CN111803489A - Application of michelia lactone and derivatives thereof in treatment of pituitary adenoma - Google Patents

Abstract

The invention relates to application of michelia lactone or a derivative thereof or a pharmaceutically acceptable salt thereof in preparing a medicament for treating pituitary adenoma, wherein the specific mechanism is that michelia lactone or a derivative thereof or a pharmaceutically acceptable salt thereof induces cells to generate apoptosis and autophagic death by promoting the expression of apoptosis and autophagy-related protein, so that the inhibition effect on pituitary adenoma such as prolactin adenoma, clinical nonfunctional pituitary adenoma, growth hormone adenoma and the like is achieved.

Description

Application of smilolide and its derivatives in the treatment of pituitary adenomas

technical field

The invention relates to the field of medicine, in particular to the new application of laugholide and its derivatives in the treatment of pituitary adenoma.

Background technique

The annual incidence of pituitary adenomas is as high as 7.5 to 15/100,000 people, accounting for 16.7% of primary intracranial tumors. Prolactinoma is the most common functional pituitary adenoma, accounting for about 40% of all pituitary adenomas. %～66%. According to the current population of 1.3 billion in my country, the annual number of new cases of prolactinoma is about 60,000 to 120,000. In fact, with the development of endocrine diagnostic and neuroimaging techniques, the number of pituitary prolactinoma cases may be higher than the above figures. The clinical manifestations of pituitary prolactinoma are mainly male impotence/hyposexual function caused by endocrine disorder, female amenorrhea lactation/infertility, and the mass effect formed by tumor enlargement, which oppresses the vision caused by adjacent structures. Symptoms such as disorders and hypopituitarism seriously endanger the life safety and quality of life of patients.

At present, dopamine agonists (DA), including bromocriptine (BRC) and cabergoline (CAB), are the first choice for prolactin adenomas, mainly by interacting with dopamine type 2 receptors on the surface of tumor cells. The combination of Dopamine receptor type 2 (D2R) can activate the downstream apoptosis signaling pathway and reduce the secretion of prolactin, and finally achieve the effect of inhibiting tumor growth. The lack of expression of D2R in tumor cells has become an important factor in the efficacy of DA-inhibited. Clinical follow-up work has shown that DA can reduce serum prolactin to normal levels in 75%-90% of patients with prolactinoma and significantly reduce tumor volume, but clinically, 10%-25% of patients are still ineffective against DA treatment, even if Surgery or stereotactic radiotherapy, the control of blood prolactin is still not ideal, called drug-resistant cases. Therefore, how to provide a new, effective and safe treatment plan for this part of DA resistant cases has become a hot issue to be solved urgently in the treatment of prolactinoma. At present, the research involving Chinese herbal medicine in the treatment of pituitary prolactinoma is still blank.

Natural products are of great significance for the discovery, design and synthesis of new drugs, and are also an important source of biologically active substances and innovative drugs. Approved drugs such as Taxol, Docetaxel, Vinorelbine ( Vinorelbine), hydroxycamptothecin (camptothecin), artemisinin (Artemisinin), etc. are all derivatives of natural products or their analogs. The advantages of natural products as drug sources are: traditional chemotherapeutic drugs for the treatment of tumors have drug resistance problems, while natural products have a unique role in the discovery of anti-cancer drugs, from which it is possible to screen out the leaders that kill tumor cells with high efficiency and low toxicity. Compounds, so as to develop new drugs for the treatment of malignant tumors, reduce the suffering of patients and improve the quality of life of patients.

Micheliolide (alias: Micheliolide; MCL, Micheliolide), is a guaiac-type extract isolated from the root bark of Michelia champaca (Michelia champaca) and Taiwan (Michelia compressa) of Magnoliaceae Hemiterpene lactone monomer of traditional Chinese medicine. In traditional Chinese medicine, it is mainly used for anti-inflammatory and analgesic, and for the treatment of fever, migraine and rheumatoid arthritis. The chemical structural formula of laugholactone is:

It was previously discovered that smilide and its derivatives are used to treat the following cancers: leukemia, breast cancer, prostate cancer, liver cancer, esophagus cancer, gastric cancer, oral cancer, Hodgkin's lymphoma, pancreatic cancer, rectal and colon cancer, cervical cancer, Non-Hodgkin's lymphoma, glioma, melanoma, bladder cancer, ovarian cancer, thyroid cancer, prostate cancer and Kaposi's sarcoma, and have applied for patents (application number: 201010153701.0; 201010153685.5, etc.). The existing method for preparing smilide is mainly through chemical synthesis. Chinese Patent Application No. 201010153685.5 discloses a preparation method of smilide, using parthenolide as a raw material, in a suitable organic solvent, rearranged under the catalysis of Lewis acid The laugholide is obtained with a yield of more than 60%. CN201711285215.2 discloses a new class of laugholide derivatives and their use in the preparation of anti-tumor and immunotherapy medicines.

In recent years, with the continuous development of traditional Chinese medicine in the motherland, the role of laurolactone (MCL) in cancer treatment has gradually emerged. Studies have confirmed that MCL can induce tumor cell apoptosis by inhibiting the NF-κB signaling pathway and regulating cellular ROS levels. . With the continuous progress of medical chemistry technology, MCL's dimethylaminoMichael adduct of MCL (DMAMCL) has higher plasma pharmacokinetic stability than MCL (its structural formula is shown below), more durable The drug release time and stronger water solubility make it have broader application prospects. In diseases of the central nervous system, it can efficiently pass through the blood- _{brain barrier} and has little side effects on nerve cells. Glioma cells die and ultimately inhibit tumor cell growth, which is considered a potential therapeutic drug for malignant gliomas. At present, MCL has entered clinical phase I trials in China (ID: 2017L04073) and Australia (ID: ACTRN12616000228482).

