JPH03366B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to an orally administered blood coagulation factor. More specifically, the present invention prevents deactivation and decomposition of blood coagulation factors by formulating blood coagulation factors using liposomes and enteric-coated capsules, and also enables efficient absorption from the epithelial cells of the small intestine mucosa. The present invention relates to a novel drug for the treatment of hemophilia, which is an orally administered drug. Hemophilia is one of the oldest known diseases caused by a congenital bleeding diathesis, and the frequency of living hemophilia patients is said to be 1 in 22,000 males, and the incidence is higher than that of a male birth. It is estimated that it affects approximately 1 in 8,000 people. One of these, called hemophilia A, is a congenital deficiency of blood clotting factors.
The basis of its treatment is said to be to supplement the coagulation factor that the patient is lacking. Historically, this factor has been replenished by transfusion of fresh blood, but highly concentrated factor preparations have been developed and have recently been developed that contain a large amount of this blood clotting factor and other factors such as fibrinogen. Concentrated formulations with low content have become widely used. However, these preparations are only administered by intravenous injection, and there has been a strong desire to develop oral preparations that are easier to handle than injections. By the way, if blood coagulation factor is simply prepared in the form of ordinary oral dosage forms such as solutions, tablets, and capsules, it can be absorbed by hydrochloric acid and pepsin in the stomach, and by trypsin and chymotrypsin in pancreatic juice in the intestine. Since it is degraded by degrading enzymes, it cannot be absorbed from the intestinal tract. Furthermore, even if the drug is encapsulated in a liposome, when it comes into contact with hydrochloric acid in the stomach, the pH inside the liposome tilts toward the acidic side, resulting in most of the drug being decomposed, so it is not absorbed from the intestinal tract. Up to now, there have been reports of oral administration of blood coagulation factors by microencapsulating them [HChemKer et al. Lancet,
8159, 70 (1980)], even if such reported cases are repeated,
Satisfactory results have not been obtained, and there has yet to be an orally administered drug for blood coagulation factors that is not degraded in the digestive system and can reach the absorption site of the small intestine. It is in. As a result of various studies aimed at realizing an orally administered agent for blood coagulation factor, the present inventors succeeded in providing a novel therapeutic agent for hemophilia A according to the present invention. The present invention will be explained in detail below. The present invention comprises: (a) blood coagulation factor added with a broad-spectrum protease inhibitor and encapsulated in liposomes; (b) the liposome encapsulated product of (a) above freeze-dried; and (c) blood. A blood coagulation method characterized in that a coagulation factor, a broad-spectrum protease inhibitor, and a liposome membrane constituent material are each freeze-dried and one selected from the group consisting of a mixture thereof is filled into an enteric-coated capsule. The present invention provides an orally administered agent for the factor. The present invention has succeeded in providing an orally administered drug that is efficiently absorbed from the epithelial cells of the small intestine mucosa while preventing the decomposition of blood coagulation factors due to the above-mentioned characteristic structure, and is applicable to the treatment of hemophilia. This provides an extremely excellent and valuable formulation for oral administration. The anti-degradation mechanism in the digestive system and the absorption mechanism from the small intestinal mucosa of the orally administered agent of the present invention will be explained. Since the oral preparation of the present invention is filled in an enteric-coated capsule, it passes through the stomach and duodenum and reaches the small intestine.When the pH in the intestinal tract becomes neutral or alkaline, the capsule disintegrates and the contents are removed. is released. Since the released contents are covered with a liposome membrane, they are prevented from coming into contact with digestive fluids in the intestinal tract and are prevented from decomposing. At that time, if a broad-spectrum protease inhibitor is encapsulated, the action of the protease will be inhibited, which will be more effective in preventing decomposition. As the protease inhibitor, for example, apronitine is preferably used. The contents thus released can reach the vicinity of the absorption site of the small intestinal mucosal epithelial cells while retaining the function of preventing the decomposition of blood coagulation factors. Blood coagulation factor coated with liposomes is taken up by absorbing cells without being degraded.
