FR2492260A1 - PHARMACEUTICAL COMPOSITION FOR ORAL ADMINISTRATION CONTAINING COAGULATION FACTOR VIII - Google Patents

Abstract

THE INVENTION RELATES TO A PHARMACEUTICAL COMPOSITION FOR ORAL ADMINISTRATION CONTAINING THE COAGULATION FACTOR VIII; A PHARMACEUTICAL COMPOSITION SUITABLE FOR THE TREATMENT AND PROPHYLAXIS OF HEMOPHILIA INCLUDING THE COAGULATION FACTOR VIII AND A PROTEASE INHIBITOR (IN PARTICULAR APROTININ) WHICH ARE INCORPORATED IN LIPOSOMES AND POTENTIALLY LYOPHILISED AND ENAPULATED. THE COMPOSITION ALLOWS TO OBTAIN A STRONG ABSORPTION OF THE COAGULATION FACTOR BY THE INTESTINAL PATHWAYS WITHOUT IT BEING DECOMPOSED IN THE GASTROINTESTINAL PATHWAYS EVEN BY ORAL ADMINISTRATION.

Description

The present invention relates to a pharmaceutical composition for

  oral administration containing the factor

coagulation VIII.

  More particularly, the invention relates to a new pharmaceutical composition suitable for the treatment or prophylaxis of hemophilia A by oral administration, which contains coagulation factor VIII and a protease inhibitor which are incorporated into liposomes and optionally lyophilized and / or encapsulated in

enteric-coated capsules.

  It is well known that hemophilia is a hereditary disorder

  coagulation disorder due to abnormalities of the anti-haemo-

  hemophilia affects 1 in 22,000 people in men and can be seen at birth in about 1 in 8,000 cases. Among these bleeding disorders, hemophilia A. is due to an inherited absence of the factor coagulation VIII. One can, to remedy this disease, provide the coagulation factor

to the sick who are missing.

  The necessary coagulation factor has been supplied by infusion of fresh blood, - but recently, since the appearance of preparations in which the factor is very concentrated, such preparations which contain large quantities of coagulation factor and are commonly used quantities

  less other factors such as fibrinogen.

  However, these preparations are generally administered intravenously and an effort is therefore being made to develop a preparation suitable for oral administration which is easier to handle.

than injections.

  When simply coagulation factor VIII is incorporated into standard oral preparations such as an aqueous preparation, tablet or capsule, it is broken down by hydrochloric acid or pepsin in the stomach or by enzymes such as trypsin or chymotrypsin in intestinal or pancreatic juice and therefore cannot be absorbed from

  effectively through the intestinal tract while retaining its activity.

  Oral administration of coagulation factor VIII was first reported by Hemker et al. who have treated factor VIII with liposomes [see H.C. Hemker et al.,

  Lancet, 8159, 70 (1980) and PCT Internation Publication no. W080 / 01456].

  The Applicant has re-examined Hemker's method but has been unable to obtain satisfactory results. Therefore, even when coagulation factor VIII is incorporated into liposomes, it is easily broken down by the various enzymes contained in gastric juice or intestinal juice when administered orally and therefore cannot not be absorbed from the intestinal tract

retaining its activity.

  Therefore, there is no known oral preparation of coagulation factor VIII that can be administered orally in order to

  that the factor be absorbed effectively through the intestinal tract

  without being broken down in the gastrointestinal tract.

  Following important studies, the applicant has discovered that an improved oral preparation can be obtained.

  coagulation factor VIII by incorporation of the factor into lipos

  somes with a protease inhibitor, possibly followed by

  lyophilization and / or encapsulation in entero capsules

soluble.

  One of the objects of the invention is a pharmaceutical composition.

  improved coagulation factor VIII therapy suitable for oral administration, which is useful for oral treatment and prophylaxis of hemophilia A. Another subject of the invention

  is a new pharmaceutical composition of coagula factor-

  tion VIII allowing the effective absorption of the factor by the

  intestinal without it being broken down in the gastrointestinal tract

  tinal. Other characteristics and advantages of the invention

  will emerge from the description which follows.

