KR19980084791A - Treatment for iron deficiency anemia in which heme is trapped in liposomes and preparation method thereof - Google Patents

Abstract

The present invention is to insert a hemin into liposomes to prevent iron from directly irritating the gastrointestinal mucosa during oral administration as well as to facilitate a sufficient supply of iron, more specifically folic acid and vitamin B ₁₂ consisting of yolk phosphatidylcholine The present invention relates to an iron preparation for treating iron deficiency anemia in which hemin is trapped in a lipophilic space of a liposome double membrane after being captured in a water-soluble space in a liposome.

Description

Treatment for iron deficiency anemia in which heme is trapped in liposomes and preparation method thereof

Since humans began to use drugs, the discovery and development of innumerable drugs have been made, but the method of administering drugs according to the drug or pathological characteristics has not been greatly improved. Improvement of the drug delivery system is essential to overcome the side effects and modifications of the drug and to obtain the maximum therapeutic effect. Liposome drug delivery system using phospholipid to improve drug delivery system has several advantages over other delivery systems. Since liposomes are composed of phosphate lipids that make up life, there are no side effects, and they have two characteristics, hydrophilic and hydrophobic, so there are no restrictions on the use of hydrophilic and hydrophobic drugs. In addition, it is possible to control the distribution in vivo differently by changing the phosphate lipid component, there are many advantages such as protecting the drug from the biological defense system and controlling the release rate of the drug to maintain the effective concentration of the drug in vivo. In addition, since liposomes can be administered in vivo, such as by intravenous, subcutaneous, intramuscular, intraperitoneal injection, oral absorption through mucosal or skin, and the method of administration, the liposomes can be used in all appropriate methods of administration for a particular drug.

Taking advantage of the characteristics of these liposomes, liposome preparations have been developed as a therapeutic agent for anemia patients among vascular diseases, which have become the focus of attention in modern serious adult diseases. Currently, mortality due to vascular disease is the second place after mortality caused by cancer, and the number of patients suffering from anemia among these vascular diseases is increasing. Anemia occurs as a multifactorial cause, but iron-deficiency anemia is the most frequent among the anemias. Iron deficiency anemia occurs when the production of red blood cells is suppressed due to the lack of iron, and there are four main causes of insufficient iron intake, insufficient absorption of iron, increased iron requirements, and increased iron loss. have. Iron deficiency should be prevented through a balanced and proper diet. However, if iron deficiency anemia has already occurred, diet alone is not enough.

The use of liposomes as a therapeutic agent for iron-deficiency anemia patients can be made by infiltrating the hydrophobic region of the phospholipid membrane, which is hemin trapping iron, to prepare heme-containing liposomes. That is, heme of heme-containing liposomes is absorbed in the duodenum or upper small intestine in the state of heme, and then stored in plasma ferritin after dissociation of porphyrine in the cells to dissociate Fe ²⁺ . This stored Fe ²⁺ is transferred to the erythroblast by transferrin. The transferred Fe ²⁺ is transported to the mitochondria and complexes with photopopyrine synthesized in the mitochondria to form heme. The heme thus synthesized is delivered to the apohemoglobin by the apohemoglobin synthesized in the endoplasmic reticulum to the mitochondria to form hemoglobin. When hemoglobin is properly formed, red blood cells are normally made, and anemia due to iron deficiency may be treated.

Ferritin, which is currently marketed as an iron product, has a protein-coated effect of iron, which has greatly resolved gastrointestinal disorders such as vomiting caused by irritation of the gastric mucosa when iron is ingested. These ferritin preparations contain 155mg of ferritin extract (20mg of ferritinic Fe ³⁺ ) in one capsule, and 500g of vitamin B ₁₂ and 800g of folic acid are combined to prevent iron deficiency anemia as well as megaloblastic anemia. It is also designed to help with healing. Folic acid and vitamin B ₁₂ are hematopoietic vitamins that play an important role in the synthesis of nucleic acids and are essential vitamins during pregnancy and lactation. In particular, the toxemia of pregnancy, childbirth complications, prenatal, so causing developmental disabilities, such as the supply of folic acid is helpful in many complications, treatment of vitamin B ₁₂ is the body use of folic acid water with a deficiency of folic acid is often associated with anemia in pregnancy Increase it.

The liposome anemia treatment drug to be developed in this study is basically designed to insert hemin into liposomes so that iron does not directly irritate the gastrointestinal mucosa during oral administration as well as to smoothly supply a sufficient amount of iron. That is, it is expected that the coating effect of protein in ferritin preparations can be obtained not only by liposomes, but also by the easy to take iron syrup. In addition, folic acid and vitamin B ₁₂ were trapped inside these liposomes to inhibit destruction by gastric acid and was designed to slow down the release rate. That is, folate and vitamin B ₁₂ were captured in the water-soluble space inside the liposome, and hemin was captured in the fat-soluble space of the phospholipid membrane.

