JPS6221335B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to pharmaceutical substance-containing fat emulsions with selective tropism. Fat emulsions used to contain pharmaceutical substances are generally O/W type emulsions, in which vegetable oils such as soybean oil are mixed with nonionic surfactants and emulsifiers such as egg yolk phospholipids, lecithin, and soybean phospholipids. It is prepared by emulsifying.
This is a nutritional infusion and is used as a heat source after administration to a living body. On the other hand, this fat emulsion has recently been developed by embedding anticancer drugs within the fat emulsion particles because the oil particles themselves have a high affinity for lymph vessels and specifically migrate to lymph nodes.
There are attempts to use fat emulsions as carriers for anticancer drugs [GANN 64 , 345-350 (1973)]. The present inventors also completed an invention in which an anti-inflammatory agent was embedded in a fat emulsion and utilized as a carrier for the anti-inflammatory agent by taking advantage of the high affinity between oil particles and inflamed local areas. (Patent application 1986-64875). The technology for using this fat emulsion as a carrier for pharmaceutical substances is
This originates from the lymphatic affinity of the oil particles themselves.
Considerable effects are expected. However, the drawback of this is that the in-vivo transport is somewhat non-specific, and the delivery of the drug substance to the diseased organ to be treated is incomplete. Usually, the degree of attainment is about 1 to 5% at most. Therefore, the present inventors investigated various methods for maximizing the effectiveness of this fat emulsion as a drug substance carrier. As a result, it was unexpectedly found that γ-globulin binds to the membrane surface of the oil particles of the fat emulsion, and the affinity between the bound γ-globulin and cells containing the corresponding antigen increases. -We have discovered that the selection-directing function of fat emulsions as pharmaceutical substance carriers can be doubled by utilizing antibody reactions, and have completed the present invention. An object of the present invention is to provide a fat emulsion containing a medicinal substance that has selective tropism to disease sites or pathogens (viruses, bacteria, etc.). Here, selective tropism refers to tropism toward cells (pathogen cells such as viruses and bacteria, diseased sites in the human body) that contain the antigen corresponding to the bound immunoglobulin. The present invention comprises a pharmaceutical substance-containing fat emulsion having selective tropism, which is characterized in that a specific immunoglobulin is bound to the surface of oil particles of the pharmaceutical substance-containing fat emulsion. The pharmaceutical substance-containing fat emulsion in the present invention is O/
Oil particles are dispersed in water, such as W type and W/O/W type. Examples of oil components in the fat emulsion of the present invention include soybean oil, cottonseed oil, sesame oil, safflower oil,
A vegetable oil such as corn oil is used, preferably
Soybean oil is used. Preferably, the vegetable oil is a highly refined vegetable oil, and specifically, refined soybean oil is processed by steam distillation [HJLipe, J.Am.Oil
Chemist.Soc., 27 , 422-423 (1950)]
Highly purified refined soybean oil (purity:
(contains 99.9% or more as triglycerides, diglycerides, and monoglycerides). The oil component is emulsified using an emulsifier. As the emulsifier, nonionic surfactants, phospholipids, lecithin, hydrogenated lecithin, etc. can be used. The origin of phospholipids, resitin, hydrogenated lecithin, etc. is not particularly limited, and for example, those derived from vegetable oils such as soybean oil, animal oils such as egg yolk, etc. are used. As a nonionic surfactant, the molecular weight is 2000.
