CN111803489B - 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 michelia lactone and derivatives thereof in treatment of pituitary adenoma

Technical Field

The invention relates to the field of medicines, in particular to a new application of michelia lactone and derivatives thereof in treating pituitary adenoma.

Background

The annual incidence rate of the pituitary adenomas of the population is as high as 7.5-15/10 thousands of people, and accounts for 16.7% of the intracranial primary tumor, wherein prolactin adenomas are the most common functional pituitary adenomas account for about 40% -66% of all the pituitary adenomas. According to the calculation of 13 hundred million people in China at present, the number of new prolactin adenoma cases is about 6-12 ten thousand. In fact, with the development of endocrine diagnosis and neuroimaging techniques, the number of cases of pituitary prolactin adenomas may be higher than the above data. The clinical manifestations of pituitary prolactin adenomas are mainly male impotence/sexual hypofunction caused by endocrine disorder, female amenorrhea/lactation/infertility, and the space occupying effect formed by tumor enlargement, visual disturbance and hypophysis hypofunction caused by pressing adjacent structures, which seriously endanger the life safety and life quality of patients.

Currently, the first choice for treating prolactin adenoma is Dopamine receptor agonists (DA), including Bromocriptine (BRC) and Cabergoline (CAB), mainly through combination with Dopamine 2 type receptor (D2R) on the surface of tumor cells, downstream apoptosis signaling pathway is activated and prolactin secretion is reduced, and finally tumor growth is inhibited. The lack of expression of the tumor cell D2R becomes an important factor for limiting the effect of DA. Clinical follow-up work shows that 75-90% of prolactin adenomas patients can reduce serum prolactin to a normal level and obviously reduce the tumor volume by DA, but 10-25% of patients still have no effect on DA treatment clinically, and the control of prolactin in blood is not ideal even through operation or stereotactic radiotherapy, so that the patients are called drug-resistant cases. Therefore, how to provide a novel, effective and safe treatment scheme for the part of DA resistant cases becomes a hot problem to be solved urgently for treating prolactin adenoma at present. The research on the Chinese herbal medicines involved in the treatment of pituitary prolactin cell adenomas still remains blank at present.

Natural products are very important for discovery, design and synthesis of new drugs, and also important sources of bioactive substances and innovative drugs, and drugs approved for the market, such as paclitaxel (Taxol), Docetaxel (Docetaxel), Vinorelbine (Vinorelbine), hydroxycamptothecin (camptothecin), Artemisinin (Artemisinin), and the like, are derivatives or analogs of natural products. The natural product as the medicine source has the following advantages: the traditional chemotherapy drugs for treating tumors have the problem of drug resistance, while natural products have unique effects in the discovery of anti-cancer drugs, and lead compounds for efficiently killing tumor cells with low toxicity can be screened out from the natural products, so that novel drugs for treating malignant tumors are developed, the pain of patients is relieved, and the life quality of the patients is improved.

Michelia lactone (alias: Michelia lactone; MCL, Micheliolide), is a guaiane type sesquiterpene lactone Chinese medicinal monomer extracted and separated from root bark of Michelia (Michelia champaca) and Michelia compressa (Michelia compressa) in magnoliaceae. In traditional Chinese medicine, the traditional Chinese medicine is mainly suitable for diminishing inflammation, relieving pain, and treating diseases such as fever, migraine, rheumatoid arthritis and the like. The chemical structural formula of the michelia lactone is as follows:

the michelia lactone and its derivatives were previously found to be useful in the treatment of the following cancers: leukemia, breast cancer, prostate cancer, liver cancer, esophageal cancer, stomach cancer, oral cancer, Hodgkin's lymphoma, pancreatic cancer, colorectal cancer, cervical cancer, non-Hodgkin's lymphoma, glioma, melanoma, bladder cancer, ovarian cancer, thyroid cancer, prostate cancer, and Kaposi's sarcoma, and have been patented (application No.: 201010153701.0; 201010153685.5, etc.). The prior method for preparing michelia lactone mainly adopts chemical synthesis, and Chinese patent application No. 201010153685.5 discloses a preparation method of michelia lactone, which uses parthenolide as a raw material, and rearranges the parthenolide in a proper organic solvent under the catalysis of Lewis acid to obtain the michelia lactone, wherein the yield reaches more than 60 percent. CN201711285215.2 discloses a new class of michelia lactone derivatives and their use in preparing anti-tumor and immunotherapy drugs.

In recent years, along with the continuous development of traditional Chinese medicine, the role of michelia lactone (MCL) in the treatment of cancer is gradually shown. The research has proved that MCL can induce tumor cell apoptosis by inhibiting NF-kB signal channel and regulating ROS level of cells, and has significant therapeutic effect on acute myelogenous leukemia, breast cancer, melanoma, intestinal cancer, pancreatic cancer and other diseases. With the continuous progress of medical chemistry technology, the Dimethylamino Michael addition compound (DMAMCL) of MCL has higher plasma pharmacokinetic stability (the structural formula is shown as below), longer drug release time and stronger water solubility than MCL, so that the MCL has wider application prospect. In central nervous system diseases, it can pass blood brain barrier efficiently with little toxic and side effects to nerve cells, and has a brain/plasma drug accumulation concentration ratio (C)_brain/C_plasma) Is 4.5 times of Temozolomide (TMZ), can efficiently induce glioma cell death and finally inhibit tumor cell growth, and is considered as a potential therapeutic drug for malignant glioma. MCL has now entered phase I clinical trials in China (ID:2017L04073) and Australia (ID: ACTRN 12616000228482).

