JPH0524133B2 - - Google Patents

Description

Claim 1: has a buoyant density lower than MLVs, has a volume about 1/3 or more larger than MLVs, contains at least one incorporated solute that is stable to autoxidation and body fluids, and contains bipolar lipids. consisting of lipid vesicles of 10,000 nm or less characterized by containing more than a few to more than 100 lipid bilayers,
Stable plurilamellar vesicles substantially free of MLVs, SUVs and REVs. 2. The stable plurilamellar vesicle according to claim 1, wherein the bipolar lipid has an acidic group and an amino group as hydrophilic groups. 3 Claim 2 in which the bipolar lipid is a phospholipid
Stable plurilamellar vesicles as described in Section. 4 Claim 3 in which the phospholipids are lecithins
Stable plurilamellar vesicles as described in Section. 5 Claim 1 containing an antioxidant,
The stable plurilamellar vesicle according to item 2, 3 or 4. 6 A vesicle that incorporates a compound selected from the group consisting of a dye, a fluorescent compound, a radioactive compound, and a radiopaque compound, has a lower buoyant density than MLVs, and has a volume that is about 1/3 or more larger than MLVs. , containing at least one incorporated solute that is stable to autoxidation and body fluids, and characterized by more than a few to more than 100 lipid bilayers containing ambipolar lipids. Consisting of vesicles, MLVs, SUVs and
Stable plurilamellar vesicles that are substantially free of REVs. 7. The stable plurilamellar vesicle according to claim 6, wherein the bipolar lipid has an acidic group and an amino group as hydrophilic groups. 8 Claim 7 in which the bipolar lipid is a phospholipid
Stable plurilamellar vesicles as described in Section. 9 Claim 8 in which the phospholipids are lecithins
Stable plurilamellar vesicles as described in Section. 10 Claim 6 containing an antioxidant,
The stable plurilamellar vesicle according to item 7, 8 or 9. 11. At least one entrapped solute comprising a biologically active compound entrapped within a vesicle, which has a lower buoyant density than the MLVs, has a volume about one-third or more greater than the MLVs, and is stable to autoxidation and body fluids. consisting of lipid vesicles of 10,000 nm or less characterized by more than a few to more than 100 lipid bilayers containing bipolar lipids,
A therapeutic agent for animals or plants containing stable plurilamellar vesicles substantially free of MLVs, SUVs and REVs. 12. The therapeutic agent for infectious diseases according to claim 11, wherein the biologically active compound is a compound selected from the group consisting of antibacterial compounds, antifungal compounds, anthelmintic compounds, and antiviral compounds. 13. The therapeutic agent according to claim 12, wherein the biologically active compound is a compound selected from the group consisting of streptomycin, dihydrostreptomycin, gentamicin, gentamicin sulfate, ampicillin, tetracycline hydrochloride, and kanamycin. 14. The therapeutic agent according to claim 11, wherein the bioactive compound is an anti-inflammatory compound, an anti-glaucoma compound, a pupil dilator or a local anesthetic compound. 15. The therapeutic agent according to claim 11, wherein the biologically active compound is a compound selected from the group consisting of antitumor compounds, toxins, cell receptor binding molecules, and immunoglobulins. 16. The therapeutic agent according to claim 11, wherein the biologically active compound is a compound selected from the group consisting of oxygen, hormones, neurotransmitters, immunomodulators, nucleotides, and cyclic adenosine monophosphate. 17 (a) forming a dispersion of at least one ambipolar lipid in an organic solvent; and (b) combining the dispersion with a sufficient amount of an aqueous phase to completely emulsify the aqueous phase. form a two-phase mixture that can, and
(c) MLVs characterized in that the organic solvent of the two-phase mixture is evaporated while simultaneously emulsifying the aqueous phase.
have a lower buoyant density than MLVs, have a volume about 1/3 more than that of MLVs, contain at least one incorporated solute that is stable to autoxidation and body fluids, and contain more than a few bipolar lipids. large to 100
characterized by more than one lipid bilayer
MLVs consisting of lipid vesicles of 10,000 nm or less,
A method for producing stable plurilamellar vesicles substantially free of SUVs and REVs. 18 The ratio of solvent volume to aqueous phase volume is approximately 3:
18. A method for producing stable plurilamellar vesicles according to claim 17, wherein the ratio is 1 to about 100:1. 19. A method for producing stable plurilamellar vesicles according to claim 17, which is carried out at a temperature of about 4 to 60°C. 20. A method for producing stable plurilamellar vesicles according to claim 17, wherein the temperature at which the method is carried out is lower than the phase transition temperature of at least one of the lipids. 21. The method for producing stable plurilamellar vesicles according to claim 17, wherein the solvent is fluorocarbon, diethyl ether, or a mixture thereof. 22. The method for producing stable plurilamellar vesicles according to claim 21, wherein the solvent contains an antioxidant. 1 Field of the Invention The present invention relates to liposomes and their use as carriers in administration systems. especially,
The present invention relates to a new type of lipid vesicles that have unique properties that give them special advantages such as increased stability and high capture efficiency. The compositions and methods described herein have wide applicability in areas such as carrier systems and targeted administration systems. The utility of the invention is illustrated by the examples of treatment of bracellosis, treatment of eye disease infections and treatment of lymphocytic meningitis virus infections. 2 BACKGROUND OF THE INVENTION 2.1 Liposomes are completely occluded bilayer membranes containing an entrapped aqueous phase. Liposomes can be unilamellar vesicles (with bilayers of monolamellar membranes) or multilamellar vesicles (onion-like structures characterized by concentric bilayers of membranes, each separated from the next by a layer of water).
It may be any variation of . The first liposome preparation by Bangham et al. involved suspending phospholipids in an organic solvent, which was then dried by evaporation, leaving a waxy deposit of phospholipids in the reaction vessel. do. An appropriate amount of aqueous phase is then added to "swell" the mixture and form multilamellar vesicles (hereinafter referred to as multilamellar vesicles).
They are called MLVs. ) The resulting liposomes are dispersed by mechanical methods. The structure of the resulting membranous bilayer is due to the hydrophobic (non-polar) nature of the lipids.
The "tail" is oriented toward the center of the bilayer, whereas the hydrophilic (polar) "head" is oriented toward the aqueous phase. This technique was developed by Papahadjopo ulos and Miller.
Millar) (1967, Biochem.Biophys.Acta.135:
small sonicated unilamellar vesicles described by (624-638)
nicated unilamellar vesicles) (hereinafter referred to as SUVs)
) provides the basis for development. However, these "classical liposomes" suffer from the considerable drawbacks of a low volume of entrained aqueous space per mole of lipid and a limited ability to encapsulate macromolecules. Efforts to increase the entrapped space have firstly been to generate reverse micelles or liposome precursors, i.e., vesicles containing an aqueous layer surrounded by a monolayer of lipid molecules, in which the polar head group is aqueous. This included being oriented toward the phase. Liposome precursors are produced by adding the aqueous solution to be entrapped to a polar lipid solution in an organic solvent and sonicating. The liposome precursor is then evaporated in the presence of excess lipid. The resulting liposomes, consisting of an aqueous phase entrapped by a bilayer of lipids, are dispersed in an aqueous phase (see U.S. Pat. No. 4,224,179, issued September 23, 1980 to M. Schneider). ). In another attempt to maximize trapping efficiency, Papahadjiyopoulos (U.S. Pat. No. 4,235,871, issued November 25, 1980) described a ``reverse phase evaporation technique'' for the production of oligolamellar lipid vesicles. ``method'' also uses reversed-phase evaporation vesicles (hereinafter referred to as
It's called REVs. ). According to this method, the aqueous substance to be captured is added to a mixture of polar lipids in an organic solvent. A homogeneous water-in-oil emulsion is then 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 REVS produced is mainly composed of monolamellar vesicles and some oligolamellar vesicles characterized by only a few concentric bilayers with large internal aqueous spaces. It has been reported that certain transmission properties of REVS are similar to those of MLVS and SUVS (Szoka and Papahajopoulos, 1978,
Proc. Natl. Acad. Sci. USA75:4194−4198). Although liposomes can be produced that entrap a variety of compounds, the stability of these liposomes during storage is always limited.
This loss in stability can result in leakage of entrapped compounds from the liposomes into the surrounding medium, and can also result in contamination of the liposomal content from the surrounding medium to the liposomes themselves due to permeation of substances. As a result, conventional liposomes have a very limited shelf life. Attempts to improve stability include adding substances that affect the physical properties of the lipid bilayer (hereinafter referred to as
It's called a "stabilizer." ) (e.g. steroid groups). however,
Many of these materials are relatively expensive and the production of such liposomes has not been cost effective. Besides the storage problems of traditional liposomes, many compounds have not been able to be contained in these vesicles. MLVS can only be produced under conditions above the phase transition temperature of lipid membranes. This precludes the introduction of thermolabile molecules into liposomes made of phospholipids that exhibit the desired properties but have long and highly saturated side chains. 2.2 Applications of Liposomes Therapeutic applications of liposomes are referred to as “Liposomes: From Physical
Structutes To Therapeutic Applications，
Knight, ed. Elsevier, North-Holland
Biomedical Press, 1981.
Much has been written about the potential use of these membrane vesicles for medical delivery systems, although a number of problems remain with such systems (e.g., Yneh-Erh and Elizabeth A.
See U.S. Pat. No. 3,993,754 issued to Cerny on November 23, 1976 and U.S. Pat. No. 4,145,410 issued to Barry D. Sears). In liposomal drug delivery systems, drugs are entrapped during liposome formation and then administered to the patient to be treated. The drug is soluble in water or non-polar solvents. Representative examples of such disclosures include Papahadjiyopoulos and Szoka.
U.S. Patent No. 25, 1980, issued November 25, 1980.
No. 4235871 and M. Schneider.
U.S. Patent No. issued September 23, 1980
No. 4244179. Some desirable features of a drug delivery system are resistance to rapid clearance of the drug accompanied by sustained release of the drug to prolong the action of the drug. These increase the effectiveness of the medicament and allow the use of smaller doses. Some problems that arise when using liposomal agents in vivo include the following. (1) Liposomes entrapping substances leak when they are incubated in body fluids. This was attributed to the scavenging of liposomal phospholipids by plasma high-density lipoproteins (HDL) or degradation of liposome membranes by phospholipases, among others. The result of degradation of liposomes in vivo is that almost all of the liposome content is released in a short time, so that sustained release and resistance to drug removal cannot be achieved. (2) On the other hand, if liposomes are used that are extremely stable in vivo (ie, if the liposomes do not leak when incubated in body fluids), the liposome content will not be released as needed. As a result, these stable liposomes are ineffective as carriers of therapeutic substances in vivo because the ability for sustained release and fractional liposomal release is not achieved when required. However, when treating intracellular infections, maintenance of stability within biological fluids is critical to the point that liposomes are internalized by infected cells. (3) cost-effectiveness of the liposomal carrier used in the delivery system; For example, an improved method of chemotherapy for Leishmania infections using liposome-containing anti-Leishmania agents was reported by Steck and Alving in US Pat. No. 4,186,183, issued January 29, 1980. Liposomes used in chemotherapy contain a number of stabilizers that increase the stability of the liposomes in vivo. However, as mentioned above, these stabilizers are expensive and the production of liposomes containing these stabilizers does not result in cost reduction. (4) Finally, a problem that arises with the use of liposomes as carriers in pharmaceutical delivery systems is that they are ineffective in aiding the treatment of the disease being treated. Besides the ineffectiveness for resistance to rapid elimination and the ineffectiveness for supporting sustained release, numerous other explanations for the ineffectiveness for disease treatment are possible. For example, if liposomes are internalized into cells of interest or into phagocytic cells (e.g. reticuloendothelial cells), they are rapidly cleared from the system, making the entrapped drug highly ineffective against disease in non-RES cells. It becomes.
After phagocytosis, the liposomes are packed into liposomes of phagocytic cells. Degradative enzymes, which are very often contained within liposomes, degrade the entrapped compound or render it inactive by cleaving it at its active site or by changing its structure. Additionally, liposomes cannot be administered in effective doses due to the low efficiency of entrapment of the active compound into the vesicles during manufacture. Liposomes have been used by researchers as a model membrane system and as "target cells" in captivity-regulated immunoassays. However, when used in such immunoassays, these assays are compatible with certain immunoglobulin classes (e.g. IgM and
Since we measure the release of liposome content as a factor in serum complement activation by immune complex products containing IgG molecules, it is important that the liposome membrane does not leak when incubated in serum. 3 Disclosure of the Invention The present invention is to provide a new and substantially improved type of lipid vesicles, hereinafter referred to as stable plurilamellar vesicles (SPLVs). Multilamellar vesicles (MLVs)
Besides being structurally different from MLVs, SPLVs are also structurally different from MLVs.
are manufactured in a different manner than MLVs, have unique properties compared to MLVs, and have various different advantages compared to such MLVs. As a result of these differences, SPLVs overcome many of the problems posed by conventional lipid vesicles. A heterogeneous mixture of lipid vesicles is generated when SPLVs are synthesized. The evidence is that the SPLVs
The results show that the lipids within are formed into a novel spramolecular structure. Many lipid vesicles have numerous bilayers, sometimes
It has as many layers as 100. Although this high degree of stratification is logical to explain, SPLVs
may contribute to a number of surprising properties possessed by Properties of SPLVs include:
(1) The ability of SPLVs to treat certain diseases that are otherwise untreatable, and (2) SPLVs while stored in buffers.
(3) can withstand harsh environments;
The large capacity of SPLVs, (4) the high trapping effect of substances;
(5) long-term adhesion to tissues and cells, (6) ability to sustain the release of entrapped substances within body fluids, (7) administration and critical dispersibility of liposome content through the cytosol of target cells, (8) ) improved cost reduction in manufacturing, and (9) release of the compound in its bioactive form in vivo. The unique properties of SPLVs make them particularly useful as carriers for administration systems in vivo, as they are resistant to excretion and are slow-release.
Methods for administering bioactive compounds in vivo and treating diseases such as infections are described.

