JP2012072077A - Inhibitor for fibroid cell proliferation - Google Patents

Abstract

PROBLEM TO BE SOLVED: To develop and provide a novel therapeutic agent for fibroid which is low invasive, is capable of a long-term administration, and has no or weak side effect.SOLUTION: There is provided an inhibitor for fibroid cell proliferation that contains a biguanide-based medicine as an active constituent, or a pharmaceutical composition that contains the inhibitor as an active constituent.

Description

  The present invention relates to a uterine fibroid cell growth inhibitor and a pharmaceutical composition containing the same.

  Uterine fibroids are benign tumors that develop in the myometrium of the uterus and are found in about 25-50% of women of reproductive age. Although it is unlikely to metastasize or destroy surrounding tissues like cancer, it may cause irregular bleeding, dysmenorrhea, compression symptoms, infertility, etc., depending on the site of occurrence, so QOL (quality of There is a problem that the quality of life is significantly reduced. Uterine fibroids are an extremely common disease, but their pathophysiology and pathogenesis are unclear except for the involvement of estrogen-mediated cell proliferation signaling mechanisms, which are effective and effective for long-term administration. Possible therapeutic agents have not yet been developed.

The main treatment methods for uterine fibroids currently performed can be broadly classified into surgical therapy and drug therapy (Non-patent Document 1).
The standard treatment method in surgical therapy is total hysterectomy. Although this method is a radical treatment with no possibility of recurrence, it can not be applied to patients who want to become pregnant, or because it loses the uterus, an important organ as a symbol of women, the patient feels lost and psychological shock There is a problem that is large. On the other hand, as a uterine preservation therapy, there is myomactomy surgery in which only the uterine fibroid nucleus is removed. This method has the advantage that it can be applied to patients who wish to become pregnant, but there is a problem that small myoma nuclei may not be completely removed, and there is a high possibility of recurrence. As other uterine preservation therapy, uterine artery embolization (UAE) and focused ultrasound surgery (FUS) have been newly developed. Uterine artery embolization is a treatment method in which an embolic substance such as a gelatin sponge is injected into a uterine artery that supplies blood to the uterine fibroid to necrotize the uterine fibroid (Non-patent Document 1). Although it can prevent and reduce myoma growth, menorrhagia and myoma compression are improved, but it is applicable to patients who want to become pregnant or who have severe allergies to embolic materials and contrast agents In addition, there is a problem that lower abdominal pain is accompanied after surgery. The focused ultrasound method is a treatment method in which uterine fibroids are incinerated by ultrasonic energy emitted from an ultrasound transmitter while confirming a lesion by MRI (Non-patent Document 1). Although there is an advantage that the invasiveness is extremely low and there are almost no side effects, there is a problem that applicable myomas are limited depending on the generation position and size.

  As drugs used in pharmacotherapy, there are GnRH agonists that can be used in Japan at present. The GnRH agonist is an agonist of GnRH (Gonadotropin-Releasing Hormone) that promotes secretion of luteinizing hormone (LH: Lutenizing Hormone) and follicle stimulating hormone (FSH). LH and FSH act on the ovaries to promote estrogen secretion. It is known that uterine fibroids usually proliferate depending on the amount of estrogen because they shrink after menopause (Non-patent Document 1). When this drug is administered, the secretion of LH and FSH is suppressed by the action of excessive GnRH, and as a result, the secretion of estrogen from the ovary is suppressed, and the growth of uterine fibroids can be suppressed. However, this drug has side effects such as ovarian function deficiency symptoms (menopausal symptoms) and bone mass loss (osteoporosis), and the effect is transient, so it is only used as escape therapy until menopause. Absent. As other drugs, selective progesterone receptor modulators and aromatase inhibitors are currently in clinical trials (Non-patent Document 1), and as with GnRH agonists, these are also uterine fibroids due to suppression of estrogen secretion or action. It is a drug that suppresses cell growth and cannot be used for a long time because it has side effects such as ovarian function deficiency and bone loss.

  As described above, none of the currently known methods for treating uterine fibroids are sufficient as treatment methods. Therefore, there is a demand for the development of a pharmacotherapy using a new treatment method or agent.

Mitsuaki Suzuki, Yasunori Yoshimura, Department of Obstetrics and Gynecology: The latest clinical practice for specialists Uterine fibroids, 2007, Chugai Medical

  An object of the present invention is to develop and provide a novel therapeutic agent for uterine fibroids that is minimally invasive, can be administered for a long term, and has no or weak side effects.

