CA1051781A - Pharmaceutical composition containing an ascochlorin derivative - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

A pharmaceutical composition for prevention and treatment of arteriosclerosis of mammals including human beings and poultry which comprises an ascochlorin derivative represented by the formula

Description

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'I'h LS irlventiorl lelates to a ~ a-:ma(~(~u~ r~osi--tion Lor pLever~tion and treatnlent of art,-riosclel-osis o[
mammals including human beings and poul-try and a mctho(~ ,'or using such a composition, particularly, it relates to a pharma-ceutical composition for prevention and treatment of arterio-sclerosis of mammals including human beings and poul-try containing an ascochlorin derivative as an effective inc~redient, and also to a method for using said composition.
It is known that, among various causes of dea-th, the proportion of deaths in human beings due to diseases caused by arteriosclerosis is high. Although the cause of arteriosclerosis is not fully unders-tood, it is generally supposed that hyperlipemia caused by excessive intake of lipid and~or hereditary factors accelerates the arterio deposition of lipid and consequently, ateromatosis is revealed on -the wall of arteries.
Further, it is also known that direct cause of death in arteriosclerosis patients is mainly due to the formation in arteries of white thrombus comprising blood platelets and fibrin.
One object of this invention is to provide a pharma-ceutical composition for prevention and treatment of ar-terio~
sclerosis of mammals including human beings and poultry having hypolipidemic activity together wit'n blood platelet an-ti-aggregation activity.
Another object of this invention is to provide a method for preventing and treating arteriosclerosis of mammals including human beings and poultry by the use of said pharma-ceutical composition.
Other objects will be self-evident from the descript-ion hereinbelow.
Ascochlorin is an antibiotic substance which is 7~L

produccd I-lom a ~i1amerlto~ls fun~us such dS ~ V~('ldC
Libert (depGsited in the culture collectiorl of the ~c~ency of Industrial Science and Technology, Fermentation Reseavch Institute, Japan, under deposit number FERM-P No. 129) and has the following formula:

OH

Cl~3 1 ` 1 The antibiotic is known to have anti-viral, anti-tumor, anti~bacterial and sedative activities and, further, cholesterol-lowering activity. The anti-viral and anti~tumor activities of the antibiotic are known from Japanese Patent No. 585,252 and anti-bacterial, sedative and cholesterol-lowering activities are disclosed in U.S. Patent 3,546,073.
However, this antibiotic is inadequate in its hypolipidemic activity, it is also strong in toxicity, its toxicity is especially noticeable when continuously administered.
The inventors of this invention have tested 4-0-methylascochlorin which is a known compound for its hypolipi-demic activity and toxicity and finally found it suitable for the purpose of this invention.
The ascochlorin derivative has excellen-t hypolipidemic activity, including cholesterol~lowering activity and blood platelet anti~aggregation activity and, also, it is low in toxicity. The inventors then continued their experiments on the derivative to complete the present invention.
Figure l is a graph showing the increase in body weight of rats to which 4-0-methylascochlorin or ascochlorin was administered every day for l0 days. In E'igure l, Curve l ..

corrc~sporlcls to the (~ontrol grou~ (no m~(lioali~,r,); Curv( 2 corresponds to -the group which received 4-0-rn(~thylascochlorin ir a dose of .10 mg/head per day, Curve 3 corresponds to th~? group which received 4-0-me-thyl.ascochlorin in a dose of 20 m~J/kg per day, and Curve 4 corresponds to the group which receiv(~d ascochl.orin in a dose of 10 mg/kg per day.
Figure 2 is a graph shc)wing the increase in body weight of rats receiving 4-0-methylascochlorin, ascochlorin or ethyl-~-(p-chlorophenoxy) isobutylate every day for 30 days.
In the F`igure, Curve 1 corresponds to the control group (no medication), Curves 2, 3, 4 and 5 correspond to -the groups received 4-0-methylascochlorin in a dose of each of 250, 500, 1,000, 2,000 mg/kg/day, respectively, Curve 6 corresponds to the group received ethyl-~-(p-chlorophenoxy) isobutylate in a dose of 500 mg/kg/day, Curve 7 corresponds to the group received : ascochlorin in a dose of 50 mg/kg/day, and symbol * shows a rat died.
One embodiment of this invention is a pharmaceu-tical composition for prevention and -treatment of arteriosclerosis of mammals including human beings and poultry which cornprises, as an effective ingredient, an ascochlorin derivative represen-t-ed by the formula OH

OH

' in an amount sufficient to exhibit desired effect.

