NL8001041A - ANTI-HYPERCHOLESTEREMIC AGENT, MONACOLIN K, AND THEIR PREPARATION. - Google Patents

Description

803048 / vdy / ea i.

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Applicant; Sankyo Company £ td, Tokyo, Japan.

Short designation: Anti-hypercholesteremic agent, monacolin K, and its preparation.

The Applicant mentions as inventor: Akira Endo

The invention relates to a new compound with anti-hypercholesteremic activity, which has been given the name Monacolin E. Monacolin® can be obtained by cultivation of various microorganisms of the genus Monascus.

The invention therefore relates to a compound,

Monacolin Z, with formula 1 of the formula sheet.

The invention also relates to a method for preparing a Monacolin called anti-hypercholesteremic agent by cultivating a Monacolin-forming microorganism 10 of the genus Monascus in a culture medium.

The invention further relates to a pharmaceutical composition containing Monacolin® in combination with a pharmacologically acceptable carrier or diluent.

It is known that high cholesterol in blood 13 is one of the main causes of heart disease, such as myocardial infarction or arteriosclerosis. In connection with this, significant studies have been conducted to find physiologically acceptable substances capable of inhibiting cholesterol biosynthesis and thus lowering blood cholesterol. One such compound is ML · 236, which is described in British Patent Specification 1,433,423 and is obtained by cultivation of microorganisms of the genus Penxcium.

When studying fungi of the genus Monascus, it was found that, in particular the strain Monascus ruber 23 1003 (PERM 4822), produced an anti-hypercholesteremic agent with significantly greater activity than ML-236. This medicine was called Monacolin®.

All microorganisms of the genus Monascus capable of producing Monacolin can be used according to the method of the invention. Strains of Monascus ruber are particularly suitable, in particular Monasous ruber strain 1003 (PERM 4822).

800 1 0 41 - 2 -

Monascus ruber strain 1005 (FEfiM 4022) is a new isolated microorganism with the microbiological properties listed below.

The micro-organism has been isolated from food products produced in Thailand and on February 16, 1979 under no. 4822 at the fermentation Research Institute Agency of Industrial Science and Technology, Ministry of International Trade and Industry, Japan and under no. NRRL 12075 at the Agricultural Research Service, northern Regional Research Laboratory, ÏÏ.SA deposited.

10 1 · Growth * Re growth on a potato 1-glucose agar medium at 25 ° C is rapid and the diameter of the colony reaches a level of 6-6.5 cm0 Re colony 10 days after inoculation and there a relatively thin base layer of hyphae develops. The development of air-15 hyphae is small. Air hyphae are white and most of them frizz. Seventh cleistothecans are formed into the base layer of the hyphae and turn red-brown upon maturation. Both the surface and the back of the colony are brown to red-brown in color.

Re-growth on a Sabourand agar medium at 25 ° C is very fast and the colony diameter reaches 6-6.5 ca. 10 days after inoculation. The colony surface is very flat and the basal hyphae and air hyphae develop better than on a potato-glucose agar-agax medium. The number of oleistotheses is small. The surface of the colony is reddish-yellow to reddish-brown and the back is reddish-brown to dark brown in color.

Re-growth on hive flour agar at 25 ° C is slow and the colony diameter reaches 1.5-2 cm 10 days after inoculation. Re colony is flat. Both the development of air hyphae and the formation of cleistothecans is very minor. Both the surface and the back of the colony are dark red to reddish-brown in color.

Re-growth on an agar medium of Czapek at 25 ° C is very slow and the diameter of the colony reaches 1.6-1.8 cm · 55 _ Se growth rates on each of the above media 10 days after inoculation. at 10010 41-3 * 37 ° C are essentially equal to that at 25 ° C.

2 «Morphologache_igenschagpea

The cleistothecans are spherical and have a diameter of 30-60 microns. Their walls are thin and membrane-shaped, their stems: 5 have septal walls and each contains a hyphe with a diameter of 3.5-4 »5 microns and a length of 13. -80 microns · The ascus consists of 8 spores and is almost spherical and disappearing · The ascospores are colorless and ovoid or ellipsoidal, they have a size of 4-5 x 4-7 microns and their surfaces are smooth · The conidia are 10 colorless and spherical or pyriform, their size is 6-9 x 6-11 microns, their bases are truncated and their walls relatively thick and smooth · The conidia are joined together near their bases like a kind of meristem arthrospore. The conidiophor resembles a vegetative hyphe and is branched or unbranched, with the conidia formed on top 15. The mycelia are colorless and branched and have septal walls. Most of them have a diameter of 3-3 microns.

Based on the above properties, this microorganism was identified as a strain of Monascus ruber van Tieghem.

