JPH0621077B2 - Hematopoietic agent - Google Patents

Description

TECHNICAL FIELD The present invention relates to a hematopoietic agent used for treating, recovering, and preventing anemia of a patient suffering from anemia due to bleeding and iron deficiency.

Conventional technology and its problems Conventionally, for the treatment and recovery of anemia caused by bleeding and iron deficiency, iron agents, for example, iron agents containing ferrous and ferric iron such as iron citrate, iron pyrophosphate, iron chloride and iron sulfate, Vitamin B ₁₂ preparations, folic acid and anabolic steroids are used. However, since these iron preparations have low absorption efficiency in the body, it is necessary to administer a relatively large amount, and when administered orally, a large amount of iron preparations causes iron (Siderosis) in the digestive tract wall.
There are various side effects such as nausea, nausea and constipation. Therefore, a sustained-release iron agent is also used, which is designed to gradually release iron in the gastrointestinal tract by ion-binding iron sulfate to a water-soluble polymer compound. However, there is a problem that it takes more than twice as long to treat and recover an anemia as a result.

On the other hand, intravenous iron preparations are used for patients who cannot take oral preparations due to strong manifestation of gastrointestinal disorders, patients with extremely weak absorption of iron from the intestinal tract, or patients requiring rapid iron saturation due to bleeding. ing. However, with this intravenous iron preparation, when a large amount of iron is injected intravenously, there is a risk that iron exceeding the iron-binding ability of serum will directly cause serious damage to the brain and large intestine, and excess iron will be deposited in the body. There is a possibility of causing tissue damage, and in addition, there is a problem that side effects such as headache, dizziness, fever, and rash may occur, as well as acute poisoning.

The vitamin B ₁₂ preparation and the folic acid mentioned above have been intramuscularly injected, but iron deficiency occurs when the anemia state recovers. Therefore, it is necessary to use the above iron agent in combination.

Under the circumstances as described above, it has been strongly desired to provide a hematopoietic agent which has a good iron absorption rate in the body and is free from the risk of side effects.

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention The present invention has been made in view of the problems found in the conventional hematopoietic agents as described above, in which the absorption efficiency of iron in the body is good and the general anemia state It is an object of the present invention to provide a hematopoietic agent which has excellent effects not only in treatment and recovery but also in early recovery of a patient who is in an extremely anemic state due to a large amount of bleeding and the like and which is free from side effects.

The present inventors have found that iron-binding lactoferrin obtained by chelating a ferric ion to lactoferrin obtained by separation from milk has a second iron-bonded lactoferrin bound to the lactoferrin.
Despite being iron (Fe ³⁺ ) and a non-heme form of iron, it is significantly absorbed by the body and has a hematopoietic effect, ie
They have found that they have an action of increasing the number of red blood cells and have completed the present invention.

By the way, [Morck TA and Cook JD, "Cereal Foods World", 26, 667-672,
1981], the iron contained in food is divided into heme iron and non-heme iron, and heme iron has a good absorption rate (absorption rate of 15 to 35%), while it is contained in food. There is a drawback that the amount is small and the taste becomes bloody when added to food and supplemented.On the other hand, non-heme iron is the main component of iron in food, but its presence has a considerable effect on absorbability. It is said to affect. For example, non-heme iron has good absorbability in the coexistence with ascorbic acid, folic acid and the like, but absorption is significantly inhibited when taken with eggs, soybean, rice, tea and the like.

In addition, according to many research reports on iron absorption, ferrous iron (Fe ²⁺ ) that is soluble in the digestive tract is generally insoluble in ferric iron that is insoluble at pH 3 or higher. It is said that the absorption rate is higher than that of (Fe ³⁺ ). That is, the pH of the contents of the upper small intestine where iron is mainly absorbed is 5 to 5
In the range of 6, ferrous iron has a high solubility as well as heme iron, and therefore has a high absorption rate.

The present invention will be described in detail below.

Composition of the Invention The compositional feature of the present invention is a hematopoietic agent containing iron-binding (iron-saturated) lactoferrin obtained by chelating a ferric ion to lactoferrin separated from milk.

