JP3040015B2 - Inclusion compound of forskolin - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an inclusion compound of forskolin, and more particularly, to an inclusion compound of forskolin having a useful physiological activity, which is included in a branched cyclodextrin.

[0002]

2. Description of the Related Art Forskolin is a labdane-type diterpene compound having a potent antihypertensive action and a potentiating action on myocardial contractility, isolated and identified from Coleusforskohlii, an Indian Lamiaceae plant. Its pharmacological action is to directly activate adenylate cyclase and increase cyclic AMP concentration without going through the β-adrenergic receptor on the cell membrane, a compound with a completely new mechanism of action that has not been seen in conventional drugs. Is said to be. However, oral administration of forskolin has poor intestinal absorption, so that pharmacological action is not sufficiently exhibited. For this reason, forskolin is expected to be used for the treatment of heart failure and the like as an injection, but its solubility in water is extremely low at room temperature of 0.0026%, so the effect is not expressed by ordinary usage There is a disadvantage that.

[0003] For this reason, attempts have been made to make water soluble in forskolin. Until now, unbranched cyclodextrin (J. Ocular Pharmacol. 5 (2), 111 (1989)) and hydroxypropyl cyclodextrin ( Molecular Pharma
col.32.133 (1987)) and other cyclodextrins (C
D) or a method using a polyoxyethylene-polyoxypropylene copolymer as a solubilizer (J.Oc
ular Phamacol. 5 (2), 111 (1989)).
However, in the case of unbranched CD, β-CD cannot obtain a sufficient amount of solubilization due to its low solubility in water, and in the case of γ-CD, water-insoluble It forms inclusion precipitates and is not water soluble at all. Therefore, it is practically impossible to make these unbranched CDs water-soluble.

[0004] On the other hand, hydroxypropyl cyclodextrin (HP-CD) has a certain degree of water solubility in HP-β-CD, and has several times higher solubility in HP-γ-CD. However, in the case of solubilization using HP-β-CD, the solubility is not sufficient, and the CD concentration becomes too high when actually used as an injection, which is not preferable in consideration of the effects on the living body such as hemolysis. Also, HP-γ-
Although CD shows sufficient solubilizing power, the price of HP-γ-CD itself is very high (usually several tens times that of β-type CD), and it is expensive to use as a drug carrier. Not practical. Moreover, HP-CD is a mixture of CDs in which a plurality of hydroxypropyl groups are substituted,
The great difficulty in isolating a single component makes it very disadvantageous in obtaining approval as a pharmaceutical. Although the use of a solubilizer such as a copolymer such as a polyoxyethylene-polyoxypropylene copolymer is also effective to some extent, the amount of solubilization is still limited, and satisfactory results have not been obtained. .

[0005] Therefore, development of water-soluble forskolin derivatives has been actively conducted, and recently, derivatives having an activity equal to or higher than that of forskolin have been developed (JP-A-63-10783, JP-A-64-9986). I came to. However, since these forskolin derivatives are synthesized using forskolin as a raw material and require a considerable number of steps, a reduction in yield is inevitable. Moreover, even if these derivatives are given water solubility, they are about 30 to 50 times the solubility of forskolin, and they do not have sufficient solubility yet, so they cannot be said to be necessarily a good method.

[0006]

Means for Solving the Problems The present inventors have conducted various studies to solve the above problems, and as a result, by enclosing forskolin with a branched cyclodextrin (branched CD), a further improvement over the conventional method has been achieved. The inventors have found that an inclusion compound with improved solubility can be obtained, and have completed the present invention. That is, the present invention is an inclusion compound comprising forskolin included in a branched CD. The inclusion compound can be manufactured in only one step, and its solubility reaches about 200 times that of forskolin.

[0007] The branched CD used in the present invention is α-C
D, β-CD, γ-CD and other CDs are linked to one or two α-1,6 molecules of small sugars such as glucose, maltose, maltotriose, etc. Glucosyl CD (hereinafter, referred to as G ₁ -CD), and two linked glucosyl CDs (hereinafter, G _1.
G ₁ -CD). Similarly, a maltose molecule bound to one maltose CD (hereinafter, referred to as G ₂ -CD) and a dimaltosyl CD (hereinafter, referred to as G _2.
G ₂ -CD).

