CN111773223A

CN111773223A - Application of dasatinib and quercetin pharmaceutical composition in preparation of drugs for preventing and/or treating glucocorticoid side effects

Info

Publication number: CN111773223A
Application number: CN202010541978.4A
Authority: CN
Inventors: 张先荣; 余斌; 苏剑文
Original assignee: Southern Hospital Southern Medical University
Current assignee: Southern Hospital Southern Medical University
Priority date: 2020-06-15
Filing date: 2020-06-15
Publication date: 2020-10-16
Anticipated expiration: 2040-06-15
Also published as: CN111773223B

Abstract

The invention belongs to the technical field of medicines, and particularly relates to application of a dasatinib and quercetin medicinal composition in preparation of a medicine for preventing and/or treating glucocorticoid side effects. Although prenatal use of dexamethasone has many benefits for newborns, its harm is not negligible. Glucocorticoids cause embryonic development retardation and related diseases after the birth of offspring. The pharmaceutical composition of the invention, namely dasatinib and quercetin, can improve the bone phenotype of offspring and the inhibition effect of glucocorticoid application on BMSCs.

Description

Application of dasatinib and quercetin pharmaceutical composition in preparation of drugs for preventing and/or treating glucocorticoid side effects

Technical Field

The invention relates to the technical field of medicines, in particular to application of a dasatinib and quercetin medicinal composition in preparation of a medicine for preventing and/or treating glucocorticoid side effects.

Background

Glucocorticoid belongs to steroid compounds, and is a metabolism regulating hormone synthesized and secreted by adrenal cortex fasciculate band. Glucocorticoids mainly affect the metabolism of sugars, proteins and fats at physiological doses, and have a variety of effects such as anti-inflammatory, immunosuppressive, antitoxin, antiallergic, antishock at pharmacological doses. Glucocorticoid has wide effects and influences on the functions of all organs of the body, for example, long-term large-dose administration can cause side effects and complications such as hypertension, hypokalemia, osteoporosis, peptic ulcer bleeding or perforation, poor wound healing, induction or aggravation of infection, mental excitation, endocrine dysfunction and the like.

Liggins and Howie1 in 1972 first reported that the survival rate of preterm infants could be improved with prenatal glucocorticoid treatment. Current prenatal glucocorticoid therapy has been prescribed and recommended by WHO. As is well known, glucocorticoids are essential for the maturation of fetal organs and tissues, and prenatal application of glucocorticoids induces fetal multi-organ maturation by various mechanisms, such as induction of specific proteins and enzyme production, which promote fetal tissue maturation, increase lung compliance and lung volume, decrease vascular permeability in fetal lung tissue by increasing tissue and alveolar surfactant production, thereby reducing the incidence of respiratory complications. A systematic review study found that treatment of pregnant women with glucocorticoids prior to delivery reduced perinatal infant mortality, and reduced incidence of various complications including respiratory distress syndrome and the like (CochraneDatabase Syst Rev.2017,3: CD 004454). Although prenatal use of dexamethasone has many benefits for newborns, its harm is not negligible. Glucocorticoids cause embryonic development retardation and diseases associated with postnatal offspring, such as cardiovascular diseases, metabolic diseases. Glucocorticoids are important regulatory signals in intrauterine development, and alter the phenotype of various cells by inducing expression changes of structural proteins, transport proteins and signaling proteins, thereby affecting the development of fetal tissues and possibly even producing wide effects on the whole organ and system level. The prenatal exposure to excessive glucocorticoid therapy in pregnant women may result in a change in fetal tissue from proliferation to differentiation, which has a long-term impact on offspring. The prenatal application of dexamethasone had a considerable impact on the skeletal development of the offspring. Korakaki et al (Calcif Tissue int.2011,88: 215-. Khan and Rodriguez et al (Paediator Perinat epidemic.2011, 15:20-36) found in a systematic review that prenatal application of dexamethasone resulted in a reduction in fetal birth weight. A fetus with a low birth weight is associated with a low peak bone mass after its adulthood. Therefore, it is of great importance to develop drugs for preventing or treating the side effects of glucocorticoids.

