Title of the Invention

Liposome preparation

Field of the invention

This invention relates to a pharmaceutical liposome preparation comprising, as an active ingredient, a tricyclic compound having a potent cholecystokinin antagonism and shown by the general formula (I) represented hereinafter or a pharmaceutically acceptable salt thereof. More particularly, the present invention relates to a liposome preparation comprising the above active ingredient stably entrapped into liposomes and as a consequence capable of maintaining stable solution in various media such as physiological saline, glucose solution for injection, water or juices and, hence, being applicable to various methods of administration including injections such as intravenous injection, intramuscular injection, and topical injections for intraarticular and the like, topical administration such as application to skin, instillation into eye, nasal administration, and inhalation, and further, oral administration and rectal administration etc.

Prior Art

It has been known that a tricyclic compound represented

by the following general formula (I) and a pharmaceutically acceptable salt thereof have a cholecystokinin antagonism and are useful as a drug for the prevention and treatment of pancreatitis and the like (Japanese Patent Application Laid-open No. HEI 2- 111774) :

wherein R ¹ is an aryl group optionally having at least one suitable substituent therein.

In addition, this tricyclic compound (I) and pharmaceutically acceptable salt thereof have been expected for an efficacy against non-cholinergic miosis (e.g. traumatic iosis, miosis after surgery of eye, etc. ) .

However, the tricyclic compound (I) and pharmaceutically acceptable salt thereof are hardly water-soluble and, accordingly, have been hardly used as a pharmaceutical solution, particularly as a solution for injection.

Disclosure of the Invention

The object of the present invention is to realize the use of the tricyclic compound (I) or pharmaceutically acceptable salt

thereof as a liquid preparation. That is, the present invention discloses a liposome preparation comprising, as an active ingredient, the above tricyclic compound (I) or pharmaceutically acceptable salt thereof entrapped into liposomes.

Best Mode of Practicing the Invention

Hereinafter, description will be made in detail to the various definitions used in the foregoing description and the description below in this specification, suitable examples and illustrations involved in the scope of the present invention.

The pharmaceutically acceptable salts of the tricyclic compound (I) are conventional non-toxic salts. Examples of such salts of the compound (I) include metal salts such as alkali metal salts, for example, sodium salt and potassium salt, and alkaline earth metal salts, for example, calcium salt and magnesium salt; ammonium salts; salts with organic bases such as amine salts, for example, trimethylamine salt, triethylamine salt, pyridine salt, picoline salt, dicyclohexyla ine salt and N , ' -dibenzylethylenediamine salt; salts with organic acids, such as acetate, maleate, tartarate, methanesulfonate , benzene- sulfonate, formate, toluenesulfonate and trifluoroacetate ; salts with inorganic acids, such as hydrochloride , hydrobromide , sulfate and phosphate; salts with amino acids such as arginine, aspartic acid and glutamic acid; and the like.

Preferred examples of the "aryl group" in the tricyclic compound (I) include, for example, phenyl, naphthyl and the like

Preferred examples of the suitable "substituents" in the "aryl group optionally having at least one suitable substituent" include, for example, halogen, amino group, lower alkoxy group, mono- (or di- or tri-) halo ( lower ) alkyl group and the like.

Preferred examples of the "halogen" and the "halogen moiety" in the "mono- (or di- or tri-) halo ( lower ) alkyl group" include, for example, chlorine, bromine, fluorine, iodine and the 1 ike .

Preferred examples of the "lower alkoxy group" include, for example, methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, tert-butoxy, pentyloxy, tert-pentyloxy , hexyloxy and the like.

Preferred examples of the "lower alkyl group" and the "lower alkyl moiety" in the "mono- (or di- or tri-) halo ( lower )alkyl group" include straight or branched chain alkyl group having 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, tert-pentyl. hexyl and the like.

The liposome preparation provided by the present invention will be described.

The liposome preparation of the present invention essentially comprises the above-described tricyclic compound (I) or pharmaceutically acceptable salt thereof [hereinaf er, simply referred to as "compound (I)"] entrapped into liposomes, and is not subject to other conditions such as the structure and composition of liposomes, method of production of the liposomes.

size thereof, the types of additives to be used, etc. , unless such conditions interfere with the stable entrapping of the tricyclic compound (I) into the liposomes.

Therefore, the structure of the liposome may be a large unilamellar vesicle (LUV) , a multilamellar vesicle (MLV) or a small unilamellar vesicle (SUV) .

As the liposome-forming substances, lipids and the like are employed. Examples of the lipids include sterols such as cholesterol, glycerides such as triglycerlde, phospholipids and nitrolipids. Among these, generally phosphollpids are preferred. For example, natural phosphollpids such as lecithins (phosphatidyl choline; e.g. , egg yolk lecithin, soybean lecithin, etc. ) , lysolecithin , sphingomyelin, phosphatidic acid, phosphatidyl serine, phosphatidyl inositol, phosphatidyl ethanolamine, phosphatidyl glycerol, diphosphatidyl glycerol, cardiolipin, plasmalogen, etc. or hydrogenation products obtainable from the above natural phosphollpids by the conventional technology, and synthetic phosphol i ids such as dicetyl phosphate, distearoylphosphatidyl choline, dipalmitoyl- phosphatidyl choline, dipalmitoylphosphatidyl ethanolamine, dipalmi toylphosphatidyl serine, eleostearoylphosphatidyl choline, eleostearoylphosphatidyl ethanolamine. eleostearoylphosphatidyl serine, etc. , can be mentioned.

