JP2837922B2 - Fat emulsion containing polyprenoid compound - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a fat emulsion containing a polyprenoid compound. More specifically, it relates to a fat emulsion suitable for administration of a polyprenoid compound useful as a pharmaceutical.

[Conventional technology]

Polyprenoid compounds exhibit a wide variety of physiological activities, and are expected to be developed as pharmaceuticals due to useful physiological actions such as inhibition of cancer growth, inhibition of cancer metastasis, induction of differentiation, improvement of blood cell production ability, and improvement of liver function. However, polyprenoid compounds generally have extremely low solubility in solvents that can be used as pharmaceuticals, and the development of a formulation method that enables effective administration has been an important issue.

Conventionally, a method of injecting a polyprenoid compound and its near-green compound as a suspension into experimental animals, and a method of dissolving them in fats and oils for oral administration [Jpn.
n), 77, pp. 351-355 (1986)], and a method of administering a drug in a liposome (JP-A-62-39521).

[Problems to be solved by the invention]

The method of administering a polyprenoid compound as a suspension has the disadvantage of diminishing the pharmacological effect of the compound, and is not suitable for intravenous administration. It is difficult to apply for administration. Furthermore, when a polyprenoid compound is used as a liposome preparation, the preparation has a drawback that its storage stability is poor and its application as a drug is limited. Therefore, there has been a strong demand for the development of a method that can safely administer a polyprenoid compound to a human body by a wide range of administration routes including intravenous administration and that has good storage stability.

[Means for solving the problem]

DISCLOSURE OF THE INVENTION The present inventors have formulated a pharmaceutical composition (eg, a cancer growth inhibitor, a cancer metastasis inhibitor, a cell differentiation inducer) that can apply a polyprenoid compound to a wide range of administration routes such as oral administration, subcutaneous administration, and intravenous administration. As a result of intensive studies on the preparation method, a compound containing a polyprenoid compound having good storage stability was successfully produced by using the compound as a fat emulsion having a small particle size, thereby completing the present invention.

That is, the present invention provides the following general formula: Is trans isoprene unit, Is a cis isoprene unit, Represents a dihydroisoprene unit. l is an integer of 2 to 8,
m represents 0 or an integer of 5 to 18, n represents 0 or 1, l
+ M is in the range of 8 to 20. X is a hydrogen atom or a formula -PO
₃ R (R represents a hydrogen atom or a cation such as sodium, potassium, calcium, and ammonium) means a group represented by R. And a fat emulsion obtained by mixing an oil component, an emulsifier, and water.

Specific examples of the above-mentioned polyprenoid compound include solanesol-diammonium phosphate (compound A) α-Dihydrodecaprenol (Compound B) α-Dihydrodecaprenol-diammonium phosphate (Compound C) Mixture of polyprenol having 45 to 60 carbon atoms (Compound D) Mixture of α-dihydropolyprenol having 50 to 60 carbon atoms (compound E) Mixture of dolichol-diammonium phosphate having 75 to 110 carbon atoms (compound F) α-Dihydrodecaprenyl-phosphate (Compound G) α-dihydrodecaprenyl-phosphate disodium salt (Compound H) And the like.

Compounds A, B and C were prepared as described in Jpn. J, Cancer Res.
ann), 76, 760-770, August, 1985.

The toxicity of these compounds was very low, for example, when administered intraperitoneally to mice (ICR male), only acute toxicity with LD ₅₀ > 1 g / kg.

As the oil component in the fat emulsion of the present invention, all pharmaceutically acceptable fats and oils usually used in the preparation of fat emulsions in the pharmaceutical technical field can be used, and specific examples of such fats and oils are as follows. , Soybean oil, cottonseed oil,
Vegetable oils such as rapeseed oil, safflower oil, sesame oil, rice bran oil, corn germ oil, sunflower oil, herring oil, sardine oil,
Examples include fish oil such as squid oil, oil obtained by partial hydrogenation of vegetable oil or fish oil, or oil obtained by transesterification.

Further, it is naturally produced or naturally produced is subjected to a fractionation treatment (eg, solvent fractionation, dewaxing treatment, etc.) and / or
Alternatively, a triglyceride of a medium-chain fatty acid having a carbon number of 8 to 12, which is generally called MCT and obtained by a transesterification treatment, for example, a liquid oil such as palm oil or fractionated palm oil can also be used as the oil component.

Furthermore, one of di- or monoglycerides of fatty acids having 6 to 18 carbon atoms or a mixture of two or more of these di- or monoglycerides can be used as the oil component.