Dasatinib (Dasainib) belongs to a polytyrosine kinase inhibitor and has the chemical name of N- (2-chloro-6-methylphenyl) -2- [ [6- [4- (2-hydroxyethyl) -1-piperazinyl]-2-methyl-4-pyrimidinyl]Amino group]-5-thiazolecarboxamide of the formula C₂₂H₂₆ClN₇O₂S, molecular weight 488.00600; can be used for treating adult chronic myelogenous leukemia, and Philadelphia chromosome positive acute lymphocytic leukemia. Quercetin (Quercetin) is a polyhydroxy flavonoid compound with the chemical name of 3,3 ', 4', 5, 7-pentahydroxyflavone, and has various biological activities and high medicinal value. Quercetin is not only widely distributed in nature, but also has wide pharmacological action, and has effects of resisting oxidation, scavenging free radicals, resisting cancer, inflammation, bacteria, virus, blood sugar and blood pressure, regulating immunity, protecting cardiovascular system, etc.

In addition, dasatinib can remove aged human adipose progenitor cells, quercetin can remove aged endothelial cells, and the combination of dasatinib and quercetin can achieve a better effect. In clinical studies, the combination of dasatinib + quercetin (D + Q) was shown to eliminate aging cells in vivo (Farr JN et al, Nat Med,2017, 23). Since each drug targets a different type of senescent cell, and the combination of the two drugs does not detract from the effect of each drug on the susceptible senescent cell type in vitro, the mice were given simultaneous D and Q treatments. Single administration of D + Q gavage reduced p16 in adipose tissue of 24-month-old mice within 5 days^Ink4aExpression and senescence-associated activity of β -galactosidase (SA- β gal) D + Q treatment also reduced p16 in older mice^Ink4amRNA⁺Expression of the cell. Inducing aging of mouse muscle and leg adipose tissue after 3 months of local ionizing radiation treatment, and treating mouse muscle p16 after D + Q treatment^Ink4amRNA and leg adipose tissue SA- β -gal⁺The cells are reduced. In another study, D + Q significantly reduced the Cell kurtosis with telomere-associated DNA damagefoci (TAF) in the aorta of 24-month old mice (Roos CM et al, Aging Cell,2016,15: 973-.While TAFs are more specific markers for senescent cells. D + Q also reduces high cholesterol apolipoprotein E^-/-TAF in mice and high fat fed young mice⁺Cell number (Roos CM et al, Aging Cell,2016,15: 973-. Furthermore, D + Q reduced bleomycin-induced accumulation of senescent cells and an increase in senescent cells in the lung of pulmonary fibrosis mice as evidenced by p16^Ink4aAnd reduced expression of the SASP component (Schafer MJ et al, Nat Commun,2017,8: 14532).

In the prior art, no relevant report that dasatinib and quercetin are used for eliminating glucocorticoid side effects exists.

Disclosure of Invention

The present invention is directed to solving at least one of the problems of the prior art. Therefore, the invention provides a new application of the dasatinib and quercetin medicine composition in medicines for preventing and/or treating glucocorticoid side effects.

The technical scheme of the invention is shown as follows.

The invention provides application of a pharmaceutical composition containing dasatinib or pharmaceutically acceptable salts thereof and quercetin or pharmaceutically acceptable salts thereof in preparing a medicament for preventing and/or treating glucocorticoid side effects.

According to some embodiments of the invention, the pharmaceutical composition consists of dasatinib or a pharmaceutically acceptable salt thereof and quercetin or a pharmaceutically acceptable salt thereof.

Through a great deal of scientific research and creative work, the inventor finds that the reduction of Bone marrow Mesenchymal Stem Cells (BMSCs) and the reduction of Bone formation induced by the PDE can be improved by administering the pharmaceutical composition of dasatinib and quercetin in the process of constructing a Dexamethasone Prenatal application (PDE) model mouse. The pharmaceutical composition is expected to become a clinical medicine for treating PDE bone development toxicity.

According to some embodiments of the invention, the dasatinib and the quercetin may be compounds themselves, in the form of pharmaceutically acceptable salts, hydrates, esters, ethers, acids, amides, racemates, isomers, enantiomers thereof, as well as in the form of sustained release formulations, and also as prodrugs or derivatives of the compounds thereof.

As used herein, the term "pharmaceutically acceptable salt" refers to a pharmaceutically acceptable and relatively non-toxic inorganic or organic acid addition salt of a compound of the present invention. The formation of the pharmaceutical salt consisted of: drug molecules that counter ion pairing acidic, basic or zwitterionic ions are used to generate salt forms of the drug. A wide variety of chemicals can be used in the neutralization reaction.

