CN112641775B - Application of brucea javanica picrol and analogues thereof in treatment of pituitary adenoma - Google Patents

Abstract

The invention discloses the use of brucei alcohol or an analog thereof or a pharmaceutically acceptable salt thereof in preparing a medicament for treating pituitary adenoma. Different from the classical pathway of DRD2-dependent induction of tumor cell apoptosis by dopamine receptor agonists, bruscoxol and its analogs mainly induce apoptosis by promoting the production of ROS to achieve the effect of drugs on pituitary adenomas such as prolactin. Inhibitory effect of adenoma, clinically nonfunctioning pituitary adenoma and growth hormone adenoma.

Description

Application of brucea javanica picrol and analogues thereof in treatment of pituitary adenoma

Technical Field

The invention relates to the field of medicines, in particular to a new application of brucea javanica extract brucea javanica picrol or analogues thereof in treating human pituitary adenoma.

Background

The pituitary adenoma is the third most common intracranial tumor, the incidence rate of the pituitary adenoma in the population is 7.5-15/10 ten thousand, the incidence rate of the pituitary adenoma is increased in recent years, the incidence rate of the pituitary adenoma in the population is 75-113/10 thousand according to recent belgium epidemiological investigation, and the incidence rate of autopsy is even 20% -30%. Pituitary adenomas are mainly classified into nonfunctional adenomas, prolactin adenomas, growth hormone adenomas, ACTH adenomas, etc., while prolactin adenomas are the most common pituitary adenomas, accounting for 40% to 45% of all pituitary adenomas. The clinical manifestations of prolactin adenoma patients are mainly amenorrhea and galactorrhea, sexual hypofunction, weight gain, infertility and the like caused by hyperprolactinemia; meanwhile, the compression effect generated by the increase of the tumor volume causes the increase of the intracranial pressure, and causes serious complications such as headache, visual deterioration, visual field change and even blindness, thereby seriously affecting the life quality and the health safety of patients.

The first-line drugs of the prior art are mainly dopamine 2 receptor agonists (DAs), and Bromocriptine (BRC) and Cabergoline (CAB) are commonly used. DAs can benefit 80-90% of patients, including restoration of normal prolactin levels to ameliorate endocrine symptoms; reducing the volume of the tumor to relieve the symptoms caused by the tumor compression, such as improving the vision and the like. However, 10-20% of patients are not sensitive or even ineffective to the drug treatment, and the patients are clinically classified as drug-resistant prolactin adenomas, and the patients are still difficult to recover the normal prolactin level after the operation and the radiotherapy. Therefore, the search for new drugs for treating pituitary prolactinoma is the focus of current clinical research, and the research of traditional Chinese medicine extracts involved in the treatment of pituitary prolactinoma is still blank.

The natural products such as Chinese medicinal extracts have important clinical and social significance for treating tumors, such as conventional Taxol and artemesinin, which are derivatives or analogs of natural products. The brucea javanica picrol related by the invention is an extract of brucea javanica in a traditional Chinese medicine in China, and brucea javanica is derived from dried mature fruits of brucea javanica in Simaroubaceae and is mainly distributed in south Linn areas such as Guangdong areas, Guangxi areas and the like in China. According to the records of Bencao gang mu Shi Yi and Ling nan Cao Yao Lu, it has the functions of clearing away heat and toxic material, stopping malaria and stopping dysentery, and the anti-tumor function of brucea javanica is studied earlier in our country and brucea javanica cream injection and soft capsules are produced.

The main components of fructus Bruceae include brucine A, B, C, D, E, F, G, H, I, bruceolactone (Bruceolide), and bruceolol (Brusatol); also contains alkaloids such as brucine (brucaraine) and brucine (Yatanine); glycosides, such as brucalin (brucalin), bruceoside (Yatanoside) and bruceol (Brucenol), brucinic acid (Bruceolic acid) and Vanillic acid (Vanillic acid).

Brucea javanica picrol, english name is brustol, CAS number: 14907-98-3, formula: c₂₆H₃₂O₁₁Molecular weight 520.54, its chemical structure is shown in formula (I), and the related preparation process can be found in Chinese patent application CN 201310384744.3.