At present, the treatment reports of laurolactone (MCL) and its derivatives in central nervous system tumors mainly focus on malignant glioma cell tumors, but there is no report on the therapeutic effect of benign tumor pituitary prolactinoma.

SUMMARY OF THE INVENTION

In view of the above problems, the inventors unexpectedly found that laugholide and its derivatives can inhibit pituitary adenomas by inducing apoptosis and autophagic death, and especially have a good therapeutic effect on pituitary prolactinomas.

In one aspect, the present invention relates to the use of a laugholide or a derivative thereof, or a pharmaceutically acceptable salt thereof, represented by the following general formula in the preparation of a medicament for the treatment of pituitary adenoma, wherein the laugholide or its derivative is Derivatives are selected from the following compounds:

in:

R ₁ is hydrogen or C _1-8 acyl, tetrahydropyrrole formyl, tetrahydrofuran formyl, Ar-C _1-4 acyl, Ar-OC _1-4 acyl, Ar-SC _1-4 acyl, YNC _1-4 acyl ; wherein C _1-8 acyl is preferably selected from straight-chain or branched alkanoyl, alkenoyl and alkynyl;

Ar is aryl or substituted aryl; Y is heterocyclic aryl or substituted heterocyclic aryl; Ar is preferably selected from phenyl, benzoyl, naphthyl, pyridyl, furyl, thienyl, pyrrolyl, isoxazole base, isothiazolyl, pyrazolyl, oxazolyl, thiazolyl, imidazolyl, pyridazinyl, pyrazinyl, benzofuranyl, benzothienyl, indolyl, quinolinyl, isoquinolinyl , purinyl, benzoxazolyl, benzothiazolyl, etc.; Y is preferably selected from uracil, tetrahydroisoquinolinyl, phthalimide, and naphthalimide. R ₂ is C _1-7 acyl group, C _1-6 alkyl group; wherein, C _1-7 acyl group is preferably selected from acetyl, propionyl, butyryl, isobutyryl, chloroacetyl, benzoyl, etc.; C _{1 -6} alkyl is preferably selected from methyl, ethyl, propyl, isopropyl, butyl, isobutyl, cyclopentyl, cyclohexyl, etc.; or R ₁ =R ₂ ;

R ₃ and R ₄ are combined into a double bond, or R ₃ is hydrogen, and R ₄ is -CH ₂ NR ₅ R ₆ , wherein R ₅ and R ₆ are respectively C _1-4 hydrocarbon groups.

In another embodiment of the present invention, the present invention relates to the use of a pharmaceutically acceptable salt of trihalide or a derivative thereof as shown above in the manufacture of a medicament for the treatment of pituitary adenoma, wherein said trihalide Esters or derivatives thereof are hydrochloride, sulfate, bromate, fumarate, acetate or citrate, and the like.

In another preferred embodiment, the structure of the laugholide or its derivative in the salt-forming compound containing laugholide or its derivative is shown by formula (I) or formula (II). Wherein, the compound represented by formula (I) is smilolide (MCL), and the compound represented by formula (II) is dimethylamino Michael addition compound DMAMCL.

In another more preferred embodiment, the laugholide or its derivative in the use is a hydrochloride or fumarate compound, and its structural formula is as follows:

Among them, DMAMCL salt-forming compounds such as DMAMCL fumarate (formula V) can slowly and steadily release DMAMCL (formula II) under normal physiological conditions, and DMAMCL further releases the active pharmaceutical ingredient MCL (formula I).

In another embodiment of the present invention, the present application discloses the use of tricholactone or a derivative thereof, or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the treatment of pituitary adenoma, wherein the pituitary gland The tumors are prolactin adenoma, clinically nonfunctioning pituitary adenoma, growth hormone adenoma, etc.

In another preferred embodiment of the present invention, the present application discloses the use of tricholactone or a derivative thereof, or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the treatment of pituitary adenoma, wherein the pituitary The adenomas are prolactin adenomas, clinically non-functioning pituitary adenomas, growth hormone adenomas, etc.; said tricholactone or its derivatives, or its pharmaceutically acceptable salts are selected from formula (I), formula (II) ), a compound represented by formula (V), formula (VI) or any combination thereof, or a traditional Chinese medicine extract comprising a compound of formula (I), formula (II), formula (V) or formula (VI).

In another preferred embodiment of the present invention, the inventors have unexpectedly discovered through a large number of experiments that laugholactone or its derivatives have a good inhibitory effect on prolactin adenomas, whether they are prolactin adenomas resistant to DA treatment or DA-sensitive prolactin adenomas have good inhibitory effect. Wherein, the laugholactone or its derivative is preferably a compound represented by formula (I), formula (II), formula (V) or any combination thereof, or a compound containing formula (I), formula (II) or formula (V) ) compounds of traditional Chinese medicine extracts.

In order to confirm the therapeutic effect of laurolactone (MCL) or its derivatives in pituitary adenomas, the inventors selected MMQ cells (highly expressing D2R, resistant to DA treatment-sensitive) and GH3 cells (low/no D2R expression, usually resistant to DA treatment) were subjected to MCL drug intervention.

The results of in vitro experiments showed that MCL had a strong cytostatic effect on both, and the GH3 cell line resistant to DA treatment was more sensitive. However, after the nude mice with subcutaneous tumor formation of GH3 cells were orally treated with DMAMCL drug, it was found that it could effectively inhibit the growth of tumors (see Figure 1). The half-inhibitory concentration (IC ₅₀ ) of MCL in GH3 cells was 10 μM, and the half-inhibitory concentration (IC ₅₀ ) of MMQ cells was 20 μM, which were both lower than the therapeutic concentration of DA in the inventor’s previous studies (the IC ₅₀ of bromocriptine was 50 μM, the _IC50 of cabergoline is 25 μM).

