15-Deoxyspergualin suppository

Abstract: A 15-deoxyspergualin suppository containing β-form crystals of 15-deoxyspergualin trihydrochloride and a lipophylic base can give high plasma concentration of the drug when administered, and has good stability and low hygroscopicity.

Description: Spergualin is an antibiotic having anticancer activity which was found by Umezawa et al. (U.S. Patent 4,416,899). Many derivatives and analogs of spergualin having similar activity are disclosed in U.S. Patent 4,518,532, U.S. Patent 4,529,549, U.S. Patent 4,556,735 and Japanese Patent Laid-open 62-48660 whereas derivatives having immunosuppressive activity are disclosed in U.S. Patent 4,851,446. Of these compounds, 15-deoxyspergualin [i.e. N-[4-(3-aminopropyl)aminobutyl]-2-(7-guanidinoheptaneamido)-2-hydroxyethaneamide)] is usable as an immunosuppressor. As preparations of spergualins, injectable preparations (U.S. Patent 4,876,244) and oral preparations for increasing the oral bioavailability by using simple or complex fats have been proposed (Japanese Patent Laid-open 2-9816 and ibid. 2-11514). 15-Deoxyspergualin is now being developed as an immunosuppressor. For the prophylaxis or therapy of the diseases wherein an immunosuppressor is needed continuous administration is required to some extent. Accordingly, it is desired to develop preparations other than injectable preparations capable of giving a high plasma concentration of the drug by easy administration at low dose. However, 15-deoxyspergualin hydrochloride shows insufficient bioavailability even when it is administered orally as mentioned above, and it is difficult to obtain a plasma concentration of the drug sufficient to impart an immunosuppresive effect by an administration route other than injection. In addition, 15-deoxyspergualin hydrochloride is hygroscopic and has a poor stability, and therefore it was difficult to obtain preparations of this drug having good storage stability. An object of the present invention is to provide a 15-deoxyspergualin preparation to solve the above-mentioned problems. The present invention relates to a novel suppository containing 15-deoxyspergualin trihydrochloride β-form crystals and lipophilic base which is solid at ambient temperature, which can be used as an anticancer agent or an immunosuppressor. As a result of the various researches, the present inventors have found that a powder or an aqueous syrup of known 15-deoxyspergualin trihydrochloride (hereinafter referred to as "15-deoxyspergualin hydrochloride") can be crystallized in the presence of a mixture of water and a water miscible solvent, and that the resulting crystals (hereinafter referred to as "β-form crystals") can be mixed with a lipophilic base which is solid at an ambient temperature to give a suppository, which imparts high plasma concentration of 15-deoxyspergualin when administered and has a good storage stability. For formulation of the suppository of the present invention, one or a mixture of two or more lipophilic bases, each of which is solid at normal ambient temperature, are melted by heating, and 15-deoxyspergualin hydrochloride β-form crystals are added thereto, and homogenized. The mixture is filled into suppository containers or gelatin capsule shells and cooled to give suppositories. 15-Deoxyspergualin hydrochloride β-form crystals are used in an amount of about 1 - 40% (all percentages are given "by weight"), preferably 3 - 25%, more preferably 10 - 20% based on the total weight of the preparation. The amount of base is 60 - 99%, preferably 75 - 97%, and more preferably 80 - 90%. For various purposes, other additives can be added to the preparation of the present invention. The amount of the additives is 0 - 0.3 part (all parts are "part by weight") relative to 1 part of the mixture of 15-deoxyspergualin hydrochloride β-form crystals and the base. The amount of the base is not less than 3 parts, preferably 4 - 100 parts relative to 1 part of the β-form crystals. β-Form crystals of 15-deoxyspergualin hydrochloride used in the present invention is prepared as follows: To about 100 parts of a powder of 15-deoxyspergualin hydrochloride obtained by a prior art method is added 500 parts of 95% ethanol, and the mixture is thoroughly mixed and allowed to stand at 5°C for 3 days, and the resulting crystals are removed by suction filtration to give the β-form crystals. Table 1 shows an X-ray diffraction pattern of β-form crystals of 15-deoxyspergualin hydrochloride used in the present invention which is measured by means of an X-ray diffraction apparatus (XD-3A, Shimadzu Co.) equipped with a Cu X-ray apparatus using graphite monochrometer and with a scintillation counter. (Electric power Cu: Graphite monochrometer, 40 KV-30 mA, λ = 1. 