JPS6137729A - Improvement of antibacterial activity by antibacterial substance mixture contained in lipid vesicle - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION (1) Introduction The present invention is directed to lipid vesicles incorporating mixtures of two or more antimicrobial substances that exert enhanced therapeutic efficacy. The present invention describes the therapeutic use of a combination of several antimicrobial substances encapsulated together in one liposomal formulation in the treatment of infections (e.g., bacterial, fungal, viral, mycoplasmal, etc.) in living organisms. The discovery that the effects are greater than those obtained when the same combination of antimicrobials is administered in solution or as a mixture of bosomal preparations each containing one of the antimicrobials. It is based on For example, gentamicin (an aminoglycoside antibiotic) and nafcillin (a beta-lactam antibiotic), or gentamicin and clindamycin (an amino acid trans-
1-4=derivative of rl-propylhycric acid) in one liposomal formulation [co
Encapuselation (the formulation is then used to treat infections in an organism) is demonstrated below by examples.

Combinations of antimicrobial substances have been widely evaluated for the treatment of bacterial infections. Simultaneous administration of more than one antimicrobial substance (
1) to prevent or minimize the emergence of resistant mutant strains, and (2) to increase pharmacological activity in the treatment of specific infections (e.g., penicillin and some 7-minoglycosides are used in Durham-negative bacteria). Recommended for the treatment of several types of infections.

), (3) to provide optimal therapy in severe infections where the causative factor has not been clearly established (e.g. in the treatment of mixed bacterial infections), (4) individual drugs by decreasing the dosage of each in combination. It has been proposed for several purposes, including for reducing the toxicity of.

Several combinations of antimicrobial agents have been demonstrated for enhanced microbial activity and clinical efficacy in the treatment of certain infections. (See Table 1).

(Left below) The antibacterial activity of the antibiotics used in the same combination is more than additive, that is, it produces a synergistic effect. For example, in the treatment of bacterial infections, combinations such as penicillin or ampicillin and streptomycin or gentamicin have been shown to have a synergistic effect against enterococcal infections. Similarly, carbenicillin or ticarcillin in combination with aminoglycosides such as gentamicin or tobramycin can be used to treat Pseudomonas phi aeruginosa [PSeUdOmOnaS aerupi
nO3a] shows synergistic effects in the treatment of infections. Combination therapy using streptomycin in combination with tetracycline is more effective in treating pulcellosis than either drug alone, and a mixture of chloramphenicol plus a sulfonamide is effective against Haemophilus. Influenza [Hemophile]
It is more effective against meningitis caused by [Us 1nfluenzae].

The use of drug combinations in antibiotic therapy of fungal infections is also recognized. Ampicillin B for 6 weeks
(2omg per day) and flucytosine (150mg per day)
Coadministration of low doses of cerebrospinal fluid (my/Kg) has been shown to be effective in the treatment of cryptococcosis as measured by a more rapid rate of sterilization of the brain cerebrospinal fluid, reduced toxicity and increased overall rate of healing. Both drugs are better than when used alone. In addition, - primary amebic meningoencephalitis [primary amebic meningoencephalitis]
encephalitis] responds to the combination of miconazole, rifampin, and intracapsular ampicillin B. Combinations of ampicillin with other drugs including flucytosine, rifampin or tetracyclines have been recognized as enhancing antifungal activity.

Similarly, the use of antiviral drug combinations (currently being explored) has recently been shown to be effective against herpes simplex virus type 1 [herpes simplex virus type 1] in hairless mice.
irus type 1] is reported to be more effective than individual drugs in attenuating the development of pathological signs of infection (Park et al., 1984, The Journal of Infectious Diseases 149
(5): 757-762 [Park, et al.
, , 1984゜The Journal of I
nfectious Diseases 149(5)
Near 57-762]). However, the drug combination is antagonistic rather than synergistic.

For example, the addition of a bacteriostatic drug (tetracytalline) to a bactericidal drug (penicillin) can only be used against microorganisms that are multiplying by penicillin.
resulting in a decrease in activity.

Therefore, several in vitro assays are used to predict the potential therapeutic efficacy of antibiotic combinations. These assays quantify the effect of antibiotics on bacterial growth in vitro.

One method used to predict the efficacy of antimicrobial substances is described by Cribner et al.
(1982, Antimicrobial Agent and Chemotherapy [Antimicrobial Ag
ent and'ChemotheraDyl, 21
(6): 939-943 > and Gutsman and Zillman (1980, The Pharmacological Basis of Therapeutics [The Phar
amacolooical Ba5is of Th
erapeuticS], 6th edition, pp, 1097-
1098) and the checkerboard assay [CheCkerboard assay].
y]. This assay involves serial two-fold dilutions of antibiotics individually and in combination in broth, which is then inoculated with the microorganism to be tested. After incubation, the minimum inhibitory concentration (MIC) of each drug used individually and in combination was determined (Note: MIC is the lowest concentration of drug that inhibits growth in the medium). Synergy is defined as the IIC of each drug when used in combination.
indicated by a decrease in . Antagonism refers to the HI of either or both drugs when used in combination.
Indicated by an increase in C.

This assay is described in more detail below and is used in the present invention to determine whether several combinations of antimicrobial substances (eg, antibacterial or antifungal) are non-antagonistic.

Other methods for evaluating drug combinations include quantifying the rate of bactericidal action.

The same medium is incubated with antibiotics added singly or in combination. Synergy is indicated when a combination of antibiotics has more rapid bactericidal activity than either agent alone.

Similarly, combinations of antiviral agents can be assayed in vitro and classified as synergistic, additive, antagonistic, and the like. Such assays are described in Park et al., 1984, The Journal of Infectious Diseases 149(5): 757-762 [Park,
et al., 1984. The Journa
of Infectious Diseases
149(5): 757-762]. Briefly, the assay involves in vitro infection of confluent host cells with a virus and treatment of the infected cells with various concentrations of antiviral agents, both individually and in combination. After incubation, the virus titer recovered from the treated cells is calculated by comparing the degree of titer reduction obtained when each drug is used alone to the amount of titer reduction obtained when the drugs are used in combination. It was measured by comparing the degree of decrease in the value of This assay is also described in more detail below and is used in the present invention to determine whether several combinations of antiviral agents are non-antagonistic.

