JPS5920218A - Liposome containing vitamin k - Google Patents

Abstract

PURPOSE:A liposome, containing vitamin K, consisting of vitamin K and phospholipid, and capable of exhibiting a sufficient effect according to the dose thereof in the oral administration. CONSTITUTION:A liposome, obtained by dissolving a vitamin K type compound, particularly vitamin K1 (phytonadione), vitamin k2 (all trans-type menatetrenone) and a phospholipid, e.g. phosphatidyl choline preferably lecithin, in ether, adding the resultant solution to a salt solution to prepare a water-in-oil type emulsion, distilling away the ether under reduced pressure while heating the emulsion at 30-40 deg.C to give a highly viscous creamy material, dispersing the creamy material in a physiological saline solution, etc. to give a solution for the internal use, or mixing the creamy material with a powder for formulation to afford a powder. The resultant liposome has the coagulating action of the vitamin K, specifically the reduction in the prothrombin time by the oral administration.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides vitamin I (containing ribbon,
-1. In more detail, this article relates to vitamin K and ribosol contained in a vitamin consisting of phospholipids, which is used to more effectively exert its pharmacological action when vitamin K is administered intraperitoneally.

Vitamins are involved in the blood coagulation system and are among the blood coagulation factors.
1r, factor 1I (prothrombin) that tends to decrease to 1°.

Factor ■ (stable factor), factor 1X (hemophilia factor B).

The activity of factor X (Stuart Blower factor) can be quickly restored. It has also been confirmed that it has the effect of activating the production of factor Xll and activating the body's hemostasis mechanism. In particular, the characteristic action of vitamin 1ζ can be clearly observed in restoring prothrombin activity. Therefore, if hypoprothrombinemia occurs, for example, in cases of obstructive jaundo, ulcerative gastroenteritis, various liver diseases, cerebral hemorrhage, leukemia, purpura, infant idiopathic vitamin deficiencies, or coumarin anticoagulants. When hypoprothrombinemia occurs during administration of antibiotics, salicylic acid drugs, etc., vitamins can be extremely effective agents for preventing or treating hypoprothrombinemia. Specifically, prolongation of prothrombin time due to decrease in prothrombin can be prevented. In addition, while hemostatic agents other than vitamins may actually disrupt the physiological hemostasis mechanism and cause serious side effects such as thrombosis and intravascular blood coagulation syndrome, vitamins are Great power for people 1
It is a highly safe drug because it does not cause any change in blood coagulation ability, fibrinolytic ability, or blood vessel properties even after injection, and it quickly activates the hemostasis mechanism only in the event of bleeding. Therefore, for example, during a surgical operation or childbirth with serious bleeding, a sufficient amount of vitamin 1 can be administered before the surgery, resulting in less bleeding.

Blood transfusions can also be saved.

Thus, although vitamins are effective and safe drugs, their more frequent use has been hindered. In other words, when the drug is administered orally, it is unable to exhibit sufficient effects depending on the dose, as shown in the experimental examples below. This is thought to be because the drug is fat-soluble, so it is solubilized by bile and absorbed into the intestinal tract, but its absorption is not sufficient to be effective.

However, the details are unknown. But either way.

Currently, orally administered vitamins are unable to exert sufficient pharmacological effects. Therefore, in order to promote the frequent use of vitamins, it is necessary to sufficiently exert the pharmacological effects of vitamins by oral administration. It is hoped that technical means will be provided that will enable them to make full use of their abilities.

In view of the above circumstances, the present inventor conducted studies with the aim of providing the technical means, and as a result, created liposomes containing vitamins and phospholipids, and by orally administering them, the intended purpose could be achieved. Knowing that this can be achieved, we have completed the present invention.

That is, two objects of the present invention are technical means devised to more effectively exert the pharmacological effects of vitamin 1 when administered orally.

Specifically, the present invention provides liposomes containing vitamins and phospholipids.

Attempts have been made to encapsulate drugs in liposomes and administer them intravenously.2 For example, Pertill and Lyman reported that a slight effect was observed when insulin was encapsulated in liposomes and administered intravenously. Ori,
Also tubocurarine.