According to some embodiments of the invention, the mass ratio of dasatinib or a pharmaceutically acceptable salt thereof to quercetin or a pharmaceutically acceptable salt thereof is 1: 5-20.

According to some embodiments of the invention, the effective dose of the pharmaceutical composition is 0.15-3 mg/day; preferably, the administration period is 3 days.

Based on the results of the present study, one skilled in the art can readily determine the effective amount. The specific dosage of the pharmaceutical composition of the present invention may be adjusted accordingly depending on various factors, including but not limited to: the severity of the condition of the subject, the age, sex, weight, route of administration and pharmaceutical dosage form of the subject, and the like.

According to some embodiments of the invention, the dasatinib, or a pharmaceutically acceptable salt thereof, and the quercetin, or a pharmaceutically acceptable salt thereof, are present in the same formulation, forming a single dosing unit.

According to some embodiments of the invention, the dasatinib or a pharmaceutically acceptable salt thereof and the quercetin or a pharmaceutically acceptable salt thereof are present as separate formulations, each being a separate administration unit.

According to some embodiments of the invention, when the dasatinib or a pharmaceutically acceptable salt thereof and the quercetin or a pharmaceutically acceptable salt thereof are present as separate formulations, they may be administered simultaneously or sequentially.

The components of the pharmaceutical composition of the present invention may be formulated separately or together. When formulated together, are present in the same formulation as a whole. When formulated separately, the components may be administered to the subject simultaneously or sequentially, or may be administered repeatedly.

According to some embodiments of the invention, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient. The pharmaceutically acceptable auxiliary materials can be any pharmaceutically acceptable auxiliary materials known in the art according to actual needs.

According to some embodiments of the invention, the pharmaceutical composition is an oral formulation, an injection formulation, an inhalation formulation, an intra-oral formulation or a topical formulation or any combination thereof.

According to some embodiments of the invention, the pharmaceutical composition further comprises an additional agent for the treatment of a glucocorticoid side effect.

According to some embodiments of the invention, the preventing and/or treating glucocorticoid side effects is achieved by eliminating senescent cells.

According to some embodiments of the invention, the glucocorticoid side effect is obesity and cushing's syndrome, hypothalamic-pituitary-adrenal axis function inhibition, depression, memory loss, hyperlipidemia, hyperglycemia, cardiovascular system diseases, catabolism of skeletal muscle, osteoporosis, femoral head necrosis, osteogenic differentiation inhibition, gastrointestinal bleeding, cortical cataract, or a side effect of prenatal glucocorticoid application on offspring.

According to some embodiments of the invention, the glucocorticoid side effect is a side effect of prenatal glucocorticoid application on progeny.

According to some embodiments of the invention, the side effect of the prenatal glucocorticoid application on progeny is suppression of bone development.

According to some embodiments of the invention, the glucocorticoid is prednisone, methylprednisolone, betamethasone, beclomethasone propionate, prednisolone, hydrocortisone, dexamethasone, preferably dexamethasone.

In a second aspect, the invention provides a kit comprising a therapeutically effective amount of a pharmaceutical composition and product instructions;

wherein the pharmaceutical composition is a pharmaceutical composition according to any one of the first aspect of the invention.

According to some embodiments of the invention, the product instructions recite instructions for the pharmaceutical composition to be used in a subject with a glucocorticoid.

According to some embodiments of the invention, the pharmaceutical composition comprises:

i) a therapeutically effective amount of dasatinib;

ii) a therapeutically effective amount of quercetin.

Based on the results of the present study, one skilled in the art can readily determine the effective amount. Generally, when used for the uses described herein, a therapeutically effective amount of dasatinib may be 0.03-0.3 mg/day, preferably 0.15 mg/day, for 3 days; the therapeutically effective amount of quercetin may be 0.3-3 mg/day, preferably 1.5 mg/day, for 3 days.

The specific dosage of the pharmaceutical composition of the present invention may be adjusted accordingly depending on various factors, including but not limited to: the severity of the condition of the subject, the age, sex, weight, route of administration and pharmaceutical dosage form of the subject, and the like.

A third aspect of the present invention provides a method for the prevention and/or treatment of glucocorticoid side effects, comprising administering to a subject in need thereof a pharmaceutically effective amount of a pharmaceutical composition according to any one of the first aspect of the present invention.