In many instances, synergistic therapy with several antimicrobial substances is further complicated by the inability of substances that exhibit synergistic effects in vitro to form into a single mixture for use in vivo. Become. A mixture of gentamicin and naphthalene at a therapeutically effective concentration results in the formation of a solution-precipitated complex and therefore cannot be administered in an organism. In fact, some drug combinations are not recommended for use in vivo due to drug incompatibility (ie, drug inactivation or precipitate formation). For example, it is recommended that the following drugs be used without mixing with other drugs: gentamicin, kanamycin, lincomycin, cephalothin, and ampicillin (Davis and Abbitt).
Ko, 1977, JAVMA 170(2):
204-207). Finally, some substances cannot be solubilized in the same medium due to chemical inhibition (eg lipophilic and water-soluble compounds). These limitations reduce the possible combinations of substances that can be used to obtain increased microbial activity in combination therapy. An overview of this view can be found in Gutsudman and Gilman, 1980, The 1 Ramacological Basis of Therapeutics, Vol. 6.
Edition pages 1080-1106 and 1239-1240 [
Goodman and Gilman, 1980°Pharmacological Ba5is
of herapeutics 5ixthEd
itio, pp, 1080-1106 and 12
39-1240] and Davis et al., 1980, Microbiology, pp. 574-583 [Davis et al.
al., 1980. Microbiology,
pp, '574-583].

(2,2) Liposomes and Uses of Liposomes Liposomes are occluded bilayer membranes containing entrapped ice blocks. Liposomes are unilamellar vesicles (with a single membranous bilayer) or multilamellar vesicles (characterized by multiple concentric lipid bilayers separated from the next bilayer by an aqueous layer). It may be of any type (like structure).

Bangam et al. (196
5, J, Mo1Bio1.13:238-252), the initial preparation of liposomes involves suspending phospholipids in an organic solvent and then drying the phospholipids by evaporation to form a waxy phospholipid in a reaction vessel. Including leaving behind deposits. An appropriate amount of water layer is then added and the mixture is "swollen";
and multilamellar vesicles (hereinafter referred to as MLV).

) are dispersed by mechanical means. The structure of the resulting membranous bilayer is such that the hydrophobic (non-polar) “tail” of the lipid is oriented toward the center of the bilayer, while the hydrophilic (polar) “head” of the lipid is directed toward the aqueous phase. Orient towards. This technique is similar to Hahaha Japrose and Miller [
Papahadjapoulos and Mille
r] (1967, Biochim, Biophr
Acta, 135: 624-628)
This is the basis for the development of small sonicated unilamellar vesicles (hereinafter referred to as SυV) described by.

However, the preparation of these “classical liposomes”
It had several drawbacks, including high heterogeneity in size distribution, number of lamellae, and low encapsulation capacity of the aqueous space, which limits the ability to encapsulate large amounts of molecules.

Efforts to increase entrapped volume are first to form reverse micelles or liposome precursors, i.e. endoplasmic reticulum (vesicles) containing an aqueous phase surrounded by a monobasic body of lipid molecules. , with the polar head groups facing the aqueous phase. Liposome precursors are produced by adding the aqueous solution to be encapsulated to an organic solvent solution of polar lipids and sonicating.

The solvent is evaporated in the presence of excess lipid.

The resulting liposomes, consisting of an aqueous phase entrapped by a lipid bilayer, are dispersed in the aqueous phase (M, Schneider [H
See U.S. Pat. )
.

In another attempt to maximize entrapment capacity, Papahadjopoulos (U.S. Pat. No. 4,235,871, issued November 25, 1980) described Evaporation method [reversephas]
evaporation process] is also known as reversed phase evaporation vesicles (hereinafter referred to as REV). According to this approach, the aqueous substance to be captured is added to a mixture of polar lipids in an organic solvent. Next, homogeneous water-in-oil [water-in-o
An emulsion of the type i l ] is formed and the organic solvent is evaporated until a gel is formed. This gel is then converted into a suspension by dispersing the gel-like mixture in an aqueous medium. The REV produced is composed of predominant unilamellar vesicles and a few oligolamellar vesicles characterized by only a few concentric bilayers with large internal aqueous spaces. Some transmission properties of REVs have been reported to be similar to those of MLVs and SUVs (Sozka and Papahadjopoulos [
5zoka and Papahadjopoulos
], 1978. Proc, Natl, Ac
ad, Sci, U, S, A, 75:4194-4
See 198. ).

Although liposomes can be prepared that entrap a variety of substances, the stability of liposomes during storage is limited. This loss in stability can result in leakage of entrapped compounds from the liposome into the surrounding medium, and also contamination of the liposome contents by permeation of substances from the surrounding medium into the liposome itself. As a result, conventional liposomes have a very limited shelf life.

Attempts to improve stability have focused on certain substances (hereinafter referred to as "stabilizers") that influence the physical properties of lipid bilayers.

) (for example, sterols) into the lipid membrane. Much has been written regarding the potential use of liposomes as drug delivery systems, despite the fact that a number of problems exist in the family system (e.g. rapid release of liposomes in organisms, instability of liposomes, etc.). In a drug delivery system, the drug is captured during pumping and then administered to the patient to be treated. Typical such statements include:
Rahman and Cerny
No. 3°993,754, issued November 23, 1976 to
US Pat. No. 235.871, Schneider [5chn
No. 4.224.179 issued September 23, 1980 to El Kelly [
U.S. Pat. No. 377, 567.

Chemotherapy for leishmania sexually transmitted infections using anti-lugemania drugs encapsulated in liposomes has been proposed by Steck and Alping [5teCk a
nd AIVing] in U.S. Pat. No. 4,186,183, issued January 29, 1980.

(3) Summary of the Invention The present invention relates to a liposome preparation in which two or more non-antagonistic antibacterial substances are encapsulated in one liposome preparation. This liposomal formulation can be administered in a single dose into an organism for greatly enhanced therapeutic efficacy. The therapeutic efficacy of antimicrobial substances co-encapsulated in liposomes is higher than when the same antimicrobial combination is used in solution or as a mixture of liposome formulations each containing one antimicrobial substance. be. Combinations of antimicrobial substances that can be co-encapsulated were tested in combination efficacy tests [the
C0IIlbinatiOn EffeCt
] is determined by testing combinations that are not included in the assay method described below. The liposomes are prepared by adding the respective antimicrobial substance to the liposome components before or during the formation of the liposome. The practice of the present invention provides a method for combining combinations such as gentamicin and naphthalene, tobramycin and ticarcillin, or cylindamycin and gentamicin, encapsulated together in one stable liposome, for example in the treatment of salmonellosis,
Exemplified herein are uses to treat a variety of infections within organisms, such as Corynebacterium pyelonephritis, Pseudomonas pyelonephritis, and Clostridial wound infections.

(4) The detailed description of the invention encompasses liposomal formulations containing combinations of antimicrobial agents and methods of use thereof. Multiple non-competitive antimicrobial substances are encapsulated together in one liposomal formulation, ie, entrapped within an aqueous compartment and/or inserted into a membranous bilayer. When this liposomal formulation is administered into an organism, a greatly enhanced therapeutic effect is obtained. The present invention allows simultaneous administration of several drug combinations within an organism.

