JPH03851B2 - - 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 the 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 a disease caused by a congenital bleeding diathesis that has been known for a long time, and the frequency of surviving hemophilia patients is said to be 1 in 22,000 males, and the incidence is approximately 1 in 22,000 males. It is estimated that it affects 1 in 8,000 people. Among these, hemophilia B is a congenital deficiency of blood coagulation factors that occurs in 15% of cases, and the basic treatment is to supplement the coagulation factor that the patient is deficient in. It is said that there is.

Historically, this factor has been replenished by transfusion of fresh blood, but highly concentrated factor preparations have been developed, and in recent years, factor preparations containing a large amount of this blood clotting factor and other substances such as fibrinogen have been developed. Concentrated preparations with low factor content have become widely used.

However, these preparations are only administered by infusion, 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. In addition, even if the dosage form is encapsulated in liposomes, if they come into contact with hydrochloric acid in the stomach, the pH inside the liposomes will tilt towards the acidic side, resulting in deactivation, so it will not be absorbed from the intestinal tract until it maintains its activity. becomes.

Until now, there has been no example of an orally administered drug by microcapsulating blood coagulation factors.
Although there are only a few reports of orally administered agents for factor [HCHemker et al., Lancet, 8159,
70 (1980)], repeated attempts at such reported cases did not yield satisfactory results. Conventionally, blood coagulation was not degraded in the digestive system and was sufficient to reach the absorption site in the small intestine. Orally administered agents have yet to emerge.

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 drug for hemophilia B according to the present invention.

The details of the present invention will be explained 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 lost. is released. The released contents are covered with a liposomal membrane, which prevents them from coming into contact with digestive fluids in the intestinal tract and preventing them from being degraded. In this case, if a broad-spectrum protease inhibitor is encapsulated, the action of the protease will be inhibited, making it more effective in preventing decomposition. Examples of protease inhibitors include aprotinin, dimethylcarbamoylmethyl-p-(p-
-guanidinobenzoyloxy)phenyl acetate mesylate 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 into cells by 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 experiments involving the fusion of model membranes or biological membranes with multilayer liposomes.

The main component of the liposome membrane is egg yolk lecithin, but any suitable material can be selected from commonly known materials and added. Blood coagulation factor is a macromolecule with a molecular weight of 70,000, exhibits an isoelectric point of 4.3 to 4.5, and is negatively charged in a neutral solution. Therefore, when phosphatidic acid is added for the purpose of improving liposome moldability and stability, the amount of factor components taken into liposomes can be increased by adding Ca ⁺⁺ ions. The amount of uptake can also be increased by adding stearylamine to lecithin, and when lecithin is the only constituent material, the same effect can be expected by increasing the ionic strength of the internal and external phase solutions.

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 liposome phase is reconstituted by the water.

(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 1 containing 10% phosphatidic acid
Dissolve g in 20 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. This is dried under reduced pressure, and 20 ml of a Ca ⁺⁺ ion-containing buffer solution with a pH of 7.0 containing 400 units of blood coagulation factor and 5 units of aprotinin is added and shaken gently to prepare a liposome suspension.
Transfer the suspension to a centrifuge tube, cool to 10℃, and incubate at 27000G.
Centrifuge for 20 minutes to obtain a creamy liposome phase at the top. The clear aqueous phase is returned to the eggplant flask coated with lecithin in step 1, and the above operation is repeated three times in total. Combine the creamy liposome phases obtained in the three operations, disperse this in 20 ml of physiological saline, wash the liposome outer phase,
Separate and collect the liposome phase by centrifuging at 40,000 G for 10 minutes while cooling at °C, and remove as much water from the outside of the liposomes as possible. The volume of the liposome obtained by this procedure is approximately 10 ml, which contains 200 units of blood coagulation factor and 15,000 units of aprotinin.

Next, a size 0 gelatin capsule with a thin coating of food oil on the inside of the capsule is filled with the liposome obtained above, 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 40 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, 1 g of egg yolk lecithin containing 10% phosphatidic acid or phosphatidic acid calcium salt was dissolved in 20 ml of ethanol, and the internal volume was 1
Place in an eggplant-shaped flask and concentrate under reduced pressure to coat the inner wall with a thin film of lecithin. This is dried under reduced pressure, and 20 ml of a pH 7.0 calcium ion-containing buffer solution containing 400 units of blood coagulation factor and 50,000 units of aprotinin is added thereto and gently shaken to prepare a liposome suspension. Transfer the suspension to a centrifuge tube and incubate for 10
Centrifuge at 27,000G for 20 minutes under cooling to obtain a creamy liposome phase at the 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 creamy liposome phases obtained in the three operations, disperse this in 30 ml of physiological saline, add an appropriate amount of distilled water to uniformly disperse the liposomes, and then -
After rapid freezing at 40°C to -60°C, lyophilization is performed by sublimating water under reduced pressure. Stabilizers such as sugars may be added to the liposome particles during lyophilization.
The total amount of liposome lyophilized product produced by this procedure contains 200 units of blood coagulation factor and aprotinin.
Contains 15000 units. After the freeze-dried liposome powder is lightly pulverized in a stream of dry nitrogen, it is filled into size 0 enteric-coated capsules, the joints are completely sealed using an acetone solution of the raw material, and the capsules are 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 A calcium ion-containing buffer (PH7.0) containing 400 units of blood coagulation factor, 1 g of egg yolk lecithin containing 10% phosphatidic acid calcium salt, and 50,000 units of aprotinin was lyophilized with the factor intact, and Lecithin was freeze-dried from a benzene solution, and an aprotinin solution was also freeze-dried to obtain three 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 capsules are sealed in the same manner as in Examples 1 and 2 using an acetone solution containing the same material. This capsule 1
Each can be filled with more than 50 units of factor. This dosage form is extremely stable and can withstand long-term storage.
Easily forms liposomes by adding a small amount of water,
The liposome contains a considerable amount of factor factor.

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.