In order to solve the above-mentioned problems, the present inventors have searched for a novel drug capable of suppressing the growth of uterine fibroid cells. As a result, it was found that metformin, a kind of biguanide-based drug, has an inhibitory effect on the growth of uterine fibroid cells. Metformin is known as a therapeutic drug for diabetes that exhibits a hypoglycemic action (Kouhei Kora et al .: Diabetes 49 (5): 325, 2006). In addition, in recent years, new effects such as breast cancer suppression effect and suppression effect of ovulation disorder in PCOS (polycystic ovary syndrome) have been found (Mol Cancer Ther. 2010; 9 (5) 1092-9) ( Ann NY Acad Sci. 2010; 1205 (1): 192-8). However, the growth-inhibitory effect of biguanides such as metformin on uterine fibroid cells has not been known so far. The present invention has been completed based on the above-mentioned new findings, and provides the following.
(1) A uterine fibroid cell growth inhibitor containing a biguanide drug as an active ingredient.
(2) The uterine fibroid cell proliferation inhibitor according to (1), wherein the biguanide drug is metformin, a derivative thereof, or a pharmaceutically acceptable salt thereof.
(3) A pharmaceutical composition comprising the uterine fibroid cell growth inhibitor according to (1) or (2) as an active ingredient.

  According to the uterine fibroid cell growth inhibitor of the present invention or a pharmaceutical composition containing the same, a novel uterine fibroid therapeutic / preventive agent capable of long-term administration that is minimally invasive, has no side effects, or is extremely weak can be provided.

It is a figure which shows the cell growth inhibitory activity specific to the uterine fibroid cell by metformin. A shows the proliferation of the cell line ELT-3 cells of the uterine fibroid model, and B shows the proliferation of the cell line HeLa cells derived from cervical cancer. It is a figure which shows the histological structure in the human uterine leiomyoma graft of the 8th week of metformin treatment. In this figure, the nucleus of the transplanted section is stained by HE staining. It is a figure which shows the proliferation cell rate in the human uterine fibroid graft | grafting of the 8th week of a metformin treatment. It is a figure which shows the apoptotic cell rate in the human uterine leiomyoma graft of the 8th week of metformin treatment.

Hereinafter, embodiments of the present invention will be described in detail.
1. 1. Uterine fibroid cell growth inhibitor 1-1. Configuration The first embodiment of the present invention is a uterine fibroid cell proliferation inhibitor. The inhibitor of the present invention is characterized by containing a biguanide drug as an active ingredient.

  As used herein, “suppression of uterine fibroid cells” refers to inhibiting or suppressing the growth of uterine fibroid cells. Therefore, “inhibition of uterine fibroid cells” is based on the inhibition or suppression of its proliferation, the apoptosis of uterine fibroid cells, and the treatment of uterine fibroids by regression or disappearance of uterine fibroids, the growth of uterine fibroids that have occurred. Suppression and prevention of the development of uterine fibroid cells shall be included.

  “Biguanide drugs” are drugs that have the effect of suppressing glucose absorption in the gastrointestinal tract while promoting glucose uptake in peripheral tissues, while suppressing gluconeogenesis from the liver without via insulin secretion. Yes, it is used as a therapeutic agent for type 2 diabetes (non-insulin-dependent diabetes), particularly as a therapeutic agent for obese diabetes. Lactic acidosis is known as a serious side effect of biguanide drugs. However, the biguanide drug of the present invention has a very low incidence of the side effects and has few other side effects, or even a minor compound such as diarrhea, nausea and abdominal pain. Particularly preferred compounds as the biguanide drug of the present invention having such properties include, for example, metformin, a derivative thereof, or a pharmaceutically acceptable salt thereof.

  As described above, “metformin” is a kind of biguanide drug and is a compound known as a hypoglycemic agent for a long time. Metformin is considered to be a highly safe compound because it rarely develops lactic acidosis and has few other side effects. Therefore, for example, in the UK, high-dose prescriptions of 750-1500 mg as the daily maintenance dose and 3000 mg as the maximum daily dose are allowed. The physiological function of metformin is known to activate AMPK (AMP-activated protein kinase), but the detailed mechanism of action is still unclear.

  “Derivatives thereof” refers to compounds that are chemically derived from metformin and that exhibit the same action and effect as metformin in the human body. Pharmaceutically non-toxic compounds that have no or little side effects on the human body are preferred. The derivative can also include a prodrug of metformin. "Prodrug" refers to a compound that is converted to an active form by undergoing a chemical change under physiological conditions. For example, in the case of the present embodiment, before administration, it exists as an inactive compound having a structure different from that of metformin, and in vivo after administration, for example, metformin or pharmaceutically by the action of digestive enzymes in the digestive tract. A compound that is converted to a non-toxic active formformin derivative.