(4,1) Selection of Antimicrobial Substances According to one embodiment of the present invention, combinations of antimicrobial substances that provide a superadditive or synergistic effect are incorporated into one liposome (as further defined in Section 4.2). , hereinafter, this will be taken as a common connotation.
It is called nl. ) to be done. Several combinations of antimicrobial agents known to be synergistic can be encapsulated in one liposomal formulation. Examples of such combinations include, but are not limited to, those listed in Table 1 and Section 2.1 above.

Still other antimicrobial combinations can be tested using the in vitro assay method already described in Section 2.1, which measures the effectiveness of antimicrobial combinations.
y ]. These test methods are (1)
(2) the Checkerboard Assay and (2) the Viral Titer Reduction Assay as defined herein, which can assess the effect of the combination on bacteria or fungi.
titer reduction assay]. These in vitro assays are detailed below, but they are also collectively referred to as the Combination Effect Test.
It is called tAs5ayl.

(A) Checkerboard Assay Method: Serial dilutions (usually 2-fold dilutions) of the anti-microbial agent are made in a checkerboard format so that many different proportions of the anti-microbial agent concentration can be tested simultaneously. This checkerboard consists of horizontal columns containing the same amount of drug A diluted along the X-axis and vertical columns containing the same amount of drug B diluted along the Y-axis. Therefore, for a given dilution range, all possible combinations of both drugs are achieved. This technique involves using a broth [brot or agar] containing the nutrients necessary to support the growth of the test microorganism (e.g. bacteria, fungi, etc.
gar] A standard inoculum of the test microorganism is then added to each dilution, and the medium is incubated under appropriate conditions and growth is observed. The minimum inhibitory concentration (MIC), ie the highest dilution of each test solution at which microbial growth was inhibited, was determined for each antimicrobial substance and combination.

Combination efficacy is defined by Scribner (supra) as follows: (1) synergy is indicated by at least a 4-fold reduction in the MIC of each antibiotic; (2) additivity is defined by Scribner (supra); or indicated by a 2-fold decrease in the HIC of both antibiotics (3
) No change is indicated by no change in the 14Ic of the antibiotic, and (4) Antagonism is indicated by no change in the 14Ic of either or both antibiotics.
indicated by a fold increase.

(B) Virus titer small assay method: confluent host cells in vitro are infected with the virus of interest. After washing the cells, media containing various concentrations of antiviral substances individually and in combination were added in duplicate to the appropriate cell culture medium. After culturing under appropriate conditions, the virus was recovered from the cell culture medium and the virus titer was assayed using, for example, a conventional plaque assay. The effect of the combination of antiviral substances in the culture medium was determined according to the following criteria (as reported by Park et al. in 1984 (mentioned above)): (1) EA (effect on drug) = (drug effect) Virus titer generated in the presence of drug B) / (Virus titer generated in the absence of drug), (2) E8 (Effect of drug B) = (Virus titer generated in the presence of drug B) (3) EA8 (effect of the combination of drugs A and B) - (virus titer produced in the presence of drugs A and B) / ( (viral titer generated in the absence of drug), (4) Eo (calculated effect of combination of drug A and drug B, i.e. additive effect of drug A and drug B) = E,
xE8° Drug synergy is defined as EAB<Eo, and additive effect is defined as EA8-Eo. Furthermore, if drug A is assumed to be more effective than drug B, an additive (subadditive) effect that does not appear to be additive is defined as Eo x EAB < FA, and an antagonistic effect is defined as Eo x EAB < FA. EAB is defined as EAB, and further interference is EA
It is defined as AB x EB.

Therefore, based on this combination effect test, it is non-antagonistic (
That is, non-encapsulated combinations deemed to be synergistic, additive, sub-additive or non-invariant can be co-encapsulated in one liposomal formulation according to the present invention.

Apart from the criteria established in vitro in the above-mentioned combinatorial efficacy tests, antimicrobial substances that exhibit an increased therapeutic index when co-encapsulated in one liposomal formulation compared to that of non-encapsulated antimicrobial substances. Any combination is also expected to fall within the scope of the present invention. The therapeutic index relates to the dose ratio between toxic and therapeutic effects. The therapeutic index in animals is the ratio LD5o/ED5o (where L
D50 is the lethal dose in 50% of the total population, and E
D5o is the therapeutically effective dose in 50% of the total number of comparables. ). Therefore, the higher the therapeutic index, the wider the margin of safety for a preferred drug.

Combinations of antimicrobial substances listed in Table 1 and Section 2.1, erythromycin + 7 minoglucoside, ampyridine + streptomycin, gentamicin decarbenicillin, gentamicin + nafcillin, chloramphenicolmycin, ethambutol, ethambutol + streptomycin, sulfonamide + tetracycline, ampholarisin B + flucytosine, sulfoamide + streptomycin, sulfoamide ampicillin, tetracycline + cycloserine, penicillin or ampicillin and gentamicin or tobramycin, steroids with water-soluble or fat-soluble antibiotics, and acyclovir and vidarabine, etc. A combination of, but certainly not limited to, can be co-encapsulated in one liposome formulation.

(4,2) Liposomal formulations According to the present invention, liposomal formulations can be prepared by adding one combination of antimicrobial substances or each antimicrobial substance into the liposome components before or during the formation of lipid vesicles. contained within. Therefore, two or more antimicrobial substances can be added to the aqueous or organic phase during liposome formation and, depending on their solubility, may be included (i.e., co-encapsulated) in the liposome bilayer or in the aqueous phase of the resulting liposome. ) to be done.

The method used to prepare liposomes depends on both the type of liposome used and the nature of the antimicrobial substance encapsulated. Stable liposomes are preferred because they entrap a high percentage of the drug.

Particularly stable liposomes that may be used in the practice of the present invention include SPLV (stable plurilamerabecil [S
table plurilamellar vesic
There is a type of lel. SPLVs are described in US patent application Ser. No. 476,496, filed March 24, 1983, which is incorporated herein by reference.

SPLVs are prepared as follows. That is,
An amphiphilic lipid or lipid mixture is dissolved in an organic solvent. Although many organic solvents are suitable, diethyl ether, fluorinated hydrocarbons and mixtures of fluorinated hydrocarbons and ethers are particularly preferred. To this solution is added the aqueous phase and the active ingredient to be captured. This two-phase mixture is converted to SPLV by emulsifying the aqueous material in a solvent and evaporating the solvent. Evaporation can be accomplished during or after sonication by any evaporation technique, such as by passing a stream of inert gas through the mixture, by heating, or by vacuum evaporation.