[Brief explanation of drawings]

Figure 1 shows MLVs treated with various concentrations and
Figure 3 is a graph showing the difference in membrane stability (expressed in % leakage) between SPLVs. Figure 2 shows SPLVs in mouse eyelid tissue.
Figure 2 is a graph showing the retention of both the lipid layer and the aqueous phase of gentamicin and the sustained release of gentamicin from SPLVs in vivo. FIG. 3 is a diagram showing the electron spin resonance absorption spectrum of SPLVs compared to the electron spin resonance absorption spectrum of MLVs. FIG. 4 is a graph showing the difference in the ability of ascorbate to reduce doxil spinprobes in SPLVs and MLVs. FIG. 5 is a graph showing the effect of two-step treatment of Brucella canis infections in mice using streptomycin-captured SPLV based on Brucella canis that can be recovered from the spleen of infected mice. FIG. 6 is a graph showing the effect of two-step treatment of Brucella canis infections in mice using SPLV capturing streptomycin based on Brucella canis that can be recovered from organs of infected mice. Figure 7 shows streptomycin captured.
Figure 2 is a graph showing the effect of two-step treatment of Brucella abortus (bovine abortus) in guinea pigs using SPLV.

[Detailed description of the invention]

SPLVs are manufactured by a method that yields a different product than any other liposomes described above. SPLVs are lipid vesicles with more than a few to more than 100 lipid bilayers. Membrane bilayers consist of a bilayer of bipolar lipids, with nonpolar hydrophobic hydrocarbon "tail" points pointing towards the center of the bilayer and polar hydrophilic "head" points pointing toward the aqueous phase. There is. The aqueous phase is occluded by a bilayer, part of which forms the lumen of the vesicle and part located between adjacent layers. The lipid bilayer can form complexes with various proteins, glycoproteins, glycolipids, mucopolysaccharides and other hydrophobic and/or ambipolar substances. SPLVs are manufactured as follows. That is, a bipolar lipid or mixture of lipids is mixed into an organic solvent. Although many organic solvents are suitable,
Diethyl ether, fluorinated hydrocarbons and mixtures of fluorinated hydrocarbons and ethers are preferred. The active ingredient to be entrapped and the aqueous phase are added to this solution. This biphasic mixture is converted to SPLVs by emulsifying the aqueous material within the solvent while evaporating the solvent. Evaporation can be performed by any evaporation technique, e.g. by passing a stream of inert gas through the mixture, during or after sonication,
This is achieved by evaporation by heating or by vacuum evaporation. The volume of solvent used should exceed sufficient aqueous volume for the aqueous substance to be completely emulsified in the mixture. In practice, about 3 or more volumes of solvent are used for each volume of aqueous phase. In fact, the ratio of solvent to aqueous phase is
Capacity can be varied below or above 100. The amount of lipid is determined by the amount of emulsion droplet (1 ml of aqueous phase).
(approximately 40 mg of lipid per coat). SPLVs can be made with 115 g of lipid per ml of aqueous phase, although the upper limit is limited only by cost-effective practicality. This method produces lipid vesicles that have a macromolecular structure, unlike conventional liposomes. According to the invention, the entire process is carried out in the temperature range from 4 to 60°C, regardless of the phase transition temperature of the lipids used. The advantage of this latter is that thermolabile products with desired properties, e.g. denatured proteins, can be combined with SPLVs made from phospholipids such as distearoylphosphatidylcholine, but only at temperatures above their phase transition temperature. The point is that it is produced in conventional liposomes. The process usually provides more than 20% of the used water-soluble substances to be included and more than 40% of the used lipid-soluble substances to be included. Most ambipolar lipids are constituents of SPLVs. Suitable hydrophilic groups include, but are not limited to, phosphate, carboxy, sulfato and amino groups. Suitable hydrophobic groups include, but are not limited to, saturated and unsaturated hydrocarbon groups and hydrocarbon groups substituted with at least one aromatic and/or alicyclic group. Preferred ambipolar compounds are phospholipids and those similar in chemical structure. Examples of these are styrene, phosphatidylethanolamine, lysolecithin, lysophatidylethanolamine, phosphatidylserine,
These include, but are not limited to, phosphatylinositol, sphingomyelin, cardiolipin, phosphatidic acid and cerebrosides. Examples of stable lipids that are particularly useful in the production of SPLVs are natural lecithins (e.g. egg lecithin or soybean lecithin) and saturated synthetic lecithins (e.g. dimyristoylphosphatidylcholine or dipalmitoylphosphatidylcholine or distearoyl phosphatidylcholine) and unsaturated synthetic lecithins (eg, dioloylphosphatidylcholine or dilinoloylphosphatidylcholine).
The bilayer of SPLVs may contain steroid compounds such as cholesterol, coprostanol, cholestanol, cholestane, etc. When using compounds with acidic hydrophilic groups (phosphate groups, sulfate groups, etc.), the resulting SPLVs will be anionic, and when using compounds with basic groups such as amino groups, the resulting SPLVs will be cationic liposomes. and when using compounds with polyethyleneoxy groups or glycol groups, natural liposomes are obtained. The size of SPLVs varies significantly. Its range is about 500-10000nm
(10 microns), but usually about 1000-4000n
It is m. Virtually any biologically active compound
can be entrapped within SPLVs (entrapment is defined as entrapment within the aqueous fraction or membrane bilayer). Such compounds include nucleic acids, polynucleotides, antibacterial compounds, antiviral compounds, antifungal compounds, anthelmintic compounds,
Antitumor compounds, proteins, toxins, enzymes, hormones, neurotransmitters, glycoproteins, immunoglobulins, immunomodulators, dyes, radioactive labels, radiopaque compounds, fluorescent compounds, polysaccharides, Examples include, but are not limited to, cell receptor binding molecules, anti-inflammatory agents, anti-glaucoma agents, mydriatic compounds, local anesthetics, and the like. Below are examples of proportions used in the synthesis of SPLVs. That is, SPLVs are prepared by adding 50 micromoles of phospholipid to 5 ml of diethyl ether containing 5 μg of BHT (butylated hydroxytoluene) and then containing the active substance to be contained.
Produce by adding 0.3 ml of aqueous phase.
The resulting solution containing the substance to be captured and the capture lipid are sonicated while passing an inert gas through the mixture, thereby removing most of the solvent. This embodiment produces particularly stable SPLVs, due in part to the incorporation of BHT into vesicles. We describe a method for preparing liposome-encapsulated antibodies by sonication and evaporation of a solution of cholesterol and phosphatidylcholine with an added aqueous phase in a mixture of chloroform and ether, but the relative proportions of lipid to aqueous phase are defined. Lenk et al.'s 1983 Eur.J.
See Biokhem. 121:475-482. 5.2 Characteristics of SPLVs SPLVs are liposomes with single or several lamellae (e.g. SUVs and REVs).
are clearly distinct in their nature. Cryofracture electron microscopy showed that the SPLV agent was virtually free of SUVs and REVs, i.e. 20% of the vesicles.
% indicates unilamellar.
However, although they differ in many of their physical properties, they can be
Indistinguishable from MLVs. Thus, the following detailed comparison focuses on differentiating SPLVs from MLVs. 5.2.1 Stability of SPLVs during storage The stability of lipid vesicles depends on the vesicle's ability to isolate its viscerated space from the external environment over long periods of time. Useful lipid vesicles are preferably stable during storage and handling. However, for some applications it is desirable for the vesicles to gradually leak their contents during use. In other applications, the vesicles remain intact after administration until the desired point of action is reached. SPLVs
exhibits these desirable characteristics whereas
MLVs are not shown. There are two reasons why vesicles leak. One is the autoxidation of lipids, whereby hydrocarbon chains generate peroxides that destabilize the bilayer. This oxidation can be drastically reduced by adding an antioxidant such as butylated hydroxytoluene (BHT) to the vesicle agent. Vesicles also leak because drugs in the external environment disrupt the lipid bilayer structure such that the lipids remain intact but the membrane forms pores. Lipid vesicle agents are white in color when first manufactured. Autoxidation causes the drug to become colored (brown). Made using the same lipid and aqueous ingredients
Comparison of MLVs to SPLVs shows that MLVs is 1
SPLVs remain white for at least two months, whereas they become colored in ~2 weeks. this is
This is supported by thin layer chromatography of lipid components showing degradation of lipids in MLVs but not SPLVs. When these vesicles are produced by the addition of BHT and other ingredients, MLVs exhibit slight coloration within one month, while SPLVs remain white and stable for at least six months. neutral in a buffer containing isotonic saline.
SPLVs containing antibiotics when placed at PH
is stable for more than four months as shown in Table 1. These data indicate that none of the original antibiotics contained within the SPLVs leaked during the experimental period. Other evidence indicates that SPLVs can sequester internalized drugs from molecules as small as calcium ions for more than six months. Arsenazo is a pigment that changes from red to blue in the presence of very small amounts of divalent cations. By incorporating this dye into SPLVs and adding calcium chloride to a storage buffer, the stability of vesicles can be measured by observing the color change. The color differed undetectably from its initial color for at least 6.5 months without exhibiting either dye leakage or ion intrusion.