  “Pharmaceutically acceptable salts thereof” are salts of metformin or derivatives thereof, and are pharmaceutically non-toxic prepared with bases or acids based on certain substituents of those compounds. Active compound. It can be classified into acid addition salts and basic addition salts according to the base or acid used.

  Examples of the “acid addition salts” include inorganic acid salts such as hydrochloride, sulfate, nitrate, phosphate, carbonate, bicarbonate, perchlorate, acetate, propionate, lactate and maleate. Acid salts, fumarate, tartrate, malate, citrate, ascorbate, etc. organic acid salts, methanesulfonate, isethionate, benzenesulfonate, p-toluenesulfonate, etc. Acidic amino acids such as acid salts, aspartates, and glutamates. In the present embodiment, metformin hydrochloride is given as a specific example.

  Examples of the “basic addition salt” include alkali metal salts such as sodium salt and potassium salt, alkaline earth metal salts such as calcium salt and magnesium salt, trimethylamine salt, triethylamine salt, dicyclohexylamine salt, ethanolamine salt and diethanolamine. Salts, aliphatic amine salts such as triethanolamine salts and brocaine salts, aralkylamine salts such as N, N-dibenzylethylenediamine, heterocyclic aromatic amine salts such as pyridine salts, picoline salts, quinoline salts and isoquinoline salts, arginine Salt, basic amino acid salt such as lysine salt, tetramethylammonium salt, tetraethylammonium salt, benzyltrimethylammonium salt, benzyltriethylammonium salt, benzyltributylammonium salt, methyltrioctylammonium salt, tetra Quaternary ammonium salts such as chill ammonium salts, ammonium salts and the like.

  The uterine fibroid cell growth inhibitor of this embodiment includes one or more biguanides, preferably metformin, derivatives thereof, or pharmaceutically acceptable salts thereof (hereinafter referred to as “metformin etc.”) as active ingredients. To do. When two or more biguanide drugs are included, the combination of biguanide drugs is not particularly limited. Moreover, the uterine fibroid cell growth inhibitor of this embodiment can also include drugs having a growth inhibitory effect on uterine fibroid cells other than biguanide drugs. Such agents include, for example, GnRH agonists, selective progesterone receptor modulator inhibitors or aromatase inhibitors.

  The uterine fibroid cell growth inhibitor of the present embodiment can be used as it is or after being dissolved in an appropriate solvent. Examples of the solvent include water, ethanol, propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, and polyoxyethylene sorbitan fatty acid esters. Such a solvent is desirably sterilized, and is preferably adjusted to be isotonic with blood as necessary.

1-2. Effective amount The effective amount of the uterine fibroid cell growth inhibitor of this embodiment will be described. In the present specification, the “effective amount” is an amount necessary for the uterine fibroid cell growth inhibitor, which is an active ingredient, to exhibit its function, that is, an amount necessary to inhibit the growth of uterine fibroid cells. And an amount that causes little or no adverse side effects on the subject to whom it is administered. When the uterine fibroid cell proliferation inhibitor in this embodiment does not contain a drug having a uterine fibroid cell proliferation inhibitory effect other than the biguanide drug, the effective amount of the uterine fibroid cell proliferation inhibitor is substantially the same as that of the biguanide drug. It is the same amount as the effective amount. This effective amount may vary depending on various conditions such as information on the subject to be administered and administration route. Here, “subject information” refers to the severity (number and size of fibroids) of uterine fibroids, general health, age, weight, race, subject drug sensitivity, presence / absence of concomitant drugs, and resistance to treatment. Etc. The “administration route” is a route through which the uterine fibroid cell growth inhibitor is administered, and includes oral, tissue routes (subcutaneous route, intramuscular route, intravenous route, etc.), percutaneous route or rectal route. Can be mentioned.

  The effective amount of the uterine fibroid cell growth inhibitor may vary depending on conditions such as the subject's information and administration route as described above, but usually 1-100 mg / kg body weight, 1.5-80 mg / kg body weight per day, It may be in the range of 2-50 mg / kg body weight, 2.5-30 mg / kg body weight, or 3-20 mg / kg body weight.

1-3. Effect As described above, the uterine fibroid cell growth inhibitor of this embodiment can be administered in a relatively large amount or for a long period of time because of its weak side effects such as a biguanide drug, preferably metformin, which is an active ingredient. is there. Furthermore, since a biguanide type | system | group drug can be orally administered, it can provide the pharmaceutical composition for uterine fibroid cell growth suppression with low invasiveness.

2. Pharmaceutical composition The second embodiment of the present invention is a pharmaceutical composition. The composition, production method, administration method and usage of the pharmaceutical composition of this embodiment will be described below.