-20,000 polymers are preferred, such as polyoxyethylene-polyoxypropylene copolymers, polyoxyethylene alkyl ethers, polyoxyethylene alkyl allyl ethers, and the like. Emulsifiers may be used alone,
In addition, they may be mixed and used as appropriate. In addition, a small amount of emulsification aid such as a known fatty acid (for example, a fatty acid having 6 to 22 carbon atoms), a salt of such a fatty acid (for example, an alkali metal salt such as a sodium salt), or a polyhydric alcohol may be added to these as desired. . There is no particular restriction on the medicinal substance contained in the fat emulsion, and both oil-soluble and water-soluble substances can be used. In general, oil-soluble substances are used as O/W type fat emulsions, and water-soluble substances are used as O/W type fat emulsions. It is formulated as a W/O/W fat emulsion. Preferred examples of medicinal substances used in the present invention include anti-inflammatory agents, anticancer agents, antiviral agents, antibiotics, and the like. The anti-inflammatory agent may be either steroidal or nonsteroidal, and preferably dexamethasone palmitate, dexamethasone stearate, dexamethasone myristate, hydrocortisone palmitate, hydrocortisone stearate, hydrocortisone myristate,
Examples include prednisolone palmitate, prednisolone stearate, prednisolone myristate, ibuprofen, flufenamic acid, and ketoprofen. Anticancer drugs include 5-FU, anthramycin, daunomycin, pleomycin, nitrogen mustard, 6-mercaptopurine, and oil-soluble derivatives thereof [oil-soluble derivatives can be prepared by adding long-chain (5-15)
[obtained by making a fatty acid derivative] can be suitably used, but the present invention is not particularly limited to these. Among the antibiotics that act on Gram-positive and Gram-negative bacteria, water-soluble or oil-soluble ones (eg, cephalosporins, penicillins, etc.) are widely used. Interferon and its oil-soluble derivatives can be used as antiviral agents. In order to impart selective directivity to fat emulsions,
A specific immunoglobulin is attached to the surface of the oil particles. Specific immunoglobulins target disease sites,
Antibodies corresponding to immunological antigens present in pathogens are used, and are appropriately selected and used depending on the medicinal substance contained in the fat emulsion. Specific immunoglobulins are currently widely known as monoclonal antibodies. For example, Hybritech has developed anti-HBs antibodies and anti-HBs antibodies.
We sell CEA antibodies, anti-IgE antibodies, and anti-T lymphocyte antibodies, as well as anti-melanoma cell antibodies [Proc. Natl. Acad. Sci., 75 (7) 3405 (1978)] and anti-malignant tumor antibodies ( There are known techniques such as JP-A-54-143513) and anti-virus antibody (JP-A-54-17185). Also,
Antibodies are by no means limited to monoclonal antibodies produced by cell culture methods; there are also anti-human lymphocyte antibodies (Japanese Patent Application Laid-Open No. 139720, 1982), anti-AFP, etc.
Antibody (Japanese Patent Publication No. 55-4306), anti-HBs antibody (Japanese Patent Application Publication No. 1989-4306)
-44620), anti-Pseudomonas antibodies, etc. may be collected by plasma fractionation technique after immunization of animals. Combinations of antibodies and pharmaceutical substances include, for example, anti-T lymphocyte antibodies for anti-inflammatory drugs, and anti-T lymphocyte antibodies for anti-cancer drugs.
CEA antibody, anti-malignant tumor antibody, anti-viral antibody,
Anti-AFP antibodies are combined with anti-viral antibodies such as anti-HBs antibodies. The selection-oriented medicinal substance-containing fat emulsion of the present invention is generally obtained by first obtaining a medicinal substance-containing fat emulsion and binding immunoglobulin thereto. The fat emulsion is prepared by forming it into an O/W type or a W/O/W type according to a known method. The oil component/water weight ratio is between 0.05 and 0.5, more preferably between 0.05 and 0.2. Preparation of fat emulsions consists of an effective amount of the selected medicinal substance, an oil component of 5-50%
(W/V), preferably 8 to 30% (W/V), weight ratio to 100 oil components is 1 to 50, preferably 5 to
Mix 30 emulsifiers and an appropriate amount of water. The medicinal substance is usually contained in the emulsion in an amount of 0.01 to 10% (W/V), preferably 0.1 to 5% (W/V), although it varies depending on the use, symptoms, body weight, etc. Since fat emulsions are generally administered intravenously, their particle size is preferably 1 μm or less. To prepare an O/W type fat emulsion, first mix the required amounts of oil components, emulsifiers, oil-soluble pharmaceutical substances, and emulsification aids if necessary, heat this to 30 to 80°C, and mix it with a homomixer. This is carried out by homogenizing and dissolving using an ultrasonic homogenizer or the like, then adding the required amount of water thereto and homogenizing using a pressure injection type homogenizer. Thus the average particle size
An extremely fine and stable O/W emulsion of 1.0μ or less is produced [J.Am.Oil.Chem.Soc., 32 , 365~
370 (1950)]. To prepare a W/O/W type fat emulsion, first, the required amounts of each oil component, a selected water-soluble pharmaceutical substance, and water are mixed, an emulsifier is added thereto, and the mixture is emulsified using a homogenizer. This is done by obtaining a type emulsion, then adding water and a suitable emulsifier, and homogenizing it with a pressure jet homogenizer to form a W/O/W type emulsion. In this way, a fine and stable emulsion with an average grain size of 1.0 μm or less is produced. By binding a specific immunoglobulin to the surface of the oil particles of the fat emulsion prepared as described above, the fat emulsion containing a medicinal substance having selective tropism of the present invention can be obtained. An example of a method for binding oil particles and specific immunoglobulin is as follows. The fat emulsion prepared as described above is centrifuged (5,000 to 20,000 rpm), for example, to separate oil particles, which are brought into contact with specific immunoglobulin. The contact may include, for example, adding 0.5 to 5 parts by weight of specific immunoglobulin to 10 parts by weight of oil particles.
