JP2000309544A - Premature or miscarriage inhibitors, cervical ripening inhibitors and inhibitors of hyaluronidase - Google Patents

Abstract

(57) [Summary] [Problems] Premature or aborted pregnant women with cervical ripening, especially
Provided are preterm or miscarriage inhibitors and inhibitors of cervical ripening that can be used to prevent preterm or miscarriage by inhibiting cervical mucus or hyaluronidase in cervical tissue of impending preterm women. Also provided is an inhibitor of cervical canal-derived hyaluronidase. (1) A premature birth or miscarriage inhibitor comprising a hyaluronidase inhibitor as an active ingredient. (2) A premature birth or miscarriage inhibitor comprising as an active ingredient at least one selected from the group consisting of sulfated glycosaminoglycans, derivatives thereof, glycyrrhizin, aglycones thereof and salts thereof. (3) A cervical ripening inhibitor comprising as an active ingredient at least one hyaluronidase inhibitor selected from the same group as (2) above. (4) An inhibitor of cervical canal-derived hyaluronidase, which comprises at least one selected from the same group as (2) above.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to an agent for preventing premature birth or miscarriage, an inhibitor for cervical ripening, and an inhibitor of hyaluronidase.

[0002]

BACKGROUND OF THE INVENTION The normal birth process is regulated mainly by two physiological processes. That is, contraction of the uterine muscles that deliver the fetus and maturation of the cervix, which is the delivery route of the fetus. If either or both processes proceed early, premature or miscarriage may be induced. Although the cause is wide-ranging, it is mainly thought to be ascending infection, intrauterine infection, chorioamnionitis, early deprivation of the placenta, and the like.

[0003] As means for preventing the above-mentioned premature birth and miscarriage, uterine motility inhibitors such as ritodrine hydrochloride (utemerin), isoxpurine hydrochloride (zufaziran), magnesium sulfate preparations, and progesterone derivatives (progehormone and progedepo) have been conventionally used. And other progestin preparations. In addition, a method of treating imminent premature birth by administering a ulinastine-like compound has been reported (Japanese Patent Application Laid-Open No. 9-505568).

However, since the uterine motility suppressant suppresses contraction of the uterine muscle at the time of premature birth and prevents premature birth or miscarriage, only premature birth accompanied by cervical ripening and cervical ripening simultaneously with contraction of the uterine muscular are performed. It is less effective for premature or miscarriage caused by In addition, there are concerns about side effects of hormone preparations on both the mother and the fetus.

On the other hand, as a means for premature birth or miscarriage caused by only cervical ripening without uterine contraction,
Surgery is performed to surgically contract the cervix. In this case, when the cervix matures beyond a certain level, uterine contraction occurs, leading to premature delivery or miscarriage.

[0006] The following has been reported on the relationship between cervical ripening and hyaluronidase.

(1) Hyaluronidase is present in the cervix of pregnant and non-pregnant humans, and the activity of hyaluronidase in cervical mucus increases with ripening of the cervix (Mikio Ohara et al., 34th Japan Newborn Society of Japan, 1998). In short, hyaluronidase activity is a good marker of cervical ripening.

(2) Administration of hyaluronidase can induce cervical ripening in humans and animals (Gupta,
T. et al., J, Indian Med.Association, 92, 47, 199
4, Li, WJ et al., Chinese Med. J., 107, 552, 1994; Li,
Z. et al., Chinese J. Obstet. Gyneocol., 28, 292, 1993
).

[0009]

An object of the present invention is to inhibit hyaluronidase in cervical mucus or cervical tissue of preterm or miscarriage pregnant women with cervical ripening, especially impending preterm women. To prevent premature birth or miscarriage and an agent for inhibiting cervical ripening that can be used to prevent premature birth or miscarriage. It is another object of the present invention to provide an inhibitor of cervical hyaluronidase.

[0010]

The above object can be easily achieved by the following present invention.

(1) An agent for preventing premature birth or miscarriage containing a hyaluronidase inhibitor as an active ingredient.