Table: These experiments demonstrate that SPLVs are sufficiently stable with respect to storage and handling issues. Stable over such a length
Although it is possible to create MLVs,
It must be made from synthetic lipids such as DSPC, which inevitably makes it expensive. 5.2.2 Stability of SPLVs in other environments Placing lipid vesicles in media containing membrane perturbants is a way to explore different molecular configurations. Depending on how their membranes are configured, different vesicles respond differently to such drugs. In the following experiments, vesicles containing a radioactive tracer molecule ( ^3H inulin) are prepared within an occluded aqueous fraction. The polysaccharide inulin partitions into the aqueous phase and is used to trace the aqueous content of lipid vesicles when radiolabeled. After exposure to a given drug at appropriate intervals, the vesicles are separated from the medium by centrifugal force and the amount of radiation that has escaped from the vesicles to the medium is measured. These results are reported in Table 2, where the values are expressed as percent leakage, meaning the proportion of radioactive material in the surrounding medium relative to the amount originally contained in the vesicles. SPLVs are more stable than MLVs in hydrochloric acid. Table 2 shows the degree of destabilization of both MLVs and SPLVs when exposed to 0.125N hydrochloric acid for 1 hour when made from egg lecithin.
However, it is noteworthy that SPLVs are significantly less acid sensitive than MLVs.
Presumably, this different response reflects the inherent differences in how lipids interact in their environment.

Table: SPLVs respond differently than MLVs when exposed to urea (Figure 1 and Table 2). Urea has a chaotropic effect
(splitting the structure of water) and a strong dipole moment. SPLVs
was observed to be much more sensitive to urea than to penetrants such as sodium chloride at the same concentration (Figure 1). MLVs are much less leaky in urea than in sodium chloride. Although the explanation for this different behavior is theoretical, this response is likely to be based on chaotropes rather than dipole effects, as guanidine, which is in close molecular proximity to urea, destabilizes SPLVs. (Table 2).
Although guanidine is also strongly chaotropic, it does not have a strong dipole moment. SPLVs are also sensitive to ammonium acetate, whereas MLVs are not (Table 2). However, neither ammonium ion (in ammonium chloride) or acetate (in sodium acetate) is particularly effective in destabilizing SPLVs. Therefore, it is clear that it is not the ion itself but the polarity of ammonium acetate that causes the induced leakage. Initially, such a result is surprising since SPLVs are much more stable than MLVs when cultured in body fluids such as serum or blood. However, theoretical explanations for such results have been proposed (other explanations are of course possible). If the stability of SPLVs is based on the unique structure of their membrane bilayer such that the polar groups of membrane lipids are hydrated by a cloud or hydration shell of oriented water molecules, then Any agent that disrupts or is incompatible with the hydration shell will promote a change to the structural membrane state and thus leak. Regardless of whether the theoretical explanation for the destabilization of SPLVs in urea is correct, MLVs and SPLVs
The above results show a characteristic difference between the structure of . This difference serves a very useful purpose in applications. As discussed below, SPLVs become progressively leaky when applied to the eye. Presumably, this desirable sustained release property is due to a similar destabilization of SPLVs when exposed to tear fluid. SPLVs are more stable than MLVs in serum. Numerous applications of lipid vesicles include administering them intraperitoneally, such as for the treatment of brucellosis. To be effective,
Vesicles must survive long enough to reach their desired mark. Both SPLVs and MLVs made from egg lecithin were exposed to fetal bovine serum containing active complement (Table 2). After being exposed to 37℃ for 48 hours,
SPLVs were much more stable than MLVs. 5.2.3 Characteristics of SPLVs administered in vivo SPLVs exhibit a number of properties that make them particularly suitable as carriers for in vivo administration systems. (A) SPLVs are resistant to evacuation.
When SPLVs are administered to an organism, the lipid components and the scavenged active components are retained within the tissues and by the cells to which they are administered. (B) SPLVs can be designed to provide sustained release. The stability of SPLVs is such that they are extremely stable during storage and in the presence of body fluids, but when administered in vivo, the slow leakage of the active ingredient provides a sustained release of the active ingredient. (C) Due to the high degree of entrapment and stability when administered, an effective dose of the active ingredient is released. (D) The production of SPLVs is inexpensive because the stability of the vesicles is achieved without the inclusion of expensive stabilizers in the bilayer. The following experiments demonstrate some of these properties of SPLVs when administered to the eyes of test animals. used in these experiments
The SPLVs were as described above, except that the reagent bilayer and the active ingredient were each radiolabeled to trace these ingredients into the ocular tissue during the period. SPLVs were manufactured using 100 mg phosphatidylcholine (EPC) and 100 mg gentamicin sulfate. Lipid components contain trace amounts of ¹²⁵ I
-phosphatidylethanolamine ( ¹²⁵ I-
PE) was radiolabeled by binding to the bilayer, and the active ingredient in the aqueous layer was radiolabeled by the addition of ¹²⁵ I-gentamicin sulfate ( ¹²⁵ I-GS). The SPLVs were washed repeatedly with buffer to effectively remove unbound or unincorporated material. A portion of the SPLVs agent was removed and extracted to remove the organic phase from the aqueous phase. The radioactivity of each phase was measured to determine the initial ratio of ¹²⁵ I-PE: ¹²⁵ I-GS that was introduced into the SPLVs (cpm (counts/min) in the lipid phase: cpm in the aqueous phase). It was done. The extraction was done as follows. 0.4M in 0.8ml
of NaCl (aqueous), 1 ml of chloroform and 2
ml of methanol were mixed to form a homogeneous phase. It was then radiolabeled with 4μ
SPLVs were added and mixed. This SPLVs
Since the fraction is dissolved in the organic and aqueous phases,
The initially cloudy mixture became clear. These phases were combined with 1 ml of 0.4 M NaCl (aqueous) and 1 ml of 0.4 M NaCl (aqueous).
ml of chloroform and mix, then
Separation was achieved by centrifugation at 2800xg for 5 minutes. A portion (1 ml) of each phase was removed and radioactivity was measured in cpm (initial ratio of ¹²⁵ I-PE: ¹²⁵ I-GS was 1.55:1). Fifteen adult female Swiss Wavestar mice were anesthetized and confined (to prevent them from wiping their eyes). Equal portions (2μ) of radiolabeled SPLVs in suspension were applied topically to each eye. Three groups of animals were then sacrificed at the following points: 1, 2, 3, 18 and 24 hours, respectively. Nine female Swiss Webster mice (controls) were treated similarly except that equal portions (2 μ) of an aqueous solution of radiolabeled gentamicin sulfate were applied topically to each eye. Immediately after sacrifice, the animal's eyelids are removed and minced;
and extracted (using the method described above) to separate the aqueous phase from the lipid components. The radioactivity of the phase was measured and the total radioactive counts were collected as well. The radiation measured in the lipid phase is an indication of the amount of SPLVs retained by the ocular tissue, the radiation measured in the aqueous phase is an indication of the amount of SPLVs retained by the ocular tissue, and the radiation measured in the aqueous phase is an indication of the amount of SPLVs retained by the ocular tissue. , an indication of the amount of gentamicin in the ocular tissue. Figure 2 is used for the eye
Figure 2 is a graph showing the abundance of each component in the eyelid tissue (expressed as a percentage of the initial cpm value). Figure 2 shows the amount of fluid in the eyelid tissue over 24 hours.
SPLVs lipid component retention and over 24 hours (expressed as percent of gentamicin retained in the eyelid tissue during the period)
The sustained release of gentamicin from SPLVs is clearly demonstrated. Figure 2 also shows that non-entrapped gentamicin (topically administered aqueous gentamicin) is rapidly cleared from the eyelid tissue. For example, gentamicin in solution (control) disappeared from the eyelid tissue within 4 hours (less than 5% gentamicin remaining in the eyelid tissue). On the other hand, more than 50% of SPLVs containing gentamicin were retained by the eyelid tissue during the last 4 hours, and in fact, after 24 hours, more than 25% of SPLVs containing gentamicin were retained in the eyelid tissue. This resulted in approximately 85% of the gentamicin-loaded SPLVs being released over 24 hours, and 95% of the unloaded gentamicin sulfate disappeared. Table 3 compares the proportion of SPLVs lipid phase and aqueous phase retained in the eyelid tissue at each point. An increase in this ratio is indicative of gentamicin release from SPLVs. The biological activity of SPLVs harboring gentamicin sulfate retained by the eyelid tissue was also evaluated. Gentamicin sulfate was recovered from eyelid tissue by removing a portion from the aqueous phase of an eyelid extract prepared 3 hours after SPLVs containing gentamicin sulfate were applied to the eye. This aqueous mixture was serially diluted and 2μ aliquots were placed on the S. aureus flora on an agar layer and the zone of inhibition was determined after 24 hours of incubation. SPLV with Gentamicin Sulfate
Gentamicin sulfate recovered from eyelid tissue extracts of animals treated with and completely retained its biological activity.

