JPH01156956A - Prostaglandin derivative ester - Google Patents

Abstract

PURPOSE:To obtain a compound consisting of an acidic polymer of prostaglandin E1, A1 or B1 and having protecting action to injury of organ cell by ischemia or anoxia, injury of nerve cell by wound and erythrocyte of drepanocythemia patient. CONSTITUTION:Prostaglandin E1, prostaglandin A1 or prostaglandin B1 or keto type compound which is a dehydrogenated product of either one thereof is polymerized under alkali conditions using potassium hydroxide, etc. Then proton of carboxyl group of polymer thereof is replaced with acetoxyalkyl group, alkoxycarbonylmethyl ester or alkyl group to provide the aimed product. The aimed compound exhibits strong affinity with cell membrane because of being extremely slightly soluble in water and is useful in remedy and prevention for cardiac infraction, cerebral apoplexy, hepatic insufficiency, acute renal insufficiency, necrocytosis occurring in accidental wound of spinal marrow, malaria, etc.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention has a protective effect on organ cell damage due to ischemia or oxygen deficiency, nerve cell damage due to trauma, and red blood cells of patients with sickle anemia. It relates to prostaglandin derivative esters.

[Conventional technology]

It is known that using prostaglandin B+ as a starting material and treating it with an alkali produces a water-soluble acidic polymer, and that this polymer has the effect of protecting the heart from ischemia (US Pat. No. 4,153). , 808).

[Problem to be solved by the invention]

However, the inventor's investigation revealed that this compound is (1) unstable, (2) water-soluble and cannot pass through biological membranes, and (3) binds to serum calcium in the blood. It has major drawbacks, such as the formation of insoluble precipitates and loss of medicinal efficacy, and it has not been put into practical use even now, about ten years after its invention.

[Means to solve the problem]

The present inventor prepared an acidic type polymer using prostaglandin El s prostaglandin A1 or prostaglandin B+ as a starting material, and treated this acidic type compound to form an acetoxyalkyl ester or an alkyl ester. It has been found that a compound free from the above-mentioned drawbacks can be obtained by preparing the above-mentioned method.

That is, the present invention provides an acidic type polymer of prostaglandin EI, prostaglandin A1, or prostaglandin B, in which the proton of the carboxyl group is substituted with an acetoxyalkyl group, an alkoxycarbonylmethyl ester group, or an alkyl group. The present invention relates to a prostaglandin derivative ester that has a protective effect on organ cell damage due to ischemia or oxygen deficiency, nerve cell damage due to trauma, and red blood cells of patients with bell-shaped anemia.

This prostaglandin derivative is produced by polymerizing a keto-type compound that is prostaglandin EI, prostaglandin A1, or prostaglandin B, or a dehydrogenated product of any of them under alkaline conditions, and then polymerizing the resulting polymer. It can be obtained by substituting a proton of a carboxyl group with an acetoxyalkyl group or an alkyl group.

The polymerization method is POLIS & POLIS (U.S. Patent 4.15
3.808). Further, as a method for introducing an acetoxyalkyl group, an alkoxycarbonylmethyl ester group, or an alkyl group into the obtained polymer, a known method for ester bonding these to a carboxyl group may be used. For example, acetoxyalkyl bromide, alkyl bromoacetate, etc. can be used.

The prostaglandin derivative ester of the present invention is prostaglandin E1% prostaglandin A.

Alternatively, it consists of a 2- to 12-mer, preferably a 5- to 7-mer, acidic type polymer of prostaglandin B+. This polymer may be a homopolymer of any of the above, or a mixed polymer of two or three types.

The carboxyl groups of this polymer are ester-bonded with acetoxyalkyl groups, alkoxycarbonylmethyl ester groups, or alkyl groups to the extent that the hydrophobicity of the polymer can be ensured, and usually 70% or more, preferably 90% or more of the carboxyl groups. is esterified. The alkyl group of the acetoxyalkyl group has 1 to 6 carbon atoms,
Preferred are methyl and ethyl groups. The alkoxyl group of the alkoxycarbonylmethyl ester group has 1 to 6 carbon atoms, and preferred are methoxy and ethoxy groups. In addition, the number of carbon atoms in the alkyl group is 1 to
There are six groups, and the preferred ones are methyl group, ethyl group and propyl group. These are usually one type, but two
More than one species may be introduced into the carboxyl group. This prostaglandin derivative ester is insoluble in water;
Soluble in alcohol such as ethanol.