  The pharmaceutical composition of the invention contains coagulation factor VIII and a protease inhibitor which are incorporated into liposomes. Preferably, the composition is encapsulated in enteric-coated capsules to be less affected by gastric juice and duodenal juice. In addition, preferably, the

  composition is lyophilized to increase storage stability.

  In another embodiment, the coagulation factor VIII, the protease inhibitor and the material used to form liposomes are lyophilized separately and the lyophilized materials

  are mixed and then encapsulated in enteric-coated capsules.

  The pharmaceutical composition of the invention may optionally contain stabilizers such as mannitol, albumin, dextran, etc., and moreover other additives to improve the fluidity of the lyophilized product such as glucose, lactose, mannitol, etc. Coagulation factor VIII comes from humans and

  other mammals such as pigs and cattle.

  The protease inhibitor used in the invention is

  consisting of aprotinin, p- (p-guanidinobenzoyloxy) mesylate -

  dimethylcarbamoylmethyl phenylacetate, etc. The protease inhibitor is preferably incorporated at a rate of 10 to 100 units per

  unit of coagulation factor VIII.

  In order for coagulation factor VIII to be efficiently absorbed into the absorption cells, the liposomes are preferably about 1 µm in size and are preferably made up of several lamellae such as two or three lamellae. In addition, the aqueous phase included in the liposomes has a higher osmotic pressure than that of the absorption cells. The liposomes can be formed from any conventional material. Examples of preferable materials are a phospholipid comprising essentially egg yolk lecithin with the addition of an appropriate amount of phosphatidic acid or phosphatidylserine to improve the liposome forming capacity or the stability of liposomes. In addition, cholesterol can also be incorporated to strengthen the liposome membrane and add lysolecithin to improve the fusion of the liposome membrane to the biomembrane of absorption cells. Phosphatidic acid increases the size of the liposome particles, which increases the amount of materials included therein. Phosphatidylserine improves the ability to

membrane formation.

  The liposomes used in the invention must have

the following properties.

  (1) Even if we separate the liposome phase by centri-

  fugation, and that the external phase has only a low water content,

  liposomes can retain their basic shape.

- 2 492260

  (2) When the liposomes obtained by centrifugation are lyophilized as in (1) above, the liposomes can easily be restored to their original state by adding water to the lyophilized product. (3) When lyophilizing coagulation factor VIII, the protease inhibitor and the liposome producing material, the mixture of these lyophilized materials can easily form

  desired liposomes by addition of water.

  One can include in the liposomes the factor of

  coagulation VIII and the protease inhibitor according to a standard process

  as described in the literature for example in "Methods in Cell Biology" published by David M. Prescott, Vol. XIV, page 34, For

  avoid an undesirable decrease in clotting factor activity

  lation VIII during incorporation into liposomes, it is preferred to use

a neutral buffer solution.

  When the pharmaceutical composition of the invention is administered orally, in which coagulation factor VIII and a protease inhibitor are incorporated into liposomes, it reaches the absorption zone (epithelial cells) of the small intestine where it is absorbed without any decomposition of factor VIII, even by contact with digestive juices in the gastrointestinal tract. When the composition is encapsulated in enteric-coated capsules, it reaches the small intestine by passage through the stomach and the duodenum, as it is and the capsule is degraded in the small intestine at a neutral or alkaline pH to release its content in the intestinal tract. Due to the coexistence of a protease inhibitor, factor VIII is protected against breakdown in the small intestine and is absorbed

effectively.

  Therefore, the pharmaceutical composition of the invention is

  useful for oral treatment and prophylaxis of helophilia.

  The dosage of the pharmaceutical composition of the invention may vary according to the age, body weight and sex of the patients as well as the severity of the symptom, but it is generally such that the active ingredient (coagulation factor VIII) is

  administered at the rate of 500 to 3,000 units per day.

  The excellent therapeutic effects of the pharmaceutical composition of the invention are confirmed by the following clinical trials. Trial 1 A patient with hemophilia A coagulation factor VIII is administered in the following preparations at

different days.

Oral administration.

  1) The composition is administered orally (20 capsules

  enteric-coated) obtained in Example 4.

  2) The composition (50 ml) is administered orally

  obtained in reference example 1.

