JPS59500952A - Stable plurilamellar vesicles - 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] Stable plurilamellar vesicles 1. Field of invention The present invention relates to liposomes and their use on carriers in administration systems. It is. In particular, they offer special advantages such as increased stability and high capture efficiency. This invention relates to a new type of lipid vesicle with unique properties.

The compositions and methods described herein include carrier systems and intended administration systems. It has wide applicability in a wide range of fields. The practicality of the present invention is to treat pulcellosis, Illustrated by the examples of 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. It is. Liposomes are unilamellar vesicles (having a bilayer of monolayers) or or multilamellar vesicles (bisected concentric membranes, each separated from the next by a layer of water) There may be any differences in the onion-like structure characterized by the child layers.

The first liposome production by B. Angham et al. containing suspended phospholipids, which are then dried by evaporation into the reaction vessel. Latter area of phospholipids 1985-500952 (3) Leaves a soot-like deposit. An appropriate amount of aqueous phase is then added to "swell" the mixture and multilamellar vesicles ) (hereinafter referred to as MLVs). Disperse by law. The structure of the resulting membranous bilayer is based on the hydrophobic (non-polar) “te” of the lipids. The hydrophilic (polar) “head” is oriented toward the center of the bilayer, whereas the “head” is oriented toward the center of the bilayer. is oriented toward the aqueous phase. This technique is based on Papahajoboulos and Miller (P apahadjop.

ulos and Millar) (1967, Biochem, 3io pbys.

Acta, 135: 624-638). Small 30nicated unila The basis for the development of mellar vesicles (hereinafter referred to as 5UVs) provides the foundation. However, these “classical liposomes” are The low volume of trapped aqueous space per mole of molecule and the ability to encapsulate macromolecules are limited. There are quite a few drawbacks. Efforts to increase captured space are primarily due to The production of micelles or liposome precursors, i.e., a single layer of lipid molecules. Vesicles containing a more surrounded aqueous layer have polar head groups directed toward the aqueous phase. The liposome precursor was oriented in an organic solvent. produced by adding the aqueous solution to be captured to a polar lipid solution and sonicating be done. The liposome precursor is then evaporated in the presence of excess lipid. lipid The resulting liposomes consist of an aqueous layer trapped by a bilayer of water. Dispersed in Shuso (September 1980 for M, 3 cln+eider (See U.S. Pat. No. 4,224,179, issued on the 23rd).

In another attempt to maximize supplementation efficiency, Papahadjopoulos <November 1980 U.S. Pat. No. 4,235,871, issued on the 25th, describes (oligolamellar 1ipid vesicles) % advancement The "reverse phase evaporation method" is also known as reverse phase evaporation vesicles (hereinafter referred to as REVs). It is stated that According to this method, the aqueous substances to be captured are Added 710 to the mixture of polar lipids in the medium. Then, a homogeneous water-in-oil emulsion A gel is formed and the organic solvent is evaporated until a gel is formed. This gel is The gel-like mixture is then converted into a suspension by dispersing it in an aqueous medium. Generate REVS mainly have monolamellar vesicles and large internal aqueous spaces. Some oligolamellar vesicles are characterized by only a few concentric bilayers. It is something that will become true. Transmission characteristics with REVS, MLVS and 5uvs It is reported that it is similar to the sex (3. zoka and p. apciha). jopoulos, 1978, proc, Naguchi, ha, c? d　 , 3ci, (J, S, A, 7 5:4, 19'I-4198・ ).

However, liposomes that entrap various compounds have permanently limited storage. During this period, stability of the liposomes is created. This loss in stability This can result in compounds trapped from the materials leaking into the surrounding media and This may also result in contamination of the liposomes from the surrounding medium to the liposomes themselves. Become. As a result, conventional liposomes have an extremely limited shelf life. . Attempts to improve stability affect the physical properties of the lipid bilayer within the liposome membrane. (hereinafter referred to as "stabilizer") (e.g. steroid group). It contained.

However, many of these materials are relatively expensive and The production of similar products was not cost efficient.

In addition to the storage problems of traditional liposomes, many compounds are It was impossible to have it.

MLVS can only be produced under conditions above the phase transition temperature of the lipid membrane. this phospholipids that exhibit the desired properties but have long and highly saturated side chains. This prevents the inclusion of thermolabile molecules within the liposomes.

2.2. Uses of liposomes Therapeutic applications of liposomes include [Liposomes]: From Physical 5 structures T.

Therapeutic Applications, Knight, ed,　E　1sevier　　North　-Holland　Biome dical Press, 1981 J. In such a system Although numerous issues remain, the use of these membrane vesicles for medical delivery systems is challenging. Much has been written about the possibility of h and EIizabeth A, against Cerny, November 1976 U.S. Patent No. 3,993゜754 issued on the 23rd and B arry D , U.S. Pat. No. 4,145,410 issued to Sears).

In liposomal drug delivery systems, the drug is entrapped during liposome formation and then , administered to the patient to be treated. The drug is soluble in water or a non-polar solvent. Ru. Representative examples of such disclosures are 19 v. Papahadjopoulos and Szoka. U.S. Patent No. 4,235, issued November 25, 1980. ．． No. 871 and M , U.S. Patent No. 4,22 issued September 23, 1980 to Schneider. No. 4,179.

Some desirable features of a drug delivery system include sustained release of the drug, which prolongs the action of the drug. resistance to rapid removal of drugs. This increases the effectiveness of medicines and Allows for use in smaller doses. Liposolysis in vivo Some problems that arise when using emollients include the following. (1) Capturing substances Entrapping liposomes leak as the liposomes grow within body fluids. child This is because, among other reasons, plasma high-density lipoprotein (HDL)-induced liposomes Causes removal of phospholipids or degradation of liposome membranes by phospholipases there was. As a result of the degradation of liposomes in vivo, almost all of the liposomes are It is released in a short time and therefore sustained release and resistance to drug removal cannot be achieved. stomach. (2) On the other hand, when using liposomes that are extremely stable in vivo, (If liposomes do not leak when they are produced in body fluids), the liposomes can be used as needed. It is not released until As a result, these stable liposomes are suitable for sustained release and release. Since the ability to release the posomal fraction is not achieved at the required time, it is possible to carry therapeutic substances in vivo. It is invalid as a body. However, when treating intracellular infections, Maintenance of quality is critical up to the point where liposomes are internalized by infected cells. be. (3) The impact of the cost of the liposome carrier used in the administration system. For example, lipo Modification of chemotherapy for Leishmanial infections using somal-containing anti-leishmanial agents The good law is described in U.S. Patent No. 4,186,183, issued January 29, 1980. reported by Steck and Alping. used in chemotherapy Liposomes contain a number of stabilizers that increase the stability of liposomes in vivo. Contains. However, as mentioned above, these stabilizers are expensive; Moreover, the production of liposomes containing these stabilizers does not result in cost reduction.

(4) Finally, we would like to address the issues that arise with the use of liposomes as carriers in pharmaceutical delivery systems. The problem is that they are not effective in helping treat the disease that is being treated. rapid evacuation In addition to being ineffective against resistance to release and ineffective in aiding sustained release, Numerous other explanations for the ineffectiveness to Qi treatment are possible. For example, liposo internalize into target cells or phagocytic cells (e.g. reticuloendothelial cells) , these are trapped medicines that are much more ineffective against disease of the cells involved than the RES. The drug is given and the system is rapidly excreted. After phagocytosis, liposomes are transferred to phagocytic cells. packed into liposomes. Very often contained within liposomes Degradative enzymes either degrade the captured compound or release the compound at their active site. make the compound inert by cleaving it or changing its structure.

Additionally, liposomes are difficult to manufacture due to low entrapment efficiency of active compounds into the vesicles. It is not possible to administer a dose that is effective for the patient.

Liposomes have been used by researchers as a model membrane system and as a transport mechanism for capture and transport. have been used as "target cells" in assays. However, this When used in immunoassays such as internalization by generation of immune complexes containing immunoclasses (e.g. I (IM) and I (IG molecules)). Since the release of liposomes is measured as a factor for complement activation, it is cultured in serum. It is important that the liposome membrane does not leak during this process.

3.Disclosure of the invention The present invention hereinafter relates to stable plurilamellar vesicles (stable plurilamellar vesicles). A new product called ellar vesicles (SPL Vs)) A substantially improved type of lipid vesicles is provided. There is a particular thing. multi lamellar vesicles (M L Besides being structurally different from VS), 5PLVs also differ from MLVs. is produced by a method that has unique properties compared to MLVs, and such ML It has various different transition points compared to Vs. As a result of these differences, 5 PLVs are represented by many conventional lipid vesicles. It solves the problem of

A heterogeneous mixture of lipid vesicles is generated when SPL'Vs are synthesized.

Evidence suggests that the lipids in the 5PLVs are formed into novel supramolecular structures. Which indicates that. Many lipid vesicles have numerous bilayers, sometimes Therefore, it has as many as 100 layers. This high degree of stratification is explained by Although theoretical, it may contribute to a number of surprising properties possessed by 5PLVS. It is possible to

The properties of 5PLVs include the following. (1) Untreatable by other methods 2. The ability of 5PLVs to treat certain diseases while stored in buffer. (3) great ability of 5PLVs to withstand harsh environments; 4) High substance trapping effect, 5) Long-term adhesion to tissues and cells. , (6) ability to sustain the release of entrapped substances within body fluids, (7) through the cytosol of target cells. (8) improved co-operation in manufacturing; (9) release of the compound in its biologically active form in vivo.

Due to the unique properties of S P L V S, they are resistant to evacuation. Moreover, since it has a sustained release property, it is particularly useful as a drug for administration in vivo.

Describes methods for administering bioactive compounds in vivo and treating diseases, e.g. infections has been done.

4. Brief explanation of the drawing Figure 1 shows the film stability between MLVs and SPL Vs treated at various concentrations. FIG.

Figure 2 shows both the lipid layer and aqueous phase of 5PLVs in mouse eyelid tissue. Groups showing retention and sustained release of gentamicin from SPL Vs in vivo It's rough.

Figure 3 shows S P compared with the electron spin resonance absorption spectrum of M L V S. FIG. 3 is a diagram showing an electron spin resonance absorption spectrum of LVs.

Figure 4 shows reduced doxil spin probes in 5PLVS and MLVs. 1 is a graph showing differences in the ability of ascorbate to

Figure 5 shows P. pursellacanis ( Canis abortus ) based on streptomycin-captured SPLV in mice. FIG.

Figure 6 shows a strain based on pulsella varnish that can be recovered from the organs of infected mice. Pulsella varnish sensitivity in mice using SPL that captures leptomycin 1 is a graph showing the effect of two-stage treatment for infectious diseases.

Figure 7 shows the results in guinea pigs using 5PLV that captured streptomycin. 2 is a graph showing the effect of two-stage treatment of Pulsella abortus (bovine abortion fungus).

5. Detailed description of the invention S P L V S produces a product that is different from any other liposomes mentioned above. Manufactured by a method.

5PLVs are lipid vesicles with several to 100 or more lipid bilayers. be. The membrane bilayer consists of a bilayer of bipolar lipids and nonpolar hydrophobic hydrocarbons. The "tail" points point toward the center of the bilayer, and the polar, hydrophilic "head" points point toward the aqueous phase. It's suitable. The aqueous phase is occluded by a double layer, part of which forms the lumen of the vesicle. and some are located between adjacent layers. The lipid bilayer contains various proteins. glycoproteins, glycolipids, mucopolysaccharides and other hydrophobic and/or can form complexes with ambipolar substances.