(2) The premature birth or miscarriage prevention according to (1), wherein the hyaluronidase inhibitor is at least one selected from the group consisting of sulfated glycosaminoglycans, derivatives thereof, glycyrrhizin, aglycones thereof and salts thereof. Agent.

(3) The agent for preventing premature birth or abortion according to (2), wherein the sulfated glycosaminoglycan is chondroitin sulfate, dermatan sulfate, keratan sulfate, heparin, heparan sulfate, sulfated hyaluronic acid or a salt thereof.

(4) An agent for preventing premature or miscarriage containing as an active ingredient at least one selected from the group consisting of sulfated glycosaminoglycans, derivatives thereof, glycyrrhizin, aglycones thereof and salts thereof.

(5) A cervical ripening inhibitor comprising as an active ingredient at least one hyaluronidase inhibitor selected from the group consisting of sulfated glycosaminoglycans, derivatives thereof, glycyrrhizin, aglycones thereof and salts thereof.

(6) An inhibitor of uterine cervix-derived hyaluronidase comprising as an active ingredient at least one selected from the group consisting of sulfated glycosaminoglycans, derivatives thereof, glycyrrhizin, aglycones thereof and salts thereof.

[0017]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.
The hyaluronidase inhibitor, which is an active ingredient of the premature birth or miscarriage inhibitor and the cervical ripening inhibitor (hereinafter collectively referred to as "agent of the present invention"), is a sulfated glycosaminoglycan or a derivative thereof. Glycyrrhizin, its aglycone or its salt. In the present invention, premature birth is a birth from 22 weeks to less than 37 weeks of gestation, and imminent premature birth is a pathological condition suggesting the onset of premature birth, that is, a parturition sign such as uterine contraction or cervical ripening. , Refers to the condition leading to premature birth in the near future. In addition, in the present invention, miscarriage refers to delivery before 22 weeks of gestation.

The sulfated glycosaminoglycan is a polysaccharide or oligosaccharide comprising a disaccharide repeating unit composed of uronic acid (or galactose) and an acetylated amino sugar, wherein the hydroxyl group and / or the amino group of the constituent saccharide are different. Refers to sulfated ones. The average molecular weight of the sulfated glycosaminoglycan is generally 2,000 to 2,000,000, preferably 5,000 to 100,000.

In the present invention, any type of sulfated glycosaminoglycan can be used as long as it inhibits mammalian hyaluronidase, particularly hyaluronidase activity in the cervix, and has an inhibitory effect on cervical ripening. However, those with low side effects such as anticoagulant activity are recommended. Particularly preferred sulfated glycosaminoglycans include various chondroitin sulfates (A, C), chondroitin polysulfate (D, E), dermatan sulfate (chondroitin sulfate B), keratan sulfate, keratan polysulfate, heparin, heparan sulfate, sulfated Hyaluronic acid. Further, salts of these sulfated glycosaminoglycans are also preferable.

Examples of the sulfated glycosaminoglycan salts include a wide range of biologically acceptable salts, and specific examples thereof include alkali metal salts such as sodium salt, lithium salt and potassium salt, calcium salts, and the like. Among salts with inorganic bases such as alkaline earth metal salts such as magnesium salts and ammonium salts, salts with organic bases such as diethanolamine salts, cyclohexyl salts and amino acid salts and double salts thereof, biologically acceptable Used salt is used.

As derivatives of sulfated glycosaminoglycans, the functional groups (hydroxyl group, carboxyl group, sulfate group, acetyl group, Amino group) is chemically modified. The type of the derivative is not particularly limited as long as it has a cervical ripening inhibitory action comparable to that of the sulfated glycosaminoglycan. Specific examples of the derivative include an ester obtained by chemically modifying a hydroxyl group, a sulfate group, or a carboxyl group of a sulfated glycosaminoglycan.