[Table] 5.2.4 Electron Spin Resonance Although SPLVs and MLVs appear identical under an electron microscope, their ESR (electron spin resonance) spectra reveal differences in their spramolecular structures. SPLVs are distinguishable from MLVs in terms of their molecular structure, as evidenced by their increased molecular order, increased molecular behavior and greater permeability to ascorbate. These differences in molecular structure are
contributing to its different biological activities. In electron spin resonance spectroscopy, 5-
Spin probes such as doxyl stearate (5DS) are incorporated into the lipid bilayer. The unpaired electrons of the doxyl group absorb microwave energy when the sample is inserted into a magnetic field. The absorption spectrum allows the determination of three experimental parameters S, the order parameter A _p , the hyperfine coupling constant and the rotational correlation time Tau. A typical reading is shown in Figure 3, where A is
Signal of SPLVs, B is signal of MLVs, both labeled with 5-doxyl stearate. Spectra were performed at room temperature and the scan range was 100 Gauss. The order parameter dependent on both 2T ₁ and 2T ₁₁ measures the error in the ESR signal from the case of perfectly uniform orientation of the probe. Uniformly oriented sample S=1.00
, the random sample is S=0. The radical coupling constant A ₀ , which can be calculated from 2T ₁ and 2T ₁₁ , is considered to represent the local polarity,
and represents the position of the spin probe within the membrane. The rotational correlation time (dependent on w ₀ , h ₀ , h−1) is
The previous spatial orientation aims at “forget”
It can be thought of as the time required for a molecule to with 5-DS as spin probe
Representative ESR determinations of the differences between SPLVs and MLVs are displayed in Table 4. Although in both cases the spin probes are reported from the same depth in the bilayer, the SPLVs
have much larger molecular order and molecular behavior than MLVs. Another explanation for the difference between SPLVs and MLVs lies in the ability of ascorbate to reduce doxyl spin probes. It is known that ascorbate often reduces the hydroxyl moiety to hydroxylamines, which probably do not absorb microwave energy in a magnetic field. In aqueous solution,
Reduction occurs rapidly with concomitant loss of ESR signal. If the Subin probe is in a protected environment such as a lipid bilayer, it is reduced more slowly or not at all by the hydrophilic ascorbate. Therefore, the rate of nitroxide reduction can be used to study the rate of ascorbate permeation into the lipid bilayer. Figure 4: Suspended in ascorbate solution
Figure 3 shows the relationship between SPLVs and MLVs residual spin percentage versus time. Ascorbate in 90 minutes
25% of probes placed in MLVs, but less than 25% of probes placed in SPLVs.
Reduce by 60%. SPLVs allow dramatically greater ascorbate penetration than MLVs.

[Table] 5.2.5 Capture of Active Substances by SPLVs Another basic example of the advantages of SPLVs over conventional MLVs is that SPLVs capture a large percentage of the active substances used, thereby retaining them (See Table 5). 5.2.6 Interaction of SPLVs with Cells Yet another advantage of SPLVs is that a relatively large portion of the material contained inside the vesicles is restricted to the cytoplasm of cells than to phagocytosed vesicles. It interacts with cells that are dispersed throughout. When SPLVs mix with cells, the two associate. Due to association, SPLVs, unlike MLVs, interact with cells in vitro, so all cells are
Contains at least some amount of the substance originally supplemented with SPLVs. This substance appears to be distributed in each cell, but is not restricted to phagocytic vesicles. This can be demonstrated by incorporating ferritin within the aqueous phase of the SPLVs agent. After association with cells in culture, ultrastructural analysis shows that ferritein is distributed throughout the cytosol and is not bounded by intracellular membranes. Although this phenomenon can occur with MLVs,
Larger amounts of material are transferred by SPLVs.

[Table] 5.2.7 Buoyant density of SPLVs Furthermore, SPLVs have a lower buoyant density than MLVs. This is measured by banding within the Ficoll gradient (see Table 6). 5.2.8 Volume of SPLVs Additionally, when collected into pellets at centrifugal forces of 1000-100000xg, SPLVs yield substantially larger pellets than MLVs, giving the same phospholipid concentration. At 16000xg centrifugal force, SPLVs become MLVs
Form pellets about 1/3 larger. 5.2.9 Osmotic properties of SPLVs Since phospholipid bilayers are water permeable, MLVs placed in a hypertonic environment will osmotically lock out water. SPLVs contract more than MLVs.
Additionally, in a buffer 20 times higher than the internal salt concentration.
After 16 hours of contraction, the SPLVs did not shrink to the same final volume as the MLVs (the pellet of SPLVs remained 1/3 larger than the pellet of MLVs). This indicates that the difference in pellet size is not due to a difference in aqueous internal volume. The penetration characteristics of MLVs are very different from those of SPLVs. The heterogeneous media concentration in the aqueous compartment of the MLV and the depletion of media in its outer layer create an osmotic gradient that compresses this MLV. In contrast, the media concentration in each compartment of SPLVs is substantially uniform with the media used to prepare SPLVs, resulting in uncompacted lipid vesicles. 5.3 Applications of SPLVs SPLVs are particularly useful in systems where the following factors are important: That is,
Stability during storage and in contact with body fluids, relatively high viscosity, low cost, and release of the scavenging compound in its biologically active form.

