JPH0551375A - Clathrate compound of forskolin - Google Patents

Abstract

PURPOSE:To obtain a soluble clathrate compound by including water-insoluble forskolin in a branched cyclodextrin. CONSTITUTION:A clathrate compound is obtained by including forskolin in a branched cyclodextrin such as glucosyl-beta-cyclodextrin (G1-beta-CD), diglucosyl-beta- cyclodextrin (G1.G1-beta-CD), maltosyl-beta-cyclodexrin (G2-beta-CD) or dimaltosyl-beta- cyclodextrin (G2.G2-beta-CD).

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 obtained by including forskolin having useful physiological activity with branched cyclodextrin.

[0002]

BACKGROUND OF THE INVENTION Forskolin is a labdane-type diterpene compound having a strong antihypertensive action and a myocardial contractile force enhancing action, which was isolated and identified from Coleus forskohlii, which is a plant belonging to the family Lamiaceae in India. Its pharmacological action is to directly activate adenylate cyclase and increase cyclic AMP concentration without passing through β-adrenergic receptors on the cell membrane, and a compound with a completely new mechanism of action not seen in conventional drugs. It is said that. However, oral administration of forskolin does not sufficiently exert its pharmacological action due to poor intestinal absorption. For this reason, forskolin is expected to be used as an injectable drug for the treatment of heart failure, etc., but its solubility in water is extremely low at 0.0026% at room temperature, and therefore it is not effective in normal use. It has the drawback of

Therefore, attempts have been made to make forskolin water-soluble, and up to 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 which is a solubilizer (J.Oc
ular Phamacol.5 (2), 111 (1989)) and the like are known.
However, in the case of unbranched CD, β-CD itself has a low solubility in water, so a sufficient amount of solubilization cannot be obtained, and in the case of γ-CD, it is insoluble in water between forskolin. It forms an inclusion precipitate and is not totally water soluble. Therefore, it is practically impossible to make them water-soluble with these unbranched CDs.

On the other hand, hydroxypropyl cyclodextrin (HP-CD) is found to be water-soluble to some extent in HP-β-CD, and is several times more soluble in HP-γ-CD. However, solubilization by HP-β-CD is not sufficient in solubility, and the CD concentration becomes too high when actually used as an injection, which is not preferable in consideration of effects on the living body such as hemolytic action. In addition, HP-γ-
Although CD has a sufficient solubilizing power, the price of HP-γ-CD itself is very high (usually said to be several tens of times that of β-type CD), and it is expensive to use as a drug carrier. It is not practical. Moreover, HP-CD is a mixture of CDs each having a plurality of hydroxypropyl groups substituted,
The significant difficulty in isolating a single component is a significant disadvantage in obtaining a pharmaceutical license. Further, the use of a solubilizing agent such as a copolymer such as a polyoxyethylene-polyoxypropylene copolymer is also effective to some extent, but the solubilizing amount is still limited, and satisfactory results have not been obtained. ..

Therefore, water-soluble forskolin derivatives have been actively developed, 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). Came to the end. However, since these forskolin derivatives are synthesized using forskolin as a raw material in a considerable number of steps, a decrease in yield cannot be avoided. Moreover, even if water-solubility is imparted to these derivatives, they are about 30 to 50 times the solubility of forskolin, and they do not always have sufficient solubility, so they cannot be said to be necessarily good methods.

[0006]

[Means for Solving the Problems] As a result of various studies conducted by the present inventors to solve the above-mentioned problems, as a result of inclusion of forskolin with branched cyclodextrin (branched CD), it was possible to further improve the results compared with the conventional method. It was found that an inclusion compound having improved solubility was obtained, and the present invention was completed. That is, the present invention is an inclusion compound obtained by including forskolin with branched CD. The inclusion compound can be produced in only one step, and its solubility reaches about 200 times that of forskolin.

The branched CD used in the present invention is α-C
D, β-CD, γ-CD, and other CDs are said to have one molecule or two molecules α-1,6 of a small sugar such as glucose, maltose, maltotriose, etc., and one glucose molecule Glucosyl CD (hereinafter referred to as G ₁ -CD), and two bound products are diglucosyl CD (hereinafter referred to as G ₁ · CD).
G ₁ -CD), similarly one with one maltose molecule bound to maltosyl CD (hereinafter referred to as G ₂ -CD), and two bound to maltosyl CD (hereinafter referred to as G ₂ · CD).
G ₂ -CD).

In the present invention, glucosyl-β-cyclodextrin (G ₁ -β-CD), which is generally branched β-cyclodextrin (branched β-CD), and diglucosyl-β-.
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 · Cd) G ₂ -γ-CD) is used.
Of these, in the present invention, the solubilizing power of forskolin and the branching C
From the viewpoint of the price of D, it is preferable to use branched β-CD such as G ₁ -β-CD and G ₂ -β-CD.

In the clathrate compound of the present invention, the branched CD and forskolin are brought into contact with each other in the presence of water to form the clathrate compound of forskolin and the branched CD. If desired, the clathrate compound is dried and powdered. It is manufactured by
The contact is usually performed by adding forskolin crystals 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, and a method of shaking in a closed container for several days or by ultrasonic waves. An inclusion method is also possible.
The amount of forskolin used is usually 0.1 to 1 for branched CD
It is a mole.