The derivative represen-ted by the general formula - disclosed above includes 4-0-methylascochlorin being a known compound. This compound may be prepared from ascochlorin by, .

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for exalllpl(~, sel~ctively met]-lyLatir-~g .Illy!~ o~ l raclic~ f 4-positiorl of the compouncl in tlle preser)ce of a basic con(lel.s-ing agent.
Because the ascochlorin derivative is almost in-soluble in water, it may be suspended in an aqueous solution of gum arabic, sucrose, etc., to form a suspension and moreovcr it may be formulated by a conven-tional way with a conventional, pharmaceutically accep-table carrier such as lac-tose, starch, crystalline cellulose, kaolin, calcium carbonate, talc and the like to form tablets, granules or powder. Further, -the result-ing granules or powder may be s-tuffed in capsules to form capsules.
Although the amount of the ascochlorin derivative in the composition depends on the kind of animal -to which it is to be administered or the form of the composition, it is an amount such that -the use of the composition realizes the administration of the derivative in an amount of usually 0.05-500, preferably 0.5-50 mg/kg body weight per day, because the asocochlorin derivative reveals the effect by continuous oral administration in a dose of 0.05 mg/kg body weight per day for several days. Other one or more medicines known to be effect-ive in treating arteriosclerosis, for example, ethyl-~-(p-chlorophenoxy) isobutylate, dextran sulfate, nico-tinic acid derivatives, dipyridamol and the like, may also be incorporated in the composition.
Another embodiment of this invention is a method for preventing and treating arteriosclerosis of mammals includ-ing human beings and poultry which comprises adminis-tering the pharmaceutical composition containing the asocochlorin derivative of this invention to an animalO
- In order to prevent and -treat arteriosclerosis by the use of the composition prepared according to the present 7~
i.nvc~nti.on, [llamrlldls dl~d poultry are orally a~ ed ~ith the pharmaceuti~al cornposition preparcd accc)~ (J to th~ Lesent invention once or more a day. E,specially, for humarl boi.n~Js, it is preferable to administer -the composi.tion -three times per day, after every meal. For animals o-ther than human beings, the composition may be administered alone or by mixing i-t wi-th feed. As stated above, the dose of the composition depends on the kind of animals to which it is to be adminis-tered and on the form of the composition. E-Iowever, the dosc ranges general-ly 0.05-500, preferably 0.5-50 mg/kg body weight per day based on the ascochlorin derivative. For human beings, although the study has not been comple-ted, a sa-tisfactory effect was obtained by the oral administration of a 50 mg dose after every meal when 4-0-methylascochlorin was used.
The mammals and poultry, arteriosclerosis of which can be prevented and treated by the administration of the com-position of this invention include, for example, human beings, monkeys, bovines, dogs, cats, pigs and chickens.
This invention is further explained by specific Examples and Experiments hereinbelow. However, it should be . understood that the Examples and Experiments do not limit this invention.
Example 1 Serum cholesterol lowerin~ activi-ty . The cholesterol lowering activi-ty of the ascochlorin derivatives of this invention was observed by the use of Wister-Imamich strain male rats weighing 230 ~ 30 g which had been bred with t.he use of high-fat feed comprising 1000 g of powdery chow CLEA CE-2* (sold by ~IH0~ CLEA I~ABUSHII~ IS~IA) admixed with 10 g of cholesterol, 95 g of hydrogena-ted coconu-t oil, 2 g of cholic acid, 150 g of milk casein and 671 g of sucrose.

* trademark The rc3ts were dividecl into grollps of 5 n~ bei-s e.lch, and orally administered with d ~-0-methylascochlorin susper~sion in gum arabic aqueous solution in a dose oL 75 mg/k(3 boc~y weight.
The rats were kep-t from eating food, and 6 hours aftc~r the ad-ministra-tion, the blood of each ra-t was sampled from its heart.
The total amount of cholesterol in serum was determine~ by a modified Zurkowski method (Clinical Chemistry, 10, 451, 1964) with the use of a colorimeter for blood analysis (sold by liYOTO
DAIICHI I~AGAI~U) . The results are shown in Table I be]ow.
Table I

