JP2000095797A - Oxytocin receptor active substance - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new endogenous and physiologically active substance acting on a human uterine muscle oxytocin receptor. SOLUTION: This new human uterine muscle oxytocin receptor has the following physicochemical and physiological properties: (1) acting on a human uterine muscle oxytocin receptor; (2) being adsorbed by a microbonder back C 18 reverse phase column; (3) being adsorbed by a cation-exchange column. The above receptor is useful for elucidation of maintenance of pregnancy, childbearing outbreak mechanism, pathological physiology of premature, or the like, is a body composition having high safety and enables us to develop medicament having diagnostic and curable effect having specific action.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to an oxytocin receptor agonist, and more particularly to a novel endogenous bioactive substance that acts on human uterine muscle oxytocin receptor.

[0002]

2. Description of the Related Art Oxytocin has a strong uterine contractile action, is a posterior pituitary hormone whose structure was determined in the 1950's, and has been widely applied clinically to induce labor, induce labor, and the like. However, the details of the relationship with the mechanism of labor including the onset of labor, miscarriage in which abnormal contraction of the uterine muscle is one of the pathophysiologies, and premature labor have not been clarified yet. Elucidation of the mechanism of pregnancy maintenance, including these mechanisms of labor, is one of the major issues of modern science. In recent years, various oxytocin antagonists (oxytocin antagonists, Oxytocin Ant,
agonists) have been chemically synthesized, but none of them have been applied clinically.

[0003]

In view of the above, substances that act on oxytocin receptors are a starting point for elucidating the mechanism of labor, the mechanism of maintaining pregnancy, the pathophysiology of premature birth, and the like. It is expected to contribute to the development of drugs or diagnostics for treatment and labor control, and screening systems, and to contribute to safer maternal and child management in the perinatal period, and also to reduce maternal mortality and morbidity. Is done. However, at present, no novel bioactive substance that acts on human uterine muscle oxytocin receptor and exists in the living body has been reported. The present inventor, in order to solve the above-described problems, as a result of eagerly searching for a substance that exists in a living body and acts on a human uterine muscle oxytocin receptor, as a result, a substance that acts on a human uterine muscle oxytocin receptor in the human placenta. The present inventors have found that the substance exists and succeeded in purifying and isolating the substance, thereby completing the present invention. That is, an object of the present invention is to provide a novel substance that acts on the human uterine muscle oxytocin receptor.

[0004]

The gist of the present invention made to solve the above problems is an oxytocin receptor active substance having the following physicochemical properties and physiological activities. Acts on human uterine muscle oxytocin receptor. Adsorb to Micro Bonder Bag C18 reverse phase column. Adsorb to cation exchange column.

[0005]

BEST MODE FOR CARRYING OUT THE INVENTION The substance of the present invention exists in a human body and can be purified and isolated from a living tissue. The living tissue as a raw material is not particularly limited as long as it is a tissue containing the substance of the present invention, but the placenta is preferably used because it is particularly present in a large amount in the placenta. The living tissue as a raw material is preferably stored in a frozen state in order to prevent denaturation and the like.

[0006] The purification and isolation of the substance of the present invention from the above-mentioned raw materials can be performed by various methods generally used for the separation and purification of biological substances, such as extraction, centrifugation, dialysis, ion exchange chromatography, and gel. It can be performed by using a method such as filtration chromatography, reverse phase chromatography, affinity chromatography, hydrophobic chromatography, high performance liquid chromatography (HPLC), ultrafiltration, or electrophoresis. Particularly preferably, the extraction is performed by extracting at least one selected from ion exchange chromatography, gel filtration chromatography, reverse phase chromatography and HPLC after the extraction from the living tissue, and preferably by appropriately combining these.

An example of a preferable separation / purification operation is as follows.
It is preferable to use a human placental tissue as a raw material that has been frozen with liquid nitrogen immediately after delivery, and the frozen tissue is lyophilized or cut into pieces in a frozen state. After heat-treating the strip in water (for example, at about 90 to 100 ° C. for about 10 minutes), acid extraction (for example, 1N acetic acid) is performed. Acetone is added to the resulting extract at a final concentration of 80% to precipitate large proteins, and the supernatant fraction is separated. The substance thus obtained can be obtained by purifying the fraction thus obtained by reverse phase HPLC and ion exchange chromatography.

