NO163168B - PROCEDURE FOR MANUFACTURING STABLE PLURILAMELA REVICES - Google Patents

Abstract

New and improved type of lipid vesicles, here called stable plurilamellar vesicles or SPLV ,. is prepared by forming a dispersion of at least one amphipathic liquid, such as phosphatidyl choline in an organic solvent, combining the dispersion with a sufficient amount of an aqueous phase to form a biphasic mixture in which the aqueous phase can be completely emulsified, and emulsifying the aqueous phase and evaporation of the organic solvent from the biphasic mixture. The SPLVs produced are stable during storage and can be used in vivo for delayed release of compounds and for the treatment of diseases.

Description

The present invention relates to the production of liposomes and their use as carriers in delivery systems. More particularly, it concerns the production of a new type of lipid vesicles with unique properties and which provide special advantages such as increased stability and high entrapment efficiency.

The vesicles produced here have a wide range of applications, for example as carrier systems and targeted delivery systems. The invention is illustrated by means of examples for the treatment of brucellosis, the treatment of ocular infections and the treatment of lymphocytic meningitis virus infections.

Liposomes are completely closed bilayer membranes containing a trapped aqueous phase. Liposomes can be any type of unilamellar vesicles (with a single membrane bilayer) or multilamellar vesicles (onion-like structures characterized by concentric membrane bilayers, each separated from the next by a layer of water).

The original liposome preparations of Bangham et al., (1965, "J. Mol. Biol.", 13:238-252) involved suspending phospholipids in an organic solvent which was then evaporated to dryness leaving a wax-like deposit of phospholipid in the reaction vessel. Then a suitable amount of aqueous phase was added, the mixture was allowed to "swell" and the resulting liposomes consisting of multilamellar vesicles (hereafter referred to as MLVs) were mechanically dispersed. The structure of the resulting membrane bilayer is such that the hydrophobic or non-polar "tails" of the lipid are oriented towards the center of the bilayer, while the hydrophilic or polar "heads" are oriented towards the aqueous phase. This technique provided the basis for the development of small sonicated unilamellar vesicles (hereinafter called SUVs) as described by Papahadjopoulos and Miller (1967, "Biochim. Biophys. Acta. 135:624-638). However, these "classical liposomes" had a number of shortcomings not the least of which was a low volume of trapped aqueous mass per mole of lipid, as well as a limited ability to encapsulate large macromolecules.

Attempts to increase the captured volume first led to the formation of inverse micelles or liposome precursors, i.e. vesicles containing an aqueous phase, surrounded by a monolayer of lipid molecules oriented so that the polar head groups are directed towards the aqueous phase. Liposome precursors are obtained by adding the aqueous solution to be captured to a solution of polar lipid in an organic solvent and sonication. The liposome precursor is then vaporized in the presence of excess lipid. The resulting liposomes, consisting of an aqueous phase captured by a lipid bisect, are dispersed in the aqueous phase, see US-PS 4,224,179 in the name of M. Schneider.

In another attempt to maximize the efficiency of entrapment, Papahadjopoulos in US-PS 4,235,871 describes a "reverse-phase evaporation process" for the production of oligolamellar lipid vesicles, also known as reverse-phase evaporation vesicles (hereinafter referred to as REV). According to this procedure, the aqueous phase to be captured is added to a mixture of polar lipid in an organic solvent. Then a homogeneous emulsion of the water-in-oil type is formed and the organic solvent is evaporated until a gel is formed. The gel is then converted into a suspension by dispersing the gel-like mixture in an aqueous medium. The REV product mainly consists of unilamellar vesicles and some oligolamellar vesicles which are characterized by only a few concentric bilayers with a large internal water space. Certain permeability properties of REV were reported to be similar to those of MLV and SUV, see Szoka and Papahadjopoulos, 1978, "Proe. Nati. Acad. Sei.", USA, 75:4194-4198.

Liposomes entrapping a number of compounds can be prepared, however, the stability of the liposomes during storage is inevitably limited. This loss of stability results in leakage of the captured compound from the liposome into the surrounding media, and can also result in contamination of the liposome contents by permeation of substances from the surrounding media into the liposome itself. As a result, the shelf life of traditional liposomes is very limited. Attempts to improve stability involved the incorporation into the liposome membrane of certain substances (hereafter called "stabilizers") that affect the physical properties of the lipid bilayers (for example steroid groups). However, many of these substances are relatively expensive and the production of such liposomes is not cost-effective.

In addition to the storage problems for traditional liposomes, a number of compounds cannot be incorporated into these vesicles. MLV can only be produced under conditions above the phase transition temperature for the lipid membrane. This interferes with the incorporation of heat-labile molecules into the liposomes which are composed of phospholipids which show desirable properties, but which have long and highly saturated side chains.

Application of liposomes for therapeutic use is described in "Liposomes: From Physical Structures To Therapeutic Applications" by Knight, published by Elsevier, North-Holland Biomedical Press, 1981. Much has been written regarding the possibilities of using these membrane vesicles for drug delivery systems although a number of problems remain in connection with the systems. See, for example, what is described in US-PS 3,993,754 as well as in US-PS 4,145,410. In a liposome drug delivery system, the drug is captured during liposome formation and then administered to the patient to be treated. The drug may be soluble in water or in a non-polar solvent. Typical of such descriptions are US-PS 4,235,871 and 4,224,179.

Certain desirable features of drug delivery systems are resistance to too rapid washout of the drug accompanied by delayed release of the drug which will prolong the drug's duration of action. This increases the effectiveness of the drug and allows the use of fewer administrations. Some of the problems associated with using liposome preparations in vivo include the following: (1) liposome-entrapped materials leak out when the liposomes are incubated in the body fluid. This is due, among other things, to removal of the liposomal phospholipids due to high-density plasma lipoproteins, HDL, or breakdown of the liposome membrane due to phospholipases. A result of the degradation of the liposomes in vivo is that virtually all liposome content is released within a short time, and therefore delayed release and resistance to

drug washout.

(2) If, on the other hand, a very stable liposome is used in vivo, (that is, liposomes that do not leak when incubated in body fluids), the liposome content will not be released as needed. As a result, these stable liposomes are ineffective as carriers for therapeutic agents in vivo because the sustained release or the ability to release the liposome contents when needed is not achieved. If, however, one is treating an intracellular infection, maintaining stability in biological fluids is up to the point where liposomes

taken up by the infected cell, significantly.

(3) The cost effectiveness of the 1iposome carriers used in the disposal system. For example, an improved method for chemotherapy of leishmanial infections using liposome-encapsulated antileishmanial drugs is disclosed in US-PS 4,186,183. The liposomes used in the chemotherapy contained a number of stabilizers which increased the stability of the liposomes in vivo. As mentioned earlier, however, these stabilizers are expensive and the preparation of the liposome containing

such is not cost effective.

(4) Finally, a problem that must be taken into account when using liposomes as carriers in drug delivery systems is the inability to cure the disease being treated. In addition to the inability to resist rapid washout and to effect delayed release, a number of other explanations for the inability to cure diseases are possible. If, for example, the liposomes are taken up in the target cells, phagocytic cells, for example reticuloendothelial cells, they are quickly flushed out of the system and make the captured drug significantly more ineffective against diseases involving cells other than REV. After phagocytosis, the liposomal contents are packed with lysosomes from part-phagocytic cells. Very often, degradative enzymes contained in lysosomes will break down the captured compound, or render the compound inactive by changing its structure, or cleave the compound at the active points. Furthermore, the liposomes cannot deliver a dose that is effective due to the low entrapment efficiency for the active compound in the vesicles during manufacture.

Liposomes have also been used by researchers as model membrane systems and have been used as "target cells" in complement-mediated iminotests. When used in such assays, however, it is important that the liposome membrane does not leak when incubating 1 sera because these assays measure release of the liposome contents as a function of serum-complement activation by immune complex formation involving certain immunoglobulin types (for example, IgM and certain IgG -molecules).

The invention provides a new and significantly improved type of lipid vesicles which will hereafter be referred to as stable plurilamellar vesicles (SPLV). Apart from being structurally different from multilamellar vesicles, MVL, SPLV according to the invention are also produced in a different way than MLV, have unique properties compared to MLV and exhibit a number of different advantages compared to MLV. As a result of these differences, SPLV overcomes many of the problems with the conventional lipid vesicles that have been available to date.

Accordingly, the present invention relates to a method for the production of stable, plurilamellar vesicles with a vesicle size within the range of 500 to 10,000 nm and a number of lipid bilayers within the range of a few to over 100; - with greater stability against autoxidation during storage in buffer; - greater stability of the body fluid;

a greater percentage of leakage of trapped, dissolved material upon exposure to urea, guanidine or

ammonium acetate;

a smaller percentage leakage of the capture dissolved

material by exposure to hydrochloric acid or serum; and

a distribution of captured content via cytosol in cells by

administration to the cells in culture;

and this method is characterized by:

a) forming a dispersion of at least one amphipathic lipid in an organic solvent; b) combining the dispersion obtained under a) with an aqueous phase to form a two-phase mixture in which the aqueous phase can be completely emulsified, the volume ratio between solvent and aqueous phase being from approx. 3:1 to 100:1; and c) emulsifying the aqueous phase and evaporating the organic solvent of the biphasic mixture,

in that steps a), b) and c) work at a temperature within the range of 4-60°C, thereby obtaining stable plurilamellar vesicles essentially free of MLV, SUV and REV.