5PLVs are manufactured as follows. i.e. bipolar lipids or lipids into an organic solvent. Many organic solvents are suitable, but diethyl ether ethers, fluorinated hydrocarbons and mixtures of fluorinated hydrocarbons and ethers are preferred. Delicious. The active ingredient to be entrapped and the aqueous phase are added to this solution. This two The layer mixture is prepared by emulsifying the aqueous material in the solvent while evaporating the solvent. Conversion to PLVS continues. Evaporation is any evaporation technique during or after sonication, For example, evaporation by passing a stream of inert gas through the mixture, evaporation by heating or is achieved by vacuum evaporation. The volume of solvent used is such that the aqueous substance is in the mixture There should be enough aqueous volume to completely emulsify. Actually, 1 volume of water About 3 or more volumes of solvent are used for the liquid phase. In fact, the ratio of solvent to aqueous phase The ratio can vary up to 100 volumes or more to 1 volume of the most aqueous phase. Wear. The amount of lipid is as follows: ■Mulsion droplet [approximately 40 mg of lipid per ml of aqueous phase] The amount must be sufficient to exceed the amount sufficient to The upper limit is the cost-effective Although limited to practicality only, SPL Vs contains 15 mg fat per mg of aqueous phase. It can be made with quality.

This method uses lipid vesicles that have a macromolecular structure, unlike conventional liposomes. generate. According to the invention, the whole method is independent of the phase transition temperature of the lipids used. It is carried out at a temperature range of 4 to 60°C. This latter advantage has the desired properties thermolabile products, such as denatured proteins that are distearoylphosphatidyl It is combined with 5PLVS made from phospholipids such as phosphorus, but its phase transition temperature The problem with conventional liposomes is that they only form at temperatures above 30°F. The no im shall contain 4'4 or more than 20% of the water-soluble substances used - and shall contain usually provides more than 40% of the lipid soluble material used.

Most ambipolar lipids are constituents of 5PLVS. Suitable hydrophilic groups include: Phosphato group, carboxyl group.

Examples include, but are not limited to, sulfato groups and amino groups. suitable Hydrophobic groups include saturated and unsaturated hydrocarbon groups and at least one aromatic group. hydrocarbon groups substituted with groups and/or alicyclic groups, but are not limited to these. It's not something you can do. Preferred ambipolar compounds are phospholipids and similar chemical structures. It is something. Examples of these are lecithin, phosphatidylethanolamine, Lysolecithin, Lysophatidylethanolamine, Phosphatidylserine, Bosu Fathylinositol, sphingomyelin, calciolibin, phosphatidine These include, but are not limited to, acids and cerebrosides. 5PLV Examples of stable lipids that are particularly useful in the production of s are natural lecithins (e.g. egg lecithin or soybean lecithin) and saturated synthetic lecithins (e.g., cimilistyl phosphatide) Fatidylcholine or dipalmitoylphosphatidylcholine or distearo Synthetic lecithins such as ylphosphatidylcholine and unsaturated synthetic lecithins (e.g. dioloyl phosphatidylcholine or silinoyl phosphatidylcholine) Lucolin). This double layer of SPLVs contains cholesterol and cholesterol. Contains steroidal compounds like stanols, cholestanol, cholestane etc.1 qru. Uses compounds with acid/semi-hydrophilic groups (phosphato group, sulfato group, etc.) In this case, the resulting S　P　Vs becomes anionic and has an amino group-like structure. When using compounds with basic groups, cationic liposomes can be obtained. , and when using compounds with polyethyleneoxy groups or glycol groups. natural liposomes can be obtained. The size of the 5 PLVs varies significantly. The range The radius is approximately 500 to 10,000 nm (10 microns), but is typically approximately 1,000 nm. 0-4. OQQnm.

Virtually any bioactive compound can be captured within 5PliVs (Entrapment is defined as entrapment within the aqueous fraction or within the membrane bilayer). like this Examples of compounds include nucleic acids, polynucleotides, antibacterial compounds, and antiviral compounds. antifungal compounds, anthelmintic compounds, antitumor compounds, proteins toxins, enzymes, holins, immune modulators, dyes, radioactive labels , radiopaque compounds, fluorescent compounds, polysaccharides, cell receptor binding molecules, anti-inflammatory agents , anti-glaucoma agents, mydriatic compounds, local anesthetics, etc., but are not limited to these. isn't it.

The following are examples of the proportions used in the synthesis of 5 PLVs.

That is, 5PLVS is 5 μ9 Bl-IT (butylated hydroxytoluene) Add 50 micromoles of phospholipid to 5 ml of diethyl ether containing Then add 0.3 ml of the aqueous phase containing the active substance to be contained. Generated by The resultant solution containing the substance to be captured and the capture lipid are The mixture is sonicated while passing an inert gas, which removes most of the solvent. removed.

This embodiment is particularly safe, in part due to the incorporation of Bl-IT into vesicles. Generate 5 fixed PLVs.

with the aqueous phase sonicated and added in a mixture of chloroform and ether. lipolysis by evaporating a solution of cholesterol and phosphatidylcholine into A method for producing somosomal antibodies is described, but the relative proportions of lipid to aqueous phase are not specified. Lenk et al.'s 1983 Eur.

See J, Biochem, 121: 475-482.

5.2. Features of 5PLVS 5PLVs have single or several lamellae, such as bosomes (e.g. 5UVs and REVS) are clearly distinguished in their properties. cryo-destruction electron microscope , the 5PLV agent is substantially free of 5UVs and REVs, i.e. vesicles. It is shown that less than 20% of the cells are unilamellar. However, these Although many of the physical properties of Can not. Thus, the following detailed comparison focuses on differentiating 5PLVS from MLVS. It becomes a point.

5.2.1. Stability of 5PLVs during storage The stability of lipid vesicles varies over long periods of time. The ability of vesicles to chelate their engulfed space from the external environment is important. do. Useful lipid vesicles should preferably be stable during storage and handling. stomach. However, some applications require that the vesicles gradually leak their contents during use. It is desirable that Other uses include post-administration until the vesicles reach the desired point of action. remain completely. 5PLVS exhibit these desirable characteristics, whereas MLVs do not. I don't.

There are two reasons why vesicles leak. One is lipid autoxidation, This causes the hydrocarbon chains to generate peroxides that destabilize the bilayer. This oxidation Adding antioxidants such as butylated hydroxytoluene (BHT) to vesicles It can be reduced rapidly by

Vesicles also have membranes that form pores, while the lipids remain intact. It disrupts the bilayer structure of lipids, resulting in leakage.

Lipid vesicle agents are white in color when first manufactured. The drug becomes colored (brown) due to autoxidation. do. MLVS for 5 PLVs made using the same lipid and aqueous components The comparison shows that MLVs become colored in 1 to 2 weeks, whereas 5PLVs become colored in at least 1 to 2 weeks. It remains white for two months. This indicates lipid degradation in MLVS, but 5P Degradation of lipids in LVs is not supported by thin layer chromatography of lipid components. It is. These vesicles are produced by adding BHT and other components. In this case, MLVs show slight coloration within 1 month, but 5PLVs remain white. Yes, and stable for at least 6 months.

Contains antibiotics when placed at neutral pH in a buffer containing isotonic saline The 5 PLVs are stable for more than 4 months as shown in Table 1. these days None of the antibiotics originally built into the 5PLVs leaked during the experiment. It shows that it did not.

Other evidence is that over 6 months, 5PLVβ has a very small amount of calcium ions. This shows that it can chelate with internalized drugs from offspring. Arsenazo■( arsenazoll [> changes from red to blue in the presence of extremely small amounts of divalent cations. It is a pigment that 5PLVS contains this dye and stores calcium chloride. The stability of vesicles can be measured by observing the color change when added to a buffer solution. can be done.

The color should be at least 6.5 without showing either dye leakage or ion intrusion. There was no detectable contribution from its initial color.

Table 1 Initial capture rate b Captured drug organism Capture agent (%) Leakage to supernatant Scientific availability (%) Streptomyces 34.1 0’Y7 sulfate Spectinomycin 37.2 0 84 Chloramphenicol 35.2 0 89 oxytetra 18.8 0 91 cycline Erythromycin 0.4 0 97 Sulfamelazine 6.3 0 93 a 5PLVs contain 127 μM egg phosph 1 tidylcollidine (EPC) and 25 manufactured using μM of the drug.

After storage for 4.5 months at 4°C, the terminal 5PLVs were removed from the storage buffer by centrifugal force. Separated. Dilutions and supernatants for 5 PLVS were compared to standard dilutions of antibiotics. was applied to the bacterial flora to assay the biological activity.

O indicates below the detectable level.

7 These experiments demonstrate that 5PLVs are sufficiently stable with respect to storage and handling issues. This shows that. Creating stable MLVs over such lengths Although it is possible, it must be made from synthetic lipids such as DSPC, and it is inevitable that It becomes expensive.

5.2.2. Stability of 5PLVS in other environments Including different vesicles is a way to explore different molecular compositions. The structure of the membrane Depending on how they are constructed, different vesicles respond differently to these drugs. .

In the following experiments, a radioactive tracer molecule (3H inulin) was present in the occluded aqueous fraction. ) are prepared.

Lipid vesicles form when inulin and polysaccharides partition into the aqueous phase and are radiolabeled. used to trace the aqueous content of exposed to a prescribed drug at appropriate intervals Afterwards, the vesicles are separated from the medium by centrifugal force, and the vesicles are released into the medium. The equivalent amount of radiation dispersed is measured. These results are reported in Table 2. , the numerical value is the amount of radioactive material in the surrounding medium relative to the amount initially incorporated in the vesicles. It is expressed as a leakage percentage, which means the quality ratio.

5PLVS is more stable than MLVs in hydrochloric acid.

Table 2 shows that both MLVs and 5PLVs when made from egg lecithin. The degree of destabilization is shown when exposed to 0.125N hydrochloric acid for 1 hour. however, It is noteworthy that 5PILVs are significantly less sensitive to acids than MLVs. Ru. Perhaps this different response reflects inherent differences in how lipids interact in their environments. It is a reflection.

Salt Acid 90.5 55.2 Urine 21.7 44.8 Guanidine 0.5M 5.7 7.4 1.0M 8.3 10.1 ammonium acetate 015M, 27,0 67.0 1.0M 25.9 54.7-63.1 Serum 76.2 57.8 a) The culture time was 2 to 4 hours except that the culture time in H(l) was 1 hour.

5PLVS respond differently than MLVs when exposed to urea (Fig. 1 and and Table 2). Urea has a chaotropic effect. ) (splits the water structure) and molecules with strong dipole moment rain sounds It is. SP　l-V　S is osmotic like sodium chloride at the same 1lii 1 degree. Figure 1. ). MLVs leak much less in urea than in naritum chloride.

Although the explanation for this different behavior is theoretical, the molecule is similar to urea. This response is more likely to be due to a dipole effect than to a dipole effect, as the guanidine makes the 5PLVs stable. This seems to be based on chaotropy (Table 2). Guanidine is also a strong force. Although it is otropic, it does not have a strong dipole moment.

5PLVs are also sensitive to ammonium acetate, whereas MLVs is not sensitive (Table 2). However, ammonium ion (ammonium chloride) (in sodium acetate) or acetate (in sodium acetate) are both particularly effective at deassembling 5PLVs. It has no effect on setting the value. Therefore, it is not the ions themselves that are the cause of induced leakage. It can be seen that this is the polarity of ammonium acetate.

Initially, 5PLVs compared to MLVs when cultured in body fluids such as serum or blood. Such a result is surprising since s is much more stable. However However, we would like to propose a theoretical explanation for these results, and of course there are other explanations as well. possible). The stability of 5PLVs is due to the cloud of water molecules oriented by the polar groups of membrane lipids. or based on the unique structure of its membrane bilayer so that it is hydrated by a hydration shell. If this is the case, then there are Any drug will promote a change to the structural membrane state and thus leak.

Regardless of the correctness of the theoretical explanation for the stabilization of 5PLVs in urea, The above results show the characteristic difference between Tachibana-zukuri of MLVS and 5PLVs. . This difference serves a very useful purpose in applications. As shown below, 5PLVs are , becomes gradually leaky when applied to the eye.

