JPS60185725A - Medicine-containing lipid emulsion - Google Patents

Abstract

PURPOSE:The titled preparation that is made by allowing the active fragments of immuno globulin to bind with the oil particle surfaces of medicine-containing lipid emulsion, thus showing selective direction. CONSTITUTION:Oil particles [o/w type or w/o type oil particles of soybean, cotton seed, sesami or safflower oil] containing medicinal substances such as antiinflammatory, carcinostatic agent, antiviral or antibiotic are allowed to bind with the active fragment of immunoglobullin on their surfaces of give a stabilized medicine-containing lipid emulsion with directionality to mal sites, viruses and bacteria. The lipid emulsion is prepared by mixing 5-50w/v% of oil component, 1-50pts.wt. of emulsifier per 100pts.wt. of oil and an appropriate amount of water. The medicinal substance content is 0.01-10w/v% in the emulsion.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a pharmaceutical substance-containing fat emulsion exhibiting selective targeting.

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 using This is a nutrient 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 cancer drugs within the fat emulsion particles, since the oil particles themselves have a high affinity for lymph vessels and specifically migrate to lymph nodes. There is an attempt to use it as (GANN Dan, 345
-350 (1973)).

The present inventors also embedded an anti-inflammatory agent in a fat emulsion,
This article relates to a fat emulsion of a steroid or a freeze-dried preparation thereof that is used as a carrier for anti-inflammatory agents and has anti-inflammatory activity by taking advantage of the high affinity between oil particles and inflamed local areas.
Completed the invention of "steroid preparation"
No. 167616).

The technique of using this fat emulsion as a carrier for medicinal substances is derived from the affinity of oil particles themselves for lymphatic vessels, and is expected to be quite effective. However, the disadvantage of this is that in vivo translocation is to some extent non-specific, and the delivery of the drug to the diseased organ to be treated is incomplete, and its reach is usually limited at best. It was about 1 to 5%.

Therefore, the present inventors have investigated various methods to maximize the effectiveness of this fat emulsion as a drug substance carrier, and have developed a method in which immunoglobulin is bound to the surface of oil particles. A fat emulsion containing a medicinal substance with selective tropism for diseased areas or pathogens (viruses, bacteria, etc.) was completed (Japanese Patent Laid-Open No. 58-99424).

However, as a result of further research, the present inventors subsequently discovered a method for binding active fragments of biologically active immunoglobulins to the surface of oil particles of fat emulsions, and the bound active fragments. It has been discovered that by utilizing the affinity with cells containing the corresponding antigen, that is, the antigen-antibody reaction, a higher selection-directing function can be added to the fat emulsion as a drug substance carrier, and the present invention completed.

An object of the present invention is to provide a stable fat emulsion containing a pharmaceutical substance that has selective tropism to diseased areas or pathogens such as viruses and bacteria.

Here, selective tropism refers to tropism toward cells (pathogen cells such as viruses and bacteria, diseased sites in the human body) containing the antigen corresponding to the bound immunoglobulin.

The present invention consists of a pharmaceutical substance-containing fat emulsion having selective tropism, characterized in that an active fragment of an immunoglobulin is bound to the surface of the oil particles of the pharmaceutical substance-containing fat emulsion.

The pharmaceutical substance-containing fat emulsion in the present invention is o/w type, w
10/w type, etc., in which oil particles are dispersed in water.

Examples of oil components in the fat emulsion of the present invention include:
Vegetable oils such as soybean oil, cottonseed oil, sesame oil, safflower and corn oil are used, preferably soybean oil is used.

Preferably, such vegetable oil is a highly refined vegetable oil, and specifically, refined soybean oil is processed, for example, by a steam distillation method (
H, J, Lipe.

J, Am, oil chemist, Soc, , 2
7. 422-423 (1950)) and further sheathed to obtain high-purity refined soybean oil (purity of nitriglycerides, diglycerides and monoglycerides of 99.9%).
% or more).

7,1 Non-lipid, lecithin, hydrogenated lecithin, etc., for which additive lecithin etc. are used, are not particularly concerned about their origin; for example, those derived from vegetable oils such as soybean oil, animal oils such as 91] yellow, etc. are used. I get irritated. As the nonionic surfactant, a polymer type surfactant having a molecular i of 2,000 to 20,000 is suitable. Oxyethylene alkyl ether etc.

