CN108685916B - Application of thiazolidinedione compounds in double-target treatment of pituitary growth hormone adenoma - Google Patents

Abstract

The invention relates to an application of thiazolidinedione compounds in double-target treatment of pituitary growth hormone adenoma. Specifically, the invention discloses application of a compound shown in a formula I, or an isomer, a solvate or a pharmaceutically acceptable salt thereof, in preparation of a preparation for inhibiting cell expression Growth Hormone Receptor (GHR), wherein the cell is a non-growth hormone secretion cell, and the definition of each group and substituent group in the compound shown in the formula I is described in the specification. The compounds are effective in effecting the treatment of pituitary growth hormone adenomas.

Description

Application of thiazolidinedione compounds in double-target treatment of pituitary growth hormone adenoma

Technical Field

The invention relates to the technical field of medicines, in particular to an application of thiazolidinedione compounds in double-target treatment of pituitary growth hormone adenoma.

Background

Pituitary Growth Hormone (GH) adenomas are functional pituitary tumors that secrete growth hormone, and are extremely dangerous neuroendocrine tumors. Epidemiological data show that GH adenoma patients have 2 times higher mortality than normal populations, with a 10 year reduction in average patient life. The risk of GH adenoma mainly includes two aspects: (1) tumor itself occupancy effects: normal pituitary tissue and surrounding tissues are affected by compression, resulting in headache, hypopituitarism, visual field defect, vision decline, eye movement disorder, etc.; (2) hazard of hypersecretion of GH: causing adverse consequences such as giant people, acromegaly, hyperglycemia, hypertension, cardiovascular diseases, organ enlargement, secondary tumors and the like, and seriously affecting the health and the life expectancy of patients.

Existing treatments for GH adenomas include surgery, medical drugs (mainly dopamine receptor agonists, somatostatin analogues, GH receptor antagonists) and radiotherapy. According to the latest guidelines at home and abroad, the goal of treatment of growth hormone adenomas is to restore normal levels of GH and IGF-1 in patients. However, the current treatment methods have limited curative effects on GH adenoma, and have no small gap from the therapeutic targets recommended by guidelines. The total cure rate of the traditional microsurgery for treating GH adenoma is 57.3%, the microadenoma is 80-91%, and the macroadenoma is 40-52%. Radiotherapy is slow to take effect (often for years), and causes problems such as hypopituitarism and secondary tumors. In the aspect of drug treatment, the curative effect of the dopamine receptor agonist for treating GH adenoma is poor, only 15% of patients can reduce GH to below 5ug/L by using bromocriptine, and the normal proportion of IGF-1 by using cabergoline is not more than 1/3; as the most effective medical drug, somatostatin analogs also normalize GH and IGF-1 levels in only about 35% of patients; GH receptor antagonists currently belong to two-line drug therapy, act on the liver, inhibit IGF-1 synthesis, but cannot inhibit pituitary secretion of GH, and have the risks of tumor enlargement and invasion; the latter two kinds of medicines are very expensive, and GH receptor antagonists are not available in China. Somatostatin analogues and dopamine receptor agonists act mainly on the pituitary gland, GH receptor antagonists act mainly on the liver, and no drug is currently available to treat pituitary growth hormone adenoma at the same time at double targets of pituitary and liver.

Therefore, it is of great importance to find drugs capable of treating pituitary growth hormone adenoma at the same time at both pituitary and liver dual targets.

Disclosure of Invention

The invention aims to provide a medicine capable of simultaneously treating pituitary growth hormone adenoma at double targets of pituitary and liver.

Specifically, the invention provides application of a compound shown in a formula I, or an isomer, a solvate or a pharmaceutically acceptable salt thereof in preparation of a preparation for inhibiting cell expression Growth Hormone Receptor (GHR).