Presumably, this desirable sustained release property is due to similar de-electrification of 5PLVS when exposed to tear fluid. This is due to

5PLVS is more stable than MLVs in serum. A large number of lipid vesicles Applications include administering these intraperitoneally, such as for the treatment of pulcellosis. ing. To be effective, vesicles must have sufficient time to reach their predetermined mark. I have to survive. 5PLVs and MLVs made from egg lecithin Both were exposed to fetal bovine serum containing active complement (Table 2). 37 After 48 hours of exposure at °C, SPL Vs was much cheaper than MIVs. It was fixed.

5.2.3. Characteristics of 5PLVs administered in vivo 5PLVS exhibits a number of properties that make it particularly suitable as a carrier for administration systems.

(Δ) SPLVs are resistant to evacuation. 5PLVS is administered to the organism If the lipid component and the scavenged active component are retained within the tissues and by the cells to which they are administered, It will be done.

(B) SPLVs can be engineered to provide sustained release. The stability of 5PLVs is S　P　l-V　S is extremely stable during storage and is not stable in the presence of body fluids. However, when administered in vivo, gradual leakage of the active ingredient may result in gradual release of the active ingredient. Gives release.

(C) effective retention of the active ingredient due to its high degree of entrapment and stability when administered; The dose is released.

(D) In the production of SPLVS, the stability of vesicles is affected by the presence of expensive stabilizers in the bilayer. It is inexpensive because it can be done without causing any damage.

The next experiment was to investigate these properties of 5PLVS when administered to the eyes of test animals. It shows. SPL VS used in these experiments is lipid The bilayer and active ingredients are used to trace these ingredients into the ocular tissue during the period. The results were as described above except that each was indicated by radiation.

5PLVs contain 1100rn of phosphatidylcholine (EPC) and 100m Produced using gentamicin sulfate of q. The lipid component is a trace amount of 1 Jf, - By bonding phosphatidylethanolamine (mer-PE) to the bilayer The active ingredient in the aqueous layer is methyl-gentamicin sulfate (I-G Radiological indexing was achieved by the addition of S). These 5 PLVs are unbound or unbound. It was washed repeatedly with buffer solution to effectively remove stored substances.

A portion of the 5PLVs agent is removed and extracted to remove the organic phase from the aqueous phase. It was done. The radioactivity of each phase was bound to 5 PLVs (cpm (cpm) in the lipid phase). 7 minutes): water rr To determine the initial ratio of l-PE:mel-G5, which is cpm during sexual phase. was measured.

The extraction was done as usual. 0.8ml 0.4M NaCl (aqueous), 1 ml chloroform and 2 ml methanol were mixed to form the lipid phase . 4 μm of radiolabeled 5PLVS was then added and mixed. child Since the 5 PLVS of 5PLVS is dissolved in the organic and aqueous phases, the initially cloudy The compound became transparent. These phases were mixed with 1 ml of 0.4 M Nac! (Aqueous) OJ: Add 1 ml of chloroform and mix, then centrifuge at 2.800xg for 5 minutes. It was isolated by heart separation. A portion (1 ml) of each phase was removed and radioactive ( The initial ratio of (1-PE:1-GS) measured at Cpm was 1.55:1. ).

Fifteen adult female Swiss Wavestar mice were protected from eye contact. In order to stop him, he was anesthetized and confined. Radiolabeled 5PLVS in suspension Equal volume sections (2 μm) were used locally for hibernation. Next, we added the three animal groups. The animals were sacrificed at 1, 2, 3, 18 and 24 hours, respectively. 9 animals Female Swiss Webster mice (control) were treated with radiolabeled gentamicin sulfate. Similarly, except that an equal portion (2 μm) of an aqueous solution of salt was used locally on each uR. Processed.

Immediately after sacrifice, the animal's eyelids are removed, minced, and the aqueous phase is separated from the lipid components. (using the method described above). The radioactivity of the phase is measured and is also radioactive. The total amount of counts was collected. The radiation measured in the lipid phase is 5P due to ocular tissue. It is an indication of the amount of LVs retained, and the radiation measured in the aqueous phase is due to the S It is an indication of the amount of P L Vs retained, and the radiation measured in the aqueous phase is This is an indication of the amount of gentamicin held. Figure 2 shows the cpm used for the eye. (expressed as a percentage of the first value of C) indicates the amount of each component in the eyelid tissue. It is a graph.

Figure 2 shows the amount of 5PLVS lipid components retained in the eyelid tissue over 24 hours and ( Expressed as the percentage of gentamicin retained in the eyelid tissue during the period ) clearly demonstrate sustained release of gentamicin from 5PLVs over 24 hours. Ru. Figure 2 also shows uncontained gentamicin (topically administered aqueous gentamicin). The results show that the spores (Syn) rapidly disappear from the eyelid tissue. For example, a gene in solution Tamycin (control) disappeared from the eyelid tissue within 4 hours (5% less than gentamicin remaining). On the other hand, it contains more than 50% gentamicin. 5 PLVs are retained by the eyelid tissue during the last 4 hours, and in fact between 24 FR Later, 5PLVS containing more than 25% gentamicin was retained in the eyelid tissue. It had been. This means that 5PLVs containing about 85% of gentamicin 95% of the unincorporated gentamicin sulfate disappeared.

Table 3 shows the ratio of 5PLVS lipid phase and aqueous phase retained in the eyelid tissue at each point. This is a comparison. The increase in this ratio was due to gentamicin from 5 PLVs. This shows the release of

SP, LV containing gentamicin sulfate retained by eyelid tissue The biological activity of S was also evaluated. Gentamicin Sulfate is Gentamicin Sulfate Eyelid extract prepared 3 hours after 5PLVs containing salts were applied to the eye was recovered from the eyelid tissue by removing a portion from the aqueous phase. This aqueous phase The mixture was serially diluted and a 2 μm portion was placed on top of the Staphylococcus aureus flora on the agar layer. After culturing for 24 hours, the inhibition zone was measured. Gentamicin sulfate built-in 5PLV and Gentamicin sulfate recovered from eyelid tissue extracts of animals treated with It retained all its biological activity.

Corner 'N3-1- Sustained release of 5PLVS loaded with gentamicin after topical application to mouse eyes. held in the eyelids 5PLVS recovered from eyelids SPLVS component lipid: water phase ratio 0’too% 1.55 1br 100% 2.1 3hr 100% 2.82 18hr 94% 6,89 24hr 85.1% 7,17 5.2.4.　Electron spin resonance 5Although PLVS and MLVS appear identically under an electron microscope, ESR (Electronic Electron Microscope) spin-resonance) spectra reveal differences in their spramolecular structure do. 5PLVs are characterized by their increasing molecular order, increasing molecular behavior and of its molecular structure, as evidenced by its large permeability to corbate. It can be distinguished from MLVs in this respect. These differences in molecular structure are due to their different biology. It contributes to physical activity.

In electron spin resonance spectroscopy, 5-doxyl stearate (5DS) Such spin probes are incorporated into lipid bilayers. The unpaired electron of doxyl group is , the sample absorbs microwave energy when inserted into a magnetic field.

The absorption spectrum is determined by three experimental parameters: S, order parameter AO, and ultrafine The fine coupling constant and rotational correlation time Tau are determined. A typical reading is 3, where A is the signal of 5PLVS, B is the signal of MLVs, and both are labeled with 5-doxyl stearate. Spectra performed at room temperature The scanning range was 100 Gauss. Depends on both 2T+ and 2T11 The order parameter is the ESR signal from the case of completely uniform orientation of the probe. measure the error of The uniformly oriented sample S-1°O○ is different from the rantom sample S-1°O○. It is 0. Hyperfine coupling that can be calculated from 2T+ and 2Tt+hsr The constant AO of the spin probe in the film is considered to be independent of local polarity. Represents location. The rotational correlation time (depending on wo, ho, h −1) is When the previous spatial orientation is required for the molecule to "forget" J It can be considered as an interval. Full 8 PL Vs with 5-DS as spin probe A typical ESR determination of the difference between .

Although in both cases the spin probes are reported from the same depth in the bilayer, , 5PLVs have much larger molecular order and molecular behavior than MLVS. ing.

Another explanation for the difference between 5PLVS and MLVs is that the doxil spin probe is reduced This is due to the ability of ascorbate to

Ascorbate is probably a hydroxyl that does not absorb microwave energy in a magnetic field. It is often known to reduce chitoxyl moieties to amines. in aqueous solution In this case, reduction occurs rapidly with concomitant loss of ESR signal. spin probe When in a protective environment such as a lipid bilayer, hydrophilic ascorbates can It gradually decreases or is not reduced at all. Therefore, nitroxide The rate of reduction can be used to study the rate of ascorbate penetration into the lipid bilayer. Ru. Figure 3 shows 5PLVS and M L suspended in ascorbate solution. The relationship between V S residual spin percentage and time is shown. Ascorbate in 90 minutes reduces 25% of probes placed in MLVs, but in 5PLVs Reduces 60% of probes inserted. 5PLVs is more than MLVS Allows dramatically greater ascorbate penetration.

S P I V S 2,65xiO’S ec O,61414,9M L Vs 3,65x10' S ec O, 56514, 95, 2, 5, 5P Trapping of active substances by LVs shows no advantage of 5PIVS over conventional MLVs. As a basic example, 5PLVs capture a large percentage of the active substances used. and thereby retain the substance (see Table 5).

5.2.6', Interaction of 5PLVs with cells Still other advantages of 5PLVs are: 5PLVS indicates that a relatively large part of the substance contained inside the vesicle is a phagocytic vesicle. Cells that are more dispersed throughout the cytoplasm of the cell than are confined to cruciforms It is to interact with φ. When 5PLVs mix with cells, the three are associated do. Due to association, SPL Vs, unlike MLVs, do not interact with cells in vitro. Since all cells are initially supplemented with 5PLVs, at least Contains some (4). This substance appears to be distributed in each cell, but it is It is not limited to only files. This is due to the ferrite in the aqueous phase of the 5PLVs agent. This can be achieved by incorporating ferritin. culture Ultrastructural analysis shows that ferritin is distributed throughout the cytosol after association with cells within the Moreover, it shows that it is not bounded by the intracellular membrane. This phenomenon is M Although it can also occur with LVs, a large amount of material is transferred by 5PLVs. .

Week K 5 R surprise- MLVs and 5PLVS,! :(7) Comparative captured effective substances (%) Built-in contents M L Vs S P L Vs Inulin aqueous space forming agent 2-6% 20-30% bovine serum albumin 15% 20-50% streptomycin 1 2-15% 20-40% polyvinylpyrrolidone (Aqueous space) 5% 23-35% 5.2.7. 5PLVS (7) Buoyant density Furthermore, 5PLVS is more than MLVs. It has a low buoyant density. This is measured by banding within the Ficoll gradient (See Table 6).

5.2.8. Volume of 5 PLVs Furthermore, 1.000 to 100. OOOxg+7) Fields collected into pellets by centrifugal force In this case, 5PLVs have substantially larger pellets than MLVs with the same phospholipid concentration. give. At a centrifugal force of 16,0 ooxa, 5PLVs are about 1/3 larger than MLVs. Forms a fine pellet.

5.2.9. 5PLVs c7) Osmotic characteristics Phospholipid bilayers are water permeable. Thus, MLVs placed in a hypertonic environment lock out water due to osmotic forces. 5PLV S contracts more than MLVS. Additionally, in a buffer that is 20 times higher than the internal salt of 8 degrees. After 16 hours of deflation, the 5PLVs did not contract to the same final volume as the MLVS. (the pellet of SPLVs remained ⅓ larger than the pellet of MLVs).

B+F, indicating that the difference in pellet size is not due to a difference in aqueous internal volume. are doing.

5.3. Applications of 3PLVs 5PLVS is particularly useful in systems where the following factors are important: . namely, stability during storage and in contact with body fluids, relatively high viscosity, and cost. reduction, and release of the scavenged compound in its biologically active form.

1% or more layer in Ficoll 1% or more layer Q 7m 1 1.071 1.0 274 Furthermore, depending on the in vivo dosage form, 5PLVs may be resistant to rapid elimination. (e.g. when sustained release is important) or to RES cells. .

