JPH03165833A - Novel suspending method and composite obtained thereby - Google Patents

Abstract

PURPOSE:To prepare stable and fine water suspension of fat-soluble material by coating the fat-soluble material which insoluble or hardly soluble in water but soluble in an org. solvent by using fatty particle membrane in the milk of mammals and then suspending the particles in water. CONSTITUTION:A fat-soluble material which is insoluble or hardly soluble in water but soluble in an org. solvent is coated with fatty particle membrane in the milk of mammals and then suspended in water. Namely, the fat-soluble material and fatty particle membrane in the milk of mammals are emulsified by a physical method to prepare fine water suspension of fat-soluble material. More concretely, after bioactive peptide is dissolved or suspended in water, neutral fat and fatty particle membrane in the milk of mammal are added thereinto and emulsified by a physical method to prepare the water suspension of the bioactive peptide.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel suspension method using fat globule membranes in mammalian milk and compositions containing the substances obtained by this method. More specifically, the present invention includes: (1) fat-soluble substances that are sparingly soluble or insoluble in water and soluble in organic solvents; (hereinafter, both of these substances are collectively referred to as "fat-soluble substances, etc."), particularly preferably fat-soluble drugs and certain steroids that are soluble in both water and organic solvents. A novel method for suspending drugs that are difficult to dissolve but whose partition coefficient is significantly tilted toward organic solvents compared to water (hereinafter, both drugs are collectively referred to as "lipid-soluble drugs, etc."), and ■
This invention relates to a novel formulation method suitable for oral administration of bioactive peptides.

Problem 4 This technique/invention aims to solve Problem 4 It has long been known from experience that it is extremely difficult to orally administer compounds that are insoluble or sparingly soluble in water and to achieve reproducible medicinal efficacy. However, recently, when lipophilic substances that are sparingly soluble or insoluble in water are administered orally, absorption in the gastrointestinal tract, distribution to various organs via the bloodstream or lymph fluid, and drug distribution within organs have recently been investigated. metabolism, and ■
Significant progress has been made in the study of the excretion of its compounds and metabolites into urine and bile. Research on drug delivery systems that aim to distribute drugs intensively to specific organs or cancer tissues is also becoming very active.

On the other hand, with the progress of pharmacology, the technology for evaluating drug efficacy has also significantly advanced, and it has become possible to measure the drug efficacy of even fat-soluble compounds that are sparingly soluble or insoluble in water with extremely high accuracy. However, when developing fat-soluble pharmacologically active substances as pharmaceuticals, it is necessary to formulate them in order to express their medicinal efficacy with good reproducibility. It has been known for a long time that it is extremely difficult to orally administer compounds that are insoluble or sparingly soluble in water to achieve reproducible drug efficacy. Therefore, the reason why reproducibility is poor when oral administration of fat-soluble drugs is the subject of active research.
Several findings regarding excretion, metabolism, etc. that differ from those for water-soluble compounds have been revealed. What has been revealed through these studies regarding the absorption, excretion, organ distribution, and metabolism of fat-soluble drugs can be summarized as follows.

■ When a lipophilic drug that is sparingly soluble or insoluble in water is orally administered, its gastrointestinal absorption rate is proportional to the surface area of the drug particles. That is, one of the factors governing the gastrointestinal absorption of fat-soluble drugs and the like is the surface area of drug particles. In other words, even when given the same amount of fat-soluble drugs, the larger the surface area, the more
In other words, the smaller the diameter of the drug particles, the faster the rate of absorption.

■ Lipid-soluble drugs are absorbed through the small intestinal mucosa by passive diffusion. No matter how poorly soluble drugs and the like are in water, once a very small amount of a single molecule is dissolved in water, they are absorbed into the body. In other words, most fat-soluble drugs are absorbed from the gastrointestinal tract and taken into the body at a slower rate than water-soluble drugs, but the process by which drugs are absorbed from the small intestine mucosa is not a rate-limiting step. This is because while fat-soluble drugs and the like dissolve in water only in very small amounts, the surface area of the small intestinal mucosa is extremely large, so the process of passive diffusion is not the rate-limiting step.

■ Even for drugs with very low solubility in water, the faster a single molecule is released from drug particles into water, the more
It is rapidly absorbed from the gastrointestinal tract. That is, another factor governing the gastrointestinal absorption of fat-soluble drugs and the like is the dissolution rate of the drug in water, not the degree of solubility.

■ When fat-soluble drugs are absorbed from the gastrointestinal tract, there are two routes by which they are taken into the body. One route is from the portal vein, through the liver, and into the general circulation, through which drugs are distributed throughout the body. Another route is absorption into the lymphatic vessels of the intestines,
Sometimes it drains into the aorta from the thoracic duct limba. When absorbed through the latter route, the drug often appears in the blood in its original form. On the other hand, the absorption route in which drugs pass from the portal vein to the liver is due to the first-pass effect (first-pass effect).
t pass effect) and are often chemically modified. Lipid-soluble drugs that are sparingly or insoluble in water vary greatly in absorption rate and absorption rate, ranging from barely absorbed to almost quantitatively absorbed even after oral administration, depending on the formulation method. Conventionally, studies have been made from two directions in order to increase the absorption rate and improve the absorption rate of fat-soluble drugs and the like.

One of these is the technology to make drugs into microparticles. For example, the average diameter of drug particles is 150-200
However, with the introduction of new equipment and new methods, it is now easy to industrially micronize drug particles to an average diameter of 2 μm. Furthermore, with regard to the technology for preparing drug formulations by suspending them in water, a technology has also been developed that allows oil droplets with a diameter of several micrometers to be stored as a stable suspension at room temperature for a long period of time. On the other hand, a method of mixing fat-soluble drugs and surfactants to form transparent micelles to increase absorption from the gastrointestinal tract has also come into practical use8.

Another direction is to increase the rate at which drug molecules are released into water from drugs or drug-containing particles, oil droplets, etc.

For example, it is well known that the dissolution rate and absorption rate of the antibiotic Novobiocin (trade name) vary greatly depending on the crystal form. In addition, research on the dissolution rate of molecules such as fat-soluble drugs in water has shown that the dissolution rate is higher when the drug is administered in the form of an amorphous powder or solid solution than in the form of crystals, which increases the absorption rate and amount of the drug. I've come to understand that I can.