As the emulsifier in the fat emulsion of the present invention, it is possible to use one or a mixture of two or more pharmaceutically acceptable phospholipids and nonionic surfactants. For example, egg yolk lecithin, soybean phospholipids such as soybean lecithin, hydrogenated products thereof, phosphatidylcholine, phosphatidylethanolamine and the like. Examples of the nonionic surfactant include high molecular weight surfactants having a molecular weight of 800 to 20,000, such as polyoxyethylene-polyoxypropylene copolymer, polyoxyethylene alkyl ether, polyoxyethylene alkylaryl ether, and hydrogenated castor oil poly. Oxyethylene derivatives and the like can be mentioned.

The fat emulsion of the present invention comprises a polyprenoid compound, an oil component, and an emulsifier, and is prepared by mixing water with the emulsifier, as described above. , An emulsifying stabilizer for improving the stability of the emulsifier, and a tonicity adjusting agent for adjusting the osmotic pressure.

Specific examples of such emulsifying aids include fatty acids having 6 to 30 carbon atoms, salts of these fatty acids or monoglycerides, such as caproic acid, capric acid, caprylic acid, lauric acid, myristic acid, palmitic acid, stearic acid, behenic acid,
Palmito oleic acid, oleic acid, linoleic acid, arachidonic acid, eicosapentaenoic acid, docosahexaenoic acid,
And salts of these carboxylic acids, such as the sodium salt,
Potassium salts, calcium salts, and monoglycerides of these carboxylic acids are included.

Specific examples of the emulsion stabilizer include cholesterol, tocopherol, albumin, fatty acid amide derivatives, polysaccharides, and fatty acid ester derivatives of polysaccharides.

Glycerin, dextran,
Albumin and the like can be used.

The fat emulsion of the present invention contains 0.001 polyprenoid compound.
-10% (all the following ratios are indicated by w / v%) preferably 0.001-1%, oil component 1-50%, preferably 1-2
0%, 0.1 to 10%, preferably 0.5 to 5%, emulsifier, and may contain an emulsifier in an amount of 0.3% or less and an emulsifier in an amount of 0.5% or less. . Then, water is added to the above-mentioned components to prepare an amount of 100%. At this time, an isotonic agent is added as needed to adjust the osmotic pressure.

The fat emulsion of the present invention contains a predetermined amount of a polyprenoid compound, an oil component, an emulsifier, and if necessary, an emulsifier,
The emulsion stabilizer and the isotonic agent are mixed, heated to a temperature of 30 to 90 ° C., a predetermined amount of water is added thereto, and the mixture is mixed with a conventional homogenizer or homomixer, for example, a pressurized injection homogenizer, ultrasonic wave. It is obtained by homogenizing using a homogenizer or a high-speed rotary mixer to obtain a coarse emulsion, adding water as required, and homogenizing with the above homogenizer.

The fat emulsion of the present invention may be added with an emulsion stabilizer or a tonicity agent after the formation of the fat emulsion depending on the production convenience, and in some cases, before or after the formation of the fat emulsion, other additives such as a coloring agent, Antioxidants, flavors, sweeteners and the like can be added.

The fat emulsion containing the polyprenoid compound of the present invention is an emulsion composed of fine particles having an average particle size of 300 nm or less, and has extremely good storage stability. When this fat emulsion is added to a culture system of cancer cells or when administered to an experimental animal, the physiological action of the polyprenoid compound can be effectively exhibited. This fat emulsion can be used, for example, as an injection for intravenous, intramuscular, subcutaneous administration and the like, and can also be used as an oral administration agent after encapsulation.

When the fat emulsion of the present invention is used as an injection, 1
10 μg / kg to 10 mg / kg as polyprenoid compound per day
Used at a dosage of In addition, when used as an oral administration agent, 0.5 mg / kg to 100 m / day as a polyprenoid compound
Used at a dose of g / kg.

〔Example〕

 Next, the present invention will be described in detail with reference to examples.

Examples 1 to 5 Each of fat emulsions containing compounds A, B, C, G or H was prepared. 50 g of soybean oil, 6 g of soybean lecithin, 12.5 g of glycerin and 2.5 g of compound A, B, C, G or H were mixed and heated to 50 to 60 ° C. for dissolution. 250 ml of distilled water was added thereto and homogenized with a mixer to obtain a crude emulsion. Distilled water was added to the crude emulsion to make the total volume 500 ml, and the mixture was placed in a liquid tank of a Manton-Gaulin type high-pressure emulsifier and circulated to produce a homogeneous fat emulsion. Each of the fat emulsions containing Compounds A, B, C, G or H thus produced had an average particle diameter of 200 nm or less. When these fat emulsions were stored at about 4 ° C. for 3 months, the average particle size of each fat emulsion was maintained at 200 nm or less.