The invention may be used to treat any subject at any stage of the disease, said subject being a patient with a glucocorticoid. The pharmaceutical composition of the present invention can prevent or ameliorate the side effects of glucocorticoid administration in the subject.

Evidence from previous experimental animal models of the present inventors indicates that prenatal application of dexamethasone (PDE) impairs long bone development in offspring mice and rats, resulting in a decrease in bone mass in offspring animals after adulthood. The birth weight of the newborn mice of PDE progeny is reduced, and the length of the femur and the length of the primary ossification center are significantly reduced (Toxicol Appl Pharmacol.2018,351: 12-20; Br J Pharmacol.2016, 173: 2250-. Compared with the single-course treatment of the 15 th day of gestation of the female mouse, the number and the thickness of the bone trabeculae of the PDE new-born mouse with multiple treatment courses are obviously reduced; in neonatal mice after multiple PDE treatments at different doses (0, 0.2, 0.8 and 1.2mg/kg/d), we observed a toxic effect of both PDE treatments at 0.8 and 1.2mg/kg/d on bone development; we found that fetal mice treated with the same dose of PDE (0.8mg/kg/d) during days 12-14 of gestation (GD12-14) developed more severe bone developmental delay than after PDE treatment during GD 15-17. In rat experiments, prenatal dexamethasone application resulted in accumulation of hypertrophic chondrocytes and delayed formation of primary ossification centers in the long bones of rats in the fetal period, and also resulted in decreased maturity of hypertrophic chondrocytes, decreased osteoclast number, down-regulation of osteocalcin (osteoprotein) and bone sialoprotein (bone sialoprotein) expression, and the like. Dexamethasone can stimulate the expression of mitogen-induced gene-6 (Mig 6) and Osteoprotegerin (OPG) in chondroprogenitor and osteoblast, and inhibit the expression of RANKL. μ CT measurements showed a decrease in bone mass of femurs from 12 week old offspring mice after PDE treatment. These data indicate that overuse of glucocorticoids during development alters skeletal system programming, resulting in reduced bone mass.

The present invention provides pharmaceutical compositions for the prevention and/or treatment of glucocorticoid side effects. More specifically, the invention combines dasatinib and quercetin (D + Q), and then the combination is administered to PDE model mice, so that the bone phenotype of the D + Q treated PDE progeny mice is obviously improved, and the inhibition effect of PDE on BMSCs is improved. Presumably, D + Q abolished senescent cells, thereby improving PDE-induced reduction of BMSCs and reduction of bone formation. Elimination of senescent cells is potentially a new approach to the treatment of PDE bone development toxicity.

Drawings

FIG. 1 is a graph of metaphyseal staining of 2 week old progeny mice;

FIG. 2 shows SA- β -Gal at metaphysis of mice in each group⁺Osteoblasts and Nestin⁺A cell number histogram;

FIG. 3 shows CD45^-Nestin⁺Group flow result graph；

FIG. 4 shows CD45^-CD29⁺CD105⁺Sca-1⁺And (4) a group flow result graph.

Detailed Description

The technical solutions and effects of the present invention will be further described and illustrated with reference to the following specific examples, but the present invention is not limited to these specific embodiments. The test methods used in the examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are commercially available reagents and materials unless otherwise specified.

Animal breeding:

the laboratory workers were trained and acquired in the southern university of medical animal center for animal laboratory procedure certificate, approved by the southern hospital animal ethics committee. The C57BL/6 mice used in this study were obtained from a unit having a production license for laboratory animals, and were issued "quality standards for laboratory animals". Mouse breeding was performed according to southern hospital laboratory animal care regulations of southern medical university. Pathogen Free (SPF) mice were bred at 25 ± 3 ℃ under a relative humidity of 40-60% for 12 hours of light and 12 hours of dark cycles, providing sufficient food and purified water, controlling the number of animals at an appropriate density, changing the padding every 3 days, maintaining a clean and dry environment. Animals were selected following the 3R principle: reducing animal use, or acquiring more data with the same animal; optimizing all experimental steps and relieving the pain of experimental animals; attempts have been made to replace animal experiments with cell experiments or other methods. Animal material taking is carried out after cervical dislocation is killed after anesthesia, and the international practice or the latest guidance scheme of euthanasia formulated by American veterinary medical society is met.

