JPS61100518A - Lyophilized ribosome pharmaceutical preparation comprising dimyristoylphosphatidylcholine as main component - Google Patents

Abstract

PURPOSE:To obtain the titled pharmaceutical preparation having improved characteristics and stability of drug, by using dimyristoylphosphatidylcholine as a main component of ribosme membrane material, blending it with a metabolic product of arachidonic acid as an active ingredient and a saccharide as a stabilizer, and lyophilizing the blend. CONSTITUTION:Dimyristoylphosphatidylcholine is used as a main component of a ribosme membrane material, blended with a metalolic product of arachidonic acid (e.g., PG, prostacyclin, thromboxane, or leukotriene), its analog structure compound (e.g., 6-keto-PGE1 derivative, carbacyclin derivative, etc.), its synthase inhibitor, and an antagonist, as active ingredients, and a saccharide (e.g., lactose, mannitol, etc.) as a stabilizer, and lyophilized, to give a ribosome pharmaceutical preparation. Stability of drug can be improved while keeping characteristics where foams will not form during redispersion, uniform particle diameters are obtained and a drug has improved inclusion ratio.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a lyophilized liposome preparation containing simyristoylphos-7-acylcholine as a product. More specifically, the present invention provides a preparation in which a poorly water-soluble arachidonic acid metabolite, a structurally similar compound thereof, a synthetic enzyme inhibitor or an antagonist thereof is incorporated into a liposome membrane, and then freeze-dried. It is related to.

[Conventional technology]

Arachidonic acid metabolites began with the discovery of prostaglandins, and in recent years their strong physiological activity has drawn attention and extensive research has been conducted, leading to the discovery of prostacyclin, thromboxane, and leukotriene.

Brostag2 and prostacyclin have effective activity and are used as medicines, but many similar compounds have been synthesized for the purpose of isolating their physiological activity, enhancing their physiological action, and improving their stability. (Japanese Unexamined Patent Publication No. 52-85151, No. 54-44639)
(See No. 54-130543). Furthermore, although thromboxane and leukotrienes themselves are thought to have little value as pharmaceuticals, a large number of their synthetic enzyme inhibitors and antagonists have been synthesized for use as pharmaceuticals (Japanese Patent Laid-Open No. 55 -313, 57-
(See No. 24338 and No. 57-193428)
.

On the other hand, research on liposomes as drug carriers has been widely conducted in recent years, but attempts have been made to encapsulate various drugs in liposomes and administer them to concentrate the drug in the target organ and maintain the drug's duration. It is.

The membrane component of liposomes is mainly phosphatidylcholine, especially natural phospholipids derived from egg yolk and soybean,
Many reports have been submitted using synthetic phospholipids such as distearoylphos-7-atedylcholine and dinormidoylphos-7-acylcholine (Eiochemistr.
y, 20, 4229-4238 (1981), J.
Pharm, Sc, 73*2, 203-206
(1984), The Lancet, 1320-13
22 (Jun.

23.1979), Annals New York
Academy of Sciences, 411-4
25 (1978)].

Many of the past applications describe a wide range of phospholipids, which are constituents of liposome membranes, and drugs contained therein. Among them, a liposome preparation containing prostaglandin as an active ingredient is described in JP-A-55-153713. Here, the addition of oil-based substances such as triglyceride and Roku to phos-7 atidylcholine improves the shortcomings of conventional liposomes, such as lack of flexibility, insufficient mechanical strength, and lack of sustained release properties. has been proposed, but there is no mention of freeze-drying, and if prostaglandins are included according to the above content, prostaglandins are unstable in water and cannot be used in practice due to storage issues. . - Fat emulsions also function as drug carriers (see JP-A-58-222014); however, fat emulsions and liposome preparations have completely different formulation forms. Liposome preparations contain the drug in a phospholipid membrane, whereas fat emulsions are made by dispersing vegetable oil with the drug dissolved in water. Therefore, liposome preparations differ from the present invention in that they can be freeze-dried, whereas fat emulsions cannot.

[Problem that the invention seeks to solve]

Natural phos-7 atidylcholine, which is a constituent of liposome membranes, does not have a constant composition and is difficult to store because it is prone to autooxidation due to oxygen and light due to double bonds in the molecule. On the other hand, synthetic phosphatidylcholine with a saturated fatty acid chain is stable against autooxidation, has 6D, and has certain physical properties because it is a single substance. It is practically impossible from the standpoint of increasing the particle size due to oxidation, oxidative decomposition of liposome constituents, leakage and decomposition of the encapsulated drug, difficulty in sterilization, etc.
In addition, frozen storage is subject to temperature restrictions, which poses major constraints on distribution and storage. Therefore, if freeze-drying storage is possible, distribution and storage will be simple and mass production will be possible, and liposome preparations will be widely used. However, liposome formulations using phosphatidylcholine, such as dipalmitoylphosphatidyl phosphorus, which is known as a common membrane material, can be freeze-dried, but when redispersed during use, the particle size becomes uneven. ], it has the disadvantage that the uptake rate of the drug contained therein is reduced.

