruction symptoms of diseases by crossing disease causes and effectively relieve pain related to fracture, pain and the like of patients. Therefore, the osteoclast differentiation inhibitor becomes a class of medicines in recent years, and can be used for various bone destruction diseases related to overactivation of osteoclasts, such as postmenopausal osteoporosis.
Although many drugs for treating the above diseases can ultimately improve the diseases by various approaches such as improvement of hormone levels, anti-inflammation, and the like, there are few osteoclast differentiation inhibitors that can be clinically directed to osteoclasts. In addition, although some drugs can be used for the treatment of bone destruction diseases such as rheumatoid arthritis against etiology such as anti-inflammation, theoretically with the possibility of directly or indirectly inhibiting osteoclastogenesis and inflammatory bone destruction, in fact many commonly used drugs have been reported to induce osteoclastogenesis or aggravate osteoporosis. Two commonly used representatives of the treatment of rheumatoid arthritis, for example: glucocorticoids (such as dexamethasone), methotrexate, both inhibit the production of peripheral or diseased joint inflammatory factors, including TNF α, but both drugs cause bone destruction. Glucocorticoids (such as dexamethasone) can promote the induction of osteoclast generation in vitro, and are easy to cause osteoporosis on animals and patients in vivo; methotrexate also promotes osteoclast formation and causes bone destruction after use.
The osteoclast differentiation inhibitor commonly used at present is a bisphosphonate (such as zoledronic acid) and an RANKL inhibitor Denosumab (Denosumab). Both of these osteoclast differentiation inhibitors were initially approved for the market as drugs for the treatment of postmenopausal osteoporosis, and have recently been approved to add a new clinical indication, tumor metastasis bone destruction. However, most bisphosphates need to be injected and easily cause serious adverse reactions such as jaw necrosis; the dinoteumab which is a biological antibody preparation aiming at the osteoclast activating factor RANKL needs subcutaneous injection, and is more expensive. Therefore, more medicaments which can be conveniently taken, are economical and effective and are used as osteoclast differentiation inhibitors are clinically needed.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide the application of the selenium-containing compound in preparing the osteoclast differentiation inhibitor.
The purpose of the invention is realized by the following technical scheme:
application of selenium-containing compound in preparation of osteoclast differentiation inhibitor, wherein the selenium-containing compound is selenadiazole (SeD, C) 6 H 3 O 2 N 3 Se), chitosan modified nano-selenium (CS-Se), lentinan modified nano-selenium (Let-Se), polyethylene glycol modified nano-selenium (Peg-Se), sodium selenite (Na) 2 SeO 3 ) And selenocysteine (SeCys) 2 One or at least two of selenicyscyscyscysses); preferably selenocysteine SeCys 2 。
The osteoclast differentiation inhibitor is a medicament for treating the clinical indications of diphosphate and denosumab.
The osteoclast differentiation inhibitor is an anti-postmenopausal osteoporosis drug, an anti-tumor metastasis bone destruction drug, an anti-rheumatoid arthritis bone destruction drug and a plurality of osteoclast over-activation related bone destruction disease drugs.
The osteoclast differentiation inhibitor comprises a selenium-containing compound and a medically acceptable pharmaceutical adjuvant.
The osteoclast differentiation inhibitor is an oral preparation, an injection preparation or an external preparation.
Compared with the prior art, the invention has the following advantages and effects:
1. the inventor of the invention discovers for the first time that different selenides have direct and different inhibitory action characteristics on osteoclastogenesis, so that the selenides can be used as osteoclast differentiation inhibitor medicines.
2. The invention provides application of different selenides in preparing an osteoclast differentiation inhibitor. The osteoclast differentiation inhibitor can be used as a medicament for treating clinical indications approved by known osteoclast differentiation inhibitor medicaments such as diphosphate and dinolizumab, for example, an anti-postmenopausal osteoporosis medicament, an anti-tumor metastasis bone destruction medicament, an anti-rheumatoid arthritis bone destruction medicament, a plurality of osteoclast over-activation related bone destruction disease medicaments and the like. The osteoclast differentiation inhibitor comprises a selenium-containing compound and medically acceptable pharmaceutical excipients, and can be prepared into oral dosage forms, injection dosage forms or external dosage forms.