Emulsifiers may be used alone or may be mixed as appropriate. In addition, these include known fatty acids (e.g. 6 carbon atoms).
If desired, emulsifying aids such as 1-22 fatty acids), salts of fatty acids (for example, alkali salts such as sodium salts), and polyhydric alcohols may be added.

There is no particular medicinal substance contained in fat emulsions, and both oil-soluble and water-soluble substances can be used; in general, oil-soluble substances can be used as o/w type fat emulsions, The liquid version is formulated as a W10/W type fat emulsion. Preferred examples of the medicinal substances used in the present invention include anti-inflammatory agents, anti-cancer agents, antiviral agents, and antibiotics, including methasone stearate, dexamethasone myritate, and hydrocortisone noglumitate. , Itrocortisone Stearate, Idrocortisone Myristate, Prednisolone Lumitate,
Examples include prednisolone stearate, prednisolone myristate, ibuprofen, flufenamic acid, and ketopifene.

Cancer-producing agents include 5-FU, anthramycin, downmycin, downmycin, vinfirastine, nitrogen mustard, and 6-mercabutoglin, but are not limited to these. In this case [oil-soluble derivatives can be obtained according to conventional techniques, for example, by converting each compound into a long-chain (5-15) fatty acid derivative].

Among the antibiotics that act on Durham-positive bacteria and Gram-negative bacteria, water-soluble or oil-soluble antibiotics (eg, cephalosporins, penicillins, etc.) are widely used. Interferon and its oil-soluble derivatives can be used as antiviral agents.

In order to impart selective targeting to the fat emulsion, active fragments of immunoglobulins are attached to the surface of the oil particles. The immunoglobulin used is an antibody corresponding to the immunological antigen present in the targeted disease site and pathogen, and is appropriately selected and used depending on the medicinal substance contained in the fat emulsion. Immunoglobulins are currently widely known as monoclonal antibodies. For example, Hybritech sells anti-HB8 antibodies, anti-CEA antibodies, anti-igE antibodies, anti-TI antibodies, etc.
Anti-melanoma cell antibodies (Proc N-atl, Acad, Sci,
75 (7) 3405 (1978)), a method for producing malignant tumor antibodies using fused cell hybrids (Japanese Unexamined Patent Publication No. 143513/1984), and furthermore, antibody culture and especially in vitro survival. There are known techniques such as a method for producing viral antibodies (Japanese Patent Application Laid-open No. 17185/1985), which involves producing antibodies using cells that contain the virus.

In addition, antibodies are not limited to monoclonal antibodies produced by cell culture methods;
-139720), an antitumor agent for the prevention and treatment of malignant tumors mainly containing antibodies against α-fetoprotein (Japanese Unexamined Patent Publication No. 1983-4306), and immunoglobulin present in plasma or serum. Among them, an immobilized vaccine effective for collecting specific antibodies corresponding to the target antigen and a method for collecting specific antibodies (% 1983-44620)
, etc. may be recovered by plasma fractionation techniques after immunization of animals.

Furthermore, immunoglobulin fractionated from normal animal or human plasma may be used.

In the present invention, active fragments of these immunoglobulins are used. In the present invention, the active fragments refer to fragments of immunoglobulins that have at least a site capable of binding to an antigen. Therefore, among the degraded products obtained by treating immunoglobulin with known proteolytic enzymes, fragments having an antigen-binding site or derivatives thereof (e.g.
alkylated derivatives, acylated derivatives, sulfonated derivatives, etc.) are used. Specifically, immunoglobulin half molecules (commonly known as H-4, bare) H chains, L chains, papain-treated, antigen-binding fragments, trypsin-treated antigen-binding fragments (commonly known as Fab fragments), begusin-treated antigen-binding dimer fragments (commonly known as (li) 'aW:b fragment) A monomer fragment (commonly known as a Fatf fragment) which is a reduced product thereof may be used, but pat1 noragment is more preferably used.