A heterogeneous mixture of lipid vesicles is realized when SPLV is synthesized. Evidence suggests that the lipids in SPLV are organized into a new supramolecular structure. Many of the lipid vesicles have a high number of bilayers, occasionally up to 100 layers. It may be possible that this high degree of layer formation contributes to many of the surprising properties that SPLV has, even if the explanations are theoretical.

The properties of SPLV include:

(1) The ability to cure certain diseases that other methodologies cannot cure; (2) Strongly increased stability of SPLV during storage in buffer; (3) The increased stability of SPLV to withstand harsh physiological environments; (4) The capture of materials with high efficiency; (5) The ability to adhere to tissues and cells for extended periods of time; (6) The ability to release entrapped substances slowly into body fluids; (7) Release and final dispersion of the liposome contents through the cytosol of the target cell;

(8) Improved cost efficiency in manufacturing; and

(9) Release of compounds in their bioactive form in vivo.

Because of the unique properties of SPLV, they are particularly useful as carriers in in vivo delivery systems, because they are resistant to shedding and are capable of sustained release. Methods for using SPLV for delivering bioactive compounds in vivo and treating pathologies, such as infections, shall be described.

The invention shall be illustrated in more detail with reference to the accompanying drawings, where: Figure 1 graphically shows the difference in membrane stability, expressed as % leakage, between MLV and SPLV, treated with different urea or NaCl concentrations; Figure 2 graphically shows the retention of both the lipid and the aqueous phase in SPLV in mouse eyelid tissue, and the delayed release of gentamycin from SPLV in vivo; Figure 3 shows the electron spin resonance absorption spectrum of SPLV, (A) compared to that of MLV, (B); Figure 4 graphically shows the differences in the ability of ascorbate to reduce the doxyl spin probes in SPLV and MLV; Figure 5 graphically shows the effectiveness of a two-step treatment of Brucella canis infections in mice using SPLV-captured streptomycin based on B. canis that could be recovered from the spleens of infected mice; Figure 6 graphically shows the effectiveness of a two-step treatment of B. canis infections in mice using SPLV-captured streptomycin, calculated for B. canis that could be recovered from organs of infected mice;

and

Figure 7 graphically shows the effectiveness of a two-trlnn treatment of Brucella abortus in guinea pigs using SPLV-captured streptomycin.

SPLV is produced by a process that results in a product that is unique to other previously described liposomes.

SPLVs are lipid vesicles that have from a few to over 100 lipid bisjlkt. The membrane bilayer consists of a bimolecular layer of an amphipathic lipid, in which the non-polar hydrophobic hydrocarbon "tails" point inwards towards the center of the bilayer, and the polar, hydrophilic "heads" point towards the aqueous phase. Occluded by the bilayers is a watery space, part of which occupies the lumen of the vesicle, and part of which lies between adjacent layers. Complexed with the lipid bilayers can be a number of proteins, glucoproteins, glucolipids, mucopolysaccharides and any other hydrophobic and/or amphipathic substances.

SPLV is prepared as follows: An amphipathic lipid or a mixture of lipids is dissolved in an organic solvent. Many organic solvents are suitable, but diethyl ether, fluorinated hydrocarbons and mixtures of fluorinated hydrocarbons and ether are preferred. An aqueous phase and the active ingredient to be captured are added to this solution. This biphasic mixture is converted to SPLV by emulsifying the aqueous material with solvent while evaporating the solvent. The evaporation can be carried out during or after sonication by any evaporation technique, for example evaporation by passing a stream of inert gas over the mixture, by heating, or by vacuum. The volume of solvent used must be in excess of the aqueous volume in an amount sufficient for the aqueous material to be emulsified in total in the mixture. In practice, a minimum of roughly 3 volumes of solvent is used for 1 volume of aqueous phase. Thus, the ratio between solvent and aqueous phase can vary up to 100 or more volumes of solvent per 1 volume of aqueous phase. The amount of lipid must be sufficient to exceed the amount necessary to coat the emulsion droplets (approx. 40 ml of lipid per mg of aqueous phase). The upper limit is limited only by the application of cost-effectiveness, as SPLV can be made from 15 g of rubber or lipid per ml aqueous phase.

The method yields lipid vesicles with a different supramolecular organization compared to conventional liposomes. According to the invention, the entire process can be carried out at a temperature within the range 4 to 60°C, regardless of the phase transformation temperature for the lipid used. The advantage of this latter point is that heat-labile products that have desirable properties, for example easily denatured proteins, can be incorporated into the SPLV preparation from phospholipid, such as distearylphosphatidylcholine, but can be converted into conventional liposomes only at temperatures above the phase transformation temperature. The method typically allows more than 20% of available water-soluble material to be incorporated, and more than 40% of available lipid-soluble material to be incorporated. With MLV, the capture of the aqueous phase is usually not more than 10%.

Most amphipathic lipids can be constituents of SPLV. Suitable hydrophilic groups are phosphate, carboxyl, sulfate and amino groups. Suitable hydrophobic groups are saturated and unsaturated aliphatic hydrocarbons and aliphatic hydrocarbons substituted with at least one aromatic and/or cycloaliphatic group. The preferred amphipathic compounds are phospholipids and closely related chemical structures. Examples of these are lecltin, phosphatidylethanolamine, lysolecithin, lysophatidylethanolamine, phosphatidic acid and cerebrosides. Specific examples of suitable lipids that can be used in the preparation of SPLV are phospholipids including natural lecithins (that is, egg lecithin or soybean lecithin) and synthetic lecithin such as saturated synthetic lecithins, such as dimyristoylphosphatidylcholine or dipalmitoylphosphatidylcholine or distearylphosphatidylcholine) and unsaturated synthetic lecithins (for for example dioloylphosphatidylcholine or dilinoloylphosphatidylcholine). The SPLV bisJikts may contain a steroid component such as cholesterol, coprostanol, cholestanol, cholestane and the like. When using compounds with acidic hydrophilic groups (phosphato, sulfato and so on), the SPLVs obtained will be anionic; with basic groups, such as amlno, cationic liposomes will be obtained; and with polyethyleneoxy or glycol groups, neutral liposomes will be obtained. The size of the SPLV varies within wide limits. The area extends from approx. 500 nm to approx. 10,000 nm (10 pm) and is usually approx. 1000 to approx. 4000 nm.

Practically any bioactive compound can be trapped inside an SPLV (entrapment is defined as trapping within the aqueous space or within the membrane target). Such compounds are nucleic acid, polynucleoids, antibacterial compounds, antiviral compounds, antifungal compounds, antiparasitic compounds, tumoricidal compounds, proteins, toxins, enzymes, hormones, neurotransmitters, glycoproteins, immunoglobulins, immunomodulators, dyes, radiolabeled substances, radio-opaque compounds, fluorescent compounds, polysaccharides, cell-receptor binding molecules, anti-inflammatory agents, antiglaucoma agents, mydriatic compounds, local anesthetics and so on.

The following is an example of the proportions that can be used in SPLV synthesis: SPLV can be obtained by adding 50 jjmol of phosphide to 5 ml of diethyl ether containing 5 pg of BHT (butylated hydroxytoluene) and then adding 0.3 ml of an aqueous phase containing the active substance to be encapsulated. The resulting solution comprising the material to be captured and the captured lipid is sonicated while passing an inert gas over the mixture so as to remove most of the solvent. This embodiment. gives particularly stable SPLV, especially due to the incorporation of BHT into the vesicles.

See also Lenk et al., 1982, "Eur. J. Biochem.", 121: 475-482, which describes a method for the preparation of liposome-encapsulated antibodies by sonication and evaporation of a solution of cholesterol and phosphatidylcholine in a mixture of chloroform and ether with added aqueous phase, but which do not give the relative ratios between lipid and aqueous phase.

SPLVs are clearly distinct in their properties in contrast to liposomes with one or more lamellae (eg SUV and REV). Freeze-fracture electron microscopy suggests that SPLV preparations are essentially free of SUV and REV, that is, less than 20% of the vesicles are unilamellar. They are therefore indistinguishable from MLV by electron microscopic techniques, even though many of the physical properties are different. However, the following detailed comparison is focused on distinguishing SPLV from MLV.

The stability of a lipid vesicle refers to the ability of the vesicle to sequester its occluded space from the external environment for a long period of time. If a lipid vesicle is to be usable, it is essential that it is stable during storage and processing. For some applications, however, it is desirable that the vesicles leak their contents slowly during use. For other applications, it is desirable that the vesicle remains intact after use until it reaches its desired point of action. It will be seen that SPLV exhibits these desired characteristics, while MLV does not.

There are two factors that cause the vesicles to leak. One is autoxidation of the lipids whereby the hydrocarbon chains form peroxides which destabilize the bilayers. Oxidation can be drastically reduced by adding antioxidants, such as butylated hydroxytoluene, BHT, to the vesicle preparation. The vesicles can also leak because agents in the external environment disrupt the bilayer organization in the lipids, so that the lipids remain intact, but the membrane develops a pore.