At present, the treatment report of michelia lactone (MCL) and derivatives thereof in central nervous system tumors mainly focuses on malignant glioma cytoma, and the treatment effect on benign tumor pituitary prolactin adenoma is not reported.

Disclosure of Invention

In view of the above problems, the inventors have unexpectedly found that michelia lactone and its derivatives can inhibit pituitary adenomas by inducing apoptosis and autophagic death, and particularly have a better therapeutic effect on pituitary prolactin adenomas.

In one aspect, the present invention relates to the use of a michelia lactone or derivative thereof, or a pharmaceutically acceptable salt thereof, represented by the following general formula for the manufacture of a medicament for the treatment of pituitary adenoma, wherein the michelia lactone or derivative thereof is selected from the group consisting of:

wherein:

R₁is hydrogen or C_1-8Acyl, tetrahydropyrroloyl, tetrahydrofuroyl, Ar-C_1-4Acyl, Ar-O-C_1-4Acyl, Ar-S-C_1-4Acyl, Y-N-C_1-4An acyl group; wherein C is_1-8The acyl group is preferably selected from the group consisting of straight or branched alkanoyl, alkenoyl and alkynoyl;

ar is aryl or substituted aryl; y is a heterocyclic aryl or substituted heterocyclic aryl; ar is preferably selected from phenyl, benzoyl, naphthyl, pyridyl, furyl, thienyl, pyrrolyl, isoxazolyl, isothiazolyl, pyrazolyl, oxazolyl, thiazolyl, imidazolyl, pyridazinyl, pyrazinyl, benzofuryl, benzothienyl, indolyl, quinolinyl, isoquinolinyl, purinyl, benzoxazolyl, benzothiazolyl, and the like; y is preferably selected from the group consisting of a uracil radical, a tetrahydroisoquinolinyl radical, a phthalimidyl radical, and a naphthalimide radical. R₂Is C_1-7Acyl radical, C_1-6An alkyl group; wherein, C_1-7The acyl group is preferably selected from acetyl, propionyl, butyryl, isobutyryl, chloroacetyl, benzoyl and the like; c_1-6The alkyl group is preferably selected from methyl, ethyl, propyl, isopropyl, butyl, isobutyl, cyclopentyl, cyclohexyl, etc.; or R₁＝R₂；

R₃、R₄Combined to a double bond, or R₃Is hydrogen, R₄is-CH₂NR₅R₆Wherein R is₅And R₆Are respectively C_1-4A hydrocarbyl group.

In another embodiment of the present invention, the present invention relates to the use of the pharmaceutically acceptable salt of michelia lactone or a derivative thereof as shown above, wherein the michelia lactone or a derivative thereof is a hydrochloride, sulfate, bromate, fumarate, acetate, citrate or the like, for the manufacture of a medicament for the treatment of pituitary adenoma.

In another preferred embodiment, the structure of the michelia lactone or its derivative in the salt-forming compound of the michelia lactone or its derivative is shown as formula (I) or formula (II). Wherein the compound shown in the formula (I) is michelia lactone (MCL), and the compound shown in the formula (II) is michelia lactone (MCL) dimethylamino Michael addition compound DMAMCL.

In another more preferred embodiment, the smilactone or derivative thereof in the use is a hydrochloride or fumarate compound having the structural formula shown below:

among them, a salt-forming compound of DMAMCL such as DMAMCL fumarate (formula V) can slowly and stably release DMAMCL (formula II) under normal physiological conditions, which further releases the pharmaceutically active ingredient MCL (formula I).

In another embodiment of the present invention, the present application discloses the use of michelia lactone or a derivative thereof, or a pharmaceutically acceptable salt thereof, for 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, and the like.

In another preferred embodiment of the present invention, the present application discloses the use of michelia lactone or a derivative 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, and the like; the michelia lactone or the derivative thereof, or the pharmaceutically acceptable salt thereof is selected from compounds shown in formula (I), formula (II), formula (V) and formula (VI) or any combination thereof, or traditional Chinese medicine extracts containing the compounds shown in formula (I), formula (II), formula (V) or formula (VI).

In another preferred embodiment of the present invention, the inventors have unexpectedly found through extensive experiments that michelia lactone or a derivative thereof has a good inhibitory effect on prolactin adenomas well as on prolactin adenomas resistant to DA treatment and on DA sensitive prolactin adenomas. Wherein, the michelia lactone or the derivative thereof is preferably a compound shown in formula (I), formula (II) or formula (V) or any combination thereof, or a traditional Chinese medicine extract containing the compound shown in formula (I), formula (II) or formula (V).

To demonstrate the therapeutic effect of michelia lactone (MCL) or its derivatives in pituitary adenomas, the inventors selected, based on the previous data from studies on pituitary prolactin cell adenoma resistance, MMQ cells (highly expressing D2R, sensitive to DA treatment) and GH3 cells (low/non-expressing D2R, normally resistant to DA treatment) with higher acceptance for MCL drug intervention.

In vitro results show that MCL has strong cytostatic effects on both, and GH3 cell line resistant to DA treatment is more sensitive. GH3 cells were shown to be effective in inhibiting tumor growth after oral therapy with DMAMCL (Methylol-N-methyl-L) drugs in nude mice. Half Inhibitory Concentration (IC) of MCL drug in GH3 cells₅₀) Half Inhibitory Concentration (IC) of MCL drug in MMQ cells at 10. mu.M₅₀) 20 μ M, which is lower than the therapeutic concentration of DA (IC of bromocriptine) in the past studies of the inventors₅₀IC at 50. mu.M, cabergoline ₅₀25 μ M).