Drawings
FIG. 1 is a photograph result graph of the effect of different selenides on mouse bone marrow macrophage differentiation into osteoclasts; wherein: RANKL is a ligand that activates nuclear factor NF-. kappa.B receptors.
FIG. 2 is a graph of the statistical results of the effect of different selenides on mouse bone marrow macrophage differentiation into osteoclasts; wherein, compared to a blank control group, ### P<0.001; p compared to RANKL group<0.05，**P<0.01，***P<0.001，ns indicates no significant difference.
Figure 3 is a graph of the results of cytotoxicity assays for different selenides.
FIG. 4 is SeCys 2 Results of in vivo experiments in mice treated with arthritis.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated. The test methods in the following examples, in which specific experimental conditions are not specified, are generally performed according to conventional experimental conditions or according to the experimental conditions recommended by the manufacturer. Unless otherwise specified, reagents and starting materials for use in the present invention are commercially available.
The following in vitro experiments were performed on selenadiazole SeD (C) 6 H 3 O 2 N 3 Se), three different modified nanoparticles (CS-Se, Let-Se, PEG-Se), and sodium selenite (Na) 2 SeO 3 Sigma) and sodium selenate (Na) 2 SeO 4 Sigma), selenocysteine (SeCys) 2 ，Selenocystine，CysSeSeCys，C 6 H 12 N 2 O 4 Se 2 Sigma), selenomethionine (SeM, C) 5 H 11 NO 2 Se, sigma) was tested for their anti-osteoblast differentiation effect.
Example 1
Synthesis of three different nano-selenium:
the elemental nano-selenium (SeNPs) has the characteristics of low toxicity, high bioavailability and easy surface modification. However, non-modified SeNPs are poor in stability and easy to aggregate, and the bioactivity of the SeNPs is greatly reduced due to the change of nanometer scale. Studies have reported that the use of polymers in the synthesis of SeNPs can prevent the aggregation of SeNPs, and increase the stability and bioactivity. Therefore, polymers (CS, Let, PEG) with different charges are selected to modify the SeNPs to synthesize three kinds of functionalized SeNPs, including chitosan nano-selenium CS-Se, lentinan nano-selenium Let-Se and polyethylene glycol nano-selenium PEG-Se.
1) The synthesis method of Let-SeNPs and CS-SeNPs comprises the following steps:
sodium selenite solution: 69mg of sodium selenite (molecular weight 172.94) was dissolved in 10mL of ultrapure water to obtain a 40mM sodium selenite solution.
Ascorbic acid solution: 352.42mg of ascorbic acid (molecular weight 176.13) were dissolved in 20mL of ultrapure water to give an ascorbic acid solution with a concentration of 100 mM.
Let solution: 75mg of Let lentinan powder (Shaanxi Senfu natural products Co., Ltd., purity 80%) is dissolved in 15mL of ultrapure water to obtain a Let solution with a concentration of 4 mg/mL.
CS solution: 75mg of CS chitosan powder (alatin, purity 80%) was dissolved in 15mL of ultrapure water, and 150. mu.L of glacial acetic acid was added to promote the dissolution of CS, resulting in a CS solution with a concentration of 4 mg/mL.
And adding 5mL of CS solution or 5mL of Let solution into a reaction bottle, then adding 2mL of sodium selenite solution, stirring, adding 1mL of ultrapure water, continuing stirring, then adding 2mL of ascorbic acid solution, and then stirring for reacting overnight. After the reaction, the CS-SeNPs or Let-SeNPs solution obtained after dialysis is finally recovered by using a 6000-8000Da dialysis bag to dialyze in distilled water overnight.
2) Synthetic method of PEG-SeNPs
Adding 10mL of PEG 400 (polyethylene glycol 400) into a 50mL beaker, then weighing 20mg of selenium powder (Aladdin, s105193), carrying out ultrasonic treatment for 10min, and stirring for 5min to completely and uniformly disperse the selenium powder in the PEG 400. And (3) placing the transfer beaker on a magnetic stirring heater, heating to about 205 ℃, stirring while heating, naturally cooling after reacting for 1 hour, transferring the transfer beaker to a centrifuge tube, centrifuging at 2000rpm for 10min, taking the upper solution, removing unreacted selenium powder, and finally obtaining the PEG-SenPs. (the stirring speeds were all 300rpm)
3) SeD synthesis method references "Selenadiazole derivative as a therapeutic agent for a simple and efficient cancer chemo-/radiotherapeutic by targeting a thioredoxin derivative, Journal of Materials Chemistry B,2015 Nov 14; 8383-8393, "Synthesis and catalysis of SeDs (1-3)".
