JP4440412B2 - Tumor metastasis inhibitor - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a tumor soft organ metastasis inhibitor comprising calcitonin as an active ingredient.
[0002]
[Prior art]
It is known that metastasis to other soft organs and bones is caused by the growth of the tumor at the primary lesion. By suppressing this growth and metastasis by an anticancer agent, the patient's life is saved and QOL is improved. . Anticancer agents such as alkylating agents, antimetabolites, immunostimulants, and anticancer antibiotics are used to suppress cancer growth and metastasis, and although they have therapeutic effects, they have hematologic toxicity, cardiotoxicity, and liver toxicity Many serious side effects are concerned (Japan Medical Newspaper; Vol. 3740, 3 (1995)). In addition, regarding the metastasis to the bone, although the therapeutic effect with the bisphosphonate preparation has been reported, the confirmation has not been obtained.
[0003]
With regard to the tumor metastasis inhibitory action of calcitonins, there has been a report (in Calcitonin 1980; 295 (1980)) in which bone metastasis due to prostate cancer has been prevented by PTH antagonistic action, and adaptation to bone destruction accompanying bone metastasis has also been considered ( The Bone; Vol. 10, 125 (1996)). For bone metastases after bone metastasis, thyroid hormone and calcitonin (elcatonin) were administered to patients with bone metastasis after thyroid cancer, and the improvement of the metastasis tumor mass and calcification to the bone defect were improved. (Hormones and clinical practice; Vol. 34 (special issue), 174 (1986)), and blood calcium levels increased in ureteral cancer, so elcatonin and prednisolone were administered. As a result, blood calcium levels Was not decreased, but there was no bone metastasis (Internal Medicine; Vol. 33, 107 (1994), and in these cases, there were no serious side effects of concern with anticancer agents. .
[0004]
Calcitonins are serum calcium-lowering hormones discovered by Copp et al. In 1962 (Endocrinology; Vol. 70, 638 (1963)) and Paget's disease of bone (Journal of Orthopedics; Vol; 58, 937 (1979)) , Osteoporosis (combined clinical; Vol. 39, 3623 (1990)), hypercalcemia (J. Bone Mineral Metab .; Vol. 6, 93 (1988)), etc., and its efficacy is established and low toxicity It is used clinically as a drug. In addition, electrolyte excretion promoting action such as sodium and phosphorus on the kidney (Am. J. Physiol .; Vol. 246, 927 (1984)), gastric juice secretion inhibiting action on the stomach (Acuta Endocrinol. (Suppl); Vol. 155, 216 (1971)), exocrine inhibitory action on pancreas (Gut; Vol. 18, 615 (1977)), analgesic action on pain of osteoporosis (Agents Actions; Vol. 41, 121 (1994)) ) Etc. are known.
[0005]
These actions are thought to be due to giving signals through calcitonin receptors present in various target cells. For osteoclasts, information is transferred into cells via the phospholipase C pathway that activates protein kinase C. (Science; Vol. 251, 1078 (1991), Proc. Natl. Acad. Sci. USA; Vol. 91, 1115 (1991)), and this action regulates the motility of osteoclasts. Receiving (J. Endocrinol .; Vol. 126, 473 (1990)) It is considered to suppress bone resorption. The presence of calcitonin receptor has also been reported in tumor cells (Endocrinology; Vol. 136, 5377 (1995)), and a regulatory function for tumor cells is expected.
[0006]
[Problems to be solved by the invention]
The present invention provides a method for suppressing metastasis of tumors to soft organs by calcitonins having low toxicity and no serious side effects, and suppresses tumor growth in organs essential for life support such as lungs and liver The purpose is to bring about a life-prolonging effect.
[0007]
[Means for Solving the Problems]
As a result of repeated studies to solve the above problems, the present inventor has surprisingly found that calcitonins are effective for suppressing metastasis of tumors to soft organs. That is, in a model system in which cultured tumor cells were inoculated subcutaneously, calcitonin suppressed metastasis to soft organs and brought a life-prolonging effect.
Tumor cell 4T1 is inoculated subcutaneously into Balb / c mice and daily administration of 1 unit of calcitonin to this substance significantly suppresses metastasis to the liver and lung, resulting in a life-prolonging effect. It was done. The present invention has been made based on the above findings, and is a tumor metastasis inhibitor containing calcitonins as active ingredients.
[0008]
The calcitonins in the present invention may be any peptide hormone that has at least a binding ability to the calcitonin receptor, and some of them are clinically used as therapeutic agents for hypercalcemia, bone paget disease and osteoporosis. Natural calcitonin or an analog peptide thereof.