The volume of solvent used should exceed the aqueous phase volume by a sufficient amount so that the aqueous substance can be completely emulsified in the mixture. In practice, a minimum of about 3 volumes of solvent are used for each volume of aqueous phase. In fact, the ratio of solvent to aqueous phase can vary up to 100 volumes or more per volume of aqueous phase. The amount of lipid must be sufficient to exceed that required to coat the emulsion droplets (approximately 40 m9 lipid per d of aqueous phase). The upper limit is limited only by cost-effective practicality, but SPLVs can be formed with 15 g of lipid per portion of aqueous phase. Therefore, lipophilic antimicrobial substances can be added directly to the organic phase of the liposome component, which ultimately forms the bilayer of the liposome. The water-soluble antimicrobial substance is added to the aqueous phase prior to the formation of the bilayer;
and can be captured. In fact, lipophilic substances added with the aqueous phase are partitioned into the lipid component of the resulting vesicles.

If the combination of antimicrobial substances in solution forms a precipitable complex, or in other words if the substances are incompatible, solutions of each substance should be prepared simultaneously but separately to prevent precipitation. It is added to the liposome components as a substance.

Other liposome formulations that can be used in the practice of the present invention include lipid vesicles (hereinafter monophasic vesicles) prepared in a monophasic solvent system.
vesicle] or RPV).

MPVs are incorporated herein by reference, 1
U.S. Patent Series No. 521,1 filed August 8, 983
It is stated in No. 76. MPVs are very stable and have high capture capacity. MPVs are prepared as follows. That is, an amphipathic lipid or lipid mixture is dissolved in an organic solvent (hereinafter referred to as a monophasic solvent) that is also miscible with water, such as an alcohol (eg, ethanol). If the antimicrobial substance to be captured is lipid-soluble, this substance is added directly to the lipid monophasic solution, whereas if the antimicrobial substance to be captured is water-soluble,
This material can be used in a small amount of aqueous solution (e.g. 0.1 to 0.1 to 10 d of ethanol containing 100 m of lipids).
37 aqueous solution). As explained above, if a combination of antimicrobial substances forms a precipitable complex, or in other words, if the antimicrobial substances are incompatible, solutions of each substance should not be present at the same time to avoid precipitation. but separately added to the monophasic solvent-lipid solution. The resulting mixture is a pure dispersion (does not separate into two phases). This solution is then heated for several minutes at a temperature dependent on the boiling point of the monophasic solvent (e.g. in the range 40° to 100° C.). It is evaporated until a transparent film (composed of lipids and antimicrobial substances) forms on the side walls of the container. A small amount of aqueous solution is then added and the mixture is resuspended and stirred to form the MPV containing the entrapped antimicrobial substance.

Besides increased stability and higher capture capacity, 5ptvs and M
PVs offer other advantages over traditional liver Vs. - For example, chemically incompatible substances such as gentamicin and naphthalene
While it can be effectively included in Class V, it cannot be included in Class MLV without first diluting the drug. Such dilution will reduce the amount of drug entrapped and therefore increase the volume required to deliver an effective dose.

(4,3) Therapeutic use of liposome formulations Antibacterial activity in the treatment of specific infections can be achieved by in vivo administration of a combination of nonantagonistic antibacterial substances co-encapsulated in one liposome. . When administered to animals,
The liposomal formulations described herein provide enhanced biological activity and pathological effects.

The liposomal formulations described herein are particularly advantageous in areas where traditional combination therapy has been unsuccessful or of limited use. For example, drugs such as isoniazizo, rifampin, ethambutol and streptomycin have been used successfully in various combinations in the treatment of tuberculosis. however,
At the same time, during therapy, multi-resistant Mycobacterium tuberculosis
ply resistout tubercle
bacillus] appears. Liposomes co-encapsulating drug combinations simultaneously provide a means to maintain effective concentrations of drugs in the body.

The liposome formulations described herein also include:
2 which usually results in synergistic or super-additive effects (described above).
It offers further advantages in combination therapy with two chemotherapeutic agents. When these two substances are present in one liposome, they are more effective as antibacterial agents in combination therapy than when they are present separately.

This enhanced effectiveness is likely due to the fact that both substances can be simultaneously applied to the site of infection. In fact, the treatment of infections with multiple synergistic drugs co-encapsulated in liposomal formulations uses the same drugs encapsulated in separate liposomal formulations that are mixed prior to administration into the organism. Therapeutically more effective than other treatments.

The liposomal formulations described herein provide advantages in the treatment of intracellular infections and provide additional advantages in the treatment of extracellular infections. Co-encapsulation of multiple drugs in one liposome formulation administered into an organism increases the possibility that a particular drug will be introduced to a specific site (intracellular or extracellular), and therefore Their biological effects and/or therapeutic effects are enhanced.

For the treatment of intracellular infections, liposomal formulations containing the drug combinations described herein in Hatake may be administered parenterally. The drug encapsulated in the liposome is delivered to the site of infection when the liposome is taken up by the infected cell. Liposomes taken into cells form cellular atrophic organs, or phagolysosomes.
[sosome] appears in [sosome]. The degree of intracellular uptake depends on the type of liposome and the type of target cell. As soon as the liposomes are internalized, the combination of co-encapsulated drugs is likely capable of fighting infection in cells.

The liposome formulations described herein can be used to treat extracellular infections within an organism. Therefore,
Parenteral administration of liposomes containing co-encapsulated drugs delivers the therapeutic substances to highly phagocytic macrophages of the reticuloendothelial system. The macrophages coalesce with the liposomes and become loaded with the substance encapsulated in the liposomes. As soon as these "loaded" macrophages reach the site of infection (eg, systemic extracellular infection), they engulf the pathogen. As a result, the pathogen will come into contact with the drug combination in the macrophages and be killed.

EXAMPLES Next, in order to explain the present invention more specifically, Examples will be shown, but the scope of the present invention is of course not limited thereto.

In the following examples, the aminoglucoside gentamicin and the benicillinase-resistant penicillin,
The antibacterial activity of various formulations of naphthalene is compared. The results are shown by the test results of this formulation, which showed that Salmonella typhimurium [Salmonella typhimurium] in mice.
Treatment of lethal infections (intracellular infections) of C. typhimurium is also effective when using a 5PLV formulation in which both gentamicin and naphthalene are included in one 5PLV formulation.

(5,1) 5PL containing gentamicin or nafcillin
Preparation of V A 5d solution of egg phosphatidylcholine (EPC, or egg lecithin) 100 (n'j) in 2 ethyl ether was prepared. This mixture was transferred into a round bottom flask.

