WO2018006689A1 - Use of fulvestrant in preparation of medicament for treating nonfunctioning pituitary adenoma - Google Patents

Abstract

The present invention provides use of fulvestrant in the preparation of a medicament for treating nonfunctioning pituitary adenoma. The present invention can effectively improve the prognosis of nonfunctioning pituitary adenoma by means of the described technical solution.

Description

Use of fulvestrant in the preparation of a medicament for treating non-functioning pituitary adenoma Technical field

The present invention relates to the field of biochemistry and medicine, and in particular to the use of fulvestrant for the preparation of a medicament for the treatment of non-functioning pituitary adenomas.

Background technique

Non-functional pituitary adenoma is the most common brain tumor in pituitary tumors. Although it is a benign tumor, it is very harmful and easily relapses. It can damage the patient's vision and cause sudden blindness and affect endocrine function. Infertility, serious can also lead to life-threatening.

The treatment methods of pituitary adenoma mainly include drug treatment, surgical treatment and radiotherapy. The current treatment is mainly surgery. A considerable number of patients have poor surgical results. Residual and recurrence often occur after surgery, which is a difficult problem for neurosurgery. . Medication is an effective way to solve this problem. However, there are still a large number of patients with residual surgical resection, postoperative recurrence, and drug resistance in the clinic. The treatment is improper and over-treatment is coexisting, which makes the treatment efficiency low, the disability rate is high, and the overall recurrence rate exceeds 30%. Dopamine receptor antagonists are a commonly used treatment for pituitary adenomas, but the overall efficiency in the treatment of nonfunctioning pituitary adenomas is only 5-10%. It can be seen that there is still no targeted and effective drug treatment. It is necessary to develop methods and drugs that can affect the differentiation, proliferation, apoptosis and invasion of pituitary adenomas, thereby providing an effective way to improve the therapeutic and prognostic effects of non-functional pituitary adenomas.

Fulvestrant group (molecular formula: C ₄₁ H ₆₅ O ₄ F ₅ S ₂ , Chinese alias: (7a, 17b)-7-[9-(4,4,5,5,5-pentafluoropentylsulfinyl)壬alkyl]-estrazine-1,3,5-(10)-triene-3,17-diol) is the first simple antiestrogens to complete phase III clinical trials without estrogen-like Acts and reduces estrogen receptor protein expression at the cellular level. Similar to the mechanism of action of tamoxifen, it competitively binds to estrogen receptors, but its binding rate is as high as 89%. Fulvestrant completely inhibits the transcription of estrogen-sensitive genes, and there is no induction of endometrial cancer. Tumor. Its efficacy in treating estrogen receptor-positive breast cancer is not worse than tamoxifen. Some studies have shown that fulvestrant can effectively inhibit tumor growth in tamoxifen-resistant breast cancer patients. In vitro tests have shown that fulvestrant can inhibit breast cancer cell growth more strongly than aromatase inhibitors. Importantly, fulvestrant is not cross-resistant to other endocrine drugs. There are no reports on the treatment of adenoma in fulvestrant.

Summary of the invention

A first object of the present invention is to provide the use of fulvestrant for the preparation of a medicament for the treatment of non-functioning pituitary adenomas.

A second object of the present invention is to provide the use of functional fragments, derivatives and their salts or solvates of fulvestrant for the preparation of a medicament for the treatment of non-functioning pituitary adenomas.

Wherein the derivative of fulvestrant is a halogenated, sulfonated, nitrated, hydroxylated, alkoxylated or esterified product of fulvestrant.

The fulvestrant derivative may be a salt or a solvate, wherein the salt includes a sodium salt, a potassium salt, etc., and the solvate means a hydrate, an ethanolate or the like. The derivative can be used to at least partially inhibit the differentiation and proliferation of non-functional pituitary adenomas. Those skilled in the art will foresee that the derivatives and functionalized fragments of fulvestrant have the same core structure as fulvestrant. Therefore, its functional fragments, derivatives, and salts or solvates thereof also have desirable effects in the preparation of a medicament for treating or preventing non-functioning pituitary adenomas.