2-1. Configuration 2-1-1. Active ingredient The pharmaceutical composition of the present invention comprises the uterine fibroid cell growth inhibitor of the first embodiment as an active ingredient. That is, the pharmaceutical composition of this embodiment is intended to treat and prevent uterine fibroids by inhibiting the growth of uterine fibroid cells and suppressing or regressing the development of fibroids. The pharmaceutical composition of the present invention may contain one or more different uterine fibroid cell growth inhibitors.

  Furthermore, the pharmaceutical composition according to the embodiment includes, as an active ingredient, a drug having a pharmacological action different from that of a uterine fibroid cell growth inhibitor within a pharmaceutically acceptable range and in a range in which the uterine fibroid cell growth inhibitor is not inactivated. Can be contained. For example, a gastrointestinal remedy for improving gastrointestinal symptoms caused by a biguanide-based drug that is an active ingredient of a uterine fibroid cell growth inhibitor may be included.

2-1-2. Carrier The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier. “Pharmaceutically acceptable carrier” refers to an additive usually used in the field of pharmaceutical technology. The carrier is mainly used for the purpose of facilitating the formation of a pharmaceutical composition and maintaining the dosage form and the drug effect. Such carriers include, for example, excipients, binders, disintegrants, fillers, emulsifiers, fluid addition modifiers, lubricants and the like. Since the carrier to be added varies depending on the dosage form and / or administration method of the pharmaceutical composition, it may be appropriately selected and added according to the conditions and necessity.

  Excipients include sugars such as monosaccharides, disaccharides, cyclodextrins and polysaccharides (more specifically, but not limited to glucose, sucrose, lactose, raffinose, mannitol, sorbitol, inositol, dextrin, malto Dextrin, starch and cellulose), metal salts (eg sodium chloride, sodium phosphate or calcium phosphate, calcium sulfate, magnesium sulfate, calcium carbonate), citric acid, tartaric acid, glycine, low, medium and high molecular weight polyethylene glycols ( PEG), pluronic, kaolin, silicic acid, or combinations thereof.

  Examples of the binder include starch paste using corn, wheat, rice, or potato starch, simple syrup, glucose solution, gelatin, tragacanth, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, shellac and / or polyvinylpyrrolidone. As mentioned.

  Disintegrants include the starch, lactose, carboxymethyl starch, cross-linked polyvinyl pyrrolidone, agar, laminaran powder, sodium bicarbonate, calcium carbonate, alginic acid or sodium alginate, polyoxyethylene sorbitan fatty acid ester, sodium lauryl sulfate, stearic acid monoglyceride Or their salts are mentioned as examples.

  Examples of the filler include the sugar and / or calcium phosphate (for example, tricalcium phosphate or calcium hydrogen phosphate).

  Examples of the emulsifier include sorbitan fatty acid ester, glycerin fatty acid ester, sucrose fatty acid ester, and propylene glycol fatty acid ester.

  Examples of the flow control agent and lubricant include silicate, talc, stearate or polyethylene glycol.

  In addition to the above additives, flavoring agents, solubilizers, suspending agents, diluents, surfactants, stabilizers, absorption promoters, extenders, moisturizers, humectants, adsorbents, if necessary Disintegration inhibitors, coating agents, coloring agents, preservatives, antioxidants, fragrances, flavoring agents, sweetening agents, buffering agents and the like can also be included. As such a carrier, those known in the pharmaceutical technical field may be appropriately used as necessary.

2-1-3. Dosage Form The dosage form of the pharmaceutical composition of the present embodiment is not particularly limited as long as it does not inactivate the uterine fibroid growth inhibitor and other additional active ingredients, which are active ingredients in the inhibitor. For example, any of liquid, solid, or semi-solid may be sufficient. Specific dosage forms include, for example, oral dosage forms such as liquids, powders, granules, tablets, capsules, sublinguals, lozenges, or injections, suspensions, emulsions, eye drops, nasal drops, Examples include parenteral dosage forms such as creams, ointments, plasters, ships, and suppositories. Since biguanides such as metformin, which is an active ingredient of a hysteromyoma growth inhibitor, can be administered orally, considering the low invasiveness, the dosage form of the pharmaceutical composition of this embodiment is oral A dosage form is preferred.

  The dosage form of the pharmaceutical composition of the present invention is finally determined based on formulation conditions and / or administration methods. The prescription conditions refer to information on a subject who administers the pharmaceutical composition, a single dosage unit amount of the pharmaceutical composition (dosage per administration), and the number of administrations per day. Moreover, although the detail of an administration method is mentioned later, normally, it can divide roughly into oral administration and parenteral administration. Therefore, here, dosage forms suitable for each administration method will be described.