In a first aspect of the present invention there is provided the use of a compound of formula I, or an isomer, or a solvate, or a pharmaceutically acceptable salt thereof, for the preparation of a formulation for inhibiting the expression of Growth Hormone Receptor (GHR) by a cell, wherein the cell is a non-growth hormone secreting cell:

wherein R is-X-Y-Z,

x is selected from the group consisting of: an unsubstituted, substituted or unsubstituted C1-C6 alkylene group;

y is selected from the group consisting of: nothing or NR ₁ Wherein R is ₁ Is H, substituted or unsubstituted C1-C6 alkyl;

z is selected from the group consisting of: a substituted or unsubstituted 5-6 membered heteroaryl group containing 1-3 heteroatoms selected from N, O, S, a substituted or unsubstituted 5-10 membered heterocyclyl group containing 1-3 heteroatoms selected from N, O, S;

wherein the substitution means substitution with one or more substituents selected from the group consisting of: halogen, C1-C6 alkyl, C1-C3 haloalkyl, C1-C4 alkoxy, hydroxy.

In another preferred embodiment, the "5-6 membered heteroaryl containing 1-3 heteroatoms selected from N, O, S" is

In another preferred embodiment, the "5-10 membered heterocyclic group containing 1-3 heteroatoms selected from N, O, S" is

In another preferred embodiment, the compound of formula I is selected from the group consisting of:

in another preferred embodiment, the formulation is a pharmaceutical formulation, or a non-pharmaceutical formulation (e.g., a test formulation).

In another preferred embodiment, the formulation is a pharmaceutical composition.

In another preferred embodiment, the formulation is used to (a) inhibit growth hormone secreting cells from expressing (or secreting) growth hormone; and (b) inhibiting expression of a growth hormone receptor by a non-growth hormone secreting cell.

In another preferred embodiment, the growth hormone secreting cells include pituitary cells.

In another preferred embodiment, the non-growth hormone secreting cells include hepatocytes.

In another preferred embodiment, the non-growth hormone secreting cells include liver cancer cells (e.g., hepG2 cells).

In another preferred embodiment, the formulation is also used for the treatment of pituitary growth hormone adenomas.

In another preferred embodiment, said pituitary growth hormone adenoma is an adenoma which is highly expressed in said non-growth hormone secreting cells.

In another preferred embodiment, the term "high expression" means: the ratio (E1/E0) of the expression level E1 of the growth hormone receptor in the non-growth hormone secreting cells (such as hepatocytes) to the expression level E0 of GHR in the normal hepatocytes is not less than 1.5, preferably not less than 2.0, more preferably not less than 2.5.

In another preferred embodiment, the formulation further has a use selected from the group consisting of: decreasing IGF1 and increasing 15-PGDH.

In another preferred embodiment, the formulation is for use in the treatment of pituitary growth hormone adenoma at a single pituitary target site; or (b)

The pharmaceutical composition or the preparation is used for treating pituitary growth hormone adenoma at a single target point of liver.

In another preferred embodiment, the formulation is used for the dual-target treatment of pituitary growth hormone adenoma in the pituitary and liver.

In another preferred embodiment, the pituitary growth hormone adenoma is selected from the group consisting of: microadenomas, macroadenomas, or combinations thereof.

In another preferred embodiment, the formulation comprises 0.001 to 99wt%, preferably 0.1 to 90wt%, more preferably 1 to 80wt% of the compound of formula I, or an isomer thereof, or a solvate thereof, or a pharmaceutically acceptable salt thereof, based on the total weight of the formulation.

In another preferred embodiment, the formulation further optionally contains a component selected from the group consisting of:

1) A second pharmaceutically active ingredient which is an anti-tumor drug, an antibody or an antisense nucleic acid;

2) A pharmaceutically acceptable carrier.

In a second aspect of the invention, there is provided a formulation comprising:

i) A compound of formula I, or an isomer thereof, or a solvate thereof, or a pharmaceutically acceptable salt thereof, as a first pharmaceutically active ingredient;

ii) a second pharmaceutically active ingredient which is different from component (i) and is intended for the inhibition of tumours;

iii) A pharmaceutically acceptable carrier.

In a third aspect of the invention, there is provided a method of non-therapeutically inhibiting growth hormone receptor expression in vitro comprising the steps of:

contacting a compound of formula I, or an isomer thereof, or a solvate thereof, or a pharmaceutically acceptable salt thereof, with a non-growth hormone secreting cell, thereby inhibiting expression of a growth hormone receptor.

In another preferred embodiment, the method is performed under GH stimulation.

In a fourth aspect of the present invention, there is provided a method of treating pituitary growth hormone adenoma, the method comprising the steps of: administering to a patient in need thereof a therapeutically effective amount of a compound of formula I, or an isomer thereof, or a solvate thereof, or a pharmaceutically acceptable salt thereof.