As described above, there have been remarkable technological advances in formulations in recent years, but the formulation technology for lipophilic drugs, etc. has not been very effective in terms of reproducibility and efficiency in terms of absorption rate and amount when administered orally to living organisms. The current situation is that it cannot be said to be sufficient in any of these respects.

Furthermore, conventionally, the administration route for bioactive peptides has been limited to injections other than oral administration. This is because bioactive peptides generally have a high molecular weight, and common sense would not suggest that even if such high molecular weight peptides were orally administered, they would be absorbed at a high rate from the intestinal tract. It is. Furthermore, orally administered bioactive peptides are easily hydrolyzed by proteolytic enzymes secreted into the gastrointestinal tract, so there is the problem that by the time they reach the absorption site, they have been hydrolyzed and reduced considerably. ing. For this reason, bioactive peptides are administered as injections, but when bioactive peptides from foreign animals are administered as injections, antibodies corresponding to the non-self antigens of the foreign protein are produced, leading to concerns about shock due to antigen-antibody reactions. There is also the problem that there is. For this reason, many attempts have been made to formulate physiologically active peptides suitable for oral administration.

These attempts can be classified into the following three categories. One approach is to provide a proteolytic enzyme inhibitor at the same time as the bioactive peptide to prevent hydrolysis of the bioactive peptide in the gastrointestinal tract as much as possible, while allowing the bioactive peptide to be absorbed through the intestinal tract. Another approach is to increase the rate of absorption into the body by simultaneously giving 10 chemicals that promote the intestinal absorption of physiologically active peptides. A third approach is to try to increase the permeation of bioactive peptides through cell membranes by temporarily damaging intestinal mucosal cells by administering surfactants simultaneously. Additionally, methods that combine these methods have also been attempted.

However, these attempts had the following drawbacks in common.

■ The amount of bioactive peptide that is absorbed while retaining its physiological effects is extremely small.

■ Absorption is often not reproducible.

■ There is a large difference in the intestinal absorption of bioactive peptides among individuals, and even if the same amount is given to some individuals, it will be barely absorbed, while in other individuals, the absorption will be so variable that it will absorb an excessive amount and cause coma. .

The present inventors have continued research on fat globules present in the milk of chewing mammals for many years. As a result, it was discovered that the fat globule membrane, obtained by removing fat from fat globules, is suitable as a material for formulating fat-soluble drugs and physiologically active peptides that are poorly soluble or insoluble in water, and the present invention was completed. I ended up doing it.

The present invention provides: 1.9 A novel suspension method characterized by using a fat globule membrane in mammalian milk as a clouding agent; 2. A fat-soluble substance characterized by coating a fat-soluble substance with a fat globule membrane in mammalian milk in order to prepare a fine aqueous suspension of a fat-soluble substance that is poorly soluble or insoluble in water and soluble in an organic solvent. A new suspension method for 3. In order to prepare a fine aqueous suspension of a substance that is sparingly soluble in both water and organic solvents, but whose partition coefficient leans toward the organic solvent, this substance was added to the fat globule coating in mammalian milk. 4. A novel method for suspending a substance that is sparingly soluble in both water and organic solvents, but whose partition coefficient between the two solvents leans toward the organic solvent; 4. In order to make a fine aqueous suspension of a fat-soluble substance that is poorly soluble or insoluble in water and soluble in organic solvents, the fat globule membrane in mammalian milk and the fat-soluble substance are emulsified in water by physical means. 5. A novel method for suspending fat-soluble substances, characterized by: In order to make a fine aqueous suspension of OI, which is sparingly soluble in both water and organic solvents, but whose partition coefficient leans toward organic solvents, the fat globule coat in the milk of mammals and the above-mentioned A novel suspension of a substance that is sparingly soluble in both water and an organic solvent, but whose partition coefficient between the two solvents leans toward the organic solvent, characterized by emulsifying the substance in water by physical means. method, 6. 7. A novel method for suspending bioactive peptides, which comprises emulsifying bioactive peptides using fat globule membranes in mammalian milk in order to prepare a fine aqueous suspension of bioactive peptides; A novel suspension method characterized by dissolving or suspending a bioactive peptide in water, adding neutral fat and fat globule membranes in mammalian milk, and emulsifying by physical means; 8. Bioactivity A novel suspension method characterized by dissolving or suspending a peptide in water, adding neutral fat, a fat globule membrane in mammalian milk, and a protease inhibitor, and emulsifying by physical means. ,9. 10. A novel suspension method according to any one of claims 1 to 8, characterized in that the fat globule membrane in clear milk is a purified milk fat globule membrane. 11. A novel suspension method according to claims 1 to 9, characterized in that a dairy product containing a milk fat globule coating is used in place of the fat globule coating in mammalian milk. 11. The novel suspension method according to claim 10, wherein the dairy product containing milk fat globule membranes is buttermilk or cream. A suspension composition suitable for oral administration, characterized in that the suspension composition prepared by the method according to any one of claims d to 11 is made into a powder as it is, or by adding a filler and spray-drying it or freeze-drying it to powder. Manufacturing method of new formulation, 13. The present invention relates to a composition prepared by the method according to any one of claims 1 to 12.

14 Milk fat globule coating is suitable as the fat globule coating in the milk of chewing mammals used in the present invention, in view of the ease of obtaining the raw material. The properties, manufacturing method, etc. of the fat globule membrane in the milk of a chewing mammal used in the present invention will be described below.

It is well known that the milk of chewing mammals is an excellent nutritional food. Although milk differs depending on the animal species,
It contains 2-18% fat and serves as an energy source for young animals. Furthermore, fat, which never mixes with water, floats in milk in the form of fat globules with a diameter of 0.1-17 μm, creating a stable suspension. When classified according to the size of fat globules, fat globules with a diameter of 1 μm or less account for more than 80% of the total number, but their fat content accounts for only a few percent of the total fat in milk. Also, since there are only a small number of fat globules with a diameter of 6 μm or more, their proportion in total fat is about 2-2 μm.
Only 3%. Most of the fat in fat globules has a diameter of 2-
6μ (on average, 3.4μ) is present in fat globules, and the fat in fat globules within this distribution range accounts for 94% of the total fat in milk.
occupies . Therefore, the fat globules of milk, which is a typical milk, have an extremely large surface area, and the surface area per gram of fat reaches as much as 2. The reason why fat globules with such a huge area are able to maintain a stable suspension form is due to the interfacial layer that partitions the highly hydrophobic lipids from the water in the outside world, that is, the fat globule membrane (milk). fatglobu
This is because MFGM) exists. As a method to separate MFGM from fat globules,
A common method is to wash the cream obtained by centrifuging milk several times with water, and then physically destroy the fat globules in a process called churning.