After the corresponding research on the mechanism of MCL drug action, it was found that, unlike the previous classic pathway that relies on the combination of DA and D2R to promote tumor cell apoptosis, MCL mainly induces apoptosis (see Figure 2) and autophagic cells. Death (see Figure 3) achieved the inhibitory effect of the drug on prolactinoma, and this effect was more pronounced in the GH3 cell line resistant to DA treatment. The above results suggest that MCL has a good inhibitory effect on prolactinoma, even DA-resistant tumor cells, and the mechanism may involve MCL-mediated apoptosis and autophagic death.

The inventors examined the GH3 and MMQ cell lines before and after drug treatment by RNA sequencing, and the results suggested that MCL may be the death of tumor cells mediated by activating the MAPK signaling pathway. Inhibition of the phosphorylation activities of ERK, JNK and P38, key genes of MAPK signaling pathway in GH3 and MMQ cells, can effectively reverse the inhibitory effect of MCL (see Figure 4). In order to further confirm that the inhibitory effect of MCL on pituitary prolactinoma is also effective in living animals, the inventors used an estrogen-induced rat pituitary prolactinoma model for drug intervention treatment. Its research found that MCL can effectively inhibit the growth of tumor tissue. Compared with the negative control group, drug treatment could effectively inhibit the volume and mass of tumor tissue. In addition, the inventors detected protein in tumor tissue samples and found that MCL can effectively activate the expression of MAPK signaling pathway in tumor tissue cells, and inhibit tumor growth through apoptosis and autophagic death (see Figure 5). Finally, in order to further explore whether MCL also has inhibitory effect on other types of pituitary tumors other than prolactinoma. The inventors cultured some tumor tissues of patients with transsphenoidal endoscopic pituitary tumor resection (see Table 1 for the basic information of patients), and treated them with MCL drugs in vitro. The results showed that 8 cases of human pituitary tumor cells (1 case of lactating (6 cases of clinically nonfunctioning pituitary adenoma, 1 case of growth hormone adenoma), 6 cases of tumor tissue were sensitive to MCL treatment, tumor tissue activity was significantly inhibited, only two cases of clinically nonfunctioning pituitary adenoma were sensitive to MCL Treatment was ineffective (see Figure 6).

In another embodiment of the present invention, the present invention provides a process for the preparation of a laugholide of the above formula (I), the process comprising subjecting parthenolide in a suitable organic solvent under the catalysis of a suitable acid catalyst Preparation of smiloid. A preferred method for preparing smilide-containing lactones includes the following steps: weighing an appropriate amount of parthenolide and an acid catalyst, adding an appropriate organic solvent, stirring and reacting for 0.5 to 48 hours, pouring the reaction solution into an appropriate amount of ice water, extracting, washing, and drying , Concentrated under reduced pressure to obtain laughing lactones. For the specific process, please refer to Chinese patent applications 201010153701.0 and 201010153685.5, etc.

For the preparation method of the smilide-containing derivatives represented by formulas (III, IV), reference may be made to Chinese patent application CN201711285215.2.

For the preparation method of the smilide-containing derivative salt represented by formula (V, VI), reference may be made to the literature Yinghong An et al, Micheliolide Derivative DMAMCL Inhibits Glioma Cell Growth In Vitro and InVivo; PLoS One.2015;10(2):e0116202. For the preparation of the smilactone-containing derivative DMAMCL fumarate represented by formula (V), further reference may be made to CN201410071673.6.

The present invention also provides a pharmaceutical composition comprising the above-mentioned laugholide or its derivative, said pharmaceutical composition comprising a therapeutically effective amount of laugholide or its derivative, or a pharmaceutically acceptable salt thereof and a pharmacy an acceptable carrier or excipient.

When laugholactone or its derivatives and pharmaceutically acceptable salts thereof are used as medicines, they can be used directly or in the form of pharmaceutical compositions. The pharmaceutical composition contains 0.1%-99%, preferably 0.5%-90%, 1%-80% or 5%-50% of laugholide or its derivatives and pharmaceutically acceptable salts thereof, and the rest are drugs Pharmaceutically acceptable carriers and/or excipients that are nontoxic and inert to humans and animals or used in combination with other anticancer drugs.

The pharmaceutical composition of the present invention can be prepared into capsules, tablets, powders, granules, syrups or similar dosage forms for oral administration, or for parenteral administration by injection, ointment, suppository or similar dosage forms. These pharmaceutical preparations can be produced by ordinary methods using well-known adjuvants in the art, such as binders, excipients, stabilizers, disintegrants, flavoring agents, lubricants, etc., and can also be prepared as controlled-release medicaments. Forms, sustained-release dosage forms, and various microparticle drug delivery systems.

Although the dosage will vary with the symptoms and age of the patient, the nature and severity of the disease or disorder, and the route and mode of administration, in the case of oral administration to an adult patient, smilolide or its derivatives, or its pharmaceutical The above acceptable salts are normally administered in a total daily dose of 1 to 1000 mg, preferably 5 to 500 mg, in single doses, or in divided doses; for example, two or three times daily; in the case of intravenous injection, one to Three doses of 0.1 to 100 mg, preferably 0.5 to 50 mg, are administered.

The present invention provides a method of treating a disease, the method comprising administering to a subject in need of treatment a therapeutically effective amount of a laugholactone or a derivative thereof of the present invention, or a pharmaceutically acceptable salt thereof. The term "subject" includes humans and non-human mammals, such as non-human primates, sheep, dogs, cats, cows and horses, etc. Preferred subjects are human patients.