Examples 6, 7 Each of fat emulsions containing compound C or G was prepared. 20 g of soybean oil, 2.4 g of egg yolk lecithin and compound C or G
0.8 g was mixed and heated to 50-60 ° C. to dissolve. to this
100 ml of distilled water was added and homogenized with a mixer to obtain a crude emulsion. Distilled water was added to the crude emulsion to make the total volume 200 ml, and the mixture was placed in a liquid tank of a Manton-Gaulin type high-pressure emulsifier and circulated to produce a homogeneous fat emulsion. The fat emulsion containing compound C or G thus prepared had an average particle size of 200 nm or less. About 4 of these fat emulsions
When stored at ℃ for 3 months, the average particle size is 200nm
It was kept below.

Example 8 A fat emulsion containing Compound F was prepared. Soybean oil 20
g, egg yolk lecithin 2.4 g, sodium palmitate 0.09 g,
5 g of glycerin and 0.5 g of compound F were mixed and heated to 50 to 60 ° C. to dissolve. 100 ml of distilled water was added thereto, and the mixture was homogenized with a mixer to obtain a crude emulsion. Distilled water was added to the crude emulsion to make the total volume 200 ml, and the mixture was placed in a liquid tank of a Manton-Gaulin type high-pressure emulsifier and circulated to produce a homogeneous fat emulsion. The fat emulsion containing compound F thus prepared had an average particle size of 300 nm or less. When this fat emulsion is stored at about 4 ° C. for 3 months, the average particle size is 300 n.
m or less.

Example 9 Using the fat emulsion containing compound C prepared in Example 3, the differentiation inducing effect on human leukemia cells HL-60 was measured using nitroblue tetrazolium (NBT) reducing activity as an index. Prior to the measurement, a fat emulsion containing Compound C was mixed with a control fat emulsion (a fat emulsion containing no polyprenoid compound prepared in the same manner as in Example 3 without adding Compound C) at an appropriate ratio, and a final concentration of 0.5 to 5 mg. A fat emulsion containing / ml of compound C was obtained.

The HL-60 cells were suspended at a density of 2 × 10 ⁵ cells / ml in RPMI 1640 medium containing 10% fetal bovine serum, and 1 ml of the suspension was plated in each well of a 24-well plastic plate. Immediately, a control fat emulsion or a fat emulsion containing compound C was added so that the final concentration of compound C in the medium was 0 to 50 μg / ml.
Plates were incubated at 37 ° C. for 4 days in a CO ₂ incubator. After incubation, centrifugation the cells were collected, at a density of 10 ⁶ cells / ml, containing 10% fetal bovine serum, 0.1% NBT and 100 ng / ml of phorbol-12-myristate-13-acetate
Suspended in RPMI 1640 medium. The cell suspension is added at 37 ° C for 20
After incubating for a minute, a portion was placed on a slide glass and fixed with methanol. The number of cells stained dark blue by NBT reduction was counted, and the percentage of the total cell number was calculated.

As is clear from the results shown in Table 1, in HL-60 cells treated with the compound C-containing fat emulsion, the percentage of NBT-reducing cells increased according to the concentration of compound C in the medium. That is, the fat emulsion containing compound C was cultured HL-
It showed a significant differentiation-inducing effect on 60 cells.

Example 10 The cancer metastasis inhibitory effect was measured using the fat emulsion containing Compound A, B or C prepared in Examples 1 to 3. Prior to the measurement, first, a fat emulsion containing Compound A, B or C was added to a control fat emulsion (a fat emulsion containing no polyprenoid compound prepared in the same manner as in Example 1 without adding any of Compounds A, B and C). To obtain a fat emulsion containing the compound A, B or C at a final concentration of 0.03 to 0.2 mg / ml.

At the time of measurement, 4 × 10 ⁵ B16F10 melanoma cells were transplanted into the left hind footpad of C57BL / 6 mice (male, 6 weeks old). A control fat emulsion or a fat emulsion containing compounds A, B or C at a final concentration of 0.03-0.2 mg / ml was administered into the tail vein nine times every third day from day 1 to day 25 after transplantation. . On day 27 after transplantation, the left hind limb was cut, and on day 41, the mouse was dissected to determine the number of metastatic nodules formed in the lung. Average number of pulmonary metastatic nodules formed in mice treated with control fat emulsion (N ₀ )
Of the number of lung metastasis nodules formed in mice to which a fat emulsion containing the compound A, B or C was administered (N)
The lung metastasis inhibition rate was calculated from the following equation.

