TWI830287B - Nicotinamide in the treatment of uterine fibroids - Google Patents

Abstract

The present invention reveals that nicotinamide can effectively inhibit the proliferation of uterine fibroid cells and the production of extracellular matrix, and can be used to treat uterine fibroids.

Description

Nicotinamide in the treatment of uterine fibroids

The present invention relates to the use of nicotinamide for the treatment of uterine fibroids.

Uterine leiomyoma (also known as uterine fibroid) is a benign tumor caused by excessive cell proliferation and extracellular matrix (ECM) deposition, and diet, Metabolism and female hormones may be factors that cause uterine fibroids. Common symptoms of uterine fibroids include uterine bleeding, pelvic pressure and pain, and reproductive dysfunction, which can even lead to infertility.

At present, no drug has been proven to be effective in treating uterine fibroids. Clinically, the only way to achieve improvement is through surgery to remove the uterus. Therefore, relevant researchers in this field are committed to developing drugs that can effectively treat uterine fibroids.

Nicotinamide has been found to enhance DNA repair in primary melanocytes with ultraviolet (UV) radiation-induced DNA damage. It is considered to be useful in the prevention and treatment of skin cancer (Thompson BC et al. (2014), Exp. Dermatol. , 23(7):509-511). In addition, nicotinamide has also been proven to reduce skin wrinkles and increase skin elasticity, and can be used to promote ECM proteins in skin fibroblasts, thereby slowing down skin aging (Ekiguchi A. et al. (2013), Skin Aging New Research ebook Nova Science , 43-58).

To the best of the applicant's knowledge, there is no literature or patent case that has ever applied nicotinamide in the treatment of uterine fibroids.

Summary of the invention

Although there are the above-mentioned previous studies on nicotine amide in the prevention and treatment of skin cancer and the promotion of extracellular matrix (ECM), in the present invention, the applicant unexpectedly discovered through experiments: nicotine amide can not only It can effectively inhibit the growth of uterine fibroids and significantly reduce the production of related extracellular matrix, so it is expected to be used to treat uterine fibroids.

Therefore, in a first aspect, the present invention provides a use of nicotinamide for preparing a pharmaceutical for treating uterine fibroids.

In a second aspect, the present invention provides a method for treating uterine fibroids, comprising administering nicotinamide to an individual in need thereof.

Detailed description of the invention

It is to be understood that if any prior publication is cited herein, that prior publication does not constitute an admission that the prior publication forms a common practice in the art in Taiwan or any other country. part of knowledge.

For the purposes of this specification, it will be clearly understood that the word "comprising" means "including but not limited to" and the word "comprises" has a corresponding meaning.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. One skilled in the art will recognize many methods and materials similar or equivalent to those described herein that can be used to practice the present invention. Of course, the present invention is in no way limited to the methods and materials described.

The present invention provides nicotinamide for use in the preparation of pharmaceuticals for treating uterine fibroids.

As used herein, "treating" or "treatment" means preventing, reducing, alleviating, ameliorating, relieving, or controlling. controlling one or more clinical signs of a disease or disorder, and lowering, stopping or reversing a condition being treated or Progression in severity of symptoms.

According to the present invention, nicotinamide may be a commercially available product, or may be prepared by synthetic techniques well known and customary to those skilled in the art.

Alternatively, nicotinamide can also be isolated and purified from a natural source using isolation and purification methods commonly used in the art.

According to the present invention, the pharmaceutical product may further comprise a pharmaceutically acceptable carrier that is widely used in pharmaceutical manufacturing technology. For example, the pharmaceutically acceptable carrier may include one or more agents selected from the group consisting of: solvent, buffer, emulsifier, suspending agent, decomposer ), disintegrating agent, dispersing agent, binding agent, excipient, stabilizing agent, chelating agent, diluent , gelling agent, preservative, wetting agent, lubricant, absorption delaying agent, liposome and the like. The selection and quantities of these reagents are within the professionalism and routine skills of those skilled in the art.

According to the present invention, the pharmaceutical product may be in a dosage form suitable for parenteral administration, oral administration or topical administration.

According to the present invention, the pharmaceutical can be manufactured into a dosage form suitable for parenteral administration [including injection, for example, sterile aqueous solution] using techniques well known to those skilled in the art. or dispersion] and is administered by a route selected from the group consisting of: intraperitoneal injection, intramuscular injection, intravenous injection , intraarterial injection, intraepidermal injection, subcutaneous injection, intradermal injection, and intralesional injection. Preferably, the pharmaceutical product is manufactured into a dosage form suitable for intravenous injection administration.