In another preferred embodiment, the "patient in need thereof" includes human and non-human mammals (e.g., mice, rats).

It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.

Drawings

Figure 1 shows the change in GH expression levels after various concentration gradients of rosiglitazone have been used to intervene in human primary GH tumor cells for 72 hours.

Figure 2 shows GH expression level changes following intervention of rosiglitazone at different concentration gradients and different time gradients in rat GH3 cells.

FIG. 3 shows the change in 15-PGDH expression levels after 72 hours of rosiglitazone intervention on human primary GH tumor cells at different concentration gradients.

FIG. 4 shows the 15-PGDH expression level change after intervention of rosiglitazone in rat GH3 cells at different concentration gradients and at different time gradients.

FIG. 5 shows the effect of rosiglitazone intervention at different concentration gradients on IGF-1 expression by human hepatocyte strains (HepG 2) under GH stimulation.

FIG. 6 shows the effect of rosiglitazone intervention at different concentration gradients on the expression of GHR in human hepatocyte strains (HepG 2) under GH stimulation.

FIG. 7 is the effect of rosiglitazone intervention at different concentration gradients on the expression of 15-PGDH in human hepatocyte strains (HepG 2) under GH stimulation.

FIG. 8 shows the construction of an efficient shRNA plasmid for 15-PGDH.

FIG. 9 is the effect of rosiglitazone on IGF-1 expression in human liver HepG2 cells following interference with 15-PGDH.

FIG. 10 is the effect of rosiglitazone on GHR expression in human liver HepG2 cells following interference with 15-PGDH.

Detailed Description

The inventor has studied intensively for a long time, and found that thiazolidinedione compounds (such as rosiglitazone) can inhibit GHR expression (or activity) of non-growth hormone secretion type cells (especially liver cells) very effectively, so as to realize the cooperative treatment of double targets (or double target organs) of pituitary growth hormone adenoma. On this basis, the inventors completed the present invention.

Active ingredient

In the invention, the main active ingredient is a compound shown in a formula I, or an isomer, a solvate or a pharmaceutically acceptable salt thereof:

wherein R is as defined above.

One representative compound of formula I is Rosiglitazone (Ros), which is a thiazolidinedione, a highly selective, potent agonist of peroxisome proliferator-activated receptor gamma (pparγ). The current clinical indication is type 2 diabetes, which acts primarily by improving insulin sensitivity, without causing hypoglycemia when taken alone.

The chemical structural formula of rosiglitazone is as follows:

in addition, analogs of rosiglitazone, such as thiazolidinediones such as Pioglitazone (Pioglitazone) and Troglitazone (Troglitazone), may also achieve dual-target effects for the treatment of pituitary growth hormone adenoma.

Specifically, the thiazolidinedione compound has the following chemical structural formula:

in terms of structural activity, A is more than or equal to B and is more than or equal to C, so that in the thiazolidinedione compound, the main active group is the structure A in the figure.

The chemical structural formula of pioglitazone is as follows:

the chemical structural formula of troglitazone is as follows:

IGF-1

insulin-like growth factor 1 (Insulin-like growth factors, IGF-1), also known as "growth hormone mediator", is an active protein polypeptide substance essential in the physiological action of growth hormone, and is also the product of autocrine and paracrine of more than ten cells in human body, mainly hepatocytes, and has the effects of reducing blood sugar, reducing blood lipid, dilating blood vessels, promoting bone metabolism synthesis, promoting growth, promoting cell differentiation, repairing wound, etc.

GHR

Growth hormone receptors (Growth Hormone Receptor, GHR) are located on the surface of cells (primarily hepatocytes), with which Growth Hormone (GH) binds in the first step to function at the tissue and cellular level, being mediated by and transmitting signals into the cells to produce a range of physiological effects. Therefore, the amount of GHR in the tissue and the normal or abnormal function will influence the physiological effect of GH.