Preparations of lipid vesicles are colored white when freshly prepared. In case of autoxidation, the preparations become discoloured, they become brownish. A comparison between MLV and SPLV, prepared using the same lipid and aqueous components, shows that MLV discolors within 1 or 2 weeks, while SPLV remains white for at least 2 months. This is supported by thin-layer chromatography of the constituent lipids, which showed degradation of the lipids in MLV, but not of the lipids in SPLV. When these vesicles are prepared by the addition of BHT, as well as the other constituents, MLV appears slightly discolored within 1 month, while SPLV remains white and appears stable for at least 6 months or longer.

Placed in a buffer containing isotonic saline at neutral pH, SPLV containing antibiotics is stable for more than 4 months as shown in Table I. These data indicate that none of the antibiotics originally encapsulated in SPLV have leaked out during the experimental period.

Other findings indicate that SPLV is able to sequester an encapsulated agent from molecules as small as calcium ion for more than 6 months. Arsenazo III is a dye that changes color from red to blue at the smallest amount of divalent cation present. By encapsulating the dye in SPLV and adding calcium chloride to the storage buffer, it is possible to measure the stability of the vesicles by looking for a color change. The color remains undetectably different from the original color for at least 6.5 months, which shows that neither the dye has leaked out nor ions have leaked in. These tests show that SPLV is sufficiently stable to withstand storage and processing problems. Although it is possible to prepare MLVs that are stable for such a long time, they must be prepared from synthetic lipids, such as DSPC, and thus become prohibitively expensive.

Placing lipid vesicles in a medium containing membrane perturbing agents is an opportunity for testing different molecular arrangements. Depending on how the membrane is organized, different vesicles will respond differently to such agents.

In the following experiments, vesicles were prepared containing a radioactive tracer molecule (<3>H-inulin) in the occluded, aqueous space. Inulln, a polysaccharide, is distributed in the aqueous phase and if the molecule is radioactively labeled, it can be used to follow the water content of the lipid vesicles. After a suitable exposure interval to any given agent, the vesicles are separated from the medium by centrifugation and the relative amount of radioactivity that has escaped from the vesicles to the medium is determined. These results are set out in Table II; the values are expressed as % leakage, which means the proportion of radioactive material in the surrounding medium in relation to the output quantity that was encapsulated in the vesicles.

SPLV is more stable than MLV in hydrochloric acid. Table II shows that both MLV and SPLV, prepared from egg lecithin, are destabilized by exposure to 0.125N hydrochloric acid for 1 hour. However, it is worth noting that SPLV is significantly less sensitive to acid than MLV. Presumably this different response reflects an inherent difference in the way the lipids react with their environment.

SPLV responds differently from MLV when exposed to urea (Figure 1 and Table II). Urea is a molecule with both a chaotropic effect (disrupts the structure in water) and a strong dipole moment. It has been observed that SPLV are far more sensitive to urea than they are to an osmotic agent, such as sodium chloride at the same concentration, see figure 1. MLV do not leak significantly more in urea than they would in sodium chloride. Although the explanation for this different behavior is theoretical, it seems that the response is due to the diol effect rather than a chaotropic property, because guanidine, which is a molecule similar to urea, does not destabilize SPLV, see Table II. Although guanidine is also strongly chaotropic, the compound has no strong dipole moment.

SPLV is also sensitive to ammonium acetate, while MLV is not, see Table II. However, neither the ammonium ion (in ammonium chloride) nor the acetate (1 sodium acetate) are particularly effective in causing destabilization of SPLV. Thus, it seems that it is not the ions themselves, but the polarity of the ammonium acetate that is responsible for the induced leakage. At first, these results seem surprising because SPLV are much more stable than MLV when incubated in body fluids such as sera or blood. However, a theoretical explanation can be proposed for these results (of course, other explanations are possible). If the stability of the SPLV is due to the unique structure of the membrane bisJicts so that the polar groups of the membrane lipids are hydrated by a cloud of oriented water molecules, or hydration shell, it is possible that any agent that interrupts or intervenes in such a hydration shell promotes changes in the structural membrane integrity, and therefore leakage.

Regardless of whether the theoretical explanations for the destabilization of SPLV in urea are correct, the results serve to show characteristic differences between the structure of MLV and SPLV. This difference has a very useful purpose in application. As described above, SPLV slowly begins to leak when applied to the eye. Presumably, this desired slow release of the contents is due to a corresponding destabilization of the SPLV upon exposure to the tear fluid.

SPLV is more stable in serum than MLV. Many applications of lipid vesicles include their administration intraperitoneally such as in the treatment of brucellosis. To be effective, the vesicles must survive for a period of time that allows them to reach the desired target. SPLV and MLV, both produced from egg lecithin, were exposed to fetal bovine serum containing active complement, see table II. After 48 hours of exposure at 37"C, SPLV was demonstrably more stable than MLV.

SPLVs exhibit a number of characteristics that make these compounds particularly suitable as carriers for delivery systems in vivo: (A) SPLVs are resistant to washout. When SPLV administered to an organism, both the lipid component and the entrapped active ingredient are retained in the tissue and by the cells into which they are administered; (B) SPLV can be designed to provide delayed release. The stability of SPLV is "adjustable" in that SPLV are very stable during storage and are stable in the presence of body fluids, but when administered in vivo, a slow leakage of the active ingredients allows the delayed release of the active ingredients; (C) Due to the high level of entrapment and stability upon administration, effective doses of the active ingredient are released; and (D) The production of SPLV is very cost-effective as the stability of the vesicles is achieved without the incorporation of

expensive stabilizers in the bilayers.

The following experiments show some of SPLV's characteristics when administered topically to the eyes of test animals. The SPLVs used in these experiments were prepared as described above, except that the lip lamp and the active ingredient were each radioactively labeled to track these components in the eye tissue over time.

SPLV became . prepared using 100 mg of egg phosphatidylcholine, EPC, and 100 mg of gentamicin sulfate. The lipid component was radioactively labeled by incorporating trace amounts of <125>I-phosphatidylethanolamine (<125>I-PE) into the bilayers, while the active component in the aqueous phase was radioactively labeled by the addition of 125I-gentamicin sulfate (125I-GS). These SPLVs were washed with buffer repeatedly to effectively remove unincorporated or unencapsulated substances.

A portion of the SPLV preparation was removed and extracted to separate the organic phase from the aqueous phase. The radioactivity for each phase was measured to determine the initial ratio 12<5>0-PE:1<25>I-GS (cpm counts per minute in the lipid phase divided by cpm in the aqueous phase) incorporated into the SPLV.

The extraction took place as follows:

0.8 ml of 0.4M NaCl (aqueous), 1 ml of chloroform and 2 ml of methanol were mixed to obtain a homogeneous phase. Then 4 µl of the labeled SPLV was added and mixed; when the SPLV components were dissolved in the organic phase and in the aqueous phase, the mixture which was originally turbid became clear. The phases were separated by the addition and mixing of 1 ml of aqueous 0.4M NaCl and 1 ml of chloroform, and were then centrifuged at 2800 g for 5 minutes. A 1 ml amount of each phase was removed and the radioactivity in cpm was measured. The original ratio 125I_PE:<125>I_GS was 1.55:1. 15 adult Swiss Webster female mice were anesthetized and restrained (to prevent them from drying their eyes). Equal amounts of 2 µl of radiolabeled SPLV in the suspension were topically applied to each eye. Groups of 3 animals were then sacrificed 1, 2, 3, 18 and 24 hours later. 9 Female Swiss Webster mice as controls were treated identically, except that the corresponding amounts of 2 µl of an aqueous solution of radiolabeled gentamicin sulfate were applied topically to each eye. Groups of 3 control animals were killed after 1, 4 and 8 hours.

Immediately after sacrifice, the animals' eyelids were removed, cut open and extracted using the previously described procedure to separate the aqueous components from the lipid components. The radioactivity of each phase was determined (as well as the total number of radioactive counts). The radioactivity measured in the lipid phase is an indication of the retention of SPLV lipids by the eye tissue, while the radioactivity measured in the aqueous phase is an indication of the retention of gentamycin in the eye tissue. Figure 2 graphically shows the retention of each component in the eyelid tissue (expressed as a percentage of the original number of counts per minute applied to the eye). Figure 2 clearly shows the retention of the SPLV lipid component in the eyelid tissue over a 24 hour period, and the delayed release of gentamycin from the SPLV over a 24 hour period (reflected by the percentage of gentamycin retained in the eyelid tissue during this time period). Figure 2 also shows that unencapsulated gentamycin (aqueous gentamycin administered topically) is quickly washed away from the eyelid tissue. For example, gentamycin in solution as a control was washed away from the eyelid tissue within 4 hours (less than 5% of gentamycin remained in the eyelid tissue). On the other hand, more than 50# of SPLV-encapsulated gentamycin was retained by the eyelid tissue during this 4 hour period; in fact, at the end of the 24-hour period, more than 15SÉ of the SPLV-encapsulated gentamycin was retained by the eyelid tissue. This suggests that approx. 8556 of the SPLV-encapsulated gentamycin was released within 24 hours, while 95% of the unencapsulated gentamicin sulfate was washed away over a 4-hour period.

Table III compares the ratio of the SPLV lipid phase to the aqueous phase retained in the eyelid tissue at any time. An increase in this ratio indicates release of gentamicin from SPLV.