According to the present invention, the pharmaceutical can be manufactured into a dosage form suitable for oral administration using techniques well known to those skilled in the art, including, but not limited to: sterile powder, tablet, and troche. ), lozenge, pellet, capsule, dispersible powder or granule, solution, suspension, emulsion, syrup , elixirs, slurries and the like.

According to the present invention, the pharmaceutical can be manufactured into an external preparation suitable for topical application to the skin using techniques well known to those skilled in the art, including, but not limited to: emulsion, Gel, ointment, cream, patch, liniment, powder, aerosol, spray, lotion, Serum, paste, foam, drop, suspension, salve and bandage.

The present invention also provides a method for treating uterine fibroids, comprising administering nicotinamide to an individual in need thereof.

As used herein, the terms "administering" and "administering" may be used interchangeably and mean introducing, providing, or delivering to an individual by any suitable route. delivering) a predetermined active ingredient to perform its intended effect.

As used herein, the term "subject" means any mammal of interest, such as humans, monkeys, cows, sheep, horses, pigs ( pigs, goats, dogs, cats, mice and rats.

According to the present invention, the dosage and frequency of administration of the pharmaceutical product will vary depending on the following factors: the severity of the disease to be treated, the route of administration, and the age, physical condition and response of the individual to be treated. Generally speaking, pharmaceuticals according to the present invention may be in the form of a single dose or divided into several doses, and may be administered orally, parenterally or topically. Detailed description of preferred embodiments

The present invention will be further described with reference to the following examples, but it should be understood that these examples are for illustrative purposes only and should not be construed as limitations on the implementation of the present invention. Examples General experimental materials: 1. Eker rat uterine leiomyoma cell line Source and culture of ELT-3:

The Eker rat uterine fibroid cell line ELT-3 used in the following examples was purchased from the American Type Culture Collection (ATCC) and cultured in Dulbecco's modified Dulbecco's Modified Eagle's Medium (DMEM)/F12 [1:1 (v/v)](Cassion) [supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (penicillin-streptomycin] in a petri dish and cultured in an incubator with culture conditions set to 37°C and 5% CO _2. After that, fresh culture medium was replaced approximately every 2-3 days. When When the cell density reaches about 80-90% confluence, subculture is performed. General experimental methods: 1. Statistical analysis:

In the following examples, the experimental data obtained were statistically analyzed using GraphPad Prism 8.0 software, and expressed as "mean ± standard deviation (SD) of the mean". The differences between each group of experimental data were evaluated by one-way analysis of variance (ANOVA) combined with Tukey's post hoc test. If the obtained statistical comparison result is p <0.05, it represents statistical significance. Example 1. Evaluation of the effectiveness of nicotinamide in inhibiting uterine fibroid cell proliferation (proliferation) : Experimental method: A. Cell crystal violet staining assay :

First, divide the ELT-3 cells that were subcultured according to item 1 of the "General Experimental Materials" above into 3 groups, including 1 control group and 2 experimental groups (i.e., experimental groups 1 and 2). Cells in each group were cultured in a 6-well culture plate containing an appropriate amount of DMEM/F12 medium (supplemented with 10% FBS and 1% penicillin-streptomycin) at a number of 1×10 ⁵ cells/well, and incubated in Culture was carried out in an incubator (37°C, 5% CO ₂ ) for 24 hours.

Next, the cell cultures of each group were replaced with fresh culture media (added with 1% FBS), and then an appropriate amount of nicotinic acid amide (purchased from Sigma-Aldrich) was added to each experimental group, so that the experimental group 1 and 2 have a final concentration of nicotinamide of 20 mM and 50 mM respectively. As for the cells in the control group, no treatment was performed.

After each group of cell cultures was cultured in an incubator (37°C, 5% CO ₂ ) for 48 hours, the liquid in each well was removed and washed several times with PBS, and then 1 mL of methanol was added to each well. Fixation lasted 10 minutes. Then, the liquid in each well was removed and stained with 5 mg/mL crystal violet for 15 minutes. Afterwards, the liquid in each well was removed and washed 3 times with _ddH2O . The stained cell culture was observed and photographed using a microscope (Olympus), and then the images were analyzed using ImageJ software to calculate the crystal violet stained area density. The higher the area density, the more surviving cells.