Cholesterol ln serum Dro~
Test Compound (mq/d~) Ratio . _ _ 4-0-methylascochlorin 76.6 -22.8 Control (ascochlorin) 101.3 -~2.0 Control (ethyl-~-(p-chloro-phenoxy) isobutylate) 103.8 +~.5 Control (gum arabic aqueous solution only) 99.3 Example 2 Aqqreqation inh_b1tion act1vity of blood platelet In this Example Wister~Imamich strain male rats weighing 230 ~ 30 g which had been bred with the use of a commercial chow, CLEA CE-2* were used.
The rats were divided into groups of 5 members each, and orally administered with a 4-0-me-thylascochlorin suspension in 2% gum arabic aqueous solution in a dose of 20 mg/kg body weight. Six hours after the administration, the blood of each rat was sampled from the heart and after the addition of citric acid, the blood sample was centrifuged at 1,500 r.p.m. for 10 minutes to obtain platelet rich plasma. The an-ti-aggregation effect when the concentration of adenosine--5'-diphosphate in - the platelet rich plasma is ul-titnately 10 M is shown in Table II.

* trademark ~ s positive controls, .~lscochlori~, e~llyl~
chloLophenoxy) isobutylate and acetyl salicyli( acid w(ue used. 'I'he agc~regation inhibition ratio was ,-~val~ated ir~
percent by comparing the increase of -transmi.ssion rate caused by the addition of adeno~in-5'-diphosphate to platele-t rich plasma of the con-trol group (no medication) wi-th -that caused on the groups administerecl with the ascochlorin derivative which is calculated for 100.
Table II
Aggregation Inhibi tion Test Compound Ratio (%) 4-0-methylascochlorin 70.5 Control (ascochlorin) 66.8 Control (ethyl-~-(p-chlorophenoxy) isobutylate) 0 Control (acetyl salicylic acid) 73.1 - Example_3 .~
Toxicity Test a) Acute Toxicity Acute toxicity of the ascochlorin deriva-tive was observed by the use of Wister-Imamichi strain male rats weigh-ing 230 + 30 g and ddY strain male and female mice ~5 weeks old) weighing 20 + 3 g which were di.vided into groups of 10 members each. Japanese na-tive male rabbits weighing 2 kg +
300 g which were divided into groups of 5 members each were also used. The test animals were orally or intraperi-toneally admlnistered with 4-0-methylascochlorin as a suspension in 5%
gum arabic aqueous solution, and the toxicity was comparecl with that of control ln which ascochlorin was used. The results are shown in Table III below.

Ta_le l-II
Test Administration LD50 Tes-t Com~ound Animal Sex Route_ _ _ (mq/~
~scochlorin Mouse Male Oral >5,000 '~ " Female " >5,000 " " Male i.p.* 102 " Female 68 " Rat Male Oral >7,000 l.p.(156-426) 4-0-methyl-ascochlorin Mouse Male Oral >7,300 " " Female " >7,300 " " Male i.p. 5,000 '~ " Female " 5,500 " Rat Male Oral >10,000 " " i.p. 3,100 " Rabbit Male Oral >2,000 * i.p.: intraperitoneal b) Toxicity by continuous administration for 10 days Wister-Imamichi strain male rats weighing 230 ~ 30 g were divided into 4 groups of 10 mernbers each, 3 groups of which were orally administered with 4-0-methylascochlorin in a dose of : 10 mg/head, 4-0 methylascochlorin in 20 mg/head and ascochlorin in 10 mg/head, respectively, as a suspension in 2% gum arabic aqueous solution once a day for ten days. The rernaining group - was administered with 2% gum arabic aqueous solution, the dose and way of administration being identical to those disclosed above. The increase in body weight of the rats of each group - was observed every day during the adrninistra-tion. The resul-ts are shown in the graph of Figure 1.
. Figure 1 clearly shows that the group to which ;~ - 8 -.'' `' `' ~

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only sli~htly in bocly we~i~ht, while the c~roup receivin~J 4-0-methylascochlorin had a normal increase in body wei~}lt almos-t comparable to that of the con-trol group receivir~g only gum arabic aqueous solution.
c) Toxicity by continuous administration for 30 days Wister strain male rats weighirlg 150 ~ 20 g were divided into groups of 10 members each, and each group was orally administered with 4-0-methylascochlorin as a suspension in 2% gum arabic aqueous solution in a predeterminec~ dose once a day for 30 days, and the increase in body weight thereof was observed each day that the administration continued- ~s con-trols, - ascochlorin (50 mg~kg/day) and ethyl-~-(p-chlorophenoxy) isobutylate (500 mg/kg/day) were used. The results are shown in the graph in Figure 2.
The groups receiving 4-0-methylascochlorin in a dose of 250 and 590 mg/kg exhibited normal increase in body weight comparative to the untreated group. In contras-t, little or no increase in body weight was observed in the group receiving ascochlorin in a dose of 50 mg/kg, and moreover they lost weight.
The three rats of the group died during the test period.
Example 4 HYPO1 ipidemic activity of_4-0 methylasco_ _orin in blood and in orqans Wister strain rats weighing 150 + 20 g were divided into groups of 10 members each and bred with high-fat feed, the same as that used in Example 1.
The rats were orally administered ascochlorin, 4-0-methylascochlorin or ethyl-~-(p-chlorophenoxy) isobu-tylate in a predetermined dose every morning for 30 days. Six hours after the last administration, liver and aorta of each rat were _ 9 _ 7~L