54051) The lipophilic base which is solid at ambient temperature for use in the present invention includes medium chain fatty acid triglycerides which are solid at an ambient temperature (e.g. capric acid triglyceride); and oil and fat suppository bases (e.g. cacao butter, laurine butter, cinnamon butter, hydrogenated oil, semi-synthetic glycerides, and the like). They can be used alone or in combination of two or more of them. Examples of the lipophilic base which is solid at normal ambient temperature are triglycerides of C₈ - C₁₈ saturated fatty acids, which preferably contain at least 50% of C₈ - C₁₄ saturated fatty acids. The fatty acids which constitute the triglycerides preferably contain one or more members selected from C₈, C₁₀, C₁₂ and C₁₄ saturated fatty acids. The fatty acids attached to glycerin preferably contain 32 to 60%, preferably 44 to 57% of a C₁₂ saturated fatty acid; 12 to 25%, preferably 17 to 23% of a C₁₄ saturated fatty acid; 9 to 20%, preferably 10 to 14% of a C₁₆ saturated fatty acid; 9 to 32%, preferably 10 to 22% of a C₁₈ saturated fatty acid; and 0 to 4%, preferably 0 to 2% of other fatty-acids based on the total weight of the fatty acids. The suppository of the present invention may be a capsule for rectal use which is prepared by molding suppositories by use of gelatin capsule shells and filling a mixture of the medicine (solution or solid) and the base therein. Besides the above components (15-deoxyspergualin and the suppository base), additives may be added to the suppository of the present invention, for example, viscosity modifying agents such as microcrystalline wax and microcrystalline cellulose; substances for reducing rectal irritation such as fractionated coconut oils; a small amount of lecithin for prevention of blooming of fats. The amount of these additives is 0 - 30% based on the total weight of the drug and the suppository base. The present invention is illustrated by the following examples. 15-Deoxyspergualin is abbreviated as "DSG". Example 1 (Formulation) The above components are for 20 suppositories. Pharmasol H-15 (proportion of fatty acids: about 54% of a C₁₂ fatty acid; about 22% of a C₁₄ fatty acid; about 12% of a C₁₆ fatty acid; and about 12% of a C₁₈ fatty acid) in a beaker was melted by heating on a water bath. Heating was stopped and 1 g of DSG was added at 40°C to the solution under stirring and the components were homogenized under sufficient mixing. The mixture was poured into suppository molds (suppository containers), cooled at room temperature then solidified by cooling in a refrigerator to give suppositories. Example 2 (Formulation) The above components are for 20 suppositories. In a similar manner to that of Example 1, suppositories were obtained. Example 3 (Formulation) β-form of DSG 5 g Pharmasol H-15 35 g The above components are for 20 suppositories. In a similar manner to that of Example 1, suppositories were obtained. Reference Example 1 A powder of DSG was dissolved in water to give a solution of 10 mg of DSG/ml. Reference Example 2 50 mg of a powder of DSG and 50 mg of Panacete 1000 were mixed and filled into No. 3 capsules (the Pharmacopoeia of Japan). Reference Example 3 (Formulation) The above components are for 20 suppositories. Macrogol 1500 and Macrogol 4000 were taken into a beaker, and melted on a warmed water bath. DSG was added little by little to the solution with stirring and homogenized with enough mixing. The mixture was filled into suppository molds, solidified by cooling to give suppositories. Test Example 1 (1) Test Method Suppositories prepared in Examples 1 and 2 were each rectally administered to male beagles weighing 9.0 - 11.0 kg (3 animals were used for each group). As control, 5 ml of a solution obtained in Reference Example 1 was administered orally or rectally, a capsule obtained in Reference Example 2 was administered orally, and the suppository of Reference Example 3 was administered rectally. In all cases, doses of DSG were 5 mg/kg. Animals were fasted from the day before the beginning of the test to the conclusion of the test. Each about 5 mg of blood samples was collected into a heparin treated syringe at 0.5, 1, 2, 4 and 8 hours after administration, and centrifuged to separate the serum. To 1 ml of the serum was added 1 µg of N-[4-(3-aminopropyl)aminobutyl]-2-(7-guanidinooctanamido)-2-hydroxyethanamide as an internal standard, and the mixture was diluted with 5 ml of distilled water and injected into CM-Sephadex® C-25 column [Na⁺ type (Pharmacia Fine Chemical Co.) 5 cm x 0.5 cm ]. The column was washed with 10 ml of 0.3 M aqueous sodium chloride solution, and then DSG and the internal standard were eluted with 10 ml of 0.5 M aqueous sodium chloride solution. After desalting by using Sep-Pak C₁₈ cartridge, the mixture was eluted with methanol, and the solvent was evaporated under reduced