This is done by mixing as a 5% aqueous solution. The contact temperature is usually 4 to 37°C, the contact time is usually 30 minutes to 2 hours, and stirring is preferred. It is also possible to bond using a crosslinking agent. After the binding process is completed, the oil phase and the aqueous phase are separated by, for example, centrifugation, and the oil particles to which the specific immunoglobulin is bound are recovered. An emulsion is obtained by mixing and homogenizing the oil particles in water, preferably containing an isotonizing agent (eg, glycerin, etc.), a stabilizer, and the like. The emulsion thus obtained was a uniform fine grain emulsion with a grain size of 1 μm or less and an average grain size of 0.1 to 0.3 μm.
The particle size was measured using a method similar to the centrifugal sedimentation method by Yokoyama et al. [Chem.Pharm.Bull. 22 (12) 2966-2971
(1974)]. The immunoglobulin was bound at a maximum of about 10-20% parts by weight based on the weight of the oil component. The amount of binding was determined by measuring free antibody in the washed fraction by the Mancini method. When the thus provided preparation of the present invention is administered into a living body, the drug is selectively delivered according to the type of specific immunoglobulin bound to the surface of the oil particles, and the oil particles are locally destroyed and the drug is absorbed into the oil particles. The medicinal substances contained in the medicine exert their medicinal effects when they come into contact with the local area. In the following, it will be explained in more detail using Examples and Experimental Examples. Example 1 100 g of purified soybean oil, 24 g of purified egg yolk phospholipid, and 0.07 g of sodium oleate are added to 2.0 g of dexamethasone palmitate, heated to 65 to 75°C, and dissolved and homogenized using a homomixer. Next, 1000 ml of distilled water for injection is added to this, and the mixture is emulsified by passing it through a Manton-Gaulin type homogenizer once at a pressure of 120 kg/cm ² in the first stage and 10 times under a pressure of 500 kg/cm ² . This emulsion was centrifuged (15,000 rpm) to separate the oil component phase. 27 mg of anti-T lymphocyte specific antibody was added to 100 g of the separated oil component, and the mixture was reacted at 36° C. for 1 hour with stirring. After the reaction, the oil component was washed by centrifugation, and 200 ml of distilled water for injection to which 7.0 g of glycerin had been added was added to the oil component.
Mix and homogenize with a homomixer to obtain an average particle size of 0.1.
An emulsion was obtained which consisted of grains of ~0.3μ and no grains larger than 1μ. As a result of measuring the amount of antibody bound by the Mancini method, it was found that 1 part by weight of antibody was bound to 10 parts by weight of the oil component. Example 2 A mixture of 0.65 g of hydrocortisone palmitate, 25.0 g of purified soybean oil, 6.0 g of purified soybean phospholipid, and 200 ml of distilled water for injection was treated in the same manner as in Example 1, and 23.5 g of the resulting oil component was 5.0 mg of T lymphocyte-specific antibody was mixed, reacted at 35°C for 2 hours, and treated in the same manner as in Example 1, followed by 1.3 g of glycerin.