The dosage form may be, for example, dissolved in ethanol or an oily solvent, or may be in the form of an ointment.

The prophylactic and therapeutic agent containing the prostaglandin derivative ester of the present invention is useful for preventing and treating damage to organ cells caused by ischemia and oxygen deficiency, and damage to nerve cells caused by spinal cord trauma, and for preventing and treating damage to red blood cells in patients with bell-shaped anemia. It is used to prevent damage to blood cell membranes due to repeated infections, and to prevent and treat malaria. For example, during an organ transplant surgery, it can be administered immediately before the surgery to protect it from damage caused by ischemia after organ removal, and it can be given to the recipient after surgery to protect the organ. It is also effective for protecting organ cells of alcoholic patients, and can also be used as a therapeutic drug. In addition, cell necrosis caused by skin ulcers and pressure sores can be prevented by applying it directly to the skin as a poultice or by mixing it with a cream.

The dosage of prostaglandin derivative ester is important for organ cell ischemia, damage due to oxygen deficiency, nerve cell damage due to spinal cord trauma, membrane damage due to repeated bell-shaped red blood cells in patients with sickle anemia, and malaria parasitism. In both cases of inhibiting the proliferation of worms in red blood cells, the dose is 1 to 30 μ/kg body weight for 7 days for oral administration, preferably 3 to 10 μ for 7 days, and 0.5 to 20 μ for intramuscular or subcutaneous injection. kg body weight/day, preferably about 2 to 6 kg body weight/7 days. Also,
The preoperative or postoperative dosage for organ transplantation may also be the same as above.

[Effect]

This prostaglandin derivative ester is extremely insoluble in water and therefore has a strong affinity with cell membranes.

Furthermore, since prostaglandin derivative esters do not have carboxyl groups, they are not precipitated by calcium in serum.

When prostaglandin derivative ester is injected orally, subcutaneously, or intravenously, this compound is easily taken up into red blood cells or tissue cells, and after being taken up, the ester bond is cut by the esterase enzyme present in the cell, resulting in acidic acid. It becomes a type polymer.

This acidic polymer binds calcium within cells and
Reduces intracellular calcium concentration. It also has properties such as inhibiting the function of phospholipid degrading enzymes or proteolytic enzymes activated by calcium.

As prostaglandin derivative esters are decomposed within cells, prostaglandin derivative esters enter the cells one after another. However, acidic polymers, which are decomposition products, are hydrophilic and therefore difficult to escape through cell membranes.Accordingly, acidic polymers accumulate internally at high concentrations and exert pharmacological effects.

〔Example〕

Example 1 Production of prostaglandin derivative (a) Production of acidic polymer Prostaglandin E+ is used as a starting material. This is P
OLIS & POLIS (US Patent 4.153,808)
is polymerized by the method used for prostaglandin B.

Briefly, 0.1 g of starting material is dissolved in 10 cc of ethanol, 1.2 g of powdered KO) is added and placed in a 50 cc flask.

The solution is heated to 70° C. with a reflux device while rotating. Add 10 cc of water to bring the final concentration of KOH to IN. Attach a reflux device to this solution and keep it at 74°C.
Heat for 6 hours. Cool the compound and add 11 cc of isobutanol, then 7.5 cc of 2.3N
Add II (i, On) to adjust the pH to 3. Discard the resulting precipitate and take the supernatant. Wash the precipitate with water and make it white with a small amount of alcohol. Combine the supernatants washed with water, add another 20cc of water,
Add 10 cc of isobutanol and extract at pH 3.

Discard the colored water layer.