After corresponding research on the action mechanism of MCL drugs, the MCL is found to achieve the inhibition effect of the drugs on prolactin adenomas by inducing apoptosis (shown in figure 2) and autophagic cell death (shown in figure 3) and is more obvious on a GH3 cell line resistant to DA treatment, unlike the traditional approach of promoting tumor cell apoptosis by relying on DA to be combined with D2R. The above results suggest that MCL has a good inhibitory effect on prolactin adenomas, even on DA-resistant tumor cells, and that the mechanism may involve MCL-mediated apoptosis and autophagic death processes.

The inventors performed RNA sequencing of GH3 and MMQ cell lines before and after drug treatment, suggesting that MCL may mediate tumor cell death by activating MAPK signaling pathway. Inhibition of phosphorylation activity of ERK, JNK and P38, which are key genes of MAPK signaling pathway, in GH3 and MMQ cells can effectively reverse the inhibition of MCL (see FIG. 4). To further demonstrate that MCL's inhibitory effect on pituitary prolactin adenomas was also effective in animal subjects, the inventors used estrogen-induced rat pituitary prolactin adenoma models for drug intervention therapy. The research shows that MCL can effectively inhibit the growth of tumor tissues. Compared with a negative control group, the drug treatment can effectively inhibit the volume of tumor tissues and the tumor mass. In addition, the inventors have found that MCL can effectively activate expression of MAPK signaling pathway in tumor tissue cells and inhibit tumor growth through apoptosis and autophagic death processes after protein detection in tumor tissue samples (see fig. 5). Finally, to further investigate whether MCL has inhibitory effects on pituitary tumors of other types than prolactin adenomas as well. The inventor carries out primary culture on partial tumor body tissues of a patient with a butterfly endoscope pituitary tumor resection (the basic information of the patient is shown in table 1), and MCL medicament in-vitro intervention treatment is carried out on the partial tumor body tissues, and the results show that 6 tumor tissues are sensitive to the treatment of MCL and obviously inhibited in the tumor tissue among 8 human pituitary tumor cell tissues (1 prolactin adenoma, 6 clinical nonfunctional pituitary adenomas and 1 growth hormone adenoma), and only two clinical nonfunctional pituitary adenomas are ineffective in the treatment of MCL (shown in figure 6).

In another embodiment of the present invention, the present invention provides a process for the preparation of the michelia lactone of formula (I) above, comprising subjecting parthenolide to catalysis by a suitable acid catalyst in a suitable organic solvent to produce the michelia lactone. The preferred method for preparing the michelia lactone comprises the following steps: weighing a proper amount of parthenolide and an acid catalyst, adding a proper amount of an organic solvent, stirring and reacting for 0.5-48 hours, pouring the reaction solution into a proper amount of ice water, extracting, washing, drying, and concentrating under reduced pressure to obtain the michelia lactone. The specific process can refer to Chinese patent applications 201010153701.0 and 201010153685.5, etc.

The preparation method of the michelia lactone derivative shown in the formulas (III and IV) can refer to Chinese patent application CN 201711285215.2.

The preparation method of the salt of the michelia lactone Derivative represented by formula (V, VI) can be referred to Yinghong An et al, Michelolide Derivative DMAMCL inhibitors Glioma Cell Growth In Vitro and In Vivo; PLoS one.2015; 10(2) e 0116202. Reference is further made to CN201410071673.6 for the preparation of michelia lactone derivative DMAMCL fumarate of formula (V).

The invention also provides a pharmaceutical composition containing the michelia lactone or the derivative thereof, and the pharmaceutical composition comprises effective amount of michelia lactone or the derivative thereof, or the pharmaceutically acceptable salt thereof and pharmaceutically acceptable carriers or excipients.

When the michelia lactone or derivative thereof and pharmaceutically acceptable salt thereof are used as medicines, the michelia lactone or derivative thereof can be directly used or used in the form of pharmaceutical compositions. The pharmaceutical composition contains 0.1-99%, preferably 0.5-90%, 1-80% or 5-50% of michelia lactone or its derivative and its pharmaceutically acceptable salt, and the rest is pharmaceutically acceptable, nontoxic and inert pharmaceutically acceptable carrier and/or excipient for human and animal or combined application with other anticancer drugs.

The pharmaceutical composition of the present invention 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 adult patients, the michelia lactone or derivative thereof, or a pharmaceutically acceptable salt thereof, is normally administered in a total daily dose of from 1 to 1000mg, preferably from 5 to 500mg, in single doses, or in divided doses; e.g., twice or three times daily; in the case of intravenous injection, a dose of 0.1 to 100mg, preferably 0.5 to 50mg, may be administered in one to three times a day.

The present invention provides a method of treating a disease comprising administering to a subject in need of treatment a therapeutically effective amount of a michelia lactone or derivative 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: the activity of michelia lactone (MCL) on GH3 and MMQ is inhibited. Wherein:

(A) results of CCK8 experiments with intervention in GH3 and MMQ cell activity under MCL drug concentration-dependent conditions. The inventor finds that the activity of GH3 and MMQ cells is obviously inhibited with the increasing concentration. Half Inhibitory Concentration (IC) after MCL intervention of GH3 cell line₅₀) About 10. mu.M (A, left), and half Inhibitory Concentration (IC) of MMQ₅₀) About 20 μ M (A, right), all below the drug semi-inhibitory concentration of Dopamine Agonist (DA) (IC of bromocriptine)₅₀IC at 50. mu.M, cabergoline ₅₀25 μ M).

(B) Results of CCK8 experiments with MCL drug time-dependent intervention in GH3(B, left, 10. mu.M) and MMQ (B, right, 20. mu.M) cell activities. The inventor finds that the activity of GH3 and MMQ cells is obviously inhibited with the increasing action time.