The principle of this method is that the cream is cooled to harden the high-melting point neutral fat that constitutes the core of the fat globules, making them brittle, and the brittle fat globules are broken by vigorous shaking.

The fat flowing out from the fat globules destroyed by this treatment method fuses with each other to form a large mass and separates onto the water layer, where MFGM remains dispersed. Therefore, when the aqueous layer (buttermilk) separated in this way is dried by an appropriate means, MFGM is obtained in the form of a white powder.The MFGM thus obtained is mainly a structural riboprotein composed of lipids and proteins. Judging from the process by which it is synthesized in vivo, it is thought that it originates from milk wAIiIII follicles.In other words, milk fat synthesized within mammary gland cells is covered by the mammary cell membrane during the process of secretion from the cells, and is contained in milk. This is because it is secreted.

The yield of MFGM varies depending on the separation method from milk, but is 0.5-1.2 from milk fat 1fI. This MFG
More than 90% of M is composed of proteins and lipids, and approximately 55% is recovered as a lipid fraction and the remaining 45% as a protein fraction.

MFGM differs from other biological membranes in that it contains high melting triglycerides in the lipid fraction.
It contains a large amount of neutral fat called lyce-ride (HMTG). The role of this high melting point neutral fat is not clear, but considering that it is present in the part of the MFGM membrane that is in contact with milk fat inside the MFGM membrane, which is protected from proteins and phospholipids, it is believed that it plays a role in maintaining the hydrophobic environment inside fat globules. It is thought to play an important role in increasing the strength of fat globules. Therefore, normal human body membranes have a double membrane structure, but the MFGM membrane, which has the role of covering the extremely hydrophobic neutral fat inside, is characterized by a single layer structure.

When this MFGM is observed with a scanning electron microscope, only a two-dimensionally spread membrane-like structure is observed, and no spherical or hemispherical structure is observed as seen in the electron microscope image of liposomes.

A. In the present invention, a lipophilic drug that is sparingly soluble or insoluble in water is suspended by reconstitution of MFGM-coated microspheres.

In order to coat a lipophilic drug that is sparingly soluble or insoluble in water with MFGM, the drug must, in principle, be in the form of an oil that is immiscible with water. Therefore, drugs that are oily at room temperature can be directly coated with MFGM. In the case of a solid, it is usually necessary to dissolve it in a suitable solvent that does not cause protein denaturation, such as vegetable oil, specifically olive oil, for example. However, even if it is solid, if it is a powder, it can be mixed well with MFGM and suspended in water without using such a solvent. In this specification, a liquid drug or drug solution will be referred to as an oil. Preparation of emulsions of oils or powders in water using MFGM can be done using, for example, MFG.
This is carried out by dispersing M in a neutral buffer solution, adding an oil and emulsifying it by physical means, such as various types of homogenizers or ultrasound. MFGM microspheres are reconstituted by such operations. For this reconstitution, for example, when a homogenizer is used, the speed of the homogenizer, time, temperature, MFGM concentration, coating oil concentration, pH, etc. are major variables, and can be selected as appropriate. For example, the MFGM concentration, that is, the amount of MFGM added to the drug, should be 1% by weight or more, and increasing the amount of MFGM added will improve the emulsifying activity, emulsifying stability, and foam stability of the suspension of MFGM spherules. can be raised. However, from a practical standpoint, MFG
The amount of M added is preferably 2 to 8% by weight. General preparation conditions are as follows.

(General preparation conditions) Disperse 270+ng of MFGM in 10+nM sodium phosphate buffer (pr{7.0) and add iog
Set to 0n.

On the other hand, 2.5 g of oil containing the drug was collected in a Pyrex test tube (2.5717 cm), and 7.5+nN (containing about 200+H of M F G M ) of the buffer solution described in "2" was added thereto. Warm at °C for 5 minutes. The content of MFCM per 1 g of oil is 80B. Then, while heating, the mixture was stirred for 1 minute using a Polytron homogenizer (using an ST20 shaft) at maximum speed (mark 11) to obtain an emulsion. Especially MFG for oily eA1g
When the content of M is 8Q+ny, a highly stable emulsion with an emulsion stability of 1 is obtained.

The emulsion thus obtained can be powdered if desired. For example, the conditions for powdering are as follows.

■ Solvent (neutral fat): Neutral fat, which has solid or semi-solid physical properties at room temperature, is selected as the solvent for dissolving the fat-soluble drug.

■ Bulking agents: milk protein, lactose, etc.

The drug-loaded MFGM microspheres can be mixed with the bulking agents described above and spray-dried or freeze-dried to obtain a stable powder containing the MFGM microspheres.

B. In the method for formulating a physiologically active peptide of the present invention,
Examples of physiologically active peptides include insulin, oxytocin, vasopressin, pituitary gonadal stimulating hormone (H
MG). Placental gonadotropin (HCG), growth hormone, corticotropin, calcitonin, gonadotropin-releasing hormone, thyroid-stimulating hormone-releasing hormone,
Interferon-α, interferon-β, erythroboetin, colony stimulating factor (GM-CSF), GM-CSF
CSF, M-CSF, epidermal growth factor (EGF)
, nerve cell growth factor (NGF), somatomedin, batroxobin, prourokinase, tissue plasminone activator, staphylokinase, superoxide disl\tase, growth hormone releasing factor, somatostati〉', atrial natriuretic peptide, protein C
, protein S, lung surfactant, ribocortin, interleukin 1, interleukin 2, interleukin 3, tumor necrosis factor (T NF ), lactoferrin, transferrin, immunoglobulin, and the like.

Formulation is carried out by dissolving or suspending the above-mentioned physiologically active peptide in water, adding neutral fat and fat globule membrane (MFGM) in the milk of chewing mammals, and emulsifying it by physical means. It will be done.