It is thought that either pinocytosis occurs in the cell membrane between the microvilli of absorbing cells, or fusion occurs between the cell membrane and the outermost liposome membrane. What is taken up by pinocytosis into the absorptive cells of the upper small intestine is released outside the cells without undergoing intracellular digestion in lysosomes, and then enters the bloodstream via the central chyle duct. It is thought that a similar route is followed when the protein is taken up into cells through fusion. In order for blood coagulation factors to be efficiently taken up into absorption cells, the size of liposomes must be 1 μm.
The front and rear membranes are preferably multilayer liposomes with two to three layers. Furthermore, it is desirable that the osmotic pressure of the aqueous phase within the liposome is higher than that of the absorbing cells. This has been proven in fusion experiments using model membranes. As the constituent material of the liposome membrane, any suitable material can be selected from commonly known materials. For example, it is preferable to use egg yolk lecithin as the main component, but adding an appropriate amount of phosphatidic acid, phosphatidyl serine, etc. to this can improve liposome forming ability and liposome stability. Addition of cholesterol strengthens the membrane, and addition of lysolecithin facilitates fusion. Phosphatidic acid is useful for increasing the amount of encapsulation by increasing the size of particles, and phosphatidylserine is useful for improving film-forming ability. Properties required of the liposome in the present invention include the following. (1) The liposome phase obtained by centrifugation and cream separation does not lose its basic form as a liposome even if the outer phase has low water content. (2) In the state of (1), when the liposome phase is freeze-dried by adding water to the outer phase, the water restores it to the original liposome. (3) Blood coagulation factor, broad-spectrum proteinase inhibitor, and liposome membrane constituent material are each freeze-dried, and a mixture of these can easily form liposomes by adding water. The present invention will be specifically explained below with reference to examples, but the present invention is not limited to the following examples. Example 1 Egg yolk lecithin 2 containing 5% phosphatidic acid
Dissolve g in 40 ml of ethanol, put into an eggplant-shaped flask with an internal volume of 1, and concentrate under reduced pressure to coat the inner wall with a thin film of lecithin. Dry this under reduced pressure,
Add 50 ml of a pH 7.0 buffer solution containing 3000 units of blood coagulation factor and 150,000 units of aprotinin, and shake gently to create a liposome suspension. Transfer the suspension to a centrifuge tube and centrifuge at 27,000G for 20 minutes while cooling to 10°C to obtain a creamy liposome phase at the top. The clear aqueous phase is coated with lecithin again, returned to the eggplant-shaped flask from step 1, and the above-mentioned operation is repeated three times in total. The cream-like liposome phases obtained in the three operations were combined, dispersed in 50 ml of physiological saline to wash the liposome outer layer, and centrifuged at 40,000 G for 10 minutes while cooling to 10°C to separate the liposome phase. Separate and collect the liposomes and remove as much water as possible from the outside of the liposomes. The volume of liposomes obtained by this operation is approximately
10ml contains 1000 units of blood coagulation factor and 50000 units of aprotinin. Next, the liposomes obtained above are filled into a size 0 gelatin capsule whose inner surface is thinly coated with food oil, and this capsule is filled into an enteric-coated capsule to form a double structure. Apply an acetone solution of the material to completely seal it. One capsule contains 50 units of factor. If stored in a cool and dark place, no decrease in activity will be observed in a short period of time. Example 2 As in Example 1, 2 g of egg yolk lecithin containing 10% phosphatidic acid was dissolved in 40 ml of ethanol,
The mixture is placed in an eggplant-shaped flask with an internal volume of 1, and concentrated under reduced pressure to coat the inner wall with a thin film of lecithin. This is dried under reduced pressure, and blood coagulation factor 3000 is added to it.
Add 50 ml of a pH 7.0 buffer solution containing 150,000 units of aprotinin and shake gently to create a liposome suspension. Transfer the suspension to a centrifuge tube and cool at 10℃.