g, and 50 c.c. of distilled water for injection were added to obtain an O/W type fat emulsion. Example 3 The same procedure as in Example 1 was carried out except that 100 g of purified safflower oil, 24 g of hydrogenated lecithin and 1000 ml of distilled water for injection were added to 2.0 g of chloramphenicol palmitate, and 10.0 g of the obtained oil component was treated with anti-HBs. 20 mg of the specific antibody was mixed, reacted at 37°C for 1 hour, and treated in the same manner as in Example 1 to obtain an O/W type fat emulsion. Example 4 A mixture of 0.5 g of dromostanolone propionate, 24.5 g of purified sesame oil, 3.6 g of purified egg yolk lecithin, 0.07 g of sodium oleate, and 100 ml of distilled water for injection was treated in the same manner as in Example 1 to obtain a product. 5.5 mg of anti-embryonic cancer antigen-specific antibody was mixed with 21.0 g of the oil component, reacted at 37°C for 2 hours, and treated in the same manner as in Example 1, followed by 1.3 g of glycerin and 50 g of distilled water for injection.
cc was added to obtain an O/W type fat emulsion. Example 5 5-FU2.0g, soybean oil 200g, Tween80 (10
g) and 1000 ml of distilled water for injection were added, and 300 mg of anti-alphaetoprotein antibody was mixed with 200 g of the obtained oil component.
The mixture was reacted at 37°C for 2 hours, and then treated in the same manner as in Example 1 to obtain an O/W fat emulsion. Example 6 2.0 g of pleomycin was dissolved in 30 ml of distilled water for injection, and 100 g of purified sesame oil and SPAN80 (10
g) is added and emulsified by ultrasonication. Next, 1000ml of distilled water for injection and PLURONICF68
(10 g) was added thereto, and emulsified by treatment using a Mantry-Gaulin type homogenizer in the same manner as in Example 1. This emulsion was centrifuged to separate the oil component.
250 mg of anti-alphafetoprotein specific antibody was added to 100 g of this oil component, and the mixture was treated in the same manner as in Example 1 to obtain a W/O/W type fat emulsion. Example 7 10 million IU of purified interferon was dissolved in 30 ml of distilled water for injection, and 100 g of purified sesame oil was added to this.
Add SPAN80 (10g) and clarify using a homomixer. Next, 1000ml of distilled water for injection,
PLURONICF68 (15 g) was added and treated in the same manner as in Example 1 using a Manton-Gaulin type homogenizer to effect emulsification, and the emulsion was centrifuged to separate the oil component. 27 mg of an anti-HBs specific antibody was added to 100 g of the oil component and treated in the same manner as in Example 1 to obtain a W/O/W type fat emulsion. Example 8 2.0 g of spadeicomycin (obtained by the method described in JP-A-56-15289) was added to 30 g of distilled water for injection.
ml, add 100g of refined safflower oil to this,
Add SPAN80 (10g) and emulsify with a homomixer. Next, 1000ml of distilled water for injection,
PLURONICF62 (30g) was added, treated with a Manton-Gaulin homogenizer in the same manner as in Example 1, emulsified, centrifuged this emulsion to separate the oil component, and 100g of the oil component was 25 mg of cell-specific antibody was added and treated in the same manner as in Example 1 to obtain a W/O/W type fat emulsion. Example 9 3′・4′-Dideoxykanamycin B (2.0 g) was dissolved in 30 ml of distilled water for injection, and 100 ml of purified soybean oil was added to this.
g, SPAN80 (10 g) and emulsify using a homomixer. Next, 1000ml of distilled water for injection,
PLURONICF68 (20 g) was added, treated with a Manton-Gaulin homogenizer in the same manner as in Example 1, emulsified, and centrifuged to separate the oil component. 250 mg of anti-Pseudomonas aeruginosa specific antibody was added to 100 g of the separated oil component, and the mixture was treated in the same manner as in Example 1 to obtain a W/O/W type fat emulsion. Example 10 (1) Preparation of pharmaceutical substance-containing fat emulsion 100 g of purified soybean oil and 24 g of purified egg yolk phospholipid were added to 2.0 g of dexamethasone palmiter.