15-PGDH

15-hydroxy prostaglandin dehydrogenase (15-Hydroxyprostaglandin Dehydrogenase, 15-PGDH) is a 29kD dehydrogenase encoded by the HPGH (Hydroxyprostaglandin Dehydrogenase) gene, which degrades prostaglandin E2 (PGE 2) in vivo, catalyzes the oxidation of the hydroxy group at position 15 to a keto group, and inactivates PGE 2. Previous studies have found that PGE2 can promote proliferation, invasion and angiogenesis of cells, inhibit apoptosis and monitor immunity. At present, 15-PGDH has been found to have the effect of inhibiting cancer in colon cancer and lung cancer.

The research of the inventor shows that the expression of 15-PGDH in GH adenoma patients is lower than that of normal people through the analysis of means such as immunohistochemical scoring, qPCR detection and the like, and the expression of 15-PGDH in GH macroadenoma is lower than that of GH microadenoma.

The research of the invention further shows that the 15-PGDH can be used as a novel therapeutic target for treating GH adenoma by improving the expression or activity of the 15-PGDH.

Use of the same

The invention provides a use of a compound of formula I, or an isomer thereof, or a solvate thereof, or a pharmaceutically acceptable salt thereof, for the preparation of a formulation for inhibiting the expression of Growth Hormone Receptor (GHR) by a cell, wherein the cell is a non-growth hormone secreting cell:

wherein R is as defined above.

The present invention provides the use of a compound of formula I (e.g. rosiglitazone) for the treatment of pituitary growth hormone adenomas, particularly for the treatment of patients who have not achieved biochemical control following surgery or radiotherapy.

Based on this, the inventors considered that: the compound of formula I (e.g. rosiglitazone) can be a treatment option for patients with growth hormone adenoma, the compound of formula I (e.g. rosiglitazone) can act on dual targets of the pituitary and liver to treat pituitary growth hormone adenoma, and 15-PGDH can be one of the target proteins on which it acts.

Pharmaceutical compositions (or formulations) and methods of administration

The invention also provides a formulation comprising:

i) A compound of formula I, or an isomer thereof, or a solvate thereof, or a pharmaceutically acceptable salt thereof, as a first pharmaceutically active ingredient;

ii) a second pharmaceutically active ingredient which is different from component (i) and is intended for the inhibition of tumours;

iii) A pharmaceutically acceptable carrier.

As used herein, the term "pharmaceutically acceptable salt" refers to a salt of a compound of formula I (e.g., rosiglitazone) with an acid or base that is suitable for use as a medicament. Pharmaceutically acceptable salts include inorganic and organic salts. One preferred class of salts is the salts of the compounds of formula I (e.g., rosiglitazone) with acids. Suitable salts forming acids include, but are not limited to: inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid, and the like; organic acids such as formic acid, acetic acid, trifluoroacetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, benzoic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, benzenesulfonic acid, naphthalenesulfonic acid, and the like; amino acids such as proline, phenylalanine, aspartic acid, and glutamic acid.

Another preferred class of salts are salts of the compounds of formula I (e.g. rosiglitazone) with bases, for example alkali metal salts (e.g. sodium or potassium salts), alkaline earth metal salts (e.g. magnesium or calcium salts), ammonium salts (e.g. lower alkanolammonium salts and other pharmaceutically acceptable amine salts), for example methylamine, ethylamine, propylamine, dimethylamine, trimethylamine, diethylamine, triethylamine, tert-butylamine, ethylenediamine, hydroxyethylamine, diethylamine, triethylamine, and amine salts formed from morpholine, piperazine, lysine, respectively.

The term "solvate" refers to a compound of formula I (e.g., rosiglitazone) coordinated to a solvent molecule to form a complex in a specific ratio.

The pharmaceutical compositions of the present invention comprise a safe and effective amount of a compound of formula I (e.g., rosiglitazone) or a pharmaceutically acceptable salt thereof in combination with a pharmaceutically acceptable excipient or carrier. Wherein "safe and effective amount" means: the amount of the compound of formula I (e.g., rosiglitazone) is sufficient to significantly improve the condition without serious side effects. Typically, the pharmaceutical compositions contain 1-2000mg of a compound of formula I (e.g., rosiglitazone) per agent, more preferably, 10-1000mg of a compound of formula I (e.g., rosiglitazone) per agent. Preferably, the "one dose" is a capsule or tablet.