Description of drawings

Figure 1: The inhibitory effect of smilolide (MCL) on the activity of GH3 and MMQ. in:

(A) The experimental results of CCK8 interfering in the activity of GH3 and MMQ cells under MCL drug concentration-dependent conditions. The inventors found that with the increasing concentration, the activity of GH3 and MMQ cells was significantly inhibited. The half-inhibitory concentration (IC ₅₀ ) of GH3 cell line after MCL intervention was about 10 μM (A, left), while the half-inhibitory concentration (IC ₅₀ ) of MMQ was about 20 μM (A, right), both lower than those of dopamine receptor agonists ( DA) ( _IC50 of ₅₀ μM for bromocriptine and 25 μM for cabergoline).

(B) The results of CCK8 experiments in which MCL drugs interfered with the activity of GH3 (B, left, 10 μM) and MMQ (B, right, 20 μM) cells in a time-dependent manner. The inventors found that the activity of GH3 and MMQ cells was significantly inhibited with the continuous increase of the action time.

(C) Inhibition of the colony formation rate of GH3 and MMQ cells by MCL. After taking the cells in the logarithmic growth phase for cell counting, 500 cells were inoculated into 6-well plates, cultured for 14 days, and then treated with different concentrations of MCL for 24 hours, and then stained with crystal violet and photographed.

(D-F) DMAMCL (100 mg/kg) was administered orally to nude mice with subcutaneous tumor formation of GH3 cells, and it was found that it could effectively inhibit the growth of tumor in nude mice. ***P<0.001; **P<0.01; *P<0.05 vs, Control group.

Figure 2: Laughtone-containing (MCL) induces apoptosis in GH3 and MMQ cells. in:

(A-B) Flow cytometric apoptosis assay showed that MCL could significantly induce early and late apoptosis in GH3 and MMQ cells.

(C-D) After GH3 and MMQ cells were treated with MCL (10 μM and 20 μM), the apoptosis-related proteins in the cells were detected by Western blot, and it was found that the expression of apoptosis-related proteins increased with MCL drug action time (C) or drug concentration ( D) changes with the increase.

Figure 3: Laughing lactone (MCL) can significantly activate autophagy in GH3 and MMQ cells. in:

(A-B) Lactone (MCL) can gradually enhance the autophagy of GH3 and MMQ cells and promote the autophagy-related proteins ATG7, P62 and LC3 under the action time-dependent condition (A) and the drug concentration-dependent condition (B). - Expression of II.

(C) After 24 hours of MCL treatment, the fluorescent spot intensity of autophagy-related protein LC3-II was significantly increased. MCL can induce the formation of LC3-II fluorescent spot sign, while in the control group, the red fluorescence of LC3-II protein is scattered and uniformly distributed in the cytoplasm.

(D) Transmission electron microscopy results showed that 24 hours after MCL drug treatment, the cytoplasmic autophagosomes and autophagolysosomes of GH3 and MMQ cells were significantly increased compared with the control group.

Fig. 4: Laughing lactone (MCL) can inhibit tumor cell growth by activating MAPK signaling pathway. in:

(A-B) RNA sequencing analysis of changes in the expression of related proteins in GH3 and MMQ cells before and after MCL drug treatment. The results of gene heat map (A) and GO analysis (B) showed that the MAPK signaling pathway was significantly activated before and after MCL drug treatment.

(C) In order to further confirm the reliability of the sequencing analysis results, the inventors performed Western blot detection of the key regulatory proteins ERK, P38 and JNK of the MAPK signaling pathway in GH3 and MMQ cells before and after drug treatment. The results showed that after MCL treatment, the key protein The phosphorylation activity level was significantly increased compared with that before treatment.

(D-E) After the inventors used ERK protein phosphatase inhibitor to inhibit its phosphorylation level (D), the inhibitory effect of MCL on GH3 and MMQ cells was significantly weakened (E).

(F-G) After the inventors used JNK protein phosphatase inhibitor to inhibit its phosphorylation level (F), the inhibitory effect of MCL on GH3 and MMQ cells was significantly weakened (G).

(H-I) After the inventors used P38 protein phosphatase inhibitor to inhibit its phosphorylation level (H), the inhibitory effect of MCL on GH3 and MMQ cells was significantly weakened (I).

Figure 5: Tricholactone (MCL) inhibits estrogen-induced growth of rat pituitary prolactinoma tumor tissue. in:

(A-B) After 6 weeks of estradiol induction, DMAMCL (200 mg/kg) drug intervention was performed on a mature rat pituitary prolactinoma model for 2 weeks. MRI examination results showed (A) that DMAMCL drug intervention could significantly inhibit the growth of tumor tissue, and the volume (B, left) and mass (B, right) of the tumor were significantly inhibited.

(C) After immunohistochemical detection of tumor tissue, it was found that the phosphorylation of key regulatory proteins of MAPK signaling pathway, ERK, P38 and JNK, was significantly increased in the tumor tissue after drug treatment.

(D) Western blot detection of tumor tissue showed that the expression of apoptosis and autophagic death-related proteins was significantly changed in the tumor tissue after drug treatment.

Figure 6: Laughing lactone (MCL) inhibits pituitary adenoma growth in other tumor types. in:

(A) After primary culture of 8 cases of human pituitary adenoma tissues, they were treated with MCL (20 μM) for 24 hours, and their cell viability was detected by CCK-8 assay. The results showed that among the 8 primary tumor cells, 6 were sensitive to MCL treatment, and 2 clinically nonfunctioning pituitary adenomas were ineffective for MCL treatment.

(B-C) Western blot detection of tissue proteins in 8 human pituitary adenomas showed that (B), in the 6 tissue samples treated by MCL, the phosphorylation of MAPK key regulatory proteins ERK, JNK, and P38 was significantly enhanced, and autophagy was significantly enhanced. The expressions of related proteins ATG7, P62 and LC3-II were significantly increased. (C) Immunohistochemical HE staining results of 8 cases of human pituitary adenoma tissue samples. NFPA, clinically nonfunctioning pituitary adenoma; GH, growth hormone adenoma; PRL, prolactin adenoma.