[Table] In addition, SPLVs depending on the in vivo dosage form
can be given to cells that are resistant to rapid elimination (eg, if sustained release is important) or RES. As a result, the SPLVs of the invention can be used in a wide range of systems. These are used to increase the medical effectiveness of treatments, to treat infectious diseases, to enhance enzyme replacement, oral medical administration, local medical administration, to introduce genetic information into cells in vitro or in vivo, and to develop vaccines. It can be used for manufacturing purposes, to introduce genetically exchanged deoxyribonucleic acid segments into cells, or as diagnostic agents for clinical trials with the release of captured "reporter" molecules. The SPLVs can also be used to incorporate cosmetics, fungicides, slow-release compounds for plant growth agents, etc. As mentioned above regarding the use of SPLVs, the method is
SPLVs or other liposomes or
It also allows the use of lipid vesicles with functionality similar to that of SPLVs. 5.3.1 Administration of biologically active compounds Administration of compounds to cells (e.g. animal cells, plant cells, protozoa, etc.) in vitro
SPLVs containing the compound in cells in culture medium
usually requires the addition of however,
SPLVs can also be used to administer the compounds into animals (including humans), plants and protozoa. Depending on the purpose of administration,
SPLVs can be administered by a number of routes.
That is, in humans and animals this includes injection (e.g. intravenous, intraperitoneal, intramuscular, subcutaneous, intraauricular, intramammary, intraurethral, etc.), local use (e.g. at the site of pain), and epithelial or mucosal ( For example, it is used in clothing epithelium, oral mucosa, rectal or vaginal mucosa, airway membranes, nasopharyngeal mucosa, intestinal mucosa, etc.).
This is not limited to, and in plants and protozoa, this is directly
It can also be carried out by dispersing it in the habitat of living things and adding it to the surrounding water or environment, but it is not limited thereto. The mode of use also determines the site or cells of the organism to which the compound is administered. For example, administration to a specific site of infection is facilitated by topical use (if the infection is external). Administration to the circulatory system (and thus to the reticulum cells) can be very easily carried out by intravenous, intraperitoneal, intramuscular or subcutaneous injection. Since SPLVs allow sustained release of the compound,
If not toxic to the organism, the dosage can be used in one or more administrations to the organism. This section provides a general description of how SPLVs can be used, but is not intended to limit the scope of the invention. 5.3.2 Disease Treatment A number of pathological conditions occurring in humans, animals and plants can be effectively treated by incorporating appropriate compounds into SPLVs. These pathological conditions include, but are not limited to, infections (intracellular or extracellular), cysts, tumors and tumor cells, allergies, and the like. Various methods are possible for using SPLVs to treat such diseases. We outline some particularly useful strategies that take advantage of the fact that SPLVs are internalized by macrophages when administered in vivo. In one plan, SPLVs are used to deliver therapeutic agents to the site of intracellular infection. Some diseases involve infection of cells of the reticuloendothelial system, such as brucellosis. These intracellular infections are difficult to treat for a number of reasons. (1) Since infectious microorganisms are present within the cells of the reticuloendothelial system, therapeutic drugs that cannot pass through the cell membrane at therapeutically sufficient concentrations are stopped from circulation;
For this reason, there is a high resistance to treatment. (2) Administering toxic levels of therapeutic agents is often necessary to prevent such infections. (3) Treatment must be completely effective because any infection remaining after treatment can reinfect the host organism or transmit it to other hosts. According to one mode of the invention, SPLVs containing suitable biologically active compounds are administered (preferably intraperitoneally or intravenously) to a host organism or potential host organism. (For example, in herds, uninfected animals can be treated in the same way as infected animals.)
Since phagocytic cells internalize SPLVs, administration of SPLVs loaded with biologically active substances against microbial infection aims to reach the biologically active compounds directly to the site of infection. Thus, the method of the present invention can be applied to various microorganisms, bacteria, parasites, fungi,
It is used to eliminate infections caused by mycoplasma, viruses, etc., and these microorganisms include Brasella spp, Mycobacterium spp, Salmonella spp, Listeria spp, Franticella spp, Histoplasma spp, Corynebacterium spp,
These include, but are not limited to, Coccidiodes spp and Lymphocytic choriomeningitidis. The therapeutic agent selected depends on the microorganism causing the infection. For example, bacterial infections can be cleared by antibiotics. The antibiotic is SPLVs
and/or inserted into the vesicle bilayer. A suitable antibiotic is
Penicillin, ampicillin, hetacillin, carbencillin, tetracycline, tetracycline hydrochloride, oxytetracycline hydrochloride, chlortetracycline hydrochloride, 7-chloro-6-dimethyltetracycline, doxycycline hydrate, methacycline hydrochloride, monocycline hydrochloride, loli Tetracycline, dihydrostreptomycin, streptomycin, gentamicin, kanamycin, neomycin, erythromycin, carbomycin, aureomycin, troreomycin, polymyxin B colistin, cephalothin sodium, cephalolidine, cephaloglycine dihydrate, and cephalexin-hydrate. . We demonstrate the effectiveness of such treatment in the treatment of brucellosis (see Examples below). The method according to the invention increases the effectiveness and duration of action. This system is effective in treating infections captured by MLVs that do not respond to known treatments such as antibiotics.
No successful treatment could be expected, since any small amount of residual infection would spread and the infection cycle would begin again. We present a successful case in lymphocytic choriomyelitis virus infection. Of course, the present invention is not limited to the treatment of intracellular infections. These SPLVs target various infection sites, whether intracellular or extracellular. For example, in other embodiments of the invention,
Macrophages are used to deliver active agents to sites of systematic extracellular infection. According to this plan, SPLVs are used in vivo to administer therapeutic substances to uninfected macrophages by administering SPLVs (preferably intraperitoneally or intravenously).
SPLVs are used. This macrophage is
They are associated with SPLVs and then "loaded" with therapeutic substances. Generally, this macrophage carries the substance at 3
Hold for ~5 days. Once the "loaded" macrophages reach the site of infection, the pathogen is internalized by the macrophages. As a result, the pathogen comes into contact with the therapeutic substance contained within the macrophage and is destroyed. This embodiment of the invention is useful in the treatment of Staphylococcus aureus in humans and livestock. SPLVs loaded with medical agents can be used locally if the site of infection or association is external or mobile. Particularly useful applications include the treatment of drowsiness. In the case of eye pain, SPLVs containing one or more suitable active ingredients are applied topically to the painful eye. Numerous microorganisms cause eye infections in animals and humans. Examples of such microorganisms include, but are not limited to, the following: Moraxel spp, Clostodium spp, Corynebacterium spp, Diprococcus spp, Erabobacterium spp, Haemofiris spp, Klebsiella spp, Restospira
spp, Mycobacterium spp, Niceceria spp,
Propionipacterium spp, Proteus spp,
Bacterium-like microorganisms including Pseudomonas spp, Cestia spp, Escherichia spp, Staphylocacchus spp, Streptococcus spp, and Mycoplasma spp and Rickettsia spp. These infections are difficult to remove by conventional methods because the infection remaining after treatment is reinfected by lacrimation. We demonstrate the use of SPLVs in curing clothing infections caused by Moraxella bovis. (See Examples below) SPLVs are resistant to excretion and are capable of sustained sustained release of their contents;
SPLVs are also useful in treating some problems where extended contact with active treatment substances is desired. For example, glaucoma is a disease characterized by a gradual increase in intraocular pressure that causes progressive loss of peripheral vision, and when ataxic, loss of central vision and eventual blindness. Medications used to treat glaucoma are primarily used as eye drops. However, sometimes treatment requires instillation of medication every 15 minutes with rapid removal of the drug from the orbit. Given that problems like glaucoma are treated with the present invention, pilocarpine fluoropyril, physostigmine, calcholine, acetazolamide, etozolamide, dichlorophenamide, carbachol,
SPLVs in which therapeutic substances such as demecarium bromide, diisopropyl phosphofluoridate, ecothioplate iodide, physostigmine or neosdigmine, etc. are given to the diseased eye.
It is possible to have it built in. Other agents that may be incorporated into SPLVs and used topically include, but are not limited to, pupil dilating agents (epinephrine, phenylebinephrine, hydroxyamphetamine, cyclopentolate, tropitucuamide). , encatropin, etc.), local anesthesia, antiviral agents (idoxuridine, adenine arabinoside, etc.), antifungal agents (amphoteracin B, natamycin, pimaricin, flucytosine, nystatin, thimerosal, sulfamerazine, thiobenzazole, tolnaftate, glyciofulvin, etc.), anthelmintic agents (sulfonamides, pyrimethamine, clindamycin, etc.) and anti-inflammatory agents (corticosteroids such as ACTH, hydrocortisone, prednisone, medrysone, betamethasone, dexamethasone, fluoromethalone, triantcinalone, etc.). The following examples are given for illustrative purposes but are not intended to limit the scope of the invention. 6 EXAMPLES Preparation of SPLVs As explained in Section 5.1, the basic method for preparing SPLVs is to dissolve a lipid or a mixture of lipids in an organic solvent, add an aqueous phase and the substance to be encapsulated, and including sonicating the mixture, with the organic solvent being removed by any evaporation method during or after the sonication. The SPLVS used in all examples of the invention is prepared as described in the next section. (However, any method of producing SPLVs can be used.) 6.1 SPLVs with antibiotics A solution of 100 mg lecithin in 5 ml diethyl ether was prepared. The mixture was placed in a round bottom flask. Next, 5mM HEPES (NEPES)
(4-[2-hydroxyethyl]piperazino2-
ethanesulfonic acid)/0.0725M NaCl/
Streptomycin sulfate 100 in 0.0725M KCl
A solution (0.3 ml) containing 1.0 mg at PH 7.4 is dropped dropwise into a glass container containing a solution of lipid in diethyl ether. The mixture was prepared using a 10536 type bath sonicator (Laboratory Supplies Company,
Incorporated [Laboratory Supplies]
Co., Inc.) for several minutes,
80kHz: output power of 80 watts), and at the same time it is dried into a sticky paste by passing a gentle vapor of nitrogen through it. To the place where the adhesive paste remains, 5
10 ml of mM Heps was added. The produced SPLV preparation containing streptomycin was suspended in a buffer solution and stirred for several minutes in a vortex mixer.
Unencapsulated streptomycin is released by centrifugation at 12,000xg for 10 minutes at 20°C. The resulting cake is 0.5 of 5mM heps.
It was suspended in ml. The above procedure was performed similarly except that streptomycin was replaced with one of dihydrostreptomycin, gentamicin sulfate, ampicillin, tetracillin hydrochloride, and kanamycin. 6.2 SPLVs Containing Other Membrane Components The procedure described in Section 6.1 was followed similarly except that any one of the following was added along with egg lecithin: (1) phosphatidic acid giving a molar ratio of 8:2 (lecithin:dicetyl phosphate);
(2) stearinamine, giving a molar ratio of 8:2 (lecithin:stearylamine), 7:2:1
(3) phosphatidic acid and cholesterol, giving a molar ratio of (lecithin; cholesterol; stearylamine); (3) phosphatidic acid and cholesterol, giving a molar ratio of 7:2:1 (lecithin: phosphatidic acid: cholesterol). 6.3 SPLVs Containing Pilocarpine The procedure of Section 6.1 was followed similarly except that the antibiotic streptomycin was replaced with pilocarpine. 6.4 SPLVs prepared with and without BHT The undistilled ether contains 1 μg/ml butyl hydroxyl toluene (BHT), an antioxidant, for storage purposes. The procedure of Section 6.1 was similarly performed using undistilled ether as the solvent to bind BHT during SPLV preparation. To prepare SPLVs without coupling to BHT, the procedure of Section 6.1 was similarly performed using distilled ether as the solvent. 7 EXAMPLE SPLV-Mediated Release in Glass Containers In the following example, SPLV-mediated release of antibiotics to macrophages in culture vessels was demonstrated. Peritoneal macrophages were obtained by peritoneal lavage from _C57 BLK adult male mice and suspended in minimum essential medium (MEM) PH7.2 containing 10% heat-inactivated fetal calf serum. It will be done. Cells are 1×10 ⁶
cells/ml in a 96-well tissue culture dish. B. canis was added to an incubator containing sticky peritoneal macrophages at a concentration of 1×10 ⁶ CFU (colony forming units)/ml. After 12 hours, bacteria that were not inhaled into the peritoneal macrophages were removed by repeated washings with MEM. After cleaning the peritoneal macrophage incubators, they were divided into 5 groups containing 12 replicate cultures per group. Group 1 represents the control standard and has no added treatment. Group 2 receives aqueous streptomycin sulfate at a concentration of 1 mg/ml. Group 3 receives SPLVs in buffer. Group 4 is aqueous streptomycin sulfate (1 mg/
ml) and placed in a pre-made buffer solution.
Receive SPLVs. Group 5 will receive SPLVs containing streptomycin sulfate (1 mg/ml).
After 24 hours, the supernatant is removed by repeated washing and the peritoneal macrophages are broken up by repeated freezing and thawing. Serial dilutions of the ground macrophages were placed on Brucella agar and after 4 days the surviving B. Canis was determined by limiting dilution. The results shown in Table 7 demonstrate that streptomycin contained in SPLV has an overall effect on killing in vitro and eliminating intracellular M. abortus infection. Experiments were conducted with B. abortus as described above, except that peritoneal macrophages were obtained from adult female albino guinea pigs by peritoneal lavage. The results are also shown in Table 7.