A solution (0.3 m) containing 200 m of gentamicin or naphthalene in phosphate buffered saline (PBS, pH 7.2) was then pipetted into the flask containing the diethyl ether solution of the lipid. The mixture was prepared using a bath sonicator (Lahoratory Subrise).
Company, Incorporated.

Type 10536 [Laboratory 5upp
lies Co., Inc.

type 10536'] for several minutes (frequency 80kHz2
9) A power output of 80 watts) was applied and at the same time a fine stream of nitrogen was passed through the mixture to dry it to a viscous paste. PBS 10d was added to the remaining viscous ingredients. The resulting naphthalene-containing 5PLV (stable plurilamellar vesicle) formulation (hereinafter referred to as 5PLV/Naf)
Alternatively, a 5PLV formulation containing gentamicin (hereinafter referred to as 5PLV/Gent) was suspended in PBS, shaken and incubated at 12,000x for 10 min at 20'C.
The antibacterial substances that were not encapsulated were removed by centrifugation at g.

The resulting pellet was washed again and PBSo, 5d
resuspended.

(5,2) Containing Gentamicin and Nafcillin To prepare 5PLV containing both gentamicin and nafcillin, the above procedure was performed with the following modifications. After being dispersed in EPC or diethyl ether, PB
Two solutions, each containing one antimicrobial substance, consisting of 100 nrg of antimicrobial substance (nafcillin or gentamicin) dissolved in S 2 O, 15d, are added quickly and simultaneously. After addition of the two solutions, the preparation was sonicated, evaporated and washed twice as described above. The obtained 5ptv captured both gentamicin and nafcillin (hereinafter referred to as 5PLV/Gent-Na
f).

(5,3) Treatment of infected mice 125 mice were treated with a lethal dose of Salmonella typhimurium (i.e., 3× 1069
A% forming units (cFU) (7) infected by intraperitoneal 11'iE (I, P ->IFi'a).

Twenty-four hours after inoculation, mice were divided into eight groups and each received the treatments indicated below. Group 1 (control) received no treatment and group 2 received aqueous nafcillin (100 mg/Kg (body weight), 1.P.
), group 3 received aqueous gentamicin (10
0mg/Kg (body weight), 1. P, ), group 4 is aqueous guntamer-sin (50IrG/Kg (body weight), '1
．． P, ) and aqueous nafcillin (50 #IN/9 (body weight)
, 1. Group 5 received a single formulation containing both nafcillin (100 mg (antibacterial substance >9
<Weight), 1. P,) is given 5PLV containing
Group 6 received SP[V containing gentamicin (100 my (antibacterial), 7 Kg (body weight), 1.
/Kg (weight), 1. P, ), and the other contains nafcillin<5Ctm! The fifth containing 'j<weight>> in i/
．． Group 8 was given the two 5ptv formulations mentioned in Section 1, and group 8 received gentamicin (50mg/Kg < body weight).
, 1, P, ) and nafcillin <50 tny/Kg (body weight), ■.

Given one ribosome as described in Section 5.2, both containing P, ). The results are shown in Table 2.

The results shown in Table 2 clearly show that 5PLV containing both gentamicin and nafcillin is the most effective in preventing deaths due to infection. In fact, not only is administration of a 5 pLvl drug containing both gentamicin and nafcillin more effective in preventing deaths than administration of both drugs in an aqueous solution, but surprisingly, gentamicin and nafcillin Treatment with a 5ptv formulation containing both was more effective in preventing deaths than simultaneous treatment with two 5ptv formulations, one containing gentamicin and the other containing nafcillin.

(Left below) 9 gu ＜X 目 ＜”tuノへ C/)
Heart Power (6) Example: Containing Gentamicin and Naphthalene In this example, the antibacterial activity and pathological effects of 5PLV containing both gentamicin and naphthalene were compared with several other formulations. Results have shown in testing these formulations that treatment of Salmonella typhimurium is most effective using the 5PLV formulation in which both gentamicin and naphthalene are contained in one liposomal formulation.

(6,1) Preparation of SPLv Drug-free 5PLVs and 5PLVs containing gentamicin or naphthalene were prepared using 20 h1 g of EPC and 200 my of drug as described in Section 5.1. 5PLV containing both gentamicin and naphthalene was prepared similarly as described in Section 5.2 using 1 g of EPC200 #1 and 2 oomg of each drug.

Each 5 pLvl agent was washed four times and resuspended in the following solution. (a) Drug-free SP[V was suspended in a total volume of 2 ml using saline.

(b) 5PLV (SPLV/NAF-GENT) containing nafcillin and guntamycin in one liposome
) t, :j,, suspended in a total volume of 2d using physiological saline. (c) Total amount of 5ptv containing nafcillin is 1m using physiological saline! ! suspended in 0 of this suspension
．． A 5m aliquot (split motion) uses 20m9 of gentamicin or saline added for a total volume of 17! (5PLV/NAF in gentamicin). (d
) 5PLV containing gentamicin was suspended in a total volume of 1 ml using saline. A 0.5 aliquot of this suspension was resuspended to a total ff11m using saline supplemented with 20 d of nafcillin (5PLV/GENT in nanocilin). (e) The remaining 0.57 aliquots of 5 PLV containing nafcillin in saline (from (c) above) were added to the 0.5 d aliquot of SP V containing gentamicin in saline (5
PLV/N8E & 5PLV/GENT). 71 [Spring 14
:5PLV formulation had the following composition per volume: 0.1%. (a) 5PLV is EPC20mg
, [b) 5PLV/NAF-GENT is EPC20
nry, nafcillin 2 Ing, gentamicin 2 mg
(c) 5PLV/NA in gentamicin (aqueous)
F is EPC20/71ff, nafcillin 2m3, gentamicin 2 rtry, (d) 5PLV/GENT in nafcillin (aqueous) is EPC20mg, gentamicin 2mF 1% nafcillin 2m3, and (e) SP
LV/NAF & 5PLV/GEN is EPC40
mg, nanosilin 2m9. Gentamicin 2 mg.

Hilltop strain mice (20 each
~30 rng) was obtained from B Broth (Plain Heart Infusion Media, BBL Mikuchi Biological Systems, Cotques Ville, Maryland [Brain Heart Infusion
Media, BBL Microbiologica
l Systems, Cockeysville.

Md, ]) in 0. D, Salmonella typhimurium medium grown to about 0.18 0.37! was infected with Salmonella typhimurium by intraperitoneal administration.

(6,3) Treatment of infected mice 27 h after infection with Salmonella typhimurium.
Mice were divided into seven groups, each group receiving a 0, 1 d inoculation (1, P, or k) as indicated below.
V, (intravenous)).