Afterwards, analyze the experimental data obtained according to the method described in item 1 of the "General Experimental Methods" above. B. Cell colony formation assay :

First, divide the ELT-3 cells that were subcultured according to item 1 of the "General Experimental Materials" above into 3 groups, including 1 control group and 2 experimental groups (i.e., experimental groups 1 and 2). Cells in each group were cultured in a 6-well culture plate containing an appropriate amount of DMEM/F12 medium (supplemented with 10% FBS and 1% penicillin-streptomycin) at a number of 5 × 10 ² cells/well. Culture was carried out in an incubator (37°C, 5% CO ₂ ) for 24 hours. Next, the cell cultures of each group were replaced with fresh culture media (added with 1% FBS), and then an appropriate amount of nicotine amide was added to each experimental group so that experimental groups 1 and 2 had a final concentration. 20 mM and 50 mM nicotinamide. As for the cells in the control group, no treatment was performed.

On the 3rd day of culture, the cell cultures in each group were replaced with fresh medium (added with 10% FBS and 1% penicillin-streptomycin), and then incubated in an incubator (37°C, 5% CO ₂ ) and continue culturing for 7-10 days. Afterwards, 95% methanol was added to fix the formed cell colony and stained with 0.5% crystal violet. The formed cell colony was then observed with a microscope (Olympus) and photographed, and then the images taken were analyzed using ImageJ software. Analysis was performed to calculate the cell colony formation density.

Afterwards, analyze the experimental data obtained according to the method described in item 1 of the "General Experimental Methods" above. Results : A. Crystal violet staining analysis of cells:

Figure 1 shows the measured area density of ELT-3 cells stained by crystal violet in each group. As can be seen from Figure 1, compared with the control group, the area density of crystal violet staining in experimental groups 1 and 2 was significantly reduced, and it became more obvious as the concentration of nicotine amide increased. B. Cell community formation analysis:

Figure 2 shows the measured cell colony formation density of ELT-3 cells in each group. As can be seen from Figure 2, compared with the control group, the density of cell community formation in experimental groups 1 and 2 was significantly reduced, and it became more obvious as the concentration of nicotine amide increased.

Based on the above, it can be seen that nicotinamide can exhibit excellent anti-proliferative activity against uterine fibroid cells. Example 2. Evaluation of the effectiveness of nicotinic acid in inhibiting the accumulation of extracellular matrix (ECM) in uterine fibroid cells: Experimental method:

First, divide the ELT-3 cells that were subcultured according to item 1 of the "General Experimental Materials" above into 3 groups, including 1 control group and 2 experimental groups (i.e., experimental groups 1 and 2). Cells in each group were cultured in a 10-cm culture dish containing an appropriate amount of DMEM/F12 medium (added with 10% FBS and 1% penicillin-streptomycin) at a quantity of 2×10 ⁵ cells/well, and incubated in Culture was carried out overnight in an incubator (37°C, 5% CO ₂ ).

Then, the cell cultures of each group were replaced with fresh medium (added with 1% FBS) and cultured for 24 hours. After that, an appropriate amount of nicotinamide was added to each experimental group, so that experimental groups 1 and 2 had a final concentration of nicotinamide of 20 mM and 50 mM respectively. As for the cells in the control group, no treatment was performed.

After the cell cultures of each group were cultured in an incubator (37°C, 5% CO ₂ ) for 72 hours, 100 μL of RIPA lysis buffer (BIOMAN) was added to the obtained cell cultures of each group. , Cat. No. 20121701) [added with protease inhibitor (Roche, Cat. No. 4693159001)] and mix well. Next, the resulting mixture was placed in a microcentrifuge tube and reacted at 4°C for 30 minutes, and then centrifuged at 12,000 rpm at 4°C for 20 minutes, and then the supernatant was collected and used as a total protein sample. Then, the total protein concentration was determined by using the bicinchoninic acid (BCA) assay kit (T-Pro Biotechnology, Cat. No. JB04-D001) and according to the manufacturer's instructions.

Afterwards, the total protein samples of each group of cell cultures were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) using techniques well known and customary to those skilled in the art. , and Western Blotting for fibronectin, α-smooth muscle actin (α-SMA), and collagen type I α1 (COL1A1). ). In addition, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control.