Specific examples of the sulfated glycosaminoglycan ester include a compound in which a sulfate group or a carboxyl group of the sulfated glycosaminoglycan is esterified with a hydroxyl group-containing compound capable of esterification. At that time, all functional groups of the sulfated glycosaminoglycan may be esterified, or a part thereof may be esterified. Examples of the hydroxyl-containing compound include aliphatic alcohols (eg, methyl alcohol, ethyl alcohol, propyl alcohol, isopropyl alcohol, etc.), aromatic alcohols (benzyl alcohol,
In addition to phenethyl alcohol and cycloaliphatic alcohol (cyclohexanol, cyclohexanediol, inositol and the like), sterol, steroid, cholic acid, alkaloid and the like can be mentioned. Another specific example is a compound in which a hydroxyl group is esterified with a carboxyl group-containing compound capable of esterification.

Glycyrrhizin is also called glycyrrhizic acid, and is usually produced from licorice (Glycyrrhizaglabra) or roots and strons of a congener plant. In the present invention, highly purified glycyrrhizin as a drug substance is used in the present invention. It is preferably used. A typical example of glycyrrhizin aglycone includes glycyrrhetinic acid obtained by alkaline hydrolysis of glycyrrhizin.

Examples of the salts of glycyrrhizin and its aglycone include salts with alkali metals such as sodium, lithium and potassium, alkaline earth metals such as calcium and magnesium, and salts with inorganic bases such as ammonium. , Diethanolamine salt, cyclohexyl salt,
Examples thereof include salts with organic bases such as amino acid salts and double salts thereof, and specific examples of preferable biologically acceptable salts include diammonium glycyrrhizinate and dipotassium glycyrrhizinate. Also, a composition of glycyrrhizin and an amino acid can be used.

The drug of the present invention contains the above-mentioned specific hyaluronidase inhibitor as an active ingredient, and one or more of the above-mentioned substances may be formulated together with a pharmaceutically acceptable auxiliary as required. I can do it. The dosage form of the drug of the present invention is not particularly limited as long as the object of the present invention can be achieved, but according to a usual method of use, a liquid preparation such as a vaginal spray liquid,
Ointments, creams, semisolids such as gels, vaginal tablets, vaginal capsules, pessaries, vaginal suppositories and other solid preparations. The desired dosage form can be achieved by blending components generally used as raw materials for pharmaceutical preparations, in addition to the aforementioned components.

Examples of the above components include animal and vegetable oils (soy oil, tallow, synthetic glyceride, etc.), hydrocarbons (liquid paraffin, squalene, solid paraffin, etc.),
Ester oil (octyldodecyl myristate, isopropyl myristate, etc.), higher alcohol (cetostearyl alcohol, behenyl alcohol, etc.), silicone resin, silicone oil, surfactant (polyethylene fatty acid ester, sorbitan fatty acid ester, glycerin fatty acid ester, polyoxyethylene) Hydrogenated castor oil, polyoxyethylene propylene block copolymer, etc.), water-soluble polymers (hydroxyethyl cellulose, polyacrylic acid, carboxyvinyl polymer, polyethylene glycol, polyvinyl pyrrolidone, methyl cellulose, etc.),
Alcohol (ethyl alcohol, isopropyl alcohol, etc.), polyhydric alcohol (glycerin, propylene glycol, dipropylene glycol, sorbitol, etc.),
Examples include sugars (glucose, sucrose, etc.), inorganic powders (silicic anhydride, aluminum magnesium silicate, aluminum silicate, etc.), purified water, and the like.

For adjusting the pH, for example, inorganic acids (such as hydrochloric acid and phosphoric acid), alkali metal salts of inorganic acids (such as sodium phosphate), inorganic bases (such as sodium hydroxide), and organic acids (lower grade) Fatty acids, citric acid, lactic acid, etc.), alkali metal salts of organic acids (sodium citrate, sodium lactate, etc.), and organic bases (arginine, ethanolamine, etc.) can be used. Further, if necessary, a preservative, an antioxidant, and the like can be added.