Figure 1 is a chart measuring the degree of leakage of folic acid contained in 60% yolk phosphatidylcholine vehicle at 4 ℃ according to the content of cholesterol (ratio of 0 and 0.5).

Figure 2 is a chart measuring the degree of leakage of folic acid contained in 60% yolk phosphatidylcholine vehicle at 37 ℃ according to the content of cholesterol (ratio of 0 and 0.5).

Figure 3 is a chart measuring the degree of leakage of folic acid contained in 100% egg yolk phosphatidylcholine vehicle at 4 ℃ according to the content of cholesterol (ratio of 0, 1.5 and 1).

4 is a chart measuring the degree of leakage of folic acid contained in 100% egg yolk phosphatidylcholine vehicle at 37 ℃ according to the content of cholesterol (ratio of 0, 1.5 and 1).

Figure 5 is a chart measuring the amount of hemin capture of liposomes by observing a decrease in fluorescence intensity with the addition of hemin to egg yolk phosphatidylcholine / β-py-C6-HPC (100/1, w / w) vehicle.

Accordingly, the present invention relates to an iron preparation for treating iron deficiency anemia and a method for preparing the same, wherein folic acid and vitamin B ₁₂ are captured in a water-soluble space in a liposome composed of yolk phosphatidylcholine, and then hemin is captured in a fat-soluble space of a liposome double membrane.

The egg yolk phosphatidylcholine is characterized in that the yolk phosphatidylcholine 60% or egg yolk phosphatidylcholine 100%, the preparation of the liposomes containing cholesterol increases the stability of the liposomes. In addition, the iron preparation for the treatment of iron deficiency of the present invention after preparing a multi-lamella bisicle (MLV) containing phospholipids, folic acid and vitamin B ₁₂ , after grinding the MLV to produce a small uni-labeled arabic (SUV) By using this, a large unilabeled biscuit (LUV) is prepared, and hemin is added thereto to capture hemin in the double membrane of the liposome. In addition, hemin capture amount of liposomes is characterized in that about 80 ~ 130㎍ per 1mg of egg yolk phosphatidylcholine.

In summary, this is as follows.

That is, a liposome composed of 60% or 100% of egg phosphatidylcholine captured folic acid and vitamin B ₁₂ dissolved in sodium bicarbonate was prepared. At this time, cholesterol was included to increase the stability of liposomes. Hemin was trapped between the phospholipid bilayers of liposomes by adding a hemin solution dissolved in sodium hydroxide. Finally, heme-containing liposomes containing folic acid and vitamin B ₁₂ in the aqueous space inside the liposomes and hemin in the phospholipid membrane of the liposomes were prepared. These heme-containing liposomes are provided as a syrup for iron-deficiency anemia patients, which is easy to take and resolves many gastrointestinal disorders that stimulate the gastric mucosa.

Looking at the characteristics of each drug trapped in liposomes in the present invention as follows.

Hemin is a compound in which heme Fe is bound to Cl ions, soluble in aqueous ammonia and sodium hydroxide, but insoluble in water. After dissolving an appropriate amount of hemin chloride in 0.1 N NaOH, the pH was adjusted to about 7.5 using 0.1 M pH 7 KPi / Cl buffer (0.02 M potassium phosphate / 0.08 M potassium chloride) in the 390 nm region. When hemin is mixed with liposomes while absorbing, hemin has been reported to migrate into the membrane of liposomes.

The solubility of folic acid is 0.0016 mg / ml, and when suspended in water, it shows acidity of pH 4 ~ 4.8. In addition, folic acid is insoluble in chloroform, ether and the like, but has a solubility in sodium hydrogen carbonate solution. The aqueous solution has a pH of 6.5 to 6.8. The folic acid solution dissolved in sodium bicarbonate showed absorbance at 284 and 362 nm and fluorescence at 446 nm when excited at 362 nm. Self quenching was observed with concentrations above 0.08 μM.

The solubility of vitamin B ₁₂ (cyanocobalamin) is about 12.5 mg / ml, the aqueous solution being neutral and insoluble in acetone, chloroform, ether and the like. The vitamin B ₁₂ showed absorbance at 278, 361 and 550 nm, and showed very weak fluorescence when excited at 361 and 550 nm, but fluorescence at 304 nm when excited at 278 nm. However, this fluorescence was also observed to be relatively very weak.