The non-functional pituitary adenomas of the present invention encompass a variety of known pituitary adenomas, including apocytic adenomas, large eosinophils, gonadotropin adenomas, resting corticosteroid adenomas, and glycoprotein secretory glands. At least one of the tumors.

In addition to the active ingredient, the medicament of the present invention also includes at least one excipient that is pharmaceutically acceptable. The excipients are understood by those skilled in the art, including but not limited to disintegration The agent, the lubricant, the dispersing agent and the like can be selected according to the actual needs of the preparation by the person skilled in the art, and the present invention is not particularly limited thereto.

The medicament of the present invention can be prepared into various common pharmaceutical dosage forms, such as tablets, capsules, pills, powders, granules, suspensions, oral solutions, powder injections or injections, etc., by a conventional method. The mode of administration can be administered orally, sublingually, intravenously, subcutaneously, transdermally or topically, etc., and administered to the animal or human in unit dosage form.

Suitable unit administration forms of the medicament of the present invention include oral dosage forms (for example, tablets, capsules, pills, powders, granules, oral solutions or suspensions), sublingual or buccal administration forms, intravenous, subcutaneous, and permeable. A dermal or intramuscular dosage form (eg, injection, powder, etc.). Further, the drug may be a general preparation, a sustained release preparation, an immediate release preparation, and a controlled release preparation.

Preferably, the medicament of the present invention is an oral dosage form, an intravenous administration form or an intramuscular administration form.

The invention also provides a desirable embodiment for the pharmaceutical preparation comprising fulvestrant, a functional fragment of fulvestrant, a fulvestrant derivative and a salt or solvate thereof, in particular when preparing a tablet or capsule form In the solid pharmaceutical composition, excipients which may be added to the active ingredient, including diluents such as lactose, dextrin, starch, pregelatinized starch, sucrose, mannitol, microcrystalline cellulose, etc.; adhesives, such as Polyvinylpyrrolidone, methylcellulose, hypromellose, etc.; disintegrants, such as sodium carboxymethyl starch, crosslinked carboxymethylcellulose, low-substituted hydroxypropylcellulose, sodium carboxymethylcellulose, Bipolyvinyl pyrrolidone, etc.; lubricants such as silica, magnesium stearate, stearate, corn starch, talc, etc., flavoring agents such as mannitol, aspartame, sodium saccharin and stevioside Further, a surfactant such as sodium dodecyl sulfate, sodium dioctyl sulfosuccinate, sucrose ester, poloxamer or the like may be added. When preparing a pharmaceutical composition in the form of a pill, excipients which may be added to the active ingredient include diluents and absorbents such as lactose, glucose, dextrin, starch, sucrose, cocoa butter, etc.; adhesives such as gum arabic , tragacanth, gelatin, honey, etc.; disintegrating agents, such as methyl cellulose, ethyl cellulose, dried starch, agar powder, alginate Wait. Oral solutions or suspensions may be added to excipients, including sweeteners such as sodium saccharin, sucrose, cyclamate, aspartame and stevioside; suspending agents such as polyvinylpyrrolidone, microcrystalline cellulose, sucrose , hydroxypropyl methylcellulose, etc.; preservatives, such as parabens, sodium benzoate, methyl paraben, propyl paraben, and the like. Excipients to which granules can be added include fillers, binders, colorants, flavors, and the like. For injection preparations, such as injections, emulsions, lyophilized powder injections, etc., various diluents commonly used in the art, such as water, ethanol, polyethylene glycol, propylene glycol, isostearyl alcohol, etc., cosolvents, buffers, can be used. Or a pH adjuster, etc. Further, in order to prepare an isotonic injection, sodium chloride, glucose or glycerin or the like may be added. The tablet of the present invention may be a pure tablet, and the tablet may be further formed into a coated tablet such as a film coat, a sugar coat or the like. Tablets can be formulated into immediate release, sustained release or controlled release dosage forms by preparing a polymeric matrix or by using a particular polymer in a film coating. The capsules may be soft or hard capsules without a film or film to provide immediate release, sustained release or controlled release properties.