The amount of the active ingredient (hyaluronidase inhibitor) in the drug of the present invention and the dose of the drug vary depending on the type of the hyaluronidase inhibitor, the method of administration, the age, weight and uterus of the pregnant woman to be administered. Can be increased or decreased. As the amount of the active ingredient in the drug of the present invention,
The amount may be, for example, about 0.1 to 10% by weight, but is not limited to such a range, and may be an amount necessary for exhibiting a desired pharmacological effect. The dosage of the drug of the present invention can be, for example, about 0.1 to 10 g per person. The administration method of the drug of the present invention is not limited as long as the object of the present invention can be achieved,
It is usually administered parenterally as a vaginal suppository or external preparation.

[0029]

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples unless it exceeds the gist of the present invention.

Example 1 (1) Sample collection and sample preparation method: First, cervical mucus of a non-pregnant woman, before term, at term, first parturition, and imminent preterm delivery was collected as follows. That is, a No. 1 cotton swab (Japan Swab Co., Ltd.) was inserted into the cervix, allowed to stand for 10 seconds, and then gently scraped and collected. Next, the cotton swab was immersed in 1 ml of Phosphate Buffer Saline (PBS), and stirred and shaken with a vibrator for 10 seconds to extract the cervical mucus absorbed by the cotton swab. After centrifugation at 3000 rpm for 10 minutes, the supernatant was recovered and used as each sample. For the measurement of chondroitin sulfate concentration, non-pregnant women, pre-term, term,
The cervical mucus of the pregnant woman in urgency and premature labor was used, and the cervical mucus of the first stage of labor was used for measurement of hyaluronidase activity.

(2) Measurement of Chondroitin Sulfate Concentration: The chondroitin sulfate concentration in the sample was determined by chondroitin (COS), chondroitin sulfate A (CS-A), and chondroitin C sulfate (CS-C) obtained by enzymatic digestion of the sample. )
Unsaturated disaccharides (ΔDi-OS, ΔDi-4S, ΔD
i-6S) by high performance liquid chromatography (HPLC)
It was calculated by quantification.

20 μl of 5 U / ml “chondroitinase ABC” (Seikagaku Corporation) was added to 100 μl of the sample and digested at 37 ° C. for 2 hours. This was combined with 5 U / ml of “chondroitinase AC II” (Seikagaku Corporation) and 1
M sodium acetate buffer (pH 6.0), 20 μl each
In addition, digestion was performed again at 37 ° C. for 2 hours. The sample after the enzyme digestion was filtered using an ultrafiltration apparatus having a molecular weight cut-off of 10,000 ("Ultrafree RC3GC" manufactured by Nippon Millipore Limited). The unsaturated disaccharide obtained by enzymatic digestion is collected in the filtrate.

ΔDi-OS is a polyamine-bonded vinyl alcohol polymer column (“NH ₂ P” manufactured by Showa Denko KK).
-50 "). For elution of ΔDi-OS, a 50 mM tetramethylammonium-acetate buffer (pH 8.5) containing 20% by weight of methanol was used.
ΔDi-4S and ΔDi-6S are dimethylamine-bonded silica gel columns (“N (NH ₃ ) ₂ −” manufactured by Chiaki Kagaku Co., Ltd.).
3151-N "). The elution of ΔDi-4S and ΔDi-6S includes 20% by weight methanol 2
0 mM Na ₂ SO ₄ was used. The flow rate when eluting ΔDi-OS is 0.5 ml / min, ΔDi-4S and ΔD
The flow rate for eluting i-6S was 0.65 ml / min. The eluate from the column was mixed with a 50 mM sodium tetraborate solution containing 0.1% by weight of 2-cyanoacetamide, and the mixture was passed through a reaction vessel set at 150 ° C. to react. The fluorescence of the reaction solution is excited at an excitation wavelength of 331 nm,
It was monitored at a fluorescence wavelength of 383 nm.

In order to avoid the influence of the dilution of the sample,
The unsaturated disaccharide concentration was corrected using the calcium concentration in the sample. All results are mean ± S.D. D,
As for the test, "Mann Whitney's U-Tes
t "was used.