It can be confirmed by a nuclear magnetic resonance apparatus (NMR) that the inclusion compound of the present invention is solubilized by inclusion of forskolin by branched CD.
Figure 2 shows Forskolin (A) and Forskolin G ₁ -β
-CD inclusion compound (B) is a NMR spectrum of the methyl group part of forskolin using chloroform as an external standard (heavy aqueous solution), but the difference between the spectra of A and B is G
It is caused by inclusion with _1- β-CD. Also,
When the NMR of the inclusion compound extracted from the aqueous solution of the inclusion compound of the present invention with an organic solvent was measured, it showed the same pattern as the NMR of forskolin. From this, it was confirmed that forskolin was included in the clathrate compound without being decomposed.

The obtained clathrate compound may be directly used as an injection in the form of an aqueous solution, or may be dried and powdered into an oral preparation, a percutaneous absorption preparation, or a dissolution injection preparation for use. At this time, additives such as starch, glucose, lactose, gums, cellulose, vitamins and the like can be added by a conventional method to prepare a desired dosage form.

[0012]

EXAMPLES Next, the present invention will be described in more detail by showing Examples and Test Examples. The content of forskolin in the clathrate compounds in Examples and Test Examples was measured as follows. That is, a fixed amount of forskolin inclusion compound aqueous solution or an aqueous solution of inclusion compound powder dissolved in an appropriate amount of water was used to extract forskolin with methylene chloride to a certain volume, and then a gas chromatograph (Shimadzu GC-14A, Chromatopack CR-4A). (Gas chromatography condition) Column = OV-1 (on Gas ChromQ) Column temperature = 235 ℃ Carrier gas = Nitrogen gas Flow rate = 40ml / min Detector = FID

Example 1 Forskolin crystals (Sigma Co., Ltd.) were added to 2 ml each of a 2,4,6,8,10,20,40% aqueous solution of G ₁ -β-CD (manufactured by Niken Chemical Co., Ltd., purity: 98.5%). 20 mg per 1 g of CD, and tightly stoppered 3
Inclusion solubilization is carried out by shaking with a shaker day and night. After filtering this shaking solution with a 0.45 μm Millipore filter, filtrate 1
Collect ml, dilute with 5-6 ml of water and extract with methylene chloride. After the volume of methylene chloride was made constant, the amount was quantified by a gas chromatograph by an absolute calibration curve method. Similarly, HP-
Inclusion solubilization was performed using β-CD (manufactured by Rocket). The results are shown in Table 1.

[0014]

[Table 1]

Example 2 Forskolin (8 mg) was added to 2 ml of 20% G ₁ -β-CD aqueous solution, sealed, shaken on a shaker for 3 days and then filtered with a 0.45 μm Millipore filter, and the filtrate was collected. It was freeze-dried to obtain an inclusion powder. 0.1 g of this powder is dissolved 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 ₂ ml of 10% aqueous solution of G ₂ -β-CD, G ₁ · G ₁ -β-CD and G ₂ · G ₂ -β-CD, 20 mg of forskolin crystals per 1 g of CD was added. Then, the container was sealed and shaken for 3 days and nights for inclusion. 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 then extracted with methylene chloride. After making it a fixed volume, it was quantified by gas chromatography. The amount of forskolin dissolved in 1 ml of each CD solution is shown below. Branched CD Forskolin dissolution (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 Chemical Co., Ltd., purity 99%)
20 mg of Forskolin crystal (manufactured by Sigma) was added to 2 ml of a 10, 20, 30% aqueous solution, and 20 mg per 1 g of CD was added, and the container was sealed and shaken for 3 days to solubilize the clathrate. After filtering this shaking solution with a 0.45 μm Millipore filter, collect 1 ml of the filtrate and collect it for 5-6 m.
It was diluted with 1 l of water and then 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 aqueous solution is shown below. G ₂ -γ-CD concentration Forskolin dissolution (mg / ml) 10% 0.88 20% 1.92 30% 3.29

Test Example A beagle dog under pentobarbital anesthesia was prepared by dissolving the inclusion powder prepared in Example 2 in physiological saline and intravenously administering 3, 10, 30 μg of forskolin per kg of body weight. The rising velocity (dp / dt) in the left ventricle, which is an index of positive inotropic force, was measured. The results are shown in Table 2. From Table 2, it can be seen that the present forskolin inclusion compound exerts a positive inotropic effect in a dose-dependent manner.

[0019]

[Table 2]

[0020]

The forskolin inclusion compound of the present invention is
The solubility is several times higher than that of the conventional inclusion compounds using unbranched CD and hydroxypropyl CD. In addition, branched CD is advantageous in obtaining a license for a drug because a single component can be used unlike a mixture of multiple components having different degrees of substitution such as HP-CD. Moreover, the inclusion compound showed the same pharmacological action as forskolin in animal experiments. Therefore, the compound of the present invention can be used as a medicine such as an injection, an oral preparation and a transdermal absorption agent.

[0021]

[Brief description of drawings]

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

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

Claims (3)

[Claims] 1. A clathrate compound obtained by clathrating forskolin with 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 inclusion compound according to claim 1, which is one compound selected from cyclodextrin, maltosyl-γ-cyclodextrin and dimaltosyl-γ-cyclodextrin. 3. The clathrate compound according to claim 1, wherein the clathrate compound is a water-soluble powder.