The study also involved the breeding and care of pregnant mice, male and female mice according to 1: 2 or 1: 3 ratio in the evening of each day and check the vaginal suppository the next morning. Female mice with vaginal emboli will be bred independently to avoid abortion in pregnant mice due to inter-mouse surprise. And eggs are added into the food of pregnant mice to supplement nutrition, so that the birth success rate and the number of offspring are improved.

In addition, all quantitative data are presented as mean ± sd in the experiments and used for independent t-test comparisons between the two groups. For multiple comparisons, one-way analysis of variance (ANOVA) and Bonferroni test were used. Statistical analysis was performed using software version SPSS20 (International Business Machines Corporation, IBM Corp.). Significance level was P < 0.05.

Example 1: construction of animal models

1. Establishment of mouse PDE model

Pregnant mice were given dexamethasone treatment during days 12-14 of gestation to establish a dexamethasone prenatal application (PDE) model. Briefly summarized, 8-12 week old C57BL/6 female mice were mated overnight with male mice. On the day of vaginal suppository appearance, gd (statistical day)0 was set, and the pregnant mice were randomized into PDE groups or control groups. Dexamethasone sodium phosphate (Cat.2392-39-4, "Tianxin") was given as a subcutaneous injection (1.2mg/kg/d) during GD12-14 to establish a PDE mouse model, and this group of mice was called the model group. To establish a control for the PDE model, control group pregnant mice, referred to as control groups, were treated with daily subcutaneous injections of an equal amount of solvent (normal saline) during GD 12-14. In order to avoid data bias caused by littermate mice, offspring of two females (the reason for selecting the same sex: avoiding the difference of sex factors influencing bone growth and development) are randomly selected from each litter to carry out the bone development research after parturition. Femurs and tibiae of female offspring mice aged 2 weeks, 4 weeks, 6 weeks and 12 weeks, respectively, were taken for further analysis.

2. Establishment of model of anti-aging drugs (senolytics) for treating PDE

D + Q treatment PDE models were established using Dasatinib (Dasatinib, S1021, Selleck Chemicals) and Quercetin (Quercetin, S2391, Selleck Chemicals), D + Q, administered orally at 5mg/kg/D and 50mg/kg/D, respectively, during GD12-14, 2 drugs suspended in 100 μ L of 1% methylcellulose (thickener) with dexamethasone treatment, and this group of mice was called the D + Q group. Femurs from 2 week old female offspring mice were taken for further analysis.

Example 2: bone morphology study in offspring mice

1. Specimen fixation and decalcification

After the offspring mice are anesthetized, cervical dislocation is sacrificed, femurs and tibias are taken out and respectively fixed in 4% paraformaldehyde, decalcification is carried out by using 0.5M ethylene diamine tetraacetic acid (EDTA, pH 7.4), and paraffin embedding/freezing embedding is carried out after dehydration is carried out by using gradient concentration ethanol/30% sucrose, so that the growth form of the long bones of the offspring mice is researched.

2. Senescence-associated beta-galactosidase (SA-beta-Gal) staining

SA-beta-Gal staining step:

(1) the frozen sections were thawed in an oven at 37 ℃ for 30 min.

(2) Preparing a beta-Galactosidase dye solution: 930 μ L of stating solution +50 μ L X-gal solution (20mg/mL, stored at-20 ℃ protected from light) +10 μ L A solution +10 μ L B solution.

(3) Sections were washed 1 time with PBS for 5 minutes each.

(4) 30-50. mu.L of β -Galactosidase staining solution was added to each specimen, and the sections were placed in a wet box in an oven (CO-free) at 37 ℃₂) The staining is carried out overnight (the staining time can be shortened according to the size and freshness of the specimen).

(5) PBS was washed 3 times.

(6) Eosin counterstain for 1 min.

(7) Gradient alcohol dehydration: dehydration was performed sequentially from 90% to 100% concentration gradient.

(8) And (3) transparency: the xylene soaking was performed 2 times for 10 minutes each.

(9) And (4) stripping the neutral resin.

Blue stained cells were identified as senescent cells under light microscopy. Aging cells (N/mm) within the range of 0-0.5mm below the growth plate²) Counting and quantitative analysis are carried out.

3. H & E staining

H & E staining procedure:

(1) and (3) baking the paraffin section in an oven at 60 ℃ for 20 minutes to prevent the specimen from being detached in the dyeing process.