Detailed ways

The preparation of embodiment 1 laughter (MCL)

Take Taiwan Hanxiao as raw material, pulverize 20-40 mesh, add it to the extraction kettle, use anhydrous methanol as the entrainer, extract with supercritical _CO2 , collect the extract, recover methanol, get the extract, and use petroleum ether to reflux to remove fat Soluble impurities, then thermally dissolve the degreasing material with low-carbon alcohol, add activated carbon to reflux for decolorization, recover the solvent to an appropriate volume from the decolorizing solution, purify it by preparing a liquid phase ODS RP-C18 reversed-phase column, and wash it with a mixed solution of methanol and water. Remove, concentrate the corresponding components, and recrystallize from petroleum ether-acetone (2:1, V/V) to obtain MCL product. The supercritical _CO extraction conditions are that the extraction temperature is 45-60°C, the extraction pressure is 23-34MPa, the temperature of the separation kettle I is 40-50°C, the pressure is 8-10MPa, and the temperature of the separation kettle II is 35-40°C, The pressure is 4-6MPa, and the extraction time is 2-6h. The petroleum ether is degreased, adding 2-5 times the volume of the extract with petroleum ether, heating and refluxing for 1-3 times, centrifuging, and separating the solid and liquid to obtain a degreased product. The low-carbon alcohol is a C1-C4 alcohol, preferably ethanol, methanol or isopropanol. The mixed solution of methanol and water refers to a mixed solution in which the volume percentage of methanol is 83% and the volume percentage of water is 17%. The MCL drug used in this experiment was recrystallized and dissolved in dimethyl sulfoxide (DMSO) to form a 20 mM stock solution, which was then stored in a -80°C refrigerator for later use.

Example 2 Preparation of smilide-containing derivatives (DMAMCL)

The preparation method of lalide-containing derivative (DMAMCL): Me ₂ NH·HCl (1.5 g, 18 mmol), K ₂ CO ₃ (5.0 g, 36 mmol) and CH ₂ Cl ₂ (100 ml) were mixed at room temperature and formed into a homogenous mixture. quality solution. MCL (300 mg, 1.2 mmol) was then mixed with the homogeneous solution, and the mixture was stirred at room temperature for 3 hours. The above reaction solution was concentrated and resuspended using CH ₂ Cl ₂ , and then separated by deionized water chromatography, and the obtained separated solution was filtered off using Na ₂ SO ₄ . The filtered material was dissolved in _{CH2Cl2 (} 5ml) and treated with fumarate (0.1N) to adjust the pH of the solution to 4. Finally, the aqueous phase was extracted with CH ₂ Cl ₂ (10 ml) and lyophilized to obtain compound DMAMCL. The DMAMCL drug used in this experiment was dissolved in deionized water to form a 400 mg/ml stock solution, which was then stored in a -80°C refrigerator for later use.

Example 3 Cytosuppressive test of lagalactone and its derivatives on prolactin adenoma

1) Take cells in logarithmic growth phase in good condition, wash 2-3 times with PBS, and culture with F12 medium at 37°C, 5% CO ₂ , medium supplemented with 2.5% fetal bovine serum, 15% horse serum and 100U/ml penicillin/streptomycin.

2) Inoculate in a culture plate at a ratio of 300,000 cells/ml medium, use a 6-well plate for culture, and the medium in each well is 3ml;

3) After an interval of one day, take the MCL stock solution out of the -80 °C refrigerator, dissolve it to a liquid state under natural conditions, and confirm that there is no precipitation and suspended matter, calculate according to the final volume of each well of the 6-well plate is 3 ml, respectively. 0 μl, 1.5 μl, 3 μl and 7.5 μl of MCL stock solution (20 mM) were added to multiple wells of GH3 and MMQ cell culture, respectively, so that the final concentrations of MCL in GH3 and MMQ cell culture medium were 0 μM, 10 μM, 20 μM and 50 μM.

4) The medium was not changed during the period, and the cells were treated for 24 hours, and then the cell activity was detected. It was confirmed that the half-inhibitory concentration (IC ₅₀ ) of MCL drugs in GH3 cells was 10 μM, and the half-inhibitory concentration (IC ₅₀ ) of MCL drugs in MMQ cells was 20 μM.

5) Take the logarithmic growth phase cells in good condition again, wash 2-3 times with PBS, and culture with F12 medium at 37°C, 5% CO ₂ , the medium is supplemented with 2.5% fetal bovine serum, 15% horse Serum and 100U/ml penicillin/streptomycin.

6) Inoculate in a culture plate at a ratio of 300,000 cells/ml medium, use a 6-well plate for culture, and the medium in each well is 3ml;

7) After an interval of one day, take the MCL stock solution out of the -80°C refrigerator, dissolve it to a liquid state under natural conditions, and confirm that there is no precipitation and suspended matter, calculate according to the final volume of each well of the 6-well plate is 3ml, respectively 1.5 μl and 3 μl of MCL stock solution (20 mM) were added to multiple wells of GH3 and MMQ cell culture, respectively, so that the final concentration of MCL in GH3 cell culture medium was 10 μM, and the final concentration of MCL in MMQ cell culture medium was 20 μM. During the period, the medium was not changed, and the cells were treated for 0, 12, 24 and 48 hours after the follow-up experiments were performed to observe the inhibition of MCL on cell activity.

Example 4 Validation test of subcutaneous tumorigenesis of prolactin adenoma in nude mice with laugholactone and its derivatives

1) 5-week-old female BALB/c(nu/nu) nude mice were purchased from Shanghai Experimental Animal Center, and were reared adaptively in SPF animal room for one week. If there was no obvious abnormality or discomfort in the nude mice, subcutaneous implantation of GH3 cells could be carried out. Tumor Experiment.

