WO2017194031A1 - Use for protein function inhibitor dapt in preparing medicine for treating adenoma - Google Patents

Abstract

Use for functional fragments, derivatives, and salts or solvates of the protein function inhibitor DAPT in preparing medicine for treating pituitary adenoma is provided in the present invention, said pituitary adenoma being functional pituitary adenoma and/or non-functional pituitary adenoma.

Description

Use of protein function inhibitor DAPT in the preparation of a medicament for treating tumor Technical field

The present invention relates to the field of biochemistry and medicine, and in particular to the use of a protein function inhibitor DAPT for the preparation of a medicament for treating tumors.

Background technique

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. Somatostatin analogues and dopamine receptor agonists are used to control a subset of patients with high-secretion GH and PRL adenomas. The level of SSTR2 expression is positively correlated with drug sensitivity. 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%. There is still a lack of targeted and effective medications.

Therefore, it is necessary to develop methods and drugs that can affect the differentiation, proliferation, apoptosis and invasion process of pituitary adenomas, so as to provide an effective way to improve the treatment and prognosis of pituitary adenomas.

Summary of the invention

A first object of the present invention is to provide a use of the protein function inhibitor DAPT for the preparation of a medicament for the treatment of pituitary adenomas.

A second object of the present invention is to provide a use of functional fragments, derivatives and their salts or solvates of the protein function inhibitor DAPT for the preparation of a medicament for the treatment of pituitary adenomas.

Among them, DAPT refers to the compound of CAS accession number 208255-80-5, Chinese name: (3,5-difluorophenylacetyl)-L-alanyl-L-2-phenylglycine tert-butyl ester; C ₂₃ H ₂₆ F ₂ N ₂ O ₄ ; is a γ-secretase inhibitor that can indirectly inhibit the activity of the γ-secretase substrate Notch, thereby affecting cell signaling and cell differentiation.

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

The DAPT 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 derivatives can be used to at least partially inhibit the differentiation, proliferation and invasion of pituitary adenomas. Those skilled in the art will foresee that the derivatives and functionalized fragments of DAPT have the same core structure as DAPT. Therefore, its functional fragments, derivatives, and salts or solvates thereof also have desirable effects in the preparation of a medicament for treating or preventing pituitary adenomas.

The pituitary adenoma of the present invention encompasses a variety of known pituitary adenomas, including functional pituitary adenomas and/or non-functional pituitary adenomas, wherein the functional pituitary adenomas include GH type pituitary adenomas, PRLs At least one of a pituitary adenoma, an ACTH-type pituitary adenoma, and a TSH-type pituitary adenoma; the non-functional pituitary adenoma including acellular adenoma, large eosinophilia, gonadotropin adenoma, static At least one of a corticosteroid adenoma and a glycoprotein secreting adenoma.

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, veins, Subcutaneous, transdermal or intramuscular dosage forms (eg, injections, powders, 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 present invention also provides a desirable embodiment for pharmaceutical preparations containing DAPT, DAPT functional fragments, DAPT derivatives and salts or solvates thereof, in particular, when preparing solid pharmaceutical compositions in the form of tablets or capsules, Excipients which may be added to the ingredients, including diluents such as lactose, dextrin, starch, pregelatinized starch, sucrose, mannitol, microcrystalline cellulose, etc.; binders such as polyvinylpyrrolidone, methylcellulose, Hypromellose or the like; a disintegrating agent such as sodium carboxymethyl starch, crosslinked carboxymethyl cellulose, low substituted hydroxypropyl cellulose, sodium carboxymethyl cellulose, crosslinked polyvinylpyrrolidone, etc.; lubricant, Such as silica, magnesium stearate, stearate, corn starch, talc, etc., flavoring agents, such as mannitol, aspartame, sodium saccharin and stevioside, in addition, surfactants can also be added For example, sodium dodecyl sulfate, sodium dioctyl sulfosuccinate, sucrose esters and poloxamers. 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 made by preparing a polymer matrix or by using a specific polymer in a film coating. Immediate release, sustained release or controlled release dosage form. 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, DAPT is typically formulated in dosage units. Each dose unit contains 5 to 10 mg of DAPT, administered once or more daily. 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 scheme, DAPT can significantly inhibit the growth of pituitary adenoma cells and transplanted tumors. Different doses of DAPT 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 5-20 ng/mL, the optimal proliferation inhibition effect can be obtained. The Transwell experiment also showed that DAPT inhibited the invasive ability of tumor cells at a dose of 2-20 ng/mL.

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, DAPT is a commercial product of Gene Operation (US), and the article number is IN01001-0005MG.

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, invasive GH type pituitary adenoma, TSH gland There were 10 cases of tumor, PRL adenoma and non-functioning 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. The tissue was 1mm ^{3 in} size and was mixed with trypsin. After the cells were well dispersed, the DMEM medium (containing 10% fetal bovine serum) was added to terminate the digestion. After filtration through a 100 mesh cell filter, centrifuge at 1000 r/min for 10 min. 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 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 DAPT at the final concentrations of 0.5, 1, 5, 10, 20, and 100 ng/mL were 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 different treatments were recorded. The time of adherence of the pituitary adenoma cells and the time of overgrowth of the monolayer were as shown in Table 1.