[Table]

Table 8 Example Treatment of Intracellular Infections The following example demonstrates how SPLVs can be used in the treatment of intracellular infections. This data is used in (1) disease treatment;
(2) represents the greater potency obtained by high dosing of SPLV preparations; used in the prescription example
A comparison of MLVs with SPLVs as excipients is described. Brucellosis is a global economic and national health problem. Brucellosis is caused by Brucella spp. It affects mammalian species, including humans, domestic animals, and a variety of wild animals. six brucellas
spp causes brucellosis in animals, and these include B. abortus bovis, B. abortus canis, and B.
melitensis), Brucella neotome (B,
neotomae), Brucella ovis (B. ovis), and B. suis. Treat both domestic and wild animals as reservoirs that can spread brucellosis to other animals and humans. Because the infected organism exists in cells of the reticuloendothelial system and has high resistance to the bactericidal action of antibiotics,
Such infections cannot be cleared by antibiotics. The amount of antibiotic required and the length of treatment result in high concentrations of antibiotic that are either toxic to the animal or unacceptable into the animal's cells. Further complicating the treatment of this disease is that any remaining infection spreads easily;
The treatment must be completely effective in order for the cycle to start repeating again. The economic impact of such diseases is illustrated by the millions of dollars worth of cattle lost each year due to spontaneous abortion. The only possible way to deal with such outbreaks is isolation and slaughter of infected animals at that point. This embodiment consists of introducing the antibiotic into the SPLVs and then dosing the animal with the encapsulated active agent by ingestion intraperitoneally into the infected animal. 8.1. Efficacy of a single treatment of M. abortus infection using antibiotics contained in SPLV Eighty adult male Swiss mice were infected intraperitoneally (IP) with M. abortus ATCC23365 (1 × 10 ⁷ CFU).
and were divided into eight groups of 10 mice each. On the 7th day after inoculation with canine abortus bacteria,
Each group received the following treatments: Group 1 represented the control standard and received no treatment. Group 2 included SPLVs in buffer (0.2 ml I.
P.) received. Group 3 is aqueous streptomycin sulfate (1 mg/Kg of total dosage of 0.2 ml I.P.
weight) was received. Group 4 included aqueous streptomycin sulfate (5 mg/ml) in the total dosage of 0.2 ml I.P.
Kg weight) received. Group 5 included aqueous streptomycin sulfate (10
mg/Kg body weight). Group 6 is 0.2mlI.P.
Streptomycin sulfate (1
mg/Kg body weight). Group 7 received SPLVs containing streptomycin sulfate (5 mg/Kg body weight) in a total dosage of 0.2 ml I.P. Group 8 contains streptomycin sulfate (10 mg/Kg body weight) in a total dosage of 0.2 ml I.P.
Received SPLVs. On the 14th day after inoculation with canine abortus bacteria,
All animals were sacrificed and the spleens were removed aseptically. Spleens are homogenized and serially diluted on Brucella agar to determine the number surviving in the spleen after treatment. The results after an incubation period of 4 days are shown in Table 8.

[Table] 8.2 Effect of multiple treatment with antibiotics contained in SPLV for infection with P. abortus 80 adult male Swiss mice were infected with P. abortus
infected with ATCC23365 (1×10 ⁷ CFU, IP);
Divided into 8 groups with 10 mice each. On days 7 and 10 after inoculation with Canis abortus, each group received the following treatments: Group 1 represents a reference standard and is not processed in any way. Group 2 is SPLVs in buffer (0.2ml, IP)
received. Group 3 is aqueous streptomycin sulfate (1 mg/Kg body weight) in a total dosage of 0.2 ml I.P.
received. Group 4 received aqueous streptomycin sulfate (5 mg/Kg body weight) in a total dosage of 0.2 mg I.P. Group 5 included aqueous streptomycin sulfate (10 mg/Kg) in a total dosage of 0.2 mg I.P.
weight) was received. Group 6 received SLPVs containing streptomycin sulfate (1 mg/Kg body weight) in a total dosage of 0.2 mg I.P. Group 7 is 0.2
mlI.P. received SLPVs containing streptomycin sulfate (5 mg/Kg body weight) in all doses. Group 8 received SPLVs containing streptomycin sulfate (10 mg/Kg body weight) in a total dosage of 0.2 ml I.P. Fourteen days after inoculation with Canis abortus, all animals were sacrificed and the spleens were aseptically removed. The spleen was homogenized and serially diluted on Brucella agar to determine the number of B. abortus surviving in the spleen after treatment. The results after 4 days of culture are shown in FIG. As shown in Figure 5, the various two-step treatment regimens for in vivo Canis abortus infection showed that the group receiving aqueous streptomycin on days 7 and 10 after inoculation showed that the group receiving aqueous streptomycin remained in the spleen. Bacteria were observed with almost no reduction. Only the group receiving SPLV-contained streptomycin dosed at 10 mg/Kg body weight on days 7 and 10 post-inoculation resulted in clearance of all viable bacteria from the spleens of inoculated animals. In addition to the above experiments, various cells from M. abortus infected mice were sampled as follows after being treated twice with streptomycin contained in SPLV. 30 adult male Swiss mice infected with Canis abortus
The cells were inoculated with ATCC23365 (1×10 ⁷ CFU, IP). Seven days after inoculation, animals were divided into three groups of 10 mice each. Group 1 represented the control standard and received no treatment. Group 2 received aqueous streptomycin sulfate (10 mg/Kg body weight) during each 0.2 ml I.P. dose (on days 7 and 10 post-inoculation). Group 3 contains streptomycin sulfate (10 mg/Kg body weight) in each dose of 0.2 ml I.P.
received SPLVs (7 and 10 days post-inoculation). From 14 to 70 days after inoculation with B. abortus, all animals were sacrificed and serially diluted on Brucella agar to isolate the heart, lungs, spleen, liver, kidneys, and testicles infected with B. abortus. did. 4
The results of canine abortus bacteria remaining per organ after 1 day of culture are shown in FIG. As shown in FIG. 6, the results of sampling various tissues in mice infected with Canis abortus after two treatments with streptomycin are as follows:
In animals treated with streptomycin contained in SPLV, all tissues sampled 14 to 75 days after inoculation with P. abortus were completely free of viable P. abortus cells. There is. In animals that were untreated or treated with aqueous streptomycin at exactly the same concentration and dosing regimen as those receiving the streptomycin contained in SPLV, viable Abortion cells ranged from 14 to 75 days after inoculation with N. Abortus. It could be isolated in all tissues sampled on the same day. 8.3 Effect of treatment with SPLVs and MLVs for comparison.
The cells were inoculated with ATCC23365 (1×10 ⁷ CFU, IP). Seven days after inoculation, animals were divided into three groups of 5 mice each. group 1
represented the reference standard and received no treatment. Group 2 is streptomycin sulfate (10
mg/Kg body weight, IP) containing MLVs (7 and 10 days post-inoculation). MLVs were prepared in a conventional manner using 2 ml of sterile heps containing 100 mg egg phosphatidylcholine (EPC) and streptomycin sulfate (100 mg/Kg). The lipid to streptomycin sulfate ratio was 28 mg streptomycin to 100 mg EPC in 2 ml of the final MLV suspension. Group 3 is 100
A modification was made in which mgEPC was used with 3 ml of heps containing 100 mg streptomycin sulfate.
SPLVs containing streptomycin sulfate (10 mg/Kg body weight, IP) prepared as described in Section 6.1
(7 and 10 days after vaccination).
The ratio of lipids in SPLVs to streptomycin sulfate was 100% in 2 ml of final SPLV suspension.
It was 28mg streptomycin sulfate for mgEPC. Fourteen days after inoculation with B. abortus, all animals were sacrificed and the pancreases were aseptically removed, homogenized, and serially diluted to isolate B. abortus on Brucella agar. . The results for viable canine abortus per organ after 4 days of culture are presented in Table 9. Table 9 Comparison of MLVs and SPLVs containing streptomycin sulfate in the killing of P. abortus in vivo after two treatments ^a. Colonization units of P. abortus per spleen ^b Reference standard 2.7 ± 1.0 × 10 ⁴ MLVs ^c 1.8±0.4×10 ⁴ SPLVs ^c 0 a Intraperitoneal injections of 10 mg/Kg body weight were performed at 3-day intervals. The reference standard was not treated in any way. b Surviving canine abortus was determined as the number of CFU isolated per spleen and expressed as the mean ± standard deviation of 5 animals per group. (Double determination per experimental animal) c The ratio of egg phosphatidylcholine to streptomycin sulfate is 100 mg lipid to 28 mg streptomycin sulfate. 8.4. Effect of various antibiotics contained in SPLV in treating infection Fifty male adult Swiss mice were inoculated with canine abortus ATCC 23365 (1×10 ⁷ CFU, IP). Seven days after inoculation, the experimental animals were divided into 10 groups with 5 mice each. Group 1 represented the control standard and received no treatment. Group 2 was placed in buffer
SPLVs (0.2 ml, IP) were received 7 and 10 days after inoculation. Groups 3, 4, 5 and 6 are
Cyhydrostreptomycin, Zentamycin, Kanamycin or Streptomycin 10mg/Kg body weight, IP aqueous injection (0.2mlI.P.)
were administered on the 7th and 10th day after vaccination. (Note: Each of these antibiotics has been shown to kill Canis abortus in vitro.) Groups 7, 8, 9 and 10 contain 10 mg/Kg body weight of dihydrostreptomycin, zentamycin, kanamycin or streptomycin. including
Receive SPLVs 7 and 10 days after inoculation.
Fourteen days after inoculation with B. abortus, all animals were sacrificed and the spleens were aseptically removed, homogenized, and serially diluted to isolate B. abortus on Brucella agar. Ta. The results for viable canine abortus per organ after 4 days of culture are presented in Table 10. The results of the tests of various antibiotics on M. abortus mice shown in Table 10 show that antibiotics that are effective in killing M. abortus in vitro (i.e., in a suspension medium) are those that are exposed to SPLVs. When packaged, it only shows the effect of killing the canine abortus bacteria in vivo. Animals receiving other aqueous antibiotics or SPLVs in buffer, or no treatment, did not undergo deletion of residual canis sulfate from infected splenic tissue.