In recent years, the antitumor effect of bruceol has been receiving more and more attention domestically and internationally. Brucea javanica picrol, as a novel NRF2 inhibitor, can effectively induce the generation of Reactive Oxygen Species (ROS) in cells and inhibit the growth of tumor cells, such as liver cancer, lung cancer, pancreatic cancer, brain glioma and the like. CN201510642651.5 reports that brucea javanica picrol medicament can promote the degradation of hypoxia inducible factor-1 alpha (HIF-1 alpha), inhibit the activation of HIF-1 signal channel and the expression of Vascular Endothelial Growth Factor (VEGF), can effectively inhibit the angiogenesis of tumor, has low toxicity and has good clinical application prospect.

CN201911337381.1 reports that trastuzumab administered in combination with brucellol shows more significant tumor progression inhibiting effect on HER2 highly expressed breast cancer BT-474 tumor-bearing mouse model than either brucellol or trastuzumab administered alone.

CN202010096435.6 reports the application of brucea javanica picrol in preparing medicaments for preventing or treating inflammatory diseases, in particular to the application in preparing medicaments for treating psoriasis.

US20020193425a1 reports the use of brucellol and analogues thereof in the prevention and/or treatment of cancer such as leukaemia or lymphoma selected from acute myeloid leukaemia, acute lymphocytic leukaemia, acute chronic myelogenous leukaemia, burkitt's leukaemia, burkitt-like leukaemia and high risk myelodysplastic syndrome.

However, at present, no report is found on the research of using brucea javanica picrol to treat pituitary adenoma at home and abroad.

Disclosure of Invention

The inventor unexpectedly found that unlike the classic route of inducing tumor cell apoptosis by DRD2 dependence of dopamine receptor agonists, brucellol and its analogs induce apoptosis mainly by inducing cell generation of ROS to achieve drug inhibition of pituitary adenomas such as prolactin adenoma, clinical nonfunctional pituitary adenoma, and growth hormone adenoma.

In one aspect, the invention discloses the use of brucellol or an analogue thereof, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of pituitary adenoma, wherein brucellol or an analogue thereof is selected from the group consisting of:

in the invention, the brucea javanica picrol shown in the formula (I) can be commercially available or prepared by self, and the preparation process refers to the method disclosed in CN 201310384744.3. The brucea javanica picrol shown in formula (II) -formula (X) can be referred to US20020193425A1, the compounds show similar pharmacological efficacy when preventing and/or treating cancers such as leukemia or lymphoma, and the compounds which have the same parent nucleus with brucea javanica picrol have similar chemical properties due to similar chemical structures, and further have similar pharmacological efficacy activity with brucea javanica picrol.

In another embodiment of the present invention, the present invention relates to the use of a pharmaceutically acceptable salt of brucellol or an analogue thereof as shown above, wherein the brucellol or an analogue thereof is the hydrochloride, sulfate, bromate, fumarate, acetate or citrate salt etc. for the manufacture of a medicament for the treatment of pituitary adenomas.

In another embodiment of the invention, the invention discloses the use of brucea javanica or an analog thereof, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of pituitary adenomas, wherein the pituitary adenomas are prolactin adenomas, clinically nonfunctional pituitary adenomas, growth hormone adenomas, ACTH adenomas, mixed adenomas, and the like.

To confirm the therapeutic effect of brucellol (Brusatol) or its analogs in pituitary adenomas, the inventors selected MMQ cells (highly expressed DRD2, sensitive to DA treatment) and GH3 cells (low/non-expressed DRD2, insensitive to DA treatment) with higher acceptance for Brusatol pharmacological intervention based on previous data from studies on pituitary prolactin cell adenoma resistance.