These liposome-forming substances can be used independ ^¬ ently or in combination of two or more of them.

The representative process for producing the liposome

preparation of the present invention is now described. For example, the compound (I) and the liposome-forming substances described above are dissolved in a non-aqueous solution [for example, an alcohol (e.g. , methanol, ethanol, etc. ) , chloroform, etc. ] , and then water was added thereto. The resultant is added and dispersed into water or an aqueous solution of a sugar (e.g. , lactose) , to thereby obtain a liposome preparation. The liposome preparation thus obtained may be once lyophilized, and re-dispersed in a suitable solvent (e.g. , water, physiological saline, a glucose solution for injection, etc. ) when it is used for therapy. The method for producing the liposome preparation of the present invention is not particularly limited to the above process, and may be any of the known methods, a method combining two or more of the known methods, or a modification of any of the known methods.

The liposome preparation of the present invention may contain suitable additives, including excipient for lyophiliza- tion such as sugars (e.g. , lactose, glucose, mannitol, sorbitol, xylitol, etc. ) , inorganic salts (e.g. , sodium chloride, disodium hydrogenphosphate, sodium dihydrogenphosphate, etc. ) and polymers [dextran, polyvinyl alcohol, polyvinyl pyrrolidone, gelatin, etc. l ; antioxidants for enhancement of the stability of the 1 iposome-forming substances and the active ingredient [compound (1)1 , such as tocopherols (e.g. , α -tocopherol , etc. ) , dibutyl- hydroxytoluene , ascorbic acid, alkali metal pyrosulfites (e.g. sodium pyrosulfite, etc. ) ; and other additives such as agents for

facilitating the formation of the liposome preparations and agents for control of the rate of release of the active compound (I) from the liposomes. There is no particular limitation on the amount ratio of the active ingredient [compound (I)] to the liposome-forming substances to be used in the liposome preparation, and the amount ratio may be suitably selected depending on the types of the active ingredient and the liposome-forming substances. Usually, the weight ratio of the active ingredient to the liposome-forming substances is within the range from 1:5 to 1:1000, preferably from 1:10 to 1:500, most preferably 1:30 to 1:300.

When the liposome preparation contains such suitable additives as described above, there is also no particular limitation on the amount ratio between the active ingredient, liposome-forming substances and the additives in the liposome preparation, and the amount ratio is may be suitably selected depending on the types of the active ingredient, the liposome- forming substances and the additives. For example, the weight ratio of the liposome-forming substances to the excipient for lyophi lization such as a sugar is usually 2:1 to 1:50, preferably 1:1 to 1:40, most preferably 1:5 to 1:20. The weight ratio of the active ingredient to the antioxidant such as a tocopherol is usually 20:1 to 1:10, preferably 10:1 to 1:5, most preferably 6:1 to 1:2.

The liposome preparation according to the present invention is useful as a cholecystokinin antagonist and,

therefore, is useful as a drug for the prevention and treatment of pancreatitis and the like.

The liposome preparation of the present invention can be administered in any form such as a solid preparation, a semi- solid preparation and a liquid preparation, and may be suitably selected depending on the routes of the administration. For example, the liposome preparation can be administered in the form of an injection, an eye drop, a nasal drop, an inhalant, a transdermal dosage, a topical injection and the like, and it is preferred to use the liposome preparation in the form of a liquid preparation, for example, for injection. The amount of the active ingredient [compound (I)] in the liposome preparation may be suitably selected depending on the routes of administration or the like. For example, when the liposome preparation is used as a liquid preparation for injection, the amount of the active ingredient is preferably 0.001 to 2 mg/ml , most preferably 0.01 to 0.5 mg/ml.

For demonstrating that the liposome preparation of the present invention is stable and pharmaceutically useful, the following tests were conducted.

[Test 1]

Test Preparation

In this test, was used a lyophilized liposome preparation obtained in Example 3 below, which comprised compound (I) (FK480) wherein R was a fluorophenyl group as an active ingredient.

FK480: (3S)-l-(2-fluorophenyl ) -3 , 4 , 6 , 7-tetrahydro-3- (2- indo lylcarbonyl amino )-4-oxopyrrolo [3,2,1- jk] [l,4]benzodiazepine.