In addition, many of the drugs proposed by the present invention are unstable in water, air, heat, etc., such as prostaglandins, prostacyclin, and leukotrienes, so they must be frozen or replaced with argon for storage. There are major constraints, such as the need to be perfect. Therefore, improvement in stability is required from the viewpoint of formulations as well.

[Means for solving problems]

The present inventors have discovered that arachidonic acid metabolites, their structurally similar compounds, their synthase inhibitors and antagonists tvyd
In the process of investigating lyophilizable membrane components for seam formulation, retention of properties, and improved stability, simyristoylphosphatidylcholine was found to be very superior. The liposome formulation prepared by incorporating the drug of the present invention into simyristoyl phosphatidylcholine and freeze-drying has very high stability, produces little foaming when redispersed, easily obtains a uniform particle size, and has the ability to incorporate the drug. The rate was also found to be very good.

The carbon number of the saturated fatty acid silver used in the present invention is 14
Compared to dipalmitoylphosphatidylcholine, which has 16 carbon atoms, there are very few reports of its use in the preparation of liposomes.
In fact, there are no reports on the application of liposome preparations in which drugs are incorporated into simyristoyl phosphatidylcholine. Although drugs are not included, there is a document using simyristoyl phosphatidylcholine in liposome membranes (Cancer Research 4C3).
75-378 (Jan, 1984). ] Its contents are simyristoylphos 7-atedylcholine: simyristoylphosphatidylglycerol-7:3 (molar ratio)
A liposome membrane was synthesized and labeled with t-
The drug was administered alone to the living body without being combined with drugs, and its persistence and organ selectivity were observed. Therefore, there is no description of particle equalization and drug stabilization by freeze-dried V4 seam preparations, which are the objects and effects of the present invention. There is no suggestion that atedylcholine exhibits particularly superior properties.

In the process of comparing cimilitoyl phosphatidylcholine and dipalmitoylphosphatidylcholine, a comparison with cifuculoylphos-7 atidylcholine, whose saturated fatty acid silver has 12 carbon atoms, may be considered, but silauroylphos-7 atidylcholine tends to have a hemolytic effect. (J.Biochem, 78,43
1-434 (1957)]. On the other hand, no hemolytic phenomenon was observed with simyristoylphos-7-atedylcholine.

The present inventors have discovered that arachidonic acid metabolites, which are lipid-soluble drugs,
Structurally similar compounds, their synthase inhibitors and antagonists have been formulated into tVbosomal formulations, and their properties include no foaming during redispersion, uniform particle diameters, and excellent drug inclusion rates. In the process of investigating membrane components that could improve stability while maintaining the same properties, we discovered that simyristoyl phosphatidylcholine showed an extremely high inclusion rate, and we also found that lyophilization using saccharides as excipients was effective. We have completed the present invention by discovering that the customization of silibosome preparations and the stability of drugs can be further improved.

The liposome formulation according to the present invention has a membrane component of 50 to 1
00%, preferably 90-100% of similistoylphos-7-acylcholine as a product, and 1-10% of saccharide as an excipient and stabilizer of the drug as an aqueous phase component at the concentration at the time of manufacture. , freeze-drying). Furthermore, depending on the desired drug, neutral lipids or charged lipids may be added alone or in combination as membrane components.

Examples of sugars as aqueous phase components include lactose, mannitol, maltose, and xylitol, with lactose and mannitol being preferred. Neutral lipids include cholesterol, triglyceride, etc., and charged lipids include phosphatidic acid, stearylamine, etc., but there are no particular limitations, and any generally medicinal lipids may be used.

Included drugs include prostaglandin (PG) A, %PGA2, PGD as arachidonic acid metabolites.
,,PGD2,PGE1. PGE2, PGro, P
Gi'2, PGI□, PG engineering 2, thrombosakin A2,
Leukotriene (LT) A, LTB 4, LTC4,
LTB4. LTB4 and the like can be mentioned, and any structurally similar compounds may be used as long as they are structurally modified compounds with arachidonic acid metabolites as the basic skeleton. Preferably, 6-keto-PGE 1 derivatives (Japanese Patent Application Laid-Open No. 1983-1992) 44
639), carbacycline conductor (Japanese Unexamined Patent Publication No. 1983)
-64541) and PGD2 derivatives (%
-164770], and the synthetase inhibitors and antagonists thereof may be any compounds as long as they inhibit or antagonize the enzyme that biosynthesizes arachiric acid metabolites, but 1. Imidazole conductor (
JP-A-55-313), aminophenol derivatives (see JP-A-58-113152), benzamide derivatives (see JP-A-59-172,570), and thromboxane A2 derivatives (see JP-A-59-119,000) )
etc.