pressure. The residue was dissolved in 250 µl of distilled water, and 100 µl of the solution was injected in a high performance liquid chromatograph (HPLC), treated with o-phthalaldehyde (OPA) to give its derivative (so-called postcolumn derivatization), which was determined by HPLC with fluorescence detector. HPLC: Shimadzu liquid chromatography apparatus Column: Cosmosil 5C₁₈P (15 cm x 4.6 mm) Column temperature: 25°C Detection wavelength: excitation wavelength 335 nm, emission wavelength 435 nm. Mobile phase: 5mM sodium heptanesulfonate was dissolved in 10 mM phosphate buffer (pH 3.0) - acetonitrile (80:20). Flow rate: mobile phase 1.0 ml/min. OPA reagent 0.5 ml/min. (2) Test Results C max : The maximum plasma concentration AUC 0-8h : Area under the plasma concentration - time curve (0 ∼ 8 hours) Test Example 2 Stability (severe Test) (Test Method) To 50 mg of exactly weighed β-form crystals of DSG (or a lyophilized powder of DSG) in a hermetic vial was added 1.25 g of Witepsol® (proportion of fatty acids: about 54% of a C₁₂ fatty acid; about 22% of a C₁₄ fatty acid; about 12% of a C₁₆ fatty acid; about 12% of a C₁₈ fatty acid) which was melted by heating on a water bath, and then the mixture was thoroughly mixed. After being tightly closed, the mixture was kept at 50°C for 2 weeks or at 35°C for 4 weeks. The amount of DGS extracted from the base was measured according to a HPLC technique. Test Example 3 Hygroscopicity Test (Test Method) Sample The above components are for 20 suppositories. The above components are for 20 suppositories. 26 g of Pharmasol® H-15 in a beaker was melted by heating on a water bath. After stopping heating, 2 g of β-form crystals of DSG (a lyophilized powder of DSG) was added to the solution at 40°C with stirring, homogenized with enough mixing, filled into suppository molds, cooled at room temperature and solidified by cooling in a refrigerator to give suppositories. The above suppositories were stored at 25°C at 75% RH, and the increase ratio of weight of the sample was measured. (Test Results) As shown in Table 2, the plasma concentration of DSG obtained by administration of the suppository of the present invention is higher than the concentrations obtained by the oral administration of DSG, by the rectal administration using the aqueous solution of DSG, and by the rectal administration of the suppository of DSG using a hydrophilic base (Macrogol). Particularly, the plasma concentration of DSG obtained by administration of the suppository using the lipophilic base in Example 1 is about 3.5 times at C max higher and about 3 times at AUC 0-8h higher than that obtained by administration of Control 1 which exhibited the highest value in the control group. As shown in Tables 3 and 4, the suppository containing β-form crystals of DSG exhibited a higher DSG stability and a lower hygroscopicity than the suppository containing a lyophilized powder of DSG. It is apparent that, according to the invention, there is provided a 15-deoxyspergualin suppository which can be obtained high plasma concentration of 15-deoxyspergualin when administered and has good storage stability.

Claims: Claims for the following Contracting States : DE, FR, GB, IT A suppository which comprises β-form crystals of 15-deoxyspergualin trihydrochloride and a lipophilic base which is solid at an ambient temperature. A suppository according to Claim 1 which comprises 3 - 25 % by weight of β-form crystals of 15-deoxyspergualin and 97 - 75 % by weight of the lipophilic base. A suppository according to Claim 1 which comprises 10 - 20% by weight of β-form crystals of 15-deoxyspergualin and 90 - 80% by weight of the lipophilic base. A suppository according to Claim 1, wherein the lipophilic base which is solid at ambient temperature is a triglyceride of C₈ - C₁₈ saturated fatty acids containing at least 50% by weight of C₈ - C₁₄ saturated fatty acids. A suppository according to Claim 4 wherein the C₈ - C₁₄ saturated fatty acids attached to glycerin are one or more members selected from C₈, C₁₀, C₁₂ and C₁₄ saturated fatty acids. A suppository according to Claim 4, wherein the saturated fatty acids attached to glycerin contain an amount of from 32 to 60% by weight of a C₁₂ of a C₁₄ saturated fatty acid; from 9 to 20% by weight of a C₁₆ saturated fatty acid; from 9 to 32% by weight of a C₁₈ saturated fatty acid; and 0 to 4% by weight of other fatty acids. A suppository according to Claim 4, wherein the saturated fatty acids attached to glycerin contain an amount of from 44 to 57% by weight of a C₁₂ saturated fatty acid; from 17 to 23% by weight of C₁₄ saturated fatty acid; from 10 to 14% by weight of a C₁₆ saturated fatty acid; from 10 to 22% by weight of a C₁₈ saturated fatty acid; and 0 to 2% by weight of other fatty acids. Process for the manufacture