The combination of antibody fragments with pharmaceutical substances can be, for example,
Anti-inflammatory agents include anti-TIJ antibody whole antibody fragments, anti-cancer agents include anti-Cl18A antibodies, anti-malignant tumor antibodies,
Each fragment of an anti-virus antibody, an anti-AFP antibody, etc. is combined with an anti-virus antibody fragment such as an anti-HBs antibody fragment to the anti-viral agent.

The fat emulsion is prepared according to known methods into 07w type or W1 type.
It is prepared by making it into a 0/W type, etc.

The weight ratio of oil component/water is 1.0.05 to 0.5, more preferably 0.05 to 0.2. For the preparation of a fat emulsion, an effective amount of the selected pharmaceutical substance, oil component 5-50% (W/V) preferably ill: 8-30% (W/V), oil component 10.0
This is carried out by mixing an emulsifier in a weight ratio of 1 to 50 degrees, preferably 5 to 3 degrees, and an appropriate amount of water. Pharmaceutical substances vary in use, symptoms, weight, etc. φ5, usually in emulsion o, oi~
io% (W/V), preferably 0°1-5% (W/V)
V) Contains. Since fat emulsions are generally administered intravenously,
The particle size is preferably 1 μm or less.

To prepare an o/w type fat emulsion, first, the required amounts of each oil component,
Mix an emulsifier, an oil-soluble pharmaceutical substance, and any necessary emulsification aids, heat this to 30 to 80°C, homogenize it with a homomixer, ultrasonic homogenizer, etc., and dissolve it.
Next, a hair's worth of water is added to this and homogenized using a pressurized jet homogenizer. In this way, an extremely fine and stable o/w type pharmaceutical substance-containing fat emulsion with an average particle size of 1.0 μm or less is produced [J, Am, Oll.
.

Chem, Soc, 32.365-370 (
1950) ).

To prepare a W10/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.
o type emulsion was obtained, then water and a suitable emulsifier were added thereto, and homogenization was performed using a pressurized jet homogenizer to obtain a W10 emulsion.
/W type emulsion. Thus,
A fine and stable fat emulsion containing a pharmaceutical substance with an average particle diameter of 1°0 μ or less is produced.

The binding method for the pharmaceutical substance-containing fat emulsion and the immunoglobulin fragment is a method in which the immunoglobulin fragment into which a lipophilic substance has been introduced is brought into contact with the pharmaceutical substance-containing fat emulsion (hereinafter referred to as method 1), and a method in which a compatible substance is used. There is a method (hereinafter referred to as method 2) in which the fat emulsion containing the medicinal substance is mixed during the preparation of the fat emulsion, and after the emulsion is prepared, it is bonded to the immunoglobulin fragment directly or via a crosslinking agent (hereinafter referred to as method 2).In method 1, a lipophilic substance is introduced. Preparation of the 7-ragment of immunoguprin can be carried out using lipophilic substances such as phospholipids,
Glycolipids, fatty acids, etc. are preferably used. Phosphatidylethanolamine (PE), a type of phospholipid
When using, it is obtained by reaction with succinimidyl 4-(P-maleimidophenol)butyrenite: (SMPH'). N-4=(P-maleimidophenol)butylphosphatylethanolamine (PE-M
PR) with an immunoglobulin fragment (J, Biol, Chem, 25
7.286. (1982)) When using ganglioside, which is a type of glycolipid, it is obtained by oxidizing it with periodic acid and then reacting with a fragment of immunoglobulin (Bio-chem Biophys, Act.
a 64066, (1981)), and for example, when using valmitic acid, which is a type of fatty acid, it can be obtained by reacting valmitic acid ester of N-hydroxysuccinimide with 7-lacment of immunoglobulin (Bio -cheITLBiophys, Aet
a, 68931. (1982)). The aqueous solution or aqueous suspension of the complex of immunoglobulin fragment and lipophilic substance thus obtained is brought into contact with the pharmaceutical substance-containing fat emulsion prepared as described above. Oil particle listing is performed by mixing as a water suspension.

