JPH03169821A - Prostaglandin i2 forming enhancer - Google Patents

Abstract

PURPOSE:To obtain a prostaglandin I2 (PGI2) forming enhancer comprising an ordinary heparin or low molecular heparin as an active ingredient. CONSTITUTION:A prostaglandin I2 (PGI2) forming enhancer comprising an unfractionated ordinary heparin having 3,000-12,000 molecular weight or a low molecular heparin which is cleft into proper molecular weight, fractionated and purified, preferably having about 4,000-6,000 average molecular weight as an active ingredient. The drug is usually administered parenterally as an injection, a dose thereof is 10-200mg per adult daily, production of PGI2 from vascular endothelial cell by thrombin stimulation is enhanced by the administration, effects by PGI2 action are exhibited and the drug is expected to treat diseases such as hypertension, bronchial asthma, gastric ulcer, etc., or to suppress metastasis of cancer.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention is directed to heparin (unfr.
actioned heparin, hereinafter [IFH
) or low molecular heparin (low molecular heparin)
lar weightheparin, hereafter LMWH
Prostaglandin 2 (prostaglandin 2) is characterized by containing as an active ingredient
1, hereinafter referred to as PGI2) production enhancer.

Prior art tIFH is a sulfonylated D-glucosamine, D-
It is a mixture of mucopolysaccharides with a molecular weight of about 3,000 to ioo, ooo, which consists of glucuronic acid and L-iduronic acid, and is known to be produced in the liver, heart, kidneys, small intestine, etc. There is.

This IJFH is known to exhibit a strong blood coagulation inhibitory effect, and is widely used as an anticoagulant in hemodialysis and the like.

The blood coagulation inhibitory effect of IJFI
fl (antithrombin [1, hereinafter referred to as ATI
llr), causing a structural change in ATI[I, and the active part of ^TII[
bin) and serine, which is the active center of factor Xa.
It is thought to be expressed by irreversibly binding with e).

Furthermore, regarding platelet aggregation, UFH is known to act rather in an aggregation-promoting manner.

On the other hand, PG12 is a type of prostaglandin, which is a metabolite of arachidonic acid, and has effects such as relaxing smooth muscle in blood vessels and bronchi, inhibiting platelet aggregation, inhibiting gastric acid secretion, and inhibiting cancer metastasis. It is known that it is produced in large quantities in cells and vascular smooth muscle cells.
Cell Engineering, Volume 17, No. 8, Page 596, 1988
.

From previous research on PGI2, arachidonic acid,
fradikinin, histamine, serotonin, thrompin,
Calcium ionophore A23187, alpha-toxin, leukotrienes C, and D, PDGF (plat
elet derived growth facto
r), I{OL-:]Resterol, human serum, nitroglycerin, angiotensin I and ■, vitamin C and other substances are known to exhibit a stimulating effect on PCI2 production.

Problems to be Solved by the Invention The purpose of the present invention is to provide IF or L that is useful as a pharmaceutical product.
MW}I as an effective component
G.I. An object of the present invention is to provide a production enhancer.

Means for Solving the Problems As a result of extensive research into the production of PGI2, the present inventors discovered that UFH or LMIII significantly enhances the production of PGI2 from vascular endothelial cells.

That is, when vascular endothelial cells are stimulated with thrombin in the presence of UPH or LMWH, the production of PGI is significantly enhanced, and this enhancement effect is also observed even when vascular endothelial cells are pretreated with UFH or LMWH. I have found things that are acceptable. The present invention is based on such knowledge.

P.G.I. As mentioned above, it is a type of substance synthesized by the arachidonic acid cascade, and has the effect of inhibiting platelet aggregation, relaxing smooth muscle, suppressing gastric acid secretion, protecting the gastric mucosa, increasing blood flow to organs, and suppressing cancer metastasis. It has been confirmed that it exhibits many physiological effects.

P.G.I. Synthetic enzymes are particularly abundant in vascular endothelial cells and vascular smooth muscle cells, and their production is promoted by the various production-stimulating substances mentioned above, but the characteristics of their production-stimulating effects are different. For example, histamine exhibits a production stimulating effect on endothelial cells but not on smooth muscle cells, and serotonin, on the other hand, exhibits a stimulating effect on smooth muscle cells but not on endothelial cells.

Among the above-mentioned PGI2 production stimulating substances, present in the living body,
Thrombin, bradykinin, and POGF exert a stimulating effect on PCI2 production in all cells.
etc. can be mentioned.