Example 2
Effect of different selenides on mouse Bone Marrow Macrophages (BMMs) differentiation into osteoclasts:
taking a femur and a tibia of a C57BL/6 mouse (Experimental animal center in Guangdong province) which is healthy for 6-8 weeks, cutting off two ends after muscle stripping, sucking alpha-MEM culture medium pre-cooled on ice by using a syringe, repeatedly flushing marrow cavities of the femur and the tibia to flush out bone marrow cells until the marrow cavities are white, filtering a tissue block by using a filter head with the size of 70 microns, transferring the filtrate into a culture dish with the size of 60cm, and standing and culturing in an incubator, wherein the culture medium is the alpha-MEM culture medium containing 30ng/mL macrophage colony stimulating factor M-CSF. After standing for 3 days, the supernatant was discarded, and the bottom cells were digested with 0.25% trypsin solution at 7X 10 3 The density of each well was inoculated in a 96-well plate, M-CSF and RANKL were added to the positive control group (RANKL group) and the drug group (drug group) except the negative control group (Blank group) at a final concentration of 50ng/mL, and different selenium-containing compounds were added to the drug group at the same time at final concentrations of 0.75. mu.M, 1.5. mu.M, 3. mu.M, 6. mu.M, 12. mu.M, 24. mu.M, and 48. mu.M (drug was prepared by dilution at double ratio), and 3 wells were added to each group. The selenium-containing compound and the medium containing the stimulating factors M-CSF and RANKL were changed 1 time every 2 days. TRAP staining was performed on day 4-5 of induction, osteoclastogenesis was observed under a microscope in a 96-well plate and photographed, and osteoclasts were counted as TRAP positive and larger than 3 nuclei (purple red, osteoclasts fused together by multiple cells). The results are shown in FIGS. 1 and 2.
Example 3
Effect of different selenides on cytotoxicity of mouse macrophage leukemia cell RAW264.7 (osteoclast precursor cell):
RAW264.7 cells (Shanghai Zhongkoji cell bank) were resuspended in phenol red-containing DMEM medium and at 1X 10 3 The density of individual cells/well was plated in 96-well plates and after overnight different selenium compounds were added at concentrations of 0.75. mu.M, 1.5. mu.M, 3. mu.M, 6. mu.M, 12. mu.M, 24. mu.M, 48. mu.M, 96. mu.M. Each group of 3 multiple wells. After 48h incubation, the supernatant was removed, 0.5mg/mL MTT 100. mu.L was added to each well, incubated in a 37 ℃ incubator for 4h, the supernatant was discarded, DMSO 150. mu.L was added, and after shaking for 10min, the enzyme was labeled with Genios Pro type Tecan enzymeThe absorbance was measured at 570nm by the instrument.
As can be seen from FIG. 3, except SeD and Na 2 SeO 3 Besides the toxicity of CS-Se and Let-Se is high, the in vitro concentration of other selenides is less than or equal to 12 mu M, and the survival of RAW264.7 cells is not obviously influenced.
Taken together the above in vitro results, SeCys in addition to SeM 2 、SeD、CS-Se、Let-Se、Peg-Se、Na 2 SeO 3 、Na 2 SeO 4 All inhibit in vitro RANKL to different degrees and induce osteoclast precursor cells to form osteoclasts (shown in fig. 2, a statistical chart of the number of osteoclasts), directly inhibit the differentiation and formation of osteoclasts, and inhibit the bone resorption function of osteoclasts.
Wherein the SeCys is selected from the group consisting of bioactivity and toxicity of the drug 2 Most preferably.