Table 1

1.4.2 Flow cytometry to detect apoptosis

Six different doses of the treatment group were set, and DAPT at the final concentrations of 0.5, 1, 5, 10, 20, and 100 ng/mL were 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 the PI hypotonic solution, FCM analyzes the cellular DNA, and the fluorescence emitted by the PI-DNA complex is analyzed by FCM, and the apoptosis is expressed by the percentage of cells having less than 2 times the body peak. The results are shown in Table 2.

Table 2

1.4.3 MTS assay for cell proliferation

Six different doses of treatment groups were set up, DAPT were added at a final concentration of 0.5, 1, 5, 10, 20 and 100 ng/mL in DMEM medium, and an equal volume of DMSO was added to the control group to culture primary pituitary adenoma cells. And GH3 cells to logarithmic growth phase, 96-well cell culture plates were taken, and each well was added with DMEM medium (plus 10% calf serum) 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 of the mixture was measured on an enzyme-linked detector at a wavelength of 570 nm to measure the light absorption value (OD) of each well. 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 Detection of tumor cell invasion ability

According to Chemicon's ECM550 series instructions, the chamber was placed in a culture plate, 300 μl of pre-warmed serum-free medium was added to the upper chamber, and allowed to stand at room temperature for 15-30 min to rehydrate the matrigel. The remaining culture solution is then aspirated.

Preparation of cell suspension: Before preparing the cell suspension, the cells can be serum-starved for 12-24 hours to further remove the serum. The cells were digested, the digestion was terminated, the culture was discarded by centrifugation, washed 1-2 times with PBS, and resuspended in serum-free medium containing BSA. Different doses of DAPT were added and the cell density was adjusted to 1-10 x 10 ⁵ .

Inoculation of cells: 200 μL of the cell suspension was added to the Transwell chamber; 500 μL of medium containing FBS or chemokine was added to the lower chamber of the 24-well plate.

Cultured cells: routinely cultured for 24 h.

The results were counted by MTT method: the matrigel and the cells in the upper chamber were wiped with a cotton swab; 500 μl of complete medium containing 0.5 mg/mL MTT was added to the 24-well plate, and the chamber was placed therein to immerse the membrane in the medium, 37 Remove after °C for 4h. 500 μl of DMSO was added to the 24-well plate, the chamber was placed therein, the membrane was immersed in DMSO, shaken for 10 min, and fully dissolved. The chamber was taken out, and the OD value (OD570) was measured on a 24-well plate on a microplate reader. The results of the treatment data are shown in Table 4.

Table 4

Treatment group (ng/mL)	0.5	1	5	10	20	100	0
Primary pituitary adenoma cells	0.379	0.334	0.296	0.223	0.178	0.142	0.457
GH3 cell	0.391	0.345	0.277	0.207	0.165	0.134	0.672

These results indicate that the application of different doses of DAPT in primary growth hormone adenoma tumor cells and GH3 cells can significantly inhibit the proliferation of primary tumor cells and GH3 cells. Especially at a treatment dose of 5-20 ng/mL, the optimal proliferation inhibition effect can be obtained. Transwell experiments show that DAPT can inhibit the invasion ability of tumor cells.

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 Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., experimental animal license number: SCXK (jing ) 2012-0001) Inoculation of rat pituitary growth hormone adenoma GH3 cells (purchased from the Cell Center of the Institute of Basic Research, Chinese Academy of Medical Sciences) under the armpits of the forelimbs, and the tumors were selected to have a uniform body weight and a tumor volume of about 120 mm ³ . 30 tumor mice, spare.

Thirty tumor-bearing mice were randomly divided into 3 groups, 10 in each group. Each group was administered different doses of DAPT via tail vein, and administered once a day, each dose was 1 mg/kg, 5 mg/ Kg, 10 mg/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 5.

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

table 5

Dosage per dose (mg/kg)	Tumor inhibition rate after 15 days of administration (%)
1	10.71
5	28.97
10	49.81

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 the protein function inhibitor DAPT in the preparation of a medicament for treating pituitary adenomas.
2. Use of functional fragments, derivatives and their salts or solvates of the protein function inhibitor DAPT for the preparation of a medicament for the treatment of pituitary adenomas.
3. The use according to claim 2, characterized in that the derivative of DAPT is a halogenated, sulfonated, nitrated, hydroxylated, alkoxylated or esterified product of DAPT.
4. The use according to claim 1 or 2, wherein the pituitary adenoma is a functional pituitary adenoma and/or a non-functional pituitary adenoma, wherein the functional pituitary adenoma comprises a GH type pituitary gland At least one of a tumor, a PRL type pituitary adenoma, an ACTH type pituitary adenoma, and a TSH type pituitary adenoma; the non-functional pituitary adenoma includes a hollow cell adenoma, a large eosinophil, a gonadotropin gland At least one of a tumor, a resting corticosteroid adenoma, and a glycoprotein-secreting adenoma.
5. The use according to any one of claims 1 to 4, characterized in that the drug is a single active ingredient or one of the active ingredients of DAPT, a functional fragment of DAPT, a derivative of DAPT, and a salt or solvate thereof. .
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.