The mixture was heated to ~75°C and dissolved and homogenized using a homomixer. Next, 1000 ml of distilled water for injection and 25 g of glycerin (9th edition Japanese Pharmacopoeia) were added to this, and the mixture was passed through the first stage once at 120 kg/cm ² using a Manton-Gaulin type homogenizer under a pressure of 500 kg/cm ^2. It was passed through 10 times and emulsified. (2) N-3-(pyridyl-2-dithio)propionyl immunoglobulin
Preparation of anti-T lymphocyte-specific antibody with N-succinimidyl-3-(2-pyridylthio)propionate (SPDP),
-dithio)propionyl-The method for preparing anti-T lymphocyte-specific antibodies (anti-T lymphocyte-specific antibodies - PDP) is the method of Martin et al. (J. Biol. Chem., 257 ,
286, 1982). After the reaction, the product was purified using Sephadex G-25 to obtain the desired product (2). (3) N-3-(pyridyl-2-dithio)propionyl phosphatidylethanolamine (PE
- PDP) Preparation Dissolve 1.5 g of egg yolk-derived phosphatidylethanolamine (Midori Juji) in 100 ml of dehydrated methanol containing 0.28 ml of triethylamine.
g of SPDP was added and reacted for 2 hours at room temperature under nitrogen gas. After the reaction was completed, the product was purified using a silica gel column (chloroform/methanol) to obtain the desired product (3). (4) Binding of PE-PDP and immunoglobulin-PDP 0.12mg PE-PDP and 0.27w/v% anti-T lymphocyte-specific antibody-PDP aqueous solution (20mM citric acid, 35%
mM disodium phosphate, 108mM NaCl, 1m
1 ml of MEDTA (pH 6.0) was added, and the mixture was reacted for 10 hours at room temperature under nitrogen gas with stirring. The obtained complex was purified by Sephadex G-25 to obtain a PE-PDP-anti-T lymphocyte specific antibody complex. (5) Binding of antibody and fat emulsion Add 0.2 ml of the PE-PDP-anti-T lymphocyte-specific antibody complex (1.38 mg/ml) obtained in (4) to 1 ml of the fat emulsion obtained in (1), The mixture was reacted for 16 hours at room temperature under nitrogen gas with shaking to prepare a fat emulsion containing dexamethasone palmitate bound to an anti-T lymphocyte-specific antibody. Example 11 Anti-melanoma cell-specific antibody-conjugated vinblastine-containing fat was prepared according to Example 10 using vinblastine instead of dexamethasone palmitate and an anti-melanoma cell-specific antibody instead of the anti-T lymphocyte-specific antibody. An emulsion was prepared. Test example 1 AH66 cells (rat ascites hepatoma)
(Odashima, 1964) in male rats (body weight 150 g±
Example 6
It was confirmed that the survival period of rats was prolonged by administering the preparation of the present invention obtained in . The inoculated cells were 5×10 ⁵ cells, and the preparation of the present invention containing 1 mg of pleomycin was administered intravenously three times every 6 hours. Administration started at the same time as vaccination, and was administered every week after vaccination until the 10th week if the animal survived. Observation is 11
I did it until the end of the week. The results showed that none of the rats in the group administered with the formulation of the present invention died, whereas all of the rats in the non-administered group died in about 20 days. Test Example 2 A male rat (body weight 150g ± 10) was used as a test animal, 1 x ¹⁰⁴ AH66 cells were transplanted into the liver of the mouse, and 1 mg of pleomycin was added at two-week intervals from immediately after the transplant until week 10. The preparation obtained in step 6 was injected intravenously, and the size of the hepatoma cell mass was examined at 11 weeks. The average of 5 animals in each group showed that the tumor size decreased in the test group, increased to 1129 ^mm3 in the non-administered control group, and there was no significant change in the group treated with 1 mg of pleomycin aqueous solution. Test Example 3 A comparative experiment regarding the in vivo activity of the formulation of the present invention was conducted. The administration preparation was [1, 2, 6, ^7-3H ]- as a steroid having anti-inflammatory activity according to Example 2.