(C) Inhibition of GH3 and MMQ cell clonality by MCL. After counting cells in logarithmic growth phase, 500 cells are inoculated into a 6-well plate, and after 14 days of culture, MCL with different concentrations is treated for 24 hours, and then crystal violet staining is carried out for photographing.

(D-F) GH3 cells were subcutaneously tumorigenic nude mice and orally treated with DMAMCL (100mg/kg) drug, which was found to be effective in inhibiting tumor growth in nude mice. P < 0.001; p < 0.01; p <0.05vs, Control group.

FIG. 2 is a drawing: michelia lactone (MCL) can induce GH3 and MMQ apoptosis. Wherein:

(A-B) after flow apoptosis detection, MCL can remarkably induce GH3 and MMQ cells to generate early and late apoptosis.

(C-D) GH3 and MMQ cells are treated by MCL (10 mu M and 20 mu M), and Western blot detection is carried out on apoptosis-related proteins in the cells, so that the expression of the apoptosis-related proteins is changed along with the increase of the MCL drug action time (C) or the drug action concentration (D).

FIG. 3: michelia lactone (MCL) significantly activates GH3 and MMQ autophagy. Wherein:

(A-B) the michelia lactone (MCL) can gradually enhance the autophagy of GH3 and MMQ cells under the action time-dependent condition (A) and the drug concentration-dependent condition (B), and promote the expression of autophagy-related proteins ATG7, P62 and LC 3-II.

(C) After 24 hours of MCL action, the intensity of the fluorescent spot of the autophagy-related protein LC3-II was significantly increased. MCL induces the formation of a fluorescent spot of LC3-II, whereas in the control group, the red fluorescence of the LC3-II protein is uniformly distributed scattered in the cytoplasm.

(D) The transmission electron microscope results show that after MCL drug treatment for 24 hours, the number of GH3 and MMQ intracellular autophagosomes and autophagosomes is obviously increased compared with that of a control group.

FIG. 4 is a drawing: michelia lactone (MCL) can inhibit tumor cell growth by activating MAPK signaling pathway. Wherein:

(A-B) RNA sequencing analysis of related protein expression changes of GH3 and MMQ cells before and after MCL drug treatment. Results from gene thermograph (a) and GO analysis (B) showed significant activation of MAPK signaling pathways before and after MCL drug treatment.

(C) In order to further confirm the reliability of the sequencing analysis result, the inventor carries out Western blot detection on key regulatory proteins ERK, P38 and JNK of MAPK signal pathway on GH3 and MMQ cells before and after drug treatment, and the result shows that the phosphorylation activity level of the key protein after MCL treatment is obviously improved compared with that before treatment.

(D-E) after the inventor adopts ERK protein phosphatase inhibitor to inhibit the phosphorylation level (D), the drug inhibition effect of MCL on GH3 and MMQ cells is obviously weakened (E).

(F-G) after the inventor adopts a JNK protein phosphatase inhibitor to inhibit the phosphorylation level (F), the drug inhibition effect of MCL on GH3 and MMQ cells is obviously weakened (G).

(H-I) after the P38 protein phosphatase inhibitor is adopted to inhibit the phosphorylation level (H) of the protein phosphatase inhibitor, the drug inhibition effect of MCL on GH3 and MMQ cells is obviously weakened (I).

FIG. 5: sphaelactone (MCL) inhibits estrogen-induced growth of rat pituitary prolactin adenoma tumor tissue. Wherein:

(A-B) 6 weeks after estradiol induction, DMAMCL (200mg/kg) drug intervention was performed on the mature rat pituitary prolactin adenoma model for 2 weeks. MRI examination results show that (A) DMAMCL drug intervention can obviously inhibit tumor tissue growth, and the volume (B, left) and the mass (B, right) of the tumor are obviously inhibited.

(C) The immunohistochemical detection of tumor body tissue shows that the phosphorylation of the key regulatory proteins ERK, P38 and JNK of the MAPK signal channel in the tumor body tissue after the drug treatment is obviously improved.

(D) Western blot detection is carried out on tumor tissues, and the expression of apoptosis and autophagic death related proteins in the tumor tissues after drug treatment is found to be obviously changed.

FIG. 6: sphaelactone (MCL) inhibits the growth of pituitary adenomas of other tumor types. Wherein:

(A)8 cases of human pituitary adenoma tissues were subjected to primary culture, then treated with MCL (20. mu.M) drug for 24 hours, and tested for cell activity in CCK-8 assay. The results showed that 6 of the 8 primary tumor cells were sensitive to MCL treatment, and 2 of the clinically non-functional pituitary adenomas were not effective for MCL treatment.

(B-C) Western blot detection is carried out on 8 cases of human pituitary adenoma tissue proteins, and then (B) is found that phosphorylation of MAPK key regulatory proteins ERK, JNK and P38 is remarkably enhanced and expression of autophagy-related proteins ATG7, P62 and LC3-II is remarkably increased in 6 cases of tissue samples with effective MCL treatment. (C) And (3) carrying out immunohistochemical HE staining detection on 8 human pituitary adenoma tissue samples. NFPA, clinical nonfunctional pituitary adenoma; GH, growth hormone adenoma; PRL, prolactin adenoma.