Example 4
SeCys 2 Effect on type ii collagen-induced arthritic mice:
mixing bovine type II collagen and complete Freund adjuvant according to the volume ratio of 1: 1, the materials are added into a mortar according to a proportion of 1, the mortar is required to be placed on ice to keep low temperature to prevent inactivation in the grinding process, the materials are fully ground for more than half an hour under the low-temperature condition until the materials become milky solution (water-in-oil), after the materials are uniformly ground, a small drop is dropped into water to detect whether the materials are qualified for grinding, and if the drops dropped into the water do not spread immediately, the materials are fully ground and can be used for injection molding. First, the hair of the tail root of a mouse (healthy 7-8 week DBA mouse, Witonglihua) was shaved with a shaver, and the ground collagen was slowly sucked up with a syringe and forcibly flicked down the wall of the syringe tube to discharge air. After the skin is disinfected by alcohol, inserting a needle into the skin (slightly picking up the skin after inserting the needle), slowly injecting collagen at a constant speed, forming a bump of a coating block at an injection position after injection, carefully drawing out a needle head (rotating and drawing out) after injection, slightly wiping the needle head by using an alcohol cotton ball to finish injection, wherein the volume of collagen injected into each mouse is 100 mu L, the mice of other groups except a normal control group are subjected to model-making immunization, and on the 21 st day after primary immunization, incomplete adjuvant is used for carrying out secondary boosting immunization in the same way. The injection site in the second immunization should avoid the ulceration site formed after the first immunization to prevent the injected collagen from overflowing from the peeping site. On days 4 and 5 after the second immunization, the mice begin to be treated by the drug when the mice have arthritis symptoms of joint red swelling. The drug was dissolved in 0.1mol/L hydrochloric acid solution to 100 Xthe mother liquor (5 mg/mL and 20mg/mL, respectively), and stored in a refrigerator at-80 ℃. On the day of administration, the drug was diluted 100 times with physiological saline and then administered, and the drug was injected into the mouse via the abdominal cavity at a rate of 10. mu.L/g body weight.
After the boost, the appearance of red and swollen symptoms can be observed in the toe and ankle joint parts of the mice, approximately on the fourth to fifth days. We observed the redness and swelling of the toes and ankle joints of each group of mice periodically and recorded their foot swelling scores, every 2 days for a period of five weeks. The criteria for foot swelling scores are shown in the following table:
TABLE 1
No red and swollen change 0 point (min)
1-toe swelling and reddening 1 minute (1)
Slight swelling of the joints of limbs or swelling of more than 2 toes 2 is divided into
Obvious red swelling of limbs and joints 3 points of
Swelling and redness of the sole and a strong and direct joint 4 is divided into
Note: the highest score per mouse was 16 points
The observation results are shown in fig. 4: compared with the normal control group, the CIA model mouse has arthrocele and obvious arthritis, and SeCys is given 2 -0.5mg/kg and SeCys 2 After treatment at-2 mg/kg, the swelling degree of arthritis can be reduced remarkably, and the loss of body weight and the arthritis score can be reduced. The results of this experiment demonstrate that SeCys 2 Has remarkable treatment effect on arthritis mice and is a candidate drug for bone destruction diseases.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims (5)

1. The application of the selenium-containing compound in preparing the osteoclast differentiation inhibitor is characterized in that: the selenium-containing compound is one or at least two of selenadiazole, chitosan-modified nano-selenium, lentinan-modified nano-selenium, polyethylene glycol-modified nano-selenium, sodium selenite and selenocysteine.
2. The use of a selenium-containing compound according to claim 1 for the preparation of an osteoclast differentiation inhibitor, wherein:
the osteoclast differentiation inhibitor is a medicine for treating the clinical indications of diphosphate and denosumab.
3. The use of a selenium-containing compound according to claim 1 for the preparation of an osteoclast differentiation inhibitor, wherein:
the osteoclast differentiation inhibitor is an anti-postmenopausal osteoporosis drug, an anti-tumor metastasis bone destruction drug, an anti-rheumatoid arthritis bone destruction drug or a plurality of bone destruction disease drugs related to osteoclast over activation.
4. The use of a selenium-containing compound according to claim 1 for the preparation of an osteoclast differentiation inhibitor, wherein:
the osteoclast differentiation inhibitor comprises a selenium-containing compound and a medically acceptable pharmaceutical adjuvant.
5. Use of a selenium-containing compound according to claim 1 for the preparation of an osteoclast differentiation inhibitor, wherein:
the osteoclast differentiation inhibitor is an oral preparation, an injection preparation or an external preparation.
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