Examples of natural calcitonin include eel calcitonin, salmon calcitonin, porcine calcitonin, chicken calcitonin, and human calcitonin. Examples of the analog peptide include synthetic derivatives in which the 1,7-position disulfide bond is replaced with an aminosuberic acid based on the structure of calcitonin, such as elcatonin ([Asu ^1.7 ] eel calcitonin). ,), [Asu ^1.7 ] human calcitonin, [Asu ^1.7 ] salmon calcitonin, [Asu ^1.7 ] chicken calcitonin, [Asu ^1.7 ] porcine calcitonin, etc. are known, and British Patent No. 1516947 discloses these substances and synthesis methods. It is described in the issue specification etc. Calcitonins other than those described above are also included in the scope of the present invention as long as they are peptide hormones having at least a binding ability to the calcitonin receptor.
[0009]
Calcitonin activity of 0.5 to 5000 units quantified by calcitonin quantified by a biological assay using the serum calcium level lowering effect of rats as an index (13th revised Japanese Pharmacopoeia Second Supplement, 27 (1999)) Is included in the scope of the present invention.
These calcitonins have extremely low toxicity. For example, even if elcatonin is administered to mice and rats by intravenous, intramuscular, subcutaneous, or oral routes at 1300 or 7400 units / kg (body weight), lethal toxicity It was not observed at all. Examples of the form of the drug in the present invention include injections, rectal absorption agents, vaginal absorption agents, nasal absorption agents, transdermal absorption agents, pulmonary absorption agents, oral absorption agents, oral administration agents, etc. The dosage form is not limited at all.
[0010]
Injections are preferably used for intramuscular or intravenous administration. Rectal and vaginal absorbents are generally used in the form of suppositories, and nasal and transdermal absorbents are used. The pulmonary absorbent is used in the form of an aerosol composition containing a suitable dispersant or water and a propellant. An oral absorption agent is used in the form of, for example, a sublingual tablet with an appropriate absorption enhancer added, and an oral administration agent is used in an oral form such as a liposome preparation or a microcapsule preparation.
[0011]
These drugs are adjusted to dosage forms suitable for usual respective drug forms. The injection can be prepared by, for example, dissolving elcatonin in a distilled water for injection in which appropriate amounts of a buffer, an isotonic agent, and a pH adjusting agent are dissolved, and dispensing the sterilized product through an antibacterial filter into an ampoule. For rectal and vaginal absorbents, for example, elcatonin can be added to distilled water or oily vehicle by appropriately using an absorption enhancer having chelating ability such as sodium bectinate and sodium alginate and hypertonic agent such as sodium chloride and glucose. It is dissolved or dispersed to prepare a rectal / vaginal injection suppository or suppository (see British Patent Nos. 20092002 and 2095994).
[0012]
The nasal absorbent is prepared, for example, as a solution or powder obtained by adding an absorption accelerator such as glucuronic acid, succinic acid, tartaric acid, etc., which are water-soluble organic acids, to elcatonin (Japanese Patent Laid-Open No. 63-243033, Japanese Patent Laid-Open No. 63-243033). JP-A-63-316737, JP-A-1-230530, JP-A-2-111, JP-A-2-104531). Furthermore, a nasal absorbent can be obtained by appropriately adding an emulsion to elcatonin (JP-A-4-99729).
[0013]
For example, a method for promoting absorption from the skin by adding an absorption enhancer such as Azone to salmon calcitonin has been reported as a transdermal absorption agent (2nd Annual Meeting of the Pharmaceutical Society of Japan, Abstracts of p57-). 58), and a percutaneous absorption agent of calcitonins may be obtained by a method based on iontophoresis (Ann. NY Acad. Sci .; Vol. 507, 32 (1988)).
The pulmonary absorbent is obtained by grinding and grinding calcitonin with a dispersing agent such as Arlacel and Span 80 into a homogeneous paste, and then dispersing this paste in a cooled propellant such as Freon 11 and Freon 12. There is a method obtained by filling a container equipped with a valve (Japanese Patent Laid-Open No. 60-161924).
[0014]
Oral absorbents include calcitonins such as ascorbic acids, acidic amino acids, citric acids, unsaturated fatty acids, salicylic acids, etc. alone or in combination of two or more, excipients such as glucose, and flavorings such as menthol It can be obtained as a troche, sublingual tablet, powder, etc. by adding an agent (JP-A-56-140924).
For oral administration, for example, calcitonin may be prepared by a method using W / O / W emulsion (Endocrinol. Japan; Vol. 23, 493 (1976)), or a liposome preparation method (Hormon Res. Vol. 16, 249 (1982)) may be used to formulate calcitonins.