The contact temperature is usually 4 to 37°C, and the contact time is usually 30 minutes to 24 hours, preferably with stirring or shaking. In method 2, for example, the above-mentioned PE-MPB
o, oi~Low when emulsifying a fat emulsion containing a pharmaceutical substance.
/v%, and by preparing an emulsion and contacting the obtained emulsion with the immunoglobulin fragment, an immunoglobulin fragment-bound fat emulsion can be obtained. This contact may be carried out, for example, from 0.01 to 10 parts by weight of the emulsion.
10 parts by weight of immunoglobulin fragment 0.1
This is done by mixing with an aqueous solution of ~10 ml. The contact temperature is usually 4 to 37℃ and the contact time is usually 30 minutes to 24℃.
It is preferable to stir, stir or shake for a period of time.

The emulsion thus obtained is 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, and is heated at 4 to 7°C.
No significant change in particle size was observed during storage under nitrogen gas for one year. The particle size was measured by a method similar to the centrifugal sedimentation method by Yasushi et al. (Chem.

pharm, Bull, 22 (12) 2966
-2971 (1974)). Immunoglobulin fragments can contain up to about lO~2 based on the weight of the oil component.
It was bonded with a 0%M lid. The bound amount was determined by measuring the free antibody in the wash fraction by the Munchie method.

When the preparation of the present invention thus provided is administered into a living body, the drug is selectively delivered according to the type of active fragment of immunoglobulin bound to the surface of the oil particle, and the oil particle is locally destroyed. The medicinal substance contained in the oil particles exerts its medicinal effect when it comes into contact with the local area.

In the following, it will be explained in more detail using Examples and Experimental Examples.

Example 1 1.) Preparation of a pharmaceutical substance-containing fat emulsion Dexamethasone palmitate 2.09 to refined soybean oil 10
09, N egg yolk phospholipid 249 was added, heated to 65-75°C, and dissolved and homogenized using a homomixer. Next, 1,000 g of distilled water for injection and glycerin (
(Japanese Bureau) Add 25g, use a Manton-Gallarin type homogenizer, pass once at 7/crll to the first stage 120,
The emulsification was carried out by passing the mixture 10 times under a pressure of 0.00 mL.

2.) Preparation of l;'at/fragment of immunoglobulin Preparation of Fad fragment of anti-TIJ tumor cell-specific antibody was carried out by the method of Martin et al. (J, Biol.
, Chem, 2nd.

286, (1982)). That is, after digesting anti-lymphocyte-specific antibodies with pepsin, DTT was added at pH 5.5.
(dithio 'l'hreitol) treatment and purification with Sephadex G25 to obtain the Fa'd fragment.

3.) Preparation of PE-PMB Dissolve PE (Green Cross) 1.5F from egg yolk oil in 100 ml of dehydrated methanol containing 0.28 g of triethylamine, add 1 g of SMPH (Pierce), and under nitrogen gas. The reaction was carried out at room temperature for 2 hours. After the reaction was completed, it was purified using a silica gel column (chloroform, methanol) to obtain PE-84.

4) Binding of PE-P■ and Fatf fragment PE-
0.27 w/v% FaR fraction aqueous solution (20fiM citric acid, 35ff1M disodium phosphate, 108mM NaCl, 1mMEDT) in FMB O, 12d.
A (pH 6.0) containing A was added thereto, and the mixture was reacted at room temperature under nitrogen gas for 10 hours with stirring. The obtained complex was purified on Sephadex G-25 and PE-Hatch-Fa
d complex.

5) Binding of antibody and fat emulsion Add the PE-PMB-Fa M complex (138 K/-) obtained in 4) above, 0.2 m'4 to the fat emulsion 1- obtained in 1) above, and shake. While at room temperature under nitrogen gas,
After reacting for 16 hours, a fat emulsion containing dexamethasone palmitate bound to an anti-T lymphocyte-specific antibody was prepared.

Example 2 Vinblastine 2. OF: N-made soybean oil 1009, purified egg yolk phospholipid 249. Then, 24 pieces of PE-PMB O prepared in 3) of Example 1 were added, heated to 65 to 75°C, and homogenized using a homomixer.

Next, 1,000 ml of distilled water for injection and 259 ml of glycerin (Japanese Pharmacopoeia) were added to this, and using a Manton-Gallin type homogenizer, the first stage was passed once at 120 fi.
Emulsification was carried out by passing the mixture under pressure of 10 times.