Means for Solving the Problems The lactoferrin used in the hematopoietic agent according to the present invention is obtained from milk by a known separation method. For example, a method of separating a fraction having a high lactoferrin concentration from milk using an ion exchange resin (Japanese Patent Laid-Open No. 58-28233) can be applied, but in order to increase the iron binding amount per protein of lactoferrin, It is desirable to use lactoferrin that is highly pure and has no deformation. For that purpose, a method of separating using an affinity column on which an anti-lactoferrin monoclonal antibody is immobilized (Kawakami et al. It is advisable to apply the "Abstracts". The ferric ions used in the present invention include ferric chloride and ferric sulfate.
A ferric compound that is harmless to food hygiene, such as iron, is suitable.

The ferric ion can be chelated to the lactoferrin by mixing the lactoferrin and the ferric compound in the presence of sodium citrate or sodium bicarbonate.

That is, lactoferrin is dissolved in a solution of sodium citrate or sodium bicarbonate, ferric chloride or ferric sulfate is added and dissolved therein, and the mixture is stirred at room temperature for about 1 hour to ferric lactoferrin. Chelate the ions. The mixing ratio of ferric iron to lactoferrin in the above chelate bond is known to be such that 2 molecules of ferric iron bind to 1 molecule of lactoferrin (unmodified), and thus the molar ratio is 2: 1. Mixing in a ratio is preferred.

Next, the iron-engagement type clatoferrin obtained as described above may be used as a hematopoietic agent in a solution form after being subjected to desalting treatment and ultrafiltration treatment, but usually the solution is concentrated. It is then dried by freeze-drying or the like and used in powder form. Further, the hematopoietic agent according to the present invention can be used as a drug product such as a sugar-coated tablet, a tablet or a capsule, and further, if there is a large amount of bleeding from the stomach wall due to a gastric ulcer, or the stomach is extremely weak due to other diseases. In the case where it is present, it can also be used by being mixed with an infusion-type nutrient for enteral administration.

Furthermore, as an application example of a hematopoietic agent, various foods and drinks, for example, soft drinks, fruit juice drinks, fermented drinks and the like, jelly, ice cream, may be used as a food material for improving anemia by adding to ice fruits, in which case Is added in an amount of 0.2% by weight or more, preferably 0.5% by weight or more.

Since lactoferrin, which is a constituent of the hematopoietic agent, is a glycoprotein, which is a kind of milk protein originally present in milk, it has no adverse effect on the human body and its intake is not particularly limited. However, when actually used as a hematopoietic agent, about 20 to 200 mg / day / kg is appropriate.

By the way, the daily iron requirement for human adult is 12m for males.
It is said to be 14 mg for women and 14 mg for women, and although a sufficient amount of iron is ingested in a normal diet, the absorption rate of iron in food is said to be 10% or less. In addition, it may be difficult to maintain the iron requirement in the body due to unbalanced diet, women's pregnancy and excretion of iron due to menstruation. In fact, iron deficiency anemia is 90% of total anemia.
%, More than 50% of the total population is said to have some anemia.

The ferric iron ion in the iron-binding lactoferrin, which is the active ingredient of the hematopoietic agent according to the present invention, is chelated to lactoferrin and therefore is not released even under acidic conditions, and therefore remains bound when administered to the human body. It can reach the intestine and be absorbed.

Effects of the Invention Next, the effects of the hematopoietic agent according to the present invention are summarized as follows.

Since it is stable under acidic conditions, iron does not become insoluble in the intestinal tract and exhibits a hematopoietic function of about 5 times that of conventional iron agents.

It is easily soluble in water, does not insolubilize due to changes in pH, does not release iron easily, and has no bloody odor found in heme iron, so it can also be used in food and drink, at which time flavors and masking agents are used. Need not be used.

Since it is one that binds iron to lactoferrin, which is a milk protein, unlike other iron agents for hematopoiesis, it can be widely used as a hematopoietic agent for patients during and after the disease, old people and newborns. The present invention and its effects will be specifically described with reference to the drawings.

Example 1 Preparation of iron-engagement type (iron-saturated) lactoferrin which is an active ingredient of the present hematopoietic agent: 100 kg of raw skim milk was passed through an affinity column on which an anti-bovine lactoferrin monoclonal antibody was immobilized, and lactoferrin I got 12g.