Detailed Description

Example 1 preparation of Sphaelactone (MCL)

Pulverizing Michelia figo of Taiwan as raw material into 20-40 mesh powder, adding into extraction kettle, using anhydrous methanol as entrainer, and supercritical CO₂Extracting, collecting extract liquor, recovering methanol to obtain extract, removing fat-soluble impurities by petroleum ether reflux, thermally dissolving degreased matters by using low-carbon alcohol, adding activated carbon for reflux decolorization, recovering a solvent from decolorized liquid to a proper volume, purifying by using a prepared liquid-phase ODS RP-C18 reverse phase column, eluting by using a mixed solution of methanol and water, concentrating corresponding components, and recrystallizing by using petroleum ether-acetone (2:1, V/V) to obtain an MCL product. The supercritical CO₂The extraction conditions comprise extraction temperature of 45-60 deg.C, extraction pressure of 23-34MPa, separation kettle I temperature of 40-50 deg.C, pressure of 8-10MPa, separation kettle II temperature of 35-40 deg.C, pressure of 4-6MPa, and extraction time of 2-6 h. Degreasing with petroleum ether, adding petroleum ether with 2-5 times of volume of the extract, heating, refluxing and degreasing for 1-3 times, centrifuging, and separating solid from liquid to obtain degreased substance. The lower alcohol is C1-C4 alcohol, preferably ethanol, methanol or isopropanol. The mixed solution of methanol and water is a mixed solution with the volume percentage of 83 percent of methanol and the volume percentage of 17 percent of water. The MCL drug used in the experiment is dissolved in dimethyl sulfoxide (DMSO) after recrystallization to form 20mM stock solution, and the stock solution is stored in a refrigerator at-80 ℃ for later use after subpackaging.

Example 2 preparation of a Sphaelactone Derivative (DMAMCL)

Preparation method of michelia lactone Derivative (DMAMCL): mixing Me with water₂NH·HCl(1.5g, 18mmol),K₂CO₃(5.0g,36mmol) and CH₂Cl₂(100ml) were mixed at room temperature to form a homogeneous solution. Then, MCL (300mg,1.2mmol) was mixed with the homogenized solution, and the mixture was stirred at room temperature for 3 hours. Using CH as the above reaction solution₂Cl₂Concentrating and re-suspending, separating with deionized water chromatography, and separating with Na₂SO₄And (5) filtering to dry. Filtering to remove residue, and adding CH₂Cl₂(5ml) was dissolved and treated with fumarate (0.1N) to adjust the pH of the solution to 4. Last using CH₂Cl₂The aqueous phase was extracted (10ml) and lyophilized to give the compound DMAMCL. The DMAMCL drug used in the experiment is dissolved in deionized water to form a stock solution of 400mg/ml, and the stock solution is subpackaged and stored in a refrigerator at minus 80 ℃ for later use.

Example 3 cytostatic test of michelia lactone and its derivatives on prolactin adenomas

1) Collecting cells in good logarithmic growth phase, washing with PBS 2-3 times, 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 3 ml;

3) after one day intervals, the MCL stock solution was removed from a refrigerator at-80 ℃ and naturally dissolved to a liquid state, and after confirming the absence of precipitates and suspended solids, 0. mu.l, 1.5. mu.l, 3. mu.l and 7.5. mu.l of the MCL stock solution (20mM) were added to each of GH3 and MMQ cell culture multiple wells, respectively, based on the final volume of 3ml per well of a 6-well plate, so that the final MCL concentrations in GH3 and MMQ cell culture medium were 0. mu.M, 10. mu.M, 20. mu.M and 50. mu.M.

4) The cells were treated for 24 hours without changing the solution, and the cell activity was examined to confirm the half Inhibitory Concentration (IC) of the GH3 cells against MCL drug₅₀) Half Inhibitory Concentration (IC) of MCL drug in MMQ cells at 10. mu.M₅₀) 20 μ M.

5) Taking the well-conditioned cells in logarithmic growth phase again, washing with PBS 2-3 times, culturing with F12 medium at 37 deg.C and 5% CO₂The culture medium is 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 3 ml;

7) after one day intervals, the MCL stock solution was removed from a refrigerator at-80 ℃ and dissolved in a liquid state under natural conditions, and after confirming the absence of precipitates and suspended solids, 1.5. mu.l and 3. mu.l of the MCL stock solution (20mM) were added to each of GH3 and MMQ cell culture multiple wells, respectively, based on the final volume of 3ml per well of a 6-well plate, so that the final concentration of MCL in GH3 cell culture medium was 10. mu.M and the final concentration of MCL in MMQ cell culture medium was 20. mu.M. During the period, the solution is not changed, and after 0, 12, 24 and 48 hours of treatment of the cells, subsequent experiments are carried out to observe the inhibition of the MCL on the cell activity.

Example 4 verification of Michellac lactone and its derivatives on subcutaneous neoplasia in nude mice with prolactin adenoma

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 can be subcutaneously planted into the nude mice to perform a tumor experiment under the condition that no obvious abnormality and discomfort are seen in the nude mice.

2) Selecting subcutaneous part of lumbar and back, avoiding forelimb, not touching muscle, inoculating 1 × 10 on one side⁷GH3 cells, one nude mouse was inoculated with one side. Cells were resuspended in F12 medium, supplemented with 2.5% fetal bovine serum, 15% horse serum and 100U/ml penicillin/streptomycin. The cell concentration is 1ml containing 1X 10⁸Cells were sterilized by wiping the needle-insertion site 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 0.1ml 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 of the experimental group are dissolved in DMAMCL waterThe liquid is fed orally, the dosage is required to be 100mg/kg, and the liquid is taken once a day. The control group was given the same amount of deionized water only. When the tumor tissue of the control group grows to 2000mm³The entire experiment was stopped. After the mice were sacrificed by dislocation, the tumors were removed for subsequent experiments.