As a result, the SPL Vs of the present invention can be used in a wide range of systems. this In order to increase the medical effectiveness of treatment and to increase the risk of infection, In order to introduce genetic information into cells within the body, we conduct gene exchange for the production of vaccines. for introducing deoxyribonucleic acid segments into cells or It can be used as a diagnostic agent for clinical trials by releasing data molecules. This 5P LVs also incorporate compounds that provide sustained release of cosmetics, fungicides, plant growth agents, etc. Can be used for.

Although described with respect to the use of 5PLVs, the method is applicable to 5PLVS or other liposols. It is also possible to use lipid vesicles with functionality similar to that of 5PLVS or 5PLVS. make it possible.

5.3.1. Administration of biologically active compounds to cells in vitro (e.g. animal cells, Administration of compounds to plant cells, protozoa, etc.) is effective when administering compounds to cells in culture. The addition of 5PLVs containing compounds is usually required.

However, 5PLVS is also used in animals (including humans), plants and protozoa. It can be used to administer the compound within production. Depending on the purpose of administration, the 5P LVS can be administered by a number of routes. i.e. human and animal odor This may be by injection (e.g. intravenously, intraperitoneally, intramuscularly).

subcutaneous, intraauricular, intramammary, intraurethral, etc.), local use (e.g., pain areas), and Epithelium or mucous membranes (e.g., clothing epithelium, mucous membranes, rectal or vaginal mucous membranes, airway membranes, nasopharyngeal mucous membranes) membranes, intestinal mucosa, etc.), but is not limited to this, and can also be used in plants and In protozoa, it is distributed directly to the organism, into its habitat, and in its surroundings. It can also be done by adding it to water or the environment, but it is not limited to these. It's not.

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 can be done locally (if the infection is external); This is easily done. Administration to the circulatory system (and therefore to the reticulum cells) can be administered intravenously, It can be very easily administered by intraperitoneal, intramuscular or subcutaneous injection.

5PLVs allow for sustained release of the compound, so if it is not toxic to organisms, The dosage can be used in one or more administrations to the organism.

Although this section provides a comprehensive description of how 5 PLVs can be used, It does not limit the scope.

5.3.2. Treatment of diseases Numerous pathological conditions occurring in humans, animals and plants can be solved with the appropriate compounds. It can be effectively treated by incorporating it into 5PLVs. These pathological conditions and Examples include infections (intracellular or extracellular), cysts, tumors and tumor cells, allergies, etc. However, it is not limited to these.

There are various ways in which 5PLVS can be used to treat such diseases. Living SPL VS is internalized by macrophages when administered into the body We outline some particularly useful plans featuring facts.

In one plan, 5PLVs could be used to deliver therapeutic agents to the site of intracellular infection. used. One disease is reticuloendothelial.

Examples include infection of pulcellosis cells. These intracellular infections are numerous. It is difficult to treat for several reasons. (1) Infectious microorganisms exist within the cells of the reticuloendothelial system. Therapeutic drugs that cannot pass through the cell membrane at therapeutically sufficient degrees are stopped from circulation, and this Therefore, there is a high resistance to treatment. (2) The toxic level of therapeutic drug administration is as follows. often necessary to prevent serious infections. (3) Does the treatment leave any residue after treatment? Infections that occur in the first place are completely infectious because they can reinfect the host organism or transmit it to other hosts. must be effective.

According to one mode of the invention, 5PLV containing a suitable biologically active compound S is administered (preferably intraperitoneally or intravenously) to a host organism or potential host organism. administered to things. (For example, in a herd, uninfected animals should be treated in the same way as infected animals.) can. ) Phagocytic cells internalize SP'LVs, making them effective against microbial infection. Administration of 5PLV, which contains biologically active substances, allows the biological activity to be delivered to the site of infection. Aiming to directly reach sexual compounds. Thus, the method of the present invention can be applied to various microorganisms, bacterium, Excludes infectious diseases caused by bacteria, parasites, mycobacteria, mycoplasma, viruses, etc. These microorganisms include Brasella s, pp, Mycobacterium spp. Mus spp, Salmonella St)+1, Listeria 5t) I), Franticella spp, Histoplasma spp, Corynebacterium spp, Coccidio des Spp and lymphocytic choriomeningitidis, but are not limited to these. do not have.

The therapeutic agent selected depends on the microorganism causing the infection. For example, bacterial infections can be removed by incorporating antibiotics. The antibiotic is present in the aqueous liquid of 5PLVS. and/or inserted within the vesicle bilayer. as a suitable antibiotic are penicillin, ampicillin, and hetacillin.

Carbencillin, Tetracycline, Tetracycline Hydrochloride, Oxytetrasa Iclin hydrochloride, chlortetracycline hydrochloride, 7-chloro-6-dimethyltetra tracycline, doxycycline hydrate, methacycline hydrochloride, monocycline Phosphorus hydrochloride, lolitetrali'icrin, dihydrostreptomycin, strep tomycin, gentamicin.

Kanamycin, neomycin, eris[1mycin, caldζmycin, ole Omycin, doloreomycin, Tsukibun IJ mixin B colistin, cephalothin cephalolidine, cephaloglycin dihydrate, and cephalexin - There is water salt.

We demonstrate the effectiveness of such treatment in treating pulcellosis (see example below). (see). The method of the invention increases the effectiveness and duration of action. This system is based on MLVs not responding to known treatments such as antibiotics (>F3 therapy for 1 infection) It is effective. Any small amount of residual infection will be widespread and the infection cycle will begin again. Therefore, no successful >fi therapy could be expected. we are lymph A successful case of choriomyelitis virus infection is shown.

Of course, the present invention is not limited to the treatment of intracellular infections. This 5 PLVs target various sites of infection, whether intracellular or extracellular. Example G f1 In another embodiment of the invention, the active agent is directed to the site of systematic extracellular infection. Macrophages are used to bring about According to this plan, 5PLVs can be administered in vivo by administering 5PLVS (preferably intraperitoneally or intravenously). 5PLVsL are used to administer therapeutic substances to infected macrophages.

This macrophage associates with 5PLVS and is then loaded with therapeutic substances. . Generally, the macrophages retain the material for 3-5 days. "Loaded" Once the macrophages reach the site of infection, the pathogen is absorbed by the macrophages. internalized. As a result, pathogens (or therapeutic agents contained within macrophages) It comes into contact with matter and is destroyed. This embodiment of the invention is applicable to Useful in the treatment of staphylococcal inflammation.

If the site of infection or association is external or mobile (, The 5 PLVs can be used locally. Particularly useful applications include the treatment of eye pain. It is. For eye pain, 5PL containing one or more suitable active ingredients Vs is applied locally to the painful eye. Numerous microorganisms are present in the eyes of animals and humans. cause infection. These microorganisms have the following lives, but are not limited to these: It is not determined. Moraxella spp, Clostidium spp, Coryne Bacterium spp, Dibrococcus spp, Erabobacterium spp , Haemophilis spp, Klebsiella spp, Restosvira spp, Miko Bacterium spp, Niceceria spp.

Propionibacterium spp, Proteus spp, Pseudomonas spp , Cestia 5 lit), ■Schelicia spp, Staphylocaccus spp , Streptococcus spp, and Mycoplasma Spp and Rickettsia Bacteria-like microorganisms including Spp. These infections are used to treat residual infections after treatment. It is difficult to remove by conventional methods because the disease is reinfected through lachrymal secretion.

We demonstrated the effectiveness of 5PLVS in curing clothing infections caused by Moraxella bovis. Indicates use. (See examples below) SPLVs are resistant to excretion and capable of sustained release of their contents. Therefore, 5PLVs may also require extended contact with active treatment substances. It is also useful in dealing with some problems. For example, glaucoma is a progressive loss of peripheral vision. It is a disease characterized by a gradual rise in intraocular pressure that causes Loss of visual field and eventual blindness. In principle, drugs used to treat glaucoma It is commonly used as eye drops. However, sometimes rapid removal of the drug from the orbit Treatment requires instillation of medication every 15 minutes. Problems like glaucoma can be solved with this invention. If treated, pilocarpine fluoropyril lucorrin, acetazolamide, etozolamide, dichlorophenamide, cal Bacol, Demecarium Bromide, Ovir Phosphofluoridate, Iodide Treatments such as cothiopret, physostigmine, or neostigmine Substances can be incorporated into SPLVS given to diseased eyes It is.

Other agents that can be incorporated into SPLVs and used locally include but are not limited to: Although it does not necessarily cause pupil dilation (epinephrine, phenylepinephrine, Roxiamphetamine, cyclobentrate, tropicamide, encatropi local anesthesia, antiviral agents (idoxuridine, adenine arabinosin), local anesthesia, antifungal agents (amphoteracin B, natamycin, pimaricin, flu Cytosine, nyscutin, thimerosal, sulfamerazine, thiobenlysole 1-lunaftate, glycioflupine, etc.), sulfonamides with anthelmintic properties , pyrimethamine.

talindamycin) and anti-inflammatory agents (corticosteroids such as ACTH) de, hydrocortiscin, prednisone, metricin, betamethacin, dexa (Methazone, fluorometalone, triancinalone, etc.)

The following examples are given for illustrative purposes, but are not intended to limit the scope of the invention. isn't it.

6. Example aM of SPLVs As explained in Section 5.1, the basic method for preparing spl-vs is dissolving a substance or a mixture of lipids in an organic solvent, the substance being encapsulated with an aqueous phase and sonicating the mixture, wherein the organic solvent is It is removed by any evaporation method during or after processing.

The SPI Vs used in all embodiments of the invention is as described in the next section. It is prepared as follows. (However, any method of producing SPLVS can be used.) It is Noh. 〉6,1. SPLVs containing antibiotics A 5 ml diethyl ether solution of ioomq lecithin was prepared. The mixture is round placed in a bottom flask. Next, 5mM HEPES (4-[2- Hydroxyethylcopiperazino 2-■ Tan Sulfone 110.0725M Na Cl　100mg of streptomycin sulfate in 10.0725M KCl The solution (0.3 ml) contained in H7.4 is a glass containing a diethyl ether solution of lipids. drips into the gas container. The mixture is passed through a 10536 type path sonicator Boratory Subrise Company, Incorporated [l-abo ratory S uppl ies C o , .

Inc. ]) for several minutes, and at (frequency 80 kHz: output 80 watts) , simultaneously dried to a sticky paste 1- by passing a gentle vapor of nitrogen through it. Ru.

To what remained of the sticky paste, 10ml of 5mM Hetubes was added. Ta. A preparation of generated SPLV containing streptomycin is suspended in a buffer solution, Mixed in a vortex mixer for several minutes and centrifuged in 12000XO for 10 minutes at 20°C. This allows unencapsulated streptomycin to be released. The resulting cake is It was suspended in 0.5ml of 5mM Hetubus.

The above procedure uses streptomycin, dihydrostreptomycin, sulfate Tamycin, ampicillin, tetracillin hydrochloride, and kanamycin It was done in the same way except that it was replaced by .

6, 2. The method described in Section 6.1 of SPLVS containing other membrane components is The same was done except that any one was added along with egg lecithin. (1) 8 :phosphatidic acid giving a molar ratio of 2 (lecithin; dicetyl phosphate) , (2>8:2 (lecithin; stearylamine)). Min, 7: 2: 1 (lecithin: cholesterol; stearylamine ) collosterol and stearylamine giving a molar ratio of (3) 7:2:1 Phosphatidine giving the molar ratio of cycine:phosphatidic acid:cholesterol) acid and cholesterol.

6, 3. The procedure in Section 6.1 for SPLVs containing pilocarbins A similar procedure was performed except that treptomycin was replaced with pilocarbin.

6, 4. SPLVs prepared with and without BHT The undistilled ether contains anti-acid T) for storage purposes. Section 6.1 The procedure is distilled as a solvent to bind BHT during SPLV preparation. The same procedure was carried out using uncontained ether.

The procedure in Section 6.1 can be used to prepare 5PLVs without binding to BHT. A similar procedure was carried out using distilled ether as the medium.