At this time, it is advantageous to add a protease inhibitor at the same time. There are no particular limitations on the protease inhibitors used, but specific examples include Trapivir, Pepscutin, and Leupeptin. Note that a neutral buffer solution can be used instead of water for dissolving or dispersing the physiologically active bebutide. Examples of neutral buffers include 1]H7 phosphate buffer or Tris/HCl buffer. The neutral fat used for suspending the physiologically active peptide of the present invention can be arbitrarily selected from, for example, those that are liquid or semi-solid at room temperature. Specifically, examples thereof include octyldesilt 22 liglyceride, which is an artificial neutral fat, vegetable oils such as coconut oil, butter oil, and animal and vegetable oils containing unsaturated fatty acids.

Note that when insulin is used as the physiologically active peptide, coconut oil is a suitable example of a neutral fat because of its excellent solubility.

The physical emulsification means used in the present invention may be carried out by using various homogenizers or by ultrasonic treatment. Fat globule membrane in the milk of chewing mammals (M
FGM), specifically, for example, the amount of milk fat globule membrane added is preferably in the range of 2 to 10% (w/w) based on the neutral fat used.

The emulsion obtained by such a method can be powdered if desired. Powderization is carried out by adding a suitable filler such as lactose, whey protein, casein, microcrystalline cellulose, etc. to the emulsion and freeze-drying or spray-drying. The powder thus obtained is itself chemically stable and can be formulated into tablets, granules, etc. as appropriate.

The characteristics of MFGM used in the present invention are that it emulsifies neutral fats more efficiently and has extremely high stability compared to artificial surfactants. In other words, artificial surfactants are a type of cytotoxin with hemolytic properties, and when ingested in large quantities, it is not uncommon to cause severe diarrhea due to detachment and sloughing of the gastrointestinal mucosa. On the other hand, MFGM has a surface derived from cell membranes, is free from cell membranes like artificial surfactants, and is characterized by extremely high stability. In the present invention, dairy products containing MFGM, such as buttermilk or cream, can be used instead of MFGH.

The present invention will be explained in more detail below with reference to Examples and Experimental Examples, but the present invention is not limited thereto.

(Example 1) Measurement of solubilization ability MFGM (provided by the Seiken Organization project of Chugai Pharmaceutical Co., Ltd.) was used, HCO60 (manufactured by Nikko Chemicals Co., Ltd.) and BL9 (manufactured by Nikko Chemicals Co., Ltd.) were each used at 100 μg. Add 25.50,100μ of vitamin A to a suspension or aqueous solution at a concentration of /ml.
The cells were added to a concentration of 1F/+a1 and subjected to ultrasonication at 30W for 5 minutes using an ultrasonic cell disrupter (manufactured by Otake Pharmaceutical Co., Ltd.). When the light transmittance of each liquid was measured at 640 nm, the results shown in FIG. 1 were obtained.

As can be seen from this figure, when using any solubilizer, as the concentration of vitamin A increases, the transmittance decreases.
That is, an increase in turbidity was observed, but when HCO60 was used, the increase in turbidity was low, whereas with BL9 and MF
When GM was used, the behavior was almost the same as when using only the buffer solution.

Therefore, it was revealed that MFGM has almost no solubilizing ability. Note that triolein was used as the oil layer.

(Example 2) Measurement of emulsifying ability 4l Straight ∆ 4: Contains 12B of vitamin A, 1g of fat and oil, and 8% of MFGM
Sodium phosphate buffer containing 0 mg/ml2
99 and heated at 40-45° C. for 5 minutes, and then stirred for 1 minute at maximum speed using a Polytron homogenizer to obtain an emulsion. (The 25 obtained here is an emulsion containing 5% by weight of oil and fat.) The above MF
Instead of GM, lecithin (manufactured by Sigma), sucrose fatty acid ester S-1170 (manufactured by Mitsubishi Kasei Foods Co., Ltd.), sucrose fatty acid ester P1670 (manufactured by Mitsubishi Kasei Foods Co., Ltd.), Tween 80 (Nikko) Chemicals Co., Ltd.) and HCO60 (Nikko Chemicals Co., Ltd.) surfactants (manufactured by Nikko Chemicals Co., Ltd.) each containing 19 g of a sodium phosphate buffer solution containing 80 IIlg/1, and 1 fat and oil containing 12+ng of vitamin A.
An emulsion containing each surfactant was prepared in the same manner as above.

Put each emulsion prepared above into a test tube, stand it vertically, and leave it at room temperature. Measure the cream layer length and the total layer length of the emulsion over time. From these measurements, (cream layer length /
The total layer length of the suspension was determined and used as an index of emulsion stability. The results are shown in Figure 2. Note that triolein was used as the oil.

As can be seen from Figure 2, the emulsion stability value of MFGM is lower than that of lecithin and Tween 80, and is approximately the same as that of HCO60, which indicates that MFGM has good emulsion stability. It shows that there is.

Each of the emulsions prepared above was placed in a test tube and stood vertically. 0.2 mI from the bottom of the test tube over time at room temperature
Collect each emulsion and mix them with 0.1z sodium dodecyl sulfate aqueous solution 3.81I+N, and add 6401l
The transmittance in ll was measured under seed conditions. These results are shown in FIGS. 3 to 6.

FIG. 3 shows the effect of each surfactant in the emulsion. According to this, the emulsion activity of MFGM was lower than that of buffer-only lecithin, and up to 30 hours later, Tween80 and HCO60
showed almost the same value.

In particular, the values up to 10 hours later are low, and it is clear that MFGM maintains its emulsion state well.

Figure 4 shows the effect of M F on emulsion activity.
This figure shows the influence of the amount of GM added. According to this, the normally used content is 80wn)7M
F GM /g
A rapid increase in emulsion activity was observed in the case of MFGM/g fat/oil, but on the other hand, in the case of high emulsion activity (160+nyMFGM/y fat), the value did not increase much even after 30 minutes. From this, it is considered that the greater the amount of MFGH added, the better the emulsified state is maintained.

FIG. 5 shows the results of a comparison of the effects on emulsion activity of sucrose fatty acid ester, which is a highly safe surfactant, and MFGM. According to this, MFGM showed an increase in emulsion activity compared to P-1670 (a sucrose fatty acid ester of H L B-16 bound to palmitic acid residues), but 81.170 (a stearic acid residue). It showed almost the same value as the sucrose fatty acid ester with HLB=11 in which the residues were bonded.