Then, the lactoferrin was dissolved in 1200 ml of 0.015 mol sodium citrate solution, and ferric chloride was added to the solution.
120 mg was added and dissolved. The obtained mixed solution was stirred at room temperature for 1 hour, desalted, concentrated by ultrafiltration, and freeze-dried to obtain 11.8 g of the desired iron-bound lactoferrin. It contained 1.4 mg of iron per 1 g of lactoferrin.

Example 2 This example shows the effect of improving the anemia state by the following administration test of the hematopoietic agent according to the present invention.

Test method Using a Wistar rat (male, 3 weeks old) as a test animal, a test group of two groups, a standard group (iron content 40 mg%) for iron-containing diet administration and a iron-free diet administration group (iron content 0.2 mg%) Both groups were given deionized water. After 18 days, the hemoglobin level (Hb level) in the rat blood of the control group was 7 g / dl or less and the red blood cell count was
Anemia was judged to have occurred when the number reached 4 million / μ or less.

Then, while allowing the anemia rat group prepared as described above to freely take the iron-free food, the rat group was further fed with Fe.
SO ₄ · 7H ₂ O (ferrous sulphate) to 50 [mu] g / mouse (10 as an iron weight
μg / animal) and 250 μg / animal (50 μg / animal as iron content)
2 groups forcibly administered daily and 7 mg / animal of iron-bound (iron-saturated) lactoferrin according to the present invention (10 μg / iron as iron amount)
, And 35 mg / mouse (50 μg / mouse as iron amount) daily forcibly administered, and a control group for maintaining anemia without iron administration. Erythrocyte counts, hematocrit values and hemoglobin values were measured daily with the above standard group. The results are shown in FIGS. 1 to 3 attached.

As can be seen in each figure, in the group in which the iron-saturated lactoferrin according to the present invention was administered in an amount of 50 μg / day / animal, blood component values were improved daily and the anemia state was recovered, as compared with the other iron-administered groups. In addition, the anemia state was improved daily also in the group administered with 10 μg / day / animal.

Example 3 This example shows an example in which the hematopoietic agent according to the present invention is applied to a food for improving anemia or preventing anemia.

Production of anemia-preventing milk drink The iron-binding lactoferrin prepared in Example 1 was blended as follows to prepare an anemia-preventing milk drink. Ingredients (wt%) Skim milk powder 7.1 Glucose 5.0 Safflower oil 2.0 Emulsifier (Shuger ester) 0.1 Iron-binding lactoferrin 1.6 Vitamin 0.02 Yogurt flavor 0.18 Water 84.0 100% Based on the above mixture, 21.3 g skim milk and glucose 15 g to 60 ° C
Dissolve in 100 ml of warm water heated to 60 ° C., while mixing 6 g of safflower oil and 0.3 g of Shuger ester (trade name DKF 160) at 60 ° C., stirring the dissolved mixture with a TK homomixer,
The non-fat emulsion was gradually added dropwise to emulsify. This was heat-sterilized at 90 ° C. for 5 minutes and then cooled to 20 ° C. Next, 4.8 g of iron-binding lactoferrin was dissolved in 125 ml of water in the obtained mixed emulsion, and 0.06 g of vitamin and 0.54 of yogurt flavor were added.
What was added g and sterilized by a 0.22 μm filter was added and mixed to obtain a product.

Next, the milk drink containing this iron-binding lactoferrin was given to rats as test animals to examine the effect of preventing anemia.

Test method: 10 Wistar rats were divided into 2 groups of 5 rats, each group was given iron-free food, and one group was given 400 mg of the above milk drink at the same time.
Was administered daily and the other group was administered daily with deionized water to measure the hemoglobin content in the blood of each group.

As a result, after 4 weeks, the hemoglobin value in the deionized water administration group was 6.3 g / dl on average, while that in the milk drink administration group was 15.5 g / dl on average.

Production of jelly for improving anemia: The iron-binding lactoferrin prepared in Example 1 was blended as follows to prepare a jelly for improving anemia. Ingredients (g) Sugar 15 Prune extract 4 Gelatin 0.5 Iron-binding lactoferrin 5 Water 75.5 Total 100 (g) With the above formulation, add sugar and gelatin to 50 ml of water and heat to 80 ° C to dissolve them. After adding and stirring, the mixture was allowed to cool to 50 ° C., iron-bound lactoferrin dissolved in 25.5 ml of warm water at 40 ° C. was added and mixed, and then the product was placed in a container and allowed to cool to obtain a product.