7. Example: In an example of the number of 5PLV-mediated releases in a glass container, the culture 5PLV-mediated release of antibiotics to microphages in culture vessels was demonstrated. It was.

Peritoneal microphages were isolated from C5ヮBLK adult male mice by peritoneal lavage. Minimum essential medium (M, E) containing 10% heat-inactivated fetal calf serum was obtained. , M) suspended in pH7°2. Cells were 96- Suspended in a 96-well tissue culture vessel. sticky peritoneal micro Add a concentration of 7ml of 1X106CFtJ (colony forming unit) to the culture vessel containing the phages. Canine abortifacients (3, canis) were added. After 12 hours, peritoneal microphone Bacteria not inhaled by the lophages are washed repeatedly with Ivl, E, and Ivl. Removed due to return. After washing the peritoneal microphage incubator, these Divided into 5 groups with 12 replicate cultures per group. Guru Group 1 represents the control standard and has not been subjected to any processing. Group 2 is aqueous Ii i! and acid streptomycin at a concentration of 1 ml/ml. glue P3 receives 5 PLVs placed in buffer. Group 4 is aqueous sulfate strep 5PL in pre-made buffer with tomycin (1111 g/ml) Receive Vs. Group 5 contains streptomycin sulfate (1 m (1/ml) Receive 5 PLVs. After 24 hours, the supernatant was removed by repeated washing. The microphages in the peritoneum are crushed by repeated freezing and thawing.

A series of dilutions of the crushed microphages were placed on Purcella agar. , after 4 days, the dog abort tubes given are determined by the limitations of the dilution method. Table 7 The results shown in 5PLV's built-in streptomycin are effective against sterilization in test tubes. and have a comprehensive impact on the elimination of intracellular P. abortus infection.

The experiment was conducted on bovine abortus bacteria (B, abortus), and was performed on an adult female albino guinea pig. The procedure was the same as described above, except that peritoneal microphages were obtained by peritoneal lavage. Ta. The results are also shown in Table 7.

(Margin below) Table 7 Intracellular view of infected microphages after treatment with streptomycin-containing 5PLVS of Reference standard 2.6± 1.13x103 3.1±0.81x104 in suspension was lost 5PLVs 2.82±0.10x103 2.9±0.17x10’1 free space Trept Mycin 3.11±0,40x103 3.3±0,25x104 Strept Mycin and placed in suspension. 5PLVS 2,76±0,20x103 2.8±0.42x104SPL Built-in Vs Streptomycin 00 a Pre-infected mouse (Cs; 7Blk) peritoneal microphages, etc. Colony forming units (CFU) of canine abortifacients isolated from numbers (mean of 12 replicates ± standard deviation) b Isolated from equal numbers of pre-infected guinea pig peritoneal microphages Colony-forming units (CFU) of B. abortus (2 standard deviations above the mean of 12 replicates) C streptomycin 1 mg/ml concentration 8, Example treatment of intracellular infection The following example demonstrates how 5PLVS treats intracellular infections. This data (1) The use of SP LV S encapsulated antibiotics in the treatment of diseases. influence and (2) greater potency obtained with higher dosages of SPLV preparations. represents. Comparison of MLVS and 5PLVS used in the documentation as an excipient It can be stated.

Pulcellosis is a global economic and national health problem.

Pulcella disease is caused by Pulcella Spp. This includes breast milk, including humans. It affects species, domestic animals, and a variety of wild animals. 6 Pulsella Spp pulled in animals These causes Bacillus abortifolia, Bacillus abortifolia, Bacillus malta fever (B.

melitensis), Pulsella neotomae (B, neotomae) , Pulsella ovis (B, ovis), Pig Abortion Tube (B, 5uis) be. Both domestic and wild animals are carriers that can spread pulcellosis to other animals and humans. shall be handled as such.

The infected organisms are present in the cells of the reticuloendothelial threads and are highly resistant to the bactericidal action of antibiotics. Because of their vigor, these infections cannot be cleared by antibiotics. Must The amount of antibiotic required and the length of treatment may be toxic to the animal or present in the animal's cells. Either there are unacceptably high concentrations of antibiotics; Where does this disease occur? To make matters even more difficult, any remaining infection can easily spread and reinitiate circulation. The treatment must be completely effective in order to begin repeating. Let's go The economic impact of this disease is that millions of dollars worth of cattle are lost each year due to spontaneous abortions. is shown by. The only possible way to deal with such outbreaks is to isolate At that point, infected animals are slaughtered.

Conjugating the antibiotic into 5PLVS is then ingested intraperitoneally into the infected animal. This embodiment consists of dosing an encapsulated active agent to an animal.

8.1. Single treatment of canine abortus infection using antibiotics contained in 5PLV effect Eighty adult male Swiss mice were infected with (1,P,) Canis abortus ΔTCC in the peritoneal cavity. 8 with 10 mice each infected with 23365 (1X 107 CFU) divided into groups. Seven days after inoculation with Canine Abortus, each group received the following treatments: Ta. Group 1 represented the control standard, no treatment received g0 Group 2 received the buffer It received 5 PLVs (0°21111 1, P,) placed in the liquid. group 3 is 1 m of the total dosage of aqueous streptomycin sulfate (0.2 ml r, P) (Received 1/K (+ body weight). Group 4 received 0.2 ml [.

received aqueous streptomycin sulfate (5 m9/9 body weight) during the total dosage of P. Ta. Group 5: 0.2ml 1. P.

received aqueous streptomycin sulfate (10 mg/K (] body weight) during the total dosage of . Group 6 is 0.2ml 1. P.

5 containing streptomycin sulfate (1n+a/K (1 body weight)) in the total dosage of I took the PLVS. Group 7 had sulfuric acid stress in a total dosage of 0.2 m11, P. Received 5 PLVs containing ptomycin (5 ma/KG body weight). Group 8 is 0.2ml 1. Streptomycin sulfate <10 mo/K ( + body weight).

Fourteen days after inoculation with Canine Abortus, all animals were sacrificed and the spleens were aseptically removed. was removed. The spleen was homogenized and serially diluted on Pulsella agar, and the spleen was removed after treatment. Determine how many survive. The results after an incubation period of 4 days are shown in Table 8.

(Margin below) Table 8 Free or built into 5PLV Streptomycin at various concentrations 5 colony-forming units of Canis abortus per spleen were added to the buffer solution. Untreated 5 PLVs Verification standard 3. '46x10±2,7x10 4.1x106±0.66xlO Go to Streb 1 Mycin concentration Free strep 5 Built-in PLV (W(1/kil1 Body weight) Mycin Streptomycin 11.5±0.45 xlo 6 1. 01±0.25X10352.12± 1.71x1051,52±0.131 xlO4109, 66±3.68x104 1,32± 1.00x104a Member Q, intraperitoneal injection of 2 ml (sterile saline), the surviving canine abortus bacteria are on the tiles [1 Determined as the number of CFU separations per m, averaged over 10 animals per experiment. It is expressed as the upper standard deviation. (Mie experiment) 8.2. Multiple treatment using antibiotics built into 5PLV for canine abortus infection effect of Eighty adult male Swiss mice were infected with Canis abortus ATCC 23365 (IX107). CFU, 1. P,) into eight groups of 10 mice each. divided. On days 7 and 10 after inoculation with Canis abortus, each group received the following treatments: Ta. Group 1 indicates verification @ standard and no processing is done. Group 2 is in buffer 5 PLVs (0°2ml, 1.P,) were received. Group 3 is 0. Aqueous streptomycin sulfate (1 m (1/kg body weight) in total dosage of 2 m, 11, P, (heavy) received. Group 4 is o, 2+z　r, P.

received aqueous streptomycin sulfate (5 mg/k (1 body weight)) during the total dosage.

Group 5 contained aqueous sulfate streptomycin in the total dosage of 0.2m(]1.P. (10 mo/kg body weight). Group 6 is 0.2m (] 1.P, all Received 5LPVS containing streptomycin sulfate (1 m9/kg body weight) in dosage. I got it. Group 7 is 0.2ml 1. P.

5 LPVs containing streptomycin sulfate (5 mg/k (] body weight) in total dosage received. Group 8 is 0.2ml.

P, 5P containing streptomycin sulfate (10 mg/k (1 body weight) in total dosage) I took LVS. Fourteen days after inoculation with Canis abortus, all animals were sacrificed. The spleen was removed aseptically. The spleens were homogenized and serially plated on Pulsella agar. The number of canine abortus bacteria remaining in the spleen after treatment was determined. 4 days The results after the incubation period are shown in Figure 5.

As shown in Figure 5, various two-stage treatment regimens for canine abortus infection in vivo. Groups receiving aqueous streptomycin 7 and 100 days after inoculation It was observed that the canine abortus bacteria that survived in the spleen was hardly reduced. . 7 and 100 days after vaccination 1Qm (1/k) Only the group receiving stored streptomycin The spleen was cleared of all viable bacteria.

In the experiment described above, 5PLV was treated with streptomycin for three times. Various cells from mice subsequently infected with Canis abortus were sampled as follows.

Thirty adult male Swiss mice were infected with Canis abortus △TC023365 (1 x 10 7 CFU, 1. P,) was inoculated.

Seven days after inoculation, animals were divided into three groups with 10 mice each. . Group 1 represented the control standard and received no treatment. Group 2 is water-based 0.2 ml of each streptomycin sulfate (10 mQ/kg body weight).

P, received during medication (7 and 100 days post-inoculation). Group 3 each has 0. Streptomycin sulfate (10 mq/k (1 body weight) during administration of 2 ml T, P, ) containing 5 PLVs (at 7 and 100 days post-inoculation). Between 14 and 70 days after inoculation with the canine abortus, all animals were sacrificed and infected with the canine abortus. Serially on Pulsera agar to separate the heart, lungs, spleens, liver, kidneys, and gonorrhea. diluted.

Figure 6 shows the results of canine abortus bacteria remaining per organ after a 4-day incubation period. .

Dog after treatment of three claws with streptomycin as depicted in Figure 6 The results of extracting samples of various tissues from mice infected with Abortus bacterium were obtained using the built-in 5PLV. In animals treated with treptomycin, 14 to 75 days after inoculation with M. canis All tissues sampled on the day were completely free of viable canine abortus bacteria. It shows that

Animals that were not treated or received streptomycin contained in the 5PLV. treated with aqueous streptomycin at exactly the same grade and dosing regimen as those treated with In the animals that were exposed to the virus, the number of viable canine abortus bacteria was 14 to 75 after inoculation with canine abortus bacteria. It could be isolated in all tissues sampled on the same day.

8.3. Effect of ita treatment using 5PLVs and Ml-VS for comparison Fifteen adult male Swiss mice were infected with Canis abortus ATCC 23365 (1 x 10 7 CFU, 1. P,) was inoculated.

Seven days after inoculation, animals were divided into three groups of 5 mice each. Group Loop 1 represented the control standard and received no treatment. Group 2 is sulfuric acid MLVS containing leptomycin (10n+a/kg body weight, r, p, > on days 7 and 100). MLVs is 100 m (] egg phosphatidylcoli Contains EPC) and streptomycin sulfate <1 oom (+/ml) It was prepared in a conventional manner using sterile Hetubus 2m+. Lipids and sulfate strepto The ratio of mycin was 28 to 100 maEPc in the final product MLV suspension of 2m+. It was m9 streptomycin. Group 3 has 100n+(l EPC) Modification used with 3 ml of Hetubes containing 100 mO streptomycin sulfate streptomycin sulfate (10 mo/ml) prepared as described in Section 6.1. SP L, Vs containing k (1 body weight, 1.P,) (7 days and 10 days after inoculation) (days later). The ratio of lipids to streptomycin sulfate in 5PIVs was 2 m 100 m (28+ ng sulfuric acid solution for IEPc) in a final product of 5 PLVsl suspension. It was treptomycin. On the 14th day after inoculation with Canine Abortus, all animals received sacrificial milk. The pancreas was aseptically removed, identified, and plated on Pulsella agar. was serially diluted to isolate Sulfuric acid bacteria. Organs after 4 days incubation period The results for the number of viable canine abortus bacteria per individual are shown in Table 9.