In addition, in the experiments related to FIGS. 3 to 5 above, triolein was used as the fat or oil.

FIG. 6 shows the effect on emulsion activity when oils other than triolein are used. Oleic acid, monoolein, and butter oil were used as fats and oils other than triolein. As a result, monoolein had the lowest increase in emulsion activity, and oleic acid had the highest increase. Butter oil and triolein showed almost the same values in between.

Next, drug-containing MFGM prepared by the method of the present invention
A test example is shown in which the drug absorption status when microspheres are administered to animals is compared with a preparation prepared by a conventional method.

(Example 3) Effect of MFG on lymphatic absorption of DDT
DDT, which is used as a model compound for fat-soluble drugs when examining drug absorption during oral administration, was used as the drug for testing the effects of M.

First, male rats of the Wista strain are fasted overnight, and cannulae are inserted into the duodenum and lymph thoracic duct by surgery. Next, an MFGM-containing microsphere emulsion prepared using an olive oil solution of DDT according to the general preparation conditions described above and a simple olive oil solution of DDT without MFGM were each administered into the duodenum of the rat through a cannula. The dose of DDT was in each case 10
my/kg.

After administration of DDT, lymph fluid flowing out from the thoracic duct limbus was collected and analyzed for DDT. The results are shown in Table 1.

Absorption rate (z) Rat**P<0.01 15.31 +7.79 6 0 6 41.16 +14.7** 4 0 4 As is clear from this table, DDT was dissolved in olive oil and used in rats. 30, the total amount of DDT absorbed within 12 hours is 15.31
However, if an olive solution of DDT is made into microspheres by MFGM and administered in the same manner, the absorbed amount is 41.g.
16 g, clearly supporting the absorption promoting effect of M F GM.

(Example 4) Effect of MF on lymphatic absorption of vitamin A
Effects of GM Male Wistar rats weighing approximately 400 g were fasted overnight. After a fasted rat was anesthetized with intraperitoneal administration of bentobarbital, the rat's abdomen was incised and a silicone cannula was inserted between the gastric pylorus and duodenum and into the ileocecal region. A drug solution (5-) consisting of each emulsion prepared in Example 2 was injected into the small intestine (-duodenum, jejunum, ileum) loop through the upper cannula, and the cannula was closed with forceps.

Blood and lymph fluid were collected over time from the groin artery and thoracic duct lymph, and the vitamin A concentration in both fluids was measured. Also,
The bile duct was cannulated and bile was excreted from the body (b
Do the same thing for rats (fistula) and leave them without such treatment (intact).
compared with rats.

The dose of vitamin A was 15IIg/rat. Quantification of vitamin A concentration was performed using an excitation wavelength of 340 nm and a measurement wavelength of 480 nm after extracting each sample with cyclohexane.
This was done by fluorescence measurement at nm.

The results are shown in FIGS. 7 to 9. It is known that vitamin A forms chylomicrons after being absorbed from the small intestine, and most of them are transferred to the lymphatic system rather than the blood system. In this study as well, the effect was determined based on the concentration in the lymph fluid, as it was hardly detected in the blood.

FIG. 7 shows the results when an emulsion prepared using sesame oil as the fat was administered to the small intestinal loop of bile fistula rats. According to this, the case using MFGM and the synthetic surfactants Tween 80 and HC
There is no large concentration difference at any time between the case of using O60 and the case of using O60. The concentration in the lymph reached its highest value 2.5 to 3.5 hours after administration, and it can be seen that the value was highest when MFGM was used.

FIG. 8 shows the results when the emulsion was administered into the small intestinal loop of intact rats under the same conditions as in FIG. According to this, MFGM and Tween80
It can be seen that the peak values in both cases increased 2 to 3 times, indicating that the amount transferred into the lymph increased.

On the other hand, when increasing the oil content from 5% to 25%,
Since the concentration of vitamin A in lymph is extremely low, it has become clear that the fat content in the emulsion greatly influences the absorption of vitamin A.

FIG. 9 shows the concentration of vitamin A in lymph depending on the type of oil in the emulsion. In addition, only in this case, vitamin A
The dosage was 2.5B/rat.

An emulsion with an oil content of 5z was used. According to FIG. 9, the concentration of vitamin A in lymph was highest when butter oil was used, and lowest when oleic acid was used. From this, it has become clear that a butter oil emulsion that closely resembles the state of natural fat globules in milk is useful for the absorption of vitamins.

33 As mentioned above, the effect of M on lymphatic absorption of DDT and vitamin A
Although the effects of FGM have been described, the absorption promoting effect of MFGM was also observed for other fat-soluble drugs.

For example, when a compound such as vitamin E, which is oily at room temperature, is turned into microspheres by MFGM, a remarkable effect of promoting absorption in the gastrointestinal tract was observed.

That is, a cottonseed oil solution of vitamin E and the solution were mixed with MFG.
When the microspheres formed by M were orally administered to rats, the absorption amount of the latter was 2.6 times that of the former. The absorption promoting effect of fat-soluble drugs when MFGM was formed into microspheres under the same conditions was also observed in fat-soluble vitamins such as vitamin D and vitamin I, and isoprene compounds such as ubiquinone. A similar phenomenon was observed for drugs such as probucol, avan, isoflavones, and prostaglandins.

Furthermore, drugs that are difficult to dissolve in both water and organic solvents, but whose partition coefficient between organic solvent (e.g., octanol) and water is large and tilt toward the organic solvent (octanol) can also be formed into microspheres by MFGM. A remarkable promotion of absorption was observed when the drug was administered as a compound.An example of a test using hydrocortisone as an example of such a drug is shown below.

(Example 5) Effect of MFGM on the absorption of hydrocortisone in the body After fasting male Wista rats weighing approximately 300 y overnight, the skin at the right common carotid artery site was incised and a cannula was inserted into this artery. On the other hand, an emulsion (prepared according to the general preparation conditions described above) in which a cottonseed oil solution of tritium (3H)-labeled hydrocortisone was turned into microspheres using MFGM was orally administered.

As a control, hydrocortisone made into transparent micelles with the surfactant T...een80 was used. The dosage of hydrocortisone is 1.5B/k! ? Met. Blood was collected periodically after administration. After collecting feces and urine and administering
After a period of time, the animals were sacrificed, and the radioactivity of hydrocortisone was measured in the digestive tract, its contents, and each organ to determine the hydrocortisone content.