(2) Dewaxing: the xylene soaking was performed 2 times for 15 minutes each.

(3) Gradient alcohol rehydration: the rehydration is carried out sequentially from a concentration gradient of 100% to 70%.

(4) Hematoxylin staining was performed for 5 minutes (time adjusted appropriately for different concentrations).

(5) Returning blue: the water was rinsed with tap water for 10 minutes.

(6) Differentiation: 1% ethanol hydrochloride is rapidly differentiated within 5 seconds, otherwise, excessive differentiation causes excessive decolorization of hematoxylin.

(7) Continuing to return blue: the tap water continued to flush for 10 minutes.

(8) Eosin staining for 2 minutes (time adjusted appropriately according to specimen size and freshness).

(9) Gradient alcohol dehydration: dehydration was performed sequentially from 90% to 100% concentration gradient.

(10) And (3) transparency: the xylene soaking was performed 2 times for 10 minutes each.

(11) And (4) stripping the neutral resin.

4. Goldner trichromatic dyeing

Goldner trichrome staining procedure:

(2) Dewaxing: the xylene soaking was performed 2 times for 15 minutes each.

(4) Weigart iron hematoxylin staining for 1 minute (time adjusted appropriately according to specimen size and freshness).

(5) Returning blue: the mixture was rinsed with tap water for 5 minutes.

(7) Continuing to return blue: the tap water was flushed for an additional 5 minutes.

(8) Acid Ponceau staining for 3 minutes (time adjusted appropriately depending on specimen size, freshness).

(9) The staining was stopped with 1% acetic acid solution and washed.

(10) Orange G staining for 5min (time adjusted as appropriate for Acid Ponceau staining).

(11) The staining was stopped with 1% acetic acid solution and washed.

(12) And (4) staining for 5 minutes in bright green (the time is properly adjusted according to the size and freshness of the specimen).

(13) The staining was stopped with 1% acetic acid solution and washed 3 times.

(14) The distilled water was slightly washed.

(15) Rapidly dehydrated with anhydrous ethanol 2 times for 2 minutes each time.

(16) And (3) transparency: the xylene soaking was performed 2 times for 2 minutes each.

(17) And (4) stripping the neutral resin.

Under light microscopy (Olympus, BX53), cells that are attached to the periphery of trabecular bone in a flat, columnar shape and abundant in cytoplasm were identified as osteoblasts. Osteoblasts (N/mm) within the range of 0-0.5mm below the growth plate²) Counting and quantitative analysis are carried out.

5. Immunofluorescence staining

And (3) immunofluorescence staining of the frozen section:

(1) the frozen sections were thawed in an oven at 37 ℃ for 30 min.

(2) Sections were washed 1 time with PBS for 5 min.

(3) And (3) sealing: blocking with 3% BSA (containing 0.1% Triton-X100) for 1 hour.

(4) Primary antibody incubation: after removing the blocking solution by pipetting, primary antibody (1% BSA, diluted with 0.1% Triton-X100) was added directly, taking care that the sections were not allowed to dry and incubated overnight at 4 ℃.

(5) The primary antibody was recovered the next day and washed 3 times with PBS to wash out the primary antibody as clean as possible, but the primary antibody was not shaken to prevent flaking for 5 min/time.

(6) And (3) secondary antibody incubation: the secondary antibody (1% BSA, diluted with 0.1% Triton-X100) was diluted in the appropriate ratio and incubated at room temperature for 1 hour, at which time it was protected from light.

(7) PBS was washed 3 times, 5 min/time, and then, the mixture was protected from light.

(8) Sealing with DAPI sealing agent, storing at-20 deg.C in dark, and taking pictures as soon as possible.

Images were taken under a fluorescence microscope (Olympus, BX 53). The area of positive staining or relative staining intensity was determined at 0-0.5mm below the growth plate for quantitative analysis.