[0015]
For example, in the case of an injection of elcatonin, such a drug is administered in an amount of 0.5 to 5000 units, preferably 1 to 400 units, as a single dose of elcatonin. Other preparations and other calcitonins may be administered according to the titration unit of elcatonin. The administration frequency may be 1 to 2 times a day, or may be daily or 1 to 3 times a week. Furthermore, a suitable amount of calcitonins may be dissolved in a suitable infusion such as Solita T-3 and administered intravenously over, for example, 1 to several hours. The amount of calcitonin in the drug can be determined as appropriate. In short, it is sufficient if the calcitonin activity per administration is determined to be equivalent to 0.5 to 5000 units of the injection.
The calcitonins produced and prepared in this manner are extremely useful in suppressing tumor metastasis and are useful as tumor metastasis inhibitors, as shown in the following Examples.
[0016]
【Example】
Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto. In addition, the abbreviation in a figure and a table | surface means the following term.
eCT; Elcatonin
[Example 1]
Dulbecco Modified Eagle Medium (hereinafter, DMEM) supplemented with mouse breast cancer cells, 4T1 cells in 10 cm cell culture dishes and supplemented with 10% fetal calf serum (hereinafter referred to as FCS) The cells were cultured until they became subconfluent. After culturing, the cells were washed twice with phosphate buffered saline and dissolved in 0.8 ml of a cell lysate (4M guanidine thiocyanate, 0.5% sodium lauroyl sarcosinate, 25 mM sodium citrate (pH 7.0)). After the collection, 1/10 amount of 1.66 M sodium acetate solution (pH 4.0) and an equal amount of PCI solution (water-saturated phenol 50: chloroform 49: isoamyl alcohol 1) were added, followed by stirring and centrifugation. The supernatant aqueous layer was collected, 0.8 ml of isopropyl alcohol was added, and the mixture was stirred and stored at -30 ° C. The resulting precipitate was spun down and dissolved again with 0.8 ml of cell lysate, an equal amount of isopropyl alcohol was added, and the mixture was stirred and stored at −30 ° C.
[0017]
The precipitate formed again was sedimented by centrifugation, rinsed with an 80% ethanol solution, air-dried, and then 10 mM Tris-HCl, 1 mM ethylenediaminetetraacetic acid (hereinafter referred to as EDTA) (pH 8.0). Was used for reverse transcription as an RNA solution. Reverse transcription reaction solution (50 mM Tris-HCl (pH 8.3), 75 mM potassium chloride, 10 mM dithiothreitol, 3 mM magnesium chloride, and 10 nM deoxyadenosine triphosphate, 10 nM deoxythymidine triphosphate, 10 nM deoxycytidine triphosphate Total RNA and 200 ng of random primer (Gibco URL) were added to 10 nM deoxyguanosine triphosphate (hereinafter, this mixture of four bases is referred to as dNTP mix), heated at 70 ° C. for 10 minutes, and then in ice Quenched to denature RNA and random primers. To this, 200 units of MMLV reverse transcriptase (GIBCO) was added and incubated at 37 ° C. for 1 hour to carry out a reverse transcription reaction.
[0018]
PCR reaction solution (60 mM Tris-HCl (pH 9.1), 18 mM ammonium sulfate, 1.6 mM magnesium sulfate, 10 nM dNTP mix) and mouse calcitonin receptor specific primer (sense; 5 ′ GTT GAC GTTTGTG CCC AAT GGA-3 ′ anti Sense; 5 ′ CCC TGG AAAGA ATC AGA GAG-3′20 pmols, reverse transcription reaction solution (corresponding to 50 ng of total RNA), and 1 unit of Elongase Enzyme mix (GIBCO) were added, and Polymerase Chain Reaction (hereinafter referred to as PCR) reaction was performed. First, after heat denaturation at 94 ° C. for 3 minutes, the reaction was carried out for 35 cycles of 94 ° C. for 10 seconds, 57 ° C. for 30 seconds and 68 ° C. for 1 minute, followed by extension reaction at 68 ° C. for 10 minutes. . The expression of calcitonin receptor was confirmed by developing 1/5 amount of the PCR reaction solution after the reaction on a 1% agarose gel containing ethidium bromide and searching for the amplified DNA fragment.
[0019]
As a result, as shown in FIG. 1, the expression of C1a type calcitonin receptor was confirmed in 4T1 cells in the same manner as the kidney, brain and growth plate cartilage tissues of mice as controls, and calcitonin directly acted on tumor cells. The possibility of bringing about was suggested.