"pharmaceutically acceptable carrier" means: one or more compatible solid or liquid filler or gel materials which are suitable for human use and must be of sufficient purity and sufficiently low toxicity. "compatibility" as used herein means that the components of the composition are capable of reacting with a compound of formula I (e.g., rosiglitazone) and their useAre blended with each other without significantly reducing the potency of the compound of formula I (e.g., rosiglitazone). Examples of pharmaceutically acceptable carrier moieties are cellulose and its derivatives (e.g., sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, and the like), gelatin, talc, solid lubricants (e.g., stearic acid, magnesium stearate), calcium sulfate, vegetable oils (e.g., soybean oil, sesame oil, peanut oil, olive oil, and the like), polyols (e.g., propylene glycol, glycerol, mannitol, sorbitol, and the like), emulsifiers (e.g.

) Wetting agents (such as sodium lauryl sulfate), coloring agents, flavoring agents, stabilizing agents, antioxidants, preservatives, pyrogen-free water and the like.

The mode of administration of the compound of formula I (e.g., rosiglitazone) or the pharmaceutical composition of the invention is not particularly limited, and representative modes of administration include (but are not limited to): oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous), and topical administration.

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In these solid dosage forms, the active compound is admixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) Fillers or compatibilizers, for example, starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) Binders, for example, hydroxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and acacia; (c) humectants, e.g., glycerin; (d) Disintegrants, for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) a slow solvent, such as paraffin; (f) an absorption accelerator, e.g., a quaternary amine compound; (g) Wetting agents, such as cetyl alcohol and glycerol monostearate; (h) an adsorbent, for example, kaolin; and (i) a lubricant, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets and pills, the dosage forms may also comprise buffering agents.

Solid dosage forms such as tablets, dragees, capsules, pills and granules can be prepared with coatings and shells, such as enteric coatings and other materials well known in the art. They may contain opacifying agents and the release of the active compound or compounds in such compositions may be released in a delayed manner in a certain part of the digestive tract. Examples of embedding components that can be used are polymeric substances and waxes. The active compound may also be in the form of microcapsules with one or more of the above excipients, if desired.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compound, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1, 3-butylene glycol, dimethylformamide and oils, in particular, cottonseed, groundnut, corn germ, olive, castor and sesame oils or mixtures of these substances and the like.

In addition to these inert diluents, the compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar-agar or mixtures of these substances, and the like.

Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and nonaqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

Dosage forms of the compounds of formula I (e.g., rosiglitazone) for topical administration include ointments, powders, patches, sprays and inhalants. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants which may be required if necessary.

The compounds of formula I (e.g., rosiglitazone) may be administered alone or in combination with other pharmaceutically acceptable other compounds (e.g., antineoplastic agents).

The methods of treatment of the present invention may be administered alone or in combination with other therapeutic means or therapeutic agents.

When a pharmaceutical composition is used, a safe and effective amount of the compound of the present invention is applied to a mammal (e.g., a human) in need of treatment, wherein the dose at the time of administration is a pharmaceutically effective dose, and the daily dose is usually 1 to 2000mg, preferably 50 to 1000mg, for a human having a body weight of 60 kg. Of course, the particular dosage should also take into account factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled practitioner.

The main advantages of the invention include:

(1) The compound of formula I (such as rosiglitazone) can simultaneously treat pituitary growth hormone adenoma at double targets of pituitary and liver;

(2) The therapeutic effect of a compound of formula I (e.g., rosiglitazone) on pituitary growth hormone adenomas is mediated by induction of an elevation of 15-PGDH.

The invention provides a new drug choice and mechanism for clinical treatment of pituitary growth hormone adenoma, namely, the compound of the formula I including the compound of the formula I (such as rosiglitazone) treats the pituitary growth hormone adenoma at double targets of pituitary and liver. On one hand, the preparation is expected to develop new medicine indications, and on the other hand, the preparation is expected to provide new therapeutic medicines for patients with growth hormone adenoma, which cannot be relieved by the existing therapeutic means, so that the relieving rate is improved, and the treatment cost is reduced.

The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions, such as, for example, sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. Percentages and parts are weight percentages and parts unless otherwise indicated.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The preferred methods and materials described herein are presented for illustrative purposes only.