Test Result

Table 1: Stability of liposome preparation

Items of Stability of : Lyophilized liposome preparation evaluation

Just after After 1 month After 3 months preparation (40 ^β C) (40 ^β C)

Appearance White mass White mass White mass

State of Translucent Translucent Translucent solution* emulsion emulsion emulsion

_P H 6.95 6.73 7.13

Particle diameter of 84 96 134 liposome (nm)

Residual percent of active 100 100.2 100.1 ingredient in liposome preparation

(%)

_• : The state of a solution in which the lyophilized liposome _p re _p aration was re-dispersed into 5 ml of water for injection

[Test 21

Test Preparation

In this test, was used a lyophilized liposome preparation obtained in Example 5 below, which comprised FK480 as an active ingredient .

Te s t Re s u l t

Table 2: Stability of liposome preparation

Items of Stability of lyophilized liposome preparation evaluation

Just after After After 3 months preparation 3 months (40"C; relative

(25°C) humidity of 75%)

Appearance Slightly Slightly Slightly yellowish yellowish yellowish white mass white mass white mass

State of Emulsion Emulsion Emulsion solution' with the with the with the above color above color above color

Time required for 15 15 15 re-dispersion

(sec. )

PH 5.97 5.78 6.65

Particle diameter of 132 126 138 liposome (nm)

Residual percent of ac ive 100 101.0 98.5 ingredient in liposome preparation

(%)

_• : The state of a solution in which the lyophilized liposome pre _p aration was re-dispersed into 5 ml of water for injection

As shown in Tables 1 and 2 above, it was found that the liposome preparation of the present invention is stable and useful as a pharmaceutical preparation, especially as a liquid preparation for injection and the like.

Hereinafter, the present invention will be described in more detail with reference to the following Examples, but they should not be construed to be limiting the scope of the present invention.

Example 1

FK480 (1 mg) and purified egg yolk lecithin (100 mg) were dissolved in ethanol to obtain 1 ml of a mixed solution, and water (0.1 ml) was further added thereto. The resultant was dispersed into water (8.9 ml) while stirring using a stirrer (Politron) to thereby obtain a liposome preparation. The content of the active ingredient (FK480) in the obtained liposome preparation was 96 wg/ml (yield: 96%).

Example 2

FK480 (10 mg) and purified egg yolk lecithin (1000 mg) were dissolved in ethanol to obtain 10 ml of a mixed solution, and a 5%-aqueous solution of lactose (1 ml) was further added thereto. The resultant was dispersed into a 55--aqueous solution of lactose (89 ml) while stirring using a stirrer (Politron) to thereby obtain a liposome preparation. The average particle diameter of the liposomes was 79 nm and the content of the active

ingredient (FK480) in the liposome preparation was 99.7 ug/ml (yield: 99.7%).

Example 3

A liposome preparation was prepared in the same manner as in Example 2. The obtained liposome preparation was then lyophilized. The obtained lyophilized liposome preparation was readily re-dispersed into both water for injection and a glucose solution for injection.

Example 4

FK480 (4 g), purified egg yolk lecithin (400 g) and α- tocopherol (2 g) were dissolved in ethanol to obtain 4 liter of a mixed solution. On the other hand, lactose (3.89 kg) was dissolved in water for injection to obtain 35 liter of an aqueous solution of lactose. Into another vessel, 31.5 liter of the resultant lactose aqueous solution was charged, and 3.5 liter of the above lecithin-ethanol solution was dispersed thereinto, to thereby obtain liquid 1.

12.5 ml of the liquid 1 was charged into a 28 ml-glass vial and then subjected to lyophilization , to thereby obtain 1.25 mg of a lyophilized liposome preparation. The content of the active ingredient (FK480) in the obtained liposome preparation was 1.25 to 1.28 mg/vial. The obtained lyophilized liposome preparation was readily re-dispersed into both water for injection and a glucose solution for injection, and the average

particle diameter of the liposomes was 115 to 130 nm.

Example 5

Substantially the same manner as in Example 4 was repeated, except 5 ml of the preparation liquid 1 obtained by the same manner as in Example 4 was charged into a 28 ml-glass vial, to thereby obtain a lyophilized liposome preparation comprising FK480 (0.5 mg) . The obtained lyophilized liposome preparation was readily re-dispersed into both water for injection and a glucose solution for injection.

Effect of the Invention

Having the above-described structure, the liposome preparation of the present invention allows the hardly water-soluble tricyclic compound (I), which has been hardly provided as a solution for injection, to be provided in the form of a stable liquid preparation. Consequently, the liposome preparation can be provided as a stable solution for injection (e.g. , intravenous injection, intramuscular injection, topical injection for intraarticular , etc.) which does not cause crystallization of the active ingredient on contact with the body fluid. Hence, by using the liposome preparation, a stable pharmaceutical preparation being safe and showing high bioavailability can be provided to therapeutic field. Besides solutions for injection, it can assume a broad variety of pharmaceutical preparations such as preparations for topical

administration including application to skin, instillation into eye, nasal administration and inhalation, oral administration and rectal administration.

Moreover, it is possible to enhance the delivery of the tricyclic compound (I) to regions where a high concentration of the compound is required, or to suppress the delivery of the compound to regions where the drug is not necessarily required. As a result, it is expected that practically excellent results such as increase in and extended duration of drug efficacy, and alleviation of adverse reactions would be obtained.

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