Then, wash the reddish-brown isobutanol layer twice with water,
Dry under a stream of nitrogen. This is called MR-256, and 0
．． Approximately 0.07 g can be obtained from 1 g.

(b) Production of prostaglandin derivative ester 50m
Dissolve g of MR-256 in a mixed solvent consisting of 0.5 d of ethyldiisopropylamine, 0.1 d of chloroform, and 2 Id of inbutanol. 0.2 Jd of acetoxymethyl bromide was added thereto, and the mixture was stirred for 20 hours in a tightly capped flask. Then, it is evaporated and solidified in a vacuum, and benzene is added and dissolved. The undissolved residue, ie, ethyl diisopropylammonium bromide, was removed with a filter paper and evaporated to dryness again to obtain prostaglandin derivative ester M.
R-356 is obtained.

It is in turn extracted several times with diethyl ether.

Combine the extracts and evaporate to dryness overnight to obtain MR-356.
Obtain a refined product. Yield is 60-70%. MR-3
56 is insoluble in water but soluble in ethyl alcohol.

(C) Thin layer chromatography When MR-256 and MR-356 are developed using a thin layer of silica gel with a developing solution of 2 volumes of benzene and 1 volume of methyl alcohol, MR-256 stays at the origin, but MR-356
is expanded and shows an Rf value of 0.69. This is MR-256
This confirms that MR-356 is an acidic type and MR-356 is a hydrophobic ester.

(d) Toxicity MR-256 in 5 mice, FI in 5 other mice
R-356 was injected intraperitoneally at 70+wg/Kg each and no toxicity was observed.

Example 2 Protective effect against cardiac ischemia (a) Protective effect against free cardiomyocytes Free cardiomyocytes were prepared by Hohl et al.
hem, Bio-phys, 221; 197 20
5.1983).

60-70% of the cells have a rectangular rod-like (ROD-like) shape indicating that the free cardiomyocytes are intact.

When the free cardiomyocytes are placed under nitrogen flow for 1 hour, they become small and spherical, indicating that the cells are damaged.

However, as shown in Figure 1, pre-addition of MR-356 prevents rounding and increases the percentage of rod-shaped cells.

(c) Perfusion model of a free heart (Langendorff model) The effectiveness of this drug was investigated using a Langendorff model of a rat heart placed in a thermostatic chamber 1 as shown in FIG.

In this experiment, the reflux solution 3 (117mM Na
(/! .

6mMKCj2.2.5mM CaCfz, 1.2m
M MgClz, 2.4mM Phosphate, 24mM Na
HCO3,5n+M glucose, temperature 37 degrees, pH 7,
4) is refluxed with pump 4 at a rate of 15/d/min. this heart 2
electrically stimulated for 1 second at a frequency of 3 times.

This system can be active for several hours. This heart beat output is continuously recorded by attaching a small rubber balloon 5 inside the left atrium and connecting it to a pressure transducer 6.

To cause cardiac ischemia, the perfusion fluid 3 is stopped for 15 minutes. A chart showing left ventricular output recorded by the recorder 7 is shown in FIG. In the figure, t indicates the time when reflux is stopped.
indicates when it has restarted.

If no drug was added, as shown in Figure 3A,
After 15 minutes, this heart resumed perfusion, but it hardly beat.
At best, only 10-15% of the original pulsatile output is restored. However, just before the start of reperfusion (white arrow), the M
When R-356 is added, the heart changes as shown in Figure 3B.
About 75% recovery.

Therefore, it can be seen that this drug protects against cell damage caused by reperfusion when added after ischemia.

(C) Keeping the CPK level down The above Langendorff model and reflux liquid were used.

It is known that when the perfusion fluid is stopped and reperfused, cardiac myocytes are damaged, and therefore CPK activity appears in the perfusion fluid and increases depending on the degree of ischemia.

Figure 4A shows the recovery rate determined by stopping the perfusion fluid for 15 minutes to induce ischemia, and measuring the left ventricular output 15 minutes after the resumption of perfusion, and the results of measuring CPK activity in the perfusion fluid are shown in the same figure. Shown in B. In the figure, the broken line indicates the non-ischemic case. As shown in these figures, 4.5 μM MR-256 or 3
．． It can be seen that if 8 μM MR-356 is added just before reperfusion, the increase can be suppressed.