Experimental cells and materials:

the primary cells of human pituitary GH tumor are from neurosurgical pituitary tumor biological sample library of the affiliated Huashan Hospital of double denier university, and the rat pituitary GH3 cell strain and the human liver HepG2 cell strain are purchased from the national academy of sciences.

Rosiglitazone (Sigma), recombinant human growth hormone (gold Sachikusetsushi), PCR primers (Shanghai), DMEM (Invitrogen) +10% FBS (Invitrogen) complete medium, horse serum (Gibco), fetal bovine serum (Gibco), hyaluronidase (Sigma), collagenase (Sigma), trizol RNA extract (Invitrogen, USA), RNA reverse transcription kit (Toyobo, japan), real-time PCR Master Mix (Roche, USA), shRNA plasmid expression Vector (pSIH 1-H1-GFP shRNA Vector, SI501A-1,System Biosciences), 15-PGDH antibody (Abcam, inc.).

The reagents and materials not specifically described are commercially available products.

Example 1 in vitro experiments

In the embodiment, in vitro experiments adopt primary cultured human pituitary GH tumor cells and rat pituitary tumor cell strain GH3 as a research model, DMSO (rosiglitazone solvent) groups are used as a control, different concentration gradients and time gradients of rosiglitazone are set for intervention, and expression differences of GH and 15-PGDH mRNA levels of each group are observed; culturing a human liver cell strain HepG2, giving GH stimulation, taking a DMSO group as a control, setting different concentration gradients of rosiglitazone for intervention, and observing the expression difference of the mRNA levels of IGF-1, GHR and 15-PGDH of each group; in addition, a shRNA plasmid of 15-PGDH was constructed, a human liver cell strain HepG2 was cultured, a control group and an intervention group were set according to different intervention conditions of GH stimulation, rosiglitazone treatment, NC plasmid, 15-PGDHshRNA1 plasmid and 15-PGDHshRNA2 plasmid, and expression differences of IGF-1 and GHR mRNA levels of the respective groups were observed.

(a) The experimental method comprises the following steps:

1. cell culture

Primary cultures of human pituitary GH tumor cells, rat pituitary GH3 cells, and human hepatocyte cell line HepG2 cells.

Primary culture of human pituitary GH tumor cells: from the biological sample library of pituitary tumors from the university of double denier affiliated Huashan Hospital, the culture medium was inoculated with DMEM (Invitrogen) +10% FBS (Invitrogen) after digestion treatment with hyaluronidase (Sigma) and collagenase (Sigma), the serum was fetal bovine serum (Gibco), the culture conditions were 37℃and 5% CO2, and the cell growth was adherent.

Rat pituitary GH3 cell culture: cell lines were purchased from the national institute and culture medium was F12K (Invitrogen) +2.5% FBS (Invitrogen) +15% horse serum (Gibco) at 37℃and 5% CO2, and cell growth was adherent growth.

Human liver HepG2 cell culture: cell lines were purchased from the national institute, using DMEM (Invitrogen) +10% FBS (Invitrogen) as medium, at 37℃and 5% CO2, and the cell growth was by adherent growth.

2. Pharmaceutical intervention

Setting up different concentration gradients and time gradients, taking a rosiglitazone solvent DMSO treatment group as a control group, taking the rosiglitazone treatment group as an intervention group, and intervening cells, wherein the cells of each group extract RNA.

Human pituitary GH tumor cells: three intervention concentrations of 10uM, 50uM and 100uM were set for rosiglitazone in the DMSO group as a control group, the intervention time was 72 hours, and cellular RNA was extracted after the intervention. The above experiment was repeated three times.

Rat pituitary GH3 cells: four intervention concentrations of 1uM, 10uM, 25uM and 50uM were set with DMSO as control, rosiglitazone, intervention times were set for three times of 24 hours, 48 hours and 72 hours, and cellular RNAs were extracted after intervention. The above experiment was repeated three times.