After adjusting the pH to 6 with IN hydrochloric acid, 2rrd! , and an aqueous solution (0.27 w
/v%) ld. Under nitrogen gas and shaking at room temperature, react for 10 hours to determine the percentage of anti-melanoma cells.
A bifracment-bound himblastine-containing fat emulsion was prepared.

Test Example 1゜AH66 cells (rat ascites hepato
rna) (Odashima, 1964) was intraperitoneally inoculated into male rats (body weight: 145±1 Oi, 5 rats in 1 group) for 7 days, and it was confirmed that the survival period of the rats was prolonged by administering the preparation of the present invention obtained in Example 2. Inoculated cells are 5 x 10
The preparation of the present invention containing vinblastine 1 was administered intravenously three times every 6 hours.

The administration time started at the same time as the inoculation, and if the animals survived, they were administered at 10-week intervals at one-week intervals after the inoculation. Observations were made by mouth for 11 weeks. As a result, the rats in the group administered with the formulation of the present invention did not die, whereas the rats in the non-administered group died in about 20 days.

Test Example 2 Male rat as test animal (body Nl 50 P±10)
The preparation obtained in Example 2 containing Chin 1 was injected, and the size of hepatoma cell clusters was examined after 11 weeks. The average of 5 animals in each group was that cancer reduction occurred in the test group, and 1 in the non-administered control group.
129-, and there was no significant change in the vinblastine 1η aqueous solution administration group.

Test example 3゜ A comparative experiment regarding the in vivo activity of the formulation of the present invention was conducted.

The administration preparation was prepared by mixing (1,2,6,7-3H)-hydrocortisone palmitate (10η: 2ItCi/-v) as a steroid with anti-inflammatory activity in accordance with Example 1, and adding anti-T lymphocyte-specific antibodies. A fat emulsion with bound Fad fragments was obtained and used.

The method of administration consisted of intravenous injection of the preparation of the present invention, oral administration of hydrocortisone tablets as a control, and administration of anti-T lymphocyte-specific antibody 7.
Intravenous injection of a fat emulsion bound to the Fak/ragment was used. In the experiment, rats were caused to develop adjuvant arthritis, and after the onset of inflammation, the radioactivity at the inflamed area was measured at 10, 50, and 100 hours after administration of 20Hi as the steroid dose, and the residual activity was determined based on the dose. Ratio fc calculated 0 Human tuberculosis killed bacteria H to liquid paraffin as an adjuvant
A suspension of s7RV at a concentration of 10% was used.

Arthritis was injected into the right hind paw skin of a 9-week-old male CD rat with the above adjuvant)'tO,06m/! Symptoms were induced by injecting each animal once.

Vo-1Hm of hindlimb swelling every 3 days after adjuvant administration
Animals that developed arthritis on the 15th day were selected using e-differentialmeter.

The results are shown in Table 2. This result showed that the in vivo activity of the preparation of the present invention was localized to the inflamed area when administered intravenously.

Table 1 Procedural amendment (voluntary) 1. Indication of the case Patent Application No. 41674 of 1982 2. Name of the invention Fat emulsion containing a pharmaceutical substance 3. Person making the amendment Relationship to the case Patent applicant address Osaka, Osaka Prefecture IL at 1-15 Imabashi, Higashi-ku, Ichi.
' 8 (Name) Midori Juji Co., Ltd. Representative Renzo Matsushita 4, Agent (1) Page 12, line 13 of the specification, ``...Chem, -...''of,"
・・・・Chem, ・−−・・−J and correct it.

(2) On page 12, line 17 of the specification, [Machi-Qcta ri'-Machi...] was replaced with ``...
...Acta, 640 y-=J is corrected.

(3) In the second line of page 13 of the specification, [...il1...] has been replaced with "...
---689-----= J, correct.

Claims (1)

[Scope of Claims] (11 A fat emulsion containing a selectively directed pharmaceutical substance, characterized in that the active fragment of an immunoglobulin is bound to the surface of the oil particles of the fat emulsion containing a pharmaceutical substance. (2+ The above-mentioned immunoglobulin) The pharmaceutical substance-containing fat emulsion according to claim 11, characterized in that the active fragment of is an immunoglobulin FaW fragment.