Intravenous injection.

  250 units of a commercially available coagulation factor VIII injection are administered intravenously:

  Concoeight (manufactured and marketed by Green Cross Co. in Japan).

  Before and after administration of the preparations, blood is drawn at fixed intervals and the factor VIII activity in the blood is determined according to the direct Matsuoka method (see Matsuoka, M .; "Laboratory methods of blood coagulation", Kanahara, pages 61-165, Tokyo, 1977) according to which the quantity of coagulation factor VIII is expressed by a percentage (%) calculated so that the quantity of coagulation factor VIII of a normal subject is 100% 7. and that of a subject having no coagulation factor VIII is 0%. The results appear in

table 1.

TABLE 1

  Timing of blood samples (hours) Preparation Route

O 2 4 6 8 10

  Composition of p.o. 2.0 3.4 3.3 3.3 - -

example 4

  Composition of p.o. 2.0 1.8 2.0 1.9 1.7 -

the example of

  reference 1 i.v. preparation 2.0 11.0O 3.0 2.0 2.0 2.0

injectable

6 2492260

  As the results show above, when coagulation factor VIII is simply incorporated into liposomes, it is not effectively absorbed by the intestinal tract while retaining its activity, whereas, when incorporated into liposomes, liposomes with aprotinin (protease inhibitor), it is well absorbed by the intestinal tract while retaining its activity. Trial 2 Coagulation factor VIII is administered to two male patients with hemophilia A

following on different days.

  1) The composition is administered orally (20 capsules

  enteric-coated) obtained in Example 4.

  2) The composition is administered orally (20 capsules

  enteric-coated) obtained in Reference Example 3.

  Before and after administration of the preparations, blood is drawn at fixed intervals and the factor VIII activity in the blood is determined in the same way as in test 1. The

  results are shown in Table 2.

TABLE 2

  The above results clearly show that the factor VIII preparation supplemented with aprotinihe is well absorbed by the intestinal tract while retaining its activity, while, on the other hand, the factor VIII preparation containing no aprotinin does not is not absorbed from the intestinal tract in

retaining its activity.

  Timing of blood samples (hours) Preparations

O 2 4 6 8

Composition of 1.4 1.8 2.1 1.8 1.9

example 4

Composition of 1.4 1.3 1.3 1.4 1.3

the example of

  reference 3 Trial 3 Coagulation factor VIII is administered to a young male patient with hemophilia A

following on different days.

  1) The composition is administered orally (20 capsules

  enteric-coated) obtained in Example 4.

  2) The composition is administered orally (20 capsules)

  obtained in reference example 2.

  Before and after administration of the preparations, blood is drawn at fixed intervals and the factor VIII activity is determined in the same way as in the test. 1. The results

are shown in Table 3.

TABLE 3

  It is clear from the above results that the preparation of factor VIII containing aprotinin is well absorbed by the intestinal tract while retaining its activity,

  while the factor VIII preparation containing no apro-

  tinine is not absorbed from the intestinal tract when stored

his activity.

  The preparation of the pharmaceutical composition of

  the invention is illustrated by the following nonlimiting examples.

EXAMPLE 1

  2 g of egg yolk lecithin containing 5% h of phospatidic acid are dissolved in 40 ml of ethanol. The solution is introduced into a 1 liter flask and concentrated under reduced pressure so that the lecithin is deposited in the form of a thin layer over the time of blood sampling (hours) Preparations _

O 2 4 6

Composition of 2.0 2.8 2.8 3.5

example 4

  Composition of example 2.0 1.9 1.9 2.0 reference 2 the inner wall of the flask. After drying under reduced pressure, 50 ml of a phosphate buffer solution (pH 7.0) containing

  - 3,000 units of coagulation factor VIII and 150,000 units of apro-

  tinine and the balloon is gently shaken to obtain a suspension of liposomes. The liposome suspension thus obtained is introduced into a centrifuge tube, cooled to 10C and then centrifuged at 27,000 G for 20 min to obtain creamy liposomes at the top. The transparent aqueous layer is again treated with lecithin and the mixture is treated in the flask as above. These operations are repeated three times. The creamy liposomes obtained during the above three operations are combined and suspended in 50 ml of physiological saline to wash the outer layer of the liposomes. After cooling to C, the suspension is centrifuged at 40,000 G for 10 min to collect the liposomes and the liposomes are dried to remove the water contained in the outer layer as much as possible. The liposomes thus obtained have a volume of approximately 10 ml and contain