Human liver HepG2 cells: human recombinant GH is used for stimulation, drug intervention is carried out after 6 hours of stimulation, DMSO group is used as a control group, three intervention concentrations of 1uM, 5uM and 10uM are set for rosiglitazone, the intervention time is 2 hours, and cellular RNA is extracted after intervention. In addition, human recombinant GH is stimulated for 6 hours for drug intervention, DMSO is used as a control group, 10uM rosiglitazone is used as a drug group, NC plasmid interference, 15-PGDHshRNA1 plasmid interference and 15-PGDHshRNA2 plasmid interference groups are set, NC plasmid interference +10uM rosiglitazone interference, 15-PGDHshRNA1 plasmid interference +10uM rosiglitazone interference and 15-PGDHshRNA2 plasmid interference +10uM rosiglitazone group interference groups are set, the interference time is 3 hours, and cellular RNA is extracted after the interference. The above experiment was repeated three times.

3. qPCR detects mRNA expression of target genes including GH, IGF-1, GHR and 15-PGDH.

The RNA extracted in the previous step is reverse transcribed into cDNA. Primers of GH, IGF-1, GHR and 15-PGDH are respectively designed and synthesized, and qPCR is performed to detect the expression level of the corresponding genes. In primary human pituitary GH tumor cells and rat GH3 cells, the expression levels of GH and 15-PGDH were mainly detected; in human HepG2 cells, the expression levels of IGF-1, GHR and 15-PGDH were mainly examined.

Specifically, extracting RNA by a Trizol method, and detecting the concentration and purity of the RNA by using a Nanodrop method;

RNA was reverse transcribed into cDNA using an RNA reverse transcription kit (Toyobo Co., ltd.) and the procedure was as described in the kit. qPCR detection of the target gene was performed using a PCR Master Mix kit (Roche Co., USA) and an ABI7500 real-time fluorescent quantitative PCR instrument. The names and primer sequences of the target genes are as follows:

TABLE 1 primer sequences

4. qPCR detection interferes with target gene mRNA expression after 15-PGDH, including IGF-1 and GHR.

In human HepG2 cells, control and intervention groups were set according to different intervention conditions of GH stimulation, rosiglitazone treatment, NC plasmid, 15-PGDHshRNA1 plasmid and 15-PGDHshRNA2 plasmid, RNA was extracted from each group of cells after the intervention, and qPCR was performed to detect the expression levels of IGF-1 and GHR.

Specifically, for construction and transfection of the shRNA plasmid of the 15-PGDH, 3 siRNA sequences aiming at the CDS region thereof are finally selected according to the 15-PGDH gene information (NM_ 000860) based on the siRNA sequence design requirement and considering various factors:

TABLE 2 inhibitory RNA and binding domain sequences for which it is intended

Note that: the sequence of each siRNA was a synthetic RNA that was fully complementary to the binding region sequence given in table 2.

Based on each siRNA sequence, a DNA template single strand is designed, synthesized and annealed to form complementary double stranded DNA. The shRNA plasmid expression Vector (pSIH 1-H1-GFP shRNA Vector, SI501A-1,System Biosciences) was subjected to an enzymatic cleavage process to ligate the desired fragment to the linearized Vector DNA. Positive clones were identified by PCR after transformation and plating. The recombinant expression vector is identified to be correct for carrying out DNA extraction of the endotoxin removal plasmid, the recombinant expression vector is transfected into HepG2 cells, and the relative content of 15-PGDH genes in the transfected cells is detected by PCR. Observed 48 hours after transfection, the transfection efficiency was approximately 90%, determining the most efficient shRNA sequence: shRNA1 and shRNA2.

Statistical analysis and mapping were performed using GraphPad Prism 6 software. The measurement data are expressed as mean ± standard error (x ± sem), the comparison between two groups is by t-test, and the difference between more than two groups is counted by single factor analysis of variance. The significance difference was defined as P <0.05.

(b) Results

The experimental results are as follows:

figure 1 shows the change in GH expression levels after various concentration gradients of rosiglitazone have been used to intervene in human primary GH tumor cells for 72 hours.

As can be seen from fig. 1: rosiglitazone down regulates GH expression in primary GH tumors, and the effect of lowering with increasing dose is more obvious.

Figure 2 shows GH expression level changes following intervention of rosiglitazone at different concentration gradients and different time gradients in rat GH3 cells.

As can be seen from fig. 2: rosiglitazone down regulates GH expression in rat GH3 cells, and the effect of decreasing with increasing dosage and prolonged action time shows a more remarkable trend.

FIG. 3 shows the change in 15-PGDH expression levels after 72 hours of rosiglitazone intervention on human primary GH tumor cells at different concentration gradients.