The bioactivities of SPLV-encapsulated gentamicin sulfate retained by the eyelid tissue were also assessed. Gentamycin sulfate was recovered from the eyelid tissue by removing an aliquot from the aqueous phase of the eyelid extracts prepared 3 hours after SPLV-encapsulated gentamicin sulfate was applied to the eye. The aqueous phase was serially diluted and 2 µl aliquots were plated on Staphylococcus aureus cultures on agar plates; After 24 hours of incubation, the zones of inhibition were measured. Gentamycin sulfate recovered from the eyelid tissue extracts of animals treated with SPLV-encapsulated gentamicin sulfate fully retained its bioactivity.

Although SPLV and MLV appear identical by electron microscopy, ESR (electron spin resonance) spectroscopy shows differences in the supramolecular structure. SPLV can be distinguished from MLV on the basis of the molar architecture, as evidenced by their increased molecular order, increased molecular motion and greater permeability to ascorbate. It is likely that these differences in molecular architecture contribute to their different biological effects.

In electron spin resonance spectroscopy, a spin sample such as 5-doxyl stearate (5 DS) is incorporated into the lipid bilayer. The unpaired electron in the doxyl group absorbs microwave energy when the sample is introduced into a magnetic field. The absorption spectrum allows the determination of three empirical parameters: S, the order parameter; AG, the hyperfine coupling constant; and t (Tau) the rotational correlation time. A typical reading is shown in Figure 3, where A is the SPLV signal and B is the MLV signal, both indicated for 5-doxyl stearate. The spectra were taken at room temperature, the scanning range was 100 Gauss. The order parameter (s) which depends on both 2T^ and 2T^ measures the deviation of the observed ESR signal from total uniform orientation in the sample. For a uniformly oriented sample, S - 1.00, a random sample, S = 0. The hyperfine coupling constant A0, which can be calculated from 2T± and 2Tn is considered to reflect local polarity and thus reflects the position of the spin sample in the membrane. The rotational correlation time (which depends on W0, h0, h-1) can be set to the time necessary for the molecules to "forget" what their more obvious spatial orientation was. A typical ESR determination for the differences between SPLV and MLV with 5-DS as spin test is summarized in table IV.

Although in both cases the spin sample rapports from the same depth in the bilayer, SPLV has a significantly greater degree of molecular order and molecular motion than MLV.

Another illustration of the difference between SPLV and MLV lies in the ability of ascorbate to reduce the doxyl spin samples. It has been known for some time that ascorbate reduces doxyl moieties, presumably to their hydroxylamine analogues which do not absorb microwave energy in a magnetic field. In aqueous solutions, the reductions occur rapidly with a simultaneous loss of the ESR signal. If the spin sample is in a protected environment, such as a lipid bilayer, it may be reduced more slowly or not at all by the hydrophilic ascorbate. Thus, the speed of the nitroxide reduction can be used to study the degree of penetration of the ascorbate into the lipid bilayer. Figure 3 shows the percentage of residual spin versus time for SPLV and MLV, suspended in an ascorbate solution. After 90 minutes, the ascorbate has reduced 25% of the sample embedded in MLV, but 6056 of the sample embedded in SPLV. SPLV allows a drastically higher permeability to ascorbate than MLV to be achieved.

Another example of the superiority of SPLV compared to traditional MLV is that SPLV captures a larger percentage of the available active material, and thereby preservative material (see table V).

A further advantage of SPLV is that SPLV has a mutual influence with the cells, so that a relatively large proportion of the material encapsulated inside the vesicles is dispersed out through the cytoplasm of the cells instead of being confined to phagocytic vesicles. When SPLV is mixed with cells the two seem to coalesce. During coalescence, unlike MLV, SPLV interacts with the cells in vitro, so that all cells contain at least some material originally captured in SPLV. This material seems to be distributed throughout every cell, and is not limited to just the phagocytic vesicles. This can be shown by incorporating ferritin into the aqueous phase of an SPLV preparation. After coalescence with a cell in culture, ultrastructural analysis shows that ferritin is distributed throughout the cytosol and is not bound to the intracellular membranes. While this phenomenon can be shown to occur with MLV, a larger amount of material can be transferred using SPLV.

In addition, SPLV has a lower buoyancy density than MLV. This is measured by "banding" in a Ficoll gradient (see Table VI).

Furthermore, SPLV, when collected in a pellet by centrifugation under from 1000 to 100,000 x g, form a pellet which is substantially larger than MLV, given the same phospholipid concentration. At a force of 16,000 x g, SPLV forms a pellet approximately 1/3 larger than MLV.

Because the phospholipid bilayers are permeable to water, when the MLV is placed in a hypertonic environment, occluded water will be expelled due to osmotic forces. SPLV shrinks more than MLV. After shrinking for 16 hours in a buffer; which is 20 times higher than the internal salt concentration, in addition, SPLV does not shrink to the same final volume as MLV (SPLV pellets remain 1/3 larger than MLV pellets). This indicates that the difference 1 pellet size is not due to differences in enclosed volume water.

SPLV is particularly applicable in systems where the following factors are important: stability during storage and contact with the body fluid; a relatively high degree of encapsulation; cost efficiency; and release of the captured compound in the biologically active form.

Furthermore, depending on the method of administration in vivo, SPLV may be resistant to rapid washout (for example, where delayed release is important) or may be delivered to the cells 1

RES.

As a result, the SPLV according to the invention can be successfully used in a wide range of systems. They can be used to increase the therapeutic effectiveness of drugs, to heal infections, to increase enzyme replacement, delivery of oral and topical drugs, to introduce genetic information into cells in vitro and in vivo, to make vaccines, to introduce recombinant deoxyribonucleic acid segments in cells, or as diagnostic reagents for clinical samples after release of captured "reporter" molecules. SPLV can also be used to encapsulate cosmetic preparations, pesticides, compounds for delayed slow release to effect plant growth and the like.

The methods that follow will, on the basis of the application of SPLV, describe the use of SPLV or any other liposome, or lipid vesicle with functional characteristics, corresponding to those of SPLV.

Delivery of compounds to cells in vitro, (eg animal cells, plant cells, protists and so on) generally requires the addition of SPLV containing the compound to the cells in culture. However, SPLV can also be used to deliver compounds in vivo in animals (including humans), plants and protists. Depending on the purpose of delivery, SPLV can be administered in a number of ways: in humans and animals this includes injection (eg intravenous, intraperitoneal, intramuscular, subcutaneous, intraauricular, intramammary, intraureteral, and so on), topical application (eg on infected areas), and by absorption through epithelial or mucocutaneous membranes, (for example ocular epithelium, oral mucosa, rectal and vaginal epithelial membranes, respiratory tract membranes, nasopharyngeal mucosa, intestinal mucosa and so on) in plants and protists this includes direct application to organisms, dlspersion in the organism's habitat , addition to the surrounding environment or surrounding water and so on.

The method of application can also determine the points and cells in the organism to which the compound is to be delivered. For example, delivery to a specific point of infection is most easily achieved by topical application (if the infection is external). Delivery to the circulatory system (and thus the reticuloendothelial cells) can most easily be achieved by intravenous, intraperitoneal, intramuscular or subcutaneous injection.

Because SPLV allows delayed release of the compound, doses that would otherwise be toxic to the organism can be used in one or more administrations to the organism.

The sections that follow below describe some overall forms in which SPLV can be used, and show the scope of the invention.

A number of pathological conditions occurring in humans, animals and plants can be treated more effectively by encapsulating the appropriate compound or compounds in SPLV. These pathological conditions include infections (intracellular and extracellular), cysts, tumors and tumor cells, allergies and so on.

Many strategies are possible for the application of SPLV in the treatment of such pathologies; a number of total schemes are set out below, which are particularly useful in that they take advantage of the fact that SPLV when administered in vivo are taken up by macrophages.

In one form, SPLV is used to deliver therapeutic agents to points with Intracellular infections. Certain diseases involve an infection of cells in the reticuloendothelial system, such as brucellosis. These intracellular infections are difficult to cure for a number of reasons: (1) because the infected organism resides within the cells of the reticuloendothelial system, where it is sequestered from circulating therapeutic agents that cannot cross the cell membranes in therapeutically sufficient concentrations and are therefore strongly resistant to treatment; (2) administration of toxic levels of therapeutic agents is often necessary to combat such infections; and (3) the treatment must be completely effective because any residual infection after treatment can lead to new infection of the host organism or can be transmitted to other hosts.

According to one embodiment of the invention, SPLV containing a suitable biologically active compound is administered (preferably intraperitoneally or intravenously) to the host organism or the potential host organism; (for example, in animal herds both the non-infected animals and the infected animals can be treated). Because phagocytic cells take up SPLV, administration of an SPLV-encapsulated substance that is biologically active against the infecting organism will result in a targeting of bioactive substance to the point of infection. Thus, the present invention can be used to eliminate infections caused by a number of microorganisms, bacteria, parasites, fungi, mycoplasma and viruses including Brucella spp., Mycobacterium spp., Salmonella spp., Listeria spp., Francisella spp., Histoplasma spp., Corynebacterium spp., Coccidiodes spp. and lymphocytic choriomeningitis virus.