taken out and lipid level ir-l t:he organs Or each of the rats was dc-termirled. Lipi,d present i.n the li.ver and aorta was extracted with methanol-chloroforrn (1:1) by allowing it -to stand overnight and the extrac-t WclS assayed as to lipid level. The results obtajned are shown in Tables IV and V
below.

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As shown :in Table~s IV and V, in those cases iri which 4-0-methylascochlorirl was administered in a close of 9 mg/kg or more, the levels of all types of lipicls were extremely lowered and besides, the amount of lipids in liver and cholesterol in aorta was also remarkably reduced. In contrast, such effec-ts were not observed in the groups receiving with ascochlGI-in or ethyl-~-(p-chlorophenoxy) isobu-tylate.
E~ample 5 Pharmaceutical preparation (a) Capsules To 100 g of pulverized 4-0-methylascochlorin were added 358 g of lactose and 2 g of magnesium s-tearate and they were thoroughly mixed, and hard gelatin capsules each weighing 65 mg were filled with 230 mg each of the mixture to form capsules.
(b) Powder To 50 g of pulverized 4-O~methylascochlorin were added 404 g of lactose, 45 g of crystalline cellulose and 1 g of magnesium stearate and they were thoroughly mixed to form powder.
(c) Tablet-I
To 100 g of pulverized 4-0-methylascochlorin were added 210 g of lactose, 72 g of microcrystalline cellulose, 14 g of corn starch and 4 g of magnesium stearate and they were thoroughly mixed; -then, with a table-t machine the mixture was formed into tablets each tablet being 8 mm in diame-ter and 200 mg in weight.
(d) Tablet-II
After being passed through a screen of 50 mesh (Tyler), 100 g of pulverized 4-0 methylascochlorin was mixed with 273 g of lactose and 20 g of calcium carboxymethyl cellulose, and then the mixture was ~neaded with a starch paste madc oL 4 g oL corn starch ~Inà watci. Thc r(~suL~:Ir,~J
mixtur(~ was g~lnl1~atc(1 by a granulating r~ }-lin(~ arld d i~d clnd -thc gl-anules Were passed through a scrcen of l4 mesh (~i~yl(-r).
After addition of and mixing with 3 g of magnesium stca-fatc, the mixturc was formed into tablets, each being 3 mm in dia-meter and 200 mg in weight.
(e) Suspension To 400 ml of solution of 200 g of sucrose in watcr were added lO g of crystalline celluLose and 0.75 g of sodium carboxymethyl cellulose to form d uniform suspension. Scparate-ly, 5 g of bulk of 4-0-methylascochlorin was pulverized in a ball mill together with 0.5 g of a fatty acid ester of sucrose and 20 ml of water. To the resulting powder was added the suspension followed by water to make the -total amoun-t 500 ml.
The suspension was stirred to form a uniform suspension.
This application is a division of Canadian Application No. 223,961, filed April 7, 1975.

Claims (4)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:-

1. A pharmaceutical composition for prevention and treatment of arteriosclerosis of mammals including human beings and poultry which comprises, as an effective ingredient, 4-0-methylascochlorin and a pharmaceutically acceptable carrier.

2. A pharmaceutical composition for prevention and treatment of arteriosclerosis of mammals including human beings and poultry as set forth in claim 1 wherein said composition is such that the use thereof realizes the administration of 4-0-methylascochlorin in an amount of 0.05-500 mg/kg body weight per day to exhibit the activity of an ascochlorin derivative.

3. A pharmaceutical composition for prevention and treatment of arteriosclerosis of mammals including human beings and poultry as set forth in claim 1 wherein said composition is such that the use thereof realized the administration of 4-0-methylascochlorin in an amount of 0.5-50 mg/kg body weight per day to exhibit the activity of an ascochlorin derivative.

4. A pharmaceutical composition for prevention and treatment of arteriosclerosis of mammals including human beings and poultry as set forth in claim 1 wherein the form of said composition is suspension, tablet, granule, powder or capsule.