[Manufacturing method]

Known techniques are used to manufacture the liposome formulation in the present invention. For example, a membrane material containing cimilistoylphos-7-atedylcholine as a product is dissolved in an organic solvent such as ethanol, methanol, diethyl ether, or chloroform, mixed with the drug t-dissolved in the above-mentioned organic solvent, and then concentrated under reduced pressure. , into a film-like dry mixture;
The aqueous solution of sugar mentioned above as an aqueous solvent has a total fat concentration t
- Adjust to 1 to 100 da/d, preferably 10 to 50 da/d, shake, perform ultrasonic treatment, filter with a membrane filter, and then dispense into vials and freeze-vacuum dry. It can be manufactured as follows.

〔Example〕

Examples and experimental examples will be shown below, but the present invention is not limited to these examples. Note that compounds a, b%C, d, e, and f used in the six examples are shown in Table 5 at the end.

Example 1 Commercially available synthetic simyristoylphosphatidylcholine 200q
The compound flO- is dissolved in chloroform, and the compound flO- is dissolved in chloroform.
Add rn9'i dissolved in ethanol 1- and add 30
The mixture was placed in a green eggplant flask, and the solvent was distilled off using a rotary evaporator. A 5% man-tol solution [10 dt] was added, shaken, and subjected to ultrasonic irradiation, filtered through a 0.2 μm polycarbonate membrane filter, dispensed into vials in 1 d portions, and then freeze-dried.

Example 2 Commercially available synthetic simyristoylphos 7 atidylcholine 500#
Dissolve klOwl in ethanol and add compound f2 to it.
~10~ dissolved in ethanol 1- is added, and 50
The mixture was placed in a ml eggplant flask, and the solvent was t-distilled off using a rotary evaporator. 5% lactose dissolved GL Qmj!
was added, shaken, and irradiated with ultrasonic waves, filtered through a 0.2 μm polycarbonate membrane filter, dispensed into vials in 1IItl portions, and then freeze-dried.

Example 3 A membrane component containing simiristoylphos-7-acylcholine listed in Table 1 was dissolved in 10-chloroform,
Compound fII to this! A solution dissolved in 19t-ethanol 1- was added, placed in a 50-bottle round bottom flask, and the solvent was removed using a rotary evaporator. Add 10 dt of 5-10% lactose solution.

After shaking and ultrasonic irradiation, the mixture was filtered through a 0.2 μm polycarbonate membrane filter, dispensed into 2 vials (1 mL each), and then freeze-dried.

Stability tests were conducted at 25°C and 40°C.

The remaining percentage of the compound in each sample was determined by HPLC over time.

The results are shown in Table 2. It is suggested that each formulation is stabilized and can be stored at room temperature.

Table 1 Table 2 Example 4 The drugs listed in Table 3 and commercially available synthetic simyristoyl phosphatidylcholine were dissolved in 400 ηtl Od of ethanol and 1
Pour into a 00m eggplant flask and remove the solvent using a rotary evaporator. rm left. 10% lactose solution/120
After adding 1 d'ft of water, shaking, and irradiating with ultrasonic waves, it was filtered with a 0.4 μm polycarbonate membrane filter.
After dispensing the solution into vials, freeze-drying was performed, and bed gas was sealed.

Distilled water was added to each lyophilized liposome preparation and redispersed by shaking gently.

Table 3 The particle diameters (volume distribution) of each are shown in Figures 1 (a) to (f) 2. All liposome formulations have a diameter of approximately 2 when redispersed.
It showed a very good particle size distribution with a volume distribution peaking in μm.

Experimental Example 1 Comparison of particle size distribution of liposomes before and after freeze-drying A control liposome formulation was obtained by carrying out the same operation as in Example 1 using commercially available synthetic dipalmitoylphos 7-acydylcholine 200~. 1 d of distilled water was added to each of the freeze-dried liposome preparations of Example 1 and the control class 21i to disperse them, and the particle size distribution (volume distribution) before and after freeze-drying was compared.

Results - As shown in Figure 2 (a) to 2), the freeze-dried liposome formulation using similistoylphos-7 atidylcholine had a significantly smaller particle size before and after redispersion than the one using dipalmitoylphos-7 atidylcholine. There was no change, and all showed a very good particle size distribution with a volume distribution in the diameter range of 0.5 to 5 μm.