The therapeutic agent chosen will depend on the organism causing the infection. For example, bacterial infections can be eliminated by encapsulating an antibiotic. This antibiotic may be contained in the aqueous fluid in the SPLV, and/or introduced into the vesicle bilayer. Suitable antibiotics include penicillin, ampicillin, hetacillin, carbencillin, tetracycline, tetracycline hydrochloride, oxytetracycline hydrochloride, chlortetracycline hydrochloride, 7-chloro-6-dimethyltetracycline, doxycycline monohydrate, methacycline hydrochloride, minocycline hydrochloride, rolitetracycline, dihydrostreptomycin, streptomycin, gentamycin, kanamycin, neomycin, erythromycin , carbomycin, oleandomycin, troleandomycin, Polymyxin B colistin, cephalothin sodium, cephaloridine, cephaloglycin dihydrate and cephalexin monohydrate.

The effectiveness of such treatment for curing brucellosis is shown in the examples below. With the help of the invention, the effectiveness and duration of action is extended. It is surprising that this system is effective for treating infections that do not respond to known treatments, such as antibiotics captured in MLV. Successful treatments unexpectedly, because any small remaining infection will spread, and the cycle of infection will begin again. Furthermore, the successful treatment of lymphocytic choriomeningitis virus infection is shown.

Of course, treatment is not limited to intracellular infections. SPLV can be directed against a number of infection points regardless of whether these are intracellular or extracellular. In another embodiment, for example, macrophages are used to carry an active agent to the point of a systemic extracellular infection. According to this scheme, SPLV is used to deliver a therapeutic agent to uninfected macrophages by administration of SPLV in vivo, preferably intraperitoneally or intravenously. The macrophages will coalesce with SPLV and then be "loaded" with therapeutic substance; in general, the macrophages will retain the substances for approx. 3-5 days. Once the "loaded" macrophages reach the point of infection, the pathogen would be taken up by the macrophages. As a result, the pathogen will come into contact with the therapeutic substance contained in the macrophage and be destroyed. This embodiment is particularly useful in the treatment of Staphylococcus aureus mastitis in humans and cattle.

If the point of infection or affliction is external or accessible, the SPLV-entrapped therapeutic agent can be applied topically. A particularly useful application involves the treatment of eye disorders. In the case of ocular disorders, SPLV containing one or more suitable active ingredients can be applied topically to the inflamed eye. A number of organisms cause eye infections in animals and humans. Such organisms include Moraxella spp., Clostridium spp., Corynebacterium spp., Diplococcus spp., Flavobacterlum spp., Hemophilus spp., Klebsiella spp., Leptospira spp., Mycobacterium spp., Neisseria spp., Propionibacterium spp., Proteus spp. , Pseudomonas spp., Serratia spp., Escherichia spp., Staphylococcus spp., Streptococcus spp. and bacteria-like organisms including Mycoplasma spp. and Rickettsia spp. These infections are difficult to clear using conventional methods because any residual infection that remains after treatment can re-infect due to lacrimal secretion. The examples below show the use of SPLV in the treatment of ocular infection caused by Moraxella bovis.

Because SPLV is resistant to excretion and is capable of delayed release of its contents, SPLV is also useful in the treatment of any disorder that requires prolonged contact with the active therapeutic agent. For example, glaucoma is a deficiency characterized by a gradual rise in intraocular pressure and causes progressive loss of peripheral vision and, if the deficiency is not controlled, loss of central vision and eventually blindness. Medicines used in the treatment of glaucoma can be administered topically as eye drops. However, the treatment requires the application of drops every 15 minutes due to the rapid excretion of the drug from the eye. If a disorder such as glaucoma is to be treated, therapeutic substances such as pilocarpine, fluoropril, physostigmine, carcholine, acetazolamide, etozolamide, dichlorphenamide, carbachol, demecarium bromide, diisopropylphosphofluorldate, ecothioplatiodide, physostigmine or neostigmine and so on can be captured in the SPLV which is then delivered to the inflamed eye.

Other agents that can be encapsulated in SPLV and used topically include mydriatics (eg, epinephrine, phenylepine-ephrine, hydroxyamphetamine, ephedrine, atropine, homatropine, scopolamine, cyclopentolate, topicalamide, encatropin, and so on), local anesthetics; antiviral agents (for example, idoxuridine, adenine arabinoside, and so on), antifungal agents (for example, amphoteracin B, natamycin, pimaricin, flucytosine, nystantine, thimerosal, sulfamerazine, thiobendazole, tolnaftate, grisiofulvin, and so on); antiparasitic agents (for example, sulfonamides, pyrimethamine, clindamycin, and so on); and anti-inflammatory agents (eg, corticosteroids such as ACTH, hydrocortisone, prednisone, medrysone, β-methasone, dexamethasone, fluoromethalone, triamcinalone, and so on).

The following examples are given to illustrate the invention.

Example 1

MANUFACTURE OF SPLV

As explained earlier, the principle method for preparing SPLV solution of a lipid or mixture of lipids in an organic solvent involves adding an aqueous phase and material to be encapsulated, and sonicating the mixture. In the preferred embodiment, the solvent is removed during or after the sonication by any evaporation technique. The SPLV used in all the examples given here was prepared as described in the following subsections (however, any method that gives SPLV can be used).

SPLV containing antibiotics

A 5 ml diethyl ether solution of 100 mg lecltin was prepared. The mixture was placed in a round bottom flask. Then 0.3 ml of a solution containing 100 mg streptomycin sulfate at pH 7.4 in 5 mmol HEPES (4-[2-hydroxyethyl]piperazine-2-ethanesulfonic acid/0.0725M NaCl/0.0725M KCl) was pipetted into the glass flask containing the diethyl ether solution of lipid. The mixture was placed in a bath sonicator (Laboratory Supplies Co., Inc.), type 10536 for several minutes;, (frequency 80 kHz: output power 80 W) with simultaneous drying to a viscous paste by pass over a gentle stream of nitrogen.

To the viscous paste that remained, 10 ml of 5 mmol HEPES was added. The resulting SPLV preparation containing streptomycin was suspended in the buffer solution, shaken for several minutes on a vortex mixer and freed from unencapsulated streptomycin by centrifugation at 12,000 x g for 10 minutes at 20°C. The resulting cake was suspended in 0.5 ml of 5 mmol HEPES.

The procedure described above was followed, except that streptomycin was replaced by the following: dihydrostreptomycin, gentamicin sulfate, ampicillin, tetracycline hydrochloride, and kanamycin.

SPLV containing other membrane constituents

The procedure just described was followed except that any of the following were added with the egg lecithin: (1) phosphate at a molar ratio of 8:2 lecithin:dicetyl phosphate; (2) stearylamine to a molar ratio of 8:2 lecithin:stearylamine; cholesterol and stearylamine to a molar ratio of 7:2:1 for lecithin:cholesterol:stearylamine; and (3) phosphatidic acid and cholesterol at a molar ratio of 7:2:1 for lecithin:phosphatidic acid:cholesterol.

SPLV containing pilocarp pln

The initially described procedure was followed, except that streptomycin as an antibiotic was replaced by pilocarplin.

SPLV prepared with and without BHT Non-distilled ether contains an antioxidant, 1 µg/ml butyl hydroxytoluene, BHT, for storage purposes. The procedure described at the outset was followed using non-distilled ether as a solvent to incorporate BHT into the SPLV preparation.

To prepare SPLV without the incorporation of BHT, the procedure described initially was followed using distilled ether as solvent.

Example 2

SPLV-MEDIATED RELEASE IN VITRO

In the following example, SPLV-mediated delivery of antibiotics to macrophages in culture was demonstrated.

Peritoneal macrophages were obtained by peritoneal secretion from C57BLK adult male mice and suspended in minimal essential medium (M.E.M.) of pH 7.2 containing 10% heat-inactivated fetal calf serum. The cells were suspended in a concentration of 1 x 10^ cells per ml in 96 well tissue culture dishes. To the cultures containing adherent peritoneal macrophages, B. canis was added at concentrations of 1 x 10^ CFU (colony forming units) per ml. After 12 hours, bacteria that were not occupied by peritoneal macrophages were removed by repeated washings with M.E.M. After washing peritoneal macrophage cultures, these were divided into 5 groups, each containing 12 replicate cultures per group.

Group 1, designated as control, received no treatment. Group 2 received aqueous streptomycin sulfate at a concentration of 1 mg/ml.

Group 3 received buffered SPLV.

Group 4 received aqueous streptomycin sulfate (1 mg/ml) plus preformed buffered SPLV.

Group 5 received SPLV containing streptomycin sulfate (1 mg/1).

After 24 hours, the supernatant was removed by repeated washings, and the peritoneal macrophages were broken up by repeated freezing and thawing. Serial dilutions of such disrupted macrophages were plated on brucella agar and after 4 days surviving B. canis were determined by limiting dilution techniques. The results shown in Table 7 suggest that SPLV-entrapped streptomycin was highly effective in killing and eliminating intracellular B. canis infection in vitro.

The experiment was repeated with B. abortus exactly as described above, except that the peritoneal macrophages were obtained by peritoneal lavage from adult albino female guinea pigs. The results are also shown in Table VII.

Example 3

TREATMENT OF INTRACELLULAR INFECTIONS

The following examples show how SPLV can be used in the treatment of intracellular infections. The data shown demonstrate: (1) the effectiveness of using antibiotics encapsulated in SPLV in the treatment of disease and (2) the greater effectiveness achieved by administration of multiple doses of the SPLV preparation. A comparison between MLV and SPLV as carriers used in the protocols is described.