  Suitable dosage forms for oral administration include, for example, solids (including tablets, pills, sublinguals, capsules, drops, troches), granules, powders, powders or liquids (syrups, dry syrups). Included). Furthermore, the solid preparation can be made into a dosage form having a drug shell known in the art, for example, a sugar-coated tablet, a gelatin-encapsulated tablet, an enteric tablet, a film-coated tablet, a double tablet, or a multilayer tablet, if necessary. When the pharmaceutical composition of this embodiment is used for oral administration, any of the above dosage forms may be used.

  As a dosage form suitable for parenteral administration, parenteral administration can be further subdivided into intra-tissue administration, topical administration and rectal administration, so that it can be made into a dosage form suitable for each administration method. Examples of dosage forms suitable for intra-tissue administration include solutions such as injections. Examples of dosage forms suitable for topical administration include solutions such as coating agents, eye drops, nasal drops and inhalants, suspensions such as emulsions and creams, nasal drops and inhalants. Powders, pastes, gels, ointments, plasters and the like. Furthermore, examples of the dosage form suitable for rectal administration include liquids, gels and solids as suppositories.

  The shape and size of each dosage form are not particularly limited as long as they are within the range known in the art for each dosage form. Taking a tablet as a solid agent as an example, the maximum length is usually in the range of 3 to 15 mm, preferably 5 to 10 mm. More specifically, for example, in the case of a substantially spherical flat plate-shaped dosage form, the diameter may be in the range of 5 to 10 mm and the maximum thickness may be in the range of 2 to 5 mm.

2-1-4. Content of inhibitor of uterine fibroid cell growth in pharmaceutical composition Since the uterine fibroid cell growth inhibitor which is an active ingredient of the pharmaceutical composition of the present embodiment has already been described in the first embodiment, here, The content of the uterine fibroid cell growth inhibitor in the pharmaceutical composition will be described.

  The content of the uterine fibroid cell growth inhibitor in the pharmaceutical composition of the present invention varies depending on the type of uterine fibroid cell growth inhibitor, its effective amount, the type of carrier used, the dosage form of the pharmaceutical composition, and the administration method. It depends on various conditions. Here, the type of the hysteromyoma cell growth inhibitor is based on, for example, the type of biguanide-based drug that is an active ingredient. In addition, the effective amount of the uterine fibroid cell growth inhibitor, the type of carrier used, and the dosage form of the pharmaceutical composition are all as described above. Furthermore, the administration method will be described later. Therefore, the content of the content of the uterine fibroid cell growth inhibitor in the pharmaceutical composition is calculated for each subject to be administered in consideration of the above conditions. This amount is ultimately determined by the judgment of the physician.

  The content of the uterine fibroid cell growth inhibitor in an individual pharmaceutical composition may vary depending on the dosage form of the pharmaceutical composition, the dosage unit, and the number of administrations per day. In general, the content of the uterine fibroid cell growth inhibitor in each individual pharmaceutical composition is not limited as long as it is adjusted to contain an effective amount of the uterine fibroid cell growth inhibitor in the total daily dose. Absent. For example, when the pharmaceutical composition of the present invention is prepared as a solid preparation such as a tablet, the solid preparation can adjust the amount of the uterine fibroid cell growth inhibitor depending on the number of the preparations. For example, in order to obtain the pharmacological effect of the pharmaceutical composition of the present invention, when it is necessary to administer a large amount of a uterine fibroid cell growth inhibitor, it is divided into several times a day in consideration of reducing the burden on the subject. You can also Therefore, the effective amount to be administered per day is not necessarily contained in one tablet. It seems that an effective amount of an inhibitor of uterine fibroid cell proliferation is contained in a total of one day by administering a plurality of tablets in one dosage unit and / or dividing the number of administrations into a plurality of times per day. You may adjust to. The dosage form that can be divided and administered in this way is convenient because it can be applied with fine adjustment according to age, weight or severity. On the other hand, when the pharmaceutical composition of the present invention is prepared as an injection, the administration by injection is usually once a day in consideration of the burden and invasiveness of the subject. It is preferable to adjust so that the effective amount per day may be contained in the agent.

  As a specific example, for example, when a pharmaceutical composition of the present invention is orally administered once a day in tablet form to a human adult female (body weight 50 kg), a uterine fibroid cell growth inhibitor is contained in one tablet. The biguanide-based drug, preferably metformin, which is an active ingredient in the above, may be contained in the range of, for example, 1 μg to 200 mg, 5 μg to 100 mg, 10 μg to 50 mg, 50 μg to 10 mg, or 1 mg to 5 mg.