Hydrocortisone palmitate (10mg: 2μ
A fat emulsion mixed with Ci/mg) and bound to an anti-T lymphocyte antibody was obtained and used. As for the administration method, the preparation of the present invention was administered intravenously, and as a control, hydrocortisone tablets were orally administered. In the experiment, rats were made to develop adjuvant arthritis, and after the onset of inflammation, the radioactive activity in the inflamed area was measured at 10, 50, and 100 hours after administering 20 mg of steroid. The ratio was calculated. As an adjuvant, a suspension of killed human tuberculosis bacteria H ₃₇ Rv in liquid paraffin at a concentration of 10 mg/ml is used. Arthritis is induced by injecting 0.06 ml of the above adjuvant into the right hind footpad of 9-week-old male CD rats once per animal. Swelling of the hind limbs every 3 days after administration of the adjuvant.
Measured using a volume differential meter, 15
Select animals that developed arthritis on day one. The results are shown in Table 1. These results showed that intravenous administration of the formulation of the present invention localized the in vivo activity to the inflamed area.

[Table] Test Example 4 A comparative experiment regarding the in vivo activity of the formulation of the present invention was conducted. (1) Dexamethasone solution Add dexamethasone phosphate to physiological saline solution
Dissolved in 0.1ml. (2) Dexamethasone-containing emulsion Add 100 g of refined soybean oil and 24 g of purified egg yolk phospholipid to 2.0 g of dexamethasone palmitate, and add 65 g of dexamethasone palmitate.
Heat to ~75°C and dissolve and homogenize using a homomixer. Next, add 22 g of glycerin and an appropriate amount of distilled water for injection to bring the total volume to 1000 ml, and use a Manton-Gaulin homogenizer to make the first stage 120 ml.
Passed once at Kg/cm ² and 10 times under pressure of 500Kg/cm ²
The emulsion was emulsified by passing through the emulsion several times to obtain an emulsion consisting of particles with an average particle size of 0.1 to 0.3 μm and no particles with a particle size exceeding 1 μm. (3) Immunoglobulin-bound dexamethasone-containing emulsion To 2.0 g of dexamethasone palmitate, add 100 g of purified soybean oil and 24 g of purified egg yolk phospholipid,
Heat to 75°C and dissolve and homogenize using a homomixer. Next, add an appropriate amount of distilled water for injection to make the total volume 1000 ml, and use a Manton-Gaulin homogenizer to pass through the first stage once at 120 kg/cm ² and 10 times under a pressure of 500 kg/cm ² to emulsify. do. Centrifuge this emulsion (15000r.pm)
Then, the oil component phase was separated. Add 27 mg of anti-T lymphocyte specific antibody to the fractionated oil component, and heat at 36°C.
The reaction was allowed to proceed for 1 hour with stirring. After the reaction is complete,
The oil component is washed by centrifugation, 22 g of glycerin and an appropriate amount of distilled water for injection are added to this oil component to make a total volume of 1000 ml, and the mixture is mixed and homogenized with a homomixer, consisting of particles with an average particle size of 0.1 to 0.3 μ.
An emulsion containing no grains with a grain size exceeding 1 μm was obtained. As a result of measuring the amount of antibody bound by the Mancini method, it was found that 1 part by weight of antibody was bound to 10 parts by weight of the oil component. Test method Animals were SD rats (male, weight 180 ± 10 g).
A group of 5 animals was used. In the experiment, carrageenan edema was induced in rats and the effects of the test drugs were compared.
Carrageenin edema was induced by administering 0.1 ml of 1% carrageenan dissolved in physiological saline under the footpad of the hind paw. The drug was administered at 0.1 mg/Kg via the tail vein one hour before carrageenin administration, and the hindlimb volume was measured 5 hours later to determine the effect. The results are shown in Table 2.

[Table] It was confirmed that the immunoglobulin-binding emulsion had smaller emulsions than the drug alone, the simple emulsion, or the untreated group, and had superior anti-inflammatory effects.
In other words, it was confirmed that the directivity to the affected area was significantly improved.

Claims (1)

[Claims] 1. A pharmaceutical substance-containing fat emulsion having selective directivity, characterized in that immunoglobulin is bound to the surface of oil particles of the pharmaceutical substance-containing fat emulsion.