Example 5 Michellac lactone and its derivatives intervention and verification test on rat prolactin adenoma model

1) 4 weeks old Fischer 344 rats are purchased from Shanghai experimental animal center, and after SPF level animal houses are bred adaptively for one week, pituitary prolactin adenoma induction formation experiment can be carried out under the condition that obvious abnormality and discomfort do not appear.

2) A1-cm silicon rubber capsule is embedded under the skin of a 4-week-old Fischer 344 rat, the capsule contains 10mg of 17 beta-estradiol hormone, and the capsule can slowly release the estradiol hormone, so that the formation of prolactin adenoma of the rat is induced.

3) After 6 weeks, the rats were examined by MRI to confirm further prolactin adenoma formation, and then divided into experimental and control groups at random. Wherein the nude mice in the experimental group are fed with DMAMCL aqueous solution orally, the dosage is required to be 200mg/kg, and the administration is once a day. The control group was given the same amount of deionized water only. After the medicine is taken for 2 weeks, the experiment is terminated, model samples are subjected to MRI examination, the growth condition of tumors is detected, and after the mice are sacrificed, the tumors are taken out for subsequent experiments.

Example 6 description of the mechanism of action of michelia lactone and its derivatives and demonstration of technical effects

A. Cell activity detection shows that MCL and derivatives thereof have obvious inhibition effect on prolactin adenoma DA drug-resistant cell strain GH3 and sensitive cell strain MMQ. (FIG. 1A) results of CCK8 experiments with intervention in GH3 and MMQ cell activity under MCL drug concentration-dependent conditions. The inventor finds that the activity of GH3 and MMQ cells is obviously inhibited with the increasing concentration. Half Inhibitory Concentration (IC) after MCL intervention of GH3 cell line₅₀) About 10. mu.M (A, left), and half Inhibitory Concentration (IC) of MMQ₅₀) About 20 μ M (A, right). (FIG. 1B) results of CCK8 experiments with MCL drug time-dependent conditions interfering with GH3(B, left, 10. mu.M) and MMQ (B, right, 20. mu.M) cell activities. The inventors have found that over timeThe activity of GH3 and MMQ cells is obviously inhibited continuously. (FIG. 1C) inhibition of the colony formation rate of GH3 and MMQ cells by MCL. After counting cells in logarithmic growth phase, 500 cells are inoculated into a 6-well plate, and after 14 days of culture, MCL with different concentrations is treated for 24 hours, and then crystal violet staining is carried out for photographing. The inventor finds that with increasing concentration, the cloning of GH3 and MMQ cells is obviously inhibited. In contrast, in the experiment of in vivo implantation of tumors subcutaneously in nude mice, the volume and mass of subcutaneous tumors of nude mice administered DMAMCL significantly decreased compared with those of the control group (FIG. 1D). The subcutaneous tumor volume of the nude mice in the DMAMCL experimental group is 154.83 +/-68.90 mm³The volume of the subcutaneous tumor of the nude mice in the control group is 517.44 +/-217.60 mm³(n ＝6,P<0.001; FIG. 1E); the subcutaneous tumor mass of the nude mice in the DMAMCL experimental group is 0.09 +/-0.03 g, the subcutaneous tumor volume of the nude mice in the control group is 0.33 +/-0.17 g (n is 6, P is 0.012; fig. 1F)

B. Flow cell apoptosis and protein detection results indicate that MCL can promote apoptosis-related protein expression and induce cells to undergo apoptosis. (FIGS. 2A and 2B) the flow detection was performed 0, 12, 24 and 48 hours after the GH3 and MMQ cells were treated with MCL by the flow apoptosis assay, and the results are shown in FIG. 2A, which shows that MCL can significantly induce early and late apoptosis in GH3 (FIG. 2A) and MMQ (FIG. 2B) cells. (FIGS. 2C and 2D) the MCL treated GH3 and MMQ cellular proteins were extracted and Western blot detection was performed, and the treated cellular proteins were extracted and added to PMSF pre-cooled cell lysate at 4 ℃ with a final concentration of 1 mmol/L. After incubation on ice for 30 minutes at 4 ℃ at 10000g, centrifugation was carried out for 15 minutes, and the supernatant was subjected to BCA protein quantification and heated at 100 ℃ for 15 minutes to denature the protein. Preparing 12% separating gel and 5% concentrated gel, placing 30-40ug protein sample in sample application hole, voltage 80V, electrophoresis 30min later voltage 120V, electrophoresis 60 min to separate protein. The proteins were transferred to 0.22um PVDF membrane by wet-spinning at a constant current of 170mA for 120 minutes, and the membrane was removed and placed in 5% skim milk and blocked at room temperature for 90 minutes. The skim milk was removed and washed three times with TBST. The membrane was placed in primary antibody dilution formulated with 5% BSA and incubated overnight at 4 ℃. Remove primary antibody, wash TBST three times, add 1: 2000 dilution of horseradish peroxidase-labeled secondary antibody, incubation for 60 minutes at room temperature, washing several times, placing the membrane in ECL luminescence solution, and color development imaging in LAS-4000 imager. The results are shown in FIGS. 2C and 2D, and the expression of apoptosis-related protein changes with the increase of the drug action time (FIG. 2C) or the drug action concentration (FIG. 2D) of MCL.