In the pharmaceutical compositions of the invention, fulvestrant is typically formulated in dosage units. Each dose unit contains 50 to 250 mg of fulvestrant, administered once or more per month. Higher or lower doses may also be employed in certain circumstances, and the appropriate dosage for each patient is ultimately determined by the physician based on the mode of administration, the age, weight and response of the patient.

The drug can be used in combination with treatments already available (eg, chemotherapy, surgery, or radiation).

In addition to the use of the drug in any of the methods outlined, the medicament of the invention may be used as an additive to other therapies. It will be appreciated that the treatment of the invention may be combined with any other known treatment method.

Through the above technical solution, fulvestrant can significantly inhibit the growth of non-functional pituitary adenoma cells and transplanted tumors. Different doses of fulvestrant in primary growth non-functional pituitary adenoma tumor cells can significantly inhibit the proliferation of primary pituitary adenoma cells. Particularly at a treatment dose of 5-10 ng/mL, an optimal proliferation inhibiting effect can be obtained.

Other features and advantages of the invention will be described in detail in the detailed description which follows.

detailed description

Specific embodiments of the present invention will be described in detail below. It is to be understood that the specific embodiments described herein are merely illustrative and not restrictive.

In the following examples, fulvestrant is a commercial product of Sigma, and the article number is i4409.

Example 1

1.1 specimen source

Specimens were taken from tumor tissue surgically removed from patients with pituitary adenomas. Preoperative diagnosis based on clinical manifestations, serum hormone levels and imaging examination, and confirmed by intraoperative findings and postoperative pathology and immunohistochemical staining, as non-functional pituitary adenoma: empty cell adenoma, large hobby Acid granuloma, gonadotropin adenoma, 30 cases of each type of non-functional pituitary adenoma.

1.2 main reagent

Trypsin, DMEM medium, fetal bovine serum, dimethyl sulfoxide, and polylysine are all commercially available products.

1.3 cell culture

The surgically removed pituitary adenoma tissue was placed in serum-free DMEM culture medium under aseptic operation, and rinsed 2-3 times with sterile PBS in a clean bench to remove connective tissue, necrotic tissue and blood clots, and the specimen was cut with ophthalmic scissors. Into 1mm ³ size tissue block, add trypsin to blow and disperse, after the cells are well dispersed, add DMEM culture solution (containing 10% fetal bovine serum) to terminate digestion, filter through 100 mesh cell filter, centrifuge at 1000r/min for 10min, discard The supernatant was added to the DMEM medium to resuspend the cells, counted by microscopy, and the cell density was adjusted to 1 × 10 ⁶ /mL. Cell viability was counted by trypan blue staining. The inoculation was carried out in a culture flask in which polylysine was previously coated. When the primary pituitary adenoma cells reached 80%-90% confluence, the cells were collected by trypsinization and resuspended in DMEM medium.

1.4 Observation indicators:

1.4.1 Morphology and cell proliferation rate observation

Six different doses of treatment groups were set for each adenoma, and fulvestrant at a final concentration of 0.5, 1, 5, 10, 20, and 100 ng/mL was added to the DMEM medium, and an equal volume of DMSO was added to the control group. Perform cell culture.

The morphological characteristics of the cultured cells were observed every day under an inverted phase contrast microscope. The time of adherence of each pituitary adenoma cells in different treatment groups and the time to fill the monolayer were recorded and the average value was calculated. The results are shown in Table 1.

Table 1

1.4.2 Flow cytometry to detect apoptosis

Six different doses of treatment groups were set, and fulvestrant at a final concentration of 0.5, 1, 5, 10, 20, and 100 ng/mL was added to the DMEM medium, and an equal volume of DMSO was added to the control group for cell culture.

The primary pituitary adenoma cells with the same culture time for 3 weeks were subcultured for 3 days, harvested, and made into a single cell suspension. After centrifugation at 1000 r/m (r=15 cm) for 5 min, the supernatant was discarded and the cells were directly suspended. In PI hypotonic solution, FCM analyzes cellular DNA, and the fluorescence of PI-DNA complex For FCM econometric analysis, apoptosis is expressed as a percentage of cells with less than 2-fold body peak. The results are shown in Table 2.