There have also been cases where Ara-C, phleomycin, and anti-hemophilic coagulation factors were made into liposomes and administered orally. However, there are still many questions about oral administration of liposomes, and it is unclear whether liposomes are absorbed intact in the gastrointestinal tract. One reference is listed below.

H, M, Pal;el and B,E,
Rymnn, FEBS Lett, 62°60 (197G,).

G, Dapergolas, E, D, Neeru
njun and G, Oregonia-dis,
FEBS Lett, 63.235 (1976
).

G, J) apergolas and G, Gra
goriatliS, Biochem.

Soc, 'l″ranS,, 5.1383 (19
77).

Y, M, Ilustom, C, Llave, E.
MayJtew and P, Papa-badj
opoulos, Cancer Research,
39.1390 (1979).

11, C, Hemker and W, T, ) Icr
mens, Lancet, 8159°'to (1
980).

R, N, Rowlind and J, F, Woo
dley, Biocbem, Biop)xys.

Actn, 673.217 (1981).

D, S, Deshmuldt, W, D, Bear
1141 (l H, Brocherhoff.

Life 5sciences, 28.239 (19
81).

M.H.Richards and C.R.Gard
uner, Biochem.

Biophys, Acta, 543.508 (1
978).

R, N, Rowland and J, F, Woo
dley, Biochetn, Biophys.

Acta, 620.400 (1980).

As interest in so-called organ-directed preparations gradually increases in the medical and pharmaceutical fields, technology that utilizes liposomes has come to be seen as a means to achieve this goal. As is well known. In other words, by focusing on the fact that liposomes exist as unique liposomes for each organ, we can deliver drugs selectively and intensively to target organs by containing a given drug in a given liposome and administering it. Technology is being developed that aims to make this happen.

An example of such an invention is JP-A-52-143218. Japanese Patent Publication No. 52151718. Japanese Patent Publication No. 53-133616. The invention in Japanese Patent Publication No. 55848B can be mentioned.

All of these inventions are related to technology for holding specific biological components or pharmaceutical components in liposomes as carriers, and it is expected that these technologies will eventually be applied to other biological components and pharmaceutical components in medical and pharmaceutical sciences. be done.

In other words, the application of liposome technology to biological components or pharmaceutical components is well known, and there are also well-known conventional methods for producing liposomes. However, by applying liposome technology to vitamin K, we succeeded in obtaining vitamin K-containing liposomes, thereby solving the aforementioned basic problems in vitamin K, and significantly increasing the utility value of vitamin K. This is the first time in the present invention that the temperature was set at °C.

The present invention will be explained in detail below.

In the present invention, "C vitamin" refers to a vitamin-like compound that exhibits a blood coagulation effect.
(phytonadione) and vitamin 2 (all-trans menatetrenone) are particularly preferred examples.

Lean lipids used in the present invention include:

Examples include phosphadylcholine, phosphatidylethanolamine, bosphatidylserine, sphingomyelin, dicetyl phosphate, stearylamine, phosphadylglycerol, phosphatidic acid, phosphatidylinositol, and mixtures thereof. However, it is not particularly limited to these.

Furthermore, 20 to 30% of cholesterol, which is a neutral lipid, may be added as a reinforcing agent for the liposome bilayer membrane. In short, any phospholipid can be used as long as it can form liposomes, and phosphatidyl coliforms such as lecithin are preferred materials as the basic constituent phospholipids of liposomes.

Ribosort in the present invention refers to a lipid multilayer structure obtained by dispersing phospholipids in a salt solution.

It is a closed vesicle, and the vitamins are evenly distributed within the vesicle. The composition ratio of vitamins and phospholipids is based on the weight ratio of vitamins and phospholipids.

It is sufficient if the weight ratio of phospholipids is 2 or more.

The liposomes of the present invention may be prepared by applying generally known methods for producing liposomes. For example, dissolve phospholipids and vitamins in ether, add this to a salt solution (for example, physiological saline), irradiate it with ultrasound to prepare a water-in-oil emulsion, and then add it to 30-40°C. All you have to do is depressurize the ether while heating it.