MR-356 is slightly more effective than MR-256.

Example 3 Protective weight against liver ischemia (liver failure) from 170
A 175 g rat is used.

Under enflucin anesthesia, the hepatic portal vein and hepatic artery are closed with clamps. Then, the patient is kept in an ischemic state for a period of 1 hour, 2 hours, 3 hours, etc. After 24 hours, blood was collected and serum glutamic acid-pyrupic acid-
Measure transaminase (SGPT level).

The results obtained are shown in FIG. As shown in the figure, it can be seen that 5GPT increases rapidly as the ischemic state increases from 2 hours to 3 hours. Therefore, the 5GPT increase is
It is thought to be related to cellular ischemic damage. The ischemia time was kept at 2 hours, MR-256 or MR-356 was injected intraperitoneally at various concentrations 30 minutes before ischemia, and 5GPT in the blood was measured 24 hours after ischemia. 256
The case of MR-356 is shown in FIG. 6A and FIG. 6B, respectively. In the figure, the broken line indicates the case without ischemia. As shown in the figure, various amounts of MR-
If 256 or MR-356 is added before ischemia, 5G
The increase in PT is significantly suppressed.

Example 4 Protection against renal ischemia Rats weighing approximately 300 g are used. Both kidneys are surgically exposed. Ischemia is induced by closing the left renal artery with a microclip for 70 minutes. Then, as the right kidney is removed, the left clip is removed to restart blood flow. The wound is then sutured and the rat is allowed to recover. meanwhile,
For a total of 3 hours, starting 30 minutes before ischemia, a total of 5
Continue to gently infuse mg/kg MR-356. The survival rate of the rats after 4 days is shown in FIG. As shown in the figure, rats that were not given the drug had a survival rate of only 17% after 4 days, whereas rats that were given the drug had a survival rate of 83%.

Example 5 Protection against cerebral ischemia (a) Surgical procedure Rat Middle developed by Chien
Cer-ebral Artery Occlusion
The on(MC^0) model was used.

3 ■/kg body weight of MR-356 or MR 30 minutes before ischemia.
Cerebral infarction is caused in the superficial layer of the right hemisphere of the brain for about 1 hour in the case where -256 is added and the case where neither of these is added. After 1 hour, blood flow is resumed and the animals are allowed to recover spontaneously.

(g)) After 3 days, the brain is removed and the wet weight of each of the left and right hemispheres is measured, and then, after drying in the open, the dry weight of each is measured. Then calculate the moisture content. The results obtained are shown in FIG.

As shown in the figure, the water content on the ischemic side (right hemisphere) increases. However, 3 mg/Kg of MR-35 30 minutes before ischemia
If 6 or MR-256 is injected, the increase in water content can be suppressed.

(C) Exercise capacity The decrease in exercise capacity of rats due to ischemia was tested by the ability to hang from the front legs, the ability to stay on a parallel bar, and the ability to stay on an inclined plate (55 degrees). The results are shown in Figure 9. Figure A shows the exercise capacity of rats subjected to ischemia after a 1-hour infarction in comparison with rats that were not subjected to ischemia, and Figure B shows the exercise capacity of rats subjected to ischemia. 0.3■/kg MR-30 minutes before infarction
356 or MR-256 injected rats. As shown on the right side of the same figure (A) (same as the left side of (B)), the motor ability of the rats that underwent infarction at -time was reduced by about half. However, 30 minutes before infarction, MR-3
In rats injected with 56 or MR-256, motor ability was almost recovered after 3 days.

(d) Memory ability test Next, a memory ability test was conducted. The following equipment was used for this test. A large room and a small room were connected, and the joints were connected by a small tunnel.The small room was kept dark and the large room was illuminated with light. The tunnel was made to be able to be opened and closed using a transparent plate. The floor of the small room was lined with metal conductors that applied electrical stimulation to the rats.