2) The inoculation site was selected under the skin of the lower back, avoiding the forelimbs and not touching the muscles, inoculating 1×10 ⁷ GH3 cells on one side, and inoculating one nude mouse on one side. Cells were resuspended in F12 medium supplemented with 2.5% fetal bovine serum, 15% horse serum and 100 U/ml penicillin/streptomycin. The cell concentration requires that 1ml contains 1×10 ⁸ cells. Before inoculation, wipe and disinfect the needle insertion site with 75% alcohol.

3) Extract the cell suspension with a disposable needle, remove the air, puncture about 1 cm forward from the needle insertion site, and perform subcutaneous injection. When injecting, the injection volume is about 0.1ml each time. After the injection, slowly withdraw the needle to avoid leakage as much as possible. During the injection process, the cells need to be kept on ice to keep the cells in a relatively low metabolic state, and try to complete within half an hour.

4) 7-10 days after the inoculation, the tumor can be seen growing up, and the vernier caliper test is carried out. When the tumor volume is mostly close to 50mm ³ , the nude mice are randomly divided into the experimental group and the control group. The nude mice in the experimental group were orally fed with DMAMCL aqueous solution at a dose of 100 mg/kg, once a day. In the control group, only the same amount of deionized water was orally fed. The whole experiment was stopped when the tumor tissue in the control group grew to 2000 mm ³ . After the mice were killed by dislocation, the tumors were removed for subsequent experiments.

Example 5 Validation test for the intervention of laughter and its derivatives on rat prolactin adenoma model

1) The 4-week-old Fischer 344 rats were purchased from Shanghai Experimental Animal Center, and they were reared adaptively in the SPF animal room for one week. If there was no obvious abnormality or discomfort, the pituitary prolactin adenoma induction experiment could be carried out.

2) 1-cm silicone rubber capsules were subcutaneously embedded in 4-week-old Fischer 344 rats, the capsules contained 10 mg of 17β-estradiol hormone, and the capsules could slowly release estradiol hormone, thereby inducing the formation of prolactin adenomas in rats .

3) After 6 weeks, the rats were examined by MRI to further confirm the formation of prolactin adenomas, and then the rats were randomly divided into the experimental group and the control group. The nude mice in the experimental group were orally fed with DMAMCL aqueous solution at a dose of 200 mg/kg, once a day. In the control group, only the same amount of deionized water was orally fed. After taking the drug for 2 weeks, the experiment was terminated, and the model samples were examined by MRI to detect the growth of the tumor. After the mice were sacrificed, the tumor was removed for subsequent experiments.

Example 6 Explanation of the mechanism of action of smilide and its derivatives and proof of technical effect

A. The detection of cell activity showed that MCL and its derivatives had a significant inhibitory effect on prolactinoma DA-resistant cell line GH3 and sensitive cell line MMQ. (FIG. 1A) The results of CCK8 experiments on MCL drug concentration-dependent intervention in GH3 and MMQ cell activity. The inventors found that with the increasing concentration, the activity of GH3 and MMQ cells was significantly inhibited. The half-inhibitory concentration (IC ₅₀ ) of GH3 cell line after MCL intervention was about 10 μM (A, left), while the half-inhibitory concentration (IC ₅₀ ) of MMQ was about 20 μM (A, right). (Fig. 1B) The results of CCK8 experiments in which MCL drugs interfered with the activity of GH3 (B, left, 10 μM) and MMQ (B, right, 20 μM) cells in a time-dependent manner. The inventors found that the activity of GH3 and MMQ cells was significantly inhibited with the continuous increase of the action time. ( FIG. 1C ) Inhibition of the colony formation rate of GH3 and MMQ cells by MCL. After taking the cells in the logarithmic growth phase for cell counting, 500 cells were inoculated into 6-well plates, cultured for 14 days, and then treated with different concentrations of MCL for 24 hours, and then stained with crystal violet and photographed. The inventors found that with the continuous increase of the concentration, the clone formation of GH3 and MMQ cells was significantly inhibited. However, in the in vivo tumor formation experiment in nude mice, the subcutaneous tumor volume and mass of nude mice in the experimental group taking DMAMCL were significantly smaller than those in the control group (Fig. 1D). The subcutaneous tumor volume of nude mice in the DMAMCL experimental group was 154.83±68.90 mm ³ , and the subcutaneous tumor volume of the nude mice in the control group was 517.44±217.60 mm ³ (n=6, P<0.001; Figure 1E); the subcutaneous tumor mass of the nude mice in the DMAMCL experimental group was 0.09±0.03g, the subcutaneous tumor volume of nude mice in the control group was 0.33±0.17g (n=6, P=0.012; Figure 1F)

B. The results of apoptosis and protein detection by flow cytometry indicated that MCL could promote the expression of apoptosis-related proteins and induce cell apoptosis. (Figure 2A and 2B) Flow cytometry was used to identify GH3 and MMQ cells after MCL treatment for 0, 12, 24 and 48 hours. The results are shown in Figure 2A. The results show that MCL can significantly induce GH3 (Fig. 2A) and MMQ (Fig. 2B) cells exhibited early and late apoptosis. (Fig. 2C and 2D) After MCL treatment, GH3 and MMQ cell proteins were extracted and detected by Western blot. The treated cell proteins were extracted and added to the 4°C pre-chilled cell lysate with a final concentration of PMSF of 1 mmol/L. After incubation on ice for 30 minutes, centrifuge at 10000g for 15 minutes at 4°C, take the supernatant for BCA protein quantification, and heat at 100°C for 15 minutes to denature the protein. Prepare 12% separating gel and 5% stacking gel, take 30-40ug protein samples in the spotting well, voltage 80V, electrophoresis for 30 minutes, voltage 120V, and electrophoresis for 60 minutes to separate the proteins. The protein was transferred to a 0.22um PVDF membrane by wet transfer under a constant current of 170mA for 120 minutes. The membrane was taken out and placed in 5% skim milk, and sealed at room temperature for 90 minutes. Skim milk was removed and washed three times with TBST. Membranes were placed in primary antibody diluent prepared in 5% BSA and incubated overnight at 4°C. Remove the primary antibody, add 1:2000 diluted horseradish peroxidase-labeled secondary antibody after washing three times in TBST, incubate at room temperature for 60 minutes, wash several times, put the membrane in ECL luminescence solution, and image on LAS-4000 Color imaging in the instrument. As a result, as shown in Figures 2C and 2D, the expression of apoptosis-related proteins changed with the increase of MCL drug action time (Fig. 2C) or drug concentration (Fig. 2D).