(Note that when fat-soluble drugs, etc. are treated with Tu+een80, they appear to be in the form of a transparent solution and the fat-soluble drugs, etc. are dissolved. However, even in such cases, fat-soluble drugs, etc. are dissolved in the molecular state. Most of the micelles exist as fine micelles, but their diameter is less than the wavelength of light, allowing light to pass through them. It is known that the absorption rate and amount of the drug are greatly increased when administered in a state where the drug is absorbed.

It is thought that the following two points are responsible for this.

■ Since the micelle diameter is smaller than the wavelength of light, the surface area of drug particles increases dramatically.

■ The surfactant used during micelle formation temporarily damages the small intestinal mucosa, causing the intestinal mucosal permeability barrier to be lost and absorption to increase.

Therefore, in this study, the absorption promoting effect of MFGM was compared using the test condition showing the highest absorption rate for hydrocortisone as a control. ) The test results are shown in Figure 10 and Table 2.

FIG. 10 shows the blood concentration of tritylim hydrocortisone after administration of this drug.

When solubilized with Tu+een80, the maximum blood concentration is reached 5 minutes after administration, and the maximum blood concentration at that time is also slightly less than twice that of J when administered as MFGM microspheres. When administered as MFGM microspheres, it takes 60 minutes to reach maximum blood concentration, and the peak height is low. However, when comparing the AUG, the two groups tend to be almost the same, or the MFGM group tends to be slightly larger. Therefore, when hydrocortisone is administered as MFGM microspheres, it exhibits an AUG equivalent to that of TIlleen 80 solubilized micelles, which are said to exhibit the highest blood concentration and AUG among the current oral preparations.

Table 2 shows the effect of M on the persistence of hydrocortisone in the body.
This shows the influence of FGM. according to this,
When administered in the form of MFGM microspheres, Tu+ee
It has been suggested that drugs are absorbed through a different route than in n80 micelles.

37 2 Hydrocozone nipple 9.6±0.2 35.3±13.5 11.6 (8.5, 14.8) 39.0 (32.3, 45.6) Stomach, small intestine, large intestine, cecum Stomach contents Small intestine contents 0.23±0.25 0.75±0.47 0.06±0.04 9.25+3.07 0.52±0.58 3.27+:1.37 0.15( 0.21, 0.08) 3.98 (5.54, 2.41) 0.38 (0.72 0.03) 18.96 (17.70, 20.21) 0.34 (0.
60, 0.07) 12.71 (16.68, 8.54) In other words, when looking at the balance of hydrocortisone 24 hours after oral administration to rats, the excretion amount and gastrointestinal residue are subtracted from the total dose. The residual amount in the body was 12.41 g in the 38 Tween 80 group, but M F G M
In the group, 53.122, or 4.3 times more, are distributed in the body. Changes in drug absorption routes are presumed to be the cause of such large differences in biodistribution. That is, Twee
When n80 micelles are administered, hydrocortisone absorbed from the intestinal mucosa flows into the general circulation via the portal vein and the liver, but when administered as MFGM microspheres, the hydrocortisone is absorbed from the intestinal mucosa and enters the general circulation. This is because the contents of the chest are thought to be taken up by the lymphatic vessels and poured into the veins via the thoracic lymphatic vessels.

That is, MFGM is difficult to dissolve in water or organic solvents like hydrocortisone, but it has the following great advantages when formulating drugs that have a larger partition coefficient to organic solvents than water.

■ Absorption rate and absorption rate are equivalent to those of surfactant-based solubilized micelles, which have extremely high absorption rates.

■ Unlike surfactants, it does not cause damage to the intestinal mucosa.

■ The residence time of the drug in the body is significantly extended due to changes in the absorption route. In other words, it shows the effectiveness of sustainability.

(Example 6) Coconut oil 3I01 was added as fat to 3.5 ml (l) of 10 mM phosphate buffer with pH 7.0 containing 80 my of MFGM, and human insulin solution (40 U/II11) was added.
6 mN of was added and heated at 45°C for 5 minutes. Next, the mixture was stirred for 1 minute using a Polytron homogenizer to completely emulsify the coconut oil. This emulsion contains 1 insulin per ml.
Contains 9.20. This preparation can be orally administered as is, but in order to maintain its preservability, lactose 'y. 5
g, and 1.2 g of whey protein were added and freeze-dried to powder. The insulin content of this powder was 20 U/g.

(Example 7) Streptozotocin in IOIIIM citric acid solution (1) H
4. An insulin-dependent diabetes model was created by injecting O> from the tail vein at 60 mg/kg.

Rats were fasted for 12 hours after being injected with strebtozotocin for 1 week, and then randomly divided into three groups: MFGM/insulin group, insulin group, and saline administration group, and used in the experiment. First, rats were subjected to laparotomy under Nembutal anesthesia, a catheter was inserted into the duodenum, and 1101 insulin (40 U) or physiological saline was injected into the intestinal tract through the catheter. Blood was collected from the tail vein 1, 2, 3, and 4 hours after injection, and blood glucose and serum insulin (immunoreac
tive insulin (IRT) was measured. First, regarding blood sugar, as shown in Figure 11, the pre-administration value was around 280 my/dl in all groups, but the blood sugar value in the insulin injection group tended to decrease for 1 to 2 hours compared to the control group. However, no significant difference was observed. On the other hand, MF
The blood glucose level in the GM/insulin group rapidly decreased until 2 hours after injection, and the 2-3 hour value was significantly lower than that in the control group. In addition, each point in FIG. 11 represents the blood glucose level of 9 to 10 diabetic rats using the average value + or one standard error. In addition, radioimmunoassay method (Dinabbot Laboratories
) Blood IRI in the control group was below the detection limit at all time points. On the other hand, blood IRI in the MFGM41/insulin group was undetectable before injection, but rose to 130 μU/II11 1 hour after injection, and gradually decreased after 2 hours. Blood IRI in this group
When compared with the insulin group, the 1-2 hour value has a higher risk rate P
<'0.01, the 3-hour value was significantly high with a risk rate P<0.05<, and a difference of more than 100 times was observed in the AUG comparison. In addition, each point in FIG. 12 represents the average value of blood insulin in 9 to 10 diabetic rats with + or one standard error. Blood insulin in the control group is below the detection limit at all time points, so it is omitted from the figure. The insulin absorption rate in the MFGM/insulin group one hour after injection is approximately 5%, assuming that the blood volume is 1/13 of the body weight, and 1/3 of the absorbed insulin is captured in the liver. Taking this into consideration, the result suggests that approximately 8% of the amount was absorbed from the intestinal tract. The intestinal absorption rate of insulin by conventional administration methods is at most 1% in the best case, and is usually less than 0.1π in most cases. Therefore, the intestinal absorption rate of the orally administered MFGM/insulin group is 50 to 100 times more efficient than the conventional method.