The instruments and reagents used for SDS-PAGE analysis and Western blot analysis are as follows: (1) SDS-PAGE analysis uses a Bio-rad Electrophoresis System (Bio-rad Electrophoresis System, Bio-Rad Laboratories, Inc. ) to proceed. (2) Protein transfer is performed using a Bio-rad wet electrophoresis transfer tank (Bio-Rad Laboratories, Inc.) and a polyvinylidene difluoride (PVDF) membrane. (3) In Western blot analysis, the primary antibody and secondary antibody used for each protein are shown in Table 1 below. Table 1. Primary and secondary antibodies for Western blot analysis protein primary antibody secondary antibody Reticulin rabbit anti fibronectin polyclonal antibody (abcam, Cat. No. ab2413) Goat anti rabbit IgG-horseradish peroxidase (HRP) antibody [Jackson ImmunoResearch Laboratories, Cat. No. 111-035-003] α-SMA rabbit anti α-SMA polyclonal antibody (Genetex, Cat. No. GTX100034) COL1A1 rabbit anti COL1A1 polyclonal antibody (Genetex, Cat. No. GTX112731) GAPDH Horseradish catalase (HRP)-conjugated mouse anti GAPDH monoclonal antibody (Proteintech, Cat. No. HRP-60004) - (4) Chemiluminescence staining was performed using ECL chromogen (T-Pro Biotechnology, Cat. No. JT96-K004M), and a luminescence imaging system (eBlot Photoelectric Technology, model Touch Imager Mi ) to detect signals.

Afterwards, ImageJ imaging software is used for analysis, whereby the protein band can be semi-quantitatively calculated to calculate the corresponding protein expression level. The expression levels of reticulin, α-SMA and COL1A1 are then corresponding. GAPDH performance level to be standardized (normalized).

Afterwards, analyze the experimental data obtained according to the method described in item 1 of the "General Experimental Methods" above. result :

Figures 3 to 5 respectively show the expression levels of reticulin, α-SMA and COL1A1 measured in ELT-3 cells in each group. From Figure 3 to Figure 5, it can be seen that compared with the control group, the expression levels of reticulin, α-SMA and COL1A1 in experimental groups 1 and 2 all showed a significant decrease. Among them, the expression levels of reticulin and α-SMA were significantly reduced. The performance level will become more significant as the concentration of nicotine amide increases. The results of this experiment show that nicotinamide has the effect of inhibiting the production of ECM by uterine fibroid cells.

Based on the above experimental results, it can be seen that nicotinamide can not only effectively inhibit the growth of uterine fibroids cells, but also significantly inhibit the production of related ECM, thereby preventing the occurrence and expansion of uterine fibroids, and is therefore expected to have the ability to develop into High potential of an anti-uterine fibroid drug.

All patents and documents cited in this specification are hereby incorporated by reference in their entirety. In the event of conflict, the detailed description of the case (including definitions) will prevail.

Although the invention has been described with reference to the specific embodiments above, it will be apparent that many modifications and changes can be made without departing from the scope and spirit of the invention. It is therefore intended that this invention be limited only as indicated by the appended claims.

The above and other objects, features and advantages of the present invention will become apparent after referring to the following detailed description and preferred embodiments and the accompanying drawings, wherein: Figure 1 shows the results of each group of ELT-3 cells The measured area density stained by crystal violet, where “***” indicates p < 0.001 when compared with the control group; Figure 2 shows the measured cell colony formation density of ELT-3 cells in each group, where “*” ” means that when compared with the control group, p <0.05; Figure 3 shows the expression level of fibroretic protein measured by ELT-3 cells in each group, where “***” means that when compared with the control group, p <0.001; Figure 4 shows the performance levels of α-SMA measured in each group of ELT-3 cells, where "**" indicates that p < 0.01 when compared with the control group; and Figure 5 shows each group of ELT-3 cells The measured performance level of COL1A1, where "**" indicates p < 0.01 when compared with the control group.

Claims (3)

A kind of nicotinamide is provided for use in preparing a medicine for treating uterine fibroids. The use of claim 1, wherein the pharmaceutical product further contains a pharmaceutically acceptable carrier. Such as the use of claim 1, wherein the pharmaceutical product is in a dosage form for oral administration, topical administration or parenteral administration.

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