In the present invention, glucosyl-β-cyclodextrin (G ₁ -β-CD), which is a branched β-cyclodextrin (branched β-CD), and diglucosyl-β-CD are generally used.
Cyclodextrin (G ₁ · G ₁ -β-CD), maltosyl-β-cyclodextrin (G ₂ -β-CD), dimaltosyl-β-cyclodextrin (G ₂ · G ₂ -β-C)
D) or branched γ-cyclodextrin (branched γ-C
D) glucosyl-γ-cyclodextrin (G
_1- γ-CD), diglucosyl-γ-cyclodextrin (G ₁ · G ₁ -γ-CD), maltosyl-γ-cyclodextrin (G ₂ -γ-CD), dimaltosyl-γ-cyclodextrin (G _2. G ₂ -γ-CD) is used.
Among them, in the present invention, the solubilizing power of forskolin and the branch C
In view of the price of D, it is preferable to use a branched β-CD such as G ₁ -β-CD or G ₂ -β-CD.

[0009] The inclusion compound of the present invention is prepared by contacting a branched CD with a forskolin in the presence of water to form an inclusion compound of the forskolin and the branched CD. It is manufactured by
The contact is usually performed by adding a forskolin crystal to an aqueous solution of branched CD and stirring the mixture for several minutes to several tens of minutes with a homogenizer, a stirrer, or the like. Inclusion methods are also possible.
The amount of forskolin used is usually 0.1-1 for the branched CD.
Is a mole.

[0010] It can be confirmed by nuclear magnetic resonance (NMR) that the solubilization of the clathrate compound of the present invention is due to the inclusion of forskolin by the branched CD.
FIG. 2 shows forskolin (A) and forskolin G ₁ -β
The NMR spectrum (heavy solution) of the methyl group portion of forskolin using chloroform of the -CD inclusion compound (B) as an external standard, the difference between the spectra of A and B is G
It is caused by inclusion by _1- β-CD. Also,
When the clathrate extracted from the aqueous solution of the clathrate of the present invention with an organic solvent is measured for NMR, this shows the same pattern as the NMR of forskolin. From this, it was confirmed that forskolin was included in the clathrate compound without being decomposed or the like.

The obtained clathrate compound may be used as an aqueous solution as an injection as it is, or may be dried and powdered to give an oral preparation, a transdermal absorption preparation, and a dissolution injection preparation at the time of use. At this time, a desired dosage form can be prepared by adding additives such as starch, glucose, lactose, gum, cellulose, vitamins and the like by a conventional method.

[0012]

Next, the present invention will be described in more detail with reference to Examples and Test Examples. The forskolin content in the clathrate compounds in the examples and test examples was measured as follows. That is, forskolin is extracted with methylene chloride from an aqueous solution in which a fixed amount of an aqueous solution of a forskolin clathrate compound or a fixed amount of a clathrate compound powder is dissolved in an appropriate amount of water to make a fixed volume, and then subjected to gas chromatography (Shimadzu). GC-14A, Chromatopack CR-4A). (Gas chromatography conditions) Column = OV-1 (on Gas ChromQ) Column temperature = 235 ° C Carrier gas = nitrogen gas Flow rate = 40 ml / min Detector = FID

Example 1 Forskolin crystals (Sigma) were added to 2 ml of 2,4,6,8,10,20,40% aqueous solution of G ₁ -β-CD (produced by Niken Chemical Co., Ltd., purity 98.5%). 20 mg / g of CD), stopper tightly and add
Shake with a shaking machine day and night to solubilize the clathrate. After filtering the shake solution through a 0.45 μm Millipore filter, the filtrate 1
Collect ml, dilute with 5-6 ml of water and extract with methylene chloride. After adjusting the volume of methylene chloride to a constant volume, it was quantified by gas chromatography using an absolute calibration curve method. Similarly, HP-
Inclusion solubilization was performed using β-CD (manufactured by Rocket). Table 1 shows the results.