2-2. Method for Producing Pharmaceutical Composition To formulate the pharmaceutical composition of the present embodiment, a general method known in the art can be used in principle. For example, when the pharmaceutical composition of the present embodiment is produced as a tablet, one or more uterine fibroid cell growth inhibitors described in the first embodiment are mixed and granulated together with a suitable pharmaceutically acceptable carrier. And can be prepared by tableting. If necessary, a coating agent such as shellac solubilized with a solvent such as ethanol is adsorbed onto the surface of the obtained uncoated tablet with a sprayer and then dried or immersed in a coating solution and then dried. By doing so, a coat (coating layer) may be formed. A specific method for such formulation can be performed in accordance with a method described in known literature, for example, Remington's Pharmaceutical Sciences (Merck Publishing Co., Easton, Pa.).

  As a specific example, when the pharmaceutical composition of the present invention is prepared as a solid preparation, the uterine fibroid cell growth inhibitor is usually 50% to 95% by weight, preferably 60% to 90%, based on the weight of one tablet. It is preferable to formulate such that it is contained in the range of 70% by weight, more preferably in the range of 70% to 85% by weight. However, as described above, it is not necessary that the weight% is an effective amount of the uterine fibroid cell growth inhibitor per day. In addition to the uterine fibroid cell growth inhibitor, the above-mentioned additives can be contained as a pharmaceutically acceptable carrier in a content known in the art.

2-3. Administration Method The pharmaceutical composition of the present embodiment can be administered to a subject in a pharmaceutically effective amount for the treatment of the target disease.

  In the present specification, the “pharmaceutically effective amount” is an effective amount of the uterine fibroid growth inhibitor contained in the pharmaceutical composition of the present embodiment.

  The administration method of the pharmaceutical composition of the present invention can be administered in a dosage unit form known in the art. Examples of the dosage unit form include oral administration and parenteral administration. Parenteral administration can be further classified into intra-tissue administration (eg, subcutaneous administration, intramuscular administration, intravenous administration, etc.), topical administration (eg, transdermal administration, etc.) or transrectal administration. Any of these dosage unit forms may be used for the pharmaceutical composition of the present embodiment.

  Oral administration is preferred for the pharmaceutical composition of the present invention. This is because the administration is easy, the invasiveness is low, and a sufficient effect can be obtained. For oral administration, the pharmaceutical composition is preferably administered in a dosage form suitable for the dosage unit form used, for example, a solid, powder or liquid.

  In addition, in order to more immediately suppress the growth of uterine fibroids, intra-tissue administration may be used. In the case of administration by injection, any method such as subcutaneous administration, intramuscular administration or intravascular administration may be used. For example, intravenous, intraarterial, intrahepatic, intramuscular (including uterus or uterine fibroids), intraarticular, intramedullary, intrathecal, intraventricular, percutaneous, subcutaneous, intradermal, intraperitoneal, intranasal, intestine Inner, sublingual and the like. Preferably, intrauterine fibroid administration or intravascular injection such as intravenous injection or intraarterial injection via blood flow.

  As described above, the dosage of the pharmaceutical composition of the present invention may be as long as a total effective amount of a uterine fibroid cell growth inhibitor is contained in a daily dosage unit (daily administration). The dose of is not particularly limited.

  When the pharmaceutical composition of the present invention is divided into a plurality of times per day, the number of divisions is not particularly limited, but since it becomes a burden on the patient if the number of times is too large, it is usually twice a day. 3 times / day or 4 times / day is preferred. In the case of divided administration, the administration time is not particularly limited, but in order to obtain a continuous effect, each administration interval is usually 1 to 24 hours, 2 to 12 hours, 3 to 10 hours, or 6 to 8 hours. It is preferable to open it. Therefore, if it is administered twice a day, for example, morning and evening, morning and afternoon, or day and evening may be used. In addition, if it is administered three times a day, it may be morning and evening. There is no particular limitation on whether to administer before or after a meal.

2-4. Usage The pharmaceutical composition of the present invention can be used for treatment of uterine fibroids and prevention of the development of uterine fibroids. In addition, since biguanide drugs are also known as therapeutic agents for type 2 diabetes (non-insulin-dependent diabetes), particularly obesity-type diabetes, the pharmaceutical composition of the present invention has an obesity tendency (or blood glucose level). It can also be used for the treatment of uterine fibroids that also serve as a treatment and prevention of diabetes and for the prevention of the onset of uterine fibroids.

<Example 1: Verification of inhibitory effect of metformin on uterine fibroid cell proliferation using uterine fibroid model cell line>
The inhibitory effect of metformin on uterine fibroid cell proliferation in vitro was verified using ELT-3 cells. ELT-3 cells are a uterine leiomyoma cell line established from Eker rats, which expresses estrogen receptor (ER) and progesterone receptor (PR) and is sensitive to these hormones. It is used as an in vitro experimental model cell line for uterine fibroids (Howe SR; Am J Pathol 1995) because it is similar to human uterine fibroid cells both biochemically and genetically. ). Moreover, the growth inhibitory activity with respect to the cervical cancer cell origin HeLa cell was simultaneously verified for the toxicity test of metformin.