In vitro experiment results show that Brusatol has strong cell inhibition effect on both GH3 cells and MMQ cells, and the Brusatol can effectively inhibit the growth of tumors after being applied to nude mice with subcutaneous tumor formation by adopting medicaments. Meanwhile, we extracted primary pituitary adenoma cells (6 cases in total, including 4 non-functional adenomas, 1 growth hormone adenoma, and 1 prolactin adenoma) from tumor specimens obtained during the operation of pituitary adenoma, and found that 5 cases were effective and 1 case was non-functional adenoma primary cells ineffective after drug treatment, which indicates that brustol may still have a killing effect on the other types of pituitary tumor subtype cells (see fig. 1 and fig. 2). Half Inhibitory Concentration (IC) of Brusatol drug in GH3 cells and MMQ cells₅₀) About 250nM, all below the therapeutic concentration of dopamine type 2 receptor agonist (IC of bromocriptine) in previous studies by the inventors₅₀IC of cabergoline at 100. mu.M ₅₀50 μ M).

After corresponding research on the action mechanism of brucellol drugs, it is found that unlike the classic route of DRD 2-dependent promotion of tumor apoptosis by dopamine receptor agonists, brucellol achieves drug inhibition of prolactin adenoma mainly by promoting cell ROS production to induce apoptosis (see figure 3), and the effect is still effective on GH3 cell line lacking DRD2 expression. The above results suggest that brucellol has a good inhibitory effect on prolactin adenomas, even on the remaining tumor subtypes resistant to dopamine agonist.

The inventor carries out Western blot experimental verification on GH3, MMQ and primary cell strains of human pituitary tumors before and after drug treatment, and the results of immunohistochemical experiments on nude mouse transplanted tumor specimens suggest that: bruatol was able to significantly inhibit phosphorylation of the downstream substrates 4E-BP1 and S6K1 of mTORC1, and therefore probably mediated tumor cell death by inhibiting the mTORC1 signaling pathway (see fig. 4). Further experiments show that the process is mainly that Brusatol promotes the generation of Reactive Oxygen Species (ROS) in cells so as to achieve the effect of inhibiting the signal path (see figure 5).

In another embodiment of the invention, the invention discloses the use of brucellol or an analogue thereof, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of pituitary adenomas, said medicament comprising a therapeutically effective amount of brucellol or an analogue thereof, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.

Brucea javanica or an analogue thereof, or a pharmaceutically acceptable salt thereof, when used as a medicament, can be used directly or in the form of a pharmaceutical composition. The pharmaceutical composition contains 0.1-99%, preferably 0.5-90%, 1-80% or 5-50% of brucea javanica picrol or analogues thereof, or pharmaceutically acceptable salts thereof, and the balance pharmaceutically acceptable carriers and/or excipients which are nontoxic and inert to human and animals.

In another embodiment of the present invention, the pharmaceutical composition may be prepared for oral administration in the form of capsules, tablets, powders, granules, syrups or the like, or for parenteral administration by injection, ointment, suppository or the like. These pharmaceutical preparations can be produced by a conventional method using auxiliary agents well known in the art, such as binders, excipients, stabilizers, disintegrants, flavors, lubricants, etc., and can also be prepared as controlled-release administration forms, sustained-release administration forms, various fine particle administration systems.

Although the dosage varies with the symptoms and age of the patient, the nature and severity of the disease or disorder and the route and manner of administration, in the case of oral administration to an adult patient, brucellol or an analogue thereof, or a pharmaceutically acceptable salt thereof, is normally administered in a total daily dose of 1-1000mg, preferably 50-500 mg, more preferably 100-200 mg. Can be administered in a single dose, or in divided doses, for example, orally or intravenously 1-3 times daily. The dosage administered to a pediatric patient may be reduced as appropriate for an adult patient.

The present invention provides a method of treating a disease comprising administering to a subject in need thereof a therapeutically effective amount of brucellol or an analog thereof, or a pharmaceutically acceptable salt thereof, as described herein. The term "subject" includes human and non-human mammals, such as non-human primates, ovines, canines, felines, bovines, and equines, with preferred subjects being human patients.