【table】

[Table] 8.5. Treatment of dogs infected with Canis abortus. Adult female beagles are infected with Canis abortus.
ATCC23365 (1×10 ⁷ CFU) was inoculated intraorally and intravaginally. Seven days after vaccination, the dogs were divided into three groups. Group 1 represented the reference standard and received no treatment. Group 2 received 10 mg/Kg body weight (each dose was 5.0 ml I.
It was P. ) received aqueous streptomycin sulfate (7 and 10 days post-inoculation). Group 3 was 10 mg/Kg body weight (each dose was 3.0
ml, IP. ) SPLVs containing streptomycin sulfate (7 and 10 days after inoculation)
I received it. Dog vaginal washes and heparinized blood samples were collected at regular intervals before, during, and at the end of the study. These were solvated on Brucella agar to isolate Canis abortus. The results are shown in Table 11. Serum samples were collected before, during, and at the end of the study for the determination of serum antibodies to M. abortus. These results are also shown in Table 11. All experimental animals were sacrificed 21 days after inoculation with Canis abortus. The following tissues were aseptically removed, homogenized, and serially diluted onto Brucella agar to isolate B. abortus. Heparinized blood, vaginal exudate,
Lungs, spleen, synovial fluid, uterus, ovaries, popliteal lymph nodes, salivary glands, tonsils, mediastinal lymph nodes, mesenteric lymph nodes, bone marrow, superficial cervical lymph nodes and accessory lymph nodes. The results of Canis abortus remaining per tissue after 4 days of culture are shown in Table 12.

【table】

【table】

[Table] The results of the serological tests of the canine abortus infected dogs before, during and after the two-step antibiotic administration of the culture results are shown in Table 11. All experimental animals were serologically negative prior to exposure to Canis abortus as determined by negative serum titers and culture negative from blood cultures and vaginal swab cultures.
All experimental animals were reported to be culture positive in both blood and vaginal cultures on days 7 and 10 prior to treatment. Dogs treated with aqueous streptomycin or receiving no treatment remained culture positive with blood and vaginal cultures during the post-treatment period before the end of the study on day 21. Group 3, which received liposomes containing streptomycin, became culture positive one day after the first treatment and remained negative throughout the post-treatment period. Dogs that receive no treatment or aqueous streptomycin develop detectable serum titers against M. abortus antigen by day 21 postinfection, but dogs that receive no treatment or aqueous streptomycin develop detectable serum titers against M. abortus antigen by day 21 postinfection, but dogs that receive no treatment or aqueous streptomycin develop detectable serum titers against M. abortus antigen by day 7 postinoculation with antibiotic-containing SPLVs. Those treated on day 10 did not develop any detectable serology for Canis abortus antigen. The results from the isolation of Bacillus abortus from infected dogs treated with two-step antibiotic treatment shown in Table 12 show that in dogs, only treatment with SPLVs containing streptomycin resulted in a significant increase in all tissues from all organ samples. It has been shown to be effective in eliminating any viable canine abortus bacteria. 8.6. Treatment of B. bovis in guinea pigs Fifteen adult female guinea pigs were infected with B. abortus in guinea pigs.
The cells were inoculated with ATCC23451 (1×10 ⁷ CFU, IP). Seven days after inoculation, the experimental animals were divided into three groups of 5 animals each. Group 1 represented the control standard and received no treatment. Group 2 received aqueous streptomycin sulfate at 10 mg/Kg body weight in IP injection (0.2 ml) on days 7 and 10 after inoculation with B. abortus. group 3
received SPLVs containing 10 mg/Kg body weight aqueous streptomycin sulfate in IP injection (0.2 ml) on days 7 and 10 after inoculation with B. abortus. All experimental animals were sacrificed 14 days after inoculation with B. abortus and the spleens were removed, aseptically homogenized, and serially diluted onto Brucella agar to isolate B. abortus. . The results for the number of viable bovine abortus bacteria per spleen after 4 days of culture are shown in FIG. Only SPLVs containing streptomycin were effective in deleting B. abortus in the spleen of guinea pigs. In laboratory animals that received aqueous streptomycin or received no treatment,
A viable bovine abortus bacterium has been identified. 8.7. Treatment of Bovine Abortus in Cattle Nine heavily infected animals were used in this experiment. Bovine abortus bacterial isolation from milk and vaginal swabs became negative after treatment with SPLVs containing streptomycin and remained negative for 6 weeks. When infection recurred in these experimental animals, cell detachment was seen only in the mammary quadrant that was positive before treatment. Nine cross-bred (Herrenford-Jersey-Brangus) cows of 22 years of age, non-pregnant, and confirmed to be culture positive for Bovine Abortus were used. At least 4 months prior to the start of the study, the animals were experimentally challenged with 1×10 ⁷ CFU of Bovine Abortus sp. 2308 in the conjunctiva prior to midterm gestation, thereby causing abortion and/or
Bovine abortus cultures of milk or uterine secretions and/or fetal tissue are positive. Cows were kept in individual isolated barns and divided into three groups.
The treatment, consisting of two dosing doses taken 3 days apart, is as follows. (1) Three cows are injected intraperitoneally with saline. (2) The three cows are
Placed in pre-made buffer with aqueous antibiotic (10mg/Kg body weight streptomycin)
SPLVs are injected intraperitoneally. (3) Three cows were injected intraperitoneally with streptomycin (10 mg/Kg body weight) contained in SPLVs. The total volume per injection was 100 ml per animal. During the first two months, replicative bacterial cultures of milk and uterine secretions were fed once a week. All cows are then killed with an overdose of sodium pentavirbatol, and the following organs are collected in duplicate from the bacterial culture. (1) Lymph nodes; left and right atlantic lymph nodes, left and right supopharyngeal lymph nodes, left and right mandibular lymph nodes, left and right parotid lymph nodes, left and right prescapular lymph nodes, left and right Prefemoral lymph nodes, left and right axillary lymph nodes, left and right popliteal lymph nodes, left and right iliac lymph nodes, left and right mammary lymph nodes, left and right kidneys, bronchi, mediastinum, mesentery and liver lymph nodes. (2) Glands: mammary glands of all four breasts, left and right adrenal glands, thymus (if present). (3) Organs and other tissues; spleen, liver, left and right uterine horns, (uterine) cervix, vagina, kidneys, tonsils. After necropsy, all tissues are frozen and handled at -70°C during transport. The tissue is frozen, exposed to alcohol flame, and aseptically shaped before weighing. Weight is recorded (0.2 to
0.1 g) and immediately remove the tissue with 1 ml of sterile saline.
The homogenate is homogenized in a medium and serially diluted with sterile saline to 1:10 ^-10 of the initial homogenate suspension.
Aliquots (20μ) of each dilution from a series of suspensions were plated on Brucella agar.
Cultured at ℃. Replication decisions were made for each tissue. Plates were read and recorded daily for bacterial growth. Before 3 days, all emerging colonies were isolated, excreted and Gram stained for identity determination. Colonies were counted and CFU/g tissue determined on days 5, 6, and 7 of culture for morphology, growth, and presence of Gram staining characteristics for B. abortus. Typical colonies were reconfirmed for bacterial confirmation of Bovine Abortus. Bacteriological isolation was performed on all tissues and the number of bacteria per gram of tissue was calculated.
The results obtained from four experimental animals (one placebo control and three animals treated with streptomycin contained in SPLVs) are shown in Table 13.