Brucellosis causes worldwide economic and health problems. Brucellosis is caused by Brucella spp. It is adapted to many mammalian species including humans, domestic animals and a number of wild animals. Six Brucella spp. cause brucellosis in animals, these are B. abortus, B. canis, B. melltensis, B. neotomae, B. ovis and B. suis. Both domestic animals and wild animals serve as a reservoir for a potential spread of brucellosis to other animals and humans.

Such infections cannot be cured with antibiotics because the infected organisms are inside the cells of the reticuloendothelial system and are highly resistant to the bactericidal activities of antibiotics. The amount of antibiotics required and the length of treatment result in either toxic effects on the animal or an unacceptable concentration of the antibiotic in question in the animal's tissues. The additional difficulty in treating this disease is that the treatment must be totally effective because any remaining infection will simply spread and the disease cycle will begin anew. The financial burden of such diseases is shown by the millions of dollars worth of cattle lost each year due to spontaneous abortion. The only potential way to combat such infectious outbreaks is to quarantine and then slaughter the animals.

The examples that follow include incorporating an antibiotic into SPLV and then administering the encapsulated active substance to the animals by inoculating the infected animals intraperitoneally.

The effect of a single treatment of B. canls infection. lon when using SPLV-captured antibiotic

80 adult male Swiss mice were infected intraperitoneally, I.P., with B. canis ATCC 23365 (1 x 10<7> CFU) and divided into 8 groups of 10 mice each. 7 days after inoculation with B. canis,

the groups treated as follows:

Group 1, designated control, received no treatment;

Group 2 received buffered SPLV (0.2 ml I.P.);

Group 3 received aqueous streptomycin sulfate (1 mg/kg body weight in a total administration of 0.2 ml I.P.);

Group 4 received aqueous streptomycin sulfate (5 mg/kg body weight) in a total administration of 0.2 ml I.P.;

Group 5 received aqueous streptomycin sulfate (10 mg/kg body weight) in a total administration of 0.2 ml I.P.;

Group 6 received SPLV containing streptomycin sulfate (1 mg/kg body weight) in a total administration of 0.2 ml I.P.

Group 7 received SPLV containing streptomycin sulfate (5 mg/kg body weight) in a total administration of 0.2 ml I.P.; and Group 8 received SPLV containing streptomycin sulfate (10 mg/kg body weight) in a total administration of 0.2 ml I.P.

On the 14th day after inoculation with B. canis, all animals were killed and the spleens removed aseptically. The spleens were homogenized and serially diluted on brucella agar to determine the number of surviving B. canis in the spleen after treatment. The results after 4 days of incubation are shown in Table VIII.

The effect of multiple treatment of B. canis infection. lon when using SPLV-captured antibiotic

80 adult male Swiss mice were infected with B. canis ATCC 23365

(1 x IO<7> CFU, I.P.) and divided into 8 groups of 10 mice each. 7 days and 10 days after inoculation with -B. canis, the groups were treated as follows: Group 1, designated as controls, received no treatment;

Group 2 received buffered SPLV (0.2 ml, I.P.);

Group 3 received aqueous streptomycin sulfate (1 mg/kg body weight) in a total administration of 0.2 ml, I.P.;

Group 4 received aqueous streptomycin sulfate (5 mg/kg body weight) in a total administration of 0.2 ml, I.P.;

Group 5 received aqueous streptomycin sulfate (10 mg/kg body weight) in a total administration of 0.2 ml, I.P.;

Group 6 received SPLV containing streptomycin sulfate (1 mg/kg body weight) in a total administration of 0.2 ml, I.P.;

Group 7 received SPLV containing streptomycin sulfate (5 mg/kg body weight) in a total administration of 0.2 ml, I.P.; and Group 8 received SPLV containing streptomycin sulfate (10 mg/kg body weight) in a total administration of 0.2 ml, I.P.

On the 14th day after inoculation with B. canis, all animals were killed and the spleens removed aseptically. The spleens were homogenized and serially diluted on brucella agar to determine the number of surviving B. canis in the spleen after treatment. The results after 4 days of incubation are shown in Figure 5. The results of different two-stage treatment regimens of B. canis infections in vivo are shown in Figure 5, and show that in groups that received aqueous streptomycin 7 and 10 days after inoculation, very little reduction of surviving B. canis in spleens observed. Only in groups receiving SPLV-captured streptomycin at a concentration of 10 mg/kg administered on days 7 and 10 after inoculation, all viable bacteria were eliminated from the spleen of infected animals. In addition to the experiment described above, various tissues from B. canls-infected mice after two treatments with SPLV-captured streptomycin were tested as follows: 30 adult male Swiss mice were inoculated with B. canis ATCC 23365 (1 x IO <7> CFU, I.P.). 7 days after inoculation, the animals were divided into 3 groups of 10 mice each.

Group 1 designated as controls received no treatment;

Group 2 received, on days 7 and 10 after inoculation, aqueous streptomycin sulfate (10 mg/kg body weight) in each administration of 2 ml, I.P. ;

Group 3 received on days 7 and 10 after inoculation SPLV-containing streptomycin sulfate (10 mg/kg body weight) in each administration of 0.2 ml, I.P.

On days 14 to 75 after inoculation with B. canis, all animals were killed and the following organs removed aseptically, homogenized and serially diluted on brucella agar for isolation of B. canis; heart, lungs, spleen, liver, kidneys, testicles. The results obtained after 4 days of incubation in terms of surviving B. canis per organ, is shown in figure 6.

The results of sampling different tissues in B. canis-infected mice after two treatment regimens, with streptomycin, as shown in Figure 6, show that in animals treated with SPLV-captured streptomycin, all tissue samples from 14 to 25 days after inoculation with B. canis totally free of any living B. canis organisms. In animals that were untreated or treated with aqueous streptomycin 1 concentrations and administration schedules identical to that of SPLV-captured streptomycin, live B. canlis organisms could be isolated in all tissues taken from 14 to 75 days after inoculation with B. canis.

Effectiveness of treatment using MLV

as a comparison to SPLV

15 adult male Swiss mice were inoculated with B. canis ATCC 23365 (1 x 10<7>CFU, I.P.). 7 days after inoculation, the animals were divided into 3 groups of 5 mice each.

Group 1, designated as control, received no treatment;

Group 2 received, on days 7 and 10 after inoculation, MLV containing streptomycin sulfate (10 mg/kg body weight, I.P.). MLV was prepared by conventional techniques using 100 mg of egg phosphatidylcholine, EPC, and 2 ml of sterile HEPES containing streptomycin sulfate (100 mg/ml). The ratio between lipid and streptomycin sulfate was 100 mg EPC per 28 mg in streptomycin sulfate in 2 ml of finished MLV suspension;

Group 3 received on day 7 and day 10 after inoculation SPLV-containing streptomycin sulfate (10 mg/kg body weight, I.P.), prepared as described in the first part of example 1 with the following modifications: 100 mg EPC and 0.3 ml were used HEPES containing 100 mg streptomycin sulfate. The ratio between lipid and streptomycin sulfate in this SPLV was 100 mg EPC per 28 mg of streptomycin sulfate in 2 ml of finished suspension.

On day 14 after inoculation with B. canis, all animals were killed and the spleens removed aseptically, homogenized and serially diluted on brucella agar for isolation of B. canis. The results of surviving B. canis per organ after 4 days of incubation, is shown in Table IX.

Efficacy of different SPLV-captured antibiotics in treating infections 50 adult male Swiss mice were inoculated with B. canis ATCC 23365 (1 x 10<7> CFU, I.P.). 7 days after inoculation, the animals were divided into 10 groups of 5 animals each.

Group 1, designated as control, received no treatment;

Group 2 received buffered SPLV (0.2 ml, I.P.) on day 7 and day 10 after inoculation;

Groups 3, 4, 5 and 6 received aqueous injections (0.2 ml I.P.) of dihydrostreptomycin, gentamycin, kanamycin or streptomycin, 10 mg/kg body weight, I.P., on days 7 and 10 after inoculation. (NB! Each of these antibiotics has been shown to kill B. canis in vitro.)

Groups 7, 8, 9 and 10 received SPLV-containing dihydrostreptomycin, gentamycin, kanamycin or streptomycin in an amount of 10 mg/kg body weight on days 7 and 10 after inoculation.

On day 14 after inoculation with B. canis, all animals were killed and the spleens removed aseptically, homogenized and serially diluted on brucella agar for isolation of B. canis. The results are surviving B. canis per organ after 4 days of incubation, is shown in Table X.

The results from tests of different antibiotics on B. canis-infected mice as found in Table X show that antibiotics which are effective for the eradication of B. canis in vitro (that is, in suspension culture) are also only effective for the eradication of B. canis infections in vivo when encapsulated in SPLV. Animals receiving either aqueous antibiotics, buffered SPLV or no treatment were in no case free of surviving B. canis in isolated spleen tissues.

Treatment of dogs infected with B. canis

Adult female beagles were inoculated with B. canis ATCC 23365

(1 x IO<7> CFU) orally and vaginally. 7 days after inoculation, the dogs were divided into 3 groups.

