WO2018006688A1 - Use of fulvestrant in the preparation of drugs for treating growth hormone adenoma - Google Patents

Abstract

Provided in the present invention is a use of a fulvestrant in the preparation of drugs for treating the growth hormone adenoma. By means of the technical solution, the present invention can effectively improve the prognosis of patients with the growth hormone adenoma.

Description

Use of fulvestrant in the preparation of a medicament for treating growth hormone 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 treating growth hormone adenoma.

Background technique

Tumor cells from patients with growth hormone adenoma (GH adenoma) secrete excessive growth hormone (GH), and their physiological effects are mainly through affecting systemic tissue metabolism and controlling liver production of somatometin (SM) to promote tissue. Growth effect. Excessive secretion of growth hormone can cause giant disease before the closure of the epiphysis, and can cause acromegaly after the closure of the epiphysis. The same patient can have both giant-acromegaly. The existing drugs that can treat growth hormone adenoma diseases are as follows:

Dopaminergic agonists: Dopaminergic agonists promote the secretion of GH in normal humans, but inhibit the secretion of GH by GH tumors. About 20% of patients with acromegaly have blood GH and insulin-like growth factor 1 (IGF-1) down to normal, while patients with pituitary tumors that secrete GH and prolactin (PRL) have the best efficacy. Although 70% of patients with acromegaly have improved their clinical condition after long-term use, only 20-50% of blood GH levels are reduced to 10 and 5μg/L, and only 10-15% of growth hormone adenomas are slightly reduced. .

Somatostatin analogue: Somatostatin (SS) is a cyclic peptide containing 14 amino acids. At present, there are three kinds of SS drugs listed at home and abroad: (1) Natural SS, its half-life is only 3 minutes, and it also has a strong inhibitory effect on insulin secretion. After cessation, GH secretion rebounds, so it cannot be used as a chronic therapeutic drug. . (2) Octapeptide SS agonist (octreotide, developed by the Swiss sandoz company). Octreotide binds more to SSTR2, and the response of octreotide to patients with acromegaly is determined by the number of SSTR2 on pituitary GH tumor cells. In 10-30% of patients, the number of tumor cells SSTR2 is reduced, the response to octreotide is low, and the curative effect is poor. (3) Sustained-release somatostatin agonist-lanreotide, blood glucose levels were inhibited to 52% before treatment in 93% of patients, and GH secretion decreased in 85% of patients after 6 months. To normal. Symptom improvement rates were 75% for night sweats, 86% for headaches, 54% for finger and joint pain, and 50% for spinal pain.

GH receptor antagonists: If the marketed Pevisol is a relatively new class of drugs, it can compete with natural GH for binding to GH receptors, directly blocking the action of GH, resulting in reduced synthesis of IGF-1. This drug has high efficiency and rapid onset of the effect of blocking GH and lowering serum IGF-1. The disadvantage is that GH does not decrease and is elevated, and some patients have enlarged tumors and increased liver enzymes. The safety has not been fully confirmed.

Due to the above-mentioned drawbacks of existing treatment methods, it is necessary to develop new methods and medicines capable of treating growth hormone adenomas, thereby providing an effective way for treating and alleviating growth hormone adenoma diseases.

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 treating growth hormone adenoma.

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]-estrone-1,3,5-(10)-triene-3,17-diol), is an antiestrogenic drug alone, has no estrogen-like effects and reduces estrogen at the cellular level Receptor protein expression. Similar to the mechanism of action of tamoxifen, it competitively binds to the estrogen receptor, and fulvestrant completely inhibits the transcription of estrogen-sensitive genes and treats patients with estrogen receptor-positive breast cancer. Importantly, fulvestrant is not cross-resistant to other endocrine drugs.

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 growth hormone 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, and the like. The solvate refers to a hydrate, an ethanolate or the like. The derivatives can be used to inhibit, at least in part, the proliferation of growth hormone adenomas and the secretion of growth hormone. 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 their salts or solvates also have ideal application effects in the preparation of drugs for the treatment of growth hormone adenomas.

The condition caused by the growth hormone adenoma is at least one of acromegaly and giant disease.

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 disintegrants, lubricants, dispersants, etc., and can be selected by those skilled in the art according to the actual needs of the preparation, and the present invention does not specifically limited.

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, Union B a pyrrolidone or the like; a lubricant such as silica, magnesium stearate, stearate, corn starch, talc, etc., flavoring agents such as mannitol, aspartame, sodium saccharin, and stevioside, etc. Surfactants such as sodium dodecyl sulfate, sodium dioctyl sulfosuccinate, sucrose esters and poloxamers may also 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, and the like. An oral solution or suspension may be added, including sweeteners such as sodium saccharin, sucrose, cyclamate, aspartame, and stevioside; suspending agents such as polyvinylpyrrolidone, microcrystalline cellulose, sucrose, Hydroxypropyl methylcellulose or the like; preservatives such as paraben, 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 50-250 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).

The medicament of the present invention can be used as other than the use of the drug in any of the methods outlined Additives for therapy. It will be appreciated that the treatment of the invention may be combined with any other known treatment method.

Through the above technical scheme, fulvestrant can significantly inhibit the growth of growth hormone adenoma cells. Different doses of fulvestrant in primary growth hormone adenoma tumor cells and GH3 cells can significantly inhibit the proliferation of primary pituitary adenoma cells and GH3 cells. Especially at a treatment dose of 5-10 ng/mL, the optimal proliferation inhibition effect can be obtained, and the secretion of growth hormone can be reduced.