This results in a creamy mass with high viscosity.

Furthermore, in order to make a preparation suitable for oral administration, the cream-like substance can be dispersed in physiological saline or the like to form an oral liquid, or mixed with a powder for preparation and separated from the powder. good. The resulting powder can be further made into granules, filled into hard capsules, or even more! It can be made into a single tablet. However, all of these pharmaceutical forms utilize the ribosort of the present invention and therefore are indirect implementations of the present invention.

As described above, the liposome of the present invention has a blood clotting effect on vitamins, specifically, a shortening of prothrombin time when administered orally. As shown in the experimental examples below, the effect is not as good as that obtained by intravenous injection, but when administered orally without being formed into liposomes.

For example, cyclodextrin, polyvinylpyrrolidone,
Compared to when administered orally in a dispersed state with polyoxyethylene hydrogenated castor oil, etc.

The effect on those things was ``1 turn''.

In order to verify this effect, for example, the following experiment may be performed. Warfarin, a coumarin-based anticoagulant, is administered to rabbits to create a hypoprothrombinemic state, and then vitamin I is administered to the rabbits, and recovery from this state is observed. According to the preliminary experiment results, warfarin 12my
When Ay is administered into the rabbit ear vein, the prothrombin time is prolonged and reaches its maximum after 21 to 24 hours, and the prothrombin activity itself decreases to 20% or less of the normal value.

after that.

It gradually recovers, and after about 80 hours, it has recovered to more than 60% of its normal value. Therefore, in the experimental example described later, warfarin 12rvAg was administered into the rabbit ear vein, and 24 hours later, a predetermined amount of vitamin K was administered via a predetermined route. PT) was measured, and each prothrombin activity (abbreviated as PCA) after a certain period of time was determined.

The present invention will be explained in detail using the following experimental examples.

Example Sample A chloroform solution containing 140 mg of lecithin was placed in a round bottom flask, dried under reduced pressure, and subjected to ultrasonication at 9 m, l for 5 minutes to form a water-in-oil emulsion.

Next, ether was heated under reduced pressure at 30° C., and the gel-like material TI obtained in 111 was dispersed in 20-proof physiological saline. As a result.

Lecithin 1807Lynol and Vitamin Kl 110L
A dispersion of 107L in a 20 rnL aqueous phase was obtained, which was used as a test sample. Separately, a 1% aqueous solution of vitamins was prepared by dissolving it in water with sesame oil and polyoxyethylene hydrogenated castor oil, and used as a control sample.

Method Warfarin 12 myAy was administered into the rabbit ear vein.

After 24 hours, the test sample was administered orally through the catheter, and the control sample was administered intravenously. After a certain period of time had elapsed since the sample administration, 1.8 raL of one blood sample was taken into 2 mL of 3.8% citrate and incubated at room temperature for 10 minutes.
Perform centrifugation at 00 rpm and collect 0JrnL of plasma.
0.1 ml of 0.9% physiological saline was added to the solution, and 0.2 ml of thrombokinase solution was added to half of this, heated at 37°C for 1 minute, and the prothrombin time was measured using a fibrometer. The rabbits weighed 3.0 to 4.5 kg, were given sufficient water and solid samples, and were raised at 25°C. Each group consisted of 6 animals.

Results The results are shown in Figure 1. The horizontal axis of the figure is the + dose of vitamins, and the vertical axis is the normal value of prothrombin activity PCA, which is 6.
Time required to recover to 096 ('I'PC!A
, abbreviated as). In the figure, the ○ marked line shows the results for the specimen sample, and the * marked line shows the results for the control sample.

From Figure 1, TPCAoo obtained when vitamin + 12 mg/hg is administered intravenously through a specimen sample corresponds to i'PcA11o obtained when vitamin 1 (13 mg/hg is intravenously administered through a control sample). Therefore, although the effect of the liposome of the present invention is not as good as that of a 1-injected vitamin, it has a bioavailability that is close to that (1 liter).
Li is understood.

Example 5 Samples Test Example 1 Same sample as the sample described in the sample section 1 sample 1
Two samples were prepared and used as test samples for the experimental example. Separately, the following ae were prepared as control samples.