[Table] Right atlantic lymph node + 0 +
Left 〃 0 0 0 +
Right axillary lymph node 0 + 0

【table】

Table 9 Example Treatment of Eye Diseases Bacterial and similar infections, like many other eye diseases, are a global economic and national problem, and if untreated or untreatable, can lead to blindness and sepsis. RISK OF DEATH. Bacterial infections of the eyes in animals and humans include Clostridium spp, Corynebacterium spp, Leptospira spp, Moraxella spp, Mycobacterium spp, Neisseria spp, Propionibacterium spp, Proteus spp, Pseudomonas spp, Serratia spp, E. coli (E.Coli)
spp., Staphylocatchus spp., and bacterium-like microorganisms including Mycoplasma spp. and Rickettsia spp. Both animals and humans are treated as reservoirs capable of spreading infectious bacteria to each other. Such bacterial infections cannot be treated with antibiotics in some infected humans without frequent treatments every 20 minutes or lengthy and cumbersome treatment regimens that result in unacceptably high concentrations of antibiotics in the tissues. Can not. Existing treatment methods are difficult for many reasons. Infectious microorganisms in the superficial tissues of the eye are in some cases highly resistant to the bactericidal action of antibiotics, and topical administration of antibiotics results in rapid removal of the drug from the orbit resulting in variable contact times. becomes. In general, treatment for eye infections must be completely effective because any residual infection can quickly become reinfected through the lachrymal secretion, starting the cycle all over again. Furthermore, in many cases, the concentrations required to clear the infection cause vision loss and, in some cases, total blindness.
The economic impact of such diseases in livestock is illustrated by the millions of dollars lost each year because the only possible way to deal with such contagious diseases is continuous therapy and isolation. has been done. The following experiment was conducted using the eye Moraxella bovis (M.
The purpose of this study is to evaluate the effectiveness of treatment with antibiotics free in glycerol on infections (B. bovis) compared to antibiotics incorporated into SPLVs. Moraxebovis causes contagious keratoconjunctivitis (pink eye) in cattle. This state is
It is characterized by blepharospasm, lacrimation, conjunctivitis, and varying degrees of septal opacification and ulceration. Adult female cows may develop a low-grade fever with some loss of appetite and reduced milk production. Although some antibiotics are effective against Moraxella bovis, they must be administered early and frequently and repeatedly by topical application or subconjunctival injection. According to the embodiments described herein, the effectiveness and duration of action of the therapeutic substance is prolonged. It is surprising that this system is effective with just one or two doses, since such infections do not respond to mere routine treatment with antibiotics. Common treatments often leave some residual infection that becomes contagious to the eye and repeats the infection cycle again if the infection is not completely eradicated by multiple treatment repetitions. 9.1 Treatment of infectious keratoconjunctivitis in mice C57 black mice (160 mice) were divided into eight groups, half of each group was exposed to UV radiation in both eyes (to cause corneal damage). All experimental animals were then inoculated with Moraxelamobis instilled into the right eye at a concentration of 1×10 ⁶ bacteria per eye. Twenty-four hours after inoculation, all experimental animals were scored for the degree of corneal opacity. Eight groups are treated with topical application to both eyes as follows. group 1
and 2 received 10μ of streptomycin (30mg/ml) contained in SPLV. Groups 3 and 4 streptomycin (30mg/ml)
received 10 μ of. Groups 5 and 6 received 10μ of buffered SPLVs suspended in aqueous streptomycin (100ml/ml).
Groups 7 and 8 received 10μ of sterile saline. (Note: The uninfected left eye was treated with the same topical solution to see if SPLVs were irritating to the eye. No irritation was observed.) Once daily, progress or regression of septal damage was recorded. , 3 and 7 days after treatment, the right eye was swabbed and Moraxella bovis isolation was performed in typical experimental animals. Moraxelamobis colonies were determined by colony morphology and reactivity to fluorescent antigens for Moraxelamobis fimbriae. No.
The results shown in Table 14 indicate that only the streptomycin contained in SPLV was effective in eliminating the infection.

[Table] Mycin ^c

Table 9.2 Treatment of rabbit conjunctiva with antibiotics contained in SPLV Moraxella bovis ATCC strain 10900 is diluted in sterile saline (0.085% NaCl) to a concentration of 1 x 10 ⁷ cells/ml. Aliquots (0.1 ml) of the bacterial suspension were locally inoculated into the eyes of 10 adult female rabbits. Culture samples were taken daily by swabbing the conjunctiva and placed on blood agar plates. On the third day after inoculation, the rabbits were divided into three groups and two experimental animals (control)
received no treatment, 4 experimental animals received streptomycin (at a concentration of 10 mg/Kg body weight) in sterile saline, and 4 experimental animals received streptomycin (contained in SPLV) in sterile saline. Concentration of 10mg streptomycin/Kg body weight)
received. All solutions were administered topically bilaterally. After 24 hours, conjunctival swabbing of all rabbits was resumed and continued daily for 7 days. The results of separation of Moraxella bovis on blood agar plates are shown in Table 15.

【table】

[Table] 9.3 Treatment of keratoconjunctivitis due to subcutaneous infection Moraxella bovis ATCC strain 10900 was diluted to 1 x 10 ⁷ cells/ml in sterile saline. Aliquots (0.1 ml) of the bacterial suspension were inoculated into the eyes of adult rabbits that had already been infected as described in Section 9.2 and were not treated with SPLVs. The right eye of all nine rabbits was inoculated subcutaneously into the conjunctival tissue with 0.1 ml of Moraxella bovis, and the left eye of all rabbits was inoculated topically with 0.1 ml of Moraxella bovis. Cultures were taken daily from the conjunctiva of both eyes of all rabbits and plated on blood agar for Moraxella bovis isolation. On the third day after infection, the rabbits
Two experimental animals received no treatment, three experimental animals received streptomycin in standard ophthalmic glycerin suspension (concentration of streptomycin of 10 mg/Kg body weight); The experimental animals received a saline suspension of streptomycin in SPLV (10 mg streptomycin/kg body weight).
The suspension or solution was administered topically (0.1 ml) to both eyes. Conjunctival swabs were performed on all rabbits after 24 hours and every 5 days thereafter. Isolated vessels of Moraxella bovis on blood agar culture are shown in Table 16. Dissection was performed on all experimental animals at the end of the experiment, and the conjunctiva was removed from all animals. These were scored for angiogenesis and minced, homogenized and placed on blood agar plates for separation of Moraxella bovis. The results are shown in Table 17.

【table】

Table 9.4 Evaluation of the effectiveness of SPLVs in the treatment of ocular infections compared to liposomal preparation Moraxella bovis (ATCC strain 10900) was diluted in sterile saline to a concentration of 1 x 10 ⁷ cells/ml. An aliquot (0.1ml) of bacterial suspension is
Conjunctival tissue from both eyes of an adult rabbit was inoculated subcutaneously. The conjunctiva of both eyes of all rabbits was swabbed daily and placed on blood agar plates for isolation of Moraxella bovis. On the 4th day after inoculation, the rabbits were divided into 6 groups, 2 animals received no treatment (control standard) and 3 animals received OD480 (480 mm Three experimental animals received a suspension of SPLV-encapsulated streptomycin (10 mg streptomycin/kg body weight) with an OD480 of 0.928 when diluted 1:100. Three experimental animals received a packaged suspension of streptomycin (10 mg streptomycin/kg body weight) with an OD480 of 0.242 when diluted 1:100.
Three experimental animals received a suspension of SPLV-encapsulated streptomycin (10 mg streptomycin/kg body weight) diluted 1:100.
Two experimental animals received a suspension of SPLV-encapsulated streptomycin (10 mg streptomycin/kg body weight) with an OD480 of 0.199, and two experimental animals received streptomycin-containing multilamellar vesicles (10 mg streptomycin/kg body weight) with an OD480 of 0.940 at a 1:100 dilution. MLVs) suspension (streptomycin 10 mg/kg body weight). MLVs are Curr.Micro.6;373 of Foutain et al.
(1981) by adding streptomycin sulfate to a dry lipid film, vortexing, and swelling for 2 hours; unincorporated streptomycin was removed by repeated centrifugation. The suspension was administered topically to both eyes. After 24 hours, swab the conjunctiva of all rabbits.
Intervals were taken daily and placed on blood agar. The results of the separation of Moraxella bovis on blood agar plates are shown in Table 18. Necropsies were performed on all animals. These were recorded for lachrymal secretion and the conjunctiva was aseptically removed from all animals. These were scored for angiogenesis and minced, homogenized and placed on blood agar plates for Moraxella bovis isolation. The results are shown in Table 19.

[Table] 2 + + + +
+ 0 0 + 0 0 0 0 0 0
3 + + + +
+ 0 0 0 0 + 0 0 0 0

【table】

[Table] 2 0 1+ 0

Mycin (1:
4 dilution)
3 + 1 + 0

[Table] 10 Examples Treatment of viral infection Lymphocytic choriomeningitis virus (LCMV), a member of the arenavirus family, is known to cause disease in humans. Being inoculated with the virus is fatal. The cause of death in this mouse was due to immune cells reacting against virus-infected cells. Since the virus does not kill the cells in which it is growing, the therapeutic agents used in mice inhibit the proliferation of the virus so that immune cells are not activated and/or kill the cells in which it is growing. activation must be inhibited. The following examples demonstrate the effectiveness of administering SPLV-encapsulated antiviral compounds in treating viral infections. 10.1 Treatment of Fatal Lymphocytic Choriomeningitis Virus Infection in Mice Two-year-old Swiss mice were intracerebrally administered a lethal dose of LCM virus, i.e., 100 plaque-forming units (PFU) in a 0.05 ml inoculum per mouse. was inoculated. Mice were divided into four groups of 7 mice each and were treated with the following intraperitoneal injections of 0.1 ml/dose/mouse on days 2, 3, and 4 after inoculation. (1) "SPLV-R group" was treated with egg phosphatidylcholine SPLVs containing 3 mg Ribavarin/ml. SPLVs are 0.3 of 100 mg lipid and 100 mg/ml drug.
ml was prepared in PBS (phosphate buffered saline) buffer and the drug loading was 10%. (2) “R
−Group” is 3 mg/mg of ribavarin in PBS.
treated with a solution of (3) “SPLV-Group”
were treated with SPLVs in buffer.
(i.e., SPLVs were made as above except without the use of ribavarin.) (4) "Reference Standard-Group" was treated with PBS. Five days after inoculation, two mice from each group were sacrificed and their spleens were homogenized. (1
Two spleens per group are homogenized in PBS at 1/20 weight per volume of buffer. ) Plague forming units (PFU)/ml were determined for each suspension. The remaining 5 mice in each group were observed every 2 days for 30 days for mortality. The results are presented in Table 20. Table 20 clearly shows the reduction in mortality from infected animals and the reduction in virus regeneration performance.
We have not yet determined whether these results are due to the antiviral activity of ribavarin released from SPLVs or to immune modulation of the murine host while undergoing release of ribavarin from SPLVs. .

【table】