The chemical names of the above unsaturated disaccharides are as follows.

Table 1 ΔDi-OS: 2-acetamido-2-deoxy-3-O- (4-deoxy-α-L-threo-hex-4-enopyranosyluronic acid) -D-galactose-ΔDi- 4S: 2-acetamido-2-deoxy-3-
O- (4-deoxy-α-L-threo-hex-4-enopyranosyluronic acid) -4-O-sulfo-D-galactose-ΔDi-6S: 2-acetamido-2-deoxy-3-
O- (4-deoxy-α-L-threo-hex-4-enopyranosyluronic acid) -6-O-sulfo-D-galactose)

(3) Results: The sum (ΔDi-CS) of ΔDi-OS, ΔDi-4S, and ΔDi-6S reflecting the total amount of chondroitin sulfate was the highest in the imminent preterm birth (33.76 ± 17. 30 μg / ml). This was significantly higher compared to pre-maturity (17.42 ± 8.42 μg / ml) and maturity (18.89 ± 6.85 μg / ml). The first stage of labor (21.71 ± 13.22μ)
g / ml) did not show a significant increase. Non-pregnant women (1
(7.53 ± 14.61 μg / ml), the same value as before the expiration. (Fig. 3)

(4) Measurement of hyaluronidase activity: 20 μl of each well-stirred sample was collected in each of two Eppendorf tubes. One sample was treated at 100 ° C. for 5 minutes to inactivate hyaluronidase in the sample. 10
20 μl of 100 mM sodium acetate buffer (pH 4.0) and hyaluronic acid (FA-HA) (10
0 μg / ml), and the final concentration was 1
Glycyrrhizin ("Glytilone No. 1" manufactured by Minophagen) was added as a hyaluronidase inhibitor in an amount of 20 μl to each of 0 mM, 1 mM, and 0.1 mM. Similarly,
Sodium chondroitin sulfate (chondron injection manufactured by Kaken Pharmaceutical Co., Ltd.) 1 mg / ml mainly composed of chondroitin sulfate C sodium, 1 mg / ml sodium dermatan sulfate
ml, 1 mg / ml sodium keratan sulfate
One by one (final concentration 0.25 mg / ml). As a control, a sample to which no inhibitor was added was used. These reaction solutions were allowed to stand at 37 ° C. for 24 hours.

Thereafter, each sample was placed in a 0.45 μm centrifugal ultrafiltration tube at 12,000 rpm for 15 minutes.
Centrifugal ultrafiltration for minutes. The obtained filtrate was applied to HPLC equipped with a gel filtration column, and FA-HA in the eluate from the column was detected with a fluorescence detector. 1 for each sample
The difference (ΔTime) between the FA-HA retention time of the 00 ° C. treatment and the untreated sample was calculated. As the hyaluronidase activity becomes stronger, FA-HA is decomposed and the molecular weight of HA becomes smaller, so that ΔTime becomes larger. The inhibition rate (%) was calculated from the ΔTime.

As inhibitors, chondroitin sodium (COS), chondroitin sulfate A sodium (CS-A), chondroitin sulfate C sodium (CS-A)
-C) (all manufactured by Seikagaku Corporation) having final concentrations of 5 μg / ml, 10 μg / ml, and 20 μg / ml, respectively.
Then, 20 μl of each was added so as to obtain a ml, and the hyaluronidase inhibition rate was calculated in the same manner as described above.