Group 1, designated as control, received no treatment; Group 2 received, on days 7 and 10 after inoculation, aqueous streptomycin sulfate 1 in an amount of 10 mg/kg body weight, each administration 5 ml, I.P.;

Group 3 received on days 7 and 10 after inoculation SPLV containing streptomycin sulfate in an amount of 10 mg/kg body weight with each administration of 3.0 ml, I.P. Vaginal scrapings from the dogs and heparinized blood samples were collected at regular intervals before, during and at the end of the study. These were cultured on brucella agar to isolate B. canis. The results are shown in Table XI. Serum samples were collected before, during and at the end of the study to determine serum antibodies against B. canis. These results are also shown in Table XI. 21 days after inoculation with B. canis, all animals were euthanized. The following tissue samples were removed aseptically, homogenized and serially diluted on brucella agar for isolation of B. canis; heparinized blood, vaginal exudate, lungs, spleen, synovial fluid, uterus, ovary, popllteal lymph nodes, salivary glands, tonsils, mediastinal lymph nodes, mesenteric lymph nodes, bone marrow, superficial cervical lymph nodes, and axillary lymph nodes. The results of surviving B. canis per tissues after 4 days of incubation are shown in Table XII. Results of culture and serological samples of dogs infected with B. canis before, during and after two-stage antibiotic administration are given in Table XI. All animals were serologically negative for previous exposure to B. canis, as measured by negative serum titers, and were culture-negative from blood cultures and cultures of vaginal scrapings. All animals were noted to be culture-positive for both blood and vaginal cultures before treatment on days 7 and 10. Dogs treated with aqueous streptomycin or dogs that received no treatment remained culture-positive for blood and vaginal cultures during the post-treatment period before termination of day 21. Group 3 which received liposomes containing streptomycin became culture-negative 1 day after the first treatment and remained negative throughout the post-treatment period. Dogs that received no treatment or aqueous streptomycin developed detectable serum titers against B. canis antigens by day 21 post-inoculation, while those treated with SPLV containing antibiotics on days 7 and 10 post-inoculation did not develop detectable antibodies to B. canis -antigens.

Results from the isolation of B. canis from infected dogs treated by two-stage antibiotic administration and given in Table XII show that in dogs only treatment with SPLV containing streptomycin was effective in eliminating any viable B. canis in all tissues from all organ samples.

Treatment of B. abortus in guinea pigs

15 adult female guinea pigs were inoculated with B. abortus ATCC 23451 (1 x 10<7> CFU.I.P.). 7 days after inoculation, the animals were divided into 3 groups of 5 animals each.

Group 1, called control, received no treatment.

Group 2 received aqueous streptomycin sulfate I.P. in injections of 0.2 ml at 10 mg/kg body weight on day 7 and day 10 after inoculation with B. abortus.

Group 3 received SPLV containing streptomycin sulfate, I.P. injections, of 0.2 ml at 10 g/kg body weight on days 7 and 10 after inoculation with B. abortus.

On day 14 after inoculation with B. abortus, all animals were sacrificed and the spleens were removed, homogenized aseptically and serially diluted on brucella agar for isolation of B. abortus. The results of surviving B. abortus per spleen after 4 days of incubation is shown in Figure 7. Only SPLV containing streptomycin was effective in eliminating B. abortus in guinea pig spleens. In animals that received aqueous streptomycin, or that received no treatment, live B. abortus bacteria could be identified.

Treatment of B. abortus infections in cows

9 heavily infected animals were used in this experiment. B. abortus bacterial isolates from milk and vaginal scrapings were and remained negative 6 weeks after treatment with SPLV containing streptomycin. When infection recurred in these animals, bacterial isolates were found only in quadrants of the udder that were positive before treatment. 9 crossbred (Hereford-Jersey-Brangus), 22-month-old non-pregnant, and confirmed B. abortus culture-positive heifers were used. At least 4 months before the start of the study, the animals were experimentally challenged per conjunctiva with 1 x 10<7 >CFU of B. abortus strain 2302 in the mid-gestation period, resulting in abortion and/or B. abortus culture-positive lacteal or uterine secretions and/or fetal tissue.

The heifers were kept in individually isolated stalls, and separated into 3 groups. The treatment comprised a two-dose schedule with 3 days' intervals as follows: (1) 3 heifers were injected Intraperitoneally with physiological saline solution. (2) 3 heifers were injected intraperitoneally with aqueous antibiotic (streptomycin in an amount of 10 mg/kg body weight) plus pre-prepared buffered SPLV. (3) 3 heifers were injected intraperitoneally with SPLV-captured streptomycin in an amount of 10 mg/kg body weight. The total volume per injection was 100 ml per animals. During the first two months, duplicate bacteriological cultures of lacteal and uterine secretions were carried out every week, provided this was available. Then all cows were euthanized with an overdose of sodium pentabarbitol and the following organs were collected in duplicate for bacteriological cultures: (1) Lymph nodes: left and right atlantal, left and right suprapharyngeal, left and right mandibular, left and right parotid, left and right prescapular, left and right prefemoral, left and right axillary, left and right popliteal, left and right internal iliac, left and right supramammary, left and right renal, bronchial, mediastinal, mesenteric and hepatic; (2) Glands: All four quarters of mammary gland, left and right adrenal glands and thymus (if present); (3) Organs and other tissues: spleen, liver, left and right horns of uterus, cervix, vagina, kidney and tonsils.

After necropsy, all tissue was frozen and kept at -70"C during transport. The tissue was thawed, alcohol-burned and aseptically trimmed before weighing. After the weights were noted, (0.2 to 1.0

g) the tissue was homogenized in 1 ml of sterile saline and serially diluted with sterile saline to 1:10"<10> of the

original homogenate suspension. Aliquots of 20 µl of each dilution from the serial suspensions were plated on brucella agar and placed at 37°C for incubation. Duplicate determinations were carried out for each tissue.

The plates were read daily and noted for bacterial growth. All colonies that appeared before 3 days were isolated, assay prepared and gram-stained to determine identity. On days 5, 6, and 7 of incubation, colonies with morphology, growth, and gram stain characteristics consistent with B. abortus were counted; CFU per g was then determined. Representative colonies were then run again for bacterial confirmation of B. abortus.

Bacteriological isolates occurred on all tissue samples and quantification of bacteria per g tissue was calculated. The results from 4 animals, one placebo control and three animals treated with SPLV-captured streptomycin are shown in Table XIII.

Example 4

TREATMENT OF OCULAR LIPS

Bacterial and similar infections as well as many other disorders of the eye cause worldwide economic and public health problems and, if untreated or treated incorrectly, can lead to loss of eye sight and possibly death due to septicemia. Bacterial infections of the eye in animals and humans have been reported to be caused by a number of bacteria including Clostridium spp., Corynebacterium spp., Leptospira spp., Moraxella spp., Mycobacterium spp., Neisseria spp., Propionibacterium spp., Proteus spp., Pseudomonas spp., Serratia spp., E. coli spp., Staphylococcus spp., Streptococcus spp. and bacteria-like organisms including Mycoplasma spp. and Sickettsia spp. Both animals and humans serve as reservoirs for the potential spread of infectious bacteria to each other.

Such bacterial infections cannot be treated with antibiotics without long-term and careful treatment plans that result in either frequent treatment, often as much as every 20 minutes in people with certain infections, or unacceptably high concentrations of the antibiotic in question in the tissue. Today's treatment methods are difficult for many other reasons. The infected organism in the surface tissue of the eye is in some cases very resistant to the bactericidal activities of antibiotics, and topical administration of antibiotics can result in rapid excretion of drugs from the eye, which gives varying contact times. As a general rule, treatment of eye diseases must be totally effective, because any remaining infection will re-infect the eye by lacrimal secretion, and the cycle begins again. Furthermore, in many cases the drug concentration necessary to eliminate the infection can cause deterioration of vision, and in certain cases, result in total blindness. The financial burden of such diseases in domestic animals is shown by the enormous sums lost each year, because the only potential way to combat such infectious diseases is extensive therapy and quarantine.

The following trials assess the effectiveness of treatment using free antibiotics in glycerin, compared to antibiotics entrapped in SPLV for M. bovis infections of the eye. M. bovis causes infectious keratoconjunctivitis (pink eye) in cattle. This condition is characterized by blepharospasms, lacrimation, conjunctivitis and varying degrees of corneal opacity and ulceration. Adult animals can develop a mild fever with slightly reduced appetite and reduced milk production. Although a number of antibiotics are effective against M. bovis, they must be administered early and repeated frequently by topical application or subconjunctival injection.

According to the examples described herein, the effectiveness and duration of the therapeutic agent is extended. It is surprising that this system is effective with only one or two administrations, because such injections do not correspond to simple ordinary treatment with antibiotics. The usual treatments often leave behind residual infections that re-infect the eye, so that the infection cycle begins again if the infection is not completely eradicated by numerous repetitions of the treatment.

Treatment of infectious keratoconus. lunctivitis in mice

160 C57 black mice were divided into 8 groups. Half of each group was exposed to ultraviolet irradiation in each eye to create corneal lesions. All animals were then inoculated with M. bovis to the right eye only at concentrations of 1 x 10<6> bacteria per eye. 24 hours after inoculation, all animals were assessed for degree of corneal opacity. The 8 groups were treated by topical application of the following drug to each eye: Groups 1 and 2 received 10 µl of SPLV-captured streptomycin, 30 mg/ml;

Groups 3 and 4 received 10 µl streptomycin, 100 mg/ml;

Groups 5 and 6 received 10 µl buffered SPLV, suspended 1 aqueous streptomycin, 100 mg/ml; and

Groups 7 and 8 received 10 pl of sterile saline solution.