Centrifuge at 27,000 for 20 min to obtain a creamy liposome phase on top. The aqueous phase is returned to the eggplant-shaped flask coated with lecithin in step 1, and the above-mentioned operation is repeated a total of three times. Combine the cream-like liposome phases obtained from the three operations, disperse this in 50 ml of physiological saline, add an appropriate amount of distilled water to uniformly disperse the liposomes, and incubate at -40° to 60°C.
After rapid freezing, the water is sublimated under reduced pressure and freeze-dried. Stabilizers such as sugars may be added to the liposome particles during lyophilization. The total amount of liposome freeze-dried product produced by this procedure contains 1000 units of blood coagulation factor and 50000 units of aprotinin. After the freeze-dried liposome powder is lightly pulverized in a stream of dry nitrogen,
It is filled into size 0 enteric-coated capsules, completely sealed using an acetone solution for the seam material, and air-dried. Each capsule can be filled with more than 50 units of blood coagulation factor and can be stored for a long time if stored in a cool, dark place. The liposomes of this product are easily restored by adding water, and almost all of the factor is contained in the liposomes. Example 3 2000 units of blood coagulation factor, 5 g of egg yolk lecithin containing 15% phosphatidic acid, and 50,000 units of aprotinin injection solution were freeze-dried with the factor intact, lecithin was freeze-dried from a benzene solution, and aprotinin injection solution was also freeze-dried. Lyophilize to obtain three types of dry powders. These three lyophilized products are mixed in a stream of dry nitrogen and filled into No. 0 enteric-coated capsules. At this time, an appropriate amount of saccharide such as glucose, lactose, mannitol, etc. may be added for the purpose of adding fluidity. The seams of the capsule were made using an acetone solution containing the same material as in Examples 1 and 2.
Seal as in the case. One capsule can be filled with 100 units of factor. This dosage form is extremely stable, can withstand long-term storage, easily forms liposomes by adding a small amount of water, and contains a considerable amount of factor factor in the liposomes. Example 4 Egg yolk lecithin 2 containing 5% phosphatidic acid
Dissolve g in 40 ml of ethanol, put into an eggplant-shaped flask with an internal volume of 1, and concentrate under reduced pressure to coat the inner wall with a thin film of lecithin. Dry this under reduced pressure,
Add 50 ml of a pH 7.0 buffer solution containing 3000 units of blood coagulation factor and 50,000 units of aprotinin, and shake gently to prepare a liposome suspension. Example 1
Treated in the same manner as 1000 units of blood coagulation factor,
Obtain 10 ml of liposomes containing 17000 units of aprotinin. The liposome thus obtained is filled into enteric-coated capsules in the same manner as in Example 1. One capsule contains 50 units of blood coagulation factor.