(Example 8) Male Sprague-Dawley rats weighing 300fI were given 50II of streptozotocin in the same manner as in Example 7.
1g/kg was administered intravenously to induce diabetes. One week after streptozotocin injection, the rats were randomly divided into three groups, one as a control group and the other two as an insulin group and an insulin/MFGM group.

After fasting rats I~ overnight, the control group (n=6) was given physiological saline, and the other groups (n-6 each) were given insulin by suspending the MFGM/insulin powder prepared in Example 1 in water. The third group (n=6) received 40 U of insulin orally. The amount of solution administered was 1-1 per 100 g of rat body weight. Rats were housed in Bollman cages, and blood was collected from the tail vein at regular intervals to measure blood sugar and serum insulin (I R I ).

As is clear from Table 3, high concentrations of insulin were detected in the blood, and blood sugar levels decreased only in the MFGM/insulin group. In the insulin group to which only the same amount of insulin solution was administered, almost no insulin was detected in the blood, and no significant drop in blood sugar was observed. Therefore, insulin is emulsified using milk fat globule membranes and fats and oils,
Furthermore, even if this emulsion is powdered by adding milk components, it is clear that a large amount is absorbed from the intestinal tract.

(Example 9) Twenty-four healthy Wista male rats weighing approximately 200 g were fasted overnight and then randomly divided into three groups, the first group being a control group (n=8) and the second group being an insulin control group. group (n=8
), the third group was the insulin fat emulsion group (n=8>).The insulin fat emulsion was the human insulin solution (4
0 U/ml) 10- was prepared by adding 10 ml of cream separated from fresh milk and mixing thoroughly.

The first group of rats was orally administered with physiological saline, the second group with insulin solution, and the third group with insulin fat emulsion. The amount of liquid administered was 20 U/k. The rats were housed in a restraint cage and were exposed to 120 minutes immediately before administration, 30 minutes after administration, and 60 minutes after administration.
After 180 minutes, blood was collected from the tail vein and blood glucose and serum insulin were measured. The results are shown in Table 4, only when insulin was mixed with cream and administered.
Large amounts of insulin appeared in the blood, resulting in a drop in blood sugar.

Example 10) An experiment was conducted by orally administering human growth hormone produced by genetic engineering to male Wistar rats.

The tail vein of a Wista strain male rat weighing approximately 200 grams was cannulated under ethereal vinegar and restrained in a Bollman cage. 110g of human growth hormone 14+nN pl+ospl+ate l+uffer
saline (PBS.+)H 7.4>, 240+ng of MFGM was added, and the mixture was heated to 40°C. Coconut oil 6]n1, previously heated and dissolved at 40°C, was added to this solution, and homogenized for 1 minute at maximum speed using a Polytron homogenizer to prepare a growth hormone emulsion (50μ2/+off). As a control, PBS and a growth hormone solution in PBS (50 Jig
/m/) was used. There were 6 rats per group. PBS was orally administered to the control group, a PBS solution of growth hormone to the growth hormone emulsion group, and an emulsion to the growth hormone emulsion group at a rate of 2+nN/rat.
Blood was collected over time from the tail vein of 3 + n 1, and the human growth polmon level in the blood was quantified by radioimmunoassay using a two-antibody method.

Table 5 Human Growth Hormone Emulsion Administered in the Blood of Rats When administered as a mature emulsion, human growth hormone appears in the blood at a high concentration from 30 minutes after administration, reaching the highest level 2 hours after administration. Ta. On the other hand, human growth hormone was not detected in the blood of the control group. Also, when dissolved in PBS and given orally, a small amount appears in the blood after 90 minutes, but
The absorption rate from the intestines is estimated to be less than 1/100 compared to emulsion. Therefore, the advantage of administration as an emulsion is clear.

(Example 11) The duodenum and tail vein of a male Wistar rat weighing approximately 200 grams were cannulated under ether anesthesia, and the rat was restrained in a Bollman cage.

On the other hand, eel calcitonin 1IIIfI is 71I11
p l+ o s pbate I+uffer
It was dissolved in saline (pH 7.4), 120 mg of MFGM was added, and the mixture was heated to 40°C. To this liquid, 3 ml of butter oil previously heated and dissolved at 40°C was added and subjected to ultrasonication for 2 minutes to form a calcitonin emulsion (
1. O O Jig/m#) was created.

0.5 ol of this calcitonin emulsion was injected into the duodenum through a cannula, and 0.3 ml was injected into the duodenum from the tail vein over time.
Blood was collected from each animal, and the level of eel calcitonin in the blood was determined by radioimmunoassay using a two-antibody method. PBS(-) for the control group, calcitonin pl+ospl+ate l1urfersa for the calcitonin control group
A line solution (100 μg/'1 nl-) was injected at 0.5+nN each, and blood was collected on the same schedule as the emulsion administration group. In addition, administering calcitonin 3
Dissolve rudium chloride in physiological saline 60 minutes before
+ngl ky was administered intraperitoneally to stimulate endogenous turzitonin secretion. -The group consists of 6 rats.

50 Table 6 Blood Calcitonin concentration of rats administered with calcitonin emulsion As is clear from the blood calcitonin concentration of rats administered with calcitonin emulsion, the blood calcitonin concentration in La Soto administered with calcitonin emulsion was overwhelmingly higher than that of the two control groups.
It is certain that a considerable amount of calcitonin in the emulsion is absorbed from the intestinal tract.