[0014]

[Table 1]

Example 2 8 mg of forskolin was added to 2 ml of a 20% G ₁ -β-CD aqueous solution, sealed, shaken with a shaking machine for 3 days and nights, filtered through a 0.45 μm Millipore filter, and then filtrated. It was freeze-dried to obtain an inclusion powder. Dissolve 0.1 g of this powder in 6-7 ml of water,
After extracting forskolin with methylene chloride, the content was measured by gas chromatography. The content of forskolin in this clathrate powder was 7.8 mg / g.

Example 3 To 20 ml of a 10% aqueous solution of each of G ₂ -β-CD, G ₁ · G ₁ -β-CD and G ₂ · G ₂ -β-CD was added 20 mg of forskolin crystal per 1 g of CD. Then, the container was sealed and shaken for 3 days and nights to perform clathrate formation. After filtration through a 0.45 μm Millipore filter, 1 ml of the filtrate was collected, diluted with 5 to 6 ml of water, and extracted with methylene chloride. After the volume was made constant, it was determined by gas chromatography. The amount of forskolin dissolved in 1 ml of each CD solution is shown below. Dissolved amount of branched CD forskolin (mg / ml) G ₂ -β-CD 0.48 G ₁ · G ₁ -β-CD 0.44 G ₂ · G ₂ -β-CD 0.38

EXAMPLE 4 Each of G ₂ -γ-CD (manufactured by Niken Kagaku Co., Ltd., purity: 99%)
Forskolin crystals (manufactured by Sigma) 20 mg per 1 g of CD were added to 2 ml of a 10, 20, or 30% aqueous solution, sealed, sealed and shaken for 3 days and nights to solubilize the clathrate. After filtering this shaking solution through a 0.45 μm Millipore filter, 1 ml of the filtrate was collected and 5 to 6 m
After dilution with 1 l of water, the mixture was extracted with methylene chloride. After adjusting the volume of methylene chloride to a constant volume, the amount was quantified by gas chromatography. The amount of forskolin dissolved in 1 ml of each concentration of the aqueous solution is shown below. G ₂ -γ-CD concentration Dissolved amount of forskolin (mg / ml) 10% 0.88 20% 1.92 30% 3.29

Test Example In a beagle dog under pentobarbital anesthesia, the clathrate powder prepared in Example 2 was dissolved in physiological saline, and 3, 10, and 30 μg of forskolin per 1 kg of body weight were intravenously administered. The rate of rise in the left ventricle (dp / dt), which is an index of positive inotropy, was measured. Table 2 shows the results. Table 2 shows that the present forskolin clathrate compound exhibits a positive inotropic effect in a dose-dependent manner.

[0019]

[Table 2]

[0020]

The forskolin clathrate of the present invention comprises
It shows several times higher solubility than the conventional inclusion compound of unbranched CD and hydroxypropyl CD. In addition, a branched CD can be used as a single component, unlike a mixture of multiple components having different degrees of substitution such as HP-CD, which is advantageous for obtaining a license for a drug. In addition, this clathrate showed similar pharmacological effects to forskolin in animal experiments. Therefore, the compounds of the present invention can be used as pharmaceuticals such as injections, oral preparations, and transdermal absorbers.

[0021]

[Brief description of the drawings]

FIG. 1 shows an ultraviolet absorption spectrum of a G ₁ -β-CD inclusion compound of forskolin (aqueous solution).

FIG. 2 is a nuclear magnetic resonance spectrum of a methyl group portion of forskolin, where A is forskolin and B is a G ₁ -β-CD inclusion compound of forskolin, and chloroform is used as an external standard (heavy solution). Is shown.

Claims (3)

(57) [Claims] 1. An inclusion compound comprising forskolin included in a branched cyclodextrin. 2. The branched cyclodextrin is glucosyl-β-cyclodextrin, diglucosyl-β-cyclodextrin, maltosyl-β-cyclodextrin, dimaltosyl-β-cyclodextrin, glucosyl-γ-cyclodextrin, diglucosyl-γ.
The clathrate according to claim 1, which is one compound selected from cyclodextrin, maltosyl-γ-cyclodextrin and dimaltosyl-γ-cyclodextrin. 3. The clathrate according to claim 1, wherein the clathrate is a water-soluble powder.