[0008] The substance obtained above is, as shown in the following Examples, an ornithine vasotocin analog (Ornithisin).
ne Vasotocin Analogue (OVTA) ¹²⁵ I
A radioreceptor assay using a radiolabeled product as a ligand in human uterine muscle (Radioreceptor assay, hereinafter referred to as RRA) was found to act on human uterine muscle oxytocin receptor. It should be noted that the RRA described above can be summarized as follows: OV, one of a series of oxytocin antagonists that have been chemically synthesized for the purpose of treating preterm labor in recent years.
The ¹²⁵ I-labeled compound of TA has high specificity and selectivity for the uterine muscle oxytocin receptor and has excellent specific radioactivity. Therefore, sufficient informed consent (inform
ed consent) to obtain human uterine muscle tissue at the end of pregnancy from the case that required hysterectomy after delivery due to obstetric complications such as cesarean section or early placenta exfoliation, flaccid hemorrhage, etc. did. ¹²⁵ I-OVTA was used as a radioligand, and RRA using human myometrial stromal fraction was used as an indicator of activity.

The above-mentioned various chromatography and RRA
When the results of the present invention were examined, the substance of the present invention was found to be oxytocin or OV which is a substance having a known human uterine muscle oxytocin receptor binding ability (oxytocin receptor agonist).
It turned out that it is a physiologically active substance different from TA and the like.
Therefore, the substance of the present invention is a novel substance characterized by the aforementioned physicochemical properties and physiological activities. In addition, as long as the substance of the present invention is characterized by the above-mentioned physicochemical properties and physiological activities, its origin and production method are not limited, and any origin and production method (including chemical method and genetic engineering method) The present invention is also included in the present invention.

The substance of the present invention is a novel physiologically active substance acting on the human uterine muscle oxytocin receptor. The substance of the present invention makes it possible to elucidate the mechanism of the onset and maintenance of human labor, the pathophysiology of miscarriage and premature birth, and the mechanism of maintenance of pregnancy, which are still unclear. In addition, many perinatal complications, such as preeclampsia, eclampsia, placental premature abrasion, flaccid hemorrhage, intrauterine fetal growth retardation, and intrauterine fetal death, which have serious consequences for mothers and children, will be revealed. Be expected. Further, the substance of the present invention is expected to have potential as a medicament in many conditions such as the following during pregnancy, labor, and postpartum.

More specifically, first of all, the substance of the present invention may be used as an agent for promoting labor or an agent for inducing labor due to uterine muscle contraction. It is necessary to induce labor (induction of labor) or enhance (induction of labor) in various conditions during pregnancy and labor, and uterine constrictors are indispensable for safety management of mothers and infants by controlling labor. The relationship between oxytocin and prostaglandins, which are conventional pharmaceuticals, and the mechanism of labor is largely unknown. Uterine contractions caused by these drugs are different from uterine contractions caused by physiological labor and are not well-regulated, so uterine rupture due to excessive labor, fetal distress, etc.
Serious side effects occur, including maternal, fetal and neonatal death. Due to the frequent occurrence of medical accidents caused by labor-promoting agents, the precautions for use in the package inserts of labor-promoting agents have been frequently revised since 1992. It has become a social problem by creating a network and collecting accident cases. Therefore,
There is a long-felt need to develop a drug having a more controllable, safer, more physiological and specific uterine contractile action that replaces the conventionally used oxytocin and prostaglandin preparations.

The substance of the present invention is a novel physiologically active substance acting on the human uterine muscle oxytocin receptor, and may have a contractile action on human uterine muscle. Since the substance of the present invention is an endogenous physiologically active substance, compared to existing labor-promoting agents,
It is possible to develop a drug that is safer, physiologically and has a specific labor promoting action. That is, by increasing labor in the range of physiological uterine contractions, it becomes possible for both mothers and infants to safely advance labor. Serious side effects such as uterine rupture due to overwork labor, fetal and neonatal asphyxia due to placental blood flow insufficiency due to overwork labor, and maternal death and fetal neonatal death, which occur with conventional pharmaceuticals, can be avoided. Also, the substance obtained by modifying the chemical structure of the substance of the present invention, compared to existing labor promoting agents,
As described above, it is possible to develop a drug which is safer, has a physiological and specific labor promoting action.