Next, the jelly containing the iron-binding lactoferrin was given to rats as test animals to examine the effect of improving anemia.

Test method: 10 Wistar rats were divided into 2 groups of 5 rats, and each group was given an iron removal diet only for 2 weeks to become anemia, and then one group had an iron removal diet and a commercially available iron preparation ( Ferrous sulfate) 180 μg (iron amount 35 μg) was given, and iron removal diet and the above-mentioned jelly 0.5 g (iron amount 35 μg) were given to the other groups and bred, and after 30 days in blood of each group The hemoglobin value and the red blood cell count were measured. As a result, the average hemoglobin value in the group given the commercially available iron preparation was 7.2 g / dl and the average number of red blood cells was 5 × 10 ⁶ cells / mm ³ , whereas the average was 12.6 g / dl in the group given jelly.
And recovered to 8.1 × 10 ⁶ pieces / mm ³ .

Example 4 This example shows an example in which the hematopoietic agent according to the present invention is applied to an enteral nutritional agent excellent in iron absorption efficiency.

Preparation of Enteral Nutrient with Good Iron Absorption The iron-binding lactoferrin prepared in Example 1 was blended according to the table below to prepare an enteral nutrient with good iron absorbability.

Based on the above formulation, MCT casein powder 653 g, low-lactose skim milk powder 270 g, whole milk powder 62 g, iron-binding lactoferrin 14.3 g,
240 mg of vitamin mixed powder and 560 mg of calcium carbonate were mixed with a powder mixer to obtain 1 kg of a product.

Further, as a comparative example, MCT casein powder 659 g, low-lactose skim milk powder 275 g, whole milk powder 65 g, sodium iron citrate succinate 200 mg, calcium carbonate 560 mg and vitamin mixed powder 24
0 mg was mixed with a powder mixer to obtain 1 kg of a product.

Next, the enteral nutrient prepared using this iron-engaging lactoferrin as an iron source, and the enteral nutrient prepared without using the iron-binding lactoferrin are given to the rat by the following, respectively, for iron absorption efficiency Examined.

Test method: 10 Wistar rats were divided into 2 groups of 5 rats each, and each group was fed with iron-free diet, and one group was simultaneously treated with 0.5 ml of 50% aqueous solution of the above iron-binding lactoferrin-containing enteral nutritional supplement. It was administered daily, and to the other group, 0.5 ml of a 50% aqueous solution of an enteral nutrient without addition of iron-binding lactoferrin was administered, and the hemoglobin level in blood of each group was examined.

As a result, the hemoglobin value of the enteral nutritional group of the comparative example to which iron-binding lactoferrin was not added after 3 weeks was 7.8 g / dl on average, while the enteral nutritional supplement containing iron-binding lactoferrin was found. The hemoglobin value of the administration group of 13.
It was 1 g / dl.

[Brief description of drawings]

1 to 3 show the improving effect of the hematopoietic agent according to the present invention on the anemia state. FIG. 1 shows the number of red blood cells, FIG. 2 shows the values of hematocrit and FIG. 3 shows the values of hemoglobin. The changes are shown respectively.

Continuation of the front page (56) Bibliography Protides Bial Fluids, 16 (1968) P. 627-632 Quarterly Journal of Experimental Physiology, 60 (2) P.P. 79-88 (1975) Biochimica et Biop hysica Acta, 588 (1979) P. 120-128 Nutrition Research, 3 (1983) P. 373-384 Journal of Pediatric Gastroenterology and Nutrition, 2 (4) P. 693 (1983)

Claims (5)

[Claims] 1. A hematopoietic agent containing iron-bound lactoferrin as an active ingredient, which is obtained by chelating ferric ion to lactoferrin separated from milk. 2. The hematopoietic agent according to claim 1, wherein 1 to 2 molecules of iron are chelated to one molecule of lactoferrin. 3. The hematopoietic agent according to claim 1, which is in the form of a sugar-coated tablet, tablet or capsule suitable for oral administration. 4. The hematopoietic agent according to claim (1), which is formulated into an infusion solution and has a form suitable for enteral administration. 5. The hematopoietic agent according to claim 1, which is in the form of a beverage or jelly.