Microbiological properties of Monascus ruber are listed in the literature below:

Takada, Transactions of the Micological Society of Japan, 9, 123-130 (1969) [Materials for the Fungus Flora of Japan. (7) J and van Tieghem, Bull · Soc. Botan · France, 31, 227 (1884) · The 23 strain ascospore formation has been reported by Cole et al in Canadian Journal of

Botany, 46, 987 (1968), "Conidium Ontogeny in hyphomycetea. The imperfect state of Monascus rubber and its meristem arthrospores" ·

While the use of Monascus ruber strain 1003 is specifically explained below, it goes without saying that all strains of the genus, Monascus including varieties and mutants, capable of producing Monacolin, can be used in the method of the invention ·

Monacolin ™ can be formed by cultivation of the selected microorganism in a culture medium under aerobic conditions using the same methods as those well known 800 1 0 41 -4 -

For cultivating fungi and other mioro-organisms. For example, the Monacolin X-forming microorganism can first be grown on a suitable medina, after which the formed microorganisms are collected, seeded and grown on another culture medium to form 5 aing of the desired Monacolin X · The culture media used for the multiplication of the microorganism and for the formation of Monacolin X can be the same or different ·

Any known culture medium for growing fungi can be used, provided it contains, as is known, the necessary nutritional components, in particular an assimilable nitrogen source. Examples of suitable sources for assimilable carbon are glucose, maltose, dextrin, starch, lactose, sucrose and glycerine. Among these sources, glucose, glycerine and starch are particularly preferred for the preparation of Monacolin X. Examples of suitable sources of assimilable nitrogen are peptone, meat extract, yeast, yeast extract, soybean meal, peanut meal, corn steep liquor, rice bran and inorganic nitrogen sources. Of these nitrogen sources, peptone is particularly preferred

In the formation of Monacolin X, an inorganic salt and / or a metal salt can be added to the culture medium if necessary. For example, a small amount of a heavy metal can also be added if necessary.

The mioro-organism is preferably cultivated under control conditions using known cultivation methods, for example a 'solid culture', a shake culture or cultivation under aeration and stirring · The micro-organism grows in a wide temperature range, for example from 7-40 ° G, but in particular for the formation of Monacolin®, the preferred culture temperature is 20-30 ° C.

During the cultivation of the microorganism, the formation of Monacolin X can be monitored by taking samples of the culture medium and determining the physiological activity of Monacolin X in the culture medium by the tests described below. 0 The cultivation can then be continued until a significant accumulation of Monacolin 35 -X in the culture medium has been achieved, after which the Monacolin X isolated ex 800 1 0 41 i 5 5 can be recovered using any suitable combination of isolation methods, taking into account the physical and chemical properties. For example, one or more of the following isolation methods can be used: extraction of the liquid from the culture with a hydrophilic solvent, eg diethyl ether, ethyl acetate, chloroform or benzene, extraction of the organism with a hydrophilic solvent such as acetone or an alcohol, concentration, dissolving in a more polar solvent, for example acetone or an alcohol, removing impurities with a less polar solvent such as petroleum ether or hexane, gel filtration through a column or a material such as Sephadex (commercially available product from Pharmacia, Co Ltd., USA), absorption chromatography with active carbon or silica gel and the like. By using an appropriate combination of these methods, the desired Monacolin K can be isolated from the culture as a pure substance.

Tan Monacolin K the following properties were found. Properties 1. Color and shape: colorless crystals.

2. Melting point: 157-159 ° G (vein decomposition).

5 Elemental analysis: C 71.56 $, H 8.85 $, 0 19.59 $ 4. Molecular weight: 404 (by mass aspect).

5. Molecular Formula: C2 ^ 6. Ultraviolet Absorption Spectrum (Methanol): As shown in Figure 1 of the accompanying drawings with maxima at 252, 258 and 246 µm.

7 «infrared absorption spectrum (£ 3r):

As shown in Fig. 2 of the accompanying drawings.

8. Keraspin magnetic resonance spectrum (60 ΜΞζ proton): 50 As shown in Figure 5 of the accompanying drawings in deuterated chloroform using tetramethylsilane as internal standard · 9 * Magnetic core spin resonance spectrum (15q) i

As shown in Figure 4 of the accompanying drawings 35 in deuterated methanol.

80 0 1 0 41 - 6 - 10. Solubility: Soluble in lower alcohols, for example methanol, ethanol and propanol, acetone, chloroform, ethyl acetate and benzene.

Insoluble in volume ther and hexane.

11. Specific rotation: [cc] - + 507 »6 (e« 1, methanol).

5 12. Thin layer chromatography:

Bf 0.47 Lh ° «57 ^ 5 Kieselgel silage gel (Merck & Cc ·,

Ltd. ·), developed with a mixture of methylene chloride and acetone in a volume ratio of 4 * 1 »discernible as an ultraviolet radiation absorbing stain, 50 $ (v / v) sulfuric acid (a light red to red-brown color develops when heated) or with iodine] ·

The compound is neutral and insoluble in neutral or acidic aqueous media. The compound is converted to an acidic substance when treated with a base and can then be dissolved in water. This acidic substance can be extracted with ethyl acetate or chloroform at an acidic pH and changes back to Monacolin on evaporation of the solvent.

The physiological efficacy of Monacolin® can be quantitatively investigated and determined by the following in vivo test. Taste in ^ viyo with. rabbits.