8. Mix vitamin 1 and polyoxyethylene hydrogenated castor oil in a weight ratio of 1 = 7, ink it in water and make an aqueous solution of 50 mp120rnl of vitamin +, vitamin and β-cyclo Dextrins were mixed at a molar ratio of 1:12.5 and dispersed in water to give vitamins 50 mg/20 rnL of aqueous liquid c.
, hitamine, and polyvinylpyrrolidone at a weight ratio of 1:30 and dispersed in water to form vitamins.
An aqueous solution of 50 mg/20 tnL was prepared.

θ, in preparing the specimen sample', instead of adding 50 mg of vitamin Kl dissolved in 9 ml of ether, 1
A mixture of this and sample (I) was prepared in the same manner except that 9 ml of ether was added thereto. The procedure was the same as described in the same section, except that the dose was administered intravenously through a catheter.All doses were 1.

12 myAy.

result The results are shown in Table 1. Table 1 shows TPCA for each sample.

It is a table showing (HR) and effectiveness (A). Note that effectiveness is defined as follows. That is, when the same shortened recovery time to the normal value was obtained when the control sample of Experimental Example 1 was administered intravenously at a dose of 12 mB 77 kg in the actual measurement 1 experiment. ,
The effectiveness is determined to be 100% and is expressed as a relative ratio. Therefore, if expressed using a formula, it becomes as shown in the following formula.

Here, the case where we wait for prothrombin activity to recover automatically after administering warfarin 12 myAy without administering vitamin II is defined as control (1).
C2 in that case to TPO80 in the above formula i'PcA6
In addition, in the experiment of Experimental Example 1, the case where the control sample of Experimental Example 1 was administered intravenously at a dose of J2mg and 2g of Lg is expressed as Funtrol (2), and TPCA and o in that case are expressed as TPCA, n (represented as 21). In the formula, TJrCAno represents each value of l'PcAl,o(HR) determined for the control sample and the specimen sample.

In addition, prothrombin time (P'I') and prothrombin activity (PCA) in the case of Funtrol (1)
Oyohi'I'PCA. The relationship between the three is not shown in Figure 2 for reference.

In addition, the following documents regarding the evaluation method in this experimental example are listed for reference.

■Basic drug development course V: Evaluation of drug efficacy) (Chijinshokan)
Pharmacological test methods (Part 2) Anticoagulants P, 1213 ■Summary of clinical test methods Blood-membrane test method VI 89■1 (Metal Publishing) Wireless prothrombin level measurement method ■Blood and blood vessels
2Nα1 (Showa 46.1) P, 35-41 Yamanaka et al. Table 1
These results demonstrate that oral administration of the liposome of the present invention exhibits superior efficacy compared to oral administration of other dispersed samples.

The present invention will be further explained in terms of airfoils with reference to Examples described below.

Example 1 140 mg of purified lecithin is dissolved in ether 9rrLL.

Furthermore, 50 mg of vitamin Kl is added and dissolved. Add 3 ml of physiological saline to this mixed solution and irradiate it with a water bath type ultrasonic irradiation device for about 5 minutes to prepare a water-in-oil emulsion.

Next, apply the emulsion to a rotary evaporator for about 30 minutes.
Remove the ether while keeping at °C. The resulting substance is a highly viscous cream. Add about 20 rnL of physiological saline to this and stir with a mixer.

[Brief explanation of drawings]

FIG. 1 corresponds to FIG. 1 described in the section of Experimental Example 1 Results. It is a graph showing the dose dependence of TPCA,o. FIG. 2 corresponds to FIG. 2 described in the section of Experimental Example 2 Results. It is a graph showing the relationship between the champions of PT, PCA and TPCA6o in the case of control (1). Special W'F Applicant Eisai A Type Yosha 1 View 1 Vitamin Administration m: (mg/51 Figure Pupil 12 1-1'P (EΔ1111〕fノΔ6゜-200204
[16LI II(+ TIMLC(Illus)

Claims (1)

[Claims] (2) A liposome containing a vitamin according to claim 1, wherein the phospholipid is lecithin.