of a suppository containing 15-deoxyspergualin trihydrochloride β-form crystals and lipophilic base which is solid at an ambient temperature, comprising a) melting by heating one or a mixture of two or more lipophilic bases which are solid at room temperature, b) adding 15-deoxyspergualin trihydrochloride β-form crystals and optionnally usual pharmaceutically acceptable additives thereto, c) homogenizing the mixture, d) filling the mixture into suppository containers or gelatin capsule shells and cooling. Process according to claim 8, wherein the 15-deoxsyspergualin trihydrochloride β-form crystals are obtained by crystallizing 15-deoxyspergualin trihydrochloride in the presence of a mixture of water and a water miscible solvent. Process according to claim 9, wherein the crystallization is carried out by adding a mixture of water and a water miscible solvent to a powder of 15-deoxyspergualin trihydrochloride, thoroughly mixing and allowing the mixture to stand for 3 days at a temperature of 5°C, and removing the resulting β-form crystals by suction filtration. Process according to claim 9 or 10, wherein the mixture of water and a water miscible solvent is 95% ethanol. Process according to claim 11, wherein the ratio of powder of 15-deoxyspergualin trihydrochloride and 95% ethanol is 1 : 5. Process according to one or more of claims 8 to 12, wherein 3 - 25% by wt. of β-form crystals of 15-deoxyspergualin hydrochloride and 97 - 75% by wt. of the lipophilic base are used. Process according to one or more of claims 8 to 13, wherein 10-20% by wt. of β-form crystals of 15-deoxyspergualin hydrochloride and 90 - 80% by wt. of the lipophilic base are used. Claims for the following Contracting State : ES Process for the manufacture of a suppository containing 15-deoxyspergualin trihydrochloride β-form crystals and lipophilic base which is solid at an ambient temperature, comprising a) melting by heating one or a mixture of two or more lipophilic bases which are solid at room temperature, b) adding 15-deoxyspergualin trihydrochloride β-form crystals and optionally usual pharmaceutically acceptable additives thereto, c) homogenizing the mixture, d) filling the mixture into suppository containers or gelatin capsule shells and cooling. Process according to claim 1, wherein the 15-deoxsyspergualin trihydrochloride β-form crystals are obtained by crystallizing 15-deoxyspergualin trihydrochloride in the presence of a mixture of water and a water miscible solvent. Process according to claim 2, wherein the crystallization is carried out by adding a mixture of water and a water miscible solvent to a powder of 15-deoxyspergualin trihydrochloride, thoroughly mixing and allowing the mixture to stand for 3 days at a temperature of 5°C, and removing the resulting β-form crystals by suction filtration. Process according to claim 2 or 3, wherein the mixture of water and a water miscible solvent is 95% ethanol. Process according to claim 4, wherein the ration of powder of 15-deoxyspergualin trihydrochloride and 95% ethanal is 1 : 5. Process according to one or more of claims 1 to 5, wherein 3 - 25% by wt. of β-form crystals of 15-deoxyspergualin hydrochloride and 97 - 75% by wt. of the lipophilic base are used. Process according to one or more of claims 1 to 6, wherein 10-20% by wt. of β-form crystals of 15-deoxyspergualin hydrochloride and 90 - 80% by wt. of the lipophilic base are used. Process according to one or more of claims 1 to 7, wherein the lipophilic base which is solid at ambient temperature is a triglyceride of C₈ to C₁₈ saturated fatty acids containing at least 50% by wt. of C₈ to C₁₄ saturated fatty acids. Process according to claim 8 wherein the C₈ - C₁₄ saturated fatty acids attached to glycerin are one or more members selected from C₈, C₁₀, C₁₂ und C₁₄ saturated fatty acids. Process according to claim 8, wherein the saturated fatty acids attached to glycerin contain an amount of from 32 to 60% by wt. of a C₁₂ saturated fatty acid; from 12 to 25% by wt. of a C₁₄ saturated fatty acid; from 9 to 20% by wt. of C₁₆ saturated fatty acid; from 9 to 32% by wt. of C₁₈ saturated fatty acid; and 0 to 4% by wt. of other fatty acids. Process according to claim 8, wherein the saturated fatty acids attached to glycerin contain an amount of from 44 to 57% by wt. a of C₁₂ saturated fatty acid; from 17 to 23% by wt. of C₁₄ saturated fatty acid; from 10 to 14% by wt. of C₁₆ saturated fatty acid; from 10 to 22% by wt. of a C₁₈ saturated fatty acid; and 0 to 2% by wt. of other fatty acids.

Inventors: HIRAGA HIRONOBU, OKUMA TAKAAKI, UMEDA YOSHIHISA, HIRAGA, HIRONOBU, OKUMA, TAKAAKI, UMEDA, YOSHIHISA
Applicants: NIPPON KAYAKU KK, TAKARA SHUZO CO, NIPPON KAYAKU KABUSHIKI KAISHA, TAKARA SHUZO CO. LTD.
Application year: 1991
Main classification: A61K31
IPC codes: A61K31, A61K9