Group 1 (control) received no treatment, group 2 received 5PLV without drug (1,P,);
Group 3 is 5PLV/GEN in nafcillin (aqueous)
1 of each antibacterial substance per K9 (body weight).
oomg, LV, ), group 4 received SPL\I/NAF in gentamicin (aqueous) and 100 m of each antimicrobial agent per <K'J <body weight)9.1. ), group 5 is a mixture of two types of liposomes, 5PLV
/NAF&5PLV/GENT (respectively antibacterial substance iomg, 1.V, per kg<body weight>)
, ri/L, -'76Ll'5 PLV/NAF-GEN (1 o of each antibacterial substance per Kg (body weight)
om3.1. ), and group 6 is 5pLV/NA
F-GENT (100 m7.1.P of each antimicrobial substance per Kg (body weight)). The results are shown in Table 3.

These results demonstrate the increased efficacy of the combination of nafcillin and gentamicin entrapped in one liposomal formulation in the treatment of Salmonella typhimurium infections in vivo, whether administered intraperitoneally or intravenously. It is.

In this example, the antibacterial activity and pathological effects of various formulations of the antibacterial gentamicin and penicillin were compared. Although the results are presented in trials of these formulations, treatment of Salmonella typhimurium is most effective using IPV formulations (gentamicin and nafcillin in one liposomal formulation).

(7,1) HPV containing gentamicin and nafcillin
Preparation of 100 mg of EPC in 10 m ethanol was prepared in a round bottom flask. The two solutions shown below were added simultaneously to this lipid solution. The two solutions were PBSl, 5
A solution of gentamicin 100a5 dissolved in d and P
A solution of 100 m5 of nafcillin dissolved in BSl, 5d. The resulting mixture (dispersion) was evaporated at 54° C. for 3 minutes until a transparent film formed on the side walls of the container. Then 310ml of PB was added and the mixture was stirred to form and resuspend the MPV.

(7,2) Treatment of Infected Mice Sixty-five mice were infected with Salmonella to cause sepsis.
Typhimurium lethal dose (i.e. 5X106 CFU)
infection by intraperitoneal inoculation. Twenty-four hours after inoculation, mice were divided into three groups and received treatments as indicated below. Group 1 (control) received no treatment and group 2 received aqueous gentamicin (100 m
3/Ny (body weight) and aqueous naphthalene (100mFI
/KFI (body weight)) (1,P,) and group 3 received gentamicin C100ml/KS (body weight>) prepared as described in Section 6.2 and nafcillin (50 mff /Kg (body weight)) (1.P,). The results are shown in Table 4.

The results shown in Table 4 clearly show that HPV containing co-encapsulated gentamicin and nafcillin was the most effective and rare in determining the number of deaths due to infection.

('JX 下 Wabisu) (8) Example: Gentamicin and nafcillin in water In the example, the antibacterial activity and pathological effects of various formulations of gentamicin and nafcillin are compared. Result is,
Corynebacterium renale (corynebacterium renale)
Treatment of pyelonephritis (m renale) is most effective with 5PLV in which both gentamicin and nafcillin are contained in one liposomal formulation.

(8,1) Preparation of 5PLVs 5PLVs containing either gentamicin or nafcillin were prepared as described in Section 5,1). 5 containing both gentamicin and nafcillin
PLV was prepared as described in Section 5.2.

(8,2) Infection of mice with Corynebacterium renale Corynebacterium renale pyelonephritis is essentially a combination of Sumon and Yanakawa [Shumono
and Ya'nagaWa] method (Infection and Immunity, April 1977, 263
- Page 267 [Infection and Immuno
nity, Aprif 1977, pp, 263-2
[67]) in adult Hilltop mice (20-309, respectively). Each mouse was anesthetized using ether and the abdominal wall was incised to isolate the bladder. Bladder contents were evacuated by applying slight pressure. Concentration 107 CFIJ (village formation unit) ln&
A suspension of Corynebacterium renale in 80ml (BBL Microbiological System, Cotface Wheel, Merriant) was administered to the bladder for about 0.1 to 0.2 d per injection or until the bladder was filled. 106 CFU of microorganisms per bladder were inoculated. The abdominal wall was then closed. This Corynebacterium renale AT in BHI broth
CC species no. 10848 [C, renale ATCC
5 train No. 10848] was cultured overnight. The bacterial cells are then 0. D, 42° suspended in saline at approximately 0.78°. A series of dilutions were plated on agar to determine the CFυ per d for each dilution.

(8,3) Treatment of Infected Mice Twenty-four hours after inoculation with Corynebacterium renale, mice were divided into seven groups, and each group received the following treatments. Group 1 (control) received no treatment, group 2 received aqueous gentamicin (100111!?/N# (body weight), 1.P
) and group 3 received SPLV containing gentamicin (100 my/Kff (body weight), 1.P
), and group 4 was given aqueous naphthalene (10
0 mFl/KEIC body weight), 1. P, ) and containing group 5 Hanafucillin (100 mg
/Kg (body weight), 1, P, ), and group 6 received gentamicin (9 (body weight), 1.
) and naphthalene (100 my/Ky (body weight), LP
), and group 7 received a single aqueous formulation containing both gentamicin C10C10O/9
(weight)〉 and naphthalene (io.

my/ni! j<body weight)))
was given an IV formulation (1.P,). The results are shown in Table 5.

(Left below) The results shown in Table 5 clearly demonstrate that 5PLV containing both gentamicin and nanocillin is the most effective in preventing the number of deaths caused by C. renare pyelonephritis. ing.

In other experiments, the effects of gentamicin and nanocillin entrapped in one 5PLV formulation were
The effects of administering these two drugs in PLV were further compared. For this, mice were infected with C. renale as described in Section 8.2. Twenty-four hours after inoculation with C. renale, mice were divided into four groups and each group received the treatments indicated below. group 1
(comparison) received no treatment, group 2 received aqueous,
Nanocillin C100m1/'j (weight), 1. P,
), and an additional 1 h of aqueous nanocillin administration (100 m'J/in).
j (weight), 1. P, ) is given (ltlAF-G
ENT (aqueous), note that the aqueous formulations of nibufucillin and gentamicin are
] Administered after 1 hour to avoid inactivation of the drug. ), Group 3 is 5 containing gentamicin.
PLV (SPLV/GENT, 100 mg/to'
j<body weight)) and the other one is 5PL containing nanosilin.
V formulation (SPLV/NAF, 100 my/Ky (
Weight)) There is.

Group 4 received a mixture of two 5PLVs (1,P,) and gentamicin (100 mFl/KF!).
(body weight)) and nanosilin (100~/hong (body weight))
T-NAF) was given (1.P,). The results shown in Table 6 show that the 5PLV/GENT-N-F formulation was the most effective in treating the infection.