On the other hand, at present, no specific and definitive therapeutic agent has been established for the mechanism of the pathological condition related to miscarriage and premature birth. Miscarriage and premature birth are one of the most frequent diseases in the obstetrics and gynecology field that require treatment. Even with the advancement of neonatal medicine, many premature baby complications such as intellectual and developmental disorders remain unresolved, and prevention of premature birth is the biggest challenge in perinatal management. All of the three currently used drugs in clinical practice, ritodrine hydrochloride, magnesium sulfate, and indomethacin, are non-specific uterine contraction inhibitors, and the side effects that frequently appear at the same time pose a problem. Among them, ritodrine hydrochloride is a non-specific drug utilizing β2 adrenergic receptor stimulating action, and is most frequently used among the three drugs. Tachycardia is almost inevitable. In addition, the appearance of serious side effects leading to maternal death due to pulmonary edema has become a problem. Furthermore, according to several meta-analysis in Europe and the United States, it is concluded that the effect of suppressing premature birth is not prolonged for more than 48 hours. Magnesium sulfate is not covered by insurance, and indomethacin is currently contraindicated in Japan due to serious side effects on the fetus and newborn. Therefore,
When the substance of the present invention has a human uterine muscle relaxation action,
Compared with ritodrine hydrochloride and the like which are currently widely used in clinical settings, it can be developed as a safer and more physiologically safe and premature birth prevention drug that relaxes human uterine muscle. That is, a long-term suppression of premature birth is expected due to a specific uterine contraction inhibitory action. At the same time, a stronger uterine contraction-suppressing action makes it possible to suppress labor in a state in which abortion prematurely progresses, which leads to prevention of premature birth and various complications of premature infants. At the same time, serious side effects on the mother, such as pulmonary edema, are avoided. Similarly, a substance obtained by modifying the chemical structure of the substance of the present invention enables the development of a safer, more physiologically specific and specific anti-migratory agent having a specific action.

Next, the possibility as a drug having an antiviral and antibacterial action is as follows. recent years,
It has become clear that the mechanism of uterine contraction or labor and the mechanism of infection in premature labor is closely related. The substance of the present invention is expected to further elucidate the relationship between premature labor, the onset of labor, and the mechanism of infection, and the possibility that drugs derived from the substance of the present invention will be developed as agents that act on infection defense mechanisms Can be considered. Further, the present invention enables the development of a drug in which a substance obtained by modifying the chemical structure of the substance of the present invention acts on an infection defense mechanism.

[0015] Possibilities as a diagnostic agent and a screening system are as follows. The substance of the present invention and the substance obtained by modifying the chemical structure thereof can be developed as a diagnostic agent or a screening system for various disease states in the perinatal period. First, the substance of the present invention and the substance obtained by modifying the chemical structure thereof have a possibility of being used as a diagnostic agent for abortion prediction and prognosis, or as a screening system. On the other hand, it can be applied as a diagnostic agent or a screening system for predicting the start of labor or predicting the progress of labor. Furthermore, it is useful as a diagnostic or screening system for a wide range of perinatal diseases, including the prediction of abnormal labor due to abnormal contractions of the myometrium, such as prolonged labor due to weak labor, excessive labor, and lax hemorrhage. Has development potential. At present, no such diagnostic agent or screening system is effective.

[0016]

Next, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples. Example 1 (Method of RRA) After obtaining a sufficient informed consent, at the time of performing cesarean section scheduled for term of pregnancy, the uterine muscle tissue of the upper margin of the uterine wall was collected. Uterine muscular tissue was washed with a small amount of NaHCO ₃ buffer (20
(mmol / l NaHCO ₃ , 5 mmol / l EDTA).
Five times the volume of NaHCO ₃ buffer was added and homogenized on ice. The mixture was centrifuged at 600 × g and 4 ° C. for 10 minutes, and the pellet was homogenized and centrifuged in the same manner. Combine the supernatant fractions
After centrifugation at 100,000 × g, 4 ° C. for 30 minutes, the pellet was suspended in Tris buffer (50 mM Tris-HCl, 10 mM MgSO ₄ , pH 7.4) and centrifuged again under the same conditions, and the pellet was suspended in Tris buffer. The crude membrane fraction was used. Some were quantified for protein by the Lowry method. ^{As a 125} I-labeled ligand, ¹²⁵ I-OVTA (NEN) having high specificity and selectivity for uterine muscle oxytocin receptor and excellent specific radioactivity was used. The reaction was performed using a multiscreen 96-well plate manufactured by Millipore, and the reaction conditions were 200 μl of total reaction solution per well and 7 μm of membrane protein.
g, ¹²⁵ I-OVTA 7000-8000 cpm. Samples were dried or desalted by HPLC fractions, and then assay buffer (50 mM Tris-HCl, 10 mM MgSO ₄ , 0.1% BSA, pH 7.4)
Was dissolved. After incubating at room temperature for 60 minutes,
The mixture was suctioned under reduced pressure, and B / F separation was performed on a Durapore membrane. As shown in FIG. 1, a sharp dose-response curve was obtained (B
_{O /} T = 16.7%).