Drawings

FIG. 1: brucellol (Brusatol) inhibits pituitary tumor cell growth and hormone secretion. Wherein: detecting the cell activity after treating GH3 and MMQ cells with different concentrations of Brusatol by using a CCK-8 cell activity detection kit, and finding that the Brusatol has cell activity inhibition effect on the MMQ and GH3 cells and shows concentration dependence with the increase of the concentration, but the effect is not stronger in 48 hours than in 24 hours; forming GH3 cells and MMQ cells into plate clone groups respectively, and adding Brusatol with a certain concentration into a culture medium during the clone group forming process, wherein the result shows that the Brusatol can inhibit the growth of GH3 and MMQ cells; obtaining different types of primary human pituitary tumor cell strains (including nonfunctional adenomas, growth hormone adenomas and prolactin adenomas) by using a primary cell culture technology, and verifying that the activity of various pituitary tumor cells can be inhibited after the treatment of the Brusatol by using a CCK-8 experiment; and F-G, collecting culture medium supernatant of the Brusatol-treated GH3 and MMQ cell lines by using a rapid centrifugation method, and verifying the change condition of prolactin and growth hormone after Brusatol treatment by using an Elisa experiment, wherein the result shows that Brusatol has a remarkable inhibitory effect on the secretion of prolactin and growth hormone. P < 0.001; p < 0.01; p <0.05vs, Control group.

FIG. 2 is a drawing: brucellol (Brusatol) inhibits the growth of nude mouse transplantable tumors. Wherein: A-C, 200 ten thousand GH3 cells are planted under the skin of the back of a nude mouse, the change of the tumor growth volume is recorded every other day, and a tumor growth curve is drawn; at the same time, the mice were sacrificed under anesthesia 24 days later, tumor tissue was obtained and weighed; D-F, planting 200 ten thousand MMQ cells under the skin of the back of the nude mouse, recording the change of the tumor growth volume every other day, and drawing a tumor growth curve; at the same time, the mice were sacrificed under anesthesia after 24 days, and tumor tissues were obtained and weighed. The results show that Brusatol has growth inhibition effect on the transplanted tumor of the ptosis nude mice

FIG. 3: brucellol (Brusatol) induces apoptosis in pituitary tumours GH3 and MMQ cells. Wherein: A-B, performing flow detection on GH3 and MMQ cells after Brusatol treatment for 24 hours by adopting a flow cytometry apoptosis identification method, wherein the results are shown in figures 3A-B, and the results show that Brusatol can remarkably induce GH3 and MMQ to generate apoptosis; and C-D, extracting GH3 and MMQ cell protein after Brusatol treatment, and carrying out Western blot detection, wherein the result is shown in figures 3C and 3D, and the expression of the apoptosis-related protein changes along with the increase of the action time of the Brusatol medicine. E-F selection of apoptosis inhibitor Z-VAD-FMK for use with Brusatol, we found that the apoptosis inhibitor Z-VAD-FMK had a tumor suppression effect that partially reversed Brusatol. The above results indicate that Brusatol has an apoptotic effect on pituitary tumour cells.

FIG. 4 is a drawing: brucellol (Brusatol) inhibits the mTORC1 signaling pathway in pituitary tumors. Wherein: a: the results of Western blot detection of GH3 and MMQ cell protein after 0h,6h,12h and 24h of Brusatol extraction are shown in FIG. 4A, and the protein phosphorylation levels of the downstream substrates S6K1 and 4E-BP1 of mTORC1 are reduced along with the increase of time. B: after treatment of primary cells without functional adenomas and GH adenomas with bruatol, Western blot assays were performed on harvested cellular proteins, and the results are shown in fig. 4B, where the levels of protein phosphorylation of the substrates S6K1 and 4EBP1 downstream of mTORC1 decreased with increasing time. C: we carried out 4% paraformaldehyde fixation on nude mouse transplantation tumor specimens in fig. 2, then made wax blocks, and then carried out pathological section, and detected the protein phosphorylation levels of S6K1 and 4E-BP1 in GH3 and MMQ transplantation tumor bodies by immunohistochemical experiments. As shown by the results, Brusatol can remarkably inhibit the protein phosphorylation levels of S6K1 and 4E-BP1

FIG. 5: brucellol (Brusatol) inhibits the mTORC1 signaling pathway via ROS. Wherein: a: GH3 cells and MMQ cells were treated with Brusatol for 24 hours, cells were stained with ROS stain and intracellular ROS levels were detected using flow cytometry, as shown, ROS rose following Brusatol treatment; b: GH3 and MMQ cellular proteins treated at different concentrations according to Brusatol were collected and tested using Western blot, and the results are shown in FIG. 5B, and the expression levels of ROS-related proteins NRF2 and Ho-1 were inhibited after Brusatol treatment. And as shown in fig. 5C, the ROS inhibitor NAC was able to reverse the cytostatic effect of brustol and reverse the protein phosphorylation levels of S6K1 and 4E-BP1 after brustol treatment (fig. 5D).