(5) Results: The hyaluronidase activity in the cervical mucus was almost completely inhibited by chondroitin sulfate ("Chondron injection" manufactured by Kaken Pharmaceutical Co., Ltd.), dermatan sulfate and keratan sulfate. In glycyrrhizin, 10
The enzyme activity was completely inhibited at a concentration of mM, and about 9% at 1 mM.
And about 40% inhibition was observed at 0.1 mM. These results are shown in FIG. Hyaluronidase activity in cervical mucus was slightly inhibited by 20 μg / ml chondroitin, but almost completely inhibited by 20 μg / ml chondroitin sulfate (CS-A and CS-C). (Fig. 2)

(6) Consideration: From the results of the above (3), the fact that the chondroitin sulfate concentration in the cervical mucus of the imminently prematurely pregnant woman is higher than that of the non-pregnant woman, before term, at term, and the first parturition. It has been shown. This result indicates that early maturing of the cervix is accelerated in imminent preterm labor compared with normal pregnant women, but in order to maintain the homeostasis of the living body, the cervix is suppressed from maturing, that is, hyaluronidase associated with cervical ripening. This indicates that it worked in the direction of suppressing the increase in activity. This suggests that inhibiting hyaluronidase involved in cervical ripening would suppress cervical ripening and, consequently, prevent premature birth or miscarriage. Further, from the result of (5), the hyaluronidase activity in cervical mucus is inhibited by hyaluronidase inhibitors such as glycyrrhizin, chondroitin sulfate (CS-A and CS-C), dermatan sulfate, and keratan sulfate. These hyaluronidase inhibitors can be used to regulate hyaluronidase activity to control cervical ripening, and furthermore, by suppressing cervical ripening, to prevent premature birth or miscarriage, especially urgency premature birth. Turned out to be possible.

[0042]

According to the present invention described above, cervical mucus comprises at least one hyaluronidase inhibitor selected from the group consisting of sulfated glycosaminoglycans, derivatives thereof, glycyrrhizin, aglycones thereof and salts thereof. By inhibiting the hyaluronidase activity in the cervix, ripening of the cervix can be suppressed and premature birth or miscarriage can be prevented.

[Brief description of the drawings]

FIG. 1 is a graph showing the inhibitory effect of chondroitin sulfate (CS), dermatan sulfate (DS), keratan sulfate (KS), and glycyrrhizin on hyaluronidase activity in cervical mucus of normal pregnant women.

FIG. 2 is a graph showing the inhibitory effects of chondroitin (COS), chondroitin sulfate A (CS-A), and chondroitin sulfate C (CS-C) on hyaluronidase activity in cervical mucus of normal pregnant women.

FIG. 3 is a graph showing changes in chondroitin sulfate concentration in cervical mucus of non-pregnant, normal pregnant women, labor and premature labor.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 43/00 111 A61P 43/00 111 C08B 37/00 C08B 37/00 H // C07H 15/256 C07H 15 / 256 A (72) Inventor Mikitaka Ohara 85-4 Nukazuka, Hiroshi-ku, Akita-shi, Akita F-term (reference) 4C090 AA09 BA62 BA65 BA66 BA67 BA68 DA09 DA23

Claims (6)

[Claims] 1. A premature birth or miscarriage inhibitor comprising a hyaluronidase inhibitor as an active ingredient. 2. The premature birth or miscarriage inhibitor according to claim 1, wherein the hyaluronidase inhibitor is at least one selected from the group consisting of sulfated glycosaminoglycans, derivatives thereof, glycyrrhizin, aglycones thereof and salts thereof. . 3. The premature birth or miscarriage inhibitor according to claim 2, wherein the sulfated glycosaminoglycan is chondroitin sulfate, dermatan sulfate, keratan sulfate, heparin, heparan sulfate, sulfated hyaluronic acid or a salt thereof. 4. A premature birth or miscarriage inhibitor comprising as an active ingredient at least one selected from the group consisting of sulfated glycosaminoglycans, derivatives thereof, glycyrrhizin, aglycones thereof and salts thereof. 5. A cervical ripening inhibitor comprising as an active ingredient at least one hyaluronidase inhibitor selected from the group consisting of sulfated glycosaminoglycans, derivatives thereof, glycyrrhizin, aglycones thereof and salts thereof. 6. An inhibitor of cervical-derived hyaluronidase, comprising as an active ingredient at least one selected from the group consisting of sulfated glycosaminoglycans, derivatives thereof, glycyrrhizin, aglycones thereof and salts thereof.