C. After a transmission electron microscope and protein detection, the MCL can promote the expression of autophagy-related protein, induce autophagosome formation, mediate autophagic death of tumor cells and achieve the effect of inhibiting prolactin adenoma. (FIGS. 3A and 3B) Western blot analysis of GH3 and MMQ cellular proteins after MCL treatment was performed, and the results are shown in FIGS. 3A and 3B, where autophagy-related protein expression increased with the increase in the drug action time (FIG. 3A) or the drug action concentration (FIG. 3B) of MCL. (FIG. 3C) cells treated with MCL were examined for the expression of autophagy-related protein LC 3-II. Attaching the treated tumor cell balls to a cover glass coated with 25ug/ml polylysine, continuously culturing for 2h, fixing with 4% paraformaldehyde for 15min, and washing with PBS for 3 times (5 min each time); perforating with 0.1% Triton for 3 times, 5min each time, rinsing with PBS for 2 times, 5min each time, and sealing with 10% goat serum for 30 min; adding rabbit anti-mouse LC3-II IgG (1: 1000) overnight at 4 deg.C, washing with PBS for 5min for 3 times; adding PE-labeled goat anti-rabbit IgG, incubating at room temperature for 2h, washing with PBS solution for 3 times, each time for 5min, sealing with glycerol, and observing under a fluorescence microscope. As shown in FIG. 3C, the intensity of the fluorescent spot of autophagy-related protein LC3-II was significantly increased after 24 hours of MCL action. MCL induces the formation of a fluorescent spot of LC3-II, whereas in the control group, the red fluorescence of the LC3-II protein is uniformly distributed scattered in the cytoplasm. (FIG. 3D) cells treated with MCL were examined for autophagosome formation by transmission electron microscopy. The cell microspheres were fixed with 2.5% glutaraldehyde in 0.1M phosphate buffer overnight and postfixed with 1% osmium tetroxide (pH 7.4) at room temperature for 2 h. The coated samples were then dehydrated in a graded ethanol series and infiltrated using a sprer resin. Then polymerizing for 48h at 60 ℃, cutting into sections with the thickness of 60nm by an LKB-i slicer, dyeing by uranyl acetate and lead citrate, and finally observing by adopting an electron microscope. The results showed that GH3 and MMQ cells were significantly increased in cytoplasmic autophagosomes and autophagosomes after 24 hours of MCL drug treatment compared to the control group.

D. To further explore the upstream mechanism of inhibition of pituitary prolactin adenomas by MCL through apoptosis and autophagic death, RNA sequencing analysis was performed on GH3 and MMQ cell lines before and after MCL treatment. The gene inclusion criteria were that the difference was greater than 1.2-fold and less than 0.05 for P. Cluster analysis suggested that MCL could significantly alter mRNA expression in GH3 and MMQ cells (fig. 4A). The results of GO enrichment analysis showed that MCL further exerted its tumor-inhibiting effect mainly by activating the MAPK signaling pathway (fig. 4B). In order to further evaluate the repeatability of RNA sequencing, Western blot detection of MAPK signal pathway key regulatory proteins ERK, P38 and JNK is carried out on GH3 and MMQ cells before and after drug treatment, and the result shows that the phosphorylation activity level of the key protein is remarkably improved after MCL treatment compared with that before treatment (figure 4C). After inhibiting the phosphorylation level of the ERK protein phosphatase inhibitor (fig. 4D), the drug inhibition effect of MCL on GH3 and MMQ cells is obviously weakened (fig. 4E). Similarly, the effect of MCL on drug inhibition of GH3 and MMQ cells was significantly reduced after inhibition of its phosphorylation levels with JNK protein phosphatase inhibitors (fig. 4F) (fig. 4G). After the P38 protein phosphatase inhibitor is adopted to inhibit the phosphorylation level (figure 4H), the drug inhibition effect of MCL on GH3 and MMQ cells is obviously weakened (figure 4I).

Example 7 Sphaelactone and its derivatives inhibit estrogen-induced growth of rat pituitary prolactin adenoma tumor tissue.

To further demonstrate that MCL's inhibitory effect on pituitary prolactin adenomas was also effective in animal subjects, the inventors used estrogen-induced rat pituitary prolactin adenoma models for drug intervention therapy. After 6 weeks of estradiol induction, the mature rat pituitary prolactin adenoma model was subjected to DMAMCL (200mg/kg) drug intervention for 2 weeks. MRI findings showed (fig. 5A) that DMAMCL drug intervention significantly inhibited tumor tissue growth. The tumor volume of the drug treatment group is 25.03 +/-4.89 mm³The tumor volume of the control group was 30.60. + -. 5.90mm³(FIG. 5B, left). In terms of tumor volume, the mass of the tumor in the drug-treated group was 12.22. + -. 0.67mg, and the mass of the tumor in the control group was 10.89. + -. 0.78mg (FIG. 5B, right). (FIG. 5C) immunohistochemical detection of tumor tissues revealed that the drug was administeredIn the tumor body tissues after treatment, the phosphorylation of the key regulatory proteins ERK, P38 and JNK of the MAPK signal pathway is obviously improved. Western blot detection of tumor tissues (FIG. 5D) revealed that the expression of apoptosis protein Bax and autophagic death-related proteins ATG7, P62 and LC3-II was increased and the expression of anti-apoptotic protein Bcl-2 was decreased in tumor tissues after drug treatment.

Example 8 Sphaelactone (MCL) inhibits the growth of pituitary adenomas of other tumor types.

To further investigate whether MCL has inhibitory effects on pituitary tumors of other types than prolactin adenomas well. The inventor carries out primary culture on partial tumor body tissues of a sphenoid endoscopic pituitary tumor resection patient (the basic information of the patient is shown in table 1). 8 cases of human pituitary adenoma tissues were subjected to primary culture, then treated with MCL (20. mu.M) drug for 24 hours, and tested for cell activity in CCK-8 assay. The results showed that 6 of the 8 primary tumor cells were sensitive to MCL treatment, and 2 of the clinically non-functional pituitary adenomas were not effective for MCL treatment (fig. 6A). Western blot detection is carried out on 8 human pituitary adenoma tissue proteins, and found that phosphorylation of MAPK key regulatory proteins ERK, JNK and P38 is remarkably enhanced and expression of autophagy-related proteins ATG7, P62 and LC3-II is remarkably increased in 6 MCL treatment sensitive tissue samples (figure 6B). (FIG. 6C) immunohistochemical HE staining assay results for 8 human pituitary adenoma tissue samples. NFPA, clinical nonfunctional pituitary adenoma; GH, growth hormone adenoma; PRL, prolactin adenoma.