(NB! The uninfected left eye was treated with the same topical solution to determine whether SPLV would irritate the eye; no irritation was observed.) Once

(legally, the animals were scored for progression or regression of corneal lesions, and on days 3, 5 and 7 after treatment, the right eyes were dried and isolates of M. bovis were performed on representative animals. M. bovis colonies were determined by colony morphology and reactivity to fluorescently labeled antibodies to M. bovis pili The results shown in Table XIV show that only SPLV-captured streptomycin was effective in eliminating infections.

Treatment of rabbit con. 1unctiva when using

of SPLV-captured antibiotics

M. bovis ATCC strain 10900 was diluted to a concentration of 1 x 10<7> cells per ml in sterile saline (0.58$ NaCl). Amounts of 0.1 ml of bacterial suspensions were inoculated topically into the eye of 10 female adult rabbits. Samples of the cultures were taken daily by drying the conjunctiva and plated on blood agar plates for isolation of M. bovis. 3 days after inoculation, the rabbits were divided into 3 groups; 2 control animals received no treatment; 4 animals received streptomycin in sterile saline, at a concentration of 10 mg/kg body weight; and 4 animals received SPLV-captured streptomycin in a sterile saline solution at a concentration of 10 mg streptomycin per kg body weight. All solutions were administered topically in each eye. After 24 hours, drying of the conjunctivae for all rabbits was resumed, and continued daily for 7 days. The results of the isolation for M. bovis on blood agar plates are shown in Table XV.

Treatment of creatoconjunctivitis from

subcutaneous infections

M. bovis ATCC strain 10900 was diluted to a concentration of 1 x 10<7> cells per ml in sterile saline solution. Aliquots of 0.1 ml of bacterial suspensions were inoculated into the islets of adult rabbits previously infected as described in the previous section and not treated with SPLV. The right eye of all nine rabbits was inoculated with 0.1 ml of M. bovis subcutaneously in the conjunctival tissue and the left eye of all rabbits was inoculated with 0.1 ml of M. bovis topically. Cultures were taken daily from the conjunctivae in both eyes from all rabbits, and plated on blood agar plates for isolation of M. bovis. 3 days after inoculation, the rabbits were divided into 3 groups; 2 animals received no treatment; 3 animals received streptomycin in a standard ophthalmic glycerin suspension (concentration of streptomycin was 10 mg/kg body weight); and 4 animals received a saline suspension of SPLV-captured streptomycin (10 mg streptomycin per kg body weight). The suspension or solution was administered topically in an amount of 0.1 ml to each eye. After 24 hours and each of the next 5 days, conjunctival samples were taken from all animals. The results of the isolation of M. bovis on blood agar plates are shown in Table XVI. Necropsy was performed on all animals at the end of the experiments, and conjunctivae were removed from all animals. These were assessed for vascularization and were chopped up, homogenized and plated on blood agar plates for isolation of M. bovis. The results are shown in Table XVII.

Assessment of the effectiveness of SPLV compared to

liposome preparations in the treatment of ocular infections

M. bovis ATCC strain 10900 was diluted to a concentration of 1 x 10<7> cells per ml in sterile saline solution. Amounts of 1 ml of bacterial suspensions were inoculated subcutaneously into the conjunctival tissue of both eyes of adult rabbits. Wipes were taken daily from the conjunctivae of both eyes of all rabbits and plated on blood agar plates for isolation of M. bovis. 5 days after inoculation, the animals were divided into 6 groups: 2 animals received no treatment, these were control animals; 3 animals received a suspension of SPLV-encapsulated streptomycin in an amount of 10 mg streptomycin sulfate per kg body weight which in the diluted state 1:100 had an O.D.480 (optical density at 480 nm) equal to 0.928; 3 animals received a suspension of SPLV-encapsulated streptomycin in an amount of 10 mg streptomycin sulfate per kg body weight which in dilution 1:100 had an O.D.480 ™ of 0.449; 3 animals received a suspension of SPLV-encapsulated streptomycin (10 mg streptomycin sulfate per kg body weight) which, diluted to 1:100, had an O.D.480 = 0.242; 3 animals received a suspension of SPLV-encapsulated streptomycin (10 mg streptomycin sulfate per kg body weight) which, diluted to 1:100, had an O.D.480 = 0.119; and 2 animals received a suspension of multilamellar vesicles, MLV, containing streptomycin (10 mg streptomycin sulfate per kg body weight) with an O.D.4<g>o of 0.940 for a 1:100 dilution = 0.940. MLV was prepared by the method according to Fountain et al. in "Curr. Micro.", 6:373 (1981) by adding streptomycin sulfate to the dried llpld film which was then vigorously stirred and allowed to swell for 2 hours; unentrapped streptomycin was removed by repeated centrifugation.

The suspensions were administered topically to each eye. After 24 hours, conjuhctival samples were taken from all rabbits daily for 9 days, and plated on blood agar. The results for the isolation of M. bovis on blood agar plates are shown in Table XVIII. Necropsy was performed on all animals. These were assessed for lacrimal secretion and the conjunctivae were removed aseptically from all animals. These were assessed for vascularization and were chopped up, homogenized and plated on blood agar plates for isolation of M. bovis. The results are shown in Table XIX.

Example 5

TREATMENT OF VIRAL INFECTIONS

Lymphocytic choriomeningitis virus, LCMV, which belongs to the Arena virus group, is known to cause disease in humans and LCMV infection is fatal in mice inoculated intracerebrally with this virus. Mouse death is caused by the immune cells reacting against virus-infected cells. The virus does not kill the cells in which it reproduces, therefore the therapeutic agent used on mice must either inhibit virus reproduction, so that the immune cells are not activated, and/or inhibit activation of the immune cells.

The following example shows the effectiveness of treatment of viral infections by administration of SPLV-encapsulated antiviral compound.

Treatment of lethal lymphocytic choriomeningitis virus infection. loners in mice

Two-month-old Swiss mice were inoculated intracerebrally with a lethal dose of LCM virus, i.e. 100 plaque-forming units, PFU, in 0.05 ml of inoculum per mouse. The mice were divided into 4 groups of 7 animals each, and were treated on days 2, 3 and 4 after infection with intraperitoneal injections of 0.1 ml per dose mice as follows: (1) "SPLV-R group" was treated with a suspension of egg phosphatidylcholine SPLV containing 3 mg RIbavarin/ml. SPLV was prepared using 100 mg lipids and 0.3 ml of 100 mg drug per ml in PBS buffer; the capture of the drug was 10%; (2) the "R group" was treated with a solution of Ribavarin 3 mg/ml in PBS; (3) the "SPLV group" was treated with buffered SPLV (that is, SPLV prepared as above but without Ribavarin); and (4) the “control group” was treated with PBS. On the 5th day after infection, 2 mice from each group were euthanized and the spleens homogenized (2 spleens per group were homogenized in PBS at 1-20 weight/volume buffer). The plate-forming units, PFU, per ml was determined for each suspension. The remaining 5 mice in each group were observed for lethality 2 hours daily for 30 days. The results are given in Table XX.

Table XX clearly suggests a reduction in lethality and a reduction in the amount of virus recoverable from the infected animals. It is not yet determined whether these results are due to the antiviral activity of Ribavarin released from SPLV or an immunomodulation of the host mouse during the delayed release of Ribavarin from SPLV.

Claims (6)

1. Process for producing stable, plurilamellar vesicles with a vesicle size in the range of 500 to 10,000 nm and a number of lipid bisects in the range of a few to over 100; - with greater stability against autoxidation during storage i buffer; greater stability of the body fluid; a greater percentage of leakage of the capture, dissolved material by exposure to urea, guanidine or ammonium acetate; - a smaller percentage of leakage of the capture dissolved material by exposure to hydrochloric acid or serum; and a distribution of captured content via cytosol in cells by administration to the cells in culture; characterized by: a) forming a dispersion of at least one amphipathic lipid in an organic solvent; b) combining the dispersion obtained under a) with an aqueous phase to form a two-phase mixture in which the aqueous phase can be completely emulsified, the volume ratio between solvent and aqueous phase being from approx. 3:1 to 100:1; and c) emulsifying the aqueous phase and evaporating the organic solvent of the two-phase mixture, in that steps a), b) and c) work at a temperature within the range 4-60°C, in order to obtain stable plurilamellar vesicles essentially free of MLV, SUV and REV. 2. Method according to claim 1, characterized in that under points a) and b). the temperature is kept below the phase transformation temperature of at least one of the lipids. 3. Method according to claim 1, characterized in that during step a) a fluorocarbon, a diethyl ether or mixtures thereof are used as solvent. 4. Method according to claim 3, characterized in that the solvent used is one which contains an antioxidant, preferably butylated hydroxytoluene. 5. Method according to claim 1, characterized in that the emulsification According to step c) is carried out before or simultaneously with the evaporation. 6. Method according to claim 1, characterized in that at the same time as the aqueous phase in step b) the material to be captured in the vesicles is added, so that at least 2056 of this material is captured in the vesicles.