According to the findings of the present inventors, when vascular endothelial cells are stimulated with thrombin in the presence of tlPH or LMWH, PCI increases compared to the absence of tlPH or LMWH. The production of is enhanced by about 1.5 times. Furthermore, PGI2 production is similarly enhanced when vascular endothelial cells are pretreated with UFH or LMW}l and then stimulated with thrombin.

Therefore, if [IP}l or LMWH is administered, the production of PGI from vascular endothelial cells due to thrombin stimulation will be enhanced, and the effects of PGI, such as vasodilation, bronchodilation, gastric acid secretion suppression, and cancer metastasis. Because it exhibits suppressive effects, it is expected to be useful in treating diseases such as hypertension, bronchial asthma, and gastric ulcers, and in suppressing cancer metastasis.

The active ingredient contained in the drug of the present invention has a molecular weight of 3.
000-12. 000 unfractionated UPI{ or LMWH that has been cut into appropriate molecular weights, fractionated, and purified can be used, but from the standpoint of activity and side effects,
LMWHs with a low average molecular weight are preferred, especially those with an average molecular weight of 4. 000~6. LMIIH of about 000 is particularly preferred.

In addition, LMW}l can be obtained by simply cutting, fractionating, and purifying OF}l to an appropriate molecular weight according to conventional methods, or by further chemically modifying it, or by using pharmacologically acceptable salts thereof. Any method may be used, but an example of this is L, which is produced by cleaving tlFH with nitrous acid, fractionating and purifying it, and reducing the terminal aldehyde group with sodium borohydride.
MI1}1 or its sodium salt (Kabi 216
5. Average molecular weight 4.000-6,000, approximately 150υ
/■) etc.

Regarding the toxicity of UFH and LMWH, their safety has already been confirmed by many people, and since they are used in actual treatments such as artificial dialysis, there are no safety issues when applying them to the human body. do not have.

When the drug of the present invention is used for actual treatment, it is usually administered parenterally in the form of an injection, and such preparations can be prepared according to conventional methods for pharmaceutical preparation.

In addition, the dosage is appropriately determined depending on the gender, weight, age, disease type and symptoms of the patient, but it is generally 10 to 200 mg (150 mg) per day per patient.
11/mg 1,500 ~ 30,000 U)
(7) Administer in one to several doses or continuously within the range.

EXAMPLES The content of the present invention will be explained in more detail by the following experimental examples and examples. Note that each example does not limit the content of the present invention.

Experimental Example 1 Thrombin-stimulated PCI. Effect on production Cell culture Method of Jaffe, E.A. et al. [Journal of Clinical Investigation (J,
CIin. Invest, ), vol. 52, 2745~
2756, 1973], a 60 mm Petri dish coated with gelatin containing vascular endothelial cells prepared by treating human axillary veins with collagenase (Taib ■, manufactured by Sigma). (manufactured by Corning Inc.) and cultured at 37°C in a humidified incubator with a stream of 5% carbon dioxide gas. As a cell culture medium, 20% fetal bovine serum (manufactured by Filtron), 200%
ECGS of x/mj2 (endothelial c
ell growthsupplement, manufactured by Sigma), 100 (1/mg penicillin and 10
RPM containing 0/n+j2 streptomycin
I 1640 culture medium was used. Cells subcultured for 6 to 9 generations under the conditions described above were used in the experiment.

Experimental procedure A collagen-coated 35m+n veterinary dish was used for the PCI production amount experiment, and a 24m+n veterinary dish was used for other experiments.
A large tissue culture dish was used. Cell density was 3.88±0.27×10′ cells per square centimeter. Cultured cells were incubated with }IBP [! S buffer (pH 7.
After washing twice with 4), add 1 mffi of HBPBS buffer (
(When using a Petri dish) or 0.251 (When using a 24-well culture dish) and cultured at 37°C.

Thrombin (manufactured by Sigma, human origin, less than INIHU/1), IIF}I (manufactured by Sigma, derived from calf lung, 1
1JsP It/mA), LMWH (manufactured by Kabi Vitram, Kabi 2165 1 1J/mj2, anti-Xa factor activity) and del-vtansulfs-}
(dermatan sulfate, hereinafter referred to as OS) (manufactured by Seikagaku Corporation, 0.2 μ/rnl) were each dissolved in the same HEP8S buffer and added to the culture buffer.

Since the USP unit and anti-factor Xa activity are almost equal, I
The activities of IFH and LMl'l}I were each expressed in units.

After culturing, collect the culture buffer and use a portion of it to incubate PGI.
．． The production amount was measured. To measure the production amount of PC+I2, use a radioimmunoassay kit (manufactured by Amersham) to measure the amount of 6-keto prostaglandin F, which is a stable metabolite of PGr (hereinafter referred to as 5-Keto-PGF, rt). It was done by doing. Significant differences with the control group were tested using the Student's t-test (student's t-test).
'sshi test).