If stored in a cool and dark place, no decrease in activity will be observed. Reference example 1 Egg yolk lecithin containing 5% phosphatidic acid
Dissolve 250 mg in 40 ml of ethanol, put into a 200 ml eggplant-shaped flask, and concentrate under reduced pressure to coat the inner wall with a thin film of lecithin. Dry this under reduced pressure, add an aqueous solution of 4000 units of blood coagulation factor,
Shake gently to create a liposome suspension. Transfer the suspension to a centrifuge tube and centrifuge at 27,000G for 20 minutes while cooling to 10°C to obtain a creamy liposome phase at the top. Clear water phase coached lecithin again
Return to the 200ml eggplant-shaped flask and repeat the above procedure twice. The cream-like liposome phases obtained in the two operations were combined, dispersed in 50 ml of physiological saline to wash the liposome outer phase, and centrifuged at 50,000 G for 20 minutes while cooling to 10°C to remove the liposome phase. Separate and collect. Dilute this with physiological saline to make a total volume of 50 ml. The suspension thus obtained contains 800 units of blood coagulation factor. Reference example 2 Egg yolk lecithin 2 containing 5% phosphatidic acid
Dissolve g in 40 ml of ethanol, put into an eggplant-shaped flask with an internal volume of 1, and concentrate under reduced pressure to coat the inner wall with a thin film of lecithin. Dry this under reduced pressure,
Add 50 ml of a pH 7.0 buffer solution containing 3000 units of blood coagulation factor and shake gently to create a liposome suspension. This is treated in the same manner as in Example 1 to obtain 10 ml of liposomes. The liposomes thus obtained are filled into gelatin capsules. One capsule contains 50 units of blood coagulation factor. Reference Example 3 In the same manner as in Reference Example 2, 10 ml of liposome containing blood coagulation factor was obtained. This liposome is filled into a size 0 gelatin capsule whose inner surface is thinly coated with food oil, and this capsule is filled into an enteric-coated capsule to form a double structure. Apply an acetone solution of the material to the seams of the outer capsule to completely seal them. One capsule contains 50 units of blood coagulation factor. Experimental Example 1 The following blood coagulation factor preparations were administered to a female patient with hemophilia A on separate days. Oral administration (1) Twenty enteric-coated capsules obtained in Example 4 were orally administered. (2) 50 ml of the suspension obtained in Reference Example 1 was orally administered. Intravenous administration Commercially available intravenous blood coagulation factor preparations:
250 units of Conchoate (manufactured by Midori Juji Co., Ltd.) was administered intravenously. Blood was collected before and at regular intervals after administration of the blood coagulation factor preparation, and blood was collected using Matsuoka's method (Matsuzo Matsuoka, "Blood Coagulation Test", pp. 61-165, published by Kanehara Publishing, 1977). Activity was measured. The amount of blood coagulation factor is expressed as a percentage calculated assuming that a healthy person is 100% and a person without factor is 0%. The results are shown in Table 1.

[Table] As is clear from the results in the above table, when factor factor is simply encapsulated in liposomes, its activity cannot be maintained and it is not efficiently absorbed from the intestinal tract. If so, it maintains its activity and is efficiently absorbed from the intestinal tract. Experimental Example 2 The following blood coagulation factor preparations were administered to a male patient with hemophilia A on separate days. (1) Twenty enteric-coated capsules obtained in Example 4 were orally administered. (2) Twenty enteric-coated capsules obtained in Reference Example 3 were orally administered. Blood was collected before and at regular intervals after the administration of blood coagulation factor, and the activity of factor factor was measured in the same manner as in Experimental Example 1. The results are shown in Table 2.

[Table] As is clear from the results in the table above, factor factor preparations with aprotinin added maintain their activity and are sufficiently absorbed from the intestinal tract, whereas factor factor preparations without aprotinin cannot maintain their activity. Not absorbed from the intestinal tract. Experimental Example 3 A young male patient with hemophilia A was administered the following blood coagulation factor preparations on separate days. (1) Twenty enteric-coated capsules obtained in Example 4 were orally administered. (2) Twenty enteric-coated capsules obtained in Reference Example 2 were orally administered. Blood was collected before and at regular intervals after the administration of blood coagulation factor, and the activity of factor factor was measured in the same manner as in Experimental Example 1. The results are shown in Table 3.

[Table] As is clear from the results in the table above, factor factor preparations with aprotinin added maintain their activity and are sufficiently absorbed from the intestinal tract, whereas factor factor preparations without aprotinin cannot maintain their activity. Not absorbed from the intestinal tract.

Claims (1)

[Scope of Claims] 1 (a) Blood coagulation factor added with a broad-spectrum protease inhibitor and encapsulated in liposomes, (b) Liposome encapsulated product of (a) above freeze-dried, and ( c) A blood coagulation factor, a broad-spectrum proteinase inhibitor, and a liposome membrane constituent material are each freeze-dried and a mixture thereof is filled into enteric-coated capsules. Orally administered blood coagulation factor.