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available products. The terms used in the following examples are abbreviated:

CCK-8: live cell count kit 8.

Brusatol: brucea javanica picrol.

The CCK-8 kit used in the examples was purchased from Beijing Biyuntian, Inc.; brusatol is purchased from MCE corporation, USA; cell culture medium and required serum are purchased from Gibco company in the United states, and other experimental consumables are conventional consumables commonly used in the laboratory.

Example 1 Brusatol assays for cellular Activity and inhibition of hormone secretion of Pituitary adenomas

1) Collecting GH3 cells and MMQ cells in logarithmic growth phase, washing with PBS for 2-3 times, and culturing in F12 medium at 37 deg.C and 5% CO₂Culturing under the condition of (1), adding 2.5% fetal calf blood into the culture mediumClear, 15% horse serum and 100U/ml penicillin/streptomycin; taking a fresh pituitary adenoma operation of neurosurgery of the first hospital affiliated to Wenzhou medical university to excise a tumor specimen, shearing the tumor tissue by using ophthalmic scissors under the aseptic condition, mechanically digesting the tumor tissue by using type IV collagenase in combination with pancreatin to obtain primary pituitary adenoma cells, and culturing in the same way as GH3 and MMQ cell culture conditions.

2) Inoculating 10-20 ten thousand cells/ml of culture medium into a culture plate, and culturing by using a 96-well plate, wherein the culture medium in each well is 100 mu l and is used for a cell activity detection experiment; cells were seeded at a concentration of 500 cells/ml in 6-well plates using polylysine-treated 6-well plates, and 2ml of medium was added for plate cloning experiments.

3) After overnight culture of the cells, the Brusatol stock solution (purchased from MCE company) is taken out from a refrigerator at minus 80 ℃, dissolved to be liquid under natural conditions, added into a 96-well plate and a 6-well plate paved with GH3 cells and MMQ cells in advance according to the working concentration of 0-1 mu M after confirmation of no precipitate and suspension, and cultured for 24 hours and 48 hours, and then the cell activity is detected by a CCK-8 experiment; meanwhile, the number of cell clones formed in the 6-well plate was measured after 3-4 weeks by methylene blue staining, and it was shown from the results of fig. 1-D that bruatol has a significant growth inhibitory effect on GH3 cells and MMQ cells.

4) In addition, the obtained pituitary adenoma primary cells of different subtypes are laid in a 96-well cell culture plate according to the cell density, Brusatol is added according to the working concentration of 0.5 mu M, after the culture is carried out for 24 hours, the activity of the primary cells is detected by a CCK-8 experiment, and the result shows that Brusatol has the function of inhibiting the cell activity on different pituitary adenoma primary cells according to the graph in FIG. 1E.

5) GH3 cells and MMQ cells in logarithmic growth phase are again collected, washed with PBS 2-3 times, and cultured in F12 medium at 37 deg.C and 5% CO₂The culture medium was supplemented with 2.5% fetal bovine serum, 15% horse serum and 100U/ml penicillin/streptomycin.

6) Inoculating 30 ten thousand cells/ml culture medium into a culture plate, and culturing by using a 6-hole plate, wherein the culture medium in each hole is 2 ml; brusatol was then added to the 6-well plate at the working concentration of 0-0.5. mu.M as described above, cultured for 24 hours, and the cells were centrifuged at 3000rpm/min to obtain a cell supernatant after Brusatol treatment.

7) The concentrations of GH3 in the culture supernatants of GH3 and MMQ cells and the concentration of PRL in the culture supernatants of MMQ cells after 24 hours of treatment with Brusatol at concentrations of 0, 50, 100, 250, 500nM were measured using an Elisa kit for rat Prolactin (PRL) and Growth Hormone (GH), respectively, and it was shown from the results of fig. 1F-1G that Brusatol has an inhibitory effect on GH hormone and PRL hormone secretion. The results are statistically significant when P is less than 0.05.