In this test the ability of Monacolin® to lower the blood cholesterol level of rabbits is determined.

The animals used must have a weight of 2.5-5.0 kg. Immediately before the start of the test, blood is collected from the vein in an ear of each rabbit and the cholesterol level in the blood serum is determined by a conventional method. A predetermined amount of Monacolin® is then continuously administered orally for 1 to 5 days and the blood serum cholesterol level determined after administration. The activity of the Monacoli® or Monacolin®-containing culture medium can be quantitatively determined from the cholesterol values obtained before and after administration of Monacolin®.

The ability of Monacolin to lower blood and liver cholesterol levels has been demonstrated in several in vivo tests.

55 Reduction of blood cholesterol levels in rats.

80 0 1 0 41 - 7 - *

Animals used were rats of strain Vistar Imamichi with a body weight of about 300 g, Se experiments were performed on groups of 5 rats each. 400 mg / kg Triton WR-1339 (a commercial product known to increase blood cholesterol) was injected intravenously into each animal while simultaneously administering 10 mg / kg Monacolin £ intraperitoneally. 14 hours after intraperitoneal administration, the rats were sacrificed by bloodletting and blood was collected and cholesterol levels determined by conventional means. It was found here that blood cholesterol levels had decreased by 23.90% compared to a group of animals to which only Triton WR-1339 had been administered,

Lowering blood cholesterol levels in rabbits.

The animals used were rabbits with a body weight of 2.7 to 2.9 kg. Each rabbit was orally administered 1 mg / kg Monacolin® twice daily for 3 days (morning and evening) · Before administration and on the third and fifth days after administration, blood was collected from a vein in the ear and determined the cholesterol level in the blood serum. It was found that the cholesterol levels on the third and fifth days after the administration of Monacolin were 13 and 29% lower, respectively, than the cholesterol level before the administration of Monacolin E,

Almost the valuable inhibitory effect on the biosynthesis of cholesterol, Monacolin E has a very low toxicity.

23 For example, the acute oral toxicity (liS ^ Q) of Monacolin E in mice is 1 g / kg body weight or more,

Monacolin E can be administered orally or parenterally in the form of capsules, tablets, injection preparations or any other known form, although oral administration is usually preferred. The dose varies depending on the age and body weight of the patient and the severity of the condition, but generally the daily dose for an adult is from 0.3 to 30 mg, as a single dose or in two or three divided doses. . Due to the low toxicity of the compound, higher doses can be used if desired. »_ 800 1 0 41 _ 8 __ é

The invention will be further illustrated by the following non-limiting example

Example.

Know Monascua ruber 1005 on a liquid culture medium, 5 containing 656 (weight / volume) glucose, 2.5 $ (weight / volume) peptone, 0.5 $ (weight / volume) corn liquor and 0.5 $ (weight / volume ) ammonium chloride contains Hen cultivates for 10 days under aerobic conditions at a temperature of 28 ° C. Hen controls the obtained filtrate (5 liters) of the culture at a pH of 5 by adding 65 salt · 10 acid and then extracts with an equal volume of ethyl acetate. The solvent evaporates from the extract under reduced pressure and the residue obtained is dissolved in 100 ml of benzene. The insolubles are filtered off.

The filtrate was washed twice with 100 ml of a 5% (weight / volume) aqueous solution of sodium bicarbonate. 100 ml 0.25 aqueous sodium hydroxide solution is added to the washed filtrate and the mixture is stirred at room temperature. After the disappearance of Monacolin K from the benzene layer has been confirmed by thin layer chromatography, the aqueous layer is separated. The pH of the aqueous layer is then adjusted to 3 by addition of 62Γ hydrochloric acid and the resulting solution is extracted twice with 100 ml of ethyl acetate.

They evaporate the extract under reduced pressure to dryness, whereby 260 mg of an oil are obtained. They dissolve this oil in benzene and allow it to crystallize. They recrystallize in a 25 ml aqueous aces solution, using 87 mg of colorless needles of Honacolin S having the previously described properties obtains -Conclusions- @ 90 1 0 41

Claims (1)

-9 -> 1 * A compound useful as an anti-hypercholesteremic and anti-hyperlipemic agent, characterized in that it has formula 1 of the formula sheet »5 2« Process for the preparation of an anti-hypercholesteremic agent designated as Monacolin K, characterized in that a Monacolin K-forming microorganism of the genus Monasous cultivates in a suitable culture medium »3 # Working method according to claim 2, characterized in that the microorganism is a strain of Monasous ruber» 4 «Method according to claim 3 , characterized in that the strain Monasous ruber strain 1003 used is »5«, Method according to claims 2-4 », characterized in that the cultivation is carried out at a temperature of 7-40 ° C 15 6 · Method according to claim 5 The characteristic is that the cultivation is carried out at a temperature of 20-35 ° C <7 * Pharmaceutical preparation containing an active ingredient mixed with a pharmacologically acceptable carrier or diluent micelle, characterized in that , Which the active ingredient 20 consists of Monacolin K * 800 1 0 41