First, when rats were placed in a large room, they spent most of the time hiding in a small room. In this memory experiment, when rats entered a small room, the tunnel path was closed off with a transparent plate, and electrical stimulation was applied for 30 seconds to create a pain sensation in the rat's paws. - Rats who received a painful sensation in their legs almost never returned to the room.

However, even after receiving electrical stimulation, the rats that had suffered a cerebral infarction forgot their memories and entered a small room.

Therefore, the length of time a person spends in a small room indicates the extent to which they have forgotten their memories due to a cerebral infarction.

The measurement results are shown in FIG. Figure A shows before stimulation (plain).
After stimulation (hatched lines), rats with and without ischemia due to cerebral infarction are compared.
Figure 1 shows rats injected with g of MR-356 or MR-256. As shown in the figure, injecting MR-356 or MR-256 before cerebral infarction significantly protects against memory loss.

Example 6 Protective effect against spinal cord trauma A rat spinal cord trauma model developed by IAra thal 1 was used.

The periosteum of T-11 was removed to expose the spinal nerve. A plastic striking rod with a diameter of 2.8 mm was placed thereon. A 10 g weight was dropped onto it from a height of 5 cm to apply an impact. The weight and striking rod were immediately removed, the wound was sutured, and the rat was returned to its breeding box.

Rat recovery was determined using Tarlov's score. A score of 0 indicates complete paralysis, and a score of 5 indicates complete recovery.

The results obtained are shown in Figure 11, in the group to which no drug was added (circle), the score was 1.5 after 4 weeks, and the rats could barely move their legs, but were unable to stand up. However, rats administered 5 mg/kg of MR-356 30 minutes, 1 hour, and 6 hours after trauma (squares) and rats administered the same amount of MR-256 (
After 4 weeks, the patient had recovered to a score of 4 to 4.5, and although some paralysis remained, the patient had recovered to the extent that he was able to walk.

Example 7 Sickle Anemia The present inventor added red blood cells taken from sickle anemia patients to
It was demonstrated that a large number of irreversibly denatured red blood cells can be produced within 2 to 3 hours by repeating the process of bell-shaped and non-bell shaped.

Since the irreversible red blood cells have a higher specific gravity, they can be easily separated and quantified. Here, we used the percentage of irreversibly degenerated red blood cells to see if the drug could protect them. As shown in Table-1, 13μH MR-356
By adding , the denaturation was reduced to about half. 40μ
On the stand, it was almost completely inhibited. On the other hand, as shown in Table 2, MR-356 had a much weaker protective effect than MR-356.

Table-1 Red blood cells that were not denatured 52.3 93.1 100
Red blood cells that have irreversibly denatured 47.7 6.9 0Table-2 Red blood cells that have not denatured 55.7 64.6 74.
8 Red blood cells with irreversible denaturation 44.3 35.4 25
゜2 Example 8 Effect on malaria Mice were used as animals, and two types of malaria parasites that infect mice, namely P. Chaubaudi and P. Vinc.
kei was used. P. Chaubaudi exhibits behavior relatively similar to malaria symptoms in humans. Mice were infected by injecting 10b P. Cha-ubaudi-infected erythrocytes and infected at 5μ/k daily from day 6 to day 9'.
g/day (black triangle) or 20■/kg/day (black angle of view)
of MR-356 or 5■/kg/day (black circle) of MR-2
56 was orally administered, and the change in parasite infection rate was investigated compared to those not administered (autotriangle).

The results are shown in FIG. As shown in the figure, the infected red blood cell rate increased to about 30% after about 8 days, and then decreased. M.R.
-256 did not have a significant protective effect. For it,? 1R-356 significantly inhibited the infection rate. Then the 10′ infected by P. Vinckei
The survival rate and the increase in the percentage of parasite-infected red blood cells in mice injected with 3 red blood cells were determined. As shown in FIG. 13, the solid line represents the control, and the dashed line represents the mice injected intraperitoneally with MR-356 once a day.