Example 2 Brusatol test for subcutaneous neoplasia of pituitary adenomas in nude mice

1) 5-week-old female BALB/c (nu/nu) nude mice are purchased from Shanghai experimental animal centers, adaptive feeding is carried out on SPF-grade animal houses for one week, and GH3 cells and MMQ cells can be subcutaneously planted to form tumor experiments under the condition that no obvious abnormality and discomfort are found in the nude mice.

2) Selecting subcutaneous part of lumbar and back, avoiding forelimb, not touching muscle, inoculating 2 × 10 on one side⁶GH3 and MMQ cells, one nude mouse was inoculated on one side. The cells were resuspended in F12 medium, 2.5% fetal bovine serum, 15% horse serum and 100U/ml penicillin/streptomycin, and the needle insertion site was wiped with 75% alcohol before inoculation.

3) The cell suspension was withdrawn from the disposable needle, evacuated of air, and punctured approximately 1cm forward from the needle insertion site for subcutaneous injection. When in injection, the injection amount is about 0.1-0.2 ml each time, and after the injection is finished, the needle head is slowly withdrawn, so as to avoid liquid leakage as much as possible. During the injection, the cells are kept on ice to keep the cells in a relatively low metabolic state, and the injection is completed within half an hour as far as possible.

4) After 7-10 days of inoculation, the tumor mass can be seen growing up, and the vernier caliper detection is carried out, when the tumor volume is mostly close to 50mm³In time, nude mice were divided randomly into experimental and control groups. Wherein the nude mice in the experimental group were gavaged with Brusatol solution (2mg/kg) once a day. The control group was given the same amount of physiological saline only by mouth feeding. Measuring the tumor volume every other day, drawing a subcutaneous tumor volume curve, and when the tumor tissue of the control group grows to about 1500mm³The entire animal experiment was stopped. After the mice were sacrificed under anesthesia, subcutaneous tumors were removed and the tumor weight was measured for subsequent experiments. According to the results shown in fig. 2, bruatol can significantly inhibit the growth of GH3 cell and MMQ cell transplanted tumor, and the difference is statistically significant.

Example 3 validation of Brusatol induced apoptosis of Pituitary adenoma cells

1) Collecting GH3 cells and MMQ cells in logarithmic growth phase, washing with PBS for 2-3 times, and culturing in F12 medium at 37 deg.C and 5% CO₂The culture medium was supplemented with 2.5% fetal bovine serum, 15% horse serum and 100U/ml penicillin/streptomycin.

2) Inoculating 30 ten thousand cells/ml culture medium into a culture plate, and culturing by using a 6-hole plate, wherein the culture medium in each hole is 2 ml; brusatol was then added to the 6-well plate at the working concentration of 0.5. mu.M as above, and cultured for 24 hours, after which the cells were collected in 1.5mL EP tubes and placed on ice.

3) The apoptotic cell ratio of GH3 and MMQ cells after Brusatol treatment was measured by Annexin-V and FITC double staining using flow cytometry, and the results showed a significant increase in the apoptotic cell ratio after Brusatol treatment.

4) GH3 cells and MMQ cells in logarithmic growth phase are again collected, washed with PBS 2-3 times, and cultured in F12 medium at 37 deg.C and 5% CO₂The culture medium was supplemented with 2.5% fetal bovine serum, 15% horse serum and 100U/ml penicillin/streptomycin.

5) Inoculating 30 ten thousand cells/ml culture medium into a culture plate, and culturing by using a 6-hole plate, wherein the culture medium in each hole is 2 ml; brusatol was then added to the 6-well plate at the working concentration of 0.5. mu.M as above, and cultured for 24 hours, after which the cells were collected in 1.5mL EP tubes and placed on ice. An appropriate amount of RIPA cell lysate was added, lysed on ice for 30 minutes, and then centrifuged at 12000rpm/min to obtain supernatant cell protein.