  1,000 units of coagulation factor VIII and 50,000 units of apro-

  tinine. The liposomes obtained above are introduced into capsules (size no 0, the inner wall of which is coated with a thin layer of edible oil) which are themselves introduced into

  enteric-coated capsules to achieve a double capsule structure.

  The seal of the outer capsules is made completely sealed by treatment with a solution of the material forming the capsules enterosoluble in acetone. The capsules each contain units of coagulation factor VIII. When the capsules are kept away from heat and in the dark, the activity of the

  factor VIII does not decrease rapidly.

EXAMPLE 2

  As described in Example 1, 2 g of egg yolk lecithin containing 10% phosphatidic acid are dissolved in 40 ml of ethanol and the solution is concentrated under reduced pressure in a 1 liter flask to deposit the lecithin as a thin layer on the inner wall of the balloon. After drying under reduced pressure, 50 ml of a phosphate buffer solution (pH 7.0) containing 3,000 units of coagulation factor VIII and 000 units of aprotinin are added and the flask is gently shaken to obtain a suspension of liposomes . The suspension of liposomes is introduced into a centrifuge tube, cooled to 100C and then centrifuged at 27,000 G for 20 min to obtain creamy liposomes at the top. The transparent aqueous layer is again treated with lecithin and the mixture is treated in the flask in the same manner as above. These operations are repeated three times. The creamy liposomes obtained in the three operations above are combined, they are suspended in 50 ml of physiological saline and an appropriate quantity of distilled water is added in which the liposomes are uniformly dispersed. The mixture is quickly frozen by cooling between -40 and -601C

  and lyophilized under reduced pressure with sublimation of the water.

  During freeze-drying, sugars can be added to the particles

liposomes as stabilizers.

  The lyophilized liposomes thus obtained contain

  1,000 units of coagulation factor VIII and 50,000 units of apro-

  tinine. The lyophilized liposomes are gently fragmented under a

  stream of dry nitrogen and then introduced into enteric capsules

  soluble (size n0 O). After making the capsule seal completely sealed by treatment with an acetone solution of the material forming the enteric-coated capsules, the capsules are air dried. Each enteric-coated capsule thus obtained contains units of coagulation factor VIII. This product can be stored for an extended period of time when stored away from heat and in the dark. The liposomes of this product can

  easily return to their original form by adding water.

EXAMPLE 3

  2,000 units of coagulation factor VIII are freeze-dried separately, 5 g of egg yolk lecithin containing 15% phosphatidic acid and an injectable liquid containing 50,000 units of aprotinin, factor VIII being lyophilized as such, lecithin being lyophilized as a solution in benzene

2492260

  and the injectable liquid containing aprotinin being lyophilized as it is. The lyophilized powders are mixed under a stream of dry nitrogen and the mixture is introduced into enteric-coated capsules (size No. 0). To improve the fluidity of the mixture, one can add to it an appropriate quantity of sugars such as glucose, lactose or mannitol. To seal the seal of the capsules, they are treated with an acetone solution of the material forming the enteric-coated capsules, as in Examples 1 and 2. Each enteric-coated capsule contains 100 units of coagulation factor VIII. This product is very stable and can be stored for an extended period. The liposomes of this product can easily return to their original form by adding a small amount of water and a fairly large amount of factor VIII is incorporated.

to liposomes.

EXAMPLE 4

  2 g of egg yolk lecithin containing 5% phosphatidic acid are dissolved in 40 ml of ethanol and the solution is concentrated under reduced pressure in a 1 liter flask to deposit the lecithin in the form of a thin layer on the inner wall of the balloon. After drying under reduced pressure, 50 ml of a phosphate buffer solution (pH 7.0) containing 3,000 units of coagulation factor VIII and 50,000 units of aprotinin are added and

  lightly shake the balloon to obtain a suspension of liposomes.