(Left below) 5P containing both gentamicin and nafcillin
Surviving mice treated with LV preparations
On day 14, animals were sacrificed and the right kidney was examined for the presence of C. renale, while the left kidney was analyzed histologically.

The right kidney was homogenized in BHI medium. This homogenate was serially diluted and plated on agar. No microbial growth was observed in the right kidney media of the eight surviving mice.

Histological experiments on the left kidney revealed no damage in 5 of the 8 kidneys sampled;
Minimized to moderate chronic inflammation in the lining of the renal kidney in two mice, and degenerative nephritis with focal necrosis in the central part of the left kidney in only one mouse. and an acute perinatal inflammatory reaction. Histological and microbiological treatment was thus demonstrated in surviving animals.

(9) Example: Tobramycin and Ticarcillin In this example, the antibacterial activity and pathological effects of various formulations of tobramycin (an aminoglucoside antibiotic) and ticarcillin (a β-lactam antibiotic) were investigated. be compared. Results are presented as test results for these formulations, but Pseudomonas aeruginosa [Pse
udomonas aerug'1nosa] Nephronephritis is treated with both tobramycin and ticarcillin.
It is most effective when using MPV formulations contained in one liposome formulation.

(9,1) Preparation of 14PV MPV containing both tobramycin and ticarcillin
was prepared as follows. First, EPC100m!
A solution of 10Id in ethanol was prepared in a round bottom flask. Next, add 1.5d of PBS to this EPC ethanol solution.
100 my of ticarcillin was added to this, and PBS 1.57 without divalent cations (PBS -) was added! tobramycin was added. The resulting mixture (dispersion) was heated for 54 hours until a film was formed on the side walls of the container.
Evaporated for 3 minutes at °C. Then 10 m of PBS was added and the mixture was stirred to form and resuspend the HPV.

1 containing either tobramycin or ticarcillin
(PV was prepared as above except that tobramycin 100=9 in PBS or ticarcillin iomg in PBS was to be added to the EPC ethanol solution.

Sprague Dori type (Spl”a (lue oaw)
+ey) rats (approximately 0.2 Ng each) were infected with Chartomonas aeruginosa by the following method.

Female rats received subcutaneously administered Brevital [Brev
ital] (10.64 mg/200 g (rat)
) was used to anesthetize the patient. The bladder was opened through a midline incision after abdominal hair removal. A small incision in the bladder was made to exclude all urea, and then a zinc pellet (3 m diameter) was inserted into the bladder. The bladder incision was sutured using silk sutures and soaked in TSB (tributicase soy broth) overnight.
o.o. of Pseudomonas aeruginosa cultured in Pseudomonas aeruginosa (Soy Broth), LB Microbiological, Maryland). iy inoculum was injected into the bladder. The abdominal incision was then closed.

(9.3) Treatment of Infected Rats Infected rats are treated with two doses of the following formulations administered intraperitoneally 4 hours and 28 hours after inoculation with Pseudomonas aeruginosa.
divided into two groups.

Group 1 (control) received no treatment, group 2 received aqueous tobramycin (4~/Kl (body weight)), and group 3 received MPVC containing tobramycin.
4In! j/ was given 9 (weight)), and group 4
is aqueous tobramycin C4001n! group 5 received tobramycin (400 g) and aqueous ticarcillin (4 to 9 bw)) and aqueous ticarcillin (4 to 9 bw);
9 (body weight) in ml/) and ticarcillin <4 mFI/
One MPV formulation (H
PV/TIC-Ding BRA).

Surviving rats were sacrificed on day 6 and each pair of kidneys was tested for the presence of Pseudomonas aeruginosa as follows.

After each kidney was removed, each kidney was transferred to a Petri dish containing ethanol, exposed to flame, and then homogenized using a SB2m knife. This homogenized product was prepared using Ding SB in the final trial 1.
Adjusted to 10d. A series of 10-fold dilutions of the homogenate were plated in duplicate on agar and CFU/m was determined for each pair of kidneys. The results are shown in Table 7.

(Margin below) These results demonstrate that the combination of tobramycin and ticarbocillin contained in one HPv formulation is the most effective in treating Pseudomonas pyelonephritis.

In this example, various formulations of gentamicin (an aminoglycoside antibiotic) and cylindamycin (a derivative of the amino acid trans-L-4-n-propylhycric acid attached to a sulfur-containing derivative of octose) Clostridium novii (clostri)
The antibacterial activity and pathological effects of P. dium novyi in the treatment of anaerobic wound infections are compared.

(10,1) 5PLVS (7) Prepared 5PLV containing gentamicin (SPLV/GE
Nymphs were prepared as described in Section 5.1 using gentamicin 1oom3. 5PLV (SPLV/CLIN) containing cylindamycin? 5PLV containing both gentamicin and cylindamycin in one liposome formulation.
3PLV/GENT-CLIN) contains 1100 In of each antibiotic, getamicin and cylindamycin.
It was prepared by the method described in Section 5.2.

All 5PLV formulations were washed three times in saline.

Adult female Swiss Websta species [5w1ss Web
ster] Twenty mice were injected into the right hind leg gravy with 0.05 d of a suspension of Clostridium novii prepared as follows. Clostridium novii is present in the stationary phase (108
-109 CEu/ml) for 1 day. The inoculum was prepared by diluting this medium 1:100 using fresh degassed 8-year old medium. This inoculum contained approximately 107 CFtl/weight.

(10,3) Treatment of Infected Mice Twenty-four hours after infection, mice were divided into four groups of five mice each and each received the following treatments.

Group 1 (control) received no treatment and group 2 received 5PLV containing gentamicin (i
oomg (gentamicin)/3 (body weight), 1. P
), group 3 contains 5 containing cylindamycin
given PLV (100 mg-(cylindamycin)
/l (g (weight), 1.P,), and group 4
were given 5PLV containing both gentamicin and cylindamycin in one liposomal formulation (
1 oom of each antibiotic per 1 KFJ (body weight)
g, 1. P.). The diameter of the infected paw was measured using a Kabbus and comparisons were made with control mice injected with fresh medium only. The results are shown in Table 8.

These results demonstrate that 5PLV containing both gentamicin and cylindamycin in one liposomal formulation
This shows that it was also effective in treating anaerobic infections of wounds.

(Hereinafter in the margin) It will be apparent to those skilled in the art that numerous additions, subtractions and changes to the above may be made without departing from the spirit and scope of the invention. The specific embodiments described above are merely examples, and the invention is limited only by the scope of the claims.

Patent Applicant: The Liposome Company, Inc.