6) After adding a proper amount of protein Loading buffer, boiling for 5 minutes at 100 ℃ to denature the protein, and then detecting the expression conditions of apoptosis-related proteins in the GH3 cell and the MMQ cell after the Brusatol treatment by using a Western blot experiment, wherein the results show that the apoptosis-indicating proteins such as c-caspase 3 and c-caspase 8 of the cell after the Brusatol treatment are increased in expression, and the Bcl-2 protein for inhibiting apoptosis is reduced in expression, which indicates that the Brusatol can induce apoptosis.

7) To better verify the role of apoptosis in pituitary tumour cells, applicants used GH3 cells and MMQ cells with Brusatol or in combination with the apoptosis inhibitor Z-VAD-FMK, and the results showed that the apoptosis inhibitor was able to partially reverse the inhibitory effect of Brusatol.

Example 4 molecular mechanism exploration and validation of Brusatol inhibition of pituitary tumor growth

1) Collecting GH3 cells and MMQ cells in logarithmic growth phase, washing with PBS for 2-3 times, and culturing in F12 medium at 37 deg.C and 5% CO₂The culture medium was supplemented with 2.5% fetal bovine serum, 15% horse serum and 100U/ml penicillin/streptomycin.

2) Inoculating 30 ten thousand cells/ml culture medium into a culture plate, and culturing by using a 6-hole plate, wherein the culture medium in each hole is 2 ml; brusatol was then added to the 6-well plate at the working concentration of 0.5. mu.M as described above, cultured for 0-24 hours, and then the cells were collected in 1.5mL EP tubes and placed on ice. An appropriate amount of RIPA cell lysate was added, lysed on ice for 30 minutes, and then centrifuged at 12000rpm/min to obtain supernatant cell protein.

3) In combination with our previous results, we used the Western blot experiment to detect changes in phosphorylation levels of intracellular mTORC1 downstream substrates following brustol treatment, suggesting that both GH3 and MMQ cells following brustol treatment and pituitary tumor primary cells mTORC1 downstream substrates 4E-BP1 and S6K1 are less phosphorylated, suggesting that brustol has the effect of inhibiting mTORC1 signaling pathway.

4) The nude mouse transplanted tumor specimen is fixed by paraformaldehyde to be made into a wax block and sliced, and the immunohistochemical experiment result proves that Brusatol can still inhibit phosphorylation of downstream substrates 4E-BP1 and S6K1 of mTORC1 in nude mice, and the result shows that the Brusatol-mediated mTORC1 signal channel inhibition has an important effect in inhibiting growth of pituitary tumor.

Example 5 upstream mechanism exploration of brustol's regulation of phosphorylation of mTORC1 downstream substrate

To validate the specific mechanism of how brustol regulates phosphorylation of mTORC1 downstream substrates, we decided to explore the effect of ROS on this effect, in conjunction with previous research efforts.

1) Collecting GH3 cells and MMQ cells in logarithmic growth phase, washing with PBS for 2-3 times, and culturing in F12 medium at 37 deg.C and 5% CO₂The culture medium was supplemented with 2.5% fetal bovine serum, 15% horse serum and 100U/ml penicillin/streptomycin.

2) Inoculating 30 ten thousand cells/ml culture medium into a culture plate, and culturing by using a 6-hole plate, wherein the culture medium in each hole is 2 ml; brusatol was then added to 6-well plates at a working concentration of 0.5. mu.M as described above, and after 24 hours of culture, cells were stained with DCFH-DA, and the stained cells were collected and positive cell distribution was examined by flow cytometry, as shown in FIG. 5A, which shows that the ratio of ROS production in pituitary tumor cells after Brusatol exposure was high.

3) In addition, the expression change of protein factors related to the production of ROS in cells is simultaneously detected by using a Western Blot experiment, as shown in FIG. 5B, the expression of Nrf-2 protein is reduced after the Brusatol treatment, and the protein expression of the downstream antioxidant factor Ho-1 is also reduced, which indicates that the Brusatol causes the increase of ROS in the cells through the Nrf-2 pathway.