C. Through transmission electron microscopy and protein detection, it was found that MCL can promote the expression of autophagy-related proteins and induce the formation of autophagosomes, mediate the autophagic death of tumor cells, and achieve the inhibitory effect on prolactinoma. (Figure 3A and 3B) GH3 and MMQ cell proteins after MCL treatment were extracted and detected by Western blot. The results were shown in Figure 3A and 3B. The expression of autophagy-related proteins increased with the time of MCL drug action (Figure 3A) or drug concentration ( Figure 3B) increased with the increase. (FIG. 3C) The expression of autophagy-related protein LC3-II was detected in cells after MCL treatment. The treated tumor cell spheroids were attached to the coverslip coated with 25ug/ml polylysine, cultured for 2h, fixed with 4% paraformaldehyde for 15min, rinsed 3 times with PBS, 5min each time; 0.1 % Triton perforated 3 times, 5 min each, rinsed 2 times with PBS, 5 min each, blocked with 10% goat serum for 30 min; added rabbit anti-mouse LC3-II IgG (1:1000) overnight at 4°C, rinsed 3 times with PBS, each 5 min; PE-labeled goat anti-rabbit IgG was added and incubated at room temperature for 2 h, washed with PBS three times for 5 min each time, mounted with glycerol and observed under a fluorescence microscope. The results are shown in Fig. 3C. After 24 hours of MCL treatment, the fluorescent spot intensity of autophagy-related protein LC3-II was significantly increased. MCL can induce the formation of LC3-II fluorescent spot sign, while in the control group, the red fluorescence of LC3-II protein is scattered and uniformly distributed in the cytoplasm. ( FIG. 3D ) The formation of autophagosomes was detected by transmission electron microscopy of MCL-treated cells. Cell microspheres were fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer overnight and post-fixed with 1% osmium tetroxide (pH 7.4) for 2 h at room temperature. The samples were then dehydrated in a graded ethanol series and infiltrated with Spurr resin. It was then polymerized at 60 °C for 48 h, cut into 60 nm thick slices with LKB-i microtome, stained with uranyl acetate and lead citrate, and finally observed by electron microscope. The results showed that after 24 hours of MCL drug treatment, the cytoplasmic autophagosomes and autolysosomes of GH3 and MMQ cells were significantly increased compared with the control group.

D. To further explore the upstream mechanism of MCL inhibiting pituitary prolactinoma through apoptosis and autophagic death, RNA sequencing analysis was performed on GH3 and MMQ cell lines before and after MCL treatment. The difference was greater than 1.2 times and the P value was less than 0.05 as the gene inclusion criteria. Cluster analysis showed that MCL could significantly alter the expression of mRNA in GH3 and MMQ cells (Fig. 4A). The results of GO enrichment analysis showed that MCL further exerted its tumor suppressor effect mainly by activating the MAPK signaling pathway (Fig. 4B). In order to further evaluate the reproducibility of RNA sequencing, Western blot detection of key regulatory proteins ERK, P38 and JNK of MAPK signaling pathway was performed on GH3 and MMQ cells before and after drug treatment. The results showed that after MCL treatment, the phosphorylation activity levels of key proteins were Significantly improved compared to before treatment (Fig. 4C). After inhibiting the phosphorylation level of ERK protein phosphatase inhibitor (Fig. 4D), the inhibitory effect of MCL on GH3 and MMQ cells was significantly weakened (Fig. 4E). Similarly, after inhibiting the phosphorylation level of JNK protein phosphatase inhibitor (Fig. 4F), the inhibitory effect of MCL on GH3 and MMQ cells was significantly weakened (Fig. 4G). However, when P38 protein phosphatase inhibitor was used to inhibit its phosphorylation level (Fig. 4H), the inhibitory effect of MCL on GH3 and MMQ cells was significantly weakened (Fig. 4I).

Example 7 Tricholactone and its derivatives can inhibit the growth of estrogen-induced pituitary prolactin adenoma tumor tissue in rats.

In order to further confirm that the inhibitory effect of MCL on pituitary prolactinoma is also effective in living animals, the inventors used an estrogen-induced rat pituitary prolactinoma model for drug intervention treatment. After 6 weeks of estradiol induction, DMAMCL (200 mg/kg) drug intervention was performed on the mature rat pituitary prolactinoma model for 2 weeks. MRI results showed (Fig. 5A) that DMAMCL drug intervention could significantly inhibit the growth of tumor tissue. The tumor volume of the drug-treated group was 25.03±4.89 mm ³ and the volume of the control group tumor was 30.60±5.90 mm ³ ( FIG. 5B , left). In terms of tumor volume, the tumor mass in the drug treatment group was 12.22 ± 0.67 mg, and the tumor mass in the control group was 10.89 ± 0.78 mg (Fig. 5B, right). (Fig. 5C) After immunohistochemical detection of tumor tissue, it was found that the phosphorylation of key regulatory proteins of MAPK signaling pathway, ERK, P38 and JNK, was significantly increased in the tumor tissue after drug treatment. (Fig. 5D) After Western blot analysis of tumor tissue, it was found that the expression of apoptosis protein Bax and autophagic death-related proteins ATG7, P62, LC3-II were increased in tumor tissue after drug treatment, while The anti-apoptotic protein Bcl-2 decreased.