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, the article number is i4409.

Example 1

1.1 Cell source

GH3 cells are from the Institute of Basic Science of the Chinese Academy of Medical Sciences.

1.2 main reagent

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

1.3 cell culture

GH3 cells were resuscitated, and when the cells reached 80%-90% confluence, the cells were collected by trypsin digestion and resuspended in DMEM culture medium.

1.4 Observation indicators:

1.4.1 Morphology and cell proliferation rate observation

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 morphological characteristics of the cultured cells were observed once a day under an inverted phase contrast microscope, and the time of adherence of GH3 cells and the time of overgrown monolayers in different treatment groups were recorded. The results are shown in Table 1.

Table 1

Treatment group (ng/mL)	0.5	1	5	10	20	100	0
Cell attachment time (hours)	14	15.5	18	19.5	20	twenty four	12
Overfull single layer time (hours)	56	60	64	72	80	96	48

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.

GH3 cells cultured for 3 weeks were subcultured for 3 days, harvested, and made into a single cell suspension, centrifuged at 1000 r / m (r = 15 cm) for 5 min, the supernatant was discarded, and the cells were directly suspended in PI hypotonic solution. In the FCM, the cellular DNA was analyzed, and the fluorescence emitted by the PI-DNA complex was analyzed by FCM, and the apoptosis was expressed as the percentage of cells having a peak of less than 2 times. The results are shown in Table 2.

Table 2

Treatment group (ng/mL)	0.5	1	5	10	20	100	0
Apoptosis as a percentage of total (%)	4.6	7.9	13.4	17.8	24.1	32.7	2.3

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 to culture the GH3 cells. In the logarithmic growth phase, 96-well cell culture plates were taken, 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. Shake the plate for 10 seconds before the test to mix the color, and measure the light absorption value (OD) of each well 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

Treatment group (ng/mL)	0.5	1	5	10	20	100	0
Percentage of cell proliferation (%)	87.6	77.3	68.3	61.9	57.2	41.4	100

The above results indicate that the application of different doses of fulvestrant in GH3 cells can significantly inhibit the proliferation of GH3 cells. Particularly at a treatment dose of 20-100 ng/mL, an optimal proliferation inhibiting effect can be obtained.

1.4.4 Detection of the effect of fulvestrant on GH secretion in GH3 cells:

The cells were seeded in a 24-well plate to a cell density of about 3 × 10 ⁵ cells/well, and cultured in a humidity chamber (containing 95% air 5% carbon dioxide) at 37 ° C for 2 days. The state of the cells was observed under the microscope, the cells adhered well, and few cells were suspended. The cells were cultured overnight in serum-free DMEM medium 12 hours before the drug experiment.

1.4.4.1Detecting the effects of different concentrations of fulvestrant on GH secretion

The working concentrations of fulvestrant were 0.5, 1, 5, 10, 20 and 100 ng/mL and a blank control. A total of 7 groups (as shown in Table 4, different concentrations of fulvestrant intervention cell grouping). After incubating for 2 hours at 37 ° C in a humidity environment (containing 95% air 5% carbon dioxide), the cell culture medium was collected, centrifuged at 12,000 rpm for 5 min, and the supernatant was retained.

GH levels were measured by enzyme-linked immunosorbent assay (ELISA) and the kit was purchased from Millipore. The absorbance at 450 and 590 nm was measured according to the kit. The GH concentration was calculated from the standard curve (as shown in Table 4).

Table 4

1.4.4.2 To determine the effect of fulvestrant on GH secretion at different working concentrations of 20 ng/mL.

Five groups (as shown in Table 5) and blank control, 7 ° C, humidity environment (95% air 5% carbon dioxide) incubator culture, the cell culture medium was collected at the corresponding time, centrifuged at 2000 rpm for 5 min, the supernatant was retained.

GH levels were measured by enzyme-linked immunosorbent assay (ELISA) and the kit was purchased from Millipore. The absorbance at 450 and 590 nm was measured according to the kit. The GH concentration was calculated from the standard curve (as shown in Table 5).

table 5

Processing group	Blank control group	Group 1	Group 2	Group 3	Group 4	Group 5
Fulvestrant	-	0.5h	1h	2h	3h	4h
GH concentration (ng/mL)	112	104	97	92	83	76

1.4.5 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 ( Beijing) 2012-0001) by the forelimb armpit inoculated subcutaneously with murine growth hormone of pituitary adenoma GH3 cells (purchased from cell Center, Institute of basic Chinese Academy of Medical Sciences), until the tumors grew to about 120mm ³ screened out of uniform weight and tumor 30 tumor-bearing mice were used.

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 inhibition rate TVI. The results are shown in Table 6.

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 6

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 (8)

1. Use of fulvestrant in the preparation of a medicament for treating growth hormone adenoma.
2. Use of functional fragments, derivatives and their salts or solvates of fulvestrant in the manufacture of a medicament for the treatment of growth hormone adenomas.
3. The use according to claim 2, characterized in that the derivative of fulvestrant is a halogenated, sulfonated, nitrated, hydroxylated, alkoxylated or esterified product of fulvestrant.
4. The use according to claim 1 or 2, wherein the condition caused by the growth hormone adenoma is at least one of acromegaly and giant disease.
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.
8. 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.