4) In addition, as shown in fig. 5C and 5D, the ROS inhibitor NAC was able to effectively reverse the cell activity inhibition and protein phosphorylation inhibition caused by brustol. In combination with the above observations, applicants have for the first time demonstrated that brucellol is able to achieve inhibition of phosphorylation of the downstream substrates 4E-BP1 and S6K1 of mTORC1 by the production of ROS in pituitary adenomas.

Table 1: clinical basic information of 6 patients with pituitary adenoma

Normal values, male: 2.63-13.08ng/ml, female 3.33-26.62ng/ml.

Normal values, male: 0.003-0.971ng/ml, female: 0.01-3.607ng/ml.

IGF-1: before operation, 688 mu g/L; three days after operation, 159 mu g/L; normal values: 109-284. mu.g/L.

d, the patient is resistant to bromocriptine drug therapy. 15mg/d bromocriptine treatment resulted in less than 20% reduction in tumor volume and maintenance of prolactin levels at 457.23ng/ml for more than 6 months.

Claims (18)

1. Use of brucea javanica picrol, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of pituitary adenoma, wherein brucea javanica picrol is represented by a compound of formula (I):

2. use according to claim 1, characterized in that: the brucea javanica picrol is hydrochloride, sulfate, bromate, fumarate, acetate or citrate.

3. Use according to claim 1, characterized in that: the pituitary adenoma is prolactin adenoma, clinical nonfunctional pituitary adenoma, growth hormone adenoma, ACTH adenoma or mixed adenoma.

4. Use according to claim 1, characterized in that: the medicine comprises brucea javanica picrol or pharmaceutically acceptable salt thereof with a therapeutically effective amount and a pharmaceutically acceptable carrier.

5. Use according to claim 4, characterized in that: the medicine contains 0.1 to 99 percent of brucea javanica picrol or pharmaceutically acceptable salts thereof, and the balance is pharmaceutically acceptable medicinal carriers which are nontoxic and inert to human and animals.

6. Use according to claim 5, characterized in that: the medicine contains 0.5 to 90 percent of brucea javanica picrol or pharmaceutically acceptable salts thereof, and the balance is pharmaceutically acceptable and nontoxic and inert medicinal carriers for human and animals.

7. Use according to claim 6, characterized in that: the medicine contains 1-80% of brucea javanica picrol or pharmaceutically acceptable salts thereof, and the balance of pharmaceutically acceptable medicinal carriers which are nontoxic and inert to human and animals.

8. Use according to claim 7, characterized in that: the medicine contains 5-50% of brucea javanica picrol or pharmaceutically acceptable salts thereof, and the balance of pharmaceutically acceptable medicinal carriers which are nontoxic and inert to human and animals.

9. Use according to any one of claims 4 to 8, characterized in that: the medicine is prepared into capsules, tablets, powder, granules or syrup for oral administration, or parenteral administration by injection, ointment or suppository.

10. Use according to claim 1, characterized in that: the normal administration of brucea javanica picrol or pharmaceutically acceptable salts thereof in the medicine is 1-1000mg of total daily dose.

11. Use according to claim 10, characterized in that: the normal administration of brucea javanica picrol or pharmaceutically acceptable salts thereof in the medicine is 50mg-500mg of total daily dose.

12. Use according to claim 11, characterized in that: the normal administration of the brucea javanica picrol or the pharmaceutically acceptable salt thereof in the medicine is 100-200mg of total dose per day.

13. Use according to any one of claims 10 to 12, characterized in that: the brucea javanica picrol or pharmaceutically acceptable salt thereof in the medicine is administered in a single dose or in a divided dose form.

14. Use according to claim 13, characterized in that: the brucea javanica picrol or pharmaceutically acceptable salt thereof in the medicine is administered by oral administration or intravenous injection 1-3 times a day.

15. Use according to any one of claims 10 to 12, characterized in that: the dose administered to pediatric patients is reduced as appropriate for adult patients.

16. Use according to claim 1, characterized in that: the use comprises administering a therapeutically effective amount of brucellol, or a pharmaceutically acceptable salt thereof, to a subject in need thereof, including humans and non-human mammals.

17. Use according to claim 16, characterized in that: the subject is a non-human primate, sheep, dog, cat, cow or horse.

18. Use according to claim 16, characterized in that: the subject is a human patient.

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