Example 8 Tricholactone (MCL) inhibits pituitary adenoma growth in other tumor types.

In order to further explore whether MCL also has inhibitory effect on other types of pituitary tumors other than prolactinoma. The inventors performed primary culture on part of the tumor tissue of patients undergoing transsphenoidal endoscopic pituitary tumor resection (see Table 1 for the basic information of the patients). 8 cases of human pituitary adenoma tissue were treated with MCL (20μM) for 24 hours after primary culture, and the cell viability was detected by CCK-8 assay. The results showed that among the 8 primary tumor cells, 6 cases were sensitive to MCL treatment, and 2 cases of clinically nonfunctioning pituitary adenomas were ineffective for MCL treatment (Fig. 6A). Western blot detection of tissue proteins in 8 cases of human pituitary adenomas showed that in 6 cases of MCL-sensitive tissue samples, the phosphorylation of MAPK key regulatory proteins ERK, JNK, and P38 was significantly enhanced, and the autophagy-related proteins ATG7, P62 and LC3 were significantly enhanced. -II protein expression was significantly increased (Fig. 6B). (Fig. 6C) Immunohistochemical HE staining results of 8 cases of human pituitary adenoma tissue samples. NFPA, clinically nonfunctioning pituitary adenoma; GH, growth hormone adenoma; PRL, prolactin adenoma.

Table 1: Basic clinical information of 8 patients with pituitary adenoma

a: normal value, male: 2.63-13.08ng/ml, female: 3.33-26.62ng/ml.

b: normal value, male: 0.003-0.971ng/ml, female: 0.01-3.607ng/ml.

c: IGF-1: preoperative, 688 μg/L; three days after operation, 159 μg/L; normal value: 109-284 μg/L.

d: This patient tolerated bromocriptine drug therapy. After 15mg/d bromocriptine treatment for more than 6 months, the tumor volume was reduced by less than 20% and the blood prolactin level was maintained at 457.23ng/ml.

Claims (10)

1. Use of smilide or a derivative thereof, or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for the treatment of pituitary adenoma, wherein smilide or its derivative is selected from the following compounds:

in: R1 is hydrogen or C1-8 acyl, tetrahydropyrrole formyl, tetrahydrofuran formyl, Ar-C1-4 acyl, Ar-O-C1-4 acyl, Ar-S-C1-4 acyl, Y-N-C1-4 acyl ; wherein C1-8 acyl is preferably selected from straight-chain or branched alkanoyl, alkenoyl and alkynyl; Ar is aryl or substituted aryl; Y is heterocyclic aryl or substituted heterocyclic aryl; Ar is preferably selected from phenyl, benzoyl, naphthyl, pyridyl, furyl, thienyl, pyrrolyl, isoxazole base, isothiazolyl, pyrazolyl, oxazolyl, thiazolyl, imidazolyl, pyridazinyl, pyrazinyl, benzofuranyl, benzothienyl, indolyl, quinolinyl, isoquinolinyl , purinyl, benzoxazolyl, benzothiazolyl, etc.; Y is preferably selected from uracil, tetrahydroisoquinolinyl, phthalimide, and naphthalimide. R2 is C1-7 acyl, C1-6 alkyl; wherein, C1-7 acyl is preferably selected from acetyl, propionyl, butyryl, isobutyryl, chloroacetyl, benzoyl, etc.; C1-6 alkyl is preferably From methyl, ethyl, propyl, isopropyl, butyl, isobutyl, cyclopentyl, cyclohexyl, etc.; or R1=R2; R3 and R4 are combined into a double bond, or R3 is hydrogen, R4 is -CH2NR5R6, wherein R5 and R6 are C1-4 hydrocarbon groups respectively. 2. purposes according to claim 1, is characterized in that, wherein said laughing lactone or its derivative is hydrochloride, sulfate, bromate, fumarate, acetate or citrate . 3. purposes according to claim 2, is characterized in that, the structure of laugholide or its derivative in described laugholide or its derivative salt-forming compound is as formula (I) or formula (II) wherein, the compound represented by the formula (I) is smilolide (MCL), and the compound represented by the formula (II) is the dimethylamino Michael addition compound DMAMCL. 4. purposes according to claim 2, is characterized in that, described laughing lactone or its derivative is hydrochloride or fumarate compound, and its structural formula is as follows:

5. purposes according to claim 4, it is characterised in that wherein said laugholide derivative DMAMCL fumarate (formula V) can release DMAMCL (formula II) slowly and stably under normal physiological environment, DMAMCL further releases the pharmaceutically active ingredient MCL (Formula I). 6 . The use according to claim 1 , wherein the pituitary adenoma is a prolactin adenoma, a clinically nonfunctional pituitary adenoma, a growth hormone adenoma, and the like. 7 . 7. purposes according to claim 1, is characterized in that, wherein said laughing lactone or its derivative or its pharmaceutically acceptable salt is selected from formula (I), formula (II), formula (V ), a compound represented by formula (VI) or any combination thereof, or a traditional Chinese medicine extract comprising a compound of formula (I), formula (II), formula (V) or formula (VI). 8. purposes according to claim 1, it is characterised in that wherein formula (I) containing laugholactone is prepared by following method, and this method comprises making parthenolide in suitable acid catalyst in suitable organic solvent Lactone was prepared under the catalysis of . 9. purposes according to claim 1, is characterized in that, described medicament is pharmaceutical composition, and it comprises the tricholactone or its derivative or its derivative and its pharmaceutically acceptable salt and A pharmaceutically acceptable carrier or excipient. 10. The use according to claim 1, characterized in that the total daily dose of laugholactone or its derivative, or its pharmaceutically acceptable salt is 1 to 1000 mg.

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