  The liposome suspension is treated as described in Example 1 to obtain approximately 10 ml of liposomes containing 1,000 units of coagulation factor VIII and 17,000 units of aprotinin. The liposomes thus obtained are introduced into enteric-coated capsules as in Example 1. Each capsule thus obtained contains 50 units of factor VIII. This product can be stored away from heat and

  in the dark without the factor VIII activity decreasing.

REFERENCE EXAMPLE 1

  250 mg of egg yolk lecithin containing% phosphatidic acid are dissolved in 40 ml of ethanol and the solution is concentrated under reduced pressure in a 200 ml flask to deposit the lecithin h1 2492260 in the form of a thin layer on the inner wall of the balloon. After drying under reduced pressure, an aqueous solution of 4,000 units of coagulation factor VIII is added and

  the mixture is gently stirred to obtain a suspension of lipo-

  somes. The suspension of liposomes is introduced into a centrifuge tube, oh cooled to 100C and then centrifuged at 27,000 G for

  min to obtain creamy liposomes at the top.

  The transparent aqueous layer is again treated with

  lecithin and treat the mixture in the flask as above.

  These operations are repeated twice. The creamy liposomes obtained in the two operations above are combined and suspended in 50 ml of physiological saline to wash the outer layer of the liposomes. While cooling to 10C, the mixture is centrifuged at 000 G for 20 min to collect the liposomes. The liposomes are diluted with physiological saline to obtain a total volume of 50 ml. The suspension thus obtained (50 ml) contains 800 units

coagulation factor VIII.

REFERENCE EXAMPLE 2

  2 g of egg yolk lecithin containing 5% are dissolved

  of phosphatidic acid in 40 ml of ethanol and the solution is concentrated

  tion under reduced pressure in a 1 liter flask to deposit the

  Lecithin as a thin layer on the inner wall of the balloon.

  After drying under reduced pressure, 50 ml of phosphate buffer solution (pH 7.0) containing 3000 units of coagulation factor VIII is added and the mixture is gently stirred to obtain a suspension of liposomes. The suspension of liposomes is treated as described in Example 1 to obtain 10 ml of liposomes. The liposomes are introduced into capsules. Each capsule contains 50 units of

coagulation factor VIII.

REFERENCE EXAMPLE 3

  Liposomes (10 ml) containing the coagulation factor VIII obtained in the same way as in reference example 2 are introduced into capsules (size No. 0, the internal wall being covered with a thin layer of edible oil) , which are themselves introduced into enteric-coated capsules to obtain capsules with a double structure. The seal of the outer capsules is made completely sealed by treatment with an acetone solution of the material forming the enteric-coated capsules. Each capsule contains 50 units of coagulation factor VIII. Of course, various modifications can be made by those skilled in the art to the devices or methods which have just been described only by way of nonlimiting examples without departing from the

part of the invention. -

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Claims (8)

  1. Pharmaceutical composition for oral administration which comprises coagulation factor VIII and an inhibitor of   proteases that are incorporated into liposomes.   2. Composition according to claim 1, characterized in   what it is encapsulated in enteric-coated capsules.   3. Composition according to claim 1, characterized in   what it is in the form of a lyophilized product.   4. Composition according to claim 3, characterized in   that the composition is encapsulated in enteric-coated capsules.   5. Pharmaceutical composition for oral administration, characterized in that it comprises coagulation factor VIII, a protease inhibitor and a material producing liposomes which are each freeze-dried separately.   6. Composition according to claim 5, characterized in that   that the lyophilized products are encapsulated in entero capsules   soluble. 7. Pharmaceutical composition for oral administration   in a form encapsulated in enteric-coated capsules, character-   laughed at in that it includes coagulation factor VIII and aprotinin which are incorporated into liposomes formed from a   mixture of a phospholipid and phosphatidic acid.   8. Freeze-dried pharmaceutical composition for administration   oral tration in a form encapsulated in enterosol capsules   ble, characterized in that it comprises coagulation factor VIII and aprotinin which are incorporated into liposomes formed at   from a mixture of a phospholipid and phosphatidic acid.