(Example 12) Under ether anesthesia, the portal vein of a male Wista strain rat weighing approximately 200 grams was cannulated, and the rat was restrained and fixed in a Bollman cage. Lactoferrin o isolated from milk. tg to 14- p I+ o s p l+
a t e1 + buffer saline (pH 7
．． 4>, add 240 mg of MFGM and heat at 40°C.
It was heated to Add to this liquid 51 Butter oil heated and dissolved at 40℃ 6 + n1
was added and subjected to 2 minutes of ultrasonication to create a lactoferrin emulsion (5 mg/me). In addition, as a control, 1+l+osl+bate of lactoferrin
A buffersaline solution (5+^#/mN) was prepared. This lactoferrin solution or emulsion was injected into the stomach of a rat in two doses through a gastric tube, and 0.3 ml of blood was collected over time from the portal vein, and the bovine lactoferrin level in the blood was measured using the two-antibody method. Quantified by radioimmunoassay. A group consists of 6 rats.

Phosphorus emulsion group *p<o. oot As is clear from Table 7, lactoferrin is phosphorylated tSm
It has been revealed that when lactoferrin is administered dissolved in a liquid, almost no lactoferrin appears in the blood, but when a lactoferrin emulsion is orally administered, it appears in the blood at a high level. That is, when bovine lactoferrin, which has a molecular weight of 87,000, is orally administered as an emulsion together with milk fat globules, it is clearly absorbed into the body from the gastrointestinal tract.

(Example 13) Against rotavirus (NCD V strain, serotype 6) (
1:31250) 8 g of bovine immunoglobulin with a Q-neutralizing antibody titer was added to a 400-pbospbate buff.
er saline (P B S , pH 7.4), heated to 40°C, added 16 g of milk fat and 800B of milk fat globule membrane, and homogenized with a Polytron homogenizer for 2 hours.
Homogenized for minutes. 200 ml of this emulsion was administered orally to 4 healthy adult male volunteers, and 5 blood samples were collected immediately before administration, 30 minutes after administration, 1.2 hours, and 3 hours after administration to obtain serum. One week later, the same four volunteers were given the same immunoglobulin 21? 20
0ml of pl+ospbate buffers
It was dissolved in aline (pH 7.4) and administered orally, blood was collected according to the same schedule as before, and serum was separated. Neutralizing antibodies against rotavirus contained in these separated sera were purified using the NCDV strain.
It was measured by the reduction method, and the anti-rotavirus neutralizing antibody titer in the serum was expressed as a dilution ratio.

54 Table 8 Neutralizing antibody titer in serum 55 As is clear from Table 8, the neutralizing antibody titer in blood was highly significantly increased when immunoglobulin was taken internally as a fat emulsion compared to when it was taken internally as a PBS solution. . Therefore,
It is clear that a significant amount of immunoglobulin is absorbed from the intestinal tract when taken internally as a fat emulsion.

As explained above, according to the method of the present invention using MFGM and the pharmaceutical composition obtained by this method, the absorption rate and absorption when lipophilic drugs, bioactive peptides, etc. are orally administered can be improved. The amount is significantly improved compared to conventional formulation techniques. Furthermore, since MFGM is a food-derived surfactant, it is also excellent in terms of safety.

[Brief explanation of the drawing]

FIG. 1 is a graph showing the solubilizing ability of MFGM used in the present invention, FIGS. 2 to 6 are graphs showing the emulsifying ability of MFGM used in the present invention under various conditions, and FIGS. 7 to 9 The figure is a graph showing the influence of MFGM on the lymphatic absorption of vitamin A, Figure 10 is a graph showing the blood concentration of hydrocortisone after oral administration of a hydrocortisone emulsion using MFGM, and Figure 11 is a graph showing diabetes. Graph showing changes over time in blood sugar levels in rats after administering insulin into the duodenum of rats. Figure 12 shows changes over time in blood insulin in rats after administering insulin into the duodenum of diabetic rats. It is a graph.

Claims (1)

[Claims] 1. A novel suspension method characterized by using a fat globule membrane in mammalian milk as a suspending agent. 2. In order to prepare a fine aqueous suspension of a fat-soluble substance that is poorly soluble or insoluble in water and soluble in an organic solvent, a fat-soluble substance is coated with a fat globule film in mammalian milk. A new method for suspending soluble substances. 3. In order to prepare a fine aqueous suspension of a substance that is sparingly soluble in both water and organic solvents, but whose partition coefficient leans toward organic solvents, this substance is added to the fat in mammalian milk. A novel method for suspending a substance that is sparingly soluble in both water and an organic solvent, but whose distribution coefficient between the two solvents is tilted towards the organic solvent, characterized by coating the substance with a spherical film. 4. In order to make a fine water suspension of a fat-soluble substance that is poorly soluble or insoluble in water and soluble in an organic solvent, the fat globule membrane and the fat-soluble substance in mammalian milk are separated in water by physical means. A novel suspension method for fat-soluble substances characterized by emulsification. 5. In order to make a fine aqueous suspension of substances that are sparingly soluble in both water and organic solvents, but whose partition coefficients tend toward organic solvents, we prepared the above substances with the fat globule coat in mammalian milk. A novel suspension of a substance that is sparingly soluble in both water and an organic solvent, but whose partition coefficient between the two solvents leans towards the organic solvent, characterized by emulsifying the substance in water by physical means. Method. 6. A novel method for suspending bioactive peptides, which comprises emulsifying bioactive peptides using fat globule membranes in mammalian milk in order to prepare a fine aqueous suspension of bioactive peptides. 7. After dissolving or suspending the bioactive peptide in water,
A novel suspension method characterized by adding neutral fat and fat globule membranes in mammalian milk and emulsifying the mixture by physical means. 8. After dissolving or suspending the bioactive peptide in water,
A novel suspension method characterized by adding neutral fat, a fat globule membrane in mammalian milk, and a protease inhibitor, and emulsifying the mixture by physical means. 9. A novel suspension method according to any one of claims 1 to 8, characterized in that the fat globule membrane in mammalian milk is a purified milk fat globule membrane. 10. A novel suspension method according to claims 1 to 9, characterized in that a dairy product containing a milk fat globule coating is used in place of the fat globule coating in mammalian milk. 11. The novel suspension method according to claim 10, wherein the dairy product containing milk fat globule membranes is buttermilk or cream. 12. For oral administration, the suspension composition prepared by the method according to any one of claims 1 to 11 is used as it is, or is powdered by spray drying or freeze drying with the addition of a filler. Method of manufacturing suitable new formulations. 13. A composition prepared by the method according to any one of claims 1 to 12.