As can be seen from fig. 3: rosiglitazone upregulates 15-PGDH expression in primary GH tumors and upregulation was more pronounced with increasing doses used.

FIG. 4 shows the 15-PGDH expression level change after intervention of rosiglitazone in rat GH3 cells at different concentration gradients and at different time gradients.

As can be seen from fig. 4: the up-regulating effect of rosiglitazone on the expression of 15-PGDH in rat GH3 cells shows more obvious trend along with the increase of the dosage and the extension of the action time.

FIG. 5 shows the effect of rosiglitazone intervention at different concentration gradients on IGF-1 expression by human hepatocyte strains (HepG 2) under GH stimulation.

As can be seen from fig. 5: the expression of human hepatocyte strain (HepG 2) IGF-1 is down-regulated by rosiglitazone under the stimulation of GH, and the down-regulating effect is more obvious with the increase of the dosage.

FIG. 6 shows the effect of rosiglitazone intervention at different concentration gradients on the expression of GHR in human hepatocyte strains (HepG 2) under GH stimulation.

As can be seen from fig. 6: under GH stimulation, rosiglitazone down regulates the expression of human liver cell strain (HepG 2) GHR, and the down regulation effect is more obvious with the increase of the dosage.

FIG. 7 is the effect of rosiglitazone intervention at different concentration gradients on the expression of 15-PGDH in human hepatocyte strains (HepG 2) under GH stimulation.

As can be seen from fig. 7: rosiglitazone up-regulates the expression of human hepatocyte strain (HepG 2) 15-PGDH, and the up-regulating effect is more obvious with the increase of the dosage.

FIG. 8 shows the presence of 15-PGDH construction of an effective shRNA plasmid.

As can be seen from fig. 8: both shRNA1 and shRNA2 plasmids were effective in down regulating 15-PGDH expression.

FIG. 9 is the effect of rosiglitazone on IGF-1 expression in human liver HepG2 cells following interference with 15-PGDH.

As can be seen from fig. 9: rosiglitazone down regulates expression of human hepatocyte strain (HepG 2) IGF-1 under GH stimulation; after inhibition of 15-PGDH, rosiglitazone down-regulates human hepatocyte strain (HepG 2) IGF-1 expression.

FIG. 10 is the effect of rosiglitazone on GHR expression in human liver HepG2 cells following interference with 15-PGDH.

As can be seen from fig. 10: under GH stimulation, rosiglitazone down regulates the expression of human hepatocyte strain (HepG 2) GHR; after inhibition of 15-PGDH, the effect of rosiglitazone on down-regulation of GHR expression in human hepatocellular strains (HepG 2) was reduced.

In summary, the experimental results show that:

(i) Rosiglitazone can significantly inhibit GH secretion and induce increased expression of 15-PGDH in primary cultured human GH tumor cells and rat GH3 cells compared to control;

(ii) Rosiglitazone can significantly inhibit GH-induced IGF-1 expression in HepG2 cells, while inhibiting GHR expression and inducing 15-PGDH expression, as compared to control groups under GH stimulation; after interference with 15-PGDH, the effect of rosiglitazone on inhibition of IGF-1 and GHR expression in human liver HepG2 cells was reduced.

The above results demonstrate that the compound of formula I represented by rosiglitazone can significantly inhibit not only GH expression and/or activity in growth hormone secreting cells or organs (such as the pituitary gland) but also GHR expression and/or activity in non-growth hormone secreting cells (especially hepatocytes, including normal cells and hepatocarcinoma cells), thereby effectively synergistically treating pituitary growth hormone adenoma in both target organs (i.e., dual targets) of the pituitary and liver.

All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the claims appended hereto.

Sequence listing

<110> university of double denier affiliated Huashan Hospital

<120> use of thiazolidinediones for the double target treatment of pituitary growth hormone adenoma

<130> P2016-2255

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Claims (1)

1. A method of non-therapeutically inhibiting growth hormone receptor expression in vitro comprising the steps of:

contacting rosiglitazone, or a pharmaceutically acceptable salt thereof, with a non-growth hormone secreting cell, thereby inhibiting expression of a growth hormone receptor by said cell under GH stimulation;

wherein the non-growth hormone secreting cells are HepG2 cells;

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