(Method) ELT-3 cells were cultured at 37 ° C. under 5% CO ₂ . DF8 medium (10% FCS + DF8 medium) containing 10% fetal calf serum (FCS) (GIBCO) was used for maintenance passage of the cells. A medium manufactured by GIBCO was used as the DF8 medium. HeLa cells were also cultured under the same conditions.

  In the treatment with metformin, the medium was cultured in a DF8 basal medium (1% BSA + DF8 basal medium) containing 1% bovine serum albumin (BSA) (SIGMA) without FCS and phenol red instead of the above medium. Metformin (Wako Pure Chemical Industries) was dissolved in distilled water and then diluted 1000 times (0.1% v / v).

The maintained passage ELT-3 cells were seeded in a 96-well cell culture plate (Corning) at about 5000 cells / well, and cultured overnight in 10% FCS + DF8 medium for attachment. On the next day, after discarding the medium, 1% BSA + DF8 basal medium was added as a new medium, and further cultured for 24 hours. Subsequently, metformin was added to a final concentration of 2.5 mM or 5.0 mM and cultured again for 72 hours, and then MTS assay (Cell Titer 96 Aqueous One Solution Cell Proliferation Assay) (Promega) was performed on the cells. It was. HeLa cells were similarly treated.
For the evaluation of cell proliferation, the number of cells for three holes was calculated for each cell, and the average value and standard deviation were calculated.

(Results) The results are shown in FIG. FIG. 1 shows the cell growth rate when the number of cells not treated with metformin (0.0 mM) is defined as 100% in ETL-3 cells (A) or HeLa cells (B). As shown in this figure, metformin significantly suppressed the proliferation of ELT-3 cells in a dose-dependent manner. On the other hand, the growth inhibitory effect on HeLa cells was not observed. That is, metformin had no growth inhibitory effect on cervical cancer cells that are malignant tumors in the same uterine tissue, and showed a growth inhibitory effect on uterine fibroid cells that were benign tumors. This suggests that the cytostatic activity of metformin is specific for uterine fibroid cells.

<Example 2: Verification of inhibitory effect of metformin on uterine fibroid cell proliferation using human uterine fibroid model mouse>
The inhibitory effect of metformin on uterine fibroid cell proliferation in vivo was examined using a human uterine fibroid model mouse. The human uterine fibroid model mouse used in this example is a model mouse prepared by the method developed by the present inventors. In addition to human uterine fibroid model mice transplanted with human uterine fibroid-derived tissue, for example, human uterine fibroid model mice transplanted with human uterine fibroid tissue into SCID mice lacking functional T cells and B cells. Is known (Hassan MH, 2008, Am J Obstet Gynecol., 199: 156.e1-8). However, this model mouse has a big problem that the transplanted tissue does not engraft because the transplanted cells begin to degenerate in 2 to 3 weeks after the tissue transplantation and the nucleus disappears in 4 weeks. In addition, the MemyI model has been developed as a similar human uterine fibroid tissue transplant mouse using SCID mice (Hassan MH, 2008, Am J Obstet Gynecol., 199: 156.e1-8). This model mouse is a human uterine fibroid model mouse in which VEGF and COX-2 are forcibly expressed in a tissue derived from human uterine fibroids using an Ad vector and the tissue is transplanted into a SCID mouse. Although it is useful as an in vivo experimental system for uterine fibroids since the nucleus of the transplanted cells does not disappear even after 4 weeks from the transplantation and the transplanted tissue is properly attached, it has the disadvantage that the production of the model animal is complicated. The human uterine fibroid model mouse used in this example is excellent in that the human uterine fibroid tissue can be easily and stably engrafted in the mouse. A more detailed method for preparing a human uterine fibroid model mouse used here is described in Japanese Patent Application No. 2009-297958.

1. Experimental method 1-1. Preparation of a human uterine fibroid model mouse Pentobarbital (SIGMA) was intraperitoneally administered to a female individual of a NOG mouse of 8 to 10 weeks old as a host animal. After the disappearance of the stimulus reflex, the posterior cervical part is dissected to approximately 2 mm wide with scissors for dissection, and the sustained-release estrogen pellet (sustained release type, 1.5 mg tablet) (Innovative Research of America) is subcutaneously injected through the opening. Administered. Thereafter, the incision was closed using Aron Alpha for medical use (Toagosei Co., Ltd.). This estrogen administration was performed 5 days before the human uterine fibroid tissue transplantation operation.