P. Vfnckei is a very strong malaria in mice and kills all mice, so it is not very suitable for drug measurements. However, even in this example, MR-356
It had the effect of extending the mortality rate by one day or suppressing the degree of red blood cell infection. MR-256 had no total thermal effect.

Example 9 Prostaglandin A1, prostaglandin B, and prostaglandin E1, prostaglandin A
Prostaglandin derivative esters were produced in the same manner as in Example 1 using the dehydrogenates (keto-type compounds) of prostaglandin B1 and Prostaglandin B1 as starting materials.
When the protective effects shown in 8 were investigated, each protective effect was observed in each case. Furthermore, in Example 1 (b), a prostaglandin derivative ester was produced from prostaglandin E+ using acetoxyethyl bromide or ethyl bromoacetate instead of acetoxymethyl bromide; Approximately equivalent protection was shown for Examples 2-8.

〔Effect of the invention〕

The prostaglandin derivative ester of the present invention prevents cell destruction caused by calcium influx into cells. For example, it prevents cell necrosis that occurs during myocardial infarction, stroke, liver failure, acute renal failure, and spinal cord trauma, and prevents irreversible membrane damage caused by repeated bell-shaped red blood cells in patients with sickle anemia. Furthermore, since prostaglandin derivative esters prevent the proliferation of malaria parasites within red blood cells, they can be used to prevent or treat malaria.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing the relationship between the survival rate of rat cardiomyocytes that were not damaged by ischemia and the concentration of MR-356, and FIG. 2 is a diagram showing a perfusion model of a free heart. FIG. 3 is a diagram comparing the time course of the cardiac output of rats after restarting perfusion after ischemia in cases where MR-356 was administered and cases where MR-356 was not administered. Figure 4 shows the results of measuring the recovery rate of rat cardiac output and CPK activity in the perfused fluid after perfusion after ischemia, and Figure 5 shows the results of rat liver blood 24 hours after changing the ischemic time. 5
The results of measuring GPT activity are shown. Figure 6 is MR
-256 and Ml? -356 was added at varying concentrations before ischemia, and the 5GPT activity of rat liver blood after ischemia was measured. FIG. 7 is a graph in which the survival rate of rats was measured by subjecting their livers to ischemia, and a comparison was made between cases in which MR-356 was administered and cases in which MR-356 was not administered. Figure 8 shows changes in water content due to ischemia in the rat brain using MR-356 or M
This is a diagram comparing the case in which R-256 was administered and the case in which it was not administered. The broken line indicates the level in a normal brain. FIG. 3 is a diagram comparing cases where the drug was administered and cases where the drug was not administered. Figure 1θ shows the decrease in memory ability caused by cerebral ischemia in rats using MR-356 or MR-25.
Figure 11 shows the recovery of nerve cell damage caused by dorsal spinal cord trauma in rats with and without administration of l-356 or MR-256. FIG. 12th
FIGS. 13A and 13B are diagrams in which the protective effects of MR-356 and MR-256 against malaria were measured. In the figure, the bars above or below the data indicate standard deviation values.

Claims (3)

[Claims] (1) The proton of the carboxyl group of the acidic type polymer of prostaglandin E_1, prostaglandin A_1, or prostaglandin B_1 is an acetoxyalkyl group,
A prostaglandin derivative substituted with an alkoxycarbonyl methyl ester group or an alkyl group that has a protective effect on organ cell damage due to ischemia or oxygen deficiency, nerve cell damage due to trauma, and red blood cells of patients with sickle anemia. ester (2) Prostaglandin E_1, prostaglandin A_1, prostaglandin B_1, or a keto-type compound that is a dehydrogenated product of any of them is polymerized under alkaline conditions, and then the protons of the carboxyl groups of the polymer are The method for producing a prostaglandin derivative according to claim (1), characterized in that the prostaglandin derivative is substituted with an acetoxyalkyl group or an alkyl group. (3) Prevention of organ cell damage due to ischemia or oxygen deficiency, nerve cell damage due to spinal cord trauma, sickle anemia, or malaria, using the prostaglandin derivative described in claim (1). , therapeutic agent