Experimental Example 2 Inclusion rate and partition coefficient during redispersion Commercially available synthetic dipalmitoyl phosphatidylcholine 500!
Similar operations were performed on n9.

Two types of lyophilized liposome preparations were each dispersed using ITnl of distilled water, and equilibrium dialysis was performed for 940 hours using a cellulose dialysis membrane. The concentration of compound f in the internal phase and external phase of the dialysis membrane = i was determined by HPLC, and the inclusion rate and partition coefficient were measured.

Table 4 shows the results. The lyophilized liposomal formulation with simyristoylphos-7-atedylcholine showed a much better partition coefficient compared to that with dipalmitoylphosphatidylcholine, indicating that the uptake of the compound was efficient. Therefore, the influence of shifts in distribution equilibrium due to dilution can be suppressed to a low level.

Table 4 "DPPC: Dipalmitoylphosphatidylcholine ^' [DMPC: Dimyristoyl phosphatidylcholine [Effects of the invention] The liposome preparation produced according to the present invention can be stored in a cool place or at room temperature61), and when used, distilled water for injection, It is used after being redispersed in physiological saline for injection, infusion for injection, etc. In this case, the solution used for redispersion can be of any volume.

Furthermore, since the drugs included in the present invention are fat-soluble, the distribution coefficient between the aqueous phase in the dispersion and the liposome membrane is a major problem. The largest value (10
0 to 5000) t''.

In addition, the liposome preparation according to the present invention can easily be made into a uniform dispersion liquid by just shaking lightly during redispersion), has almost no foaming, and has a liposome particle size with a peak of 0.5 to 5.0 μmK volume distribution. It is something.

It has a size that poses no problem as an injection for intravenous administration, and is therefore useful as an injection for intravenous administration.

Although there is no regulation regarding the particle size for intravenous administration, the liposome preparation of the present invention fully satisfies this condition, which requires that it be smaller than blood cells.

By using dipalmitoyl phosphatidyl coli as a membrane component, arachidonic acid metabolites, structurally similar compounds thereof, and their synthetase inhibitors and antagonists are formulated into liposome formulations and lyophilized. This makes it possible for the first time to create practical liposome formulations. Became.

This preparation satisfies all of the conditions necessary for use as a pharmaceutical, namely mass production, distribution, storage, and easy usage, and therefore the value of the use of the present invention as a preparation is extremely high for commercialization in the future. expensive.

Furthermore, the liposome preparation according to the present invention selectively transports these drugs to the liver, pancreas, lungs, and other areas of inflammation, and can be expected to have various main effects with a small dose without causing side effects. can.

[Brief explanation of drawings]

Figures 1 (a) to (f) are graphs showing the particle size distribution (volume distribution) of a lyophilized liposome preparation upon redispersion using simyristoyl phosphatidylcholine containing the five types of drugs listed in Table 5; Figure 2 is a graph showing the particle size distribution (volume distribution) t of liposome preparations before and after freeze-drying. The symbol "-4" indicates the distribution of di/falcolumitoyl phosphatidylcholine before freeze-drying, and (-4 indicates the distribution after freeze-drying. (3 people) 1st (a) Compound a Liigo Gu7At#L%d(pm) (c) Compound -C Rifuosome no Suidate) 11 (ri^) Figure (b) Compound b ShiJ? Thor 4's palm t is good ◇#)L) (d) Formation 4-Things d Riwaimurim'/@(7ttS(E)Combination #I?)e Ritamatsum, 0 palm ff) (ban (, u 夙) 1 figure

Claims (1)

[Scope of Claims] 1) Dimyristoylphosphatidylcholine is used as a component of the liposome membrane material, and arachidonic acid metabolites, structurally similar compounds thereof, and synthetic enzyme inhibitors and antagonists thereof are contained as active ingredients, Liposome preparations are freeze-dried with the addition of sugars. 2) The liposome preparation according to claim 1, wherein the arachidonic acid metabolite is prostaglandin, prostacyclin, thromboxane, or leukotriene. 3) Arachidonic acid metabolite structurally similar compounds are 6-keto-PGE_1 derivatives, carbacycline derivatives or
The liposome preparation according to claim 1, which is a GD_2 derivative. 4) The liposome preparation according to claim 1, wherein the saccharide is lactose or mannitol. 5) The liposome preparation according to any one of claims 1 to 4, wherein the liposome membrane material further contains a neutral lipid or a charged lipid. 6) The liposome preparation according to claim 5, wherein the neutral lipid is cholesterol or triglyceride.