  Subsequently, uterine fibroids removed by uterine fibroid nuclear surgery from a 45-year-old uterine fibroid patient who obtained informed consent at the Department of Obstetrics and Gynecology at Tohoku University Hospital were cut into about 3 mm x about 3 mm x about 2 mm, and human uterine fibroid tissue Pieces were prepared.

  The six estrogen-administered NOG mice were anesthetized, then the hairs on both flank sides were shaved, and one shaved portion was incised as a transplant site. The prepared human uterine fibroid tissue fragment from the same patient was immersed in BD Matrigel Basement Membrane Matrix (BD Biosciences) for 1 minute, and then one by one with tweezers subcutaneously from the incision on each side of each mouse. Transplanted. An additional 0.5 ml of BD Matrigel was added to the implantation site with a syringe, and then the incision was closed with medical Aron Alpha.

2. Verification of histological structure in human uterine leiomyoma graft (Method) Six transplanted mice were divided into 2 groups, and one group was intraperitoneally administered metformin (Wako Pure Chemical Industries) daily at 1 mg / day. To the group, physiological saline was intraperitoneally administered as a control. Eight weeks after the start of administration, human uterine fibroid grafts were removed from each mouse and examined histologically. For the preparation of thin-layer sections, the excised tissue pieces were treated with 10% formalin for 24 hours and fixed, and then paraffin infiltrated for 24 hours and embedded. Next, it was sliced using a microtome (Daiwa Kogyo REM-700-AN) to prepare a thin layer slice. Subsequently, the obtained thin-layer sections were stained with hematoxylin-eosin (HE) (Wako Pure Chemical Industries). Nucleophilic nuclei can be confirmed by the presence of hematoxylin due to the bluish purple color.

(Results) The results are shown in FIG. A shows HE staining results of grafts of human uterine fibroids extracted from control individuals not treated with metformin and B from individuals treated with metformin. Innumerable spindle-shaped spots and round spots that can be observed in the control are both nuclei of human uterine fibroid cells stained with HE. The difference in shape depends on whether the nucleus is observed from the long axis or the short axis. On the other hand, in human uterine fibroids derived from individuals administered with metformin, it can be seen that most cells are enucleated and degenerated compared to controls. This indicates that the proliferation of uterine fibroid cells is histologically inhibited.

3. Evaluation of cell proliferation in human uterine fibroid grafts (Method) Cell division in human uterine fibroid grafts excised from six transplanted mice in “2. Verification of histological structure in human uterine fibroid grafts” above Human uterine fibroid cells were detected by immunostaining using a mouse anti-human Ki-67 monoclonal antibody (#M 7240; Dako Cytomation), and cell proliferation in human uterine fibroid grafts was evaluated. Since Ki67 is a dividing nuclear-specific protein, the presence of dividing cells can be confirmed by antibody staining of this protein. Immunostaining was performed using Nichirei immunostaining kit # 424022 and Histofine SAB-PO (M) kit according to the attached protocol.

(Results) The results are shown in FIG. The ratio of Ki67 antibody-positive cells in the figure is the ratio of all cells per field, and each graft was counted in 4 fields, and the average value was taken. Human uterine fibroid cells in human uterine leiomyoma grafts removed from individuals treated with metformin compared to the growth of human uterine fibroid cells in human uterine leiomyoma grafts removed from control individuals. It was shown that.

4). Verification of apoptosis in human uterine fibroid grafts (Method) Apoptosis occurred in grafts of human uterine fibroids extracted from 6 transplanted mice in “2. Verification of histological structure in human uterine fibroid grafts” above. Human uterine fibroid cells were evaluated using TUNEL staining. TUNEL staining was performed with In Situ Cell Detection Kit (Roche Diagnostics).

(Results) The results are shown in FIG. The proportion of TUNEL positive cells in the figure is the proportion of all cells per field, and each graft was counted in 4 fields and the average value was taken. Compared to human uterine fibroid grafts removed from control individuals, human uterine fibroid cells removed from individuals treated with metformin clearly have more human uterine fibroid cells undergoing apoptosis. Indicated. This is because 2. It is not different from the results of verification of histological construction in human uterine fibroid grafts.

  From the results 2 to 4 above, it was demonstrated that metformin can suppress the proliferation of uterine fibroid cells even in vivo.

Claims (3)

  A uterine fibroid cell growth inhibitor containing a biguanide drug as an active ingredient.   The uterine fibroid cell proliferation inhibitor according to claim 1, wherein the biguanide drug is metformin, a derivative thereof, or a pharmaceutically acceptable salt thereof.   A pharmaceutical composition comprising the uterine fibroid cell growth inhibitor according to claim 1 or 2 as an active ingredient.

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