Claims (56)

[Claims] (1) Liposome-drug preparations consisting of at least two non-antagonistic antimicrobial substances co-encapsulated in lipid vesicles. (2) The liposome-drug preparation according to claim 1, wherein the lipid vesicle is a stable plurilamellar vesicle. (3) The liposome-drug preparation according to claim 1, wherein the lipid vesicle is a monophasic vesicle. (4) The liposome-drug preparation according to claim 1, wherein the antibacterial substance is synergistic in co-encapsulated form. (5) The liposome-drug preparation according to claim 1, wherein the antibacterial substance exhibits synergism in the co-encapsulated form, and this synergism is determined by a combination effect test. thing. (6) The liposome-drug preparation according to claim 1, wherein the antibacterial substance exhibits additivity in a co-encapsulated form, and this additivity is determined by a combination effect test. . (7) The liposome-drug according to claim 1, wherein the antibacterial substance exhibits unchanging properties in a co-encapsulated form, and this unchanging property is determined by a combination effect test. Preparation. (8) a liposome-drug preparation consisting of at least two antimicrobial substances co-encapsulated in lipid vesicles, and wherein the therapeutic index of the liposome-drug preparation is greater than in the non-encapsulated form of the combined antimicrobial substance; Liposomal-drug preparation characterized in that it has a greater than therapeutic index. (9) The liposome-drug preparation according to claim 1, wherein the at least one antibacterial substance is antibacterial. (10) The liposome-drug preparation according to claim 8, wherein the at least one antibacterial substance is antibacterial. (11) The liposome-drug preparation according to claim 1, wherein the at least one antibacterial substance is antifungal. (12) The liposome-drug preparation according to claim 8, wherein the at least one antibacterial substance is antifungal. (13) The liposome-drug preparation according to claim 1, wherein the at least one antibacterial substance is antiviral. (14) The liposome-drug preparation according to claim 8, wherein the at least one antibacterial substance is antiviral. (15) The liposome-drug preparation according to claim 1, wherein the antibacterial substance is an aminoglycoside antibiotic and a β-lactam antibiotic. (16) The liposome-drug preparation according to claim 15, wherein the aminoglycoside antibiotic is gentamicin. (17) The liposome-drug preparation according to claim 15, wherein the aminoglycoside antibiotic is tobramycin. (18) The liposome-drug preparation according to claim 15, wherein the β-lactam antibiotic is nafcillin. (19) The liposome-drug preparation according to claim 15, wherein the β-lactam antibiotic is ticarcillin. (20) The liposome-drug preparation according to claim 15, wherein the aminoglucoside antibiotic is gentamicin and the β-lactam antibiotic is nafcillin. (21) The liposome-drug preparation according to claim 15, wherein the aminoglucoside antibiotic is tobramycin and the β-lactam antibiotic is ticarcillin. (22) The liposome-drug preparation according to claim 1, wherein the antibacterial substances are gentamicin and cylindamycin. (23) The liposome-drug preparation according to claim 20, wherein the lipid vesicles are stable plurilamellar vesicles. (24) The liposome-drug preparation according to claim 21, wherein the lipid vesicle is a stable plurilamellar vesicle. (25) The liposome-drug preparation according to claim 22, wherein the lipid vesicles are stable plurilamellar vesicles. (26) The liposome-drug preparation according to claim 20, wherein the lipid vesicle is a monophasic vesicle. (27) The liposome-drug preparation according to claim 21, wherein the lipid vesicle is a monophasic vesicle. (28) The liposome-drug preparation according to claim 22, wherein the lipid vesicle is a monophasic vesicle. (29) A method for treating infection, which comprises administering the liposome-drug preparation according to claim 1 to an organism. (30) The method according to claim 29, wherein the administration is parenteral. (31) The method according to claim 29, wherein the administration is local. (32) The method according to claim 29, wherein the administration is ocular. (33) A method for treating infection, which comprises administering the liposome-drug preparation according to claim 4 to an organism. (34) A method for treating infection, which comprises administering the liposome-drug preparation according to claim 5 to an organism. (35) A method for treating infection, which comprises administering the liposome-drug preparation according to claim 6 to an organism. (36) A method for treating infection, which comprises administering the liposome-drug preparation according to claim 7 to an organism. (37) A method for treating infection, which comprises administering the liposome-drug preparation according to claim 8 to an organism. (38) A method for treating infection, which comprises administering the liposome-drug preparation according to claim 9 or 10 to an organism. (39) Liposome according to claim 23 -
A method of treating an infection comprising administering a drug preparation to an organism. (40) Liposome according to claim 24 -
A method of treating an infection comprising administering a drug preparation to an organism. (41) Liposome according to claim 25 -
A method of treating an infection comprising administering a drug preparation to an organism. (42) Liposome according to claim 26 -
A method of treating an infection comprising administering a drug preparation to an organism. (43) Liposome according to claim 27 -
A method of treating an infection comprising administering a drug preparation to an organism. (44) Liposome according to claim 28 -
A method of treating an infection comprising administering a drug preparation to an organism. (45) If the infection is caused by Salmonella spp.
spp. ] The third claim arising from
The method according to paragraph 9 or paragraph 42. (46) The method according to claim 45, wherein the administration is parenteral. (47) If the infection is caused by Corynebacterium species [Coryneb
acterium spp. ] The method according to claim 39 or 42, which occurs by. (48) The method according to claim 47, wherein the administration is parenteral. (49) If the infection is caused by Pseudomonas spp.
asspp. ] The method according to claim 40 or 43, which occurs by. (50) The method according to claim 49, wherein the administration is parenteral. (51) If infection is caused by Clostridium species [Clostridium spp.
ium spp. ] The method according to claim 41 or 44, which occurs according to the method. (52) The method according to claim 51, wherein the administration is parenteral. (53) (a) forming a dispersion of at least one amphipathic lipid in an organic solvent; and (b) an amount sufficient to form a biphasic mixture in which the aqueous phase is capable of being completely emulsified. mixing this dispersion with an aqueous phase, and (c) emulsifying the aqueous phase and further evaporating the organic solvent of the biphasic mixture, and the antimicrobial substance is added with the aqueous phase and substantially forms multilamellar vesicles; The method for preparing stable plurilamellar vesicles according to claim 2, which does not include unilamellar vesicles and reverse phase evaporation vesicles. (54) (a) forming a dispersion of at least one amphiphilic lipid and an antimicrobial substance in a monophasic solvent; (b) dispersing the antimicrobial substance at a temperature that maintains the monophasic substance until a film is formed and facilitates evaporation; A method for preparing monophasic vesicles according to claim 3, comprising: evaporating the compatible solvent; (c) adding an aqueous phase to the film; and stirring to form monophasic vesicles. (55) The method according to claim 54, wherein the monophasic solvent is ethanol. (56) The method according to claim 55, wherein the evaporation is carried out at 54°C.