Table 2

1.4.3 MTS assay for cell proliferation

Six different doses of the treatment group were set, and the fulvestrant at the final concentrations of 0.5, 1, 5, 10, 20, and 100 ng/mL was added to the DMEM medium, and the control group was added with an equal volume of DMSO. Functional pituitary adenoma cells were cultured in 96-well cells in a logarithmic growth phase, and DMEM medium (plus 10% calf serum) was added to each well for 24 hours in a saturated water vapor carbon dioxide incubator at 37 ° C in 5% CO ₂ . Add 20 μl of MTS/PMS mixture to each well and continue to culture for 3-4 hours. The plate was shaken for 10 seconds before the test, and the color was mixed, and the light absorption value (OD) of each well was measured at a wavelength of 570 nm on an enzyme-linked detector. A curve was plotted against the OD value (OD570) using sample dilution. The statistical cell proliferation was as shown in Table 3.

table 3

1.4.4 nude mice transplant tumor experiment

SPF BALB/c-nu mice (variety), female, 5-7 weeks old, body weight (18±2) g (purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., experimental animal license number: SCXK (京)2012-0001) Inoculation of GH3 cells (purchased from the Cell Center of the Institute of Basic Research, Chinese Academy of Medical Sciences) under the forage of the forelimbs, and the tumors were grown to a length of about 120 mm ³ to screen 30 tumor-bearing mice with uniform body weight and tumors. .

Thirty tumor-bearing mice were randomly divided into 3 groups, 10 in each group. Each group was administered with different doses of fulvestrant via tail vein, and administered once a day, each dose was 1 mg/kg. ,

5mg/kg, 10mg/kg. After 15 days of administration, the animals were sacrificed, and the tumor tissue was stripped to calculate the tumor suppressing rate TVI. The results are shown in Table 4.

TVI% = (tumor volume on the day of the blank group - tumor volume on the day of the administration group) / (tumor volume on the day of the blank group) × 100%.

Table 4

Dosage per dose (mg/kg)	Tumor inhibition rate after 15 days of administration (%)
1	17.7
5	36.1
10	48.4

The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solutions of the present invention within the scope of the technical idea of the present invention. These simple variants All fall within the scope of protection of the present invention.

It should be further noted that the specific technical features described in the above specific embodiments may be combined in any suitable manner without contradiction. To avoid unnecessary repetition, the present invention has various possibilities. The combination method will not be described separately.

In addition, any combination of various embodiments of the invention may be made as long as it does not deviate from the idea of the invention, and it should be regarded as the disclosure of the invention.

Claims (7)

1. Use of fulvestrant in the manufacture of a medicament for the treatment of non-functioning pituitary adenomas.
2. Use of functional fragments, derivatives and their salts or solvates of fulvestrant in the manufacture of a medicament for the treatment of non-functioning pituitary adenomas.
3. The use according to claim 2, characterized in that the derivative is a halogenated, sulfonated, nitrated, hydroxylated, alkoxylated or esterified product of fulvestrant.
4. The use according to claim 1 or 2, wherein the non-functional pituitary adenoma comprises a hollow cell adenoma, a large eosinophilic tumor, a gonadotropin adenoma, a resting corticosteroid adenoma and a sugar. At least one of a protein-secreting adenoma.
5. The use according to any one of claims 1 to 4, wherein the drug is fulvestrant, a functional fragment of fulvestrant, a derivative of fulvestrant, and a salt or solvate thereof. A single active ingredient or as one of the active ingredients.
6. The use according to claim 5, wherein the medicament further comprises at least one of a pharmaceutically acceptable excipient, a carrier and a diluent.
7. The use according to any one of claims 1 to 6, wherein the drug is a tablet, a capsule, a pill, a powder, a granule, a suspension, an oral solution, a powder injection or an injection.

   The use according to claim 7, wherein the medicament is an oral dosage form, a sublingual or buccal administration dosage form, an intravenous, subcutaneous, transdermal or intramuscular dosage form.