Lung metastasis inhibition rate (%) = (1−N / N ₀ ) × 100 The number of animals used in each experimental group was 10 in each case.

As is apparent from the results shown in Table 2, 0.3 to 2.0 mg / k
Lung metastases were significantly suppressed in mice administered with a fat emulsion containing g of Compound C or 0.8 mg / kg of Compound A or B as compared with mice administered with a control fat emulsion.

〔The invention's effect〕

As shown in Examples 1 to 8, the fat emulsion containing the polyprenoid compound of the present invention is an emulsion composed of fine particles having an average particle diameter of 300 nm or less, and can be stored at about 4 ° C. for 3 months. The fine particles are stably maintained. When this fat emulsion is administered to an experimental animal, the physiological action of the polyprenoid compound is effectively exhibited. Example 9
Showed that when a fat emulsion containing polyprenoid compound C was added to a culture system of human leukemia cells, differentiation of the cells was induced. Example 10 further showed that when a fat emulsion containing the polyprenoid compound A, B or C was administered intravenously to mice, a remarkable effect of suppressing cancer metastasis was exhibited.

From the above facts, it can be seen that the fat emulsion containing the polyprenoid compound of the present invention is a formulation extremely suitable for use as a pharmaceutical.

Comparative Example 1 A suspension of Compound A was prepared. 1 ml of dimethyl sulfoxide was added to 200 mg of compound A, and the mixture was homogenized as much as possible using an agate mortar. Further, physiological saline was added to make the total volume 100 ml, and a suspension of compound A was prepared. The suspension thus prepared consisted of inhomogeneous particles when viewed under an optical microscope. Precipitation of the particles occurred when the suspension was allowed to stand at 4 ° C. for about 1 hour.

Example 11 Using the fat emulsion containing compound A prepared in Example 1 and the suspension of compound A prepared in Comparative Example 1, the effect of suppressing cancer metastasis was compared. Prior to the measurement, the fat emulsion containing Compound A was mixed with a control fat emulsion at an appropriate ratio to obtain a fat emulsion containing Compound A at a final concentration of 0.25-1 mg / ml. Further, a suspension of compound A and a physiological saline solution containing 1% dimethyl sulfoxide are mixed at an appropriate ratio to obtain a final concentration.
A suspension of Compound A at 0.25-1 mg / ml was obtained.

At the time of measurement, cancer cells were implanted into mice in the same manner as described in Example 6. Control fat emulsion or final concentration 0.25 ~
Fat emulsion containing 1 mg / ml Compound A (Experiment I) or saline containing 1% dimethylsulfoxide or suspension of Compound A at a final concentration of 0.25-1 mg / ml (Experiment I)
II) was administered intraperitoneally 14 times every other day from day 1 to day 27 after transplantation. On day 28 after transplantation, the left hind limb was cut, and on day 42 the mouse was dissected to determine the number of metastatic nodules formed in the lung. In Experiment I, the rate of inhibition of lung metastasis was calculated in the same manner as described in Example 6. In Experiment II, 1
% Dimethylsulfoxide saline lung metastatic nodules average number of formed in mice administered containing de and (M _0), Compound A suspension pulmonary metastatic nodules number of formed in mice treated with the The lung metastasis inhibition rate was calculated from the average value (M) according to the following equation.

Lung metastasis suppression rate (%) = (1−M / M ₀ ) × 100 The number of animals used in each experimental group was 10 in each case.

As is evident from the results shown in Table 3, the mice administered with the fat emulsion containing Compound A showed a greater effect than those administered as a suspension.

────────────────────────────────────────────────── ─── Continued on the front page (58) Fields surveyed (Int.Cl. ⁶ , DB name) A61K 31/045 A61K 31/66 A61K 31/765 A61K 31/80 A61K 9/107 CA (STN) REGISTRY (STN ) WPIDS (STN)

Claims (1)

(57) [Claims] 1. The following general formula: Cis isoprene unit, Represents a dihydroisoprene unit. l is an integer of 2 to 8,
m represents 0 or an integer of 5 to 18, and n represents 0 or 1. l
+ M is in the range of 8 to 20. X is a hydrogen atom or a formula -PO
₃ R (R represents a hydrogen atom or a cation such as sodium, potassium, calcium, and ammonium) means a group represented by R. A fat emulsion obtained by mixing the polyprenoid compound represented by the formula (1), an oil component, an emulsifier and water.