Example 2 Placental tissue delivered during normal term delivery at human term was immediately frozen in liquid nitrogen and lyophilized. The dried tissue was finely ground and heated at 95 ° C. for 10 minutes in 4 times the volume of water. After cooling,
Homogenized with a Polytron as a 1 M acetic acid solution. Acetone at a final concentration of 80% was added to remove large molecular weight proteins, concentrated by a rotary evaporator, and freeze-dried to obtain a coarse powder. A part of the crude powder was separated by reverse phase HPLC, and the activity of each fraction was measured by RRA. The activity corresponding to the fraction of reverse phase HPLC is shown in FIG. As a comparative example, synthetic oxytocin and OVTA were separated under the same conditions, and the activity values were measured similarly. Figure 2 shows the result.
Are also shown. As shown in FIG. 2, the placental crude extract, synthetic oxytocin and OVTA were suggested to be different substances. The analysis and separation conditions are as follows.

(Analysis conditions) Column: YMC-PACK ODS-AM (4.6 mm × 250 m)
m, YMC) Detection wavelength: UV210 nm Detection sensitivity: 2.0 AUFS Flow rate: 1.0 ml / min A solvent: 0.1% TFA B solvent: 100% acetonitrile

Example 3 A placenta delivered by normal vaginal delivery (without a labor-promoting agent or uterine-contracting agent) was immediately frozen in liquid nitrogen, stored at -80 ° C., and subjected to the following extraction. The frozen placenta is crushed with a hammer and a small amount of dry ice is mixed with a waling blender.
And shred. Remove unshredded tissue and heat in boiling water at 100 ° C. for 10 minutes. After cooling in ice, acetic acid was added (final concentration IN) and homogenized with a Polytron. Acetone was added to a final concentration of 80%, concentrated, freeze-dried, and subjected to the following purification.

Example 4 The crude powder obtained in Example 3 (corresponding to 500 ml of acetic acid-extracted supernatant) was separated by reversed-phase HPLC, and the activity of each fraction was measured by RRA. The analysis and separation conditions are as follows. (Analysis conditions) Column: Micro Bonder Pack C18 (7.8 mm x 300 mm,
Waters) Detection wavelength: UV210 nm Detection sensitivity: 2.56 AUFS Flow rate: 2.0 ml / min A solvent: 0.1% TFA B solvent: 100% acetonitrile FIG. 3 shows the results of graphically illustrating the relative activity values by RRA in each fraction on a chromatogram.

Example 5 The fraction 39 showing the maximum activity value by the separation by reverse phase HPLC in Example 4 was separated by cation exchange HPLC, and each fraction was subjected to RRA. The analysis and separation conditions are as follows. (Analysis conditions) Column: TSK gel CM 2SW (4.6 mm × 250 mm) Detection wavelength: UV 280 nm Detection sensitivity: 0.04 AUFS Flow rate: 1.0 ml / min A solvent: 10 mM ammonium formate / 10% acetonitrile B solvent: 1.0 M Ammonium formate / 10% acetonitrile FIG. 4 shows the results of graphically illustrating the relative activity values by RRA in each fraction on a chromatogram.

Example 6 One-fourth of the fractions 61 and 62 which showed the maximum activity in the ion exchange chromatography of Example 5 was subjected to reverse phase HP
Each fraction was separated by LC and subjected to RRA for each fraction. The analysis and separation conditions are as follows. (Analysis conditions) Column: YMC-PACK C8-AP (4.6 mm × 250 mm,
YMC) Detection wavelength: UV210 nm Detection sensitivity: 0.64 AUFS Flow rate: 1.0 ml / min A solvent: 0.1% TFA B solvent: 70% acetonitrile / 0.085% TF
A FIG. 5 shows the results of the relative activity values by RRA in each fraction shown on a chromatogram.

[0023]

The substance of the present invention is a novel physiologically active substance that acts on the human uterine muscle oxytocin receptor, and has many unclear points such as maintenance of pregnancy, the mechanism of delivery, the pathophysiology of miscarriage, etc. Useful for elucidation. In addition, since it is a biological component, it is possible to develop a drug having higher safety and a diagnostic and therapeutic effect having a specific action than conventional drugs.

[Brief description of the drawings]

FIG. 1 is a dose-response curve of RRA using ¹²⁵ I-OVTA as a radioligand using human pregnant uterine muscle.

FIG. 2. Placental extract (a), oxytocin (b) and O
It is a figure which shows the isolation | separation in reverse phase HPLC of VTA (c), and the activity in RRA.

FIG. 3. Purification of substances of the invention by reverse phase HPLC and R
It is a figure which shows activity in RA.

FIG. 4 is a cation exchange HP of the maximum active fraction in FIG.
It is a figure which shows purification by LC and activity in RRA.

FIG. 5 is a graph showing purification of the maximum active fraction in FIG. 4 by reverse phase HPLC and activity in RRA.

Claims (1)

[Claims] 1. An oxytocin receptor active substance having the following physicochemical properties and physiological activities. Acts on human uterine muscle oxytocin receptor. Adsorb to Micro Bonder Bag C18 reverse phase column. Adsorb to cation exchange column.