[0020]
[Example 2]
4T1 cells are seeded in a 24-well plate at a density of 10,000 cells / well, and citrate buffer containing 0.1% bovine serum albumin (hereinafter referred to as BSA) in DMEM medium containing 10% FCS. Each concentration of elcatonin diluted in the solution was added from the day of sowing to confirm the effect on monolayer culture. The medium is changed every day, and after culturing for 5 days, the cells are washed twice with phosphate buffered saline, recovered with 0.25% trypsin, 0.02% EDTA, and the number of cells is counted on a hemocytometer. Measured. As a result, as shown in FIG. 2, cell growth was suppressed at a concentration of 10 ⁻¹⁰ molar or higher (equivalent to 0.02 units / ml).
[0021]
Further, 4T1 cells diluted with DMEM containing 5% calf serum and 5% FCS were suspended in 0.3% soft agar and seeded in a 24-well plate at 1000 cells / well. After the agar gelled, each concentration of elcatonin was added onto the soft agar, and elcatonin was similarly added on the seventh day of culture. At this time, on the 14th day of culture, the number of colonies consisting of 50 or more cells was counted under a phase contrast microscope. As a result, as shown in FIG. 3, cell growth was suppressed at 10 ⁻¹⁰ molar concentration or more of elcatonin.
These facts indicate that elcatonin directly exerts a growth inhibitory action on tumor cells, suggesting that this is at least one mechanism that acts as an inhibitor of metastasis to soft organs.
[0022]
[Example 3]
A 4T1 cell into which a luciferase gene was introduced was transplanted subcutaneously into the right breast of a 6-week-old female Balb / c mouse (body weight: about 20 g) at 1 million cells / individual. One week later, after confirming the formation of the primary tumor, 1 unit of elcatonin was dissolved in a citrate buffer containing 0.1% BSA and administered intraperitoneally every day. Only the citrate buffer containing 0.1% BSA was administered to the control group.
On the 20th day after transplantation, the mice were sacrificed, lung and liver luciferase activities were measured, and the effect of inhibiting metastasis to tumor organs was evaluated by luciferase activity per organ wet weight. The luciferase activity was measured with a chemiluminometer using Dual-Luciferase Reporter Assay System (Promega). As a result, as shown in FIG. 4, the metastasis to the organ was markedly suppressed.
[0023]
[Experimental Example 4]
After transplanting 1 million cells / individual 4T1 cells subcutaneously into the right breast of a 6-week-old female Balb / c mouse (body weight: about 20 g), and confirming that a primary tumor was formed one week later, One unit of elcatonin was dissolved in citrate buffer containing 0.1% BSA and administered daily into the peritoneal cavity of mice. Only the citrate buffer containing 0.1% BSA was administered to the control group. When the survival rate of the mice was evaluated by the Kaplan-Meier survival rate curve, a significant difference was observed between the two groups as shown in FIG. 5 in the Wilcoxon test, and the life-prolonging effect of elcatonin was confirmed.
[0024]
【The invention's effect】
From the above, the present invention has shown that calcitonin can suppress tumor metastasis and further exert an effect on life extension, and also because calcitonin has low toxicity, Thus, a useful tumor metastasis inhibitor including the above range can be obtained.
[Brief description of the drawings]
FIG. 1 shows the expression of calcitonin receptor in 4T1 cells.
FIG. 2 shows the inhibitory effect of elcatonin on proliferation in monolayer culture of 4T1 cells.
FIG. 3 shows the inhibitory effect of elcatonin on the proliferation of 4T1 cells in soft agar.
FIG. 4 shows the inhibitory effect of elcatonin on organ metastasis of 4T1 cells.
FIG. 5 shows the survival effect of elcatonin by inhibiting 4T1 cell organ metastasis.

Claims (4)

Tumor soft organ metastasis inhibitor containing elcatonin as an active ingredient.  The tumor metastasis inhibitor according to claim 1, wherein the tumor metastasis inhibitor is an injection, a rectal suppository, a vaginal suppository, a nasal absorption agent, a transdermal absorption agent, a pulmonary absorption agent, an oral absorption agent or an oral administration agent. Agent. The tumor metastasis inhibitor according to claim 1, wherein elcatonin has a calcitonin activity of 0.5 to 5000 units quantified by a biological measurement method using serum calcium level lowering action as an index. The tumor metastasis inhibitor according to any one of claims 1 to 3, wherein 0.5 to 5000 units of elcatonin per day are administered daily or 1 to 3 times a week.

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