As shown in FIG. 1, which is a graph of femoral metaphysis staining of offspring female mice of 2 weeks of age in offspring, the results show that dasatinib and quercetin (D + Q) can inhibit senescence of cells in the long metaphysis of offspring caused by PDE, improve the loss of PDE osteoblasts, and improve bone phenotype, wherein, A is a graph of senescence-associated β -galactosidase (SA- β -Gal) staining, and from left to right are a control group, a model group, and a D + Q group, it can be seen that the number of cells in the metaphysis of the offspring mice treated with D + Q is significantly lower than that of the model group, and B is hematoxylin-eosin (H + Q)&E) And a staining graph C is a Goldner's-trycrome staining graph, and a control group, a model group and a D + Q group are sequentially arranged from left to right, so that the number of metaphyseal bone trabeculae and the number of formed bones of the PDE progeny mice after D + Q treatment are obviously increased compared with the model group. D is an immune fluorescence (green) image of Nestin, and D + Q-treated PDE progeny mouse Nestin⁺The number of cells was significantly increased compared to the model group.

As shown in FIG. 2, the histograms of the A, B and C plots show SA- β -Gal at the metaphysis of each group of mice⁺Osteoblasts and Nestin⁺Cell number, A: SA- β -Gal in a unit area of femoral metaphyseal tissue⁺Quantitative analysis of the relative number of cells. B: quantitative analysis of the number of osteoblasts per unit area of the metaphyseal tissue of the femur. C: femoral metaphyseal tissue unit area Nestin⁺Quantitative analysis of cells it can be seen that the D + Q group compares with SA- β -Gal of the model group mice⁺Decreased cell number, osteoblasts and Nestin⁺The number of cells is increased, which shows that the elimination effect of dasatinib and quercetin (D + Q) on aged cells can improve the reduction of the mouse osteoblastic lineage cells of offspring caused by PDE.

Example 3 flow cytometric and statistical analysis of PDE progeny mice

Flow cytometry

Nestin⁺Cell detection:

(1) separating femur and tibia, removing soft tissue, and removing cartilage at both ends. The surface of the bone is wiped clean by sterile gauze, and periosteum and connective tissue are removed.

(2) Preparing a protease digestive juice: collagenase A at 2.0mg/mL and trypsin at 2.5 mg/mL. Adding a little digestive juice into the treated bone tissue, and shearing with scissors to form chyle shape.

(3) Sufficient digest was added, protected from light, shaken, and digested at 37 ℃ for 20 minutes.

(4) Digestion was stopped by adding 2 times complete medium (containing 15% FBS), tissue-sieving, centrifugation at 1200rpmin for 5min, and the precipitate was collected and washed 2 times with PBS.

(5) Adding 1mL of erythrocyte lysate for resuspension, and incubating for 0.5-1 min.

(6) After centrifugation at 1200rpm, 4 ℃ for 5min, PBS was washed once, centrifuged again, resuspended in PBS, and the cells were counted.

(7) At 10⁶Cell/100 μ L system CD45 (cell surface marker) antibody was incubated in flow tube for 30min on ice in a light-tight shaker.

(8) Washing with 1mL PBS 1-2 times (1200rpm, 4 ℃,5 min).

(9) Cells were resuspended in 100. mu.L of 2% paraformaldehyde and fixed for 10-15 min.

(10)1200rpmin, 5min centrifugation, PBS wash 1-2 times. Add 100. mu.L of PBST (0.5% Tween-20) to rupture the membrane and incubate on ice for 10-15 min.

(11)1200rpmin, 5min centrifugation, PBS wash 1-2 times.

(12) At 10⁶Cells/100. mu.L system Nestin (intracellular antigen) antibody was incubated in flow tube and on ice in dark shaker for 30 min.

(13) 1mL of PBS was added and washed 1 time (1200rpm, 4 ℃ C., 5min), and 500. mu.L of PBS was used to resuspend the cells.

(14) And (6) performing detection on the machine.

Mesenchymal stem cell detection:

(1) separating femur and tibia, removing soft tissue, and cutting cartilage at both ends. Cells in the medullary cavity were flushed out with a syringe and stored on ice.

(2) Adding 1mL of erythrocyte lysate for resuspension, and incubating for 0.5-1 min.

(3) After centrifugation at 1200rpm, 4 ℃ for 5min, PBS was washed once, centrifuged again, resuspended in PBS, and the cells were counted.

(4) At 10⁶Cell/100. mu.L System CD45, CD29, CD105, Sca-1 (all of which areCell surface marker) antibody, incubated on ice in a dark shaker for 30 min.

(5) 1mL of PBS was added and washed 1 time (1200rpm, 4 ℃ C., 5min), and 500. mu.L of PBS was used to resuspend the cells.

(6) And (6) performing detection on the machine.