There are many methods for preparing liposomes to capture these drugs, but the most efficient method for capturing water-soluble drugs is the freeze and thawing method, and low-cost egg (egg) as a phospholipid component of liposomes. Sulfur phosphatidylcholine 60% and 100% were selected. To this, cholesterol was added to improve the stability of liposomes. In order to capture folic acid and vitamin B ₁₂ in the liposomes thus constructed, these two vitamins must be present in the solution phase, and sodium bicarbonate buffer, a buffer for dissolving folic acid, was used as a buffer for liposome preparation.

Leakage tests were performed to measure the stability of folate and vitamin B ₁₂ trapped inside the liposomes. Folic acid dissolved in sodium bicarbonate showed fluorescence emitted at 446 nm when excited at 362 nm, so that the leak test could be used as a fluorescent probe. On the other hand, vitamin B ₁₂ was relatively weak in fluorescence and could not be used as a fluorescent probe. However, since vitamin B ₁₂ has a much higher solubility in aqueous solution than folic acid and a much higher molecular weight, the stability of liposomes trapping vitamin B ₁₂ is satisfactory if the stability of the liposomes trapping folic acid is adequate for a given purpose. Measuring will not have much meaning.

A leak test for folate was performed on liposomes consisting of 100% yolk phosphatidylcholine (egg PC) and 60% yolk phosphatidylcholine. In all cases it was more stable at 37 ° C than 4 ° C, and was observed to be stable with increasing cholesterol content (FIGS. 1-4). 100% egg yolk phosphatidylcholine was found to be slightly more stable than 60% egg yolk phosphatidylcholine, but satisfactory stability was also observed when 60% yolk phosphatidylcholine was used. From these results, it was observed that the stability of the liposomes trapping folic acid was very high, and the stability of the liposomes trapping vitamin B ₁₂ is expected to be very high.

In order to measure the amount of hemin capture of liposomes, in the preparation of liposomes, a fluorescent probe called β-py-C ₆ -HPC was included in egg yolk phosphatidylcholine at a weight ratio of 1/100 to finally produce LUV. The hemin dissolved in 0.01N sodium hydroxide solution and measuring the fluorescence energy transfer occurring between the egg yolk phosphatidylcholine was added to a liposome solution containing the _{β-py-C 6 -HPC β} -py-C 6 -HPC with hemin. That is, the decrease in fluorescence intensity at 377 nm after excitation at 343 nm was measured according to the addition of the hemin solution (FIG. 5). As a result, it was observed that hemin intercalated between the membranes of liposomes at a very high rate, at which time about 50 μg of hemin per 0.45 mg of egg yolk phosphatidylcholine was captured (FIG. 5).

The present invention will be described in more detail with reference to the following examples. However, these examples do not limit the present invention.

Example 1 Preparation of Liposomes

As a syrup for liposome anemia, liposomes composed of inexpensive 60% yolk phosphatidylcholine or 100% yolk phosphatidylcholine were used, and the content of cholesterol was changed. To prepare these liposomes, a freezing and dissolving method was used. After dissolving an appropriate amount of phospholipid in chloroform, put it in a vial and blow organic solvent with nitrogen gas to form a thin film on the glass wall. An appropriate amount of sodium bicarbonate buffer containing folic acid and vitamin B ₁₂ was added thereto, followed by vigorous stirring to form a multilamellar vesicle (MLV). The MLV was ultrasonically pulverized for 30 minutes to prepare a small unilamellar vesicle (SUV), followed by freezing and dissolving it with liquid nitrogen three to four times to prepare LUV. Hemin was added to capture hemin inside the double membrane of liposomes.

Example 2 Stability Test of Liposomes

Liposomes consisting of 60% yolk phosphatidylcholine or 100% yolk phosphatidylcholine and cholesterol were prepared using the freezing and dissolution methods. After dissolving an appropriate amount of phospholipid in chloroform, put it in a vial and blow organic solvent with nitrogen gas to form a thin film on the glass wall. To this was added a suitable amount of sodium hydrogen carbonate buffer solution (15 mM Na ₂ CO ₃ /34.88 mM NaHCO ₃ pH 9.0) containing 5 mg / ml folic acid as a fluorescent probe, followed by vigorous stirring to prepare a multilamella bisicle (MLV). The MLV was ultrasonically pulverized for 30 minutes to prepare a Small Unilabeled Bisicle (SUV), and then freeze and dissolve it in liquid nitrogen three to four times to prepare LUV. A -50 column (lx20 cm) was passed through to remove uncaptured fluorescent probes in liposomes.

The stability of liposomes was investigated by measuring the degree of leakage of LUV prepared according to the composition and temperature of phospholipids. Leakage of folic acid trapped in liposomes was measured as an increase in fluorescence intensity using JASCO FP-770 spectrofluorometa. Leakage measurements of folic acid were made at excitation at 362 nm and emission at 446 nm. The prepared liposomes were stored at 4 ° C. and 37 ° C. under the same conditions, and were recorded by measuring the degree of leakage over time.