Table 9 Streptomycin sulfate for sterilization of in-vivo dog abortion after two treatments The amount of canine abortus bacteria per spleen Verification standard 2.7± 1,0XIO4 M1-Vso 1,8±0.4X104SPLVsc O a. Intraperitoneal injection of 1 Qmg/kg body weight was maintained at 3 eye intervals. What is the verification standard? No treatment was given.

b Surviving canine abortus bacteria was determined as the number of CFU isolates per spleen, 1 Expressed as standard deviation above the mean of 5 animals per group. (2 per experimental animal weight determination) The ratio of C egg phosphatidylcholine to streptomycin sulfate is 100. n+g lipid 28 mg streptomycin sulfate.

8.1 Effect of various antibiotics contained in 5PLV in infection treatment Fifty adult male Swiss mice were infected with canine abortus ATCC 23365 (IX1). inoculated with 07 CFU, [,p,). Seven days after inoculation, experimental animals each received five The mice were divided into 10 groups. Group 1 represents the reference standard , received no treatment. Group 2 contained 5 PLVs (0 , 2ml, 1. P,) were received 7 and 10 days after inoculation. Group 3, 4.5 and 6 are dihydrostreptomycin, zentamycin, kanamycin or streptomycin (1/kg body weight, iP) in an aqueous injection (0 , 2 ml [, p,) were received on the 7th and 10th day after inoculation. (Note: These Each antibiotic has been shown to kill the canine abortus bacteria in vitro. )G Loops 7.8.9 and 10 contain dihydrostreptomycin, centamycin, 5PL containing 1On+o/kg body weight of kanamycin or streptomycin Vs on days 7 and 10 after inoculation. On the 14th day after inoculation with canine abortifacients, all All animals were sacrificed and the pancreas was aseptically removed, homogenized, and pulled. It was serially diluted to isolate B. abortus on Sera agar. 4 days incubation The results of viable canine abortus bacteria per organ after the period are shown in Table 10. Ru.

The results of tests of various antibiotics on mice with canine abortus are shown in Table 10. Antibiotics that are effective in killing P. abortus in a test tube (i.e., in a suspension medium) are When these are encapsulated in 5PLVs, they are effective in killing the canine abortus bacteria in vivo. Shows only the effect. Receive other aqueous antibiotics or 5PLVs in buffer Animals that have not been treated or have received no treatment should be treated with residual canine sulfur in infected spleen tissue. No deletion of acid bacteria occurred.

(Margin below) Table 10 Formation of a colony of Canine abortus bacteria per spleen in vivo after two treatments Encapsulated in unit 5 PLV Aqueous solution antibiotic Untreated 3.93±1.51x106 4.66±0.87x106 Antibiotics 0 Dihydrost Leptomycin 1.13±0.30×1050 Zentamycin 7.06±2.5 3X1050 Kanamycin 2.72±0.91x1050 Streb+ Mycin 1.01±0.17xlO50810m (in 1/! 11 body weight peritoneal cavity Internal injections were kept at 3 eye intervals. The reference standard was not treated in any way.

b The canine abortus bacteria that survive per living organism is the number of CFU isolated per spleen. and is expressed as the standard deviation above the mean of 5 animals per group.

(Double determination per experimental animal) Effect of antibiotics on killing B. canis in suspension medium 8.5. Treatment of dogs infected with Canine Abortion Bacteria Adult female beagles are infected with Canine Abortion Bacterium AT. CC23365 (1x107 CFU) was inoculated intrabuccally and vaginally.

Seven days after vaccination, the dogs were divided into three groups. Group 1 uses the reference standard and received no treatment. Group 2 received 10 mg/kg body weight, respectively. Administration: 5.0 m, 1. It was P. ) aqueous streptomycin sulfate Group 3 received 10+z/ko body weight (7 and 10 days after vaccination). Each administration is 3. Oml, I.

It was P. ) containing streptomycin sulfate (7 days after inoculation). and on the 10th day). Canine membrane wash and heparinized blood samples were collected before and during the study. and collected at regular intervals at termination. These are used to isolate canine abortus bacteria. solvated on Purcella agar medium. The results are shown in Table 11. Serum sample was from a canine abortion. were collected before, during and at the end of the study for determination of serum antibodies against the fungus. child The results are also shown in Table 11. On the 21st day after inoculation with Canine Abortus, all experimental Things were sacrificed. The following tissues were aseptically removed, homogenized, and Serial dilutions were made onto Purcella agar to isolate B. abortus. heparin Turned blood, l! Exudate, lungs, spleen, synovial fluid, uterus, ovaries.

Popliteal lymph nodes, salivary glands, tonsils, septal lymph nodes, mesenteric lymph nodes, bone marrow, superficial cervical lymph nodes lymph nodes and accessory lymph nodes. Dog flow remaining per tissue after 4 day incubation period The results of bacterial production are shown in Table 12.

(ymatao margin) Engraving (no changes to the content) Table 12 Antibiotic treatment was performed twice. Tissue samples from canine abortifacient infection streptomycin strepto 11 Nitrogen cotton swab Q 10 Lung O+ + Zou Q + + Synovial peritoneum N, D, + + Uterus Q Juju Ovary Q + + popliteal fossa Lymph nodes N, D, + 10 Salivary line OOO Tonsil Q + + Mediastinal lymph nodes Q N, D, + mesentery :Lymph nodes N, D, O0 Table 12 (continued) streptomycin strepto 5 PLVs including tissue Mycin reference standard bone marrow 0 + + a. Experimental animals treated on the 7th and 10th day after infection b. Samples taken at necropsy Diluted on Purcella agar. + is equal to or less than 1 CFU If the number is also large, 0 indicates there is no village.

C streptomycin sulfate 1On+o/9 times, ■.

P, includes S, PLVS d 10m(]/kg body weight, 1.P, M acid streptomycin e. The reference standard has not undergone any treatment.

Culture results and serum from canine abortus infection before and after two-step antibiotic treatment The results of the academic examination are shown in Table 11. All experimental animals were tested by negative serum titers. Therefore, the serology was negative prior to exposure to the canine abortus bacterium, and the surface culture surface and Cultures from vaginal swab exploitation cultures are negative. All experimental animals were tested for 7 days and Both blood and vaginal cultures were reported to be culture positive on day 1 and 10. aqueous strike Dogs treated with leptomycin or those that received no treatment had a 21-day oral Blood and vaginal cultures were maintained during the post-treatment period prior to the end of the study. strike Group 3 received liposomes containing leptomycin (the first treatment was Cultures became positive after 1 day and remained negative throughout the post-treatment period. What kind of treatment will I get? Dogs that received aqueous streptomycin or aqueous streptomycin should not have oral infections 21 days after infection. 5PLV, which contains antibiotics, develops to a serum titer that can be detected against bacterial antigens. 7 and 10 days after inoculation with S. There was no development of detectable serum titers.

Canine abortions from infected dogs treated with two-stage antibiotic treatment as shown in Table 12 Results from bacterial isolation indicate that treatment of 5PLVs containing streptomycin in dogs Only all organ samples and all tissues of any viable dog can be analyzed. It has been shown that it is also effective in eliminating miscarriage bacteria.

8.6. Treatment of bovine abortion bacteria in guinea pigs 15 adult female guinea pigs , inoculated with Bovine Abortus ATCC23451 (lx 107 CFU, E, P,). It was.

Seven days after inoculation, the experimental animals were divided into three groups of 5 animals each. Guru Group 1 represented the control standard and received no treatment. Group 2 is 1. P, note Aqueous streptomycin sulfate was added to the water (0.2 ml) at 9 body weight in 10 m (]/ The mice received the test on the 7th day and 10th day after the inoculation with the Abortus bacterium. Group 3 is 1. P, injection (0 , 2 ml) containing 10 mo/ko body weight of aqueous streptomycin sulfate. PLVs were received 7 days and 10 days after inoculation with Bovine Abortus. Bovine abortion inoculation On day 14 post-inoculation, all experimental animals were sacrificed, the spleen removed and aseptically removed. Homogenized and serially diluted onto Purcella agar to isolate B. abortus It will be done. The results of the viable bovine abortus bacteria per spleen after the 4-day incubation period are It is shown in FIG. Only 5PLVs containing streptomycin are guinea pig tiles. It was effective in eliminating bovine febrile bacteria present in the viscera. aqueous streptomycin In laboratory animals that have received treatment or have not received any treatment, viable The Abortus bacterium was identified.

8.7. Treatment of bovine abortion bacteria in cows Nine heavily infected animals were used in this experiment. Bovine flow from breast and vaginal swab exploitation Bacterial isolation became negative after treatment with 5PLVs containing streptomycin6. It remained negative for the week. If infection recurs in these experimental animals, bacterial isolation It was seen only in the breast quadrant that was previously positive.

9 cross-breeders (Helenford breed - Jersey breed - Plancus [3rangu s]), a 22-year-old non-pregnant cow whose abortive bacteria culture was confirmed to be correct. used. At least 4 months before the start of the study, the animals were Experimentally tested in the conjunctiva with IX 107 CFU of Abortus bacterium species 2308, This can lead to miscarriage and/or milk or uterine secretions and/or fetal tissue. The culture of bovine abortion bacteria was positive. Cows are kept in individual isolated barns and divided into three groups. Divided. A procedure consisting of two drug intakes given three doses apart is as follows: be. (1) Three cows are injected intraperitoneally with saline. (2) The three cows are , pre-made with an aqueous antibiotic (10 nz/kg body weight St. Leptomycin). SPLVs in buffered solution is injected intraperitoneally. (3)3 The head of the cow was treated with streptomycin (10 mg/kg body weight) in 5PLVS. Injected intraluminally. The total volume per injection was 100 ml per animal.

Replicating bacterial cultures of the first two months' open secretions and uterine secretions are given once a week. He was forced to secrete his secretions. Next, all cows were overdosed with pentabicibar sodium. They are killed with drugs and the next organs are harvested for reproduction from bacterial cultures. (1) Lymph nodes; Left and right parotid lymph nodes, left and right suprapharyngeal lymph nodes, left and right parotid lymph nodes , left and right parotid lymph nodes.

Left and right prescapular lymph nodes, left and right prefemoral lymph nodes.

Left and right infratid lymph nodes, left and right infratid lymph nodes, left and right iliac lymph nodes, Left and right breast tissue, left and right kidney, 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 organization is It is frozen before lightweighting, exposed to alcohol, and shaped aseptically. weight As soon as a The initial homogenate suspension was homogenized in 110-1 of the sterile saline solution. diluted to An aliquot (20 μm) of each dilution from a series of suspensions was The cells were placed on Lucella agar medium and cultured at 37°C. For each organization, The decision was made.

Plates were read and recorded daily for bacterial growth.

Before 3 days, all emergent colonies were separated, excreted and polluted for identity determination. Mu-stained. Morphology, growth rate, and B. abortus on days 5.6 and 7 during culture Colonies were counted and CFU/(I tissue determined) by the presence of Durham staining characteristics at .

Typical colonies were reconfirmed for bacterial confirmation of Bovine Abortus.

Bacteriological isolation was performed on all tissues and the number of bacteria per duram of tissue was counted. calculated. Four experimental animals (one placebo (ρIacebo) control standard and three Obtained from animals treated with streptomycin contained in SPL Vs The results are shown in Table 13.

(T-breath mortar) Table 13 Tissue samples from cows infected with Bovine Abortus Culture results from Built into 5PLV Left adrenal gland o oo.

Right 1) + + OO1 Right atlantal lymph node 10 + + 0 10 Right n OOO ten Right axillary lymph node +++ Q -1-Q left tt ++OOO Bronchial lymph nodes o oo.

(Uterus) Neck ooo.

Tonsil lymph node +++++ OOO Left uterine horn o o’ o + 6〃〃　0　0　0 Right intracervical bone lymph node ++ 000 Left n +++++ O+ 0 Kidney o o’o.