Flow cytometry analysis was performed on a BD-LSRFortessa flow cytometer (BD Biosciences, San Jose) and analyzed using FlowJo software (BD Life Sciences, San Jose).

As shown in fig. 3, is CD45^-Nestin⁺Group flow result graph, wherein, A: flow cytometry analysis of sets of CD45^-Nestin⁺Representative pictures of osteoprogenitor cells. B: the results of the flow cytometric analysis were compared quantitatively. The results showed CD45 in bone marrow of PDE progeny mice^-Nestin⁺The percentage of the population is obviously reduced, which shows that the elimination effect of the dasatinib and the quercetin (D + Q) on the aged cells can improve the reduction of the mouse osteoprogenitor cells of the offspring caused by PDE.

As shown in fig. 4, is CD45^-CD29⁺CD105⁺Sca-1⁺Group flow result graph, wherein, A: flow cytometry analysis of sets of CD45^-CD29⁺CD105⁺Sca1⁺Representative pictures of bone marrow mesenchymal stem cells. B: the results of the flow cytometric analysis were compared quantitatively. The results showed CD45 in bone marrow of PDE progeny mice^-CD29⁺CD105⁺Sca-1⁺The percentage of the population is significantly reduced, indicating that the elimination of senescent cells by dasatinib and quercetin (D + Q) can improve the reduction of BMSCs in progeny mice caused by PDE.

Flow analysis results show that D + Q treatment significantly improves the inhibition of BMSCs by PDE.

In conclusion, PDE may induce the aging of bone local cells of the offspring mice to cause the change of bone local microenvironment, which results in the reduction of the number of H-type blood vessels, the reduction of the proliferation capacity and osteogenic differentiation capacity of bone marrow mesenchymal stem cells (BMSCs), and the reduction of the bone growth amount of the offspring mice. And the current clinical results also show that the prenatal use of glucocorticoids can improve the survival rate of premature infants, but also cause a lot of adverse effects on offspring. The research of the invention shows that the elimination of aged cells is a potential new method for treating PDE bone developmental toxicity, and the dasatinib and the quercetin are expected to be used for preventing or treating the side effect of glucocorticoid taking during pregnancy.

While the invention has been disclosed with reference to specific embodiments, it will be apparent that other embodiments and variations of the invention may be devised by those skilled in the art without departing from the true spirit and scope of the invention, and it is intended that the following claims be interpreted to include all such embodiments and equivalent variations. In addition, the contents of all references cited herein are hereby incorporated by reference.

Claims

1. Use of a pharmaceutical composition comprising dasatinib or a pharmaceutically acceptable salt thereof and quercetin or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the prevention and/or treatment of glucocorticoid side effects.

2. The use according to claim 1, wherein the mass ratio of dasatinib or the pharmaceutically acceptable salt thereof to quercetin or the pharmaceutically acceptable salt thereof is 1: 5-20.

3. The use according to claim 1 or 2, wherein the effective dose of the pharmaceutical composition is 0.15-3 mg/day.

4. The use according to claim 1 or 2, wherein the pharmaceutical composition is any one of an oral formulation, an injectable formulation, an inhaled formulation, an intra-oral formulation, a topical formulation or any combination thereof.

5. Use according to claim 1 or 2, wherein the pharmaceutical composition further comprises other drugs for the treatment of glucocorticoid side effects.

6. Use according to claim 1 or 2, wherein the prevention and/or treatment of glucocorticoid side effects is achieved by the elimination of senescent cells.

7. The use according to claim 1 or 2, wherein the glucocorticoid side effect is obesity and cushing's syndrome, hypothalamic-pituitary-adrenal axis function inhibition, depression, memory loss, hyperlipidemia, hyperglycemia, cardiovascular diseases, skeletal muscle catabolism, osteoporosis, femoral head necrosis, osteogenic differentiation inhibition, gastrointestinal bleeding, cortical cataract or the side effect of prenatal glucocorticoid application on offspring.

8. Use according to claim 1 or 2, wherein the glucocorticoid side effect is a side effect of prenatal glucocorticoid application on the offspring.

9. The use according to claim 8, wherein the side effect of prenatal glucocorticoid application on the offspring is suppression of bone-in-bone development.

10. Use according to claim 1 or 2, wherein the glucocorticoid is prednisone, methylprednisolone, betamethasone, beclomethasone dipropionate, prednisolone, hydrocortisone or dexamethasone, preferably dexamethasone.