Table 1: clinical basic information of 8 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 (24)

1. Use of sphaelactone or a derivative thereof, or a pharmaceutically acceptable salt thereof, as a single active ingredient in the manufacture of a medicament for the treatment of pituitary adenoma, wherein the sphaelactone or the derivative thereof is

Shown; wherein the compound shown in the formula (I) is michelia lactone (MCL), and the compound shown in the formula (II) is michelia lactone (MCL) dimethylamino Michael addition compound DMAMCL.

2. The use according to claim 1, wherein said sphaelactone or derivative thereof is a hydrochloride, sulfate, bromate, fumarate, acetate or citrate salt.

3. The use according to claim 2, wherein said sphaelactone or derivative thereof is a hydrochloride or fumarate compound having the formula:

4. the use of claim 3, wherein the michelia lactone derivative DMAMCL fumarate (formula V) slowly and stably releases DMAMCL (formula II) which further releases MCL (formula I) as a pharmaceutically active ingredient under normal physiological conditions.

5. The use according to claim 1, wherein the pituitary adenoma is prolactin adenoma, clinically nonfunctional pituitary adenoma or growth hormone adenoma.

6. The use of claim 1, wherein the michelia lactone or derivative thereof, or pharmaceutically acceptable salt thereof is a compound of formula (I), formula (II), formula (V), formula (VI), or any combination thereof.

7. The use according to any one of claims 1 to 6, wherein said michelia lactone or derivative thereof, or a pharmaceutically acceptable salt thereof, has a good inhibitory effect on prolactin adenomas.

8. The use according to claim 7, wherein said michelia lactone or derivative or pharmaceutically acceptable salt thereof has good inhibitory effect on prolactin adenomas resistant to treatment with dopamine agonist or prolactin adenomas sensitive to dopamine agonist.

9. The use of claim 7, wherein the sphaelactone or derivative thereof, or pharmaceutically acceptable salt thereof is a compound of formula (I), formula (II), formula (V), or any combination thereof.

10. Use according to claim 1, wherein the michelia lactone of formula (I) is prepared by a process comprising subjecting parthenolide to catalysis by a suitable acid catalyst in a suitable organic solvent to give the michelia lactone.

11. The use according to claim 10, wherein the method for preparing michelia lactone comprises the steps of: weighing a proper amount of parthenolide and an acid catalyst, adding a proper amount of an organic solvent, stirring and reacting for 0.5-48 hours, pouring the reaction solution into a proper amount of ice water, extracting, washing, drying, and concentrating under reduced pressure to obtain the michelia lactone.

12. The use according to claim 1, wherein the medicament is a pharmaceutical composition comprising a therapeutically effective amount of michelia lactone or a derivative thereof and a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or excipient.

13. The use according to claim 12, wherein the pharmaceutical composition comprises 0.1% to 99% of michelia lactone or a derivative thereof and a pharmaceutically acceptable salt thereof, the remainder being pharmaceutically acceptable, non-toxic and inert pharmaceutically acceptable carriers and/or excipients for humans and animals.

14. The use of claim 13, wherein the pharmaceutical composition comprises 0.5% to 90%, 1% to 80%, or 5% to 50% of michelia lactone or derivative thereof and a pharmaceutically acceptable salt thereof.

15. Use according to any one of claims 12 to 14, wherein the pharmaceutical composition is prepared for oral administration in the form of capsules, tablets, powders, granules, syrups, or parenterally by injection, ointments, suppositories.

16. The use according to claim 15, wherein the pharmaceutical composition is produced by a conventional method using a binder, excipient, stabilizer, disintegrant, flavoring agent or lubricant, or is prepared in a controlled release dosage form, a sustained release dosage form, or various microparticle delivery systems.

17. Use according to claim 1, wherein the michelia lactone or derivative thereof, or a pharmaceutically acceptable salt thereof, is administered in a total daily dose of from 1 to 1000 mg.

18. The use of claim 17, wherein the michelia lactone or derivative thereof, or a pharmaceutically acceptable salt thereof, is administered in a total daily dose of 5 to 500 mg.

19. Use according to any one of claims 17 to 18, wherein the michelia lactone or derivative thereof, or a pharmaceutically acceptable salt thereof, is administered in a single dose or in divided doses.

20. Use according to claim 19, wherein the administration form of the michelia lactone or derivative thereof, or a pharmaceutically acceptable salt thereof, is two or three times daily.

21. Use according to claim 19, wherein the administration form of the michelia lactone or derivative thereof, or a pharmaceutically acceptable salt thereof, is intravenous injection, administered in amounts of 0.1 to 100mg one to three times a day.

22. Use according to claim 21, wherein the administration form of the michelia lactone or derivative thereof, or a pharmaceutically acceptable salt thereof, is intravenous injection, administered in a dose of 0.5 to 50mg divided into one to three times a day.

23. The use of claim 1, wherein a therapeutically effective amount of michelia lactone or a derivative thereof, or a pharmaceutically acceptable salt thereof, is administered to a subject in need of treatment, said subject being a human or non-human mammal.

24. The use of claim 23, wherein said non-human mammal is a non-human primate, sheep, dog, cat, cow, and horse.

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