PGI2 production due to thrombin stimulation was performed using zone vascular endothelial cells.
The amount of GI2 produced was measured. 0.01, 0.1 for endothelial cells
and 1,ON[H U/1 thrombin were added and cultured at 37°C, and the amount of PCI2 produced at each culture time was determined by 6-Keto-PGF, . It was measured by radioimmunoassay method using No. 31 as an index. The result was 1.0
It was confirmed that NIH tl/d thrombin significantly promoted PGI production, and the production amount reached a plateau in about 20 minutes.

Based on this result, all subsequent experiments were conducted at 0.5 NIH t
Culture was performed for 20 minutes under thrombin stimulation at l/ml2.

Using human A-band vascular endothelial cells, P by thrombin stimulation
G.I. IIFH, LMIIIH and O for production
We investigated the influence of S.

0.5 N[}I O
/1 volume of thrombin was added, and cultured at 37°C for 20 minutes.
2165, 1υ/1) or OS (0.2 mg/d
) was measured in the presence and absence of tenoPGI2, and the respective effects were investigated.

The results were as follows.

6-Keto-PGF +rt (1) g/well)
CONT UFH LMWH OS 70.3 103. 4 102. 2 79,3 (mean + S.E. (n=8), ± 5.0
0 ± 4.61 10 ± 4.32 ”” ± 4.57 This book: p<0.0013 In the table, CONT is the control group, tlFl{, I, M
ltll{ and OS are [IFH, LMWH
and a group to which OS was added (the same applies hereinafter).

Experimental Example 2 PGr2 Production in Pretreated Cells UFH, LMW}I (Kabi 2165) Furthermore, the amount of PGI2 produced by thrombin stimulation was measured using human astringent vascular endothelial cells precultured with HaDs.

[IFH (I U/mj!) in human clay vascular endothelial cells
, LMWH (Kabi 2165. I U/of)
Or OS (0.2 mg/ml) was added and cultured at 37°C for 1 hour, then the cells were washed with buffer, thrombin (0.5 NIH U/+++1) was added, and the cells were incubated at 37°C.
The amount of PGr2 produced after culturing at ℃ for 20 minutes was 61 ke.
to-PGF,. It was measured by the amount of t. The results were as follows.

6-keto-PGF IIE (pg/Well)C
ONT tlFH LMWH OS 9.37±2.66 16.96±2.00 12.55±2.20 5.92±2.06 (+++ean +S, B, (n=3)) Experimental example 3 Experimental example l and UFH, LM for PGr2 production induced by calcium ionophore A 23187 and arachidonic acid (hereinafter referred to as AA) stimulation using the same procedure as in 2.
The effects of WH and DS were measured.

^23187 (10 μM) or AA (25 μM) was added to human emotional vascular endothelial cells and cultured at 37°C for 20 minutes. t'L LIFH (I U/1)
, PCI in the presence and absence of LMWH (Kabi 2165, II1/d) or OS (0.2 mg/me).
1, and the effects of each were investigated. The results were as follows.

6 keto PGF+a (lag/well)
CONT UFH LMW}I DS 113.0±8.4 133.1±7.8 138.9±8.0 ° 114.2±4.8 [mean +S. E, (n=6). 378.4±2
0.2 393.5±17,4 402. 7±13,1 416.9±35,3 pieces: ρ<0. 05) Example 1 150 U/mg LMW}l CKabi 2165
> 5,000.000 11 and sodium chloride 11um4
Dissolve 5 g in an appropriate amount of distilled water and make the total amount 5,000mj.
After that, 5.000 tl (1.000 (1.0
1,000 5-ampoules containing the active ingredient of /1) were obtained.

Example 2 150 U/mg (D LMWH (Kabi 216
5) Dissolve 50,000,000 [J and 450 g of sodium chloride in an appropriate amount of distilled water, and reduce the total amount to 50,000,000 [J] and 450 g of sodium chloride.
After making 000 ml, fill each 50 ml into vials.
Sterilized using conventional methods, 50,000 U in one vial.
(1,000 U/nffl) (1,000 50 ml vials containing D-active fraction were obtained.

Claims (2)

[Claims] (1) Prostaglandin I_, which is characterized by containing normal heparin or low-molecular-weight heparin as an active ingredient
2 production enhancer. (2) The drug according to claim 1, which contains low molecular weight heparin having an average molecular weight of about 4,000 to 6,000 as an active ingredient.