Liposomes containing each fluorescent probe were passed through a Sephadex G-50 column to obtain a fluorescence intensity of 0% leakage immediately after removal of uncaptured fluorescent probes in liposomes, and 1% SDS was added to destroy liposomes. Fluorescence intensity was taken as 100% leak and 100% leak was measured and corrected each time.

In order to develop liposomes as a syrup preparation, the stability of liposomes composed only of yolk phosphatidylcholine was measured. First, liposomes were prepared with 100% yolk phosphatidylcholine and 60% yolk phosphatidylcholine, respectively, and a leak test for folate was performed. In all cases, it was more stable at 37 ° C than 4 ° C, and was observed to be stable as the cholesterol content was increased (1 to 4 degrees). After one month, 60% of egg yolk phosphatidylcholine showed 26-37% leakage at 4 ° C and 21-27% leakage at 37 ° C (FIGS. 1 and 2). On the other hand, 100% of egg yolk phosphatidylcholine showed 23-28% leakage at 4 ° C and 19-25% leakage at 37 ° C (FIGS. 3 and 4). The use of 100% yolk phosphatidylcholine was found to be slightly more stable than the use of 60% yolk phosphatidylcholine, but satisfactory stability was also observed with 60% yolk phosphatidylcholine.

Example 3 Measurement of Hemin Capture Amount of Liposomes

For the preparation of liposomes for measuring the hemin capture amount of liposomes, a fluorescent probe called β-py-C ₆ -HPC was used. 9 mg of yolk phosphatidylcholine dissolved in chloroform was mixed with 0.09 mg of β-py-C ₆ -HPC, followed by blowing nitrogen gas chloroform to form a thin film on the vial wall. 3 ml of Tris buffer (50 mM Tris, pH 7.5) was added thereto, followed by stirring to make MLV. Ultrasonic grinding was performed here for about 1 minute using an ultrasonic grinder to finally produce LUV.

A hemin solution was prepared on the day of the experiment by dissolving hemin at 1 mg / ml in 0.01 N sodium hydroxide solution. To 0.1 ml of egg phosphatidylcholine liposome (3 mg / ml) solution containing β-py-C ₆ -HPC, 1.9 ml of Tris buffer is added and mixed well (final concentration 0.15 mg / ml). The solution is placed in a fluorescent cell and kept well mixed at 37 ° C. with a small magnetic bar. In order to measure the amount of hemin trapped, fluorescence energy transfer between β-py-C ₆ -HPC and hemin was used. That is, the decrease in fluorescence intensity at 377 nm after excitation at 343 nm was measured in accordance with the addition of the hemin solution (FIG. 5). As a result, it was observed that hemin intercalated between the membranes of liposomes at a very high rate at which about 50 μg of hemin was captured per 0.45 mg of egg yolk phosphatidylcholine (FIG. 5).

The effect of the present invention is that liposome anemia treatment agent basically inserts hemin into liposomes so that iron does not directly stimulate gastrointestinal mucosa during oral administration, and smooth supply of sufficient amount of iron. In other words, the coating effect of the protein in the ferritin formulation can be obtained as a liposome as well as easy to take iron syrup developed. In addition, folate and vitamin B ₁₂ were trapped inside these liposomes to inhibit destruction by gastric acid, and was developed to slow down the release rate. That is, folate and vitamin B ₁₂ were captured in the water-soluble space inside the liposome, and hemin was captured in the fat-soluble space of the phospholipid membrane.

Claims (5)

Iron preparation for iron deficiency anemia in which folic acid and vitamin B ₁₂ are captured in a water-soluble space in a liposome composed of yolk phosphatidylcholine, and then hemin is captured in a fat-soluble space of a liposome double membrane The iron preparation for treating iron deficiency anemia according to claim 1, wherein the yolk phosphatidylcholine is 60% yolk phosphatidylcholine or 100% yolk phosphatidylcholine. The iron preparation for treating iron deficiency anemia according to claim 1, wherein the liposome contains cholesterol to stabilize the preparation. The iron preparation for treating iron deficiency anemia according to claim 1, wherein the amount of hemin captured by liposomes is about 80 to 130 µg per 1 mg of egg yolk phosphatidylcholine. After preparing a multilamella bisicle (MLV) containing phospholipids, folic acid and vitamin B ₁₂ , the MLV is pulverized to produce a small unilabeled biscuit (SUV), which is used to produce a large unilabeled bicyclic (LUV). After the preparation of the iron preparation for iron deficiency anemia, characterized in that the addition of hemin to capture the hemin in the double membrane of liposomes