Liver o o-o.

Lungs ooo.

Left anterior mammary gland 0 +-jQ Left rear n Q Q O+ Front right n 10 + OOO Right rear n + + OOO Right mandibular lymph node +++000 Left mandibular lymph node 10++ Q’OO Mediastinal lymph node ++o-1-0 Intestinal delymph node +++ Q Q Q Left subauricular lymph node +++ 000 Right Il 10++ o o o.

Left popliteal lymph node +000 Right // + OOO Left anterior thigh lymph node +000 Right II Oo o.

Table 13 (continued) Built into SPLV Left prescapular lymph node 0 0. 0 + right u +++ + OOO Kidney lymph nodes o oo.

Juju + OOO Left breast lymph node 10+→-+OO Right Il OOOO Left suprapharyngeal lymph node 10　OOOO right ! j　OOOO Thymus ooo.

Vagina +++ OOO O: No identified bacteria in tissue culture medium of 0.3 to iqm 10: 200 colonies/9 m (group) woven) less.

++: More than 300 villages/qm (organization).

+++++ More than 1000 villages/rim (organization).

+++++: More than 10,000 villages/glIl (organization).

9. Example Treatment of eye diseases Bacterial and similar infections, like many other eye diseases, are a global economic and national problem. If no treatment is given or treatment is possible, there is a risk of blindness and death from sepsis. occurs.

Bacterial infections of the eyes of animals and humans are caused by Clostridium spp, Corynebacterium spp, Leptospira spp, Moraxella spp, My] Bacterium spp , Neisseria 5t)l), Propionibacterium spp, Proteus 5 t)l), pu is Idomonas spp.

Serratia spp, E. coli (E, Co11) 5lip, Stuff Bacteria including, but not limited to, Irocatchus spp. and bacterial-like microorganisms including Mykop, Plasma spp, and Rickettsia spρ. It has been reported that it is caused by living organisms. Both animals and humans are infectious Treated as a reservoir capable of spreading bacteria to each other. These 5 types of bacterial infections may be treated on a daily basis, every 20 minutes, or in combination in some infected humans. A redundant complication that results in unnecessarily high concentrations of antibiotics in textiles. It cannot be treated with antibiotics without a detailed treatment plan. Existing treatment methods are This is difficult for a number of reasons. Infectious microorganisms in the surface tissues of eye diseases can be treated with antibiotics in some cases. Highly resistant to the bactericidal action of high quality bacteria, topical antibiotics are highly resistant to uneven contact. This results in rapid removal of the drug from the orbit over time. Generally speaking The treatment for eye infections is that if there is even a small amount of infection left, it will be immediately re-sensed through lachrymal secretion. It must be completely effective, as it will cause the dyeing process to repeat once again.

Furthermore, in many cases, the degree of acuity needed to clear the infection may lead to loss of vision. In some cases, it can cause complete blindness. Economics of such diseases in livestock The impact is that the only possible way to deal with such a contagious disease is through continuous therapy. This is illustrated by the millions of dollars lost each year due to segregation.

The following experiments will be carried out to investigate the effects of infection of the eye with Moraxella bovis (M. 1) ovis. The effect of treatment with antibiotics released in serine was compared with the antimicrobials built into 5PLVS. It is a comparative evaluation of biological substances.

Moraxebovis causes contagious keratoconjunctivitis (pink eye) in cattle. . This condition causes blepharospasm and lacrimation.

It is characterized by variable degrees of conjunctivitis and septal opacification and ulceration. of the mansion Adult cows may develop a low-grade fever with some loss of appetite and reduced milk production. go Although some antibiotics are effective against Moraxella bovis, these or should be given early and frequently by repeated mucosal injections. Must be. According to the examples described here, the effect and duration of the action of therapeutic substances will be extended. Such infections do not respond to just routine treatment with antibiotics. Therefore, it is surprising that the main system is effective with just a few doses. That is. A common treatment is that if the infection becomes completely severe after many repeated treatments. If not eradicated, there will often be some residual infection that infects the eye and repeats the infection cycle again. Leave it alone.

9.1. Treatment of infectious proconjunctivitis in mice C57 black mice (160 ) were divided into eight groups, half of each group had both eyes exposed to UV light. (for the purpose of causing corneal damage). Next, all experimental animals The patient was inoculated with Moraxelamobis instilled into the right eye at a concentration of 1 x 106 bacteria.

Twenty-four hours after inoculation, all experimental animals were scored for the degree of corneal opacity. 8 One group is treated with topical application to both eyes as follows: Group 1 and and 2 are streptomycin (30m (] /1111) built into 5PLV. 10 μm was received.

Groups 3 and 4 received 10 μm of streptomycin (30 m9/ml). Ta. Groups 5 and 6 are aqueous streptomycin (100ml/n1l) received 10 μm of SPI Vs in buffer suspended in water. Group 7 and 8 received 10 μm of sterile saline. (Note: The uninfected left eye has 5 PLVs. were treated with the same topical solution to see if it was irritating. No irritation observed There wasn't. ) The progression or regression of diaphragm damage is recorded once daily and 3 times after treatment. , and on day 7, the right eye was swabbed and the Moraxera laboratory The screws were separated. Moraxelamobis settlements are village morphology and moragra labs. It was determined by the reactivity of bis fimbriae to fluorescent antigens. The results shown in Table 14 The result is that only the streptomycin contained in 5PLV is effective in eliminating infection. It represents that.

(Margin below) Engraving (contents (no changes)) Clean (no summer content) Bow ΩΦ 9.2. Treatment of rabbit mucosa using antibiotics built into 5PLV Moraxella bovis ATCC strain 10900 was mixed with sterile saline (0,085% Naci ) to a concentration of 1×10 7 cells/−IrLl. Alico of bacterial suspension (0.1 ml) was inoculated topically into the eyes of 10 adult female rabbits.

Culture samples were taken daily by swabbing the conjunctiva and plated on blood agar. placed on top. On the third day after inoculation, the rabbits were divided into three groups and two The test animals (control standard) received no treatment, and four test animals received stock treatment in sterile saline. Four experimental animals received leptomycin (concentration of 10 m(]/kg body weight) and Streptomycin (101+1!+) incorporated into 5PLV in sterile saline solution Streptomycin/concentration of 1 kg body weight) was administered. All solutions are topical The drug was administered to both eyes. After 24 hours, swab the conjunctiva of all rabbits. was started again and continued daily for 7 days. The results of the separation are shown in Table 15.

Engraving (no changes to the content) ii store (inside't, (this change is 1-) Nori 0 fu 0 9.3. Treatment of keratoconjunctivitis caused by subcutaneous infection Moraxella smallis ATCC strain 1090 0 was diluted in sterile saline to 1×10 7 cells/bar. Aliquot of bacterial suspension (0,1i1) in rabbits that were previously infected as described in Section 9.2. were inoculated into adult eyes and were not treated with SPLVs. all 9 animals The right eye of a rabbit was inoculated subcutaneously into the conjunctival tissue with 0.1 nl of Moraxella Bovis. The left eye of all rabbits was topically inoculated with 0.1 nl of Moraxella bovis. It was. Bacterial cultures were taken daily from the conjunctiva of both eyes of all rabbits and were placed on blood agar for separation of bis. On the third day after infection.

The rabbits were divided into three groups, two of which received no treatment, and three of which received no treatment. Experimental animals were treated with streptomycin (10 mfl/kg body weight of streptomycin), and four experimental animals received 5P Streptomycin saline suspension contained in the LV 1 to mycin 10nz).

The suspension or solution is administered topically to both eyes (0,1mff1). Ta. Swabbing the conjunctiva after 24 hours and every 5 days thereafter is all that is needed. was conducted on rabbits. Isolation of Moraxella bovis on blood agar culture ground Table 16 shows the blood vessels of Dissections were performed on all experimental animals at the end of the experiment. The mucosa was removed from all animals. These have been recorded regarding angiogenesis. , and chopped into small pieces, homogenized and blood for the separation of moracle bovis Liquid agar was placed on a plate. The results are shown in Table 17.

(Margin below) Table 16 Inoculation of Moraxella bovis into the conjunctiva and inoculation of onosalmine in glycerin solution. in streptomycin or saline 5PLV-encapsulated S [Check standard with hereptomycin 1 ++ +++ 5PI-V++ Sahtae 1 + + OO(-) Streptomycin 2 + 4-　〇〇〇〇a　i8 culture is at 37℃, 2411 crazy, 11? blood agar The presence of the Moraxela Bovis settlement on the board was recorded. Plus (→-) is one isolate Or Moraxela Bovis is 1 CFU, J: indicates more than or equal to, O is the category. This shows that there are no viable villages.

b All experimental animals were locally inoculated with Moraxella Bovis 1X 106 CFU in both eyes. However, in the right eye, 1 x 106 CFU of moraxelapovis was observed. 5 During silk weaving In the left eye, Moraxela Bovis 1x106 CFU was locally deposited. It was.

Topically treated with pumicin (10 mQ/kg body weight).

e Experimental animals received 5PLV-encapsulated streptomyces in sterile saline solution in both eyes. (10 mg/kc + body weight) was treated topically with t2.

Table 17 Inoculation of molaxone 1 s His into conjunctival tissue and ophthalmic glycerol dissolution Streptomycin in liquid or reference standard A+2+ a. The explanation of the codes used is the same as in Table 12, and it was carried out on the 5th day after inoculation.

b. Angiogenesis is the next step.'': The score for the sea urchin was lowered. O - standard blood vessel, 1 - few blood vessels Those that expand in a limited manner and are invaded by minute blood vessels, 2. Individual blood vessels are easily classified 3- Dark red hyperemia due to blood vessel leakage and blood seepage into the mucous membranes. of.

9.4. Efficacy of 5PLVS in the treatment of ocular infections compared to bosomal preparations evaluation of Moraxella bovis (ATCCTl2O300) was prepared from lX107 cells in sterile saline. It was diluted to a degree of 1/7rL. Bacterial % suspension aliquot 1~(0,1mff1 ) was inoculated subcutaneously in the conjunctival tissue of both eyes of an adult rabbit. all the rabbits Swabbing of the conjunctiva of both eyes is done daily to isolate moraxelapovis. were placed on a blood agar plate. On the fourth day after inoculation, the Urigi were divided into six groups. Two experimental animals received no treatment (control standard), and three experimental animals received no treatment. : When diluted to 100, ○, [), 480 (480mmT17) optical @ degree ) of 5 PLV-encapsulated streptomycin with a weight of 0.928 (body weight 1 Three experimental animals received 1:10 oz of streptomycin at 1:100 p.m. 5PLV encapsulated Saleta Streb with 0.499T at o, D, '480 when diluted 1 to receive a suspension of mycin (streptomycin 10 mg/ka body weight) , three experimental animals were diluted 1:100, ○, D, 0.242 at 480. Suspension of Strep-1 to Mycin encapsulated in 5PLV (strep/kg body weight) tomycin (10 mQ) and three experimental animals received 0.0 mQ when diluted 1:100. D, the suspension of SP LV encapsulated streptomycin which is 0.199 at 480 1 to 10 mg of mycin per gram of body weight) and two fruit For test animals, streptomycin with Q, [), 480 of 0.940 at 1:100 dilution Suspension of syn-containing multilamellar vesicles (MLVs) (body weight 11 (grams per gram) received treptomycin 10+2). MLVs are fountain (Fou) tain) et al., Curr, Micro, 6:373 (1981), Streptomycin 1III acid was added to the dry lipid film in a vortex motion, and Streptomycin, which was made by incubation for 2 hours and was not incorporated, was The heart was removed by repeated heart isolation.

The suspension was administered topically to both eyes. After 24 hours, remove the conjunctiva from all rabbits. Testing was performed daily for 9 days and plated on blood agar. on blood agar plates The results of the separation of Moraxelahohis are shown in Table 18. Dissection in all animals It was done. These were recorded for lachrymal secretion and the conjunctiva was isolated from all animals. Removed by bacterial treatment.

These were scored for angiogenesis and then minced, homogenized, and Therabovis was placed on a blood agar plate for isolation. The results are shown in Table 19. Ru.

Engraving (contents (no changes)) Table 19 Lep1hemycin or 5PLV encapsulated streptoMLV encapsulated 1 + 1 + 1 + Streptomycin 2 0 1-1- 03 PLV encapsulated 1000 Streb 1 to Mycin 2. 0 1 + 0 (undiluted) 3 0 00 SPLV encapsulated 1 + 2-1- 2 + streptomycin 2 0 0 0 (1:2 dilution) 3 0 1+0 3PLV encapsulated 1000 Streptomycin 20 100 <1:4-#! Interpretation) 3 + 1 + 08PLV encapsulated 1 + 1 + 1 + Streb 1 Hemycin 2 0 1 + 0 <1:6 dilution > 3 + 1 + O a. The code description is the same as in Table 14, and the test was carried out on the 14th day after inoculation.

b. Vascularization was plotted as follows: 1a - standard surface tube, 1 - some blood vessels swollen and infiltrated by minute blood vessels, 2- red color in which individual blood vessels cannot be easily identified. 3- Deep red hyperemia due to blood vessel leakage and blood outflow into the conjunctiva.

C secretion was scored as follows: 〇-no secretion, 1-eyelid and adjacent eyelid hair. 2 - Enough secretion to wet the eyelids and the hair adjacent to the eyes and their area. Secretion.

10. Example: Treatment of viral infection Lymphocytic choriomeningitis virus (LCMV), a member of the arenavirus family, is a human virus. LCMV infection is known to cause illness in the brain. It is lethal in mice inoculated internally. The cause of death of this mouse is This is due to immune cells reacting against virus-infected cells. The virus proliferates Therapeutic agents used in mice are unable to kill immune cells, so the therapeutic agents used in mice are suppress the proliferation of the virus so that it does not become sexually active, and/or activate the immune cells. must be suppressed.

The following examples demonstrate how to treat viral infections by administering 5PLV-encapsulated antiviral compounds. This represents the effect of the treatment.

10.1 Treatment of lethal lymphocytic choriomeningitis virus infection in mice Two-year-old Swiss mice received a lethal dose of 10M virus, two or zero per mouse. ．． Inoculated intracerebrally with 100 plaque-forming units (PFU) in a 05mff inoculum. Ta. Mice were divided into 4 groups of 7 mice each, 2 days, 3 days and 3 days after inoculation. and on day 4 by intraperitoneal injection of 0.17/Ll/dose/mouse as follows: Ta. (1) "rSPLV-R group" is 3m9ribavarin (R1bavarin) ) / vL (l containing egg phosph 1 tidylcholine 5 PLVsT-treated.5 PLVS PB 0.3*1 of lipid 100++z+ and 10CHI1g/nu drug Prepared in S (phosphate buffered saline) buffer and contains 10% of the drug. Ta. <2) rR~ group” was treated with a solution of Norivabalin 3 mg/vt# in PBS. was managed.

(3) rsPLV-group' was treated with 5 PLVs in buffer.

(In other words, 5PIVs were made in the same manner as above, except without using Ribabalin. Ta. ) (4) [Reference Standard-Group] was treated with PBS. Each in the 5th white of the eye after inoculation. Two mice from each group were sacrificed and their spleens were homogenized. ( 2 tiles per group is 1 loose! ie, in PBS at 1/20 weight per volume of homogenized by ) For plaque formation (IN(PFU)/mλ is the respective suspension It was decided regarding. The remaining five mice in each group were Observations were made every 2 days for 30 days.

The results are presented in Table 20.

Table 20 reveals reduced mortality from infected animals and reduced viral reactivation. It shows. The present inventors believe that these results indicate that the liver damage released from 5PLVs Whether this is due to the antiviral activity of the 5PLVs or the release of babarin from the 5PLVs. It has not yet been determined whether this is due to immune modulation in the murine host during the release of .

Table 20 Virus recovered from spleen Verification standard 515 7.0 Croup 515 6.9 R-Group 515 5.2 a 2 month old mice each received a lethal dose, i.e. no l-C in 0.05 ml inoculum. They were inoculated intraperitoneally with 10 PFU of M virus.

b. Mortality rate is expressed as number of deaths/total number in group.

Two mice from each croup were sacrificed at day 58 post-infection with C. The spleens were homogenized in 1 mO/2O-IrLl, 1 m to homosyne-1.

Purification (no change in content) . . 1 011 2nd figure 5th rin MG7KG Figure 6 A: Jousu Takumi, B: Ztref・Fmycin C: E; PLV (SuFshi)・Lmazzu Figure 7 Procedural City Official Book (Method) March 15, 1982 Mr. Kazuo Wakasugi, Commissioner of the Patent Office 1.Display of the incident PCT/1Js83100419 2. Name of defense Stable plurilamellar vesicles 3. Person who makes corrections Case related to patent applicant Address: New Chassis, United States 08540 Prince (Hemp) Linst Schiff 4 Restal Center One Research Way (No address) Name: The Liposome Company, Inc. Representative: Motsuku Kenneth Ai 4. Agent Address〒102 Dia Palace Niban-cho 5, 11-9 Niban-cho, Chiyoda-ku, Tokyo, on the day of the correction order.'' February 9, 1980 (Shipping date: February 14, 1980) 6, subject to amendment (1) A document pursuant to Article 184-5, Paragraph 1 of the Patent Law [Representative of the patent applicant, 1 and [Column (2) of inventor's address J] Power of attorney, corporate citizenship certificate and its translation ( 3) Translation of the drawing (4) Translation of Mei 1f III book 7. Contents of correction (1> The revised document pursuant to the provisions of Article 184-5, Paragraph 1 of the Patent Act attached to the attached sheet) Ori.

(2) As shown in the attached power of attorney, corporate citizenship certificate and its translation.

<3> As shown in the engraving of the translation of the drawing (no changes to the content).

(4) Translation of the specification, page 58, table 11, page 72, table 14, page 74, table 15 The table and Table 18 on page 81 will be amended as shown in the attached sheets.

Claims (1)

[Claims] 1. Stable plurilamellar vesicles. 2. Stable pluri-lame substantially free of MLVs, 5UVs and REVs Labesicles. 3. Has a lower buoyant density and a volume approximately one-third larger than MLVs, and has the same composition Stable plurilamellar vesicles according to claim 1 made from. 4. More stable against auto-oxidation while stored in buffer and from the same ingredients Stable plurilamellar hesicles according to claim 1, which are produced. 5.MtVs, 5UVs and REVs made from the same ingredients 2. The stable plurilamellar vesicles of claim 1, which are more stable within. 6. Patent claims that release trapped compounds when exposed to urea Stable plurilamellar hesicles according to item 1. 7. Gradual release of any trapped compounds when administered in vivo Stable plurilamellar vesicles according to claim 1. 8. When administered to cells in culture, the contents of vesicles are Stable plurilamellar vesicles according to claim 1, which are divided into two groups. . 9.Claim No. 1 that vesicles become oily when administered to cells in a living body. Stable plurilamellar vesicles according to item 1. 10. Claim No. 1, wherein the main lipid component of the vesicles is phosphatidylcholine. Stable plurilamellar vesicles according to item 1. 11. The stable pull according to claim 1, wherein the antioxidant is a component of vesicles. Lilamellar vesicles. 12. The antioxidant according to claim 1, wherein the antioxidant is butylated hydroxytoluene. Stable plurilamellar vesicles. 13. The stable protein according to claim 1, wherein the protein is trapped in a vesicle. Plurilamellar vesicles. 14. Antibacterial, antifungal, anthelmintic and antiviral compounds Claim 1, wherein a compound selected from the group consisting of: is trapped in a vesicle. Stable plurilamellar vesicles described in . 15. Anti-tumor compounds, toxins, cell receptor binding molecules and immunoglobulins. Claim 1, wherein a compound selected from the group consisting of: Stable plurilamellar vesicles described. 16. anti-inflammatory compounds, anti-glaucoma compounds, pupil dilating compounds and local anesthetics; Claim 1, wherein a compound selected from the group consisting of: is trapped in a vesicle. Stable plurilamellar vesicles described in . 17. Enzymes, hormones, neurotransmitters, immune converters, nucleotides and cyclic amino acids Compounds selected from the group consisting of zon and cin monophosphates are trapped within the vesicles. Stable plurilamellar vesicles according to claim 1, comprising: 18. The group consisting of dyes, fluorescent compounds, radioactive compounds and radiopaque compounds The stable compound according to claim 1, wherein a compound selected from the above is trapped in a vesicle. plurilamellar vesicles. 19. (a) producing a dispersion of at least one ambipolar lipid in an organic solvent; (b) the dispersion is mixed with a sufficient amount of an aqueous phase to completely emulsify the aqueous phase; forming a mixture; and (C) emulsifying the aqueous phase and dissolving the organic solvent of the two-phase mixture. substantially free of MLVs, 5UVs and REVs characterized by evaporation; Method for producing stable plurilamellar vesicles 20, wherein the ratio of the volume of the solvent to the volume of the aqueous phase is from about 3:1 to about 100:1. The method described in Scope No. 1. 21. According to claim 19, the temperature at which the method is carried out is about 4-60°C. the method of. 22. The temperature at which the method is carried out is below the phase transition temperature of the at least one lipid. 20. The method of claim 19. 23. The solvent is fluorocarbon or diethyl ether or a mixture thereof A method according to claim 19. 24. The method according to claim 23, wherein the solvent contains an antioxidant. 25. The person according to claim 24, wherein the antioxidant is butylated hydroxytoluene. Law. 26. The method according to claim 19, wherein the emulsification is carried out before evaporation. . 27. The method according to claim 19, wherein emulsification is carried out simultaneously with evaporation. Law. 28. The substance to be trapped within the vesicles is added together with the aqueous phase. A method according to claim 19. 29. Claim No. 2, wherein at least 20% of the compound is entrapped within vesicles. The method described in Section 28. 30. The method according to claim 28, wherein the substance is a protein. 31. The stable plurilamellar vesicle according to claim 1 containing the above compound. A method for administering a compound to cells in a living body, the method comprising administering a compound to an organism. 32. Stable plurilamellar vesicles can be administered locally, intraperitoneally, intravenously, intramuscularly, subcutaneously. The method according to claim 31, wherein the method is administered intraaurally. 33. The stable protein according to claim 1 containing a compound effective for treating infectious diseases. Treatment of infectious diseases in animals or plants, characterized by administering Lulilamella vesicles Treatment method. 34. The method according to claim 33, wherein the infection is intracellular. 35. Claim 34, wherein the infectious disease is caused by a parasite. How to put it on. 36. Claim No. 3 in which the infectious disease is caused and caused by Pulsella spp. Method described in Section 35. 37. The method of claim 36, wherein the administration is intraperitoneal. 38. The method according to claim 33, wherein the infection is extracellular. 39. Claim 38, wherein the infectious disease is caused by bacteria. The method described in. 40. Claim No. 4 in which the infectious disease is caused by Staphylococcus aureus The method described in paragraph 39. 41. The method of claim 40, wherein the administration is intraperitoneal. 42. The method of claim 33, wherein the infection is an eye infection. 43. Claim No. 4 in which the infectious disease is caused by Moraxella Spp. The method according to paragraph 42. 44. The method of claim 43, wherein the infection is localized. 45. According to claim 33, the infectious disease is caused by a virus. How to put it on. 46. Claim that @ infection is caused by lymphocytic choriomyelitis virus The method according to item 45. 47. The method of claim 46, wherein the administration is intraperitoneal. 48. The stable plurilamellar vesicle according to claim 1 containing said compound. Necessary sustained release of a compound effective in treating pain characterized by administering quince A method of treating pain in animals or plants. 49. The method according to claim 48, wherein the pain is an eye disease. 50. The method according to